TW202309102A - Cd8-targeted modified il-2 polypeptides and uses thereof - Google Patents

Cd8-targeted modified il-2 polypeptides and uses thereof Download PDF

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TW202309102A
TW202309102A TW111127072A TW111127072A TW202309102A TW 202309102 A TW202309102 A TW 202309102A TW 111127072 A TW111127072 A TW 111127072A TW 111127072 A TW111127072 A TW 111127072A TW 202309102 A TW202309102 A TW 202309102A
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polypeptide
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約翰 Ｃ 湯門
威廉 奎葛
佛羅里安 薩梅爾
盧卡斯 瑞斯肯
伯瑞登 Ｐ 艾克曼
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美商英伊布里克斯公司
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Abstract

Provided herein are polypeptides comprising at least one VHH domain that binds CD8 and a modified IL-2. Uses of the polypeptides are also provided.

Description

靶向CD8之經修飾IL-2多肽及其用途Modified IL-2 polypeptides targeting CD8 and uses thereof

本發明係關於靶向CD8之經修飾IL-2多肽及使用此類多肽調節CD8+細胞之生物活性的方法。該等方法包括但不限於治療癌症之方法。在一些實施例中，靶向CD8之經修飾IL-2多肽為包含CD8結合多肽及經修飾IL-2的融合多肽。The present invention relates to modified IL-2 polypeptides targeting CD8 and methods of using such polypeptides to modulate the biological activity of CD8+ cells. Such methods include, but are not limited to, methods of treating cancer. In some embodiments, a modified IL-2 polypeptide targeting CD8 is a fusion polypeptide comprising a CD8 binding polypeptide and modified IL-2.

CD8為跨膜醣蛋白，其表現於細胞毒性T細胞(CD8+ T細胞)以及淋巴系統之其他細胞(包括自然殺手細胞、γδ T細胞、皮質胸腺細胞及樹突狀細胞亞群)之表面上。CD8通常為由CD8α鏈及CD8β鏈構成之雜二聚體，但在一些情況下可以CD8α均二聚體形式存在。在細胞毒性T細胞上，CD8充當T細胞受體(TCR)之共受體以增強抗原識別及T細胞活化。細胞毒性T細胞活化係藉由TCR與結合至I類主要組織相容複合體(MHC)蛋白質之肽抗原的相互作用來支配。CD8經由結合至I類MHC蛋白之恆定區來幫助使TCR/肽-MHC相互作用穩定。CD8亦藉由將Lck募集至CD8α之細胞質域來增強TCR信號傳導，從而產生放大T細胞活化信號之級聯。CD8 is a transmembrane glycoprotein that is expressed on the surface of cytotoxic T cells (CD8+ T cells) and other cells of the lymphatic system (including natural killer cells, γδ T cells, cortical thymocytes and dendritic cell subsets). CD8 is usually a heterodimer composed of CD8α chain and CD8β chain, but in some cases it can exist as a CD8α homodimer. On cytotoxic T cells, CD8 serves as a co-receptor for the T cell receptor (TCR) to enhance antigen recognition and T cell activation. Cytotoxic T cell activation is governed by the interaction of the TCR with peptide antigens bound to class I major histocompatibility complex (MHC) proteins. CD8 helps stabilize TCR/peptide-MHC interactions by binding to the constant regions of class I MHC proteins. CD8 also enhances TCR signaling by recruiting Lck to the cytoplasmic domain of CD8α, thereby creating a cascade that amplifies T cell activation signals.

T細胞之活化亦受其他分子控制，該等其他分子包括由活化T細胞自身合成及分泌的細胞介素介白素-2 (IL-2)。IL-2被發現為T細胞生長因子，係一種多效性細胞介素，調節輔助T細胞之分化，加強自然殺手細胞之溶胞活性且調節CD8+ T細胞產生。IL-2結合至由T細胞表面上之三個次單元(IL-2α、IL-2β及γc)構成之高親和性受體。經由IL-2受體複合物之信號傳導觸發T細胞經由細胞分裂進行，驅動活化T細胞之純系擴增。IL-2即使在低濃度下亦具有強效作用，因此其產生受精確控制且需要經由TCR/共受體(CD8)複合物遞送之信號及經由CD28遞送之共刺激信號。Activation of T cells is also controlled by other molecules, including the interleukin-2 (IL-2) synthesized and secreted by the activated T cells themselves. IL-2 was discovered as a T cell growth factor, a pleiotropic interleukin that regulates the differentiation of helper T cells, enhances the lytic activity of natural killer cells and regulates the production of CD8+ T cells. IL-2 binds to a high-affinity receptor composed of three subunits (IL-2α, IL-2β, and γc) on the surface of T cells. Signaling through the IL-2 receptor complex triggers T cells to proceed through cell division, driving the homologous expansion of activated T cells. IL-2 is potent even at low concentrations, so its production is precisely controlled and requires signaling via the TCR/co-receptor (CD8) complex and costimulatory signaling via CD28.

因此，使IL-2靶向細胞毒性CD8+ T細胞可提高細胞毒性T細胞反應之效能及選擇性，且存在對靶向CD8之經修飾IL-2多肽之需求。Therefore, targeting IL-2 to cytotoxic CD8+ T cells may increase the potency and selectivity of cytotoxic T cell responses, and a need exists for modified IL-2 polypeptides that target CD8.

本文提供靶向CD8之經修飾IL-2多肽及使用靶向CD8之經修飾IL-2多肽治療例如癌症之方法。在一些實施例中，靶向CD8之經修飾IL-2多肽包含一或多個額外結合域及/或細胞介素序列。下文列出某些實施例。 實施例1. 一種多肽，其包含至少一個結合CD8之VHH域及經修飾IL-2，其中至少一個結合CD8之VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列，且其中該經修飾IL-2相對於包含SEQ ID NO: 71之胺基酸序列的野生型人類IL-2而言包含T3A、H16A、P65R、C125S突變及D84S或D84Y突變。 實施例2. 如實施例1之多肽，其中該經修飾IL-2包含T3A、H16A、P65R、C125S及D84S突變。 實施例3. 如實施例1之多肽，其中該經修飾IL-2包含T3A、H16A、E61R、P65R、C125S及D84Y突變。 實施例4. 如實施例1或2之多肽，其中該經修飾IL-2包含SEQ ID NO: 79之胺基酸序列。 實施例5. 如實施例1或3之多肽，其中該經修飾IL-2包含SEQ ID NO: 78之胺基酸序列。 實施例6. 如實施例1至5中任一項之多肽，其中至少一個VHH域包含CDR1、CDR2及CDR3，其分別包含胺基酸序列SEQ ID NO: 3、4及5；3、12及5；3、14及5；3、4及16；3、4及18；3、22及5；3、14及18；3、27及5；3、29及5；3、31及5；80、14及18；81、14及18；3、82及18；3、83及18；3、84及18；3、85及18；3、86及18；或3、87及18。 實施例7. 如實施例1至6中任一項之多肽，其中至少一個VHH域包含CDR1，其包含SEQ ID NO: 3之胺基酸序列；CDR2，其包含SEQ ID NO: 85之胺基酸序列；及CDR3，其包含SEQ ID NO: 18之胺基酸序列。 實施例8. 如實施例1至7中任一項之多肽，其中至少一個VHH域或各VHH域經人類化。 實施例9. 如實施例1至8中任一項之多肽，其中至少一個VHH域包含與SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列至少85%、90%、95%或至少99%一致的胺基酸序列。 實施例10. 如實施例1至9中任一項之多肽，其中至少一個VHH域包含SEQ ID NO: 6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列。 實施例11. 如實施例1至10中任一項之多肽，其中至少一個VHH域包含SEQ ID NO: 99或109之胺基酸序列。 實施例12. 如實施例1至11中任一項之多肽，其包含兩個VHH域。 實施例13. 如實施例1至11中任一項之多肽，其包含三個VHH域。 實施例14. 如實施例1至13中任一項之多肽，其中該多肽包含Fc區。 實施例15. 如實施例14之多肽，其中該Fc區包含選自SEQ ID NO: 32-70或112-122之胺基酸序列。 實施例16. 如實施例14或實施例15之多肽，其中該經修飾IL-2融合至該Fc區之C端。 實施例17. 如實施例1至11、14及15中任一項之多肽，其中該多肽包含一個結合CD8之VHH域、Fc區及經修飾IL-2。 實施例18. 如實施例1或實施例17之多肽，其中該多肽包含SEQ ID NO: 72、74、76、107或110之胺基酸序列。 實施例19. 如實施例1至18中任一項之多肽，其中該多肽包含至少一個結合除CD8以外之抗原的抗原結合域。 實施例20. 如實施例19之多肽，其包含至少一個結合以下之抗原結合域：TGFBR1、TGFBR2、Fas、TNFR2、1-92-LFA-3、5T4、α-4整合素、α-V整合素、α4β1整合素、α4β7整合素、AGR2、抗Lewis-Y、Apelin J受體、APRIL、B7-H3、B7-H4、B7-H6、BAFF、BCMA、BTLA、C5補體、C-242、CA9、CA19-9、(Lewis a)、碳酸酐酶9、CD2、CD3、CD6、CD9、CD11a、CD19、CD20、CD22、CD24、CD25、CD27、CD28、CD30、CD33、CD38、CD39、CD40、CD40L、CD41、CD44、CD44v6、CD47、CD51、CD52、CD56、CD64、CD70、CD71、CD73、CD74、CD80、CD81、CD86、CD95、CD117、CD123、CD125、CD132、(IL-2RG)、CD133、CD137、CD138、CD166、CD172A、CD248、CDH6、CEACAM5 (CEA)、CEACAM6 (NCA-90)、CLAUDIN-3、CLAUDIN-4、cMet、膠原蛋白、Cripto、CSFR、CSFR-1、CTLA4、CTGF、CXCL10、CXCL13、CXCR1、CXCR2、CXCR4、CYR61、DL44、DLK1、DLL3、DLL4、DPP-4、DSG1、EDA、EDB、EGFR、EGFRviii、內皮素B受體(ETBR)、ENPP3、EpCAM、EPHA2、EPHB2、ERBB3、RSV之F蛋白質、FAP、FcRH5、FGF-2、FGF8、FGFR1、FGFR2、FGFR3、FGFR4、FLT-3、葉酸受體α (FRα)、GAL3ST1、G-CSF、G-CSFR、GD2、GITR、GLUT1、GLUT4、GM-CSF、GM-CSFR、GP IIb/IIIa受體、Gp130、GPIIB/IIIA、GPNMB、GPRC5D、GRP78、HAVCAR1、HER2/neu、HER3、HER4、HGF、hGH、HVEM、玻尿酸酶、ICOS、IFNα、IFNβ、IFNγ、IgE、IgE受體(FceRI)、IGF、IGF1R、IL1B、IL1R、IL2、IL11、IL12、IL12p40、IL-12R、IL-12Rβ1、IL13、IL13R、IL15、IL17、IL18、IL21、IL23、IL23R、IL27/IL27R (wsx1)、IL29、IL-31R、IL31/IL31R、IL2R、IL4、IL4R、IL6、IL6R、胰島素受體、鋸齒狀配位體、鋸齒狀1、鋸齒狀2、KISS1-R、LAG-3、LIF-R、Lewis X、LIGHT、LRP4、LRRC26、Ly6G6D、LyPD1、MCSP、間皮素、MICA、MICB、MRP4、MUC1、黏蛋白-16 (MUC16、CA-125)、Na/K ATP酶、NGF、Nicastrin、Notch受體、Notch 1、Notch 2、Notch 3、Notch 4、NOV、OSM-R、OX-40、PAR2、PDGF-AA、PDGF-BB、PDGFRα、PDGFRβ、PD-1、PD-L1、PD-L2、磷脂醯絲胺酸、P1GF、PSCA、PSMA、PSGR、RAAG12、RAGE、SLC44A4、神經鞘胺醇1磷酸酯、STEAP1、STEAP2、TAG-72、TAPA1、TEM-8、TGFβ、TIGIT、TIM-3、TLR2、TLR4、TLR6、TLR7、TLR8、TLR9、TMEM31、TNFα、TNFR、TNFRS12A、TRAIL-R1、TRAIL-R2、運鐵蛋白、運鐵蛋白受體、TRK-A、TRK-B、TROP-2 uPAR、VAP1、VCAM-1、VEGF、VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGFR1、VEGFR2、VEGFR3、VISTA、WISP-1、WISP-2或WISP-3。 實施例21. 如實施例19或20之多肽，其中至少一個結合除CD8以外之抗原的抗原結合域為VHH域。 實施例22. 如實施例21之多肽，其中各結合除CD8以外之抗原的抗原結合域為VHH域。 實施例23. 如實施例19至21中任一項之多肽，其中至少一個結合除CD8以外之抗原的抗原結合域包含重鏈可變區及輕鏈可變區。 實施例24. 如實施例23之多肽，其中各結合除CD8以外之抗原的抗原結合域包含重鏈可變區及輕鏈可變區。 實施例25. 一種包含第一多肽及第二多肽之複合物，其中該第一多肽為如實施例14至24中任一項之多肽，其中該第一多肽包含第一Fc區，且其中該第二多肽包含至少一個結合CD8之VHH域及第二Fc區，其中該等第一與第二Fc區相同或不同。 實施例26. 一種包含第一多肽及第二多肽之複合物，其中該第一多肽包含至少一個結合CD8之VHH域及第一Fc區，且該第二多肽包含第二Fc區及經修飾IL-2，其中至少一個結合CD8之VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列；且其中該經修飾IL-2相對於包含SEQ ID NO: 71之胺基酸序列的野生型人類IL-2而言包含T3A或T3G、H16A、P65R、C125S及D84S或D84Y突變。 實施例27. 如實施例26之複合物，其中該CDR1包含SEQ ID NO: 3之胺基酸序列；該CDR2包含SEQ ID NO: 85之胺基酸序列；且該CDR3包含SEQ ID NO: 18之胺基酸序列。 實施例28. 如實施例26或實施例27之複合物，其中該經修飾IL-2包含T3A、H16A、P65R、C125S及D84S突變。 實施例29. 如實施例26或實施例27之複合物，其中該經修飾IL-2包含T3A、H16A、E61R、P65R、C125S及D84Y突變。 實施例30. 如實施例26至29中任一項之複合物，其中該經修飾IL-2包含與SEQ ID NO: 78或79之胺基酸序列至少90%、至少95%、至少97%、至少98%、至少99%或100%一致的胺基酸序列。 實施例31. 如實施例26、27及29中任一項之複合物，其中該經修飾IL-2包含SEQ ID NO: 78之胺基酸序列。 實施例32. 如實施例26至31中任一項之複合物，其中至少一個VHH域或各VHH域經人類化。 實施例33. 如實施例26至32中任一項之複合物，其中至少一個VHH域包含與選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列至少85%、至少90%、至少95%或至少99%一致的胺基酸序列。 實施例34. 如實施例26至33中任一項之複合物，其中至少一個VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列。 實施例35. 如實施例26至34中任一項之複合物，其中至少一個VHH域包含SEQ ID NO: 99或109之胺基酸序列。 實施例36. 如實施例25至35中任一項之複合物，其中該第二多肽包含至少一個抗原結合域。 實施例37. 如實施例36之複合物，其中該第二多肽之至少一個抗原結合域為VHH域。 實施例38. 如實施例37之複合物，其中該第二多肽包含至少一個結合CD8之VHH域。 實施例39. 如實施例25至38中任一項之複合物，其中該第二多肽包含至少一個結合CD8之VHH域，該VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列。 實施例40. 如實施例39之複合物，其中該第二多肽包含至少一個VHH域，該VHH域包含CDR1、CDR2及CDR3，其分別包含胺基酸序列SEQ ID NO: 3、4及5；3、12及5；3、14及5；3、4及16；3、4及18；3、22及5；3、14及18；3、27及5；3、29及5；3、31及5；80、14及18；81、14及18；3、82及18；3、83及18；3、84及18；3、85及18；3、86及18；或3、87及18。 實施例41. 如實施例39或實施例40之複合物，其中該第二多肽包含至少一個VHH域，該VHH域包含CDR1，其包含SEQ ID NO: 3之胺基酸序列；CDR2，其包含SEQ ID NO: 85之胺基酸序列；及CDR3，其包含SEQ ID NO: 18之胺基酸序列。 實施例42. 如實施例39至41中任一項之複合物，其中該第二多肽之至少一個VHH域或各VHH域經人類化。 實施例43. 如實施例39至42中任一項之複合物，其中該第二多肽之至少一個VHH域包含與SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列至少85%、90%、95%或至少99%一致的胺基酸序列。 實施例44. 如實施例39至43中任一項之複合物，其中該第二多肽之至少一個VHH域包含SEQ ID NO: 6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列。 實施例45. 如實施例39至44中任一項之複合物，其中該第二多肽之至少一個VHH域包含SEQ ID NO: 99或109之胺基酸序列。 實施例46. 如實施例25至45中任一項之複合物，其中該第二多肽包含一個結合CD8之VHH域。 實施例47. 如實施例25至46中任一項之複合物，其中該第一Fc區包含至少一個杵突變且該第二Fc區包含至少一個臼突變；或其中該第一Fc區包含至少一個臼突變且該第二Fc區包含至少一個杵突變。 實施例48. 如實施例47之複合物，其中該第一或第二Fc區包含T366W突變且該第一或第二Fc區中之另一者包含T366S、L368A及Y407V突變。 實施例49. 如實施例48之複合物，其中包含該等T366S、L368A及Y407V突變之該Fc區進一步包含H435R或H435K突變。 實施例50. 如實施例25至49中任一項之複合物，其中該第一多肽包含SEQ ID NO: 72、74、76、107或110之胺基酸序列。 實施例51. 如實施例25至50中任一項之複合物，其中該第二多肽包含SEQ ID NO: 73、75、108或111之胺基酸序列。 實施例52. 如實施例51之複合物，其中該第一多肽包含SEQ ID NO: 72之胺基酸序列且該第二多肽包含SEQ ID NO: 73之胺基酸序列；該第一多肽包含SEQ ID NO: 74之胺基酸序列且該第二多肽包含SEQ ID NO:75之胺基酸序列；該第一多肽包含SEQ ID NO: 76之胺基酸序列且該第二多肽包含SEQ ID NO:75之胺基酸序列；該第一多肽包含SEQ ID NO: 107之胺基酸序列且該第二多肽包含SEQ ID NO:108之胺基酸序列；或該第一多肽包含SEQ ID NO: 110之胺基酸序列且該第二多肽包含SEQ ID NO:111之胺基酸序列。 實施例53. 如實施例25至52中任一項之複合物，其中該複合物在生理條件下形成。 實施例54. 如實施例1至53中任一項之多肽或複合物，其中該CD8為人類CD8。 實施例55. 如實施例54之多肽或複合物，其中該人類CD8包含SEQ ID NO: 1之序列。 實施例56. 一種醫藥組合物，其包含如實施例1至55中任一項之多肽或複合物及醫藥學上可接受之載劑。 實施例57. 一種經分離核酸，其編碼如實施例1至55中任一項之多肽或複合物。 實施例58. 一種載體，其包含如實施例57之核酸。 實施例59. 一種宿主細胞，其包含如實施例57之核酸或如實施例58之載體。 實施例60. 一種宿主細胞，其表現如實施例1至55中任一項之多肽或複合物。 實施例61. 一種產生如實施例1至55中任一項之多肽或複合物之方法，其包含在適合於表現該多肽或複合物之條件下培育如實施例59或實施例60之宿主細胞。 實施例62. 如實施例61之方法，其進一步包含分離該多肽或複合物。 實施例63. 一種增加CD8 +T細胞增殖之方法，其包含使T細胞與如實施例1至55中任一項之多肽或複合物接觸。 實施例64. 如實施例63之方法，其中該等CD8 +T細胞係在活體外。 實施例65. 如實施例63之方法，其中該等CD8 +T細胞係在活體內。 實施例66. 一種治療癌症之方法，其包含向患有癌症之個體投與醫藥學上有效量之如實施例1至55中任一項之多肽或複合物或如實施例56之醫藥組合物。 實施例67. 如實施例66之方法，其中該癌症係選自基底細胞癌、膽道癌；膀胱癌；骨癌；腦及中樞神經系統癌；乳癌；腹膜癌；子宮頸癌；絨毛膜癌；大腸直腸癌；結締組織癌；消化系統癌；子宮內膜癌；食道癌；眼癌；頭頸癌；胃癌；胃腸癌；神經膠母細胞瘤；肝癌瘤；肝腫瘤；上皮內贅瘤；腎臟癌或腎癌；喉癌；肝癌；肺癌；小細胞肺癌；非小細胞肺癌；肺腺癌；鱗狀細胞肺癌(squamous carcinoma of the lung)；黑素瘤；骨髓瘤；神經母細胞瘤；口腔癌；卵巢癌；胰臟癌；前列腺癌；視網膜母細胞瘤；橫紋肌肉瘤；直腸癌；呼吸系統癌；唾液腺癌；肉瘤；皮膚癌；鱗狀細胞癌；胃癌；睪丸癌；甲狀腺癌；子宮或子宮內膜癌；泌尿系統癌；外陰癌；淋巴瘤；霍奇金氏淋巴瘤(Hodgkin's lymphoma)；非霍奇金氏淋巴瘤；B細胞淋巴瘤；低惡性度(low grade)/濾泡型非霍奇金氏淋巴瘤(NHL)；小淋巴球性(SL) NHL；中惡性度/濾泡型NHL；中惡性度彌漫性NHL；高惡性度免疫母細胞NHL；高惡性度淋巴母細胞NHL；高惡性度小型無裂隙細胞NHL；腫塊性病變NHL (bulky disease NHL)；套細胞淋巴瘤；AIDS相關淋巴瘤；瓦爾登斯特倫氏巨球蛋白血症(Waldenstrom's macroglobulinemia)；慢性淋巴球性白血病(CLL)；急性淋巴母細胞白血病(ALL)；毛細胞白血病及慢性骨髓母細胞白血病。 實施例68. 如實施例66或67之方法，其進一步包含投與額外治療劑。 實施例69. 如實施例68之方法，其中該額外治療劑為抗癌劑。 實施例70. 如實施例69之方法，其中該抗癌劑係選自化學治療劑、抗癌生物製劑、放射線療法、CAR-T療法及溶瘤病毒。 實施例71. 如實施例68之方法，其中該額外治療劑為抗癌生物製劑。 實施例72. 如實施例71之方法，其中該抗癌生物製劑為抑制PD-1及/或PD-L1之藥劑。 實施例73. 如實施例71之方法，其中該抗癌生物製劑為抑制VISTA、gpNMB、B7H3、B7H4、HHLA2、CTLA4或TIGIT之藥劑。 實施例74. 如實施例69之方法，其中該抗癌劑為抗體。 實施例75. 如實施例71之方法，其中該抗癌生物製劑為細胞介素。 實施例76. 如實施例69之方法，其中該抗癌劑為CAR-T療法。 實施例77. 如實施例69之方法，其中該抗癌劑為溶瘤病毒。 實施例78. 如實施例66至77中任一項之方法，其進一步包含腫瘤切除及/或放射線療法。 Provided herein are modified IL-2 polypeptides that target CD8 and methods of using modified IL-2 polypeptides that target CD8 to treat, for example, cancer. In some embodiments, modified IL-2 polypeptides targeting CD8 comprise one or more additional binding domains and/or interleukin sequences. Some examples are listed below. Example 1. A polypeptide comprising at least one CD8-binding VHH domain and modified IL-2, wherein at least one CD8-binding VHH domain comprises CDR1, which comprises the amino acid sequence of SEQ ID NO: 3, 80 or 81 ; CDR2, which includes the amino acid sequence of SEQ ID NO: 4, 12, 14, 22, 27, 29, 31, 82, 83, 84, 85, 86 or 87; and CDR3, which includes SEQ ID NO: 5 , 16 or 18 amino acid sequence, and wherein the modified IL-2 includes T3A, H16A, P65R, C125S mutations and D84S or D84Y mutation. Embodiment 2. The polypeptide of embodiment 1, wherein the modified IL-2 comprises T3A, H16A, P65R, C125S and D84S mutations. Embodiment 3. The polypeptide of embodiment 1, wherein the modified IL-2 comprises T3A, H16A, E61R, P65R, C125S and D84Y mutations. Embodiment 4. The polypeptide of embodiment 1 or 2, wherein the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 79. Embodiment 5. The polypeptide of embodiment 1 or 3, wherein the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 78. Embodiment 6. The polypeptide of any one of embodiments 1 to 5, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3, which respectively comprise the amino acid sequences SEQ ID NO: 3, 4 and 5; 3, 12 and 5; 3, 14 and 5; 3, 4 and 16; 3, 4 and 18; 3, 22 and 5; 3, 14 and 18; 3, 27 and 5; 3, 29 and 5; 3, 31 and 5; 80, 14 and 18; 81, 14 and 18; 3, 82 and 18; 3, 83 and 18; 3, 84 and 18; 3, 85 and 18; 3, 86 and 18; or 3, 87 and 18. Embodiment 7. The polypeptide of any one of embodiments 1 to 6, wherein at least one VHH domain comprises CDR1, which comprises the amino acid sequence of SEQ ID NO: 3; CDR2, which comprises the amino group of SEQ ID NO: 85 Acid sequence; and CDR3, which includes the amino acid sequence of SEQ ID NO: 18. Embodiment 8. The polypeptide of any one of embodiments 1 to 7, wherein at least one VHH domain or each VHH domain is humanized. Embodiment 9. The polypeptide of any one of embodiments 1 to 8, wherein at least one VHH domain comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, An amino acid sequence that is at least 85%, 90%, 95% or at least 99% identical to the amino acid sequence of 105, 106 or 109. Embodiment 10. The polypeptide of any one of embodiments 1 to 9, wherein at least one VHH domain comprises SEQ ID NO: 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21 ,23,24,25,26,28,30,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106 Or the amino acid sequence of 109. Embodiment 11. The polypeptide of any one of embodiments 1 to 10, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99 or 109. Embodiment 12. The polypeptide of any one of embodiments 1 to 11, comprising two VHH domains. Embodiment 13. The polypeptide of any one of embodiments 1 to 11, comprising three VHH domains. Embodiment 14. The polypeptide of any one of embodiments 1 to 13, wherein the polypeptide comprises an Fc region. Embodiment 15. The polypeptide of embodiment 14, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 32-70 or 112-122. Embodiment 16. The polypeptide of embodiment 14 or embodiment 15, wherein the modified IL-2 is fused to the C-terminus of the Fc region. Embodiment 17. The polypeptide of any one of embodiments 1 to 11, 14 and 15, wherein the polypeptide comprises a VHH domain that binds CD8, an Fc region and modified IL-2. Embodiment 18. The polypeptide of embodiment 1 or embodiment 17, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 72, 74, 76, 107 or 110. Embodiment 19. The polypeptide of any one of embodiments 1 to 18, wherein the polypeptide comprises at least one antigen-binding domain that binds an antigen other than CD8. Embodiment 20. The polypeptide of embodiment 19, comprising at least one antigen-binding domain binding to: TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin protein, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9 , CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L , CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137 , CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3 , RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptor, Gp130, GPIIB/IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2/neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18 , IL21, IL23, IL23R, IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, serrated ligand, serrated 1, serrated 2. KISS1-R, LAG-3, LIF-R, Lewis 125), Na/K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα , PDGFRβ, PD-1, PD-L1, PD-L2, phospholipid serine, P1GF, PCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72 , TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin Receptor, TRK-A, TRK-B, TROP-2 uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP- 1. WISP-2 or WISP-3. Embodiment 21. The polypeptide of embodiment 19 or 20, wherein at least one antigen-binding domain that binds an antigen other than CD8 is a VHH domain. Embodiment 22. The polypeptide of embodiment 21, wherein each antigen-binding domain that binds an antigen other than CD8 is a VHH domain. Embodiment 23. The polypeptide of any one of embodiments 19 to 21, wherein at least one antigen-binding domain that binds an antigen other than CD8 comprises a heavy chain variable region and a light chain variable region. Embodiment 24. The polypeptide of embodiment 23, wherein each antigen-binding domain that binds an antigen other than CD8 includes a heavy chain variable region and a light chain variable region. Embodiment 25. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is the polypeptide of any one of embodiments 14 to 24, wherein the first polypeptide comprises a first Fc region , and wherein the second polypeptide comprises at least one CD8-binding VHH domain and a second Fc region, wherein the first and second Fc regions are the same or different. Embodiment 26. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least one CD8-binding VHH domain and a first Fc region, and the second polypeptide comprises a second Fc region and modified IL-2, wherein at least one CD8-binding VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 80 or 81; CDR2 comprising SEQ ID NO: 4, 12, 14, 22 , the amino acid sequence of SEQ ID NO: 5, 16 or 18; and wherein the modified IL -2 contains T3A or T3G, H16A, P65R, C125S and D84S or D84Y mutations relative to wild-type human IL-2 comprising the amino acid sequence of SEQ ID NO: 71. Embodiment 27. The complex of embodiment 26, wherein the CDR1 includes the amino acid sequence of SEQ ID NO: 3; the CDR2 includes the amino acid sequence of SEQ ID NO: 85; and the CDR3 includes SEQ ID NO: 18 The amino acid sequence. Embodiment 28. The complex of embodiment 26 or embodiment 27, wherein the modified IL-2 comprises T3A, H16A, P65R, C125S and D84S mutations. Embodiment 29. The complex of embodiment 26 or embodiment 27, wherein the modified IL-2 comprises T3A, H16A, E61R, P65R, C125S and D84Y mutations. Embodiment 30. The complex of any one of embodiments 26 to 29, wherein the modified IL-2 comprises at least 90%, at least 95%, or at least 97% the amino acid sequence of SEQ ID NO: 78 or 79 , an amino acid sequence that is at least 98%, at least 99%, or 100% identical. Embodiment 31. The complex of any one of embodiments 26, 27 and 29, wherein the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 78. Embodiment 32. The complex of any one of embodiments 26 to 31, wherein at least one or each VHH domain is humanized. Embodiment 33. The complex of any one of embodiments 26 to 32, wherein at least one VHH domain comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17 ,19,20,21,23,24,25,26,28,30,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103 , the amino acid sequences of 104, 105, 106 and 109 are at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequences. Embodiment 34. The complex of any one of embodiments 26 to 33, wherein at least one VHH domain comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, Amino acid sequences of 104, 105, 106 and 109. Embodiment 35. The complex of any one of embodiments 26 to 34, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99 or 109. Embodiment 36. The complex of any one of embodiments 25 to 35, wherein the second polypeptide comprises at least one antigen binding domain. Embodiment 37. The complex of embodiment 36, wherein at least one antigen-binding domain of the second polypeptide is a VHH domain. Embodiment 38. The complex of embodiment 37, wherein the second polypeptide comprises at least one VHH domain that binds CD8. Embodiment 39. The complex of any one of embodiments 25 to 38, wherein the second polypeptide comprises at least one VHH domain that binds CD8, the VHH domain comprising CDR1 comprising SEQ ID NO: 3, 80 or 81 The amino acid sequence of SEQ ID NO: 4, 12, 14, 22, 27, 29, 31, 82, 83, 84, 85, 86 or 87; and CDR3, which includes Amino acid sequence of SEQ ID NO: 5, 16 or 18. Embodiment 40. The complex of embodiment 39, wherein the second polypeptide comprises at least one VHH domain, and the VHH domain comprises CDR1, CDR2 and CDR3, which respectively comprise the amino acid sequences SEQ ID NO: 3, 4 and 5. ;3, 12 and 5; 3, 14 and 5; 3, 4 and 16; 3, 4 and 18; 3, 22 and 5; 3, 14 and 18; 3, 27 and 5; 3, 29 and 5; 3 , 31 and 5; 80, 14 and 18; 81, 14 and 18; 3, 82 and 18; 3, 83 and 18; 3, 84 and 18; 3, 85 and 18; 3, 86 and 18; or 3, 87 and 18. Embodiment 41. The complex of embodiment 39 or embodiment 40, wherein the second polypeptide comprises at least one VHH domain, and the VHH domain comprises CDR1, which comprises the amino acid sequence of SEQ ID NO: 3; CDR2, which Comprising the amino acid sequence of SEQ ID NO: 85; and CDR3 comprising the amino acid sequence of SEQ ID NO: 18. Embodiment 42. The complex of any one of embodiments 39 to 41, wherein at least one or each VHH domain of the second polypeptide is humanized. Embodiment 43. The complex of any one of embodiments 39 to 42, wherein at least one VHH domain of the second polypeptide comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13 ,15,17,19,20,21,23,24,25,26,28,30,88,89,90,91,92,93,94,95,96,97,98,99,100,101 , an amino acid sequence that is at least 85%, 90%, 95% or at least 99% identical to the amino acid sequence of 102, 103, 104, 105, 106 or 109. Embodiment 44. The complex of any one of embodiments 39 to 43, wherein at least one VHH domain of the second polypeptide comprises SEQ ID NOs: 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, Amino acid sequence 103, 104, 105, 106 or 109. Embodiment 45. The complex of any one of embodiments 39 to 44, wherein at least one VHH domain of the second polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 109. Embodiment 46. The complex of any one of embodiments 25 to 45, wherein the second polypeptide comprises a VHH domain that binds CD8. Embodiment 47. The complex of any one of embodiments 25 to 46, wherein the first Fc region comprises at least one hammer mutation and the second Fc region comprises at least one hammer mutation; or wherein the first Fc region comprises at least one cleavage mutation and the second Fc region contains at least one knuckle mutation. Embodiment 48. The complex of embodiment 47, wherein the first or second Fc region comprises the T366W mutation and the other of the first or second Fc region comprises the T366S, L368A and Y407V mutations. Embodiment 49. The complex of embodiment 48, wherein the Fc region comprising the T366S, L368A and Y407V mutations further comprises an H435R or H435K mutation. Embodiment 50. The complex of any one of embodiments 25 to 49, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 72, 74, 76, 107 or 110. Embodiment 51. The complex of any one of embodiments 25 to 50, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 73, 75, 108 or 111. Embodiment 52. The complex of embodiment 51, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 72 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 73; the first The polypeptide includes the amino acid sequence of SEQ ID NO: 74 and the second polypeptide includes the amino acid sequence of SEQ ID NO: 75; the first polypeptide includes the amino acid sequence of SEQ ID NO: 76 and the second polypeptide includes the amino acid sequence of SEQ ID NO: 76. The two polypeptides comprise the amino acid sequence of SEQ ID NO: 75; the first polypeptide comprises the amino acid sequence of SEQ ID NO: 107 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108; or The first polypeptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 111. Embodiment 53. The complex of any one of embodiments 25 to 52, wherein the complex is formed under physiological conditions. Embodiment 54. The polypeptide or complex of any one of embodiments 1 to 53, wherein the CD8 is human CD8. Embodiment 55. The polypeptide or complex of embodiment 54, wherein the human CD8 comprises the sequence of SEQ ID NO: 1. Embodiment 56. A pharmaceutical composition comprising the polypeptide or complex of any one of embodiments 1 to 55 and a pharmaceutically acceptable carrier. Embodiment 57. An isolated nucleic acid encoding the polypeptide or complex of any one of embodiments 1 to 55. Embodiment 58. A vector comprising the nucleic acid of embodiment 57. Embodiment 59. A host cell comprising the nucleic acid of embodiment 57 or the vector of embodiment 58. Embodiment 60. A host cell expressing the polypeptide or complex of any one of embodiments 1 to 55. Embodiment 61. A method of producing a polypeptide or complex as in any one of embodiments 1 to 55, comprising culturing a host cell as in embodiment 59 or embodiment 60 under conditions suitable for expressing the polypeptide or complex. . Embodiment 62. The method of embodiment 61, further comprising isolating the polypeptide or complex. Embodiment 63. A method of increasing the proliferation of CD8 + T cells, comprising contacting the T cells with the polypeptide or complex of any one of embodiments 1 to 55. Embodiment 64. The method of embodiment 63, wherein the CD8 + T cells are in vitro. Embodiment 65. The method of embodiment 63, wherein the CD8 + T cells are in vivo. Embodiment 66. A method of treating cancer, comprising administering to an individual suffering from cancer a pharmaceutically effective amount of a polypeptide or complex as in any one of embodiments 1 to 55 or a pharmaceutical composition as in embodiment 56 . Embodiment 67. The method of Embodiment 66, wherein the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma. ;Colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; gastrointestinal cancer; glioblastoma; liver cancer; liver tumors; intraepithelial neoplasms; kidney Cancer or kidney cancer; laryngeal cancer; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; squamous cell carcinoma of the lung; melanoma; myeloma; neuroblastoma; oral cavity Cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or Endometrial cancer; Urinary tract cancer; Vulvar cancer; Lymphoma; Hodgkin's lymphoma; Non-Hodgkin's lymphoma; B-cell lymphoma; low grade/follicular type Non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastoid NHL; highly malignant small non-cleft cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytes leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia and chronic myeloblastic leukemia. Embodiment 68. The method of embodiment 66 or 67, further comprising administering an additional therapeutic agent. Embodiment 69. The method of embodiment 68, wherein the additional therapeutic agent is an anti-cancer agent. Embodiment 70. The method of embodiment 69, wherein the anti-cancer agent is selected from the group consisting of chemotherapeutic agents, anti-cancer biological agents, radiotherapy, CAR-T therapy and oncolytic viruses. Embodiment 71. The method of embodiment 68, wherein the additional therapeutic agent is an anti-cancer biologic. Embodiment 72. The method of embodiment 71, wherein the anti-cancer biological agent is an agent that inhibits PD-1 and/or PD-L1. Embodiment 73. The method of embodiment 71, wherein the anti-cancer biological agent is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4 or TIGIT. Embodiment 74. The method of embodiment 69, wherein the anti-cancer agent is an antibody. Embodiment 75. The method of embodiment 71, wherein the anti-cancer biological agent is an interleukin. Embodiment 76. The method of embodiment 69, wherein the anti-cancer agent is CAR-T therapy. Embodiment 77. The method of embodiment 69, wherein the anti-cancer agent is an oncolytic virus. Embodiment 78. The method of any one of embodiments 66 to 77, further comprising tumor resection and/or radiation therapy.

相關申請案之交叉引用Cross-references to related applications

本申請案主張2021年7月20日申請之美國臨時申請案第63/223,798號、2021年12月10日申請之美國臨時申請案第63/288,113號；及2022年1月5日申請之美國臨時申請案第63/296,775號之優先權，該等申請案各自出於任何目的以全文引用的方式併入本文中。This application claims US Provisional Application No. 63/223,798 filed on July 20, 2021, US Provisional Application No. 63/288,113 filed on December 10, 2021; and US Provisional Application No. 63/288,113 filed on January 5, 2022 Priority to Provisional Application No. 63/296,775, each of which is hereby incorporated by reference in its entirety for any purpose.

本文所提供之實施例係關於靶向CD8之經修飾IL-2多肽及其在治療例如癌症之各種方法中之用途。 定義及各種實施例 Examples provided herein relate to modified IL-2 polypeptides targeting CD8 and their use in various methods of treating, for example, cancer. Definitions and various examples

本文使用之章節標題僅出於組織目的而不應被視為限制所述標的物。The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

本文所引用之所有參考文獻(包括專利申請案、專利公開案及Genbank寄存編號)均以引用方式併入本文中，如同各個別參考文獻具體地且單獨地指出以全文引用的方式併入本文中一般。All references (including patent applications, patent publications, and Genbank deposit numbers) cited herein are hereby incorporated by reference as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety. generally.

本文所描述或提及之技術及程序大體上由熟習此項技術者充分瞭解且通常使用習知方法而採用，諸如以下中所描述之廣泛利用的方法：Sambrook等人, Molecular Cloning: A Laboratory Manual 第3版(2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel等人編, (2003))；METHODS IN ENZYMOLOGY系列(Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames及G. R. Taylor編(1995)), Harlow及Lane編(1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney編(1987))；Oligonucleotide Synthesis (M. J. Gait編, 1984)；Methods in Molecular Biology, Humana Press；Cell Biology: A Laboratory Notebook (J. E. Cellis編, 1998) Academic Press；Animal Cell Culture (R. I. Freshney)編, 1987)；Introduction to Cell and Tissue Culture (J. P. Mather及P. E. Roberts, 1998) Plenum Press；Cell and Tissue Culture Laboratory Procedures (A. Doyle、J. B. Griffiths及D. G. Newell編, 1993-8) J. Wiley and Sons；Handbook of Experimental Immunology (D. M. Weir及C. C. Blackwell編)；Gene Transfer Vectors for Mammalian Cells (J. M. Miller及M. P. Calos編, 1987)；PCR: The Polymerase Chain Reaction, (Mullis等人編, 1994)；Current Protocols in Immunology (J. E. Coligan等人編, 1991)；Short Protocols in Molecular Biology (Wiley and Sons, 1999)；Immunobiology (C. A. Janeway及P. Travers, 1997)；Antibodies (P. Finch, 1997)；Antibodies: A Practical Approach (D. Catty等人, IRL Press, 1988-1989)；Monoclonal Antibodies: A Practical Approach (P. Shepherd及C. Dean編, Oxford University Press, 2000)；Using Antibodies: A Laboratory Manual (E. Harlow及D. Lane (Cold Spring Harbor Laboratory Press, 1999)；The Antibodies (M. Zanetti及J. D. Capra編, Harwood Academic Publishers, 1995)；及Cancer: Principles and Practice of Oncology (V. T. DeVita等人編, J.B. Lippincott Company, 1993)；及其更新版本。The techniques and procedures described or referred to herein are generally well understood by those skilled in the art and are commonly employed using conventional methods, such as the widely used methods described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., (2003)); METHODS IN ENZYMOLOGY Series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (edited by M. J. MacPherson, B. D. Hames and G. R. Taylor (1995)), Harlow and Lane (edited (1988)) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (edited by R. I. Freshney (1987)); Oligonucleotide Synthesis (edited by M. J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J. E. Cellis, 1998) Academic Press; Animal Cell Culture (edited by R. I. Freshney), 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (edited by A. Doyle, J. B. Griffiths and D. G. Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (edited by D. M. Weir and C. C. Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by J. M. Miller and M. P. Calos, 1987); PCR: The Polymerase Chain Reaction, (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by J. E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty et al., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M . Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993); and their updated editions.

除非另外定義，否則結合本發明使用之科學與技術術語將具有一般技術者通常理解之含義。此外，除非情景另有需要或另外明確地指出，否則單數術語應包括複數且複數術語應包括單數。關於各種來源或參考文獻之間在定義方面之任何衝突，將以本文所提供之定義為凖。Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires or otherwise expressly indicates otherwise, singular terms shall include the plural and plural terms shall include the singular. In the event of any conflict in definitions between various sources or references, the definitions provided herein will prevail.

一般而言，免疫球蛋白重鏈中之殘基編號係如Kabat等人, Sequences of Proteins of Immunological Interest, 第5版Public Health Service, National Institutes of Health, Bethesda, Md.(1991)中之EU索引之編號。「如Kabat中之EU索引」係指人類IgG1 EU抗體之殘基編號。 Generally speaking, residue numbers in immunoglobulin heavy chains are based on the EU index in Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991) number. "EU index in Kabat" refers to the residue number of the human IgG1 EU antibody.

應瞭解，本文所述之本發明實施例包括「由實施例組成」及/或「基本上由實施例組成」。除非另外指示，否則如本文所用，單數形式「一(a/an)」及「該(the)」包括複數個參考物。術語「或」在本文中之使用不意圖暗示替代方案係互相排斥的。It should be understood that embodiments of the invention described herein include "consisting of embodiments" and/or "consisting essentially of embodiments." As used herein, the singular forms "a/an" and "the" include plural references unless otherwise indicated. The use of the term "or" herein is not intended to imply that the alternatives are mutually exclusive.

在本申請案中，除非明確地敍述或如熟習此項技術者所瞭解，否則「或」之使用意謂「及/或」。在多重附屬項之情況下，「或」之使用重新提及一個以上前述獨立項或附屬項。In this application, the use of "or" means "and/or" unless explicitly stated or otherwise understood by those skilled in the art. In the case of multiple items, the use of "or" refers back to more than one of the preceding independent items or items.

片語「參考樣品」、「參考細胞」或「參考組織」表示可用作與具有至少一種未知特徵之樣品進行比較的具有至少一種已知特徵之樣品。在一些實施例中，參考樣品可用作陽性或陰性指示物。參考樣品可用於確定例如在健康組織中存在之蛋白質及/或mRNA之含量，與在具有未知特徵之樣品中存在之蛋白質及/或mRNA之含量形成對比。在一些實施例中，參考樣品來自同一個體，但來自所測試個體之不同部位。在一些實施例中，參考樣品來自癌周圍或鄰近癌之組織區域。在一些實施例中，參考樣品並非來自所測試個體，而是來自已知患有或不患有所討論病症(例如特定癌症或CD8相關病症)之個體的樣品。在一些實施例中，參考樣品係來自同一個體，但來自在個體罹患癌症前之時間點。在一些實施例中，參考樣品係來自相同或不同個體之良性癌樣品。在使用陰性參考樣品進行比較時，陰性參考樣品中所討論的分子之表現量或量將指示出熟習此項技術者將瞭解之含量，假定本發明中無分子及/或具有低含量之分子。在使用陽性參考樣品進行比較時，陽性參考樣品中所討論的分子之表現量或量將指示出熟習此項技術者將瞭解之含量，假定本發明中存在一定含量之分子。The phrase "reference sample", "reference cell" or "reference tissue" means a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to determine, for example, the amount of protein and/or mRNA present in healthy tissue, in contrast to the amount of protein and/or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is from the same individual, but from a different part of the individual being tested. In some embodiments, the reference sample is from a tissue region surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the individual being tested, but is a sample from an individual known to have or not have the disorder in question (eg, a specific cancer or a CD8-related disorder). In some embodiments, the reference sample is from the same individual, but from a time point before the individual develops cancer. In some embodiments, the reference sample is a benign cancer sample from the same or a different individual. When a negative reference sample is used for comparison, the amount or amount of expression of the molecule in question in the negative reference sample will be indicative of what one skilled in the art would understand, assuming no molecules and/or low levels of the molecules in the present invention. When a positive reference sample is used for comparison, the amount or amount of expression of the molecule in question in the positive reference sample will be indicative of the amount that one skilled in the art would understand, assuming a certain amount of the molecule is present in the present invention.

如本文在受益於治療劑投與或對治療劑投與有反應之上下文中所用之術語「益處」、「臨床益處」、「反應」及「治療反應」可藉由評定各種評估指標來量測，例如在一定程度上抑制疾病進展，包括減緩及完全遏止；減少疾病發作及/或症狀之數量；減小病變大小；抑制(即，減少、減緩或完全停止)疾病細胞浸潤至鄰近周邊器官及/或組織；抑制(即，減少、減緩或完全停止)疾病擴散；在一定程度上減輕與該病症相關之一或多種症狀；增加在治療後呈現無病(例如，無進展存活期)之時長；增加總體存活率；反應率更高；及/或減小在治療後給定時間點之死亡率。「無反應」或「無法反應」之個體或癌症係無法滿足關於「反應」之上述限制條件之個體或癌症。As used herein, the terms "benefit," "clinical benefit," "response," and "therapeutic response" in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various assessment metrics. , such as inhibiting disease progression to a certain extent, including slowing down and completely arresting; reducing the number of disease attacks and/or symptoms; reducing the size of lesions; inhibiting (i.e., reducing, slowing or completely stopping) the infiltration of disease cells into adjacent peripheral organs and or tissue; inhibits (i.e., reduces, slows, or completely stops) the spread of disease; alleviates to some extent one or more symptoms associated with the condition; increases the length of time one remains disease-free (e.g., progression-free survival) after treatment ;increased overall survival; higher response rate; and/or reduced mortality at a given time point after treatment. An individual or cancer that is "non-responsive" or "unresponsive" is an individual or cancer that is unable to meet the above restrictions on "response."

術語「核酸分子」、「核酸」及「聚核苷酸」可互換使用，且係指核苷酸聚合物。此類核苷酸聚合物可含有天然及/或非天然核苷酸且包括但不限於DNA、RNA及PNA。「核酸序列」係指包含於核酸分子或多核苷酸中之核苷酸線性序列。The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to polymers of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

術語「多肽」與「蛋白」可互換使用以指胺基酸殘基之聚合物，且不限於最小長度。此類胺基酸殘基之聚合物可含有天然或非天然胺基酸殘基且包括但不限於胺基酸殘基構成之肽、寡肽、二聚體、三聚體及多聚體。全長蛋白質與其片段皆涵蓋於該定義中。術語亦包括多肽之表現後修飾，例如糖基化、唾液酸化、乙醯化、磷酸化及類似修飾。此外，出於本發明之目的，「多肽」係指蛋白質，其包括對天然序列之修飾，諸如缺失、添加及取代(實際上通常係保守的)，只要該蛋白質保持所需活性即可。此等修飾可為有意的，如經由定點突變誘發進行；或可為偶然的，諸如經由產生蛋白質之宿主的突變或由於PCR擴增所引起的錯誤引起。在一些實施例中，多肽為第一多肽及第二多肽之「複合物」。The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers composed of amino acid residues. Full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. Furthermore, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (which are generally conservative in practice), so long as the protein retains the desired activity. Such modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the host in which the protein is produced or due to errors in PCR amplification. In some embodiments, the polypeptide is a "complex" of a first polypeptide and a second polypeptide.

術語「CD8a」及「CD8」在本文中可互換使用，指代由細胞中處理CD8前驅體產生的任何天然成熟CD8。除非另外規定，否則該術語包括來自任何脊椎動物來源之CD8，包括哺乳動物，諸如靈長類動物(例如，人類及食蟹獼猴或恆河猴)及嚙齒動物(例如，小鼠及大鼠)。該術語亦包括CD8之天然存在之變異體，諸如剪接變異體或對偶基因變異體。非限制性例示性成熟人類CD8胺基酸序列展示於例如NCBI寄存編號NP_001369627.1中。參見SEQ ID NO. 1。 The terms "CD8a" and "CD8" are used interchangeably herein to refer to any naturally occurring mature CD8 produced by processing a CD8 precursor in a cell. Unless otherwise specified, the term includes CD8 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus macaques or rhesus monkeys) and rodents (eg, mice and rats) . The term also includes naturally occurring variants of CD8, such as splice variants or allele variants. A non-limiting exemplary mature human CD8 amino acid sequence is shown, for example, in NCBI Accession No. NP_001369627.1. See SEQ ID NO. 1.

如本文所用之「IL-2」或「介白素-2」係指由在細胞中加工IL-2前驅體產生之任何天然成熟IL-2。除非另外規定，否則該術語包括來自任何脊椎動物來源之IL-2，包括哺乳動物，諸如靈長類動物(例如，人類及食蟹獼猴或恆河猴)及嚙齒動物(例如，小鼠及大鼠)。該術語亦包括IL-2之天然存在之變異體，諸如剪接變異體或對偶基因變異體。非限制性例示性人類IL-2胺基酸序列示於例如GenBank寄存編號NP_000577.2中。參見SEQ ID NO. 71 (成熟形式)。 "IL-2" or "interleukin-2" as used herein refers to any naturally occurring mature IL-2 produced by processing of an IL-2 precursor in a cell. Unless otherwise specified, the term includes IL-2 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus macaques or rhesus monkeys) and rodents (e.g., mice and rats). mouse). The term also includes naturally occurring variants of IL-2, such as splice variants or allelogenic variants. A non-limiting exemplary human IL-2 amino acid sequence is shown, for example, in GenBank accession number NP_000577.2. See SEQ ID NO. 71 (mature form).

如本文所用之「經修飾IL-2」係指因至少一個胺基酸位置處之取代而不同於野生型IL-2胺基酸序列的多肽。 "Modified IL-2" as used herein refers to a polypeptide that differs from the amino acid sequence of wild-type IL-2 due to a substitution at at least one amino acid position.

如本文所用之術語「IL-2活性」或IL-2之「生物活性」包括任一生物作用或IL-2之生物學相關功能中之至少一者。在一些實施例中，IL-2活性包括IL-2誘導T細胞增殖及/或活化自然殺手(NK)細胞之能力。非限制性例示性IL-2活性包括增加pSTAT5表現、增加CD4 +及/或CD8 +T細胞之增殖、增加CD71在T細胞上之表現及減少Treg細胞對CD4 +及CD8 +T細胞活化及增殖之抑制活性。 The term "IL-2 activity" or "biological activity" of IL-2 as used herein includes at least one of any biological effect or biologically relevant function of IL-2. In some embodiments, IL-2 activity includes the ability of IL-2 to induce T cell proliferation and/or activate natural killer (NK) cells. Non-limiting exemplary IL-2 activities include increasing pSTAT5 expression, increasing CD4 + and/or CD8 + T cell proliferation, increasing CD71 expression on T cells, and reducing CD4 + and CD8 + T cell activation and proliferation by Treg cells its inhibitory activity.

術語「特異性結合」至抗原或抗原決定基係此項技術中充分瞭解之術語，且用於測定此特異性結合之方法亦為此項技術中所熟知。若分子與特定細胞或物質之反應或締合比其與替代性細胞或物質更頻繁、更快速，持續時間更長及/或親和力更大，則稱其呈現「特異性結合」或「較佳結合」。若單域抗體(sdAb)或含VHH多肽與目標之結合比其與其他物質之結合親和力、親合力更大、更容易及/或持續時間更長，則其「特異性結合」或「優先結合」於目標。舉例而言，特異性或優先結合至CD8抗原決定基之sdAb或含VHH多肽為與此抗原決定基之結合比其與其他CD8抗原決定基或非CD8抗原決定基之結合親和力、親合力更大、更容易及/或持續時間更長的sdAb或含VHH多肽。藉由閱讀此定義，亦應理解，例如特異性或優先結合至第一目標之sdAb或含VHH多肽可能或可能不特異性或優先結合至第二目標。因此，「特異性結合」或「優先結合」未必需要(儘管其可包括)獨佔式結合。一般而言，但不一定，提及結合意謂優先結合。「特異性」係指結合蛋白選擇性結合抗原之能力。The term "specific binding" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "better binding" if it reacts or associates with a specific cell or substance more frequently, more rapidly, for a longer period of time, and/or with greater affinity than with an alternative cell or substance. combination". A single domain antibody (sdAb) or VHH-containing polypeptide "binds specifically" or "binds preferentially" if it binds to a target with greater affinity, avidity, easier and/or longer duration than with other substances. ” at the target. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a CD8 epitope binds to this epitope with greater affinity or avidity than to other CD8 epitopes or non-CD8 epitopes. , easier and/or longer lasting sdAb or VHH-containing peptides. By reading this definition, it will also be understood that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to union means preferential union. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

術語「抑制(inhibition)」或「抑制(inhibit)」係指任何表型特徵減少或停止，或彼特徵之發生率、程度或可能性降低或停止。「降低」或「抑制」係使活性、功能及/或量相較於參考物減小、降低或停滯。在一些實施例中，「降低」或「抑制」意謂整體減少10%或更多之能力。在一些實施例中，「降低」或「抑制」意謂整體減少50%或更多之能力。在一些實施例中，「減小」或「抑制」意謂能夠引起總體減少75%、85%、90%、95%或更大。在一些實施例中，一段時間內上文所提及之量相對於同一段時間內之對照而言受到抑制或減少。The term "inhibition" or "inhibit" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the occurrence, extent, or likelihood of that characteristic. "Reducing" or "inhibiting" means reducing, reducing or stagnating activity, function and/or quantity compared to a reference. In some embodiments, "reducing" or "inhibiting" means an overall reduction in ability by 10% or more. In some embodiments, "reducing" or "inhibiting" means an overall reduction of capability by 50% or more. In some embodiments, "reduce" or "inhibit" means capable of causing an overall reduction of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amounts noted above are inhibited or reduced over a period of time relative to a control over the same period of time.

如本文所用，術語「直接抑制」及類似術語係指其中增加的抗體濃度導致抑制增加的抑制特徵。在一些實施例中，在特定濃度之後，達至最大抑制且抑制概況平穩。最大抑制無需為100%抑制，但可為至少50%、55%、60%、65%、70%、75%、80%、85%或90%。As used herein, the term "direct inhibition" and similar terms refer to inhibitory characteristics in which increasing antibody concentration results in increased inhibition. In some embodiments, after a specific concentration, maximum inhibition is achieved and the inhibition profile plateaus. Maximum inhibition need not be 100% inhibition, but may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

如本文所用，術語「抗原決定基」係指抗原結合分子(例如sdAb或含VHH多肽)在目標分子(例如抗原，諸如蛋白質、核酸、碳水化合物或脂質)上結合之位點。抗原決定基常常包括一組表面具有化學活性之分子，諸如胺基酸、多肽或糖側鏈，且具有特定三維結構特徵以及荷質比特徵。抗原決定基可由目標分子之相鄰及/或併接非相鄰殘基(例如，胺基酸、核苷酸、糖、脂質部分)形成。由相鄰殘基(例如，胺基酸、核苷酸、糖、脂質部分)形成之抗原決定基通常在暴露於變性溶劑時得以保留，而藉由三級摺疊形成之抗原決定基通常在經變性溶劑處理時丟失。抗原決定基可包括但不限於至少3個、至少5個或8至10個殘基(例如，胺基酸或核苷酸)。在一些實施例中，抗原決定基之長度小於20個殘基(例如，胺基酸或核苷酸)、小於15個殘基或小於12個殘基。若兩個抗體對一抗原展現競爭性結合，則其可結合該抗原內之同一個抗原決定基。在一些實施例中，可根據相對於抗原結合分子上之CDR殘基的某一最小距離鑑別抗原決定基。在一些實施例中，抗原決定基可藉由以上距離鑑別，且進一步受限於抗原結合分子之殘基與抗原殘基之間的一鍵(例如，氫鍵)所涉及之彼等殘基。抗原決定基亦可藉由各種掃描來鑑別，例如丙胺酸或精胺酸掃描可指示可與抗原結合分子相互作用之一或多個殘基。除非明確表示，否則作為抗原決定基之一組殘基不排除其他殘基作為特定抗原結合分子之抗原決定基的一部分。實際上，此類集合之存在表示最小的抗原決定基系列(或物種集合)。因此，在一些實施例中，鑑別為抗原決定基之殘基集合表示該抗原之最小相關抗原決定基，而非抗原上之抗原決定基的排他性殘基清單。As used herein, the term "epitope" refers to the site on a target molecule (eg, an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) that an antigen-binding molecule (eg, an sdAb or VHH-containing polypeptide) binds. Epitopes often include a group of molecules with chemically active surfaces, such as amino acids, polypeptides or sugar side chains, and have specific three-dimensional structural characteristics and charge-to-mass ratio characteristics. Epitopes may be formed from adjacent and/or conjoined non-adjacent residues (eg, amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally retained upon exposure to denaturing solvents. Lost during processing with denaturing solvents. Epitopes may include, but are not limited to, at least 3, at least 5, or 8 to 10 residues (eg, amino acids or nucleotides). In some embodiments, the epitope is less than 20 residues (eg, amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. If two antibodies exhibit competitive binding to an antigen, they can bind to the same epitope within the antigen. In some embodiments, epitopes can be identified based on a certain minimum distance relative to CDR residues on the antigen-binding molecule. In some embodiments, epitopes can be identified by the above distances and are further limited to those residues involved in a bond (eg, a hydrogen bond) between the residues of the antigen-binding molecule and the residues of the antigen. Epitopes can also be identified by various scans, for example alanine or arginine scans may indicate one or more residues that may interact with the antigen-binding molecule. Unless expressly stated, reference to a group of residues as an epitope does not exclude other residues from being part of the epitope of a particular antigen-binding molecule. In fact, the existence of such a collection represents a minimal epitope series (or collection of species). Thus, in some embodiments, the set of residues identified as epitopes represents the minimal relevant epitopes of the antigen, rather than an exclusive list of residues for epitopes on the antigen.

「非線性抗原決定基」或「構形抗原決定基」包含在對抗原決定基具有特異性之抗原結合分子所結合之抗原蛋白質內的非相鄰多肽、胺基酸及/或糖。在一些實施例中，至少一個殘基將與抗原決定基之其他所指出之殘基不相鄰；然而，一或多個殘基亦可與其他殘基相鄰。"Nonlinear epitopes" or "configurational epitopes" include non-contiguous polypeptides, amino acids and/or sugars within the antigenic protein bound by an antigen-binding molecule specific for the epitope. In some embodiments, at least one residue will not be adjacent to other indicated residues of the epitope; however, one or more residues may also be adjacent to other residues.

「線性抗原決定基」包含在對抗原決定基具有特異性之抗原結合分子所結合之抗原蛋白質內的相鄰多肽、胺基酸及/或糖。應注意，在一些實施例中，並非線性抗原決定基內之每一個殘基均需要與抗原結合分子直接結合(或與鍵相關)。在一些實施例中，線性抗原決定基可來自用有效地由線性抗原決定基之序列組成之肽免疫，或來自蛋白質中與蛋白質之其餘部分相對分離之結構部分(使得抗原結合分子可至少首先與彼序列部分相互作用)。"Linear epitopes" include contiguous polypeptides, amino acids and/or sugars within the antigenic protein bound by an antigen-binding molecule specific for the epitope. It should be noted that in some embodiments, not every residue within a linear epitope is required to directly bind (or be associated with a bond) to an antigen-binding molecule. In some embodiments, the linear epitope may result from immunization with a peptide that effectively consists of the sequence of the linear epitope, or from a structural portion of the protein that is relatively isolated from the rest of the protein (such that the antigen-binding molecule can at least first bind to The sequence partially interacts).

術語「抗體」以最廣泛意義使用且涵蓋包含抗體樣抗原結合域之各種多肽，包括但不限於習知抗體(通常包含至少一條重鏈及至少一條輕鏈)、單域抗體(sdAb，包含至少一個VHH域及Fc區)、含VHH多肽(包含至少一個VHH域之多肽)及前述中之任一者之片段，只要其展現所需抗原結合活性即可。在一些實施例中，抗體包含二聚域。此等二聚域包括但不限於重鏈恆定域(包含CH1、鉸鏈、CH2及CH3，其中CH1通常與輕鏈恆定域CL配對，而鉸鏈介導二聚化)及Fc區(包含鉸鏈、CH2及CH3，其中鉸鏈介導二聚化)。The term "antibody" is used in the broadest sense and encompasses various polypeptides containing antibody-like antigen-binding domains, including but not limited to conventional antibodies (usually containing at least one heavy chain and at least one light chain), single domain antibodies (sdAb, containing at least A VHH domain and an Fc region), a VHH-containing polypeptide (a polypeptide comprising at least one VHH domain), and fragments of any of the foregoing, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, the heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 typically pairs with the light chain constant domain CL, and the hinge mediates dimerization) and the Fc region (comprising hinge, CH2 and CH3, where the hinge mediates dimerization).

術語抗體亦包括但不限於嵌合抗體、人類化抗體及諸如駱駝(包括駱馬)、鯊魚、小鼠、人類、食蟹獼猴等各種物種之抗體。The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as camels (including llamas), sharks, mice, humans, macaques, and the like.

如本文所用，術語「抗原結合域」係指抗體足以結合抗原之一部分。在一些實施例中，習知抗體之抗原結合域包含三個重鏈CDR及三個輕鏈CDR。因此，在一些實施例中，抗原結合域包含：重鏈可變區，其包含CDR1-FR2-CDR2-FR3-CDR3及維持與抗原結合所需的FR1及/或FR4之任何部分；及輕鏈可變區，其包含CDR1-FR2-CDR2-FR3-CDR3及維持與抗原結合所需的FR1及/或FR4之任何部分。在一些實施例中，sdAb或含VHH多肽之抗原結合域包含VHH域之三個CDR。因此，在一些實施例中，sdAb或含VHH多肽之抗原結合域包含VHH域，該VHH域包含CDR1-FR2-CDR2-FR3-CDR3及維持與抗原之結合所需的FR1及/或FR4之任何部分。As used herein, the term "antigen-binding domain" refers to a portion of an antibody sufficient to bind an antigen. In some embodiments, the antigen-binding domain of a conventional antibody includes three heavy chain CDRs and three light chain CDRs. Thus, in some embodiments, the antigen binding domain includes: a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and/or FR4 required to maintain binding to the antigen; and a light chain Variable region, which includes CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and/or FR4 required to maintain binding to the antigen. In some embodiments, the sdAb or antigen-binding domain containing a VHH polypeptide comprises three CDRs of the VHH domain. Thus, in some embodiments, the sdAb or antigen-binding domain containing a VHH polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any of FR1 and/or FR4 required to maintain binding to the antigen. part.

如本文中所用之術語「VHH」或「VHH域」或「VHH抗原結合域」係指單域抗體之抗原結合部分，該單域抗體諸如駱駝抗體或鯊魚抗體。在一些實施例中，VHH包含三個CDR及四個構架區，稱為FR1、CDR1、FR2、CDR2、FR3、CDR3及FR4。在一些實施例中，VHH可在N端或C端截短，使得其僅包含部分FR1及/或FR4，或缺乏彼等構架區中之一者或兩者，只要VHH實質上維持抗原結合及特異性即可。The term "VHH" or "VHH domain" or "VHH antigen-binding domain" as used herein refers to the antigen-binding portion of a single domain antibody, such as a camel antibody or a shark antibody. In some embodiments, a VHH contains three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH can be truncated at the N- or C-terminus such that it contains only a portion of FR1 and/or FR4, or lacks one or both of these framework regions, as long as the VHH substantially maintains antigen binding and Specificity is enough.

術語「單域抗體」及「sdAb」在本文中可互換使用，指代包含至少一個無輕鏈單體域(諸如VHH域)及Fc區之抗體。在一些實施例中，sdAb為兩種多肽之二聚物，其中各多肽包含至少一個VHH域及Fc區。如本文所用，術語「單域抗體」及「sdAb」涵蓋包含多個VHH域之多肽，諸如具有結構VHH 1-VHH 2-Fc或VHH 1-VHH 2-VHH 3-Fc之多肽，其中VHH 1、VHH 2及VHH 3可相同或不同。 The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one light chain-free monomer domain (such as a VHH domain) and an Fc region. In some embodiments, the sdAb is a dimer of two polypeptides, wherein each polypeptide includes at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, such as polypeptides having the structure VHH 1 -VHH 2 -Fc or VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 , VHH 2 and VHH 3 can be the same or different.

術語「含VHH多肽」係指包含至少一個VHH域之多肽。在一些實施例中，VHH多肽包含兩個、三個或四個或更多個VHH域，其中各VHH域可相同或不同。在一些實施例中，含VHH多肽包含Fc區。在一些此類實施例中，含VHH多肽可稱為sdAb。另外，在一些此類實施例中，VHH多肽可形成二聚體。含VHH多肽之非限制性結構(其亦為sdAb)包括VHH 1-Fc、VHH 1-VHH 2-Fc及VHH 1-VHH 2-VHH 3-Fc，其中VHH 1、VHH 2及VHH 3可相同或不同。在此類結構之一些實施例中，一個VHH可藉由連接子連接至另一VHH，或一個VHH可藉由連接子連接至Fc。在一些此類實施例中，連接子包含1-20個胺基酸，較佳為1-20個主要由甘胺酸且視情況由絲胺酸構成之胺基酸。在一些實施例中，連接子包含：Gly-Gly-Gly-Gly (SEQ ID NO: 123)、Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO:124)及/或Gly-Gly-Ser-Ser-Gly-Ser (SEQ ID NO:125)。在一些實施例中，當含VHH多肽包含Fc時，其形成二聚體。因此，若結構VHH 1-VHH 2-Fc形成二聚體，則認為其為四價的(亦即，二聚體具有四個VHH域)。類似地，若結構VHH 1-VHH 2-VHH 3-Fc形成二聚體，則認為其為六價的(亦即，二聚體具有六個VHH域)。 The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, a VHH polypeptide contains two, three, or four or more VHH domains, where each VHH domain can be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as an sdAb. Additionally, in some such embodiments, VHH polypeptides can form dimers. Non-limiting structures of VHH-containing polypeptides (which are also sdAbs) include VHH 1 -Fc, VHH 1 -VHH 2 -Fc, and VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 , VHH 2 and VHH 3 can be the same Or different. In some embodiments of such structures, one VHH can be connected to another VHH via a linker, or one VHH can be connected to Fc via a linker. In some such embodiments, the linker contains 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. In some embodiments, the linker includes: Gly-Gly-Gly-Gly (SEQ ID NO: 123), Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 124), and/or Gly-Gly- Ser-Ser-Gly-Ser (SEQ ID NO: 125). In some embodiments, when a VHH-containing polypeptide includes an Fc, it forms a dimer. Therefore, if the structure VHHi - VHH2 -Fc forms a dimer, it is considered tetravalent (ie, the dimer has four VHH domains). Similarly, the structure VHHi - VHH2 - VHH3 -Fc is considered to be hexavalent if it forms a dimer (ie, the dimer has six VHH domains).

術語「單株抗體」係指實質上均質之抗體群的抗體(包括sdAb或含VHH多肽)，即，包含此群體之個別抗體除可能存在少量天然產生之突變以外均相同。單株抗體為高度特異性的，其針對單一抗原位點。此外，與典型地包括針對不同決定子(抗原決定基)之不同抗體的多株抗體製劑相反，各單株抗體針對抗原上之單個決定子。因此，單株抗體之樣品可結合於抗原上之同一抗原決定基。修飾語「單株」指示抗體之特徵為自實質上均質之抗體群獲得，且不應理解為需要藉由任何特定方法來產生該抗體。舉例而言，單株抗體可藉由Kohler及Milstein, 1975, Nature 256:495首次描述之融合瘤方法製造，或可藉由諸如美國專利第4,816,567號中所述之重組DNA方法製造。舉例而言，單株抗體亦可自使用McCafferty等人，1990, Nature 348:552-554中所描述之技術生成的噬菌體庫中分離。The term "monoclonal antibody" refers to a population of antibodies (including sdAbs or VHH-containing polypeptides) that are substantially homogeneous, that is, the individual antibodies comprising such a population are identical except for the possible presence of a small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. Therefore, samples of monoclonal antibodies can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method to produce the antibody. For example, monoclonal antibodies can be produced by the fusionoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from phage libraries generated using techniques described in McCafferty et al., 1990, Nature 348:552-554.

術語「CDR」表示互補決定區，如由熟習此項技術者藉由至少一種鑑別方式所定義。在一些實施例中，CDR可根據Chothia編號方案、Kabat編號方案、Kabat與Chothia之組合、AbM定義及/或接觸定義中之任一者來定義。VHH包含三個CDR，稱為CDR1、CDR2及CDR3。在某些實施例中，CDR係根據AbM定義來定義。The term "CDR" means complementary determining region, as defined by one skilled in the art by at least one means of identification. In some embodiments, CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, the combination of Kabat and Chothia, the AbM definition, and/or the contact definition. VHH contains three CDRs, called CDR1, CDR2 and CDR3. In certain embodiments, the CDRs are defined according to the AbM definition.

如本文所用，術語「重鏈恆定區」係指包含至少三個重鏈恆定域C H1、鉸鏈、C H2及C H3之區域。當然，除非另外指示，否則此等域中之非功能改變性缺失及修改係涵蓋於術語「重鏈恆定區」之範疇內。非限制性例示性重鏈恆定區包括γ、δ及α。非限制性例示性重鏈恆定區亦包括ε及μ。各重鏈恆定區對應於一種抗體同型。舉例而言，包含γ恆定區之抗體為IgG抗體，包含δ恆定區之抗體為IgD抗體，且包含α恆定區之抗體為IgA抗體。此外，包含μ恆定區之抗體為IgM抗體，且包含ε恆定區之抗體為IgE抗體。某些同型可進一步再分為亞類。舉例而言，IgG抗體包括但不限於IgG1 (包含γ 1恆定區)、IgG2 (包含γ 2恆定區)、IgG3 (包含γ 3恆定區)及IgG4 (包含γ 4恆定區)抗體；IgA抗體包括但不限於IgA1 (包含α 1恆定區)及IgA2 (包含α 2恆定區)抗體；且IgM抗體包括但不限於IgM1及IgM2。 As used herein, the term "heavy chain constant region" refers to a region containing at least three heavy chain constant domains, CH1 , hinge, CH2 , and CH3 . Of course, unless otherwise indicated, non-functionally altering deletions and modifications in these domains are encompassed by the term "heavy chain constant region". Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, and an antibody that includes an alpha constant region is an IgA antibody. Furthermore, antibodies containing the mu constant region are IgM antibodies, and antibodies containing the epsilon constant region are IgE antibodies. Certain isotypes can be further subdivided into subtypes. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ 1 constant region), IgG2 (comprising a γ 2 constant region), IgG3 (comprising a γ 3 constant region), and IgG4 (comprising a γ 4 constant region) antibodies; IgA antibodies include But are not limited to IgA1 (comprising α 1 constant region) and IgA2 (comprising α 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

如本文所用，「Fc區」係指包含CH2及CH3之重鏈恆定區之部分。在一些實施例中，Fc區包含鉸鏈、CH2及CH3。在各種實施例中，當Fc區包含鉸鏈時，鉸鏈介導兩個含Fc之多肽之間的二聚化。Fc區可為本文所論述之任何抗體重鏈恆定區同型。在一些實施例中，Fc區為IgG1、IgG2、IgG3或IgG4。在一些實施例中，當Fc區包含鉸鏈時，該鉸鏈具有與Fc區相同的同型。在一些實施例中，IgG4鉸鏈包含S228P穩定突變。As used herein, "Fc region" refers to the portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgGl, IgG2, IgG3 or IgG4. In some embodiments, when the Fc region includes a hinge, the hinge has the same isotype as the Fc region. In some embodiments, the IgG4 hinge contains the S228P stabilizing mutation.

如本文所論述，如本文所用之「受體人類構架」為包含源自人類免疫球蛋白構架或人類共同構架之重鏈可變域(V H)構架之胺基酸序列的構架。源自人類免疫球蛋白構架或人類共同構架之受體人類構架可包含與其相同之胺基酸序列，或其可含有胺基酸序列改變。在一些實施例中，跨單一抗原結合域(諸如VHH)中之所有人類構架，胺基酸改變之數目少於10，或少於9，或少於8，或少於7，或少於6，或少於5，或少於4，或少於3。 As discussed herein, an "acceptor human framework" as used herein is a framework comprising amino acid sequences derived from the heavy chain variable domain ( VH ) framework of the human immunoglobulin framework or the human consensus framework. Receptor human frameworks derived from human immunoglobulin frameworks or human consensus frameworks may comprise the same amino acid sequence therefor, or they may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6 across all human frameworks in a single antigen binding domain (such as VHH) , or less than 5, or less than 4, or less than 3.

「親和力」係指分子(例如抗體，諸如sdAb，或含VHH多肽)之單一結合位點與其結合搭配物(例如抗原)之間的非共價相互作用之總和之強度。分子X對其搭配物Y之親和力或表觀親和力通常可分別由解離常數(K D)或K D - 表觀來表示。親和力可藉由此項技術中已知之常用方法(諸如ELISA K D、KinExA、流動式細胞測量術及/或表面電漿子共振裝置)，包括本文所描述之彼等方法來量測。此類方法包括但不限於涉及BIAcore®、Octet®或流動式細胞測量術之方法。 "Affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (eg, an antibody, such as an sdAb, or a VHH-containing polypeptide) and its binding partner (eg, an antigen). The affinity or apparent affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) or K D -apparent , respectively. Affinity can be measured by common methods known in the art, such as ELISA KD , KinExA, flow cytometry, and/or surface plasmon resonance, including those described herein. Such methods include, but are not limited to, methods involving BIAcore®, Octet®, or flow cytometry.

如本文所用之術語「K D」係指抗原結合分子/抗原相互作用之平衡解離常數。當本文使用術語「K D」時，其包括K D及K D- 表觀。 The term " KD " as used herein refers to the equilibrium dissociation constant of the antigen-binding molecule/antigen interaction. When the term " KD " is used herein, it includes both KD and KD- apparent .

在一些實施例中，抗原結合分子之K D使用表現抗原之細胞株且將在各抗體濃度下量測之平均螢光擬合至非線性單點結合方程式(Prism Software graphpad)藉由流動式細胞測量術來量測。在一些此類實施例中，K D為K D- 表觀。 In some embodiments, the K D of the antigen-binding molecule is determined using a cell line expressing the antigen and the average fluorescence measured at each antibody concentration is fit to a nonlinear single-point binding equation (Prism Software graphpad) by flow cytometry To measure using metrology. In some such embodiments, KD is KD- apparent .

術語「生物活性」係指分子之任一或多種生物特性(無論係在活體內發現為天然存在的，或藉由重組方式提供或實現的)。生物特性包括但不限於結合配體、誘導或增加細胞增殖(諸如T細胞增殖)及誘導或增加細胞介素之表現。The term "biological activity" refers to any one or more biological properties of a molecule (whether found naturally occurring in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding of ligands, inducing or increasing cell proliferation (such as T cell proliferation), and inducing or increasing expression of interleukins.

「促效劑」或「活化」抗體為增加及/或活化目標抗原之生物活性的抗體。在一些實施例中，促效劑抗體結合於抗原且使其生物學活性增加至少約20%、40%、60%、80%、85%或更多。"Agonist" or "activating" antibodies are antibodies that increase and/or activate the biological activity of a target antigen. In some embodiments, the agonist antibody binds to the antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85%, or more.

「拮抗劑」，一種「阻斷」或「中和」抗體，為抑制、降低及/或不活化目標抗原之生物活性的抗體。在一些實施例中，中和抗體結合於抗原且使其生物學活性降低至少約20%、40%、60%、80%、85%、90%、95%、99%或更多。"Antagonist" is a "blocking" or "neutralizing" antibody that inhibits, reduces and/or does not activate the biological activity of the target antigen. In some embodiments, the neutralizing antibody binds to the antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99%, or more.

「親和力成熟」sdAb或含VHH多肽係指相較於在一或多個CDR中不具有一或多個改變之親本sdAb或含VHH多肽，具有此類改變之sdAb或含VHH多肽，此類改變改良sdAb或含VHH多肽對抗原之親和力。"Affinity matured" sdAb or VHH-containing polypeptide means an sdAb or VHH-containing polypeptide that has one or more changes in one or more CDRs compared to a parent sdAb or VHH-containing polypeptide that does not have such changes, such Change the affinity of the modified sdAb or VHH-containing polypeptide for the antigen.

如本文所用，「人類化VHH」係指其中一或多個構架區已實質上經人類構架區置換之VHH。在一些情況下，人類免疫球蛋白之某些構架區(FR)殘基經對應非人類殘基置換。此外，人類化VHH可包含既不存在於原始VHH亦不存在於人類構架序列中的殘基，但包括該等殘基在內以進一步改進及優化sdAb含VHH多肽效能。在一些實施例中，人類化sdAb或含VHH多肽包含人類Fc區。如將瞭解，人類化序列可藉由其一級序列鑑別且不一定表示創建抗體之過程。As used herein, a "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced with a human framework region. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Additionally, a humanized VHH may contain residues that are neither present in the original VHH nor in the human framework sequence, but are included to further improve and optimize sdAb VHH-containing polypeptide potency. In some embodiments, the human sdAb or VHH-containing polypeptide comprises a human Fc region. As will be understood, humanized sequences can be identified by their primary sequence and do not necessarily represent the process of creating antibodies.

「效應子陽性Fc區」具有天然序列Fc區之「效應功能」。例示性「效應功能」包括Fc受體結合；Clq結合及補體依賴性細胞毒性(CDC)；Fc受體結合；抗體依賴性細胞介導之細胞毒性(ADCC)；吞噬作用；細胞表面受體(例如B細胞受體)之下調；以及B細胞活化等。該等效應功能一般需要Fc區與結合域(例如抗體可變域)組合且可使用各種分析評定。The "effector-positive Fc region" has the "effector function" of the native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors ( For example, B cell receptor) downregulation; and B cell activation, etc. Such effector functions generally require an Fc region in combination with a binding domain (eg, an antibody variable domain) and can be assessed using a variety of assays.

「天然序列Fc區」包含與自然界中存在之Fc區之胺基酸序列一致的胺基酸序列。天然序列人類Fc區包括天然序列人類IgG1 Fc區(非A及A異型)；天然序列人類IgG2 Fc區；天然序列人類IgG3 Fc區；及天然序列人類IgG4 Fc區，以及其天然存在之變異體。"Native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc region includes native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

「變異Fc區」包含與天然序列Fc區之胺基酸序列相差至少一個胺基酸修飾的胺基酸序列。在一些實施例中，「變異Fc區」包含與天然序列Fc區之胺基酸序列相差至少一個胺基酸修飾，但保留天然序列Fc區之至少一種效應功能的胺基酸序列。在一些實施例中，變異Fc區相較於天然序列Fc區或相較於親本多肽之Fc區具有至少一個胺基酸取代，例如在天然序列Fc區中或在親本多肽之Fc區中約一個至約十個胺基酸取代，且較佳約一個至約五個胺基酸取代。在一些實施例中，本文中的變異型Fc區與天然序列Fc區及/或親本多肽之Fc區具有至少約80%序列一致性、與其具有至少約90%序列一致性、至少約95%、至少約96%、至少約97%、至少約98%，或與其至少約99%序列一致性。A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide, e.g., in the native sequence Fc region or in the Fc region of the parent polypeptide From about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions. In some embodiments, a variant Fc region herein has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity with the native sequence Fc region and/or the Fc region of the parent polypeptide. , at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

「Fc受體」或「FcR」描述與抗體Fc區結合之受體。在一些實施例中，FcγR為天然人類FcR。在一些實施例中，FcR為結合IgG抗體之FcR (γ受體)且包括FcγRI、FcγRII及FcγRIII亞類之受體，包括彼等受體之對偶基因變異體及交替剪接形式。FcγRII受體包括FcγRIIA (「活化受體」)及FcγRIIB (「抑制受體」)，兩者具有主要在其細胞質域方面不同的類似胺基酸序列。活化受體FcγRIIA在其細胞質域中含有基於免疫受體酪胺酸之活化模體(ITAM)。抑制受體FcγRIIB在其細胞質域中含有基於免疫受體酪胺酸之抑制模體(ITIM)。(參見例如Daeron, Annu. Rev. Immunol. 15:203-234 (1997))。FcR評述於例如Ravetch及Kinet, Annu . Rev . Immunol9:457-92 (1991)；Capel等人, Immunomethods4:25-34 (1994)；及de Haas等人, J . Lab . Clin . Med .126:330-41 (1995)中。其他FcR，包括待將來鑑別出之彼等FcR，為本文中之術語「FcR」所包涵。舉例而言，術語「Fc受體」或「FcR」亦包括新生兒受體FcRn，其負責將母體IgG轉移至胎兒(Guyer等人, J . Immunol .117:587 (1976)及Kim等人, J . Immunol .24:249 (1994))且調節免疫球蛋白之穩態。與FcRn之結合之量測方法為已知的(參見例如Ghetie及Ward, Immunol . Today18(12):592-598 (1997)；Ghetie等人, Nature Biotechnology, 15(7):637-640 (1997)；Hinton等人, J . Biol . Chem .279(8):6213-6216 (2004)；WO 2004/92219 (Hinton等人))。 "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcyR is a native human FcR. In some embodiments, the FcR is an FcR (gamma receptor) that binds an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelogenic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, eg, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR is reviewed, for example, in Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991); Capel et al . , Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab . Clin . Med . 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol . 117 : 587 (1976) and Kim et al., J. Immunol . 24:249 ( 1994 )) and regulate immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, eg, Ghetie and Ward, Immunol . Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology , 15(7):637-640 ( 1997 ); Hinton et al., J. Biol . Chem . 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.)).

如本文所用，術語「實質上類似」或「實質上相同」表示兩個或兩個以上數值之間之相似度足夠高，以使得在藉由該值量測生物特徵之情況下，熟習此項技術者將認為該兩個或兩個以上值之間的差異具有極小或不具有生物及/或統計顯著性。在一些實施例中，兩個或更多個實質上相似的值相差大致不超過以下中之任一者：5%、10%、15%、20%、25%或50%。 As used herein, the terms "substantially similar" or "substantially the same" mean that the similarity between two or more values is high enough to allow familiarity with the measurement of a biometric characteristic by that value. A skilled artisan would consider the difference between the two or more values to be of minimal or no biological and/or statistical significance. In some embodiments, two or more substantially similar values differ by approximately no more than any of: 5%, 10%, 15%, 20%, 25%, or 50%.

多肽「變異體」意謂在比對序列且必要時引入間隙以達到最大序列一致性百分比之後，且不考慮任何保守取代為序列一致性之一部分，與天然序列多肽具有至少約80%胺基酸序列一致性的生物活性多肽。該等變異體包括例如在多肽之N末端或C末端添加或缺失一或多個胺基酸殘基之多肽。在一些實施例中，變異體將具有至少約80%胺基酸序列一致性。在一些實施例中，變異體將具有至少約90%胺基酸序列一致性。在一些實施例中，變異體與天然序列多肽將具有至少約95%胺基酸序列一致性。 A "variant" of a polypeptide means a polypeptide that has at least about 80% of the amino acids of the native sequence polypeptide after aligning the sequences and introducing gaps where necessary to achieve the maximum percent sequence identity, without regard to any conservative substitutions that are part of the sequence identity. Biologically active peptides with sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the polypeptide. In some embodiments, variants will have at least about 80% amino acid sequence identity. In some embodiments, variants will have at least about 90% amino acid sequence identity. In some embodiments, a variant will have at least about 95% amino acid sequence identity to a native sequence polypeptide.

如本文所用，關於肽、多肽或抗體序列之「胺基酸序列一致性百分比(%)」及「同源性」定義為在比對序列且必要時引入間隙以達到最大序列一致性百分比之後，且不考慮任何保守取代為序列一致性之部分，候選序列中與特定肽或多肽序列中之胺基酸殘基一致的胺基酸殘基之百分比。用於確定胺基酸序列一致性百分比目的之比對可由此項技術範圍內之各種方式來達成，例如使用公開可用之電腦軟體，諸如BLAST、BLAST-2、ALIGN或MEGALIGNTM (DNASTAR)軟體。熟習此項技術者可確定適用於量測比對之參數，包括在所比較之序列全長內達成最大比對所需之任何演算法。 As used herein, "percent amino acid sequence identity (%)" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined after the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum percent sequence identity. The percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished by various means within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. One skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared.

胺基酸取代可包括但不限於將多肽中之一個胺基酸置換為另一個胺基酸。例示性取代展示於表1中。可向所關注之抗體中引入胺基酸取代，且針對所需活性來篩選產物，該所需活性例如抗原結合保留/改良、免疫原性減小或ADCC或CDC改良。 表1 原始殘基 例示性取代 Ala (A) Val；Leu；Ile Arg (R) Lys；Gln；Asn Asn (N) Gln；His；Asp, Lys；Arg Asp (D) Glu；Asn Cys (C) Ser；Ala Glu (E) Asp；Gln Gly (G) Ala His (H) Asn；Gln；Lys；Arg Ile (I) Leu；Val；Met；Ala；Phe；正白胺酸 Leu (L) 正白胺酸；Ile；Val；Met；Ala；Phe Lys (K) Arg；Gln；Asn Met (M) Leu；Phe；Ile Phe (F) Trp；Leu；Val；Ile；Ala；Tyr Pro (P) Ala Ser (S) Thr Thr (T) Val；Ser Trp (W) Tyr；Phe Tyr (Y) Trp；Phe；Thr；Ser Val (V) Ile；Leu；Met；Phe；Ala；正白胺酸 Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the antibodies of interest and the products screened for desired activity, such as retention/improvement of antigen binding, reduction in immunogenicity, or improvement in ADCC or CDC. Table 1 original residue illustrative substitution Ala (A) Val;Leu;Ile Arg(R) Lys; Gln; Asn Asn(N) Gln; His; Asp, Lys; Arg Asp(D) Glu;Asn Cys(C) Ser;Ala Glu(E) Asp;Gln Gly(G) Ala His (H) Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; norleucine Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg; Gln; Asn Met(M) Leu;Phe;Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ser(S) Thr Thr(T) Val;Ser Trp(W) Tyr; Phe Tyr(Y) Trp;Phe;Thr;Ser Val(V) Ile; Leu; Met; Phe; Ala; norleucine

胺基酸可根據共有側鏈特性來進行分組： ( 1 ) 疏水性 ： 正白胺酸、 Met 、 Ala 、 Val 、 Leu 、 Ile ； ( 2 ) 中性親水性 ： Cys 、 Ser 、 Thr 、 Asn 、 Gln ； ( 3 ) 酸性 ： Asp 、 Glu ； ( 4 ) 鹼性 ： His 、 Lys 、 Arg ； ( 5 ) 影響鏈取向之殘基： Gly 、 Pro ； ( 6 ) 芳族 ： Trp 、 Tyr 、 Phe 。 Amino acids can be grouped according to shared side chain characteristics: ( 1 ) Hydrophobicity : Norleucine, Met , Ala , Val , Leu , Ile ; ( 2 ) Neutral hydrophilicity : Cys , Ser , Thr , Asn , Gln ; ( 3 ) Acidic : Asp , Glu ; ( 4 ) Basic : His , Lys , Arg ; ( 5 ) Residues that affect chain orientation: Gly , Pro ; ( 6 ) Aromatic : Trp , Tyr , Phe .

非保守取代將引起此等類別中之一者的成員換成另一個類別。A non-conservative substitution would cause a member of one of these classes to be exchanged for another class.

術語「載體」用於描述可經工程改造為含有可在宿主細胞中繁殖之一或多個經選殖之聚核苷酸的聚核苷酸。載體可包括以下元件中之一或多個：複製起點、一或多個調控所關注多肽之表現的調控序列(諸如啟動子及/或強化子)及/或一或多個可選擇標記基因(諸如抗生素抗性基因及可用於比色分析中之基因，例如β-半乳糖)。術語「表現載體」係指用於表現宿主細胞中之所關注多肽之載體。The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more selected polynucleotides that can be propagated in a host cell. The vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate expression of the polypeptide of interest (such as a promoter and/or enhancer), and/or one or more selectable marker genes ( Such as antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

「宿主細胞」係指可為或已為載體或經分離聚核苷酸之接受者的細胞。宿主細胞可為原核細胞或真核細胞。例示性真核細胞包括哺乳動物細胞，諸如靈長類動物或非靈長類動物細胞；真菌細胞，諸如酵母；植物細胞；以及昆蟲細胞。非限制性例示性哺乳動物細胞包括但不限於NSO細胞、PER.C6 ®細胞(Crucell)以及293及CHO細胞，及其衍生物，諸如293-6E、CHO-DG44、CHO-K1、CHO-S及CHO-DS細胞。宿主細胞包括單個宿主細胞之後代，且後代可能歸因於自然、偶然或故意突變而不一定與原始母細胞完全一致(在形態或基因體DNA補體方面)。宿主細胞包括在活體內經本文提供之多核苷酸轉染之細胞。 "Host cell" refers to a cell that is or has been the recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include progeny of a single host cell, and progeny may not necessarily be identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with the polynucleotides provided herein.

如本文所用，術語「經分離」係指已與自然界中通常一起發現或產生之至少一些組分分離之分子。舉例而言，當多肽與產生多肽之細胞的至少一些組分分離時，稱該多肽「經分離」。若多肽在表現後由細胞分泌，則以物理方式將含有該多肽之上清液與產生其之細胞分離視為「分離」多肽。類似地，當聚核苷酸不為自然界中通常發現其之較大聚核苷酸(諸如在DNA聚核苷酸之情況下為基因體DNA或粒線體DNA)之部分，或與產生其之細胞之至少一些組分分離(例如在RNA聚核苷酸之情況下)時，該聚核苷酸稱為「經分離」。因此，宿主細胞內部之載體中所含的DNA聚核苷酸可稱為「經分離」。As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components normally found or produced together in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some components of the cell in which the polypeptide was produced. If a polypeptide is secreted by a cell after expression, the physical separation of the supernatant containing the polypeptide from the cell that produced it is considered to be an "isolated" polypeptide. Similarly, when the polynucleotide is not part of a larger polynucleotide from which it is normally found in nature (such as genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or is associated with the gene from which it is produced, When at least some components of a cell are separated (for example, in the case of an RNA polynucleotide), the polynucleotide is said to be "isolated." Therefore, the DNA polynucleotide contained in the vector inside the host cell can be said to be "isolated."

術語「個體(individual)」與「個體(subject)」在本文中可互換使用以指代動物；例如哺乳動物。在一些實施例中，提供治療哺乳動物之方法，該等哺乳動物包括但不限於人類、嚙齒動物、猿猴、貓科動物、犬科動物、馬科動物、牛科動物、豬科動物、綿羊、山羊、哺乳類實驗室動物、哺乳類農畜、哺乳類運動動物及哺乳類寵物。在一些實例中，「個體」或「個體」係指需要治療疾病或病症之個體或個體。在一些實施例中，接受治療之個體可為患者，表明以下事實：該個體已鑑別為患有與該治療有關聯之病症，或有極大的風險感染該病症。The terms "individual" and "subject" are used interchangeably herein to refer to animals; for example, mammals. In some embodiments, methods are provided for treating mammals including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, Goats, mammalian laboratory animals, mammalian farm animals, mammalian sporting animals and mammalian pets. In some examples, "individual" or "individual" refers to an individual or individuals in need of treatment of a disease or condition. In some embodiments, the individual receiving treatment may be a patient indicative of the fact that the individual has been identified as having a condition associated with the treatment, or is at substantial risk for contracting the condition.

如本文所用，「疾病」或「病症」係指需要及/或期望治療之病況。As used herein, "disease" or "disorder" means a condition for which treatment is required and/or desired.

除非另有指示，否則術語「腫瘤細胞」、「癌細胞」、「癌症」、「腫瘤」及/或「贅瘤」在本文中可互換使用，且係指展現生長不受控及/或細胞存活率異常增加及/或抑制細胞凋亡之細胞，其干擾身體器官及系統之正常功能。此定義中包括良性及惡性癌症、息肉、增生以及潛伏性腫瘤或微小轉移灶。Unless otherwise indicated, the terms "tumor cell", "cancer cell", "cancer", "tumor" and/or "neoplasia" are used interchangeably herein and refer to cells exhibiting uncontrolled growth and/or Abnormally increased survival rate and/or inhibition of cell apoptosis, which interferes with the normal function of body organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, and latent tumors or micrometastases.

術語「癌症」及「腫瘤」涵蓋實體癌症及血液/淋巴癌，且亦涵蓋惡性、癌前及良性生長，諸如發育異常。例示性癌症包括但不限於：基底細胞癌、膽道癌；膀胱癌；骨癌；腦及中樞神經系統癌；乳癌；腹膜癌；子宮頸癌；絨膜癌；大腸及直腸癌；結締組織癌；消化系統癌；子宮內膜癌；食道癌；眼球癌；頭頸癌；胃癌(包括胃腸癌)；神經膠母細胞瘤；肝癌瘤；肝癌；上皮內腫瘤；腎臟癌或腎癌；喉癌；白血病；肝癌；肺癌(例如小細胞肺癌、非小細胞肺癌、肺腺癌及肺之鱗狀癌瘤)；黑素瘤；骨髓瘤；神經母細胞瘤；口腔癌(唇、舌、口及咽)；卵巢癌；胰臟癌；前列腺癌；視網膜母細胞瘤；橫紋肌肉瘤；直腸癌；呼吸系統癌；唾液腺癌瘤；肉瘤；皮膚癌；鱗狀細胞癌；胃癌；睪丸癌；甲狀腺癌；子宮或子宮內膜癌；泌尿系統癌；外陰癌；淋巴瘤，包括霍奇金氏及非霍奇金氏淋巴瘤以及B細胞淋巴瘤(包括低惡性度/濾泡型非霍奇金氏淋巴瘤(NHL)；小淋巴球性(SL) NHL；中惡性度/濾泡NHL；中惡性度彌漫性NHL；高惡性度免疫母細胞NHL；高惡性度淋巴母細胞NHL；高惡性度小非裂解細胞NHL；腫塊性病變NHL；套細胞淋巴瘤；AIDS相關淋巴瘤；及瓦爾登斯特倫氏巨球蛋白血症；慢性淋巴球性白血病(CLL)；急性淋巴母細胞白血病(ALL)；毛細胞白血病；慢性骨髓母細胞白血病；以及其他癌瘤及肉瘤；及移植後淋巴增生病症(PTLD)，以及異常血管增殖伴母斑(細胞)病、水腫(諸如伴有腦瘤之水腫)及梅格斯氏症候群(Meigs' syndrome)。The terms "cancer" and "tumor" encompass solid cancers and hematological/lymphoid cancers, and also encompass malignant, precancerous and benign growths, such as dysplasia. Exemplary cancers include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer ; Digestive system cancer; Endometrial cancer; Esophageal cancer; Eyeball cancer; Head and neck cancer; Gastric cancer (including gastrointestinal cancer); Glioblastoma; Liver cancer; Liver cancer; Intraepithelial tumors; Kidney or renal cancer; Laryngeal cancer; Leukemia; liver cancer; lung cancer (such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx) ); Ovarian cancer; Pancreatic cancer; Prostate cancer; Retinoblastoma; Rhabdomyosarcoma; Rectal cancer; Respiratory system cancer; Salivary gland carcinoma; Sarcoma; Skin cancer; Squamous cell carcinoma; Gastric cancer; Testicular cancer; Thyroid cancer; Uterine cancer. or endometrial cancer; urinary tract cancer; vulvar cancer; lymphoma, including Hodgkin's and non-Hodgkin's lymphoma, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate diffuse NHL; high immunoblastic NHL; high lymphoblastic NHL; high small nonlytic NHL Cellular NHL; Massive NHL; Mantle cell lymphoma; AIDS-associated lymphoma; and Waldenstrom's macroglobulinemia; Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Hairy leukemia; chronic myeloblastic leukemia; and other cancers and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation with maculopathy, edema (such as that associated with brain tumors), and leucocytosis Meigs' syndrome.

如本文所用，術語「非腫瘤細胞」係指正常細胞或組織。示例性非腫瘤細胞包括但不限於：T細胞、B細胞、自然殺手(NK)細胞、自然殺手T (NKT)細胞、樹突狀細胞、單核球、巨噬細胞、上皮細胞、纖維母細胞、肝細胞、間質性腎細胞、纖維母細胞樣滑膜細胞、骨母細胞以及位於乳房、骨骼肌、胰臟、胃、卵巢、小腸、胎盤、子宮、睪丸、腎臟、肺、心臟、腦、肝、前列腺、大腸、淋巴器官、骨及骨來源間葉幹細胞中之細胞。如本文所用，術語「位於周邊之細胞或組織」係指並非位於腫瘤細胞附近及/或腫瘤微環境內部之非腫瘤細胞。As used herein, the term "non-tumor cells" refers to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts , liver cells, interstitial renal cells, fibroblast-like synoviocytes, osteoblasts, and cells located in the breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testicle, kidney, lung, heart, brain , cells in liver, prostate, large intestine, lymphoid organs, bone and bone-derived mesenchymal stem cells. As used herein, the term "peripheral cells or tissues" refers to non-tumor cells that are not located near tumor cells and/or within the tumor microenvironment.

如本文所用，術語「腫瘤微環境內之細胞或組織」係指圍繞及/或餵飼腫瘤細胞之細胞、分子、細胞外基質及/或血管。腫瘤微環境內之例示性細胞或組織包括但不限於腫瘤血管結構；腫瘤浸潤淋巴細胞；纖維母細胞網狀細胞；內皮先驅細胞(EPC)；癌症相關纖維母細胞；外被細胞；其他基質細胞；細胞外基質之組分(ECM)；樹突狀細胞；抗原呈遞細胞；T細胞；調節性T細胞(Treg細胞)；巨噬細胞；嗜中性白血球；骨髓衍生之抑制細胞(MDSC)及位於靠近腫瘤處之其他免疫細胞。如下文中所描述，此項技術中熟知用於鑑別腫瘤細胞及/或位於腫瘤微環境內之細胞/組織的方法。As used herein, the term "cells or tissues within the tumor microenvironment" refers to the cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed tumor cells. Exemplary cells or tissues within the tumor microenvironment include, but are not limited to, tumor vasculature; tumor infiltrating lymphocytes; fibroblast reticular cells; endothelial pioneer cells (EPC); cancer-associated fibroblasts; coat cells; other stromal cells ; Extracellular matrix components (ECM); dendritic cells; antigen-presenting cells; T cells; regulatory T cells (Treg cells); macrophages; neutrophils; bone marrow-derived suppressor cells (MDSC) and Other immune cells located near tumors. As described below, methods for identifying tumor cells and/or cells/tissues located within the tumor microenvironment are well known in the art.

在一些實施例中，「增加」或「減少」分別係指統計學上顯著之增加或減少。如熟習此項技術者將顯而易見，「調節」亦可包括相較於相同條件但不存在測試劑之情形，對目標或抗原之配體、結合搭配物、搭配物中之一或多者結合為均多聚體或雜多聚體形式或受質之親和力、親合力、特異性及/或選擇性達成改變(可為增加或減少)；對目標或抗原對該目標或抗原所存在之介質或環境中之一或多種條件(諸如pH、離子強度、輔因子之存在等)的敏感度達成改變(可為增加或減少)；及/或細胞增殖或細胞介素產生。視所涉及之目標而定，此可藉由任何合適方式及/或使用本身已知或本文所描述之任何合適分析來確定。In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be apparent to those skilled in the art, "modulation" may also include binding one or more of a ligand, binding partner, or partner to a target or antigen as compared to the same conditions but in the absence of the test agent. A change (either an increase or a decrease) in the affinity, avidity, specificity and/or selectivity of a homomultimeric or heteromultimeric form or substrate; for a target or antigen, for a medium in which the target or antigen is present, or A change in sensitivity (which may be an increase or decrease) to one or more conditions in the environment (such as pH, ionic strength, presence of cofactors, etc.); and/or cell proliferation or interleukin production. Depending on the objectives involved, this may be determined by any suitable means and/or using any suitable analysis known per se or described herein.

如本文所用，「免疫反應」意謂涵蓋足以抑制或阻止疾病發作或改善疾病症狀(例如癌症或癌轉移)之細胞及/或體液免疫反應。「免疫反應」可涵蓋先天性及後天性免疫系統兩者之態樣。As used herein, "immune response" is meant to encompass cellular and/or humoral immune responses sufficient to inhibit or prevent the onset of a disease or ameliorate symptoms of a disease (eg, cancer or cancer metastasis). "Immune response" can encompass both the innate and acquired immune systems.

如本文所用，「治療」為用於獲得有利的或所期望的臨床結果之途徑。如本文所用，「治療」涵蓋對於包括人類在內之哺乳動物之疾病進行之任何投藥或施用治療方案。出於本發明之目的，有益或所需臨床結果包括但不限於以下中之任何一或多者：緩解一或多種症狀、減弱疾病程度、預防或延緩疾病擴散(例如轉移，例如轉移至肺或淋巴結)、預防或延緩疾病復發、延緩或減緩疾病進展、改善疾病狀態、抑制疾病或疾病進展、抑制或減緩疾病或其進展、遏制其發展，及緩解(無論部分或完全)。「治療」亦涵蓋增殖性疾病之病理後果減輕。本文所提供之方法涵蓋該等治療態樣中之任一種或多種。根據以上所述，術語治療不需要百分之一百移除病症之所有態樣。As used herein, "treatment" is an approach used to obtain a beneficial or desired clinical outcome. As used herein, "treatment" encompasses any administration or regimen of treatment for a disease in mammals, including humans. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease severity, prevention or delay of disease spread (e.g., metastasis, e.g., to the lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing disease progression, improving disease status, inhibiting disease or disease progression, inhibiting or slowing disease or its progression, arresting its development, and remission (whether partial or complete). "Treatment" also covers the alleviation of the pathological consequences of proliferative diseases. The methods provided herein cover any one or more of these treatment modalities. According to the above, the term treatment does not require 100% removal of all aspects of the disease.

「緩解」意謂相較於未投與治療劑，一或多種症狀得以減輕或改善。「改善」亦包括縮短或減少症狀之持續時間。"Remission" means a reduction or improvement in one or more symptoms compared to where the therapeutic agent would not have been administered. "Improvement" also includes shortening or reducing the duration of symptoms.

術語「抗癌劑」在本文中以其最廣含義用於指用於治療一或多種癌症之藥劑。此等藥劑之例示性種類包括但不限於化學治療劑、抗癌生物製劑(諸如細胞介素、受體細胞外域-Fc融合體及抗體)、放射線療法、CAR-T療法、治療性寡核苷酸(諸如反義寡核苷酸及siRNA)及溶瘤病毒。The term "anticancer agent" is used herein in its broadest sense to refer to an agent used to treat one or more cancers. Exemplary types of such agents include, but are not limited to, chemotherapeutic agents, anti-cancer biologics (such as interleukins, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides acids (such as antisense oligonucleotides and siRNA) and oncolytic viruses.

術語「生物樣品」意謂一定量的來自活物或先前為活物之物質。該等物質包括但不限於血液(例如全血)、血漿、血清、尿液、羊水、滑液、內皮細胞、白細胞、單核細胞、其他細胞、器官、組織、骨髓、淋巴結及脾臟。The term "biological sample" means a quantity of material derived from a living organism or a previously living organism. Such substances include, but are not limited to, blood (such as whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

術語「對照」或「參考」係指已知不含分析物之組合物(「陰性對照」)或已知含有分析物之組合物(「陽性對照」)。陽性對照可包含已知濃度之分析物。The term "control" or "reference" refers to a composition known to contain no analyte ("negative control") or to a composition known to contain the analyte ("positive control"). Positive controls may contain known concentrations of the analyte.

如本文所用，「延遲疾病發展」意謂延緩、阻礙、減緩、扼止、穩定、遏制及/或推遲疾病(諸如癌症)之發展。此延遲可具有不同時間長度，視所治療之疾病及/或個體之病史而定。如熟習此項技術者顯而易見，充分或顯著延遲可實際上涵蓋預防，使得該個體不發展該疾病。舉例而言，可延遲晚期癌症，諸如癌轉移發展。As used herein, "delaying disease progression" means delaying, hindering, slowing down, arresting, stabilizing, containing and/or postponing the development of a disease (such as cancer). This delay can be of varying lengths, depending on the disease being treated and/or the individual's medical history. As will be apparent to those skilled in the art, sufficient or significant delay may actually encompass prevention such that the individual does not develop the disease. For example, the development of advanced cancer, such as cancer metastases, may be delayed.

如本文所用，「預防」包括在個體疾病出現或復發方面提供預防作用，該個體可能易患該疾病但尚未診斷患有該疾病。除非另有規定，否則術語「降低」、「抑制」或「預防」不表示或不要求一直完全預防，而僅在所量測之時間段內。As used herein, "prevention" includes providing prevention of the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms "reduce," "suppress," or "prevent" do not imply or require complete prevention at all times, but only during the time period being measured.

物質/分子(促效劑或拮抗劑)之「治療有效量」可根據以下因素改變：諸如個體之疾病病況、年齡、性別及體重、及物質/分子(促效劑或拮抗劑)在個體體內引發所要反應之能力。治療有效量亦係治療之有利作用超過該物質/分子(促效劑或拮抗劑)之任何有毒或有害作用的量。治療有效量可經一或多次投與來傳遞。「治療有效量」係指在必需劑量下且在必需時間內有效實現所需治療及/或預防結果的量。The "therapeutically effective amount" of a substance/molecule (agonist or antagonist) may vary depending on factors such as the individual's disease condition, age, gender and weight, and the presence of the substance/molecule (agonist or antagonist) in the individual's body The ability to elicit the desired response. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the substance/molecule (agonist or antagonist). A therapeutically effective amount can be delivered via one or more administrations. "Therapeutically effective amount" means an amount effective, at doses and for times necessary, to achieve the desired therapeutic and/or prophylactic results.

術語「醫藥調配物」及「醫藥組合物」可互換地使用，且係指所呈形式准許活性成分之生物活性有效，且不含對調配物將投與之個體具有不可接受毒性之額外組分的製劑。此類調配物可為無菌的。The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a form that permits the biological activity of the active ingredient to be effective and does not contain additional components that would be unacceptable toxicity to the individual to whom the formulation is to be administered. preparations. Such formulations can be sterile.

「醫藥學上可接受之載劑」係指此項技術中習知之無毒固體、半固體或液體填補劑、稀釋劑、囊封材料、調配助劑或載劑，其與治療劑一起使用，一起構成向個體投與之「醫藥組合物」。醫藥學上可接受之載劑在所用劑量及濃度下對接受者無毒且與調配物之其他成分相容。醫藥學上可接受之載劑適於所用之調配物。"Pharmaceutically acceptable carrier" means non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials, formulation aids or vehicles commonly known in the art, which are used with therapeutic agents, together with constitutes the administration of a "pharmaceutical composition" to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the doses and concentrations employed and is compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulations employed.

與一或多種其他治療劑「組合」投與包括同時(並行)及以任何次序依序投與。Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

術語「並行地」在本文中用以指代投與兩種或更多種治療劑，其中投與之至少部分在時間上重疊，或其中一個治療劑之投與落入針對另一治療劑之投與的一較短時間段內，或其中兩種藥劑之治療效果重疊至少一個時間段。The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents with which the administration at least partially overlaps in time, or with which the administration of one therapeutic agent falls within the context of another therapeutic agent. within a short period of time of administration, or the therapeutic effects of the two agents overlap for at least one period of time.

術語「依序」在本文中用以指代兩種或更多種治療劑之投與在時間上不重疊，或其中該等藥劑之治療作用不重疊。The term "sequential" is used herein to mean that the administration of two or more therapeutic agents does not overlap in time, or wherein the therapeutic effects of the agents do not overlap.

如本文中所用，「結合」係指除一種處理形式以外亦投與另一種處理形式。因此，「結合」係指在向個體投與一種處理形式之前、在投與一種處理形式期間或在投與一種處理形式之後投與另一種處理形式。As used herein, "combine" means to subject one form of processing to another form of processing in addition to another. Thus, "in conjunction with" means administering to an individual one form of treatment before, during, or after one form of treatment.

術語「藥品說明書」用以指通常包括於治療性產品之商業包裝中的說明，其含有關於與使用此類治療性產品有關之適應症、用法、劑量、投與、組合療法、禁忌及/或警告的資訊。The term "package insert" is used to mean the instructions typically included in the commercial packaging of therapeutic products containing information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or relevant to the use of such therapeutic products. Warning information.

「製品」係包含至少一種試劑，例如用於治療疾病或病症(例如，癌症)之藥物之任何製品(例如，包裝或容器)，或用於特異性偵測本文所述之生物標記之探針。在一些實施例中，製品或套組係以用於執行本文所描述之方法之單元形式推銷、分銷或出售。An "article of manufacture" is any article of manufacture (eg, a package or container) containing at least one agent, such as a drug for treating a disease or condition (eg, cancer), or a probe for specifically detecting a biomarker described herein . In some embodiments, articles or kits are marketed, distributed, or sold in unit form for performing the methods described herein.

術語「標記」及「可偵測標記」意謂例如附著至抗體或抗原以使得特異性結合對之成員之間的反應(例如，結合)可偵測之部分。特異性結合對之經標記成員稱為「可偵測地標記」。因此，術語「經標記之結合蛋白」係指併入標記以便鑑別結合蛋白之蛋白質。在一些實施例中，標記為可產生可藉由目視或儀器方式偵測的信號之可偵測標記，例如併入經放射性標記之胺基酸或連接至可藉由經標記之抗生物素蛋白(例如含有可藉由光學或比色方法偵測之螢光標記或酶促活性的鏈黴抗生物素蛋白)偵測之生物素基部分的多肽。用於多肽之標記的實例包括但不限於以下：放射性同位素或放射性核素(例如 3H、 14C、 35S、 90Y、 99Tc、 111In、 125I、 131I、 177Lu、 166Ho或 153Sm)；色素原、螢光標記(例如FITC、若丹明(rhodamine)、鑭系磷光體(lanthanide phosphor))、酶標記(例如辣根過氧化酶、螢光素酶、鹼性磷酸酶)；化學發光標記；生物素基；由二級報導子識別之預定多肽抗原決定基(例如白胺酸拉鏈對序列、二級抗體之結合位點、金屬結合域、抗原決定基標籤)；及磁化劑，諸如釓螯合物。常用於免疫檢定之標記之代表性實例包括產生光之部分，例如吖錠化合物，及產生螢光之部分，例如螢光素。就此而言，該部分自身可能並非可偵測標記的，但可在與又一部分反應之後變為可偵測的。 例示性靶向 CD8 之經修飾 IL - 2 多肽 The terms "label" and "detectable label" mean, for example, a moiety attached to an antibody or antigen such that a reaction (eg, binding) between members of a specific binding pair is detectable. The labeled member of a specific binding pair is called a "detectable label". Thus, the term "tagged binding protein" refers to a protein that incorporates a label to identify the binding protein. In some embodiments, the label is a detectable label that produces a signal that can be detected visually or instrumentally, such as incorporating a radioactively labeled amino acid or linking to a labeled avidin. (e.g., polypeptides containing a biotinyl moiety detectable by a fluorescent label or enzymatically active streptavidin detectable by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153 Sm); chromogens, fluorescent labels (such as FITC, rhodamine, lanthanide phosphor), enzyme labels (such as horseradish peroxidase, luciferase, alkaline phosphate enzyme); chemiluminescence label; biotinyl; predetermined polypeptide epitope recognized by the secondary reporter (such as leucine zipper pair sequence, secondary antibody binding site, metal-binding domain, epitope tag); and magnetizing agents, such as chromium chelates. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as azine compounds, and fluorescent-generating moieties, such as luciferin. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with another moiety. Exemplary modified IL - 2 polypeptides targeting CD8

本文提供靶向CD8之經修飾IL-2多肽。在各種實施例中，靶向CD8之經修飾IL-2多肽包含至少一個結合CD8之VHH域及經修飾IL-2。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽包含一個、兩個、三個、四個、五個、六個、七個或八個結合CD8之VHH域。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽包含一個、兩個、三個或四個結合CD8之VHH域。Provided herein are modified IL-2 polypeptides that target CD8. In various embodiments, a modified IL-2 polypeptide targeting CD8 comprises at least one VHH domain that binds CD8 and modified IL-2. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that bind CD8. In some embodiments, modified IL-2 polypeptides that target CD8 provided herein comprise one, two, three, or four VHH domains that bind CD8.

在一些實施例中，靶向CD8之經修飾IL-2多肽包含至少一個結合CD8之VHH域、Fc區及經修飾IL-2。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽包含一個、兩個、三個或四個結合CD8之VHH域、Fc區及經修飾IL-2。在一些實施例中，Fc區介導靶向CD8之經修飾IL-2多肽在生理條件下之二聚化，使得形成使CD8結合位點數目加倍的二聚體。舉例而言，包含三個結合CD8之VHH域、Fc區及經修飾IL-2的靶向CD8之經修飾IL-2多肽作為單體為三價，但在生理條件下，Fc區可介導二聚化，使得靶向CD8之經修飾IL-2多肽在此類條件下以六價二聚體形式存在。In some embodiments, a modified IL-2 polypeptide targeting CD8 comprises at least one VHH domain that binds CD8, an Fc region, and modified IL-2. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein comprise one, two, three, or four CD8-binding VHH domains, an Fc region, and modified IL-2. In some embodiments, the Fc region mediates dimerization of a modified IL-2 polypeptide targeting CD8 under physiological conditions, such that dimers are formed that double the number of CD8 binding sites. For example, a modified IL-2 polypeptide that targets CD8 that includes three CD8-binding VHH domains, an Fc region, and modified IL-2 is trivalent as a monomer, but under physiological conditions, the Fc region can mediate Dimerization such that the modified IL-2 polypeptide targeting CD8 exists as a hexavalent dimer under such conditions.

在一些實施例中，靶向CD8之經修飾IL-2多肽為包含結合CD8之第一VHH域、第一Fc域及經修飾IL-2多肽的第一多肽與包含結合CD8之第二VHH域及第二Fc域的第二多肽的複合物。在一些此類實施例中，第一或第二Fc域包含「杵」突變且另一Fc域包含「臼」突變。因此，在一些實施例中，靶向CD8之經修飾IL-2多肽為第一多肽與第二多肽之複合物，其中該複合物包含兩個CD8結合VHH域及一個經修飾IL-2多肽。在一些實施例中，靶向CD8之經修飾IL-2多肽為包含結合CD8之第一VHH域及第一Fc域的第一多肽與包含第二Fc域、經修飾IL-2多肽及視情況存在之第二VHH域的第二多肽的複合物。在一些此類實施例中，第一或第二Fc域包含「杵」突變且另一Fc域包含「臼」突變。因此，在一些實施例中，靶向CD8之經修飾IL-2複合物在複合物之不同多肽內包含CD8結合VHH域及一個經修飾IL-2多肽。In some embodiments, the modified IL-2 polypeptide that targets CD8 is a first polypeptide comprising a first VHH domain that binds CD8, a first Fc domain, and a modified IL-2 polypeptide and a second VHH that includes a CD8-binding A complex of a second polypeptide of a second Fc domain and a second Fc domain. In some such embodiments, the first or second Fc domain contains a "stick" mutation and the other Fc domain contains a "mortar" mutation. Accordingly, in some embodiments, the CD8-targeting modified IL-2 polypeptide is a complex of a first polypeptide and a second polypeptide, wherein the complex includes two CD8-binding VHH domains and one modified IL-2 Peptides. In some embodiments, a modified IL-2 polypeptide targeting CD8 is a first polypeptide comprising a first VHH domain and a first Fc domain that binds CD8 and a second Fc domain, a modified IL-2 polypeptide and a visual The situation exists where a second VHH domain is present in a complex with a second polypeptide. In some such embodiments, the first or second Fc domain contains a "stick" mutation and the other Fc domain contains a "mortar" mutation. Thus, in some embodiments, a modified IL-2 complex targeting CD8 includes a CD8 binding VHH domain and a modified IL-2 polypeptide within different polypeptides of the complex.

在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽包含CD8結合VHH及結合除CD8以外之抗原的抗原結合域。在一些此類實施例中，抗原係Lag3、CTLA4、TGFBR1、TGFBR2、Fas、TNFR2、PD1、PDL1或TIM3。在一些實施例中，該抗原為1-92-LFA-3、5T4、α-4整合素、α-V整合素、α4β1整合素、α4β7整合素、AGR2、抗Lewis-Y、Apelin J受體、APRIL、B7-H3、B7-H4、B7-H6、BAFF、BCMA、BTLA、C5補體、C-242、CA9、CA19-9、(Lewis a)、碳酸酐酶9、CD2、CD3、CD6、CD9、CD11a、CD19、CD20、CD22、CD24、CD25、CD27、CD28、CD30、CD33、CD38、CD39、CD40、CD40L、CD41、CD44、CD44v6、CD47、CD51、CD52、CD56、CD64、CD70、CD71、CD73、CD74、CD80、CD81、CD86、CD95、CD117、CD123、CD125、CD132、(IL-2RG)、CD133、CD137、CD138、CD166、CD172A、CD248、CDH6、CEACAM5 (CEA)、CEACAM6 (NCA-90)、CLAUDIN-3、CLAUDIN-4、cMet、膠原蛋白、Cripto、CSFR、CSFR-1、CTLA4、CTGF、CXCL10、CXCL13、CXCR1、CXCR2、CXCR4、CYR61、DL44、DLK1、DLL3、DLL4、DPP-4、DSG1、EDA、EDB、EGFR、EGFRviii、內皮素B受體(ETBR)、ENPP3、EpCAM、EPHA2、EPHB2、ERBB3、RSV之F蛋白質、FAP、FcRH5、FGF-2、FGF8、FGFR1、FGFR2、FGFR3、FGFR4、FLT-3、葉酸受體α (FRα)、GAL3ST1、G-CSF、G-CSFR、GD2、GITR、GLUT1、GLUT4、GM-CSF、GM-CsFR、GP IIb/IIIa受體、Gp130、GPIIB/IIIA、GPNMB、GPRC5D、GRP78、HAVCAR1、HER2/neu、HER3、HER4、HGF、hGH、HVEM、玻尿酸酶、ICOS、IFNα、IFNβ、IFNγ、IgE、IgE受體(FceRI)、IGF、IGF1R、IL1B、IL1R、IL2、IL11、IL12、IL12p40、IL-12R、IL-12Rβ1、IL13、IL13R、IL15、IL17、IL18、IL21、IL23、IL23R、IL27/IL27R (wsx1)、IL29、IL-31R、IL31/IL31R、IL2R、IL4、IL4R、IL6、IL6R、胰島素受體、鋸齒狀配位體、鋸齒狀1、鋸齒狀2、KISS1-R、LAG-3、LIF-R、Lewis X、LIGHT、LRP4、LRRC26、Ly6G6D、LyPD1、MCSP、間皮素、MICA、MICB、MRP4、MUC1、黏蛋白-16 (MUC16、CA-125)、Na/K ATP酶、NGF、Nicastrin、Notch受體、Notch 1、Notch 2、Notch 3、Notch 4、NOV、OSM-R、OX-40、PAR2、PDGF-AA、PDGF-BB、PDGFRα、PDGFRβ、PD-1、PD-L1、PD-L2、磷脂醯絲胺酸、P1GF、PSCA、PSMA、PSGR、RAAG12、RAGE、SLC44A4、神經鞘胺醇1磷酸酯、STEAP1、STEAP2、TAG-72、TAPA1、TEM-8、TGFβ、TIGIT、TIM-3、TLR2、TLR4、TLR6、TLR7、TLR8、TLR9、TMEM31、TNFα、TNFR、TNFRS12A、TRAIL-R1、TRAIL-R2、運鐵蛋白、運鐵蛋白受體、TRK-A、TRK-B、TROP-2 uPAR、VAP1、VCAM-1、VEGF、VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGFR1、VEGFR2、VEGFR3、VISTA、WISP-1、WISP-2或WISP-3。在一些實施例中，靶向CD8之經修飾IL-2多肽包含CD8結合VHH及結合腫瘤細胞抗原之抗原結合域。 結合 CD8 之例示性 VHH 域 In some embodiments, modified IL-2 polypeptides that target CD8 provided herein comprise an antigen-binding domain of CD8 that binds VHH and binds an antigen other than CD8. In some such embodiments, the antigen is Lag3, CTLA4, TGFBR1, TGFBR2, Fas, TNFR2, PD1, PDL1, or TIM3. In some embodiments, the antigen is 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor , APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90 ), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4 , DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3 , FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CsFR, GP IIb/IIIa receptor, Gp130, GPIIB/IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2/neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27/IL27R (wsx1), IL29, IL-31R, IL31 /IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MICA, MICB, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2. Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phospholipid serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, TROP-2 uPAR, VAP1, VCAM-1 , VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2 or WISP-3. In some embodiments, modified IL-2 polypeptides targeting CD8 comprise an antigen-binding domain of CD8 that binds VHH and binds a tumor cell antigen. Exemplary VHH domains that bind CD8

在各種實施例中，結合CD8之VHH域包含選自SEQ ID NO: 3、80及81之CDR1序列；選自SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86及87之CDR2序列；及選自SEQ ID NO: 5、16及18之CDR3。在各種實施例中，結合CD8之VHH域包含選自以下之CDR1、CDR2及CDR3序列：SEQ ID NO: 3、4及5；SEQ ID NO: 3、12及5；3、14及5；3、4及16；3、4及18；3、22及5；3、14及18；3、27及5；3、29及5；3、31及5；80、14及18；81、14及18；3、82及18；3、83及18；3、84及18；3、85及18；3、86及18；以及3、87及18。在各種實施例中，將VHH域人類化。In various embodiments, the CD8-binding VHH domain comprises a CDR1 sequence selected from the group consisting of SEQ ID NO: 3, 80, and 81; selected from the group consisting of SEQ ID NO: 4, 12, 14, 22, 27, 29, 31, 82, 83 , the CDR2 sequences of 84, 85, 86 and 87; and the CDR3 selected from SEQ ID NO: 5, 16 and 18. In various embodiments, the CD8-binding VHH domain comprises CDR1, CDR2, and CDR3 sequences selected from: SEQ ID NO: 3, 4, and 5; SEQ ID NO: 3, 12, and 5; 3, 14, and 5; 3 , 4 and 16; 3, 4 and 18; 3, 22 and 5; 3, 14 and 18; 3, 27 and 5; 3, 29 and 5; 3, 31 and 5; 80, 14 and 18; 81, 14 and 18; 3, 82 and 18; 3, 83 and 18; 3, 84 and 18; 3, 85 and 18; 3, 86 and 18; and 3, 87 and 18. In various embodiments, the VHH domain is humanized.

在一些實施例中，結合CD8之VHH域包含與選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、至少99%一致的胺基酸序列。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30之胺基酸序列，其中殘基XX不存在。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30之胺基酸序列，其中殘基XX為Gly-Gly。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105及106之胺基酸序列，其中VHH域包含突變K117D、K117E或K117R。In some embodiments, the CD8-binding VHH domain comprises a sequence selected from SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25 , 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 and 109 amino acids An amino acid sequence whose sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, Amino acid sequences of 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 and 109 . In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, Amino acid sequences 26, 28, and 30, in which residue XX does not exist. In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, Amino acid sequences 26, 28, and 30, in which residue XX is Gly-Gly. In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, The amino acid sequences of 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 and 106, among which The VHH domain contains mutations K117D, K117E or K117R.

在一些實施例中，結合CD8之VHH域包含SEQ ID NO: 3之CDR1序列、SEQ ID NO: 14之CDR2序列及SEQ ID NO: 18之CDR3。在一些實施例中，結合CD8之VHH域包含SEQ ID NO: 3之CDR1序列、SEQ ID NO: 85之CDR2序列及SEQ ID NO: 18之CDR3。在一些實施例中，結合CD8之VHH域包含與胺基酸序列SEQ ID NO: 25至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、至少99%一致的胺基酸序列。在一些實施例中，結合CD8之VHH域包含SEQ ID NO: 25之胺基酸序列。在一些實施例中，結合CD8之VHH域包含與胺基酸序列SEQ ID NO: 99或109至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、至少99%一致的胺基酸序列。在一些實施例中，結合CD8之VHH域包含SEQ ID NO: 99之胺基酸序列。在一些實施例中，結合CD8之VHH域包含SEQ ID NO: 109之胺基酸序列。In some embodiments, the VHH domain that binds CD8 includes the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 14, and the CDR3 of SEQ ID NO: 18. In some embodiments, the VHH domain that binds CD8 includes the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 85, and the CDR3 of SEQ ID NO: 18. In some embodiments, the CD8-binding VHH domain comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence SEQ ID NO: 25 amino acid sequence. In some embodiments, the CD8-binding VHH domain comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VHH domain that binds CD8 comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 the amino acid sequence SEQ ID NO: 99 or 109 %identical amino acid sequence. In some embodiments, the CD8-binding VHH domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD8-binding VHH domain comprises the amino acid sequence of SEQ ID NO: 109.

在各種實施例中，CD8結合多肽包含一個、兩個、三個或四個結合CD8之VHH域。In various embodiments, a CD8-binding polypeptide comprises one, two, three, or four VHH domains that bind CD8.

在一些實施例中，結合CD8之VHH域可經人類化。人類化抗體(諸如sdAbs或含VHH多肽)適用作治療性分子，此係因為人類化抗體減少或消除對非人類抗體之人類免疫反應，人類免疫反應可引起對抗體治療劑之免疫反應且減小治療劑之有效性。一般而言，人類化抗體包含一或多個可變域，其中CDR (或其部分)來源於非人類抗體，且FR (或其部分)來源於人類抗體序列。人類化抗體視情況亦將包含人類恆定區之至少一部分。在一些實施例中，人類化抗體中之一些FR殘基經來自非人類抗體(例如用於獲得CDR殘基之抗體)之對應殘基取代，從而例如恢復或改良抗體特異性或親和力。In some embodiments, the VHH domain that binds CD8 can be humanized. Humanized antibodies (such as sdAbs or VHH-containing peptides) are suitable for use as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies that can cause and reduce the immune response to antibody therapeutics. Effectiveness of therapeutic agents. Generally, humanized antibodies comprise one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies will optionally also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues were derived), thereby, for example, restoring or improving antibody specificity or affinity.

人類化抗體及其製造方法綜述於例如Almagro及Fransson, (2008) Front. Biosci. 13: 1619-1633中，且進一步描述於以下各者中：例如Riechmann等人, (1988) Nature332:323-329；Queen等人, (1989) Proc . Natl Acad . Sci . USA86: 10029-10033；美國專利第5,821,337號、第7,527,791號、第6,982,321號及第7,087,409號；Kashmiri等人, (2005) Methods36:25-34；Padlan, (1991) Mol . Immunol .28:489-498 (描述「表面再塑(resurfacing」)；Dall'Acqua等人, (2005) Methods36:43-60 (描述「FR改組」)；及Osbourn等人, (2005) Methods36:61-68及Klimka等人, (2000) Br . J . Cancer, 83:252-260 (描述FR改組之「導向選擇」方法)。 Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and further described in, for example, Riechmann et al., (1988) Nature 332:323- 329; Queen et al., (1989) Proc . Natl Acad . Sci . USA 86: 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36 :25-34; Padlan, (1991) Mol . Immunol . 28:489-498 (describing "resurfacing");Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling""); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br . J. Cancer , 83: 252-260 (describing the "guided selection" method of FR shuffling).

可用於人類化之人類構架區包括但不限於：使用「最佳擬合(best-fit)」法選擇之構架區(參見例如Sims等人 (1993) J . Immunol .151 :2296)；來源於具有重鏈可變區之特定子組之人類抗體的共同序列之構架區(參見例如Carter等人 (1992) Proc . Natl . Acad . Sci . USA, 89:4285；及Presta等人 (1993) J . Immunol, 151:2623)；人類成熟(體細胞突變)構架區或人類生殖系構架區(參見例如Almagro及Fransson, (2008) Front . Biosci .13:1619-1633)；及來源於篩選FR庫之構架區(參見例如Baca等人, (1997) J . Biol . Chem .272: 10678-10684及Rosok等人, (1996) J . Biol . Chem .271 :22611-22618)。通常，VHH之FR區經人類FR區置換以製備人類化VHH。在一些實施例中，人類FR之某些FR殘基經置換以便改良人類化VHH之一或多個特性。具有此等經置換殘基之VHH域在本文中仍稱為「人類化」。 例示性 經修飾 IL - 2 多肽 Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using "best-fit" methods (see, e.g., Sims et al . (1993) J. Immunol . 151:2296); Framework regions of consensus sequences for human antibodies with specific subsets of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc . Natl . Acad . Sci . USA , 89:4285; and Presta et al. (1993) J . Immunol , 151:2623); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front . Biosci . 13:1619-1633); and derived from screening FR libraries The framework region (see, for example, Baca et al., (1997) J. Biol . Chem . 272: 10678-10684 and Rosok et al . , (1996) J. Biol . Chem . 271: 22611-22618 ). Typically, the FR region of a VHH is replaced with a human FR region to create a humanized VHH. In some embodiments, certain FR residues of the human FR are substituted to improve one or more properties of the humanized VHH. VHH domains with these substituted residues are still referred to herein as "humanized." Exemplary modified IL - 2 polypeptides

本文提供包含CD8a結合VHH及經修飾IL-2的靶向CD8之經修飾IL-2多肽。在一些此類實施例中，靶向CD8之經修飾IL-2多肽包含CD8a結合VHH、Fc區及經修飾IL-2。在一些實施例中，相較於野生型IL-2，經修飾IL-2包含至少一個降低經修飾IL-2對IL-2受體之親和力的胺基酸取代。在各種實施例中，本文提供的包含經修飾之IL-2之多肽為IL-2R之促效劑。在一些實施例中，經修飾IL-2為經修飾人類IL-2，且IL-2R為人類IL-2R。在一些實施例中，經修飾IL-2對人類IL-2R之結合親和力比人類野生型IL-2對IL-2R之親和力低至少2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、至少10倍、至少20倍、至少30倍、至少50倍或至少100倍。Provided herein are CD8-targeting modified IL-2 polypeptides comprising CD8a binding VHH and modified IL-2. In some such embodiments, a modified IL-2 polypeptide targeting CD8 comprises CD8a binding VHH, an Fc region, and modified IL-2. In some embodiments, modified IL-2 includes at least one amino acid substitution that reduces the affinity of modified IL-2 for the IL-2 receptor compared to wild-type IL-2. In various embodiments, polypeptides provided herein comprising modified IL-2 are agonists of IL-2R. In some embodiments, modified IL-2 is modified human IL-2 and IL-2R is human IL-2R. In some embodiments, the binding affinity of modified IL-2 for human IL-2R is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold lower than the affinity of human wild-type IL-2 for IL-2R. times, 8 times, 9 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times or at least 100 times.

在各種實施例中，經修飾IL-2包含在至少一個選自T3、H16、E61、P65、D84及C125之胺基酸位置處的至少一個取代。在一些實施例中，經修飾IL-2包含在胺基酸位置T3、H16、E61、P65、D84及C125處之取代。在一些實施例中，經修飾IL-2包含在胺基酸位置T3、H16、P65、D84及C125處之取代。在一些實施例中，經修飾IL-2包含取代T3A、H16A、E61R、P65R、D84Y及C125S。在一些實施例中，經修飾IL-2包含取代T3A、H16A、P65R、D84S及C125S。在一些實施例中，經修飾IL-2包含與SEQ ID NO: 78或79至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致之胺基酸序列。在一些此類實施例中，經修飾IL-2包含SEQ ID NO: 78之胺基酸序列。在一些此類實施例中，經修飾IL-2包含SEQ ID NO: 79之胺基酸序列。In various embodiments, modified IL-2 comprises at least one substitution at at least one amino acid position selected from T3, H16, E61, P65, D84, and C125. In some embodiments, modified IL-2 includes substitutions at amino acid positions T3, H16, E61, P65, D84, and C125. In some embodiments, modified IL-2 includes substitutions at amino acid positions T3, H16, P65, D84, and C125. In some embodiments, modified IL-2 includes substitutions T3A, H16A, E61R, P65R, D84Y, and C125S. In some embodiments, modified IL-2 includes substitutions T3A, H16A, P65R, D84S, and C125S. In some embodiments, modified IL-2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 78 or 79 Identical amino acid sequence. In some such embodiments, the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 78. In some such embodiments, the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 79.

在一些實施例中，靶向CD8之經修飾IL-2多肽包含CD8a結合VHH、Fc區及經修飾IL-2。在一些此類實施例中，Fc區包含選自SEQ ID NO: 32-70之胺基酸序列。在一些實施例中，經修飾IL-2融合至Fc區之C端，該Fc區繼而融合至CD8a結合VHH之C端。在一些實施例中，CD8a結合VHH及Fc區藉由連接子連接，且Fc區及經修飾IL-2藉由連接子連接。在一些此類實施例中，連接子包含1-20個胺基酸，較佳為1-20個主要由甘胺酸且視情況由絲胺酸構成之胺基酸。在一些實施例中，連接子包含：Gly-Gly-Gly-Gly (SEQ ID NO: 123)、Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO:124)及/或Gly-Gly-Ser-Ser-Gly-Ser (SEQ ID NO:125)。在一些實施例中，Fc區包含至少一個杵或臼突變。在一些此類實施例中，靶向CD8之經修飾IL-2多肽為包含第二多肽之複合物的一部分，該第二多肽包含CD8a結合域及第二Fc區。在一些實施例中，靶向CD8之經修飾IL-2多肽包含SEQ ID NO: 72、74、76、107或110之胺基酸序列。在一些實施例中，複合物包含有包含SEQ ID NO: 72、74、76、107或110之胺基酸序列的第一多肽及包含CD8a結合域及Fc區之第二多肽。在一些此類實施例中，第二多肽包含SEQ ID NO: 73、75、108或111之胺基酸序列。In some embodiments, a modified IL-2 polypeptide targeting CD8 comprises CD8a binding VHH, an Fc region, and modified IL-2. In some such embodiments, the Fc region comprises an amino acid sequence selected from SEQ ID NO: 32-70. In some embodiments, modified IL-2 is fused to the C-terminus of the Fc region, which in turn is fused to the C-terminus of the CD8a-binding VHH. In some embodiments, CD8a binds VHH and the Fc region are connected by a linker, and the Fc region and modified IL-2 are connected by a linker. In some such embodiments, the linker contains 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. In some embodiments, the linker includes: Gly-Gly-Gly-Gly (SEQ ID NO: 123), Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 124), and/or Gly-Gly- Ser-Ser-Gly-Ser (SEQ ID NO: 125). In some embodiments, the Fc region contains at least one knob or mortar mutation. In some such embodiments, the modified IL-2 polypeptide targeting CD8 is part of a complex comprising a second polypeptide comprising a CD8a binding domain and a second Fc region. In some embodiments, a modified IL-2 polypeptide targeting CD8 comprises the amino acid sequence of SEQ ID NO: 72, 74, 76, 107, or 110. In some embodiments, the complex includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 72, 74, 76, 107 or 110 and a second polypeptide comprising a CD8a binding domain and an Fc region. In some such embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 73, 75, 108, or 111.

在一些實施例中，第一多肽包含SEQ ID NO: 72之胺基酸序列且第二多肽包含SEQ ID NO: 73之胺基酸序列。在一些實施例中，第一多肽包含SEQ ID NO: 74之胺基酸序列且第二多肽包含SEQ ID NO: 75之胺基酸序列。在一些實施例中，第一多肽包含SEQ ID NO: 76之胺基酸序列且第二多肽包含SEQ ID NO: 75之胺基酸序列。在一些實施例中，第一多肽包含SEQ ID NO: 107之胺基酸序列且第二多肽包含SEQ ID NO: 108之胺基酸序列。在一些實施例中，第一多肽包含SEQ ID NO: 110之胺基酸序列且第二多肽包含SEQ ID NO: 111之胺基酸序列。In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 72 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 74 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 76 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 107 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 111.

在一些實施例中，靶向CD8之經修飾IL-2多肽為包含第一多肽及第二多肽之複合物，其中第一多肽包含至少一個結合CD8之VHH域及第一Fc區，且第二多肽包含第二Fc區及經修飾IL-2。在一些此類實施例中，至少一個結合CD8之VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列。在一些實施例中，至少一個VHH域或各VHH域經人類化。在一些實施例中，靶向CD8之經修飾IL-2複合物之至少一個VHH域包含與選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、109之胺基酸序列至少85%、至少90%、至少95%或至少99%一致的胺基酸序列。在一些此類實施例中，至少一個VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30之胺基酸序列，其中殘基XX不存在。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30之胺基酸序列，其中殘基XX為Gly-Gly。在一些實施例中，結合CD8之VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105及106之胺基酸序列，其中VHH域包含突變K117D、K117E及K117R。In some embodiments, the modified IL-2 polypeptide targeting CD8 is a complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least one VHH domain that binds CD8 and a first Fc region, And the second polypeptide includes a second Fc region and modified IL-2. In some such embodiments, at least one CD8-binding VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 80 or 81; CDR2 comprising SEQ ID NO: 4, 12, 14, 22 , the amino acid sequence of SEQ ID NO: 5, 16 or 18. In some embodiments, at least one or each VHH domain is humanized. In some embodiments, at least one VHH domain of the modified IL-2 complex targeting CD8 comprises a compound selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, Amino acid sequences that are at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequences of 104, 105, 106 and 109. In some such embodiments, at least one VHH domain comprises SEQ ID NOs: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25 , 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 and 109 amino acids sequence. In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, Amino acid sequences 26, 28, and 30, in which residue XX does not exist. In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, Amino acid sequences 26, 28, and 30, in which residue XX is Gly-Gly. In some embodiments, the VHH domain that binds CD8 includes a VHH domain selected from the group consisting of SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, The amino acid sequences of 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 and 106, among which The VHH domain contains mutations K117D, K117E and K117R.

在一些實施例中，靶向CD8之經修飾IL-2複合物之經修飾IL-2相對於野生型人類IL-2包含T3A或T3G、H16A、P65R、C125S及D84S或D84Y突變。在一些實施例中，經修飾IL-2包含T3A、H16A、P65R、C125S及D84S突變。在一些實施例中，經修飾IL-2包含T3A、H16A、E61R、P65R、C125S及D84Y突變。在一些實施例中，經修飾IL-2包含與SEQ ID NO: 78或79之胺基酸序列至少90%、至少95%、至少97%、至少98%、至少99%或100%一致的胺基酸序列。在一些實施例中，經修飾IL-2包含SEQ ID NO: 78之胺基酸序列。In some embodiments, the modified IL-2 of the CD8-targeting modified IL-2 complex includes T3A or T3G, H16A, P65R, C125S, and D84S or D84Y mutations relative to wild-type human IL-2. In some embodiments, modified IL-2 includes T3A, H16A, P65R, C125S, and D84S mutations. In some embodiments, modified IL-2 includes T3A, H16A, E61R, P65R, C125S, and D84Y mutations. In some embodiments, modified IL-2 comprises an amine that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 78 or 79 amino acid sequence. In some embodiments, modified IL-2 comprises the amino acid sequence of SEQ ID NO: 78.

在一些實施例中，靶向CD8之經修飾IL-2複合物的第二多肽包含至少一個抗原結合域。在一些此類實施例中，第二多肽之至少一個抗原結合域為VHH域。在一些實施例中，第二多肽包含至少一個結合CD8之VHH域。In some embodiments, the second polypeptide of the modified IL-2 complex targeting CD8 comprises at least one antigen binding domain. In some such embodiments, at least one antigen-binding domain of the second polypeptide is a VHH domain. In some embodiments, the second polypeptide comprises at least one VHH domain that binds CD8.

在本文中描述之任一實施例中，經修飾IL-2可為經修飾人類IL-2。在各種實施例中，該等取代之胺基酸位置對應於SEQ ID NO: 71中之胺基酸位置。In any of the embodiments described herein, the modified IL-2 can be modified human IL-2. In various embodiments, the substituted amino acid positions correspond to the amino acid positions in SEQ ID NO: 71.

在各種實施例中，靶向CD8之經修飾IL-2多肽中所包括之Fc區為人類Fc區，或來源於人類Fc區。In various embodiments, the Fc region included in the modified IL-2 polypeptide targeting CD8 is a human Fc region, or is derived from a human Fc region.

在一些實施例中，包括於靶向CD8之經修飾IL-2多肽中之Fc區來源於人類Fc區，且在低級鉸鏈中包含對應於IgG1 E233、L234及L235之三個胺基酸缺失，本文中稱為「Fc xELL」。Fc xELL多肽不接合FcγR，且因此稱為「效應子靜默」或「效應子空缺」，然而在一些實施例中，xELL Fc區結合FcRn且因此具有延長之半衰期及與FcRn介導之再循環相關的胞吞轉送。In some embodiments, the Fc region included in the modified IL-2 polypeptide targeting CD8 is derived from a human Fc region and contains three amino acid deletions in the lower hinge corresponding to IgG1 E233, L234, and L235, It is called "Fc xELL" in this article. Fc xELL polypeptides do not bind FcγR and are therefore referred to as "effector silent" or "effector vacant", however in some embodiments, the xELL Fc region binds FcRn and therefore has an extended half-life and is associated with FcRn-mediated recycling transcytosis.

在一些實施例中，包括於靶向CD8之經修飾IL-2多肽中的Fc區來源於人類Fc區且包含突變M252Y及M428V，本文中稱作「Fc-YV」。在一些實施例中，此等突變在核內體之酸性pH (接近6.5)下增強與FcRn之結合，而在中性pH (約7.2)下失去可偵測之結合，使得FcRn介導之再循環增強且半衰期延長。In some embodiments, the Fc region included in the modified IL-2 polypeptide targeting CD8 is derived from a human Fc region and includes mutations M252Y and M428V, referred to herein as "Fc-YV." In some embodiments, these mutations enhance binding to FcRn at the acidic pH of endosomes (nearly 6.5) and lose detectable binding at neutral pH (about 7.2), allowing FcRn-mediated regeneration. Circulation is enhanced and half-life is prolonged.

在一些實施例中，包括於靶向CD8之經修飾IL-2多肽中之Fc區來源於人類Fc區且包含針對雜二聚化設計之突變，本文中稱為「杵」及「臼」。在一些實施例中，「杵」Fc區包含突變T366W。在一些實施例中，「臼」Fc區包含突變T366S、L368A及Y407V。在一些實施例中，用於雜二聚化之Fc區包含額外突變，諸如位於形成不對稱二硫鍵之雜二聚體Fc對之第一成員上之突變S354C及位於雜二聚體Fc對之第二成員上之對應突變Y349C。在一些實施例，雜二聚體Fc對之一個成員包含修飾H435R或H435K以阻止蛋白質A結合，同時維持FcRn結合。在一些實施例中，雜二聚體Fc對之一個成員包含修飾H435R或H435K，而雜二聚體Fc對之第二成員在H435處不經修飾。在各種實施例中，固持Fc區包含修飾H435R或H435K (在一些情況下，當修飾為H435R時，稱為「臼-R」)，而杵Fc區不包含修飾。在一些情況下，相對於可能存在之均二聚體臼Fc區，臼-R突變改良雜二聚體之純化。In some embodiments, the Fc region included in the modified IL-2 polypeptide targeting CD8 is derived from a human Fc region and contains mutations designed for heterodimerization, referred to herein as "pestle" and "mortar." In some embodiments, the "杵" Fc region contains mutation T366W. In some embodiments, the "acetyl" Fc region includes mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization includes additional mutations, such as mutation S354C located on the first member of the heterodimeric Fc pair that forms an asymmetric disulfide bond and located on the heterodimeric Fc pair The corresponding mutation Y349C on the second member. In some embodiments, one member of the heterodimeric Fc pair contains modifications H435R or H435K to prevent Protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair includes the modification H435R or H435K, while the second member of the heterodimeric Fc pair is unmodified at H435. In various embodiments, the anchor Fc region contains the modification H435R or H435K (in some cases, when modified to H435R, referred to as "H435R"), while the anchor Fc region contains no modification. In some cases, the acetaminophen-R mutation improves purification of heterodimers relative to the homodimeric acetaminophen Fc region that may be present.

在一些實施例中，包括於靶向CD8之經修飾IL-2多肽中的Fc區來源於人類Fc區且缺乏C端離胺酸殘基(稱為「ΔK447」)。In some embodiments, the Fc region included in the modified IL-2 polypeptide targeting CD8 is derived from a human Fc region and lacks a C-terminal lysine residue (referred to as "ΔK447").

可用於靶向CD8之經修飾IL-2多肽之非限制性例示性Fc區包括包含SEQ ID NO: 32-70及112-122之胺基酸序列的Fc區。在一些實施例中，靶向CD8之經修飾IL-2多肽包括Fc區，該Fc區包含選自SEQ ID NO: 33及36-52、68-70及112-122之胺基酸序列。在一些實施例中，靶向CD8之經修飾IL-2多肽包括Fc區，該Fc區包含選自SEQ ID NO: 41-52、58-70及112-122之胺基酸序列。在一些實施例中，靶向CD8之經修飾IL-2多肽包括Fc區，該Fc區包含選自SEQ ID NO: 41、48、70及112之胺基酸序列。 靶向 CD8 之經修飾 IL - 2 多肽的例示性活性 Non-limiting exemplary Fc regions of modified IL-2 polypeptides useful for targeting CD8 include Fc regions comprising the amino acid sequences of SEQ ID NOs: 32-70 and 112-122. In some embodiments, a modified IL-2 polypeptide targeting CD8 includes an Fc region comprising an amino acid sequence selected from SEQ ID NO: 33 and 36-52, 68-70, and 112-122. In some embodiments, a modified IL-2 polypeptide targeting CD8 includes an Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-52, 58-70, and 112-122. In some embodiments, a modified IL-2 polypeptide targeting CD8 includes an Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, 48, 70, and 112. Exemplary activities of modified IL - 2 polypeptides targeting CD8

在各種實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體外及/或活體內刺激CD8+細胞。在一些實施例中，使用本文實例中所提供之方法可測定活體外及/或活體內CD8+細胞之刺激或活性。在一些實施例中，經修飾IL-2之CD8+細胞刺激活性在融合至CD8結合VHH時比在單獨使用時更大及/或更特異性地靶向細胞毒性T細胞。在一些實施例中，IL-2之毒性藉由使其特異性靶向CD8+ T細胞來降低。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體外及/或活體內增加T細胞活化及/或增殖。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體內刺激CD8+細胞之活化及/或增殖，且因此靶向CD8之經修飾IL-2多肽可用於治療癌症之方法中。In various embodiments, modified IL-2 polypeptides targeting CD8 provided herein stimulate CD8+ cells in vitro and/or in vivo. In some embodiments, the stimulation or activity of CD8+ cells in vitro and/or in vivo can be determined using the methods provided in the examples herein. In some embodiments, the CD8+ cell stimulating activity of modified IL-2 when fused to a CD8-binding VHH is greater and/or more specifically targets cytotoxic T cells than when used alone. In some embodiments, the toxicity of IL-2 is reduced by specifically targeting it to CD8+ T cells. In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase T cell activation and/or proliferation in vitro and/or in vivo. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein stimulate the activation and/or proliferation of CD8+ cells in vivo, and thus the CD8-targeting modified IL-2 polypeptides can be used in methods of treating cancer. middle.

在各種實施例中，本文提供之靶向CD8之經修飾IL-2多肽為IL-2R活性之促效劑。在一些實施例中，可使用本文實例中提供之方法，諸如使用293F細胞或類似細胞測定促效劑活性。在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽在靶向CD8+ T細胞時為IL-2R活性之促效劑，但在不存在靶向的情況下展示極少或不展示促效活性。In various embodiments, the CD8-targeting modified IL-2 polypeptides provided herein are agonists of IL-2R activity. In some embodiments, agonist activity can be determined using methods provided in the examples herein, such as using 293F cells or similar cells. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein are agonists of IL-2R activity when targeting CD8+ T cells, but display little or no display in the absence of targeting. agonistic activity.

在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體外及/或活體內增加CD8 +T細胞之增殖。在一些實施例中，多肽在Treg細胞存在下增加CD8 +T細胞增殖。在一些此類實施例中，CD8 +T細胞為活化CD8 +T細胞。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體外增加活化CD8 +T細胞增殖。在一些實施例中，靶向CD8之經修飾IL-2多肽使活化CD8 +T細胞增殖相對於在不存在多肽之情況下的CD8 +T細胞增殖增加至少1.5倍、至少2倍、至少3倍或至少5倍。在一些實施例中，相對於在不存在多肽的情況下所觀測到的增殖，靶向CD8之經修飾IL-2多肽使活化CD8 +T細胞增殖增加至少1.5倍、至少2倍、至少3倍或至少5倍，且不實質上增加靜息CD8 +T細胞的增殖。 In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase the proliferation of CD8 + T cells in vitro and/or in vivo. In some embodiments, the polypeptide increases CD8 + T cell proliferation in the presence of Treg cells. In some such embodiments, the CD8 + T cells are activated CD8 + T cells. In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase activated CD8 + T cell proliferation in vitro. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases activated CD8 + T cell proliferation by at least 1.5-fold, at least 2-fold, at least 3-fold relative to CD8 + T cell proliferation in the absence of the polypeptide. Or at least 5 times. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases activated CD8 + T cell proliferation by at least 1.5-fold, at least 2-fold, at least 3-fold relative to the proliferation observed in the absence of the polypeptide. or at least 5-fold without substantially increasing the proliferation of resting CD8 + T cells.

活化CD8 +T細胞之增殖的增加可藉由此項技術中之任何方法，諸如本文實例中提供的方法測定。一種非限制性例示性分析如下。CD8 +T細胞可自一或多個健康人類供體分離。T細胞用CellTrace紫(CTV)染色且藉由抗CD3抗體活化，與包含經修飾IL-2多肽接觸，且隨後藉由FACS分析。CTV染色損失指示增殖。在一些實施例中，諸如藉由量測自不同健康人類供體分離之CD8 +T細胞之增殖，以一組實驗或合併T細胞之平均值形式測定CD8 +T細胞增殖之增加。在一些實施例中，以使用來自至少五個或至少十個不同健康供體之T細胞或來自至少五個或至少十個不同健康供體之T細胞池進行的實驗的平均值形式測定CD8 +T細胞增殖之增加。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽甚至在Treg細胞存在下增加CD8 +T細胞之增殖。 Increased proliferation of activated CD8 + T cells can be determined by any method in the art, such as those provided in the Examples herein. A non-limiting illustrative analysis follows. CD8 + T cells can be isolated from one or more healthy human donors. T cells were stained with CellTrace Violet (CTV) and activated by anti-CD3 antibodies, contacted with polypeptides containing modified IL-2, and subsequently analyzed by FACS. Loss of CTV staining indicates proliferation. In some embodiments, the increase in CD8 + T cell proliferation is determined as an average of a set of experiments or pooled T cells, such as by measuring the proliferation of CD8 + T cells isolated from different healthy human donors. In some embodiments, CD8 + is determined as an average of experiments performed using T cells from at least five or at least ten different healthy donors or a pool of T cells from at least five or at least ten different healthy donors. Increased T cell proliferation. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein increase the proliferation of CD8 + T cells even in the presence of Treg cells.

在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體外及/或活體內增加CD8 +T細胞上之CD71表現。CD71表現指示T細胞活化。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽活體外增加CD8 +T細胞上之CD71表現。在一些實施例中，靶向CD8之經修飾IL-2多肽使CD8 +T細胞上之CD71表現相對於在不存在多肽的情況下之CD71表現增加至少1.5倍、至少2倍、至少3倍或至少5倍。在一些實施例中，靶向CD8之經修飾IL-2多肽使活化CD8 +T細胞上之CD71表現相對於在無多肽存在下觀測到之CD71表現增加至少1.5倍、至少2倍、至少3倍或至少5倍，且實質上不增加靜息CD8 +T細胞上之CD71表現。在一些實施例中，靶向CD8之經修飾IL-2多肽在Treg細胞之存在下增加CD8 +T細胞上之CD71表現。 In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase CD71 expression on CD8 + T cells in vitro and/or in vivo. CD71 expression indicates T cell activation. In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase CD71 expression on CD8 + T cells in vitro. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases CD71 expression on CD8 + T cells by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 3-fold relative to CD71 expression in the absence of the polypeptide. At least 5 times. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases CD71 expression on activated CD8 + T cells by at least 1.5-fold, at least 2-fold, at least 3-fold relative to CD71 expression observed in the absence of the polypeptide. or at least 5-fold without substantially increasing CD71 expression on resting CD8 + T cells. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases CD71 expression on CD8 + T cells in the presence of Treg cells.

CD8 +T細胞上之CD71表現之增加可藉由此項技術中之任何方法測定，諸如本文實例中所提供之方法。一種非限制性例示性分析如下。CD8 +T細胞可自一或多個健康人類供體分離且用抗CD3抗體刺激，與靶向CD8之經修飾IL-2多肽接觸，且隨後藉由FACS分析CD71表現。在一些實施例中，CD8 +T細胞上之CD71表現之增加諸如藉由量測自不同健康人類供體分離之CD8 +T細胞上之CD71表現以一組實驗或合併T細胞之平均值形式測定。在一些實施例中，以使用來自至少五個或至少十個不同健康供體之T細胞或來自至少五個或至少十個不同健康供體之T細胞池進行的實驗的平均值形式測定CD8 +T細胞上CD71表現之增加。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽甚至在Treg細胞存在下增加CD8 +T細胞上之CD71表現。 Increased expression of CD71 on CD8 + T cells can be determined by any method in the art, such as that provided in the Examples herein. A non-limiting illustrative analysis follows. CD8 + T cells can be isolated from one or more healthy human donors and stimulated with anti-CD3 antibodies, contacted with modified IL-2 polypeptides targeting CD8, and subsequently analyzed for CD71 expression by FACS. In some embodiments, the increase in CD71 expression on CD8 + T cells is determined, such as by measuring CD71 expression on CD8 + T cells isolated from different healthy human donors as an average of a set of experiments or pooled T cells. . In some embodiments, CD8 + is determined as an average of experiments performed using T cells from at least five or at least ten different healthy donors or a pool of T cells from at least five or at least ten different healthy donors. Increased expression of CD71 on T cells. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein increase CD71 expression on CD8 + T cells even in the presence of Treg cells.

在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽活體外及/或活體內增加CD8 +T細胞中之pSTAT5表現。pSTAT5表現指示T細胞活化。在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽活體外增加CD8 +T細胞中之pSTAT5表現。在一些實施例中，靶向CD8之經修飾IL-2多肽使CD8 +T細胞上之pSTAT5表現相對於在無多肽存在下之pSTAT5表現增加至少1.5倍、至少2倍、至少3倍或至少5倍。在一些實施例中，靶向CD8之經修飾IL-2多肽在Treg細胞之存在下增加CD8 +T細胞上之pSTAT5表現。CD8 +T細胞中之pSTAT5表現的增加可藉由此項技術中之任何方法，諸如本文實例中所提供之方法測定。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽甚至在Treg細胞存在下增加CD8 +T細胞中之pSTAT5表現。 In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase pSTAT5 expression in CD8 + T cells in vitro and/or in vivo. pSTAT5 expression indicates T cell activation. In some embodiments, modified IL-2 polypeptides targeting CD8 provided herein increase pSTAT5 expression in CD8 + T cells in vitro. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases pSTAT5 expression on CD8 + T cells by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold relative to pSTAT5 expression in the absence of the polypeptide. times. In some embodiments, a modified IL-2 polypeptide targeting CD8 increases pSTAT5 expression on CD8 + T cells in the presence of Treg cells. Increased expression of pSTAT5 in CD8 + T cells can be determined by any method in the art, such as that provided in the Examples herein. In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein increase pSTAT5 expression in CD8 + T cells even in the presence of Treg cells.

在一些實施例中，本發明所提供之含經修飾IL-2多肽降低或減弱調控性T細胞(Treg)之抑制活性。在一些實施例中，靶向CD8之經修飾IL-2多肽使CD8 +T細胞上之Treg抑制活性降低至少10%、至少20%、至少30%或至少50%。習知CD8 +T細胞上之Treg抑制活性之降低可藉由此項技術中之任何方法，諸如本文實例中提供之方法來測定。在一些實施例中，本文所提供之靶向CD8之經修飾IL-2多肽在Treg細胞存在下增加CD8 +T細胞活化及增殖，例如，相比於在存在Treg細胞但不存在本文所提供之靶向CD8之經修飾IL-2多肽的情況下的CD8 +T細胞活化及增殖。 多肽表現及產生 In some embodiments, the modified IL-2-containing polypeptide provided by the invention reduces or weakens the suppressive activity of regulatory T cells (Treg). In some embodiments, a modified IL-2 polypeptide targeting CD8 reduces Treg suppressive activity on CD8 + T cells by at least 10%, at least 20%, at least 30%, or at least 50%. It is known that the reduction of Treg suppressive activity on CD8 + T cells can be measured by any method in the art, such as the method provided in the examples herein. In some embodiments, CD8-targeting modified IL-2 polypeptides provided herein increase CD8 + T cell activation and proliferation in the presence of Treg cells, e.g., compared to in the presence of Treg cells but in the absence of CD8 + T cell activation and proliferation in the presence of modified IL-2 polypeptides targeting CD8. Peptide expression and production

提供包含編碼靶向CD8之經修飾IL-2多肽之聚核苷酸的核酸分子。在一些實施例中，核酸分子亦可編碼引導靶向CD8之經修飾IL-2多肽之分泌的前導序列，該前導序列通常裂解使得其不存在於所分泌之多肽中。前導序列可為天然重鏈(或VHH)前導序列，或可為另一種異源前導序列。Nucleic acid molecules comprising polynucleotides encoding modified IL-2 polypeptides targeting CD8 are provided. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of a modified IL-2 polypeptide targeting CD8, which leader sequence is normally cleaved such that it is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

核酸分子可使用此項技術中習知之重組DNA技術來構築。在一些實施例中，核酸分子為適於在所選宿主細胞中表現的表現載體。Nucleic acid molecules can be constructed using recombinant DNA techniques known in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in the host cell of choice.

提供包含編碼本文所述之靶向CD8之經修飾IL-2多肽之核酸的載體。此類載體包括但不限於DNA載體、噬菌體載體、病毒載體、逆轉錄病毒載體等。在一些實施例中，選擇經最佳化以在所需細胞型(諸如CHO或CHO衍生之細胞)或NSO細胞中表現多肽之載體。例示性此類載體描述於例如Running Deer等人, Biotechnol . Prog. 20:880-889 (2004)中。 Vectors comprising nucleic acids encoding CD8-targeting modified IL-2 polypeptides described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, vectors are selected that are optimized for expression of the polypeptide in a desired cell type (such as CHO or CHO-derived cells) or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol . Prog . 20:880-889 (2004).

在一些實施例中，靶向CD8之經修飾IL-2多肽可在原核細胞中表現，諸如細菌細胞；或在真核細胞中表現，諸如真菌細胞(諸如酵母)、植物細胞、昆蟲細胞及哺乳動物細胞。此類表現可例如根據此項技術中已知的程序進行。可用於表現多肽的例示性真核細胞包括但不限於COS細胞，包括COS 7細胞；293細胞，包括293-6E細胞；CHO細胞，包括CHO-S、DG44. Lec13 CHO細胞，及FUT8 CHO細胞；PER.C6 ®細胞(Crucell)；及NSO細胞。在一些實施例中，靶向CD8之經修飾IL-2多肽可在酵母中表現。參見例如美國公開案第US 2006/0270045 A1號。在一些實施例中，特定的真核宿主細胞係基於其對多肽產生所需轉譯後修飾的能力來選擇。舉例而言，在一些實施例中，CHO細胞產生多肽，該等多肽之唾液酸化程度高於293細胞中所產生之相同多肽。 In some embodiments, modified IL-2 polypeptides targeting CD8 can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. animal cells. Such performance may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, modified IL-2 polypeptides targeting CD8 can be expressed in yeast. See, for example, US Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell line is selected based on its ability to produce the desired post-translational modification of the polypeptide. For example, in some embodiments, CHO cells produce polypeptides that are more sialylated than the same polypeptides produced in 293 cells.

將一或多種核酸(諸如載體)引入至所需宿主細胞中可藉由任何方法完成，包括但不限於磷酸鈣轉染、DEAE-聚葡萄糖介導之轉染、陽離子脂質介導之轉染、電穿孔、轉導、感染等。非限制性例示性方法描述於例如Sambrook等人, Molecular Cloning, A Laboratory Manual, 第3版 Cold Spring Harbor Laboratory Press (2001)中。核酸可根據任何適合方法短暫或穩定轉染於所需宿主細胞中。Introduction of one or more nucleic acids (such as vectors) into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-polydextrose-mediated transfection, cationic lipid-mediated transfection, Electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into desired host cells according to any suitable method.

亦提供包含本文所描述之任何核酸或載體之宿主細胞。在一些實施例中，提供表現本文所描述之靶向CD8之經修飾IL-2多肽的宿主細胞。宿主細胞中表現之靶向CD8之經修飾IL-2多肽可藉由任何適合方法純化。此類方法包括但不限於使用親和基質或疏水性相互作用層析。適合之親和配體包括ROR1 ECD及結合Fc區之藥劑。舉例而言，蛋白質A、蛋白質G、蛋白質A/G或抗體親和管柱可用於結合Fc區及純化包含Fc區之靶向CD8之經修飾IL-2多肽。疏水相互作用層析，例如丁基或苯基管柱，亦可適用於純化一些多肽，諸如抗體。離子交換層析(例如，陰離子交換層析及/或陽離子交換層析)亦可適用於純化一些多肽，諸如抗體。混合模式層析(例如逆相/陰離子交換、逆相/陽離子交換、親水相互作用/陰離子交換、親水相互作用/陽離子交換等)亦適合於純化一些多肽，諸如抗體。此項技術中已知多種用於純化多肽之方法。Host cells containing any nucleic acid or vector described herein are also provided. In some embodiments, host cells expressing a CD8-targeting modified IL-2 polypeptide described herein are provided. The CD8-targeting modified IL-2 polypeptide expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind the Fc region. For example, Protein A, Protein G, Protein A/G, or antibody affinity columns can be used to bind the Fc region and purify CD8-targeting modified IL-2 polypeptides that include the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for the purification of some peptides, such as antibodies. Ion exchange chromatography (eg, anion exchange chromatography and/or cation exchange chromatography) may also be suitable for purifying some polypeptides, such as antibodies. Mixed mode chromatography (eg reverse phase/anion exchange, reverse phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.) is also suitable for the purification of some polypeptides, such as antibodies. Various methods for purifying polypeptides are known in the art.

在一些實施例中，靶向CD8之經修飾IL-2多肽在無細胞系統中產生。非限制性例示性無細胞系統描述於例如Sitaraman等人, Methods Mol . Biol. 498: 229-44 (2009)；Spirin, Trends Biotechnol. 22: 538-45 (2004)；Endo等人, Biotechnol . Adv. 21: 695-713 (2003)中。 In some embodiments, modified IL-2 polypeptides targeting CD8 are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol . Biol . 498: 229-44 (2009); Spirin, Trends Biotechnol . 22: 538-45 (2004); Endo et al., Biotechnol . Adv . . 21: 695-713 (2003).

在一些實施例中，提供藉由上文所描述之方法製備之靶向CD8之經修飾IL-2多肽。在一些實施例中，靶向CD8之經修飾IL-2多肽係在宿主細胞中製備。在一些實施例中，靶向CD8之經修飾IL-2多肽係在無細胞系統中產生。在一些實施例中，純化靶向CD8之經修飾IL-2多肽。在一些實施例中，提供包含靶向CD8之經修飾IL-2多肽之細胞培養基。In some embodiments, modified IL-2 polypeptides targeting CD8 prepared by the methods described above are provided. In some embodiments, modified IL-2 polypeptides targeting CD8 are prepared in host cells. In some embodiments, modified IL-2 polypeptides targeting CD8 are produced in a cell-free system. In some embodiments, modified IL-2 polypeptides targeting CD8 are purified. In some embodiments, cell culture medium comprising a modified IL-2 polypeptide targeting CD8 is provided.

在一些實施例中，提供包含藉由上文所述方法製備之抗體的組合物。在一些實施例中，組合物包含在宿主細胞中製備之靶向CD8之經修飾IL-2多肽。在一些實施例中，組合物包含在無細胞系統中製備之靶向CD8之經修飾IL-2多肽。在一些實施例中，組合物包含經純化之靶向CD8之經修飾IL-2多肽。 使用靶向CD8之經修飾IL-2多肽治療疾病之例示性方法 In some embodiments, compositions comprising antibodies prepared by the methods described above are provided. In some embodiments, the composition comprises a CD8-targeting modified IL-2 polypeptide prepared in a host cell. In some embodiments, the composition comprises a modified IL-2 polypeptide targeting CD8 prepared in a cell-free system. In some embodiments, the composition comprises a purified modified IL-2 polypeptide targeting CD8. Exemplary methods of treating disease using modified IL-2 polypeptides targeting CD8

在一些實施例中，提供治療個體之疾病之包含投與靶向CD8之經修飾IL-2多肽的方法。此類疾病包括將得益於CD8 +T細胞之增殖及活化增加的任何疾病。在一些實施例中，CD8+ T細胞之增殖或活化增加係由靶向CD8之經修飾IL-2多肽誘導。在一些實施例中，提供用於治療個體之癌症的方法。 In some embodiments, methods of treating a disease in an individual are provided comprising administering a modified IL-2 polypeptide that targets CD8. Such diseases include any disease that would benefit from increased proliferation and activation of CD8 + T cells. In some embodiments, increased proliferation or activation of CD8+ T cells is induced by modified IL-2 polypeptides targeting CD8. In some embodiments, methods are provided for treating cancer in an individual.

所述方法包含向個體投與有效量的本文所提供之靶向CD8之經修飾IL-2多肽。此類治療方法可係針對人類或動物。在一些實施例中，提供治療人類之方法。可用本發明提供之靶向CD8之經修飾IL-2多肽治療之非限制性例示性癌症包括基底細胞癌、膽道癌；膀胱癌；骨癌；腦及中樞神經系統癌；乳癌；腹膜癌；子宮頸癌；絨膜癌；大腸及直腸癌；結締組織癌；消化系統癌；子宮內膜癌；食道癌；眼球癌；頭頸癌；胃癌(包括胃腸癌)；神經膠母細胞瘤；肝癌瘤；肝癌；上皮內腫瘤；腎臟癌或腎癌；喉癌；白血病；肝癌；肺癌(例如小細胞肺癌、非小細胞肺癌、肺腺癌及肺之鱗狀癌瘤)；黑素瘤；骨髓瘤；神經母細胞瘤；口腔癌(唇、舌、口及咽)；卵巢癌；胰臟癌；前列腺癌；視網膜母細胞瘤；橫紋肌肉瘤；直腸癌；呼吸系統癌；唾液腺癌瘤；肉瘤；皮膚癌；鱗狀細胞癌；胃癌；睪丸癌；甲狀腺癌；子宮或子宮內膜癌；泌尿系統癌；及外陰癌；淋巴瘤；霍奇金氏淋巴瘤；非霍奇金氏淋巴瘤；B細胞淋巴瘤；低惡性度/濾泡型非霍奇金氏淋巴瘤(NHL)、小淋巴球性(SL) NHL；中惡性度/濾泡型NHL；中惡性度彌漫性NHL；高惡性度免疫母細胞NHL；高惡性度淋巴母細胞NHL；高惡性度小型無裂隙細胞NHL；腫塊性病變NHL (bulky disease NHL)；套細胞淋巴瘤；AIDS相關淋巴瘤；瓦爾登斯特倫氏巨球蛋白血症；慢性淋巴球性白血病(CLL)；急性淋巴母細胞白血病(ALL)；毛細胞白血病及慢性骨髓母細胞白血病。The methods comprise administering to an individual an effective amount of a CD8-targeting modified IL-2 polypeptide provided herein. Such treatments may be for humans or animals. In some embodiments, methods of treating humans are provided. Non-limiting exemplary cancers that can be treated with the modified IL-2 polypeptide targeting CD8 provided by the invention include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; Cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eyeball cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer ; Liver cancer; intraepithelial neoplasm; kidney cancer or kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous carcinoma of the lung); melanoma; myeloma ; Neuroblastoma; Oral cancer (lip, tongue, mouth and pharynx); Ovarian cancer; Pancreatic cancer; Prostate cancer; Retinoblastoma; Rhabdomyosarcoma; Rectal cancer; Respiratory system cancer; Salivary gland carcinoma; Sarcoma; Skin Cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; and vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B cells Lymphoma; low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL; intermediate-grade/follicular NHL; intermediate-grade diffuse NHL; high-grade immune Blastic NHL; Highly malignant lymphoblastic NHL; Highly malignant small non-cleft cell NHL; Bulky disease NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenström's macroglobulin blood; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia and chronic myeloblastic leukemia.

可視需要向個體投與靶向CD8之經修飾IL-2多肽。可由熟習此項技術者，諸如主治醫師，基於考慮所治療病況、所治療個體之年齡、所治療病況之嚴重度、所治療個體之一般健康狀況及其類似因素來決定投與頻率。在一些實施例中，向個體投與有效劑量之靶向CD8之經修飾IL-2多肽一或多次。在一些實施例中，每天、每半週、每週、每兩週、每月一次等向個體投與有效劑量的靶向CD8之經修飾IL-2多肽。向個體投與有效劑量之靶向CD8之經修飾IL-2多肽至少一次。在一些實施例中，可多次，包括在至少一個月、至少六個月或至少一年之時程內多次投與有效劑量之靶向CD8之經修飾IL-2多肽。Modified IL-2 polypeptides targeting CD8 can be administered to an individual if desired. Frequency of administration may be determined by one skilled in the art, such as an attending physician, based on consideration of the condition being treated, the age of the individual being treated, the severity of the condition being treated, the general health of the individual being treated, and the like. In some embodiments, an effective dose of a CD8-targeting modified IL-2 polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of a CD8-targeting modified IL-2 polypeptide is administered to the subject daily, biweekly, weekly, biweekly, monthly, etc. An effective dose of a CD8-targeting modified IL-2 polypeptide is administered to the subject at least once. In some embodiments, an effective dose of a CD8-targeting modified IL-2 polypeptide can be administered multiple times, including multiple times over the course of at least one month, at least six months, or at least one year.

在一些實施例中，以有效治療(包括預防)癌症及/或增加T細胞增殖之量投與醫藥組合物。治療有效量通常取決於所治療個體之體重、其生理或健康狀況、所治療病狀之延伸性或所治療個體之年齡。一般而言，可以每次給藥約0.05 mg/kg體重至約100 mg/kg體重範圍內之量投與抗體。In some embodiments, the pharmaceutical composition is administered in an amount effective to treat (including prevent) cancer and/or increase T cell proliferation. The therapeutically effective amount will generally depend on the weight of the individual being treated, his/her physiological or health condition, the extent of the condition being treated, or the age of the individual being treated. In general, the antibody may be administered in an amount ranging from about 0.05 mg/kg to about 100 mg/kg of body weight per administration.

在一些實施例中，靶向CD8之經修飾IL-2多肽可在活體內藉由各種途徑投與，該等途徑包括但不限於靜脈內、動脈內、非經腸、腹膜內或皮下。適當調配物及投與途徑可根據預期應用選擇。In some embodiments, modified IL-2 polypeptides targeting CD8 can be administered in vivo by various routes including, but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Appropriate formulations and routes of administration can be selected based on the intended application.

在一些實施例中，使用靶向CD8之經修飾IL-2多肽之治療性治療藉由增加T細胞增殖及/或活化及/或藉由使CD8+ T細胞與癌細胞接觸達成。在一些實施例中，增加T細胞增殖及/或活化抑制癌症生長。 醫藥組合物 In some embodiments, therapeutic treatment using modified IL-2 polypeptides targeting CD8 is achieved by increasing T cell proliferation and/or activation and/or by contacting CD8+ T cells with cancer cells. In some embodiments, increasing T cell proliferation and/or activation inhibits cancer growth. Pharmaceutical composition

在一些實施例中，包含靶向CD8之經修飾IL-2多肽之組合物與廣泛多種醫藥學上可接受之載劑一起提供於調配物中(參見例如Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 第20版. (2003); Ansel等人, Pharmaceutical Dosage Forms and Drug Delivery Systems, 第7版, Lippencott Williams and Wilkins (2004); Kibbe等人, Handbook of Pharmaceutical Excipients, 第3版, Pharmaceutical Press (2000))。各種醫藥學上可接受之載劑，包括媒劑、佐劑及稀釋劑係可用的。此外，各種醫藥學上可接受之輔助物質，諸如pH調節劑及緩衝劑、張力調節劑、穩定劑、濕潤劑及類似物，亦係可用的。非限制性例示性載劑包括鹽水、緩衝鹽水、右旋糖、水、丙三醇、乙醇及其組合。In some embodiments, compositions comprising modified IL-2 polypeptides targeting CD8 are provided in a formulation with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 7th ed. 3rd edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers, including vehicles, adjuvants and diluents are available. In addition, various pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

在一些實施例中，醫藥組合物包含濃度為至少10 mg/mL之靶向CD8之經修飾IL-2多肽。 組合療法 In some embodiments, the pharmaceutical composition comprises a CD8-targeting modified IL-2 polypeptide at a concentration of at least 10 mg/mL. combination therapy

靶向CD8之經修飾IL-2多肽可單獨或與其他治療模式，諸如其他抗癌劑組合投與。其可在其他治療模式之前、實質上同時或之後(亦即，並行或依序)提供。在一些實施例中，本文所描述之治療方法可進一步包括投與：放射線療法、化學療法、疫苗接種、靶向腫瘤療法、CAR-T療法、溶瘤病毒療法、癌症免疫療法、細胞介素療法、手術切除、染色體修飾、消融、冷凍療法、針對腫瘤目標的反義藥劑、針對腫瘤目標的siRNA藥劑、針對腫瘤目標的微小RNA藥劑或抗癌/腫瘤藥劑，或生物製劑，諸如抗體、細胞介素或受體胞外域-Fc融合體。Modified IL-2 polypeptides targeting CD8 can be administered alone or in combination with other treatment modalities, such as other anti-cancer agents. It may be provided before, substantially simultaneously with, or after other treatment modalities (ie, concurrently or sequentially). In some embodiments, the treatment methods described herein may further comprise administering: radiation therapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, interleukin therapy , surgical resection, chromosomal modification, ablation, cryotherapy, antisense agents for tumor targets, siRNA agents for tumor targets, microRNA agents or anti-cancer/tumor agents for tumor targets, or biologics such as antibodies, cell mediators protein or receptor extracellular domain-Fc fusion.

在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽與第二治療劑(例如PD-1或PD-L1療法)同時給予。PD-1/PD-L1療法之實例包括納武單抗(nivolumab)(BMS)；皮立珠單抗(pidilizumab)(CureTech，CT-011)、派立珠單抗(pembrolizumab)(Merck)；德瓦魯單抗(durvalumab)(Medimmune/AstraZeneca)；阿特珠單抗(atezolizumab) (Genentech/Roche)；艾維路單抗(avelumab)(Pfizer)；AMP-224 (Amplimmune)；BMS-936559；AMP-514 (Amplimmune)；MDX-1105 (Merck)；TSR-042 (Tesaro/AnaptysBio，ANB-011)；STI-A1010 (Sorrento Therapeutics)；STI-A1110 (Sorrento Therapeutics)；以及針對程式化死亡-1 (PD-1)或程式化死亡配位體1 (PD-L1)之其他藥劑。In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein are administered concurrently with a second therapeutic agent (eg, PD-1 or PD-L1 therapy). Examples of PD-1/PD-L1 therapies include nivolumab (BMS); pidilizumab (CureTech, CT-011), pembrolizumab (Merck); durvalumab (Medimmune/AstraZeneca); atezolizumab (Genentech/Roche); avelumab (Pfizer); AMP-224 (Amplimmune); BMS-936559 ; AMP-514 (Amplimmune); MDX-1105 (Merck); TSR-042 (Tesaro/AnaptysBio, ANB-011); STI-A1010 (Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and for programmed death- 1 (PD-1) or other agents of programmed death ligand 1 (PD-L1).

在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽與免疫刺激劑(例如腫瘤壞死因子受體超家族(TNFRSF)之成員的促效劑或B7家族之成員)同時給予。免疫刺激TNFRSF成員之非限制性實例包括OX40、GITR、41BB、CD27及HVEM。B7家族成員之非限制性實例包括CD28及ICOS。因此，在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽與OX40、GITR、41BB、CD27、HVEM、CD28及/或ICOS之促效劑(諸如促效劑抗體)同時給予。In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein are administered concurrently with an immunostimulatory agent, such as an agonist of a member of the tumor necrosis factor receptor superfamily (TNFRSF) or a member of the B7 family. Non-limiting examples of immunostimulatory TNFRSF members include OX40, GITR, 41BB, CD27, and HVEM. Non-limiting examples of B7 family members include CD28 and ICOS. Accordingly, in some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein are administered simultaneously with an agonist (such as an agonist antibody) of OX40, GITR, 41BB, CD27, HVEM, CD28, and/or ICOS .

在一些實施例中，本文提供之靶向CD8之經修飾IL-2多肽與CAR-T (嵌合抗原受體T細胞)療法、溶瘤病毒療法、細胞介素療法及/或靶向其他檢查點分子之藥劑(諸如VISTA、gpNMB、B7H3、B7H4、HHLA2、CTLA4、TIGIT等)同時給予。 非限制性例示性診斷及治療方法 In some embodiments, the CD8-targeting modified IL-2 polypeptides provided herein are combined with CAR-T (chimeric antigen receptor T cell) therapy, oncolytic virus therapy, interleukin therapy, and/or targeted other tests Drugs with specific molecules (such as VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, TIGIT, etc.) are administered simultaneously. Non-limiting exemplary diagnostic and therapeutic methods

在一些實施例中，本文所描述之方法適用於評估個體及/或來自個體(例如，癌症患者)之試樣。在一些實施例中，評估係診斷、預後及/或對治療之反應中之一或多者。In some embodiments, the methods described herein are suitable for use in evaluating individuals and/or samples from individuals (eg, cancer patients). In some embodiments, the assessment is one or more of diagnosis, prognosis, and/or response to treatment.

在一些實施例中，本文所描述之方法包含評估蛋白質之存在、不存在或含量。在一些實施例中，本文所描述之方法包含評估核酸之存在、不存在或表現量。本文所描述之組合物可用於該等量測。舉例而言，在一些實施例中，本文所描述之方法包含使腫瘤或由腫瘤培養之細胞試樣與如本文所描述之治療劑接觸。In some embodiments, methods described herein include assessing the presence, absence, or amount of a protein. In some embodiments, methods described herein include assessing the presence, absence, or amount of nucleic acid expressed. The compositions described herein can be used for these measurements. For example, in some embodiments, the methods described herein include contacting a tumor or a sample of cells cultured from the tumor with a therapeutic agent as described herein.

在一些實施例中，評估可為直接治療(包括用本文所述之抗體治療)。在一些實施例中，評估可導引輔助療法在切除之後的使用或保留。輔助療法(亦稱作輔助護理)為除初級、主要或初步治療以外給與之治療。藉助於非限制性實例，輔助療法可為通常在手術後當所有可偵測疾病已移除，但由於隱性疾病而仍存在統計學復發風險時所給與之額外治療。在一些實施例中，抗體用作癌症治療中之輔助療法。在一些實施例中，抗體用作癌症治療中唯一之輔助療法。在一些實施例中，本文所描述之抗體被拒絕作為癌症治療中之輔助療法。舉例而言，若患者不大可能對本文所描述之抗體起反應或將具有最低反應，則為生活品質起見及為避免來自無效化學療法之不必要毒性，可不投與治療。在此等情況下，可使用姑息性照護。In some embodiments, assessment may be direct treatment (including treatment with an antibody described herein). In some embodiments, assessment may guide the use or retention of adjuvant therapy after resection. Complementary therapy (also called complementary care) is treatment given in addition to primary, main, or initial treatment. By way of non-limiting example, adjuvant therapy may be additional treatment usually given after surgery when all detectable disease has been removed but there is still a statistical risk of recurrence due to occult disease. In some embodiments, antibodies are used as adjuvant therapy in cancer treatment. In some embodiments, the antibody is used as the sole adjuvant therapy in cancer treatment. In some embodiments, the antibodies described herein are contemplated for use as adjuvant therapy in the treatment of cancer. For example, if a patient is unlikely to respond or will have a minimal response to an antibody described herein, treatment may be withheld for quality of life reasons and to avoid unnecessary toxicity from ineffective chemotherapy. In these situations, palliative care may be used.

在一些實施例中，投與該等分子作為切除前之新輔助療法。在一些實施例中，新輔助療法係指在任何手術之前使腫瘤縮小及/或降等之療法。在一些實施例中，新輔助療法意謂在手術之前向癌症患者投與之化學療法。在一些實施例中，新輔助療法意謂在手術之前向癌症患者投與抗體。通常考慮新輔助化學療法之癌症類型包括例如乳腺癌、大腸直腸癌、卵巢癌、子宮頸癌、膀胱癌及肺癌。在一些實施例中，抗體用作癌症治療中之新輔助療法。在一些實施例中，在切除之前使用。In some embodiments, the molecules are administered as neoadjuvant therapy prior to resection. In some embodiments, neoadjuvant therapy refers to therapy that shrinks and/or downgrades tumors before any surgery. In some embodiments, neoadjuvant therapy means administering chemotherapy to a cancer patient prior to surgery. In some embodiments, neoadjuvant therapy means administering antibodies to cancer patients prior to surgery. Cancer types for which neoadjuvant chemotherapy is commonly considered include, for example, breast, colorectal, ovarian, cervical, bladder, and lung cancers. In some embodiments, antibodies are used as neoadjuvant therapy in cancer treatment. In some embodiments, it is used prior to resection.

在一些實施例中，本文所述方法中涵蓋之腫瘤微環境係以下中之一或多者：腫瘤血管結構；腫瘤浸潤淋巴細胞；纖維母細胞網狀細胞；內皮先驅細胞(EPC)；癌症相關纖維母細胞；外被細胞；其他基質細胞；胞外基質之組分(ECM)；樹突狀細胞；抗原呈遞細胞；T細胞；調節性T細胞；巨噬細胞；嗜中性白血球；及位於靠近腫瘤處之其他免疫細胞。 套組 In some embodiments, the tumor microenvironment encompassed by the methods described herein is one or more of the following: tumor vasculature; tumor infiltrating lymphocytes; fibroblastic reticular cells; endothelial pioneer cells (EPC); cancer-associated Fibroblasts; coat cells; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen-presenting cells; T cells; regulatory T cells; macrophages; neutrophils; and located in other immune cells near the tumor. set

亦提供包括如本文所描述之任何靶向CD8之經修飾IL-2多肽及合適包裝的製品及套組。在一些實施例中，本發明包括具有(i)靶向CD8之經修飾IL-2多肽及(ii)關於使用套組向個體投與靶向CD8之經修飾IL-2多肽之說明書的套組。Articles and kits including any modified IL-2 polypeptide targeting CD8 as described herein and suitable packaging are also provided. In some embodiments, the invention includes a kit having (i) a modified IL-2 polypeptide that targets CD8 and (ii) instructions for using the kit to administer the modified IL-2 polypeptide that targets CD8 to an individual. .

適用於本文所描述之組合物的包裝為此項技術中已知，且包括例如小瓶(例如，密封小瓶)、容器、安瓿、瓶子、罐、可撓性包裝(例如，密封聚酯薄膜(Mylar)或塑料袋)及其類似物。此等製品可進一步經滅菌及/或密封。亦提供包含本文所描述之組合物之單位劑型。該等單位劑型可按單個或多個單位劑量儲存於適合包裝中且亦可經進一步滅菌及密封。本發明套組中供應之說明書為通常在標記或藥品說明書(例如，套組中包括之紙片)上之書面說明書，但機器可讀說明書(例如，磁化或光學儲存盤上載有的說明書)亦為可接受的。與使用抗體相關之說明一般包括關於用於預期治療或工業用途之劑量、給藥時程及投與途徑之資訊。套組可進一步包含關於選擇適合之個體或治療的描述。Packaging suitable for use in the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar ) or plastic bags) and the like. Such articles may further be sterilized and/or sealed. Unit dosage forms containing the compositions described herein are also provided. Such unit dosage forms may be stored in suitable packaging in single or multiple unit doses and may also be further sterilized and sealed. The instructions supplied in the kit of the invention are written instructions, usually on label or package insert (e.g., a piece of paper included in the kit), but also machine-readable instructions (e.g., instructions contained on a magnetized or optical storage disk). acceptable. Instructions related to the use of the antibodies generally include information regarding dosage, schedule of administration, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting appropriate individuals or treatments.

容器可為單位劑量、散裝(例如，多劑量包裝)或次單位劑量。舉例而言，亦可提供含有足夠劑量之本文所揭示分子以對個體提供延長週期之有效治療的套組，該延長週期諸如約1週、2週、3週、4週、6週、8週、3個月、4個月、5個月、6個月、7個月、8個月、9個月或更多個月中之任一者。套組亦可包括多個單位劑量之分子及使用說明且以對於在藥房(例如醫院藥房及配藥房)中儲存及使用而言足夠之量進行包裝。在一些實施例中，套組包括乾燥(例如凍乾)組合物，其可經復原、再懸浮或復水以形成一般穩定之抗體水性懸浮液。 實例 Containers can be unit dose, bulk (eg, multi-dose packaging), or sub-unit dose. For example, kits may also be provided that contain a sufficient dose of a molecule disclosed herein to provide an individual with effective treatment for an extended period of time, such as about 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks. , any of 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. Kits may also include multiple unit doses of the molecule and instructions for use and be packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and dispensing pharmacies. In some embodiments, the kit includes a dry (eg, lyophilized) composition that can be reconstituted, resuspended, or reconstituted to form a generally stable aqueous antibody suspension. Example

下文所述之實例僅意欲例示本發明，且不應視為以任何方式限制本發明。該等實例並非意欲表示下述實驗為所進行之所有實驗或唯一實驗。已儘力確保關於所用數字(例如量、溫度等)之準確度，但應考慮一些實驗誤差及偏差。除非另外指明，否則份為重量份，分子量為平均分子量，溫度以攝氏度計，且壓力為大氣壓或接近大氣壓。 實例 1 ： CD8a 結合 VHH 域之產生 The examples described below are merely intended to illustrate the invention and should not be construed as limiting the invention in any way. These examples are not intended to represent that the experiments described below are all or the only experiments performed. Every effort has been made to ensure accuracy with respect to the figures used (e.g. quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Example 1 : Generation of CD8a- binding VHH domain

經由用融合至駱馬Fc (SEQ ID NO: 77)之人類CD8a之胞外域免疫接種駱馬來產生靶向人類CD8a之單域抗體。產生特定抗CD8a抗體效價之後，自經免疫接種之動物的500 mL血液中分離出駱馬周邊血液單核細胞(PBMC)，且使用Qiagen RNeasy Maxi套組分離總mRNA，且隨後使用Thermo Superscript IV逆轉錄酶及寡聚dT引發(oligo-dT priming)轉化為第一股cDNA。VHH序列經由PCR使用cDNA作為模板特異性擴增且選殖至酵母表面顯示載體中作為VHH-Fc-AGA2融合蛋白。Fc為人類IgG1 Fc (SEQ ID NO: 32)，或在一些情況下，具有降低之效應功能的變異IgG1 Fc (例如Fc xELL；SEQ ID NO: 33)。Single domain antibodies targeting human CD8a were generated by immunizing vicuñas with the extracellular domain of human CD8a fused to vicuña Fc (SEQ ID NO: 77). After generation of specific anti-CD8a antibody titers, vicuña peripheral blood mononuclear cells (PBMC) were isolated from 500 mL of blood from immunized animals, and total mRNA was isolated using the Qiagen RNeasy Maxi kit, followed by Thermo Superscript IV Reverse transcriptase and oligo-dT priming convert into the first strand of cDNA. The VHH sequence was specifically amplified via PCR using cDNA as a template and cloned into a yeast surface display vector as a VHH-Fc-AGA2 fusion protein. The Fc is a human IgG1 Fc (SEQ ID NO: 32) or, in some cases, a variant IgG1 Fc with reduced effector function (eg, Fc xELL; SEQ ID NO: 33).

顯示VHH-Fc-AGA2融合蛋白之酵母庫使用CD8a ECD之重組形式，經由磁珠分離，接著進行螢光活化細胞分選(FACS)來富集。使分選的酵母析出，且挑選分離的群落置於96孔板中且使其在培養基中生長，使表面顯示之VHH-Fc表現轉變成分泌至培養基中。將來自96孔酵母菌分泌培養物之上清液施加至經CD8a短暫轉染之293F細胞(CD8a陽性)或未經轉染之293F細胞(CD8a陰性)，洗滌，用螢光團標記之抗人類IgG1 Fc二級抗體處理，且藉由96孔流動式細胞測量術分析。Yeast libraries displaying VHH-Fc-AGA2 fusion proteins were enriched using recombinant forms of CD8a ECD via magnetic bead isolation, followed by fluorescence-activated cell sorting (FACS). The sorted yeasts are pelleted, and isolated colonies are picked and placed in 96-well plates and grown in culture medium, converting surface-displayed VHH-Fc expression into secretion into the culture medium. Supernatants from 96-well yeast secretion cultures were applied to 293F cells transiently transfected with CD8a (CD8a positive) or untransfected 293F cells (CD8a negative), washed, and fluorophore-labeled anti-human IgG1 Fc secondary antibody treatment and analysis by 96-well flow cytometry.

編碼結合於CD8a陽性細胞及不結合於CD8a陰性細胞之VHH的核酸序列用人類Fc xELL編碼區同框選殖至哺乳動物表現載體中，且藉由在HEK293 Freestyle細胞(293F細胞)或CHO細胞中使用聚乙烯亞胺瞬時轉染表現。在3-7天之後收集上清液，藉由蛋白質A層析純化所分泌之重組蛋白，且由280 nm下之吸光度及消光係數計算濃度。The nucleic acid sequence encoding VHH that binds to CD8a-positive cells and does not bind to CD8a-negative cells was cloned in-frame with the human Fc xELL coding region into a mammalian expression vector, and expressed in HEK293 Freestyle cells (293F cells) or CHO cells. Transient transfection performance using polyethylenimine. The supernatant was collected after 3-7 days, and the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance and extinction coefficient at 280 nm.

使結合CD8a之一個VHH域(純系B7)人類化。簡言之，B7之各種人類化形式係基於人類重鏈構架製成。某些胺基酸回復突變成供體胺基酸，且例如在CDR中對某些突變針對其結合特性進行測試。B7之胺基酸序列及各種人類化形式提供於下文之某些序列表格中。應注意B7 VHH之序列(SEQ ID NO: 2)及人類化形式hzB7v1-hzB7v 18 (SEQ ID NO: 6-30可在其C端包括視情況存在之Gly-Gly (GG)連接子(由某些序列表格中之XX表示)。另外，其限制條件為所揭示之VHH域中之任一者中殘基117處的離胺酸(K117)可經天冬胺酸(K117D)、麩胺酸(K117E)或精胺酸(K117R)取代。命名為hzB7v41 (SEQ ID NO:109)之人類化VHH包含K117R取代(在某些序列表格中以粗體展示且加下劃線)。Humanizing a VHH domain (clone B7) that binds CD8a. In short, the various humanized forms of B7 are based on the human heavy chain architecture. Certain amino acids are backmutated to donor amino acids, and certain mutations are tested for their binding properties, for example in CDRs. The amino acid sequence of B7 and various humanized forms are provided in certain sequence tables below. It should be noted that the sequence of B7 VHH (SEQ ID NO: 2) and the humanized forms hzB7v1-hzB7v 18 (SEQ ID NO: 6-30) may include an optional Gly-Gly (GG) linker at their C-terminus (provided by a (represented by XX in these sequence tables). In addition, the restriction is that the lysine (K117) at residue 117 in any of the disclosed VHH domains can be passed through aspartic acid (K117D), glutamic acid (K117E) or arginine (K117R) substitution. The humanized VHH designated hzB7v41 (SEQ ID NO:109) contains the K117R substitution (shown in bold and underlined in some sequence tables).

藉由流動式細胞測量術在經分離之人類CD8+ T細胞上評定格式化為CD8a VHH-xELL-Fc之CD8a結合多肽的結合。將經分離T細胞以30,000個細胞/孔塗鋪於96孔盤之FACS緩衝液(PBS，1% BSA，0.1% NaN 3，pH 7.4)中。未經轉染之HEK293F細胞用作CD8a陰性對照且以30,000個細胞/孔塗鋪於單獨的盤中。隨後將測試多肽稀釋至2×最終濃度1000 nM，且進行3、4及5倍連續稀釋。無多肽之FACS緩衝液用作僅二級抗體之對照。將多肽稀釋液添加至等體積之細胞中，且將分析盤在4℃下培育30分鐘。用150 μL FACS緩衝液/孔洗滌兩次之後，將細胞再懸浮於FACS緩衝液中，其中螢光標記抗人類Fc抗體以1:2000稀釋以偵測結合，且螢光標記抗CD4抗體(純系OKT4)以1:100稀釋作為複染劑。在4℃下培育分析盤20分鐘。再一次用150 μL FACS緩衝液/孔洗滌之後，藉由流動式細胞測量術對CD4-細胞偵測結合多肽。CD8a結合在此群體上量測為647nm下之中值螢光形式。繪製資料且使用GraphPad Prism分析軟體分析。在Intellicyt iQue Plus上進行流動式細胞測量術偵測。所得最大結合(Bmax)值及結合親和力(K d)展示於表2及表3中，且結合曲線展示於圖1A-1B及圖2A-2B中。較高Bmax值通常指示較慢之解離速率，較低Bmax值可能指示較快之解離速率。 表2 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. B7-xELL-Fc 50903 0.06072 2, 33 hzB7v2-xELL-Fc 51439 0.06946 7, 33 hzB7v3-xELL-Fc 50103 0.05792 8, 33 hzB7v4-xELL-Fc 50839 0.05671 9, 33 hzB7v6-xELL-Fc 43658 0.09356 11, 33 hzB7v7-xELL-Fc 58151 0.08346 13, 33 hzB7v8-xELL-Fc 53344 0.05797 15, 33 hzB7v9-xELL-Fc 57324 0.05084 17, 33 表3 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. B7-xELL-Fc 39806 0.06663 2, 33 hzB7v2-xELL-Fc 38832 0.05951 7, 33 hzB7v10-xELL-Fc 26561 0.1065 19, 33 hzB7v11-xELL-Fc 41041 0.06089 20, 33 hzB7v12-xELL-Fc 29460 0.05847 21, 33 hzB7v13-xELL-Fc 31494 0.05805 23, 33 hzB7v14-xELL-Fc 36208 0.06564 24, 33 hzB7v15-xELL-Fc 39345 0.09653 25, 33 Binding of CD8a-binding polypeptide formatted as CD8a VHH-xELL-Fc was assessed by flow cytometry on isolated human CD8+ T cells. The isolated T cells were plated in FACS buffer (PBS, 1% BSA, 0.1% NaN 3 , pH 7.4) in a 96-well plate at 30,000 cells/well. Untransfected HEK293F cells were used as a CD8a negative control and were plated in separate dishes at 30,000 cells/well. Test peptides were then diluted to 2× final concentration of 1000 nM and serial dilutions of 3, 4 and 5 times were performed. FACS buffer without peptide was used as a secondary antibody only control. The polypeptide dilution was added to an equal volume of cells and the assay plate was incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer/well, the cells were resuspended in FACS buffer with a fluorescently labeled anti-human Fc antibody diluted 1:2000 to detect binding and a fluorescently labeled anti-CD4 antibody (pure strain OKT4) was diluted 1:100 as a counterstain. Incubate the assay plate for 20 minutes at 4°C. After washing again with 150 μL FACS buffer/well, CD4-cells were probed for bound peptides by flow cytometry. CD8a binding on this population was measured as the median fluorescence pattern at 647 nm. Data were plotted and analyzed using GraphPad Prism analysis software. Flow cytometry detection on Intellicyt iQue Plus. The resulting maximum binding (Bmax) values and binding affinities (K d ) are shown in Table 2 and Table 3, and the binding curves are shown in Figures 1A-1B and 2A-2B. Higher Bmax values generally indicate slower dissociation rates, lower Bmax values may indicate faster dissociation rates. Table 2 fusion protein Bmax(MFI) K d (nM) SEQ ID NO. B7-xELL-Fc 50903 0.06072 2, 33 hzB7v2-xELL-Fc 51439 0.06946 7, 33 hzB7v3-xELL-Fc 50103 0.05792 8, 33 hzB7v4-xELL-Fc 50839 0.05671 9, 33 hzB7v6-xELL-Fc 43658 0.09356 11, 33 hzB7v7-xELL-Fc 58151 0.08346 13, 33 hzB7v8-xELL-Fc 53344 0.05797 15, 33 hzB7v9-xELL-Fc 57324 0.05084 17, 33 table 3 fusion protein Bmax(MFI) K d (nM) SEQ ID NO. B7-xELL-Fc 39806 0.06663 2, 33 hzB7v2-xELL-Fc 38832 0.05951 7, 33 hzB7v10-xELL-Fc 26561 0.1065 19, 33 hzB7v11-xELL-Fc 41041 0.06089 20, 33 hzB7v12-xELL-Fc 29460 0.05847 21, 33 hzB7v13-xELL-Fc 31494 0.05805 23, 33 hzB7v14-xELL-Fc 36208 0.06564 24, 33 hzB7v15-xELL-Fc 39345 0.09653 25, 33

如圖1A及2A及上表中所示，所測試CD8結合多肽以低於0.2 nM且在大多數實例中低於0.1 nM之親和力結合人類CD8+ T細胞。如圖1B及圖2B中所示，除親本B7-xELL-Fc之外的所有測試多肽不展現與293對照細胞之顯著結合，且B7-xELL-Fc以相比於結合於CD8+ T細胞而言減少超過2,000倍之親和力結合對照細胞。此等結果表明CD8a結合多肽特異性結合CD8。 實例 2 ： CD8 結合多肽與人類及食蟹獼猴 CD8 之結合 As shown in Figures 1A and 2A and the table above, the CD8-binding polypeptides tested bound human CD8+ T cells with an affinity below 0.2 nM, and in most cases below 0.1 nM. As shown in Figure 1B and Figure 2B, all test polypeptides except the parental B7-xELL-Fc did not exhibit significant binding to 293 control cells, and B7-xELL-Fc bound to CD8+ T cells at a lower rate than Said to reduce the affinity of binding control cells by more than 2,000-fold. These results indicate that the CD8a-binding polypeptide specifically binds CD8. Example 2 : Binding of CD8- binding polypeptides to human and cynomolgus monkey CD8

藉由流動式細胞測量術對經轉染之HEK293F細胞評定上文所描述之親本及兩種人類化CD8a結合多肽的結合。HEK293F細胞用編碼全長人類或食蟹獼猴CD8a之質體，接著用IRES及GFP瞬時轉染。將經轉染的細胞以30,000個細胞/孔塗鋪於96孔盤之FACS緩衝液(PBS，1% BSA，0.1% NaN 3，pH 7.4)中。隨後將測試多肽稀釋至2×最終濃度500 nM，且進行3、4及5倍連續稀釋。無多肽之FACS緩衝液用作僅二級抗體之對照。將測試多肽添加至等體積之細胞中，且將分析盤在4℃下培育30分鐘。用150 μL FACS緩衝液/孔洗滌兩次之後，將細胞再懸浮於FACS緩衝液中，其中螢光標記抗人類Fc抗體以1:2000稀釋。在4℃下培育分析盤20分鐘。再一次用150 μL FACS緩衝液洗滌之後，藉由流動式細胞測量術偵測結合多肽。在Intellicyt iQue Plus上進行流動式細胞測量術偵測。表現CD8a之經轉染細胞作為GFP陽性閘控且多肽結合量測為647 nm下之中值螢光形式。繪製資料且使用GraphPad Prism分析軟體分析。結果展示於表4及表5以及圖3A-3B中。 表4：人類CD8a轉染之HEK293F細胞上之結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. B7-xELL-Fc 189563 0.1448 2, 33 hzB7v10-xELL-Fc 171061 0.5654 19, 33 hzB7v15-xELL-Fc 187850 0.1784 25, 33 表5：食蟹獼猴CD8a轉染之HEK293F細胞上之結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. B7-xELL-Fc 82948 0.03533 2, 33 hzB7v10-xELL-Fc 69358 0.09725 19, 33 hzB7v15-xELL-Fc 82954 0.04056 25, 33 Binding of the parental and two humanized CD8a binding polypeptides described above was assessed by flow cytometry on transfected HEK293F cells. HEK293F cells were transiently transfected with plasmids encoding full-length human or cynomolgus CD8a, followed by IRES and GFP. Transfected cells were plated in FACS buffer (PBS, 1% BSA, 0.1% NaN 3 , pH 7.4) in a 96-well plate at 30,000 cells/well. Test peptides were then diluted to 2× final concentration of 500 nM, and serial dilutions of 3, 4, and 5 times were performed. FACS buffer without peptide was used as a secondary antibody only control. Test polypeptide is added to an equal volume of cells and the assay plate is incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer/well, cells were resuspended in FACS buffer with fluorescently labeled anti-human Fc antibody diluted 1:2000. Incubate the assay plate for 20 minutes at 4°C. After washing again with 150 μL FACS buffer, bound peptides were detected by flow cytometry. Flow cytometry detection on Intellicyt iQue Plus. Transfected cells expressing CD8a served as GFP positive gates and peptide binding was measured as median fluorescence at 647 nm. Data were plotted and analyzed using GraphPad Prism analysis software. The results are shown in Tables 4 and 5 and Figures 3A-3B. Table 4: Binding on human CD8a transfected HEK293F cells fusion protein Bmax(MFI) K d (nM) SEQ ID NO. B7-xELL-Fc 189563 0.1448 2, 33 hzB7v10-xELL-Fc 171061 0.5654 19, 33 hzB7v15-xELL-Fc 187850 0.1784 25, 33 Table 5: Binding on cynomolgus monkey CD8a transfected HEK293F cells fusion protein Bmax(MFI) K d (nM) SEQ ID NO. B7-xELL-Fc 82948 0.03533 2, 33 hzB7v10-xELL-Fc 69358 0.09725 19, 33 hzB7v15-xELL-Fc 82954 0.04056 25, 33

如圖3A及表4中所示，所測試CD8結合多肽結合經轉染之HEK293F細胞，該細胞表現人類CD8a，其親和力低於0.6 nM。如圖3B及表5中所示，所測試的CD8結合多肽以低於0.1 nM的親和力結合表現食蟹獼猴CD8a的HEK293F細胞。 實例 3 ： CD8 結合多肽結合於人類及食蟹獼猴免疫細胞 As shown in Figure 3A and Table 4, the CD8-binding polypeptides tested bound transfected HEK293F cells expressing human CD8a with an affinity below 0.6 nM. As shown in Figure 3B and Table 5, the CD8-binding polypeptides tested bound HEK293F cells expressing cynomolgus CD8a with an affinity below 0.1 nM. Example 3 : CD8- binding polypeptide binds to human and cynomolgus monkey immune cells

藉由流動式細胞測量術對經分離人類T細胞及經分離食蟹獼猴PBMC評定上文所描述之親本及兩種人類化CD8a結合多肽的結合。對於食蟹獼猴PBMC，將經分離細胞以每孔200,000個細胞，且對於人類T細胞，以每孔50,000個細胞塗鋪於96孔盤中FACS緩衝液(PBS，1% BSA，0.1% NaN 3，pH 7.4)中。隨後將測試多肽稀釋至2×最終濃度250 nM且進行4倍連續稀釋。單獨的FACS緩衝液用作僅二級抗體之對照。將多肽稀釋液添加至等體積之細胞中，且將分析盤在4℃下培育30分鐘。用150 μL FACS緩衝液/孔洗滌兩次之後，將細胞再懸浮於FACS緩衝液中，其中螢光標記抗人類抗體以1:1000稀釋以偵測CD8a結合，且螢光標記抗CD3抗體(純系SP34.2)以1:40稀釋且抗CD4抗體(純系OKT4)以1:100稀釋。在4℃下培育分析盤20分鐘。再一次用150 μL FACS緩衝液洗滌之後，藉由流動式細胞測量術對CD3+ CD4-細胞偵測CD8a結合。在此等細胞群上之結合經量測為647 nm下之平均螢光形式。繪製資料且使用GraphPad Prism分析軟體分析。在ACEA Biosciences Novocyte-Quanteon流動式細胞儀上進行流動式細胞測量術偵測。結果展示於表6及表7及圖4A-4B中。 表6：人類CD3+ CD4- T細胞上之結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. B7-xELL-Fc 111947 0.02335 2, 33 hzB7v10-xELL-Fc 102521 0.08498 19, 33 hzB7v15-xELL-Fc 112777 0.02296 25, 33 表7：食蟹獼猴CD3+ CD4-細胞上之結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. B7-xELL-Fc 180823 0.04832 2, 33 hzB7v10-xELL-Fc 142617 0.07370 19, 33 hzB7v15-xELL-Fc 181851 0.05399 25, 33 Binding of the parental and two humanized CD8a binding polypeptides described above was assessed by flow cytometry on isolated human T cells and isolated cynomolgus monkey PBMC. Dissociated cells were plated in 96-well plates in FACS buffer (PBS, 1% BSA, 0.1% NaN 3 , pH 7.4). Test peptides were then diluted to 2× final concentration of 250 nM and 4-fold serial dilutions were performed. FACS buffer alone was used as a secondary antibody only control. The polypeptide dilution was added to an equal volume of cells and the assay plate was incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer/well, the cells were resuspended in FACS buffer with a fluorescently labeled anti-human antibody diluted 1:1000 to detect CD8a binding and a fluorescently labeled anti-CD3 antibody (clone SP34.2) was diluted 1:40 and the anti-CD4 antibody (clone OKT4) was diluted 1:100. Incubate the assay plate for 20 minutes at 4°C. After washing again with 150 μL FACS buffer, CD3+ CD4- cells were assayed for CD8a binding by flow cytometry. Binding on these cell populations was measured as the mean fluorescence at 647 nm. Data were plotted and analyzed using GraphPad Prism analysis software. Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. The results are shown in Tables 6 and 7 and Figures 4A-4B. Table 6: Binding on human CD3+ CD4- T cells fusion protein Bmax(MFI) K d (nM) SEQ ID NO. B7-xELL-Fc 111947 0.02335 2, 33 hzB7v10-xELL-Fc 102521 0.08498 19, 33 hzB7v15-xELL-Fc 112777 0.02296 25, 33 Table 7: Binding on cynomolgus monkey CD3+ CD4- cells fusion protein Bmax(MFI) K d (nM) SEQ ID NO. B7-xELL-Fc 180823 0.04832 2, 33 hzB7v10-xELL-Fc 142617 0.07370 19, 33 hzB7v15-xELL-Fc 181851 0.05399 25, 33

如圖4A及表6中所示，所測試的CD8結合多肽以低於0.1 nM的親和力結合人類CD3+ CD4- T細胞。如圖4B及表7中所示，所測試的CD8結合多肽以低於0.08 nM的親和力結合食蟹獼猴CD3+ CD4-細胞。As shown in Figure 4A and Table 6, the CD8-binding polypeptides tested bound human CD3+ CD4- T cells with an affinity below 0.1 nM. As shown in Figure 4B and Table 7, the CD8-binding polypeptides tested bound cynomolgus monkey CD3+ CD4- cells with an affinity below 0.08 nM.

藉由流動式細胞測量術對人類leukopak T細胞及食蟹獼猴PBMC評定CD8a結合多肽hzB7v15-xELL-Fc之結合。將leukopak T細胞用CTL抗聚集體洗滌解凍溶液解凍且塗鋪於96孔U底分析盤中。細胞在400× g下離心5分鐘，且丟棄上清液。跨10個孔自200 nM之初始濃度以1:3連續稀釋hzB7v15-xELL-Fc。FACS緩衝液用作非結合對照，且將該盤在4℃下培育30分鐘。以400× g離心分析盤5分鐘且丟棄上清液。洗滌細胞一次且在4℃下使其再懸浮於染色組(抗CD3抗體純系OKT3-BV605 (1:200)、抗CD4抗體純系OKT4-BV785 (1:200)及螢光標記抗人類Fc抗體(1:500))中30分鐘。以400× g離心分析盤5分鐘且丟棄上清液。細胞用150 μL FACS緩衝液洗滌，且用70 μL FACS緩衝液再懸浮以便在Novocyte流動式細胞儀上讀數。結果展示於下表8及圖5A中。Binding of the CD8a-binding peptide hzB7v15-xELL-Fc was assessed by flow cytometry on human leukopak T cells and cynomolgus monkey PBMC. Leukopak T cells were thawed with CTL anti-aggregate wash thawing solution and plated in 96-well U-bottom assay plates. Cells were centrifuged at 400 × g for 5 min, and the supernatant was discarded. hzB7v15-xELL-Fc was serially diluted 1:3 across 10 wells from an initial concentration of 200 nM. FACS buffer was used as a non-binding control and the plate was incubated at 4°C for 30 minutes. Centrifuge the assay plate at 400×g for 5 minutes and discard the supernatant. The cells were washed once and resuspended at 4°C in the staining group (anti-CD3 antibody pure line OKT3-BV605 (1:200), anti-CD4 antibody pure line OKT4-BV785 (1:200) and fluorescently labeled anti-human Fc antibody ( 1:500)) for 30 minutes. Centrifuge the assay plate at 400×g for 5 minutes and discard the supernatant. Cells were washed with 150 μL FACS buffer and resuspended with 70 μL FACS buffer for reading on the Novocyte flow cytometer. The results are shown in Table 8 below and Figure 5A.

食蟹獼猴PBMC用CTL抗聚集體洗滌解凍溶液解凍且以每孔500,000個細胞塗鋪於96孔U底分析盤中。細胞在400× g下離心5分鐘且丟棄上清液。跨10個孔自30 nM之初始分析濃度以1:3連續稀釋經Alexa Fluor 647化學標記之hzB7v15-xELL-Fc (AF647-hzB7v15-xELL-Fc)。FACS緩衝液用作非結合對照，且將該盤在4℃下培育20分鐘。以400× g離心分析盤5分鐘且丟棄上清液。細胞用150 μL PBS緩衝液洗滌，且用40 μL FACS緩衝液、10 μL BV染色緩衝液(Brilliant Stain Buffer Plus；BD Biosciences)再懸浮，且將抗體(抗CD3抗體純系SP34-BV421 (1:25)、抗CD4抗體純系OKT4-BV785 (1:100)及抗CD16抗體純系3G8-PE (1:100))於FACS緩衝液中之50 μL混合物添加至細胞。細胞在4℃下染色20分鐘。以400× g離心分析盤5分鐘且丟棄上清液。細胞用150 μL FACS緩衝液洗滌，且用70 μL FACS緩衝液再懸浮以便在Novocyte流動式細胞儀上讀數。結果展示於下表9及圖5B中。 表8：人類CD3+ CD4- T細胞上之結合 融合蛋白 K d(nM) SEQ ID NO. hzB7v15-xELL-Fc 0.09465 25, 33 表9：食蟹獼猴CD3+ CD4- CD16-細胞上之結合 融合蛋白 K d(nM) SEQ ID NO. hzB7v15-xELL-Fc 0.05412 25, 33 Cynomolgus macaque PBMC were thawed with CTL anti-aggregate wash thawing solution and plated at 500,000 cells per well in 96-well U-bottom assay plates. Cells were centrifuged at 400 × g for 5 min and the supernatant discarded. Alexa Fluor 647 chemically labeled hzB7v15-xELL-Fc (AF647-hzB7v15-xELL-Fc) was serially diluted 1:3 across 10 wells from an initial assay concentration of 30 nM. FACS buffer was used as a non-binding control and the plate was incubated at 4°C for 20 minutes. Centrifuge the assay plate at 400×g for 5 minutes and discard the supernatant. The cells were washed with 150 μL PBS buffer, resuspended with 40 μL FACS buffer, 10 μL BV staining buffer (Brilliant Stain Buffer Plus; BD Biosciences), and the antibody (anti-CD3 antibody pure line SP34-BV421 (1:25) ), anti-CD4 antibody pure line OKT4-BV785 (1:100) and anti-CD16 antibody pure line 3G8-PE (1:100)) in FACS buffer was added to the cells. Cells were stained for 20 min at 4°C. Centrifuge the assay plate at 400×g for 5 minutes and discard the supernatant. Cells were washed with 150 μL FACS buffer and resuspended with 70 μL FACS buffer for reading on the Novocyte flow cytometer. The results are shown in Table 9 below and Figure 5B. Table 8: Binding on human CD3+ CD4- T cells fusion protein K d (nM) SEQ ID NO. hzB7v15-xELL-Fc 0.09465 25, 33 Table 9: Binding on cynomolgus monkey CD3+ CD4- CD16- cells fusion protein K d (nM) SEQ ID NO. hzB7v15-xELL-Fc 0.05412 25, 33

如圖5A及表8中所示，hzB7v15-xELL-Fc以低於0.1 nM之親和力結合人類CD3+ CD4- T細胞。如圖5B及表9中所示，hzB7v15-xELL-Fc以低於0.06 nM之親和力結合食蟹獼猴CD3+ CD4- CD16-細胞。 實例 4 ：經修飾 IL-2 之 CD8a 靶向恢復活性 As shown in Figure 5A and Table 8, hzB7v15-xELL-Fc bound human CD3+ CD4- T cells with an affinity below 0.1 nM. As shown in Figure 5B and Table 9, hzB7v15-xELL-Fc bound cynomolgus monkey CD3+ CD4- CD16- cells with an affinity below 0.06 nM. Example 4 : CD8a- targeted restoration of activity by modified IL-2

在IL-2報導細胞中評定包含CD8a結合VHH hzB7v15或VHH hzB7v31域、Fc區及融合至Fc區之C端的經修飾IL-2的多肽的靶向CD8a之IL-2活性。融合蛋白為二聚的，包含融合至杵Fc區及經修飾IL-2之VHH hzB7v15或VHH hzB7v31域以及融合至臼Fc區之VHH hzB7v15或VHH hzB7v31域。因此，二聚融合蛋白包含兩個CD8a結合VHH域、兩個Fc區及一個經修飾IL-2。經修飾IL-2包含突變T3A、H16A、E61R、P65R、D84Y及C125S (SEQ ID NO: 78)；或突變T3A、H16A、P65R、D84S及C125S (SEQ ID NO: 79)。分離HEK-Blue IL2報導細胞或表現CD8a之HEK-Blue IL2報導細胞，將其轉移至50 mL錐形管，以400× g集結5分鐘，且以0.5×10^6個細胞/ml之密度再懸浮於新鮮預溫熱之分析培養基(DMEM + 4.5g/L葡萄糖、2 mM L-麩醯胺酸 + 10%熱滅活之FBS + 100U/mL青黴素 + 100µg/mL鏈黴素 + 100µg/mL normocin)中。以2×最終濃度在分析培養基中製備多肽稀釋系列，且每孔添加100 µL。將100 µL之50,000個細胞添加至平底96孔經組織培養物處理之盤的各孔中。將該盤在CO 2培育箱中在37℃下培育20小時。遵循製造商說明書製備Quanti-Blue溶液(再懸浮於水中且在水浴中升溫至37℃持續30分鐘)。在400× g下短暫離心分析盤5分鐘。將100 µL上清液轉移至新的平底96孔組織培養盤中，且添加100 µL/孔之Quanti-Blue溶液，且在5% CO 2培育箱中在37℃下培育1-2小時。在EMax讀盤儀上在650 nm下讀取吸光度。 The CD8a-targeting IL-2 activity of a polypeptide comprising a CD8a-binding VHH hzB7v15 or VHH hzB7v31 domain, an Fc region, and a modified IL-2 fused to the C-terminus of the Fc region was assessed in IL-2 reporter cells. The fusion protein is dimeric and includes the VHH hzB7v15 or VHH hzB7v31 domain fused to the cleavage Fc region and modified IL-2 and the VHH hzB7v15 or VHH hzB7v31 domain fused to the cleavage Fc region. Therefore, the dimeric fusion protein contains two CD8a-binding VHH domains, two Fc regions and a modified IL-2. Modified IL-2 includes mutations T3A, H16A, E61R, P65R, D84Y, and C125S (SEQ ID NO: 78); or mutations T3A, H16A, P65R, D84S, and C125S (SEQ ID NO: 79). Isolate HEK-Blue IL2 reporter cells or HEK-Blue IL2 reporter cells expressing CD8a, transfer them to a 50 mL conical tube, assemble at 400×g for 5 minutes, and repeat at a density of 0.5×10^6 cells/ml. Suspended in fresh pre-warmed analytical medium (DMEM + 4.5g/L glucose, 2 mM L-glutamine + 10% heat-inactivated FBS + 100U/mL penicillin + 100µg/mL streptomycin + 100µg/mL normocin). Prepare a peptide dilution series in assay medium at 2× final concentration and add 100 µL per well. Add 100 µL of 50,000 cells to each well of a flat-bottom 96-well tissue culture-treated plate. The plate was incubated in a CO2 incubator at 37°C for 20 hours. Quanti-Blue solution was prepared following the manufacturer's instructions (resuspended in water and warmed to 37°C in water bath for 30 minutes). Centrifuge the assay plate briefly at 400 × g for 5 min. Transfer 100 µL of the supernatant to a new flat-bottomed 96-well tissue culture plate, add 100 µL/well of Quanti-Blue solution, and incubate in a 5% CO2 incubator at 37°C for 1-2 hours. Read absorbance at 650 nm on an EMax plate reader.

如圖6A中所示，在不表現CD8a之細胞上，包含經修飾IL-2之靶向CD8a之多肽展現比包含非靶向VHH域及野生型IL-2之多肽顯著較低的活性。如圖6B及圖6C中所示，在表現CD8a之細胞中，包含CD8a結合VHH及經修飾IL-2之多肽展現穩定的IL-2活性，類似於包含非靶向VHH域及野生型IL-2之多肽的活性。包含非靶向VHH域及經修飾IL-2之多肽在表現CD8a之報導細胞上展現顯著較低的活性。此等結果顯示IL-2活性可在較寬濃度範圍內特異性地靶向表現CD8a之細胞，其範圍在此報導分析中大致落在0.01至1 nM之間。 實例 5 ： 由包含 CD8a 結合 VHH 及 經修飾 IL - 2 之多肽誘導的 T 細胞增殖 As shown in Figure 6A, CD8a-targeting polypeptides containing modified IL-2 exhibited significantly lower activity than polypeptides containing the non-targeting VHH domain and wild-type IL-2 on cells that did not express CD8a. As shown in Figures 6B and 6C, in cells expressing CD8a, peptides containing CD8a-binding VHH and modified IL-2 exhibited stable IL-2 activity, similar to those containing the non-targeting VHH domain and wild-type IL-2. 2. Activity of polypeptides. Polypeptides containing non-targeting VHH domains and modified IL-2 exhibited significantly lower activity on reporter cells expressing CD8a. These results demonstrate that IL-2 activity can specifically target CD8a-expressing cells over a wide concentration range, which in this reported assay fell roughly between 0.01 and 1 nM. Example 5 : T cell proliferation induced by polypeptides containing CD8a binding VHH and modified IL - 2

在非人類靈長類動物中測試融合蛋白對CD8+ T細胞擴增的影響，該融合蛋白包含有hzB7v15及融合至CD8a結合VHH hzB7v15之C端的包含突變T3A、H16A、P65R、D84S及C125S (SEQ ID NO: 79)的經修飾IL-2。食蟹獼猴以0.3 mg/kg靜脈內推注注射投與融合蛋白。在融合蛋白投與之前及融合蛋白投與之後7天，自研究動物收集全血樣品。在Lymphoprep™中使用密度離心來分離各時間點的周邊血液單核細胞(PBMC)，且用螢光標記細胞類型特異性抗體組合來染色細胞。將T細胞分類為表現CD4或CD8a、不表現B細胞標記CD20的CD3+細胞。調節性T細胞(「Treg」)定義為亦表現CD25且具有降低之CD127含量的CD4+ T細胞。CD4+習知T細胞(「CD4+ Tcon」)定義為不表現CD25且具有正常CD127含量的CD4+ T細胞。NK細胞定義為表現NKG2A之非T及非B細胞。針對CD20染色呈陽性之群體歸類為B細胞。使用流動式細胞測量術測定各PBMC亞群之絕對細胞計數，且藉由用給藥後7天絕對細胞計數除以給藥前基線計數來計算擴增。The effect of a fusion protein containing hzB7v15 and mutations T3A, H16A, P65R, D84S and C125S fused to the C-terminus of the CD8a-binding VHH hzB7v15 on CD8+ T cell expansion was tested in non-human primates (SEQ ID NO: 79) modified IL-2. Cynomolgus macaques were administered the fusion protein as an intravenous bolus injection of 0.3 mg/kg. Whole blood samples were collected from study animals before and 7 days after fusion protein administration. Peripheral blood mononuclear cells (PBMC) at each time point were isolated using density centrifugation in Lymphoprep™ and cells were stained with a combination of fluorescently labeled cell type-specific antibodies. T cells are classified as CD3+ cells that express CD4 or CD8a and do not express the B cell marker CD20. Regulatory T cells ("Tregs") are defined as CD4+ T cells that also express CD25 and have reduced CD127 content. CD4+ conventional T cells (“CD4+ Tcon”) are defined as CD4+ T cells that do not express CD25 and have normal CD127 content. NK cells are defined as non-T and non-B cells expressing NKG2A. Populations that stain positive for CD20 are classified as B cells. Absolute cell counts of each PBMC subpopulation were determined using flow cytometry, and expansion was calculated by dividing absolute cell counts 7 days post-dose by pre-dose baseline counts.

如圖7中所示，0.3 mg/kg之單次劑量之靶向CD8a之經修飾IL-2引起CD8+ T細胞之5.6倍擴增及NK細胞之2.8倍擴增，同時不顯著影響CD8-細胞群，包括Treg、CD4+習知T細胞及B細胞。較高數目之CD8+ T細胞亦導致總T細胞之3.2倍增加，且總PBMC數目相比於給藥前細胞計數增加2.7倍。此等數據顯示靶向CD8a之經修飾IL-2特異性地誘導活體內CD8+細胞群之細胞增殖。 實例 6 ： CD8a 結合多肽與 293F 細胞上表現之人類 CD8 鏈的結合 As shown in Figure 7, a single dose of 0.3 mg/kg modified IL-2 targeting CD8a caused a 5.6-fold expansion of CD8+ T cells and a 2.8-fold expansion of NK cells without significantly affecting CD8- cells. group, including Treg, CD4+ T cells and B cells. Higher numbers of CD8+ T cells also resulted in a 3.2-fold increase in total T cells, and a 2.7-fold increase in total PBMC numbers compared to predose cell counts. These data show that modified IL-2 targeting CD8a specifically induces cell proliferation of CD8+ cell populations in vivo. Example 6 : Binding of CD8a- binding polypeptides to human CD8 chains expressed on 293F cells

藉由流動式細胞測量術，在經過編碼人類CD8a或CD8b鏈之質體瞬時轉染之HEK293F細胞上評定包含人類化CD8a結合VHH域、Fc區、及在某些多肽中包含有融合至Fc區之C端的包含T3A、H16A、E61R、P65R、D84Y及C125S突變(SEQ ID NO: 78)的減毒IL-2的多肽之結合。經標記之複合物或多肽「KiH」包含杵臼雜二聚體Fc區，其中所指示之CD8a結合VHH域融合至各Fc區之N端，且突變IL-2僅融合至「杵」Fc區之C端。在生理條件下未經標記之「KiH」的複合物或多肽形成均二聚體。將經轉染細胞以每孔50,000個細胞塗鋪於96孔盤之FACS緩衝液(PBS，1% BSA，0.1% NaN 3，pH 7.4)中。隨後將測試多肽稀釋至2×最終濃度500 nM，且進行6倍連續稀釋。無多肽之FACS緩衝液用作僅二級抗體之對照。將測試多肽添加至等體積之細胞中，且將分析盤在4℃下培育30分鐘。用150 μL FACS緩衝液/孔洗滌兩次之後，將細胞再懸浮於FACS緩衝液中，其中螢光標記抗人類Fc抗體以1:1000稀釋以偵測CD8結合。分析盤在4℃下培育30分鐘。再用150 μL FACS緩衝液洗滌一次之後，藉由流動式細胞測量術在對轉染標記citrine呈陽性之細胞上偵測結合於CD8之多肽。在此等細胞群上之結合經量測為647 nm下之平均螢光強度(MFI)。在IntelliCyt iQue Screener Plus上進行流動式細胞測量術偵測。使用GraphPad Prism分析軟體繪製及分析資料。結果展示於表10及圖8A-8B中。 表10：與經轉染之HEK293F細胞上表現之人類CD8a的結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. hzB7v19-xELL-P329G-KiH Fc-IL-2突變體 14162685 7.495 88、68；及88、69、78 hzB7v20-xELL-P329G-KiH Fc-IL-2突變體 11553318 4.482 89、68；及89、69、78 hzB7v21-xELL-P329G-KiH Fc-IL-2突變體 14220676 5.234 90、68；及90、69、78 hzB7v23-xELL-P329G-KiH Fc-IL-2突變體 14341168 3.897 91、68；及91、69、78 hzB7v24-xELL-P329G-KiH Fc-IL-2突變體 12760589 6.543 92、68；及92、69、78 hzB7v25-xELL-P329G-KiH Fc-IL-2突變體 14542422 3.279 93、68；及93、69、78 hzB7v26-xELL-P329G-KiH Fc-IL-2突變體 15247397 4.546 94、68；及94、69、78 hzB7v27-xELL-P329G-KiH Fc-IL-2突變體 14456494 3.179 95、68；及95、69、78 hzB7v28-xELL-P329G-KiH Fc-IL-2突變體 13748843 2.838 96、68；及96、69、78 hzB7v15-xELL Fc 14220016 0.8207 25、33 Containing humanized CD8a-binding VHH domains, Fc regions, and fusion to the Fc region in certain polypeptides, as assessed by flow cytometry on HEK293F cells transiently transfected with plasmids encoding human CD8a or CD8b chains Binding of the C-terminal attenuated IL-2 polypeptide containing T3A, H16A, E61R, P65R, D84Y and C125S mutations (SEQ ID NO: 78). The labeled complex or polypeptide "KiH" contains a N-terminal heterodimeric Fc region in which the indicated CD8a-binding VHH domain is fused to the N-terminus of each Fc region and mutant IL-2 is fused only to the N-terminus of the "N-terminal" Fc region. C end. Under physiological conditions, unlabeled "KiH" complexes or peptides form homodimers. Transfected cells were plated in FACS buffer (PBS, 1% BSA, 0.1% NaN 3 , pH 7.4) in a 96-well plate at 50,000 cells per well. The test peptides were then diluted to 2× final concentration of 500 nM and 6-fold serial dilutions were performed. FACS buffer without peptide was used as a secondary antibody only control. Test polypeptide is added to an equal volume of cells and the assay plate is incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer/well, cells were resuspended in FACS buffer with fluorescently labeled anti-human Fc antibody diluted 1:1000 to detect CD8 binding. The assay plate was incubated at 4°C for 30 minutes. After another wash with 150 μL FACS buffer, peptides bound to CD8 were detected by flow cytometry on cells positive for the transfection marker citrine. Binding on these cell populations was measured as mean fluorescence intensity (MFI) at 647 nm. Flow cytometry detection on the IntelliCyt iQue Screener Plus. Use GraphPad Prism analysis software to draw and analyze data. The results are shown in Table 10 and Figures 8A-8B. Table 10: Binding to human CD8a expressed on transfected HEK293F cells fusion protein Bmax(MFI) K d (nM) SEQ ID NO. hzB7v19-xELL-P329G-KiH Fc-IL-2 mutant 14162685 7.495 88, 68; and 88, 69, 78 hzB7v20-xELL-P329G-KiH Fc-IL-2 mutant 11553318 4.482 89, 68; and 89, 69, 78 hzB7v21-xELL-P329G-KiH Fc-IL-2 mutant 14220676 5.234 90, 68; and 90, 69, 78 hzB7v23-xELL-P329G-KiH Fc-IL-2 mutant 14341168 3.897 91, 68; and 91, 69, 78 hzB7v24-xELL-P329G-KiH Fc-IL-2 mutant 12760589 6.543 92, 68; and 92, 69, 78 hzB7v25-xELL-P329G-KiH Fc-IL-2 mutant 14542422 3.279 93, 68; and 93, 69, 78 hzB7v26-xELL-P329G-KiH Fc-IL-2 mutant 15247397 4.546 94, 68; and 94, 69, 78 hzB7v27-xELL-P329G-KiH Fc-IL-2 mutant 14456494 3.179 95, 68; and 95, 69, 78 hzB7v28-xELL-P329G-KiH Fc-IL-2 mutant 13748843 2.838 96, 68; and 96, 69, 78 hzB7v15-xELL Fc 14220016 0.8207 25, 33

如圖8A及表10中所示，所測試CD8a結合多肽以低奈莫耳範圍內之親和力結合人類CD8a。圖8B展示多肽以低至可忽略的親和力結合於人類CD8b。 實例 7 ： CD8a 結合多肽與 T 細胞之結合 As shown in Figure 8A and Table 10, the tested CD8a-binding polypeptides bound human CD8a with affinities in the low nanomolar range. Figure 8B shows that the polypeptide binds to human CD8b with negligible affinity. Example 7 : Binding of CD8a- binding polypeptide to T cells

藉由流動式細胞測量術在經分離之人類T細胞上評定多肽之結合，該等多肽包含人類化CD8a結合VHH域、Fc區且在某些多肽中包含有融合至Fc區之C端的包含T3A、H16A、E61R、P65R、D84Y及C125S突變(SEQ ID NO: 78)的減毒IL-2。經標記之複合物或多肽「KiH」包含杵臼雜二聚體Fc區，其中所指示之CD8a結合VHH域融合至各Fc區之N端，且突變IL-2僅融合至「杵」Fc區之C端。在生理條件下未經標記之「KiH」的複合物或多肽形成均二聚體。將經分離細胞以每孔50,000個細胞塗鋪於96孔盤之FACS緩衝液(PBS，1% BSA，0.1% NaN 3，pH 7.4)中。隨後將測試多肽稀釋至2×最終濃度200 nM，且進行5倍連續稀釋。無多肽之FACS緩衝液用作僅二級抗體之對照。將測試多肽添加至等體積之細胞中，且將分析盤在4℃下培育30分鐘。用150 μL FACS緩衝液/孔洗滌兩次之後，將細胞再懸浮於具有以1:1000稀釋以偵測CD8a結合的螢光標記抗人類IgG抗體及螢光標記抗CD4抗體(純系OKT4，1:200)的FACS緩衝液中。以1:2000添加碘化丙錠(PI)以區分活細胞與死細胞。分析盤在4℃下培育30分鐘。在用150 μL FACS緩衝液再洗滌一次之後，藉由流動式細胞測量術在PI- CD4-細胞及PI- CD4+細胞上偵測結合於CD8a之多肽。在此等細胞群上之結合經量測為647 nm下之平均螢光強度(MFI)。在ACEA Biosciences Novocyte-Quanteon流動式細胞儀上進行流動式細胞測量術偵測。使用GraphPad Prism分析軟體繪製及分析資料。結果展示於表11及圖9A-9B中。 表11：與經轉染之HEK293F細胞上表現之人類CD8a的結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. hzB7v29-xELL-KiH Fc-IL-2突變體 287960 0.0606 97、48；及97、70、78 hzB7v30-xELL-KiH Fc-IL-2突變體 246762 0.09433 98、48；及98、70、78 hzB7v31-xELL-KiH Fc-IL-2突變體 276473 0.1288 99、48；及99、70、78 (108及107) hzB7v33-xELL-KiH Fc-IL-2突變體 212072 0.1392 100、48；及100、70、78 hzB7v34-xELL-KiH Fc-IL-2突變體 76068 4.049 101、48；及101、70、78 hzB7v36-xELL-KiH Fc-IL-2突變體 245259 0.07723 103、48；及103、70、78 hzB7v37-xELL-KiH Fc-IL-2突變體 272085 0.08843 104、48；及104、70、78 hzB7v39-xELL-KiH Fc-IL-2突變體 209090 0.1484 105、48；及105、70、78 hzB7v40-xELL-KiH Fc-IL-2突變體 71704 4.434 106、48；及106、70、78 hzB7v15-xELL Fc 266083 0.07576 25、33 Binding of polypeptides comprising a humanized CD8a binding VHH domain, an Fc region and, in some polypeptides, a T3A-containing polypeptide fused to the C-terminus of the Fc region was assessed by flow cytometry on isolated human T cells , H16A, E61R, P65R, D84Y and C125S mutations (SEQ ID NO: 78) attenuated IL-2. The labeled complex or polypeptide "KiH" contains a N-terminal heterodimeric Fc region in which the indicated CD8a-binding VHH domain is fused to the N-terminus of each Fc region and mutant IL-2 is fused only to the N-terminus of the "N-terminal" Fc region. C end. Under physiological conditions, unlabeled "KiH" complexes or peptides form homodimers. The isolated cells were plated in FACS buffer (PBS, 1% BSA, 0.1% NaN 3 , pH 7.4) in a 96-well plate at 50,000 cells per well. The test peptides were then diluted to 2× final concentration of 200 nM and 5-fold serial dilutions were performed. FACS buffer without peptide was used as a secondary antibody only control. Test polypeptide is added to an equal volume of cells and the assay plate is incubated at 4°C for 30 minutes. After washing twice with 150 μL FACS buffer/well, the cells were resuspended in 1:1000 dilution of fluorescently labeled anti-human IgG antibody and fluorescently labeled anti-CD4 antibody (pure OKT4, 1:1000) to detect CD8a binding. 200) in FACS buffer. Propidium iodide (PI) was added at 1:2000 to distinguish live from dead cells. The assay plate was incubated at 4°C for 30 minutes. After another wash with 150 μL of FACS buffer, peptides bound to CD8a were detected on PI-CD4- cells and PI-CD4+ cells by flow cytometry. Binding on these cell populations was measured as mean fluorescence intensity (MFI) at 647 nm. Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Use GraphPad Prism analysis software to draw and analyze data. The results are shown in Table 11 and Figures 9A-9B. Table 11: Binding to human CD8a expressed on transfected HEK293F cells fusion protein Bmax(MFI) K d (nM) SEQ ID NO. hzB7v29-xELL-KiH Fc-IL-2 mutant 287960 0.0606 97, 48; and 97, 70, 78 hzB7v30-xELL-KiH Fc-IL-2 mutant 246762 0.09433 98, 48; and 98, 70, 78 hzB7v31-xELL-KiH Fc-IL-2 mutant 276473 0.1288 99, 48; and 99, 70, 78 (108 and 107) hzB7v33-xELL-KiH Fc-IL-2 mutant 212072 0.1392 100, 48; and 100, 70, 78 hzB7v34-xELL-KiH Fc-IL-2 mutant 76068 4.049 101, 48; and 101, 70, 78 hzB7v36-xELL-KiH Fc-IL-2 mutant 245259 0.07723 103, 48; and 103, 70, 78 hzB7v37-xELL-KiH Fc-IL-2 mutant 272085 0.08843 104, 48; and 104, 70, 78 hzB7v39-xELL-KiH Fc-IL-2 mutant 209090 0.1484 105, 48; and 105, 70, 78 hzB7v40-xELL-KiH Fc-IL-2 mutant 71704 4.434 106, 48; and 106, 70, 78 hzB7v15-xELL Fc 266083 0.07576 25, 33

如圖9A及表11中所示，所測試CD8a結合多肽以低奈莫耳濃度至次奈莫耳濃度範圍內之親和力結合人類CD8 T細胞。圖9B展示多肽不結合於人類CD4 T細胞。 實例 8 ： CD8a 結合多肽與人類及食蟹獼猴 CD8a 之結合 As shown in Figure 9A and Table 11, the tested CD8a-binding polypeptides bound human CD8 T cells with affinities ranging from low nemolar to sub-nemolar concentrations. Figure 9B shows that the polypeptide does not bind to human CD4 T cells. Example 8 : Binding of CD8a- binding polypeptide to human and cynomolgus monkey CD8a

藉由流動式細胞測量術在經分離人類T細胞及經分離人類及食蟹獼猴周邊血液單核細胞(PBMC)上評定包含融合至xELL Fc區之人類化CD8a結合VHH域之四種多肽的結合。對於食蟹獼猴PBMC，將經分離細胞以每孔200,000個細胞，且對於人類T細胞，以每孔100,000個細胞塗鋪於96孔盤中FACS緩衝液(PBS，1% BSA，0.1% NaN 3，pH 7.4)中。隨後將測試多肽稀釋至2×最終濃度25 nM或50 nM，且製備3或5倍連續稀釋液。單獨的FACS緩衝液用作僅二級抗體之對照。將多肽稀釋液添加至等體積之細胞中，且將分析盤在4℃下培育30分鐘。用150 µL FACS緩衝液/孔洗滌兩次之後，將細胞再懸浮於FACS緩衝液中，其中該緩衝液具有1:1000稀釋以偵測CD8a結合之螢光標記抗人類IgG抗體、螢光標記抗CD3抗體(僅針對PBMC製劑，純系SP34.2，1:50)及螢光標記抗CD4抗體(純系OKT4，1:100)。以1:2000添加碘化丙錠(PI)以區分活細胞與死細胞。分析盤在4℃下培育30分鐘。再一次用150 μL FACS緩衝液洗滌之後，藉由流動式細胞測量術對PI- (CD3+) CD4-細胞偵測CD8a結合。在此等細胞群上之結合經量測為647 nm下之平均螢光強度(MFI)。在ACEA Biosciences Novocyte-Quanteon流動式細胞儀上進行流動式細胞測量術偵測。使用GraphPad Prism分析軟體繪製及分析資料。結果展示於表12及表13及圖10A-10D中。 表12：與人類CD8 T細胞(CD4富集T細胞)之結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. hzB7v15-xELL-Fc 287001 0.09133 25, 33 hzB7v29-xELL-Fc 292513 0.07587 97, 33 hzB7v31-xELL-Fc 269349 0.09089 99, 33 hzB7v35-xELL-Fc 278470 0.07665 102, 33 hzB7v41-xELL-Fc 363856 0.1093 109, 33 表13：食蟹獼猴CD8 T細胞(CD3+ CD4- PBMC)上之結合 融合蛋白 Bmax (MFI) K d(nM) SEQ ID NO. hzB7v15-xELL-Fc 121170 0.02514 25, 33 hzB7v29-xELL-Fc 123694 0.03172 97, 33 hzB7v31-xELL-Fc 103591 0.03887 99, 33 hzB7v35-xELL-Fc 110657 0.02359 102, 33 hzB7v41-xELL-Fc 99723 0.04993 109, 33 Binding of four polypeptides containing humanized CD8a-binding VHH domains fused to the Fc region of xELL assessed by flow cytometry on isolated human T cells and isolated human and cynomolgus monkey peripheral blood mononuclear cells (PBMC) . Dissociated cells were plated in 96-well plates in FACS buffer (PBS, 1% BSA, 0.1% NaN 3 , pH 7.4). Test peptides were then diluted to 2× final concentration of 25 nM or 50 nM, and 3- or 5-fold serial dilutions were prepared. FACS buffer alone was used as a secondary antibody only control. The polypeptide dilution was added to an equal volume of cells and the assay plate was incubated at 4°C for 30 minutes. After washing twice with 150 µL FACS buffer/well, cells were resuspended in FACS buffer with a 1:1000 dilution of fluorescently labeled anti-human IgG antibody to detect CD8a binding, fluorescently labeled anti- CD3 antibody (only for PBMC preparations, pure line SP34.2, 1:50) and fluorescently labeled anti-CD4 antibody (pure line OKT4, 1:100). Propidium iodide (PI) was added at 1:2000 to distinguish live from dead cells. The assay plate was incubated at 4°C for 30 minutes. After washing again with 150 μL FACS buffer, CD8a binding was detected by flow cytometry on PI- (CD3+) CD4- cells. Binding on these cell populations was measured as mean fluorescence intensity (MFI) at 647 nm. Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Use GraphPad Prism analysis software to draw and analyze data. The results are shown in Tables 12 and 13 and Figures 10A-10D. Table 12: Binding to human CD8 T cells (CD4 enriched T cells) fusion protein Bmax(MFI) K d (nM) SEQ ID NO. hzB7v15-xELL-Fc 287001 0.09133 25, 33 hzB7v29-xELL-Fc 292513 0.07587 97, 33 hzB7v31-xELL-Fc 269349 0.09089 99, 33 hzB7v35-xELL-Fc 278470 0.07665 102, 33 hzB7v41-xELL-Fc 363856 0.1093 109, 33 Table 13: Binding on cynomolgus monkey CD8 T cells (CD3+ CD4- PBMC) fusion protein Bmax(MFI) K d (nM) SEQ ID NO. hzB7v15-xELL-Fc 121170 0.02514 25, 33 hzB7v29-xELL-Fc 123694 0.03172 97, 33 hzB7v31-xELL-Fc 103591 0.03887 99, 33 hzB7v35-xELL-Fc 110657 0.02359 102, 33 hzB7v41-xELL-Fc 99723 0.04993 109, 33

如圖10A及圖10E以及表12中所示，所測試CD8a結合多肽以等於或低於0.1 nM之親和力結合人類CD8 T細胞。圖10B及圖10F展示此等多肽不結合於人類CD4 T細胞。如圖10C及表13中所示，所測試CD8a結合多肽以低於0.05 nM之親和力結合食蟹獼猴CD8 T細胞。圖10D及圖10H展示多肽不結合於食蟹獼猴CD4 T細胞。 實例 9 ： 由包含 CD8a 結合 VHH 及減毒 IL - 2 之多肽誘導的特異性 IL - 2 信號傳導 As shown in Figures 10A and 10E and Table 12, the tested CD8a-binding polypeptides bound human CD8 T cells with an affinity equal to or less than 0.1 nM. Figures 10B and 10F show that these polypeptides do not bind to human CD4 T cells. As shown in Figure 10C and Table 13, the tested CD8a-binding polypeptides bound cynomolgus monkey CD8 T cells with an affinity below 0.05 nM. Figures 10D and 10H show that the polypeptide does not bind to cynomolgus monkey CD4 T cells. Example 9 : Specific IL - 2 signaling induced by polypeptides comprising CD8a- binding VHH and attenuated IL - 2

在pSTAT5分析中評定多肽之靶向CD8a之IL-2活性，該多肽包含有CD8結合VHH域hzB7v31或hzB7v41、「杵臼」雜二聚體Fc區及融合至Fc區之C端(SEQ ID NO: 107及108；或110及111)的包含T3A、H16A、E61R、P65R、D84Y及C125S突變的減毒IL-2。對照蛋白包括包含CD8a結合VHH域及Fc區但無IL-2之多肽、包含非靶向VHH、Fc區及突變減毒IL-2及野生型IL-2之融合蛋白。磷酸化STAT5 (pSTAT5)含量或表現pSTAT5之細胞百分比之增加係藉由細胞內流動式細胞測量術量測為IL-2受體接合及信號傳導之近端讀數形式。將富集之人類T細胞以500,000個細胞/孔塗鋪於96孔盤之完全生長培養基(RPMI，10% FBS，1%抗生素-抗黴劑(anti-anti))中。隨後將測試多肽稀釋至2×最終濃度200 nM或50 nM，且進行4倍連續稀釋。將連續稀釋液添加至細胞中且在37℃下培育15分鐘。隨後將細胞在4℃下於100 µL之Cytofix固定緩衝液(BD)中固定30分鐘。隨後將細胞在200 µL FACS緩衝液中洗滌一次且在4℃下於Perm緩衝液III (BD Phosflow)中滲透30分鐘。將經滲透細胞在1×滲透緩衝液(eBioscience)中洗滌總共三次，且隨後在4℃下於含有針對以下之螢光標記抗體的1×滲透緩衝液中培育隔夜：CD4 (OKT4, 1:100)、CD3 (SP34-2, 1:50)、FoxP3 (236A/E7，1:40)、pSTAT5 (SRBCZX，1:70)、CD25 (M-A251，1:500)及CD8 (RPA-T8，1:4000)。次日，細胞用150 µL FACS緩衝液洗滌且使用ACEA Biosciences Novocyte-Quanteon流動式細胞儀分析。經由用偵測CD8 T細胞(CD3+CD8+)或調節性T細胞(Treg，CD3+CD4+FoxP3+)上之pSTAT5的螢光標記抗體染色的陽性細胞之中值螢光強度或百分比之增加來定量IL-2信號傳導。使用GraphPad Prism分析軟體繪製及分析資料。The IL-2 activity targeting CD8a of a polypeptide comprising a CD8-binding VHH domain hzB7v31 or hzB7v41, a "Pestlet" heterodimer Fc region, and a C-terminus fused to the Fc region (SEQ ID NO: 107 and 108; or 110 and 111) attenuated IL-2 containing T3A, H16A, E61R, P65R, D84Y and C125S mutations. Control proteins include polypeptides containing CD8a binding VHH domain and Fc region but no IL-2, fusion proteins containing non-targeting VHH, Fc region and mutated attenuated IL-2 and wild-type IL-2. Increases in phosphorylated STAT5 (pSTAT5) content or the percentage of cells expressing pSTAT5 are measured by intracellular flow cytometry as a proximal readout of IL-2 receptor engagement and signaling. Enriched human T cells were plated in 96-well plates in complete growth medium (RPMI, 10% FBS, 1% antibiotic-anti-anti) at 500,000 cells/well. Test peptides were then diluted to 2× final concentration of 200 nM or 50 nM, and 4-fold serial dilutions were performed. Serial dilutions were added to cells and incubated at 37°C for 15 minutes. Cells were then fixed in 100 µL of Cytofix fixation buffer (BD) for 30 minutes at 4°C. Cells were then washed once in 200 µL FACS buffer and permeabilized in Perm buffer III (BD Phosflow) for 30 min at 4°C. Permeabilized cells were washed a total of three times in 1× permeabilization buffer (eBioscience) and subsequently incubated overnight at 4°C in 1× permeability buffer containing fluorescently labeled antibodies against: CD4 (OKT4, 1:100 ), CD3 (SP34-2, 1:50), FoxP3 (236A/E7, 1:40), pSTAT5 (SRBCZX, 1:70), CD25 (M-A251, 1:500) and CD8 (RPA-T8, 1:4000). The next day, cells were washed with 150 µL FACS buffer and analyzed using an ACEA Biosciences Novocyte-Quanteon flow cytometer. Quantified by increase in median fluorescence intensity or percentage of positive cells stained with a fluorescently labeled antibody that detects pSTAT5 on CD8 T cells (CD3+CD8+) or regulatory T cells (Treg, CD3+CD4+FoxP3+) IL-2 signaling. Use GraphPad Prism analysis software to draw and analyze data.

如圖11A及圖11C中所示，包含CD8a結合VHH hzB7v31或VHH hzB7v41域、Fc區及融合至Fc區之C端的減毒IL-2的所測試多肽以濃度依賴性方式且以0.03 nM或更低之EC 50誘導pSTAT5含量增加或pSTAT5陽性CD8 T細胞之百分比升高。野生型IL-2 (非靶向)展現下降約50倍的強效活性，其中EC 50為大致1.6 nM。野生型IL-2亦以大致2.5 pM之EC 50誘導Treg上之IL-2受體信號傳導，而Treg pSTAT5或pSTAT5陽性CD4 T細胞百分比之可偵測增加不由靶向CD8a之減毒IL-2誘導(圖11A-11D)。包含CD8a-hzB7v31而無IL-2之多肽或包含非靶向VHH、Fc區及突變減毒IL-2之多肽均不誘導任何測試細胞型中pSTAT5含量之可偵測增加，指示需要減毒IL-2靶向細胞以便誘導IL-2受體信號傳導活性(圖11A-11D)。 實例 10 ： 由包含 CD8a 結合 VHH 及減毒 IL - 2 的多肽誘導之人類腫瘤浸潤 T 細胞及健康供體 T 細胞之 T 細胞增殖 As shown in Figure 11A and Figure 11C , the tested polypeptides comprising CD8a bound the VHH hzB7v31 or VHH hzB7v41 domain, the Fc region, and attenuated IL-2 fused to the C-terminus of the Fc region in a concentration-dependent manner and at 0.03 nM or more. A low EC 50 induces an increase in pSTAT5 content or an increase in the percentage of pSTAT5-positive CD8 T cells. Wild-type IL-2 (untargeted) exhibited approximately 50-fold reduced potent activity with an EC50 of approximately 1.6 nM. Wild-type IL-2 also induces IL-2 receptor signaling on Tregs with an EC50 of approximately 2.5 pM, and a detectable increase in the percentage of Treg pSTAT5 or pSTAT5-positive CD4 T cells was not induced by attenuated IL-2 targeting CD8a. Induced (Figures 11A-11D). Neither peptides containing CD8a-hzB7v31 without IL-2 nor peptides containing non-targeting VHH, Fc region, and mutated attenuated IL-2 induced a detectable increase in pSTAT5 content in any of the cell types tested, indicating the need for attenuated IL. -2 targets cells to induce IL-2 receptor signaling activity (Figures 11A-11D). Example 10 : T cell proliferation of human tumor-infiltrating T cells and healthy donor T cells induced by peptides containing CD8a - binding VHH and attenuated IL - 2

在用來自人類癌症患者之解離腫瘤細胞(DTC)樣品或來自健康人類供體血液之PBMC進行增殖分析中進一步評定包含CD8a結合VHH hzB7v31域、Fc區及融合至Fc區之C端的包含T3A、H16A、E61R、P65R、D84Y及C125S突變(SEQ ID NO: 78)的減毒IL-2的多肽之活性。使用人類腫瘤解離套組(Miltenyi Biotec)由頭頸、腎臟或大腸腫瘤之活組織切片產生DTC單細胞懸浮液。DTC或PBMC隨後根據製造商建議方案用增殖染料CellTrace紫(Thermo)標記。在補充有10 nM測試多肽或測試多肽以200 nM濃度開始之5倍稀釋液的完全生長培養基(RPMI，10% FBS，1%抗生素-抗黴劑)中培育細胞。對照蛋白包括包含格式化為VHH-xELL Fc之CD8a-hzB7v31的多肽、包含非靶向VHH-xELL Fc及突變減毒IL-2及野生型IL-2的融合蛋白。在培養六天或七天之後，用針對CD3 (Hit3，1:100)、CD4 (OKT4，1:200)、CD8 (RPA-T8，1:200)及CD45 (HI30，1:100)之螢光標記抗體以及碘化丙錠(PI，1:2000)來標記細胞亞群以區分活細胞與死細胞。將T細胞分類為表現CD4或CD8a之CD45+ CD3+ PI-細胞。在第六或七天使用流動式細胞測量術定量此等T細胞亞群之細胞數。在ACEA Biosciences Novocyte-Quanteon流動式細胞儀上進行流動式細胞測量術偵測。使用GraphPad Prism分析軟體繪製及分析資料。細胞數以用CD8a-hzB7v31-Fc-xELL處理之樣品標準化，來確定相對於不包含IL-2且不引起細胞增殖之對照多肽之細胞計數的增加倍數。藉由定量具有比親本未分裂細胞峰值低的CellTrace紫螢光強度之細胞的百分比來確定增殖百分比。The CD8a-binding VHH hzB7v31 domain, the Fc region, and the C-terminus fused to the Fc region, including T3A, H16A, were further evaluated in proliferation assays using dissociated tumor cell (DTC) samples from human cancer patients or PBMC from healthy human donor blood. , E61R, P65R, D84Y and C125S mutations (SEQ ID NO: 78) attenuated IL-2 polypeptide activity. DTC single cell suspensions were generated from biopsies of head and neck, kidney, or colorectal tumors using a human tumor dissociation kit (Miltenyi Biotec). DTC or PBMC were subsequently labeled with the proliferation dye CellTrace Violet (Thermo) according to the manufacturer's recommended protocol. Cells were grown in complete growth medium (RPMI, 10% FBS, 1% antibiotic-antimycotic) supplemented with 10 nM test peptide or 5-fold dilutions of test peptide starting at 200 nM. Control proteins included polypeptides containing CD8a-hzB7v31 formatted as VHH-xELL Fc, fusion proteins containing non-targeting VHH-xELL Fc and mutant attenuated IL-2 and wild-type IL-2. After six or seven days of culture, fluorescence was used for CD3 (Hit3, 1:100), CD4 (OKT4, 1:200), CD8 (RPA-T8, 1:200), and CD45 (HI30, 1:100). Labeled antibodies and propidium iodide (PI, 1:2000) were used to label cell subpopulations to differentiate between live and dead cells. T cells were classified as CD45+ CD3+ PI- cells expressing CD4 or CD8a. Cell numbers of these T cell subsets were quantified using flow cytometry on days six or seven. Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Use GraphPad Prism analysis software to draw and analyze data. Cell numbers were normalized to samples treated with CD8a-hzB7v31-Fc-xELL to determine the fold increase in cell count relative to a control polypeptide that did not contain IL-2 and did not cause cell proliferation. Percent proliferation was determined by quantifying the percentage of cells with a lower CellTrace violet fluorescence intensity than the peak value of parental undivided cells.

如圖12A及圖12C中所示，包含CD8a結合VHH hzB7v31域、Fc區及融合至Fc區之C端的突變減毒IL-2的所測試多肽誘導CD8 T細胞在解離腫瘤樣品及健康PBMC中之增殖。野生型IL-2亦誘導CD8 (圖12A及圖12C)及CD4 (圖12B) T細胞之增殖，然而靶向CD8a之突變減毒IL-2不誘導CD4 T細胞之增殖。包含CD8a-hzB7v31而無IL-2之多肽及包含非靶向VHH、Fc區及突變減毒IL-2之多肽均不誘導CD8或CD4 T細胞增殖之可偵測增加，指示需要減毒IL-2靶向細胞以便誘導IL-2受體信號傳導活性，諸如增殖。 實例 11 ： 由包含 CD8a 結合 VHH 及減毒 IL - 2 之多肽誘導的食蟹獼猴 PBMC 亞群之細胞擴增 As shown in Figures 12A and 12C, the tested polypeptides comprising the CD8a binding VHH hzB7v31 domain, the Fc region, and mutant attenuated IL-2 fused to the C-terminus of the Fc region induced CD8 T cells in dissociated tumor samples and healthy PBMCs. proliferation. Wild-type IL-2 also induced the proliferation of CD8 (Fig. 12A and Fig. 12C) and CD4 (Fig. 12B) T cells, whereas mutant attenuated IL-2 targeting CD8a did not induce the proliferation of CD4 T cells. Neither peptides containing CD8a-hzB7v31 without IL-2 nor peptides containing non-targeting VHH, Fc region, and mutant attenuated IL-2 induced a detectable increase in CD8 or CD4 T cell proliferation, indicating the need for attenuated IL-2 2 targets cells to induce IL-2 receptor signaling activity, such as proliferation. Example 11 : Cell expansion of cynomolgus monkey PBMC subpopulations induced by polypeptides containing CD8a- binding VHH and attenuated IL - 2

在非人類靈長類動物中測試融合蛋白對活體內細胞擴增的影響，該融合蛋白包含CD8a結合VHH hzB7v15、xELL P329G、杵臼雜二聚體Fc區及融合至「杵」Fc (SEQ ID NO: 74及75)之C端的減毒IL-2。向食蟹獼猴以1.0 mg/kg靜脈內推注注射投與融合蛋白。在融合蛋白投與之前及之後七天自研究動物收集全血樣品。在Lymphoprep™中使用密度離心分離各時間點的PBMC且用螢光標記細胞類型特異性抗體組合染色細胞。將T細胞分類為表現CD4或CD8a、不表現B細胞標記CD20的CD3+細胞。調節性T細胞(「Treg」)定義為亦表現CD25且具有降低之CD127含量的CD4+ T細胞。CD4+習知T細胞(「CD4+ Tcon」)定義為不表現CD25且具有正常CD127含量的CD4+ T細胞。NK細胞定義為表現NKG2A之非T且非B細胞，且對於CD16呈陽性或陰性。針對CD20染色呈陽性之群體歸類為B細胞。使用流動式細胞測量術測定各PBMC亞群之絕對細胞計數，且藉由用給藥後7天絕對細胞計數除以給藥前基線計數來計算擴增。使用額外固定、滲透及染色步驟在上文所述之PBMC亞群中量測Ki67表現。簡言之，針對細胞表面標記，細胞用螢光標記細胞類型特異性抗體組合染色，接著使用FoxP3轉錄因子染色緩衝液組(eBioscience)固定及滲透。FoxP3及Ki67接著用特異性螢光標記抗體偵測。T細胞分類為表現CD4或CD8a、不表現NK細胞標記NKG2A的CD3+細胞。調節性T細胞(「Treg」)定義為亦表現CD25及FoxP3之CD4+ T細胞。CD4+習知T細胞(「CD4+ Tcon」)定義為不表現CD25或FoxP3之CD4+ T細胞。NK細胞定義為非T且表現NKG2A且對於CD16呈陽性或陰性。在ACEA Biosciences Novocyte-Quanteon流動式細胞儀上進行流動式細胞測量術偵測。使用GraphPad Prism分析軟體繪製及分析資料。藉由用第七天每mL全血中之細胞計數除以基線(給藥前)處每mL全血中之細胞計數來計算變化倍數。The effect of a fusion protein containing CD8a-binding VHH hzB7v15, xELL P329G, the PESTLE heterodimer Fc region and fused to the "PEST" Fc (SEQ ID NO. : 74 and 75) C-terminal attenuated IL-2. The fusion protein was administered to cynomolgus macaques as an intravenous bolus injection of 1.0 mg/kg. Whole blood samples were collected from study animals before and seven days after fusion protein administration. PBMC at each time point were isolated using density centrifugation in Lymphoprep™ and cells were stained with a combination of fluorescently labeled cell type-specific antibodies. T cells are classified as CD3+ cells that express CD4 or CD8a and do not express the B cell marker CD20. Regulatory T cells ("Tregs") are defined as CD4+ T cells that also express CD25 and have reduced CD127 content. CD4+ conventional T cells (“CD4+ Tcon”) are defined as CD4+ T cells that do not express CD25 and have normal CD127 content. NK cells are defined as non-T and non-B cells that express NKG2A and are positive or negative for CD16. Populations that stain positive for CD20 are classified as B cells. Absolute cell counts of each PBMC subpopulation were determined using flow cytometry, and expansion was calculated by dividing absolute cell counts 7 days post-dose by pre-dose baseline counts. Ki67 expression was measured in the PBMC subpopulations described above using additional fixation, permeabilization and staining steps. Briefly, cells were stained with a combination of fluorescently labeled cell type-specific antibodies for cell surface markers, followed by fixation and permeabilization using the FoxP3 transcription factor staining buffer set (eBioscience). FoxP3 and Ki67 were then detected using specific fluorescently labeled antibodies. T cells are classified as CD3+ cells that express CD4 or CD8a and do not express the NK cell marker NKG2A. Regulatory T cells (“Treg”) are defined as CD4+ T cells that also express CD25 and FoxP3. CD4+ conventional T cells (“CD4+ Tcon”) are defined as CD4+ T cells that do not express CD25 or FoxP3. NK cells are defined as non-T cells that express NKG2A and are either positive or negative for CD16. Flow cytometry detection was performed on an ACEA Biosciences Novocyte-Quanteon flow cytometer. Use GraphPad Prism analysis software to draw and analyze data. Fold change was calculated by dividing the cell count per mL of whole blood at day 7 by the cell count per mL of whole blood at baseline (predose).

如圖13A中所示，單次劑量1 mg/kg的靶向CD8a之減毒IL-2分別引起CD8 T細胞之5倍擴增，以及表現CD8a之CD16+或CD16-NK細胞的3.9倍及4.7倍擴增。CD8a陰性細胞群，包括Treg、CD4+習知T細胞及B細胞之數目在給藥前抽血與第七天之間未顯著增加。圖13B展示活體內表現CD8a之細胞群的特異性擴增伴隨增殖標記Ki67的特異性增加。Ki67+增殖CD8 T細胞之百分比自基線處6%增加至第七天之58%，而CD16+及CD16-NK細胞群在相同時間範圍中展示Ki67+細胞平均增加40-53%。CD8a陰性細胞群(包括Treg及CD4+習知T細胞)內Ki67+群體的百分比不變。此等資料展示靶向CD8a之減毒IL-2特異性地誘導活體內CD8a陽性細胞群之細胞增殖。 實例 12 ： 由包含 CD8a 結合 VHH 及減毒 IL - 2 之多肽誘導的 CD8 T 細胞之細胞毒活性及針對人類癌細胞之抗體依賴性細胞毒性的增強 As shown in Figure 13A, a single dose of 1 mg/kg of attenuated IL-2 targeting CD8a caused a 5-fold expansion of CD8 T cells, and a 3.9-fold and 4.7-fold expansion of CD8a-expressing CD16+ or CD16-NK cells, respectively. times amplification. The number of CD8a negative cell groups, including Tregs, CD4+ conventional T cells and B cells, did not increase significantly between the blood draw before administration and the seventh day. Figure 13B shows specific expansion of the CD8a expressing cell population in vivo accompanied by a specific increase in the proliferation marker Ki67. The percentage of Ki67+ proliferating CD8 T cells increased from 6% at baseline to 58% at day seven, while the CD16+ and CD16-NK cell populations showed an average increase in Ki67+ cells of 40-53% over the same time frame. The percentage of Ki67+ population within the CD8a negative cell population (including Tregs and CD4+ conventional T cells) remained unchanged. These data demonstrate that attenuated IL-2 targeting CD8a specifically induces cell proliferation of CD8a-positive cell populations in vivo. Example 12 : Enhancement of cytotoxic activity of CD8 T cells and antibody-dependent cytotoxicity against human cancer cells induced by polypeptides containing CD8a -binding VHH and attenuated IL - 2

在用富集CD8 T細胞之腫瘤細胞殺死分析及與西妥昔單抗組合之抗體依賴性細胞毒性(ADCC)分析中進一步評定多肽之活性，該多肽包含CD8a結合VHH hzB7v31域、Fc區及包含融合至Fc區之C端(SEQ ID NO: 107及108)的包含T3A、H16A、E61R、P65R、D84Y及C125S突變(SEQ ID NO: 78)的減毒IL-2。對照蛋白包括包含非靶向VHH-xELL Fc及突變減毒IL-2及野生型IL-2的融合蛋白。對於CD8 T細胞殺死分析，來自健康人類供體血液之PBMC用於分離CD8 T細胞，且在培養盤上，在存在或不存在來自野生型IL-2或包含CD8a結合VHH hzB7v31-xELL Fc及突變減毒IL-2之融合蛋白(各1nM)的額外細胞介素支持下，用以1 µg /ml塗佈的針對CD3之抗體(純系：OKT3)刺激所富集細胞3天。在目標細胞殺死分析當天，用CYTO-ID紅長期細胞示蹤劑(Enzo)標記A431細胞，接著在96孔平底培養盤中以4,000個細胞/孔塗鋪100 µL，且使其黏附4小時。將預刺激之CD8 T細胞在PBS中洗滌一次且如所指示以不同效應子與目標細胞比(20:1、10:1及5:1)添加至經標記之A431目標細胞中。將凋亡蛋白酶-3/7綠色染料(Sartorius)添加至各孔中以偵測細胞死亡。20小時後藉由使用Incucyte成像器定量凋亡蛋白酶-3/7及CYTO-ID紅之重疊來測定A431殺死。The activity of the polypeptide comprising the CD8a binding VHH hzB7v31 domain, Fc region and Attenuated IL-2 comprising T3A, H16A, E61R, P65R, D84Y and C125S mutations (SEQ ID NO: 78) fused to the C-terminus of the Fc region (SEQ ID NO: 107 and 108). Control proteins included fusion proteins containing non-targeting VHH-xELL Fc and mutant attenuated IL-2 and wild-type IL-2. For CD8 T cell killing assays, PBMC from healthy human donor blood were used to isolate CD8 T cells and cultured on culture plates in the presence or absence of IL-2 from wild-type or containing CD8a-binding VHH hzB7v31-xELL Fc and The enriched cells were stimulated with an antibody against CD3 (clone: OKT3) coated at 1 µg/ml with the support of additional interleukin of fusion proteins of mutated attenuated IL-2 (1 nM each) for 3 days. On the day of target cell killing analysis, A431 cells were labeled with CYTO-ID Red long-term cell tracer (Enzo), then plated in 100 µL at 4,000 cells/well in a 96-well flat-bottom culture dish and allowed to adhere for 4 hours. . Prestimulated CD8 T cells were washed once in PBS and added to labeled A431 target cells at different effector to target cell ratios (20:1, 10:1 and 5:1) as indicated. Apoptase-3/7 green dye (Sartorius) was added to each well to detect cell death. A431 killing was determined after 20 hours by quantifying the overlap of apoptotic proteinase-3/7 and CYTO-ID red using an Incucyte imager.

對於ADCC分析，用CYTO-ID紅長期細胞示蹤劑(Enzo)標記A431細胞，接著在96孔平底培養盤中以10,000個細胞/孔塗鋪100 µL，且使其黏附4小時。將人類PBMC解凍且藉由流動式細胞測量術測試NK細胞頻率。對於各孔而言，25 µL Incucyte®針對細胞凋亡的凋亡蛋白酶-3/7綠色染料(Sartorius)之最終稀釋度為1:2000，25 µL培養基或ADCC抗體西妥昔單抗之最終濃度為20 nM，25 µL培養基，野生型重組IL-2之最終濃度為1 nM，或IL-2變異體融合多肽之最終濃度為1 nM，及25 µL人類PBMC的濃度調整至每1個A431細胞10或5個NK細胞。使細胞在室溫下靜置10分鐘，隨後將培養盤置於37℃下之Incucyte成像器中進行成像。15小時後藉由定量凋亡蛋白酶-3/7及CYTO-ID紅之重疊來測定A431殺死，其中最大殺死由20 nM西妥昔單抗界定。使用GraphPad Prism分析軟體繪製及分析所有資料。For ADCC analysis, A431 cells were labeled with CYTO-ID Red long-term cell tracer (Enzo), then plated in 100 µL at 10,000 cells/well in a 96-well flat-bottom culture dish and allowed to adhere for 4 hours. Human PBMC were thawed and tested for NK cell frequency by flow cytometry. For each well, 25 µL of Incucyte® Apoptosis-3/7 Green Dye for Apoptosis (Sartorius) at a final dilution of 1:2000, 25 µL of media or final concentration of the ADCC antibody cetuximab is 20 nM, 25 µL culture medium, the final concentration of wild-type recombinant IL-2 is 1 nM, or the final concentration of IL-2 variant fusion peptide is 1 nM, and the concentration of 25 µL human PBMC is adjusted to per 1 A431 cell 10 or 5 NK cells. The cells were allowed to stand at room temperature for 10 minutes, and then the culture plate was placed in an Incucyte imager at 37°C for imaging. A431 killing was determined after 15 hours by quantifying the overlap of apoptotic proteinase-3/7 and CYTO-ID red, with maximum killing defined by 20 nM cetuximab. All data were plotted and analyzed using GraphPad Prism analysis software.

如圖14A及圖14B中所示，包含CD8a結合VHH hzB7v31域、Fc區及融合至Fc區之C端之突變減毒IL-2的所測試多肽在不同效應子與目標細胞比下增強CD8 T細胞之相對細胞毒性(圖14A)，且有助於以次佳子與目標細胞比增加PBMC針對EGFR陽性A431目標細胞之西妥昔單抗驅動之ADCC活性(圖14B)。CD8 T細胞活性程度比在野生型IL-2下觀測到之程度高3至4倍，但在ADCC分析中為相當的。包含非靶向VHH-xELL Fc及突變減毒IL-2的融合蛋白不能夠增加較低效應子與目標細胞比之ADCC活性，指示需要減毒IL-2靶向效應細胞以便誘導IL-2受體信號傳導活性及增強之細胞毒性。As shown in Figures 14A and 14B, tested polypeptides comprising the CD8a binding VHH hzB7v31 domain, the Fc region, and mutant attenuated IL-2 fused to the C-terminus of the Fc region enhanced CD8 T The relative cytotoxicity of the cells (Figure 14A) and contributed to increased cetuximab-driven ADCC activity of PBMCs against EGFR-positive A431 target cells at a suboptimal daughter-to-target cell ratio (Figure 14B). The degree of CD8 T cell activity was 3- to 4-fold greater than that observed with wild-type IL-2, but was comparable in the ADCC assay. Fusion proteins containing non-targeting VHH-xELL Fc and mutant attenuated IL-2 were unable to increase ADCC activity at lower effector to target cell ratios, indicating the need for attenuated IL-2 to target effector cells in order to induce IL-2 receptor body signaling activity and enhanced cytotoxicity.

在不偏離本發明之精神或基本特徵之情況下，本發明可以其他特定形式體現。前述實施例因此在所有態樣中均欲視為說明性而非限制本發明。本發明之範疇因此由所附申請專利範圍而非由前述描述來指示，且具有申請專利範圍等效性之含義及範圍內的所有變化因此均意欲包括於本文中。 某些序列之表格 SEQ ID NO 描述 序列 1 人類CD8a，前驅體(信號序列為胺基酸1-21) MALPVTALLL PLALLLHAAR PSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYV 77 用於免疫接種之CD8a-IgV LFc抗原(信號序列為胺基酸1-21) MALPVTALLL PLALLLHAAR PSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKTGGS GGGGCPPCPA PELPGGPSVF VFPPKPKDVL SISGRPEVTC VVVDVGKEDP EVNFNWYIDG VEVRTANTKP KEEQFNSTYR VVSVLPIQHQ DWLTGKEFKC KVNNKALPAP IERTISKAKG QTREPQVYTL APHREELAKD TVSVTCLVKG FYPADINVEW QRNGQPESEG TYANTPPQLD NDGTYFLYSK LSVGKNTWQR GETLTCVVMH EALHNHYTQK SISQSLGK 2 B7 VHH QVQLVQSGGGLVRPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMNGLKSEDTAVYVCAKGSPELQYDSWGQGTQVTVKPXX 其中各X係Gly或不存在 3 B7、hzB7v1、hzB7v2、hzB7v3、hzB7v4、hzB7v5、hzB7v6、hzB7v7、hzB7v8、hzB7v9、hzB7v10、hzB7v11、hzB7v12、hzB7v13、hzB7v14、hzB7v15、hzB7v16、hzB7v17、hzB7v18、hzB7v19、hzB7v20、hzB7v21、hzB7v25、hzB7v26、hzB7v27、hzB7v28、hzB7v29、hzB7v30、hzB7v31、hzB7v33、hzB7v34、hzB7v35、hzB7v36、hzB7v37、hzB7v39及hzB7v40之CDR1 GFTFDDYAMS 80 hzB7v23之CDR1 GFTFDSYAMS 81 hzB7v24之CDR1 GFTFSSYAMS 4 B7、hzB7v1、hzB7v2、hzB7v3、hzB7v4、hzB7v5、hzB7v8、hzB7v9、hzB7v13及hzB7v14之CDR2 TITWDGEGTD 12 hzB7v6及hzB7v10之CDR2 TITWEGEGTD 14 hzB7v7、hzB7v11、hzB7v15、hzB7v19、hzB7v20、hzB7v21、hzB7v23、hzB7v24及hzB7v28之CDR2 TITWDAEGTD 22 hzB7v12之CDR2 TITWSGEGTD 27 hzB7v16之CDR2 TITWEGESTD 29 hzB7v17之CDR2 TITWEGEGTY 31 hzB7v18之CDR2 TITWEGESTY 82 hzB7v25之CDR2 TITWSAEGTD 83 hzB7v26、hzB7v30及hzB7v36之CDR2 TITWDAGGTD 84 hzB7v27、hzB7v29及hzB7v35之CDR2 TITWDAEGTY 85 hzB7v31及hzB7v37之CDR2 TITWDAGGTY 86 hzB7v33及hzB7v39之CDR2 TITWSAEGTY 87 hzB7v34及hzB7v40之CDR2 TITWSAGGTD 5 B7、hzB7v1、hzB7v2、hzB7v3、hzB7v4、hzB7v5、hzB7v6、hzB7v7、hzB7v10、hzB7v11、hzB7v12、hzB7v16、hzB7v17及hzB7v18之CDR3 GSPELQYDS 16 hzB7v8及hzB7v13之CDR3 GSPELQYES 18 hzB7v9、hzB7v14、hzB7v15、hzB7v19、hzB7v20、hzB7v21、hzB7v23、hzB7v24、hzB7v25、hzB7v26、hzB7v27、hzB7v28、hzB7v29、hzB7v30、hzB7v31、hzB7v33、hzB7v34、hzB7v35、hzB7v36、hzB7v37、hzB7v39及hzB7v40之CDR3 GSPELQYDT 6 hzB7v1 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 7 hzB7v2 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 8 hzB7v3 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 9 hzB7v4 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 10 hzB7v5 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 11 hzB7v6 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 13 hzB7v7 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 15 hzB7v8 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYESWGQGTLVTVKPXX 其中各X係Gly或不存在 17 hzB7v9 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDTWGQGTLVTVKPXX 其中各X係Gly或不存在 19 hzB7v10 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 20 hzB7v11 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 21 hzB7v12 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 23 hzB7v13 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYESWGQGTLVTVKPXX 其中各X係Gly或不存在 24 hzB7v14 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPXX 其中各X係Gly或不存在 25 hzB7v15 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPXX 其中各X係Gly或不存在 26 hzB7v16 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGESTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 28 hzB7v17 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGEGTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 30 hzB7v18 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGESTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX 其中各X係Gly或不存在 88 hzB7v19 VHH QVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 89 hzB7v20 VHH EVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 90 hzB7v21 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 91 hzB7v23 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 92 hzB7v24 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 93 hzB7v25 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 94 hzB7v26 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 95 hzB7v27 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 96 hzB7v28 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 97 hzB7v29 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 98 hzB7v30 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 99 hzB7v31 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 100 hzB7v33 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 101 hzB7v34 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 102 hzB7v35 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 103 hzB7v36 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 104 hzB7v37 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 105 hzB7v39 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 106 hzB7v40 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 109 hzB7v41 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTV R P 32 人類IgG1 Fc區 DKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 33 人類IgG1 Fc xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 34 Fc區M252Y及M428V (YV) S354C T366W杵 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVVHE ALHNHYTQKS LSLSPGK 35 Fc區M252Y、M428V、H435R (YVR) T366S、L368A、Y407V臼 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVVHE ALHNRYTQKS LSLSPGK 36 Fc區xELL H435R DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 37 Fc區xELL M252Y及M428V (YV) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 38 Fc區xELL M252Y及M428L (YL) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 39 Fc區xELL M252Y、M428L、H435R (YLR) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 40 Fc區xELL M252Y、M428V、H435R (YVR) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 41 Fc區xELL S354C T366W杵 DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 42 Fc區xELL H435R S354C T366W杵 DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 43 Fc區xELL M252Y及M428V (YV) S354C T366W杵 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPGK 44 Fc區xELL M252Y及M428L (YL) S354C T366W杵 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK 45 Fc區xELL M252Y、M428L、H435R (YLR) S354C T366W杵 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSLSPGK 46 Fc區xELL M252Y、M428V、H435R (YVR) S354C T366W杵 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPGK 47 Fc區xELL T366S、L368A、Y407V臼 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 48 Fc區xELL H435R、T366S、L368A、Y407V臼 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 49 Fc區xELL M252Y及M428V (YV) T366S、L368A、Y407V臼 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPGK 50 Fc區xELL M252Y及M428L (YL) T366S、L368A、Y407V臼 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK 51 Fc區xELL M252Y、M428L、H435R (YLR) T366S、L368A、Y407V臼 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSLSPGK 52 Fc區xELL M252Y、M428V、H435R (YVR) T366S、L368A、Y407V臼 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPGK 53 Fc區H435R DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 54 Fc區M252Y及M428V (YV) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 55 Fc區M252Y及M428L (YL) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 56 Fc區M252Y、M428L、H435R (YLR) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 57 Fc區M252Y、M428V、H435R (YVR) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 58 Fc區S354C T366W杵 DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 59 Fc區H435R S354C T366W杵 DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 60 Fc區M252Y及M428L (YL) S354C T366W杵 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK 61 Fc區M252Y、M428L、H435R (YLR) S354C T366W杵 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSLSPGK 62 Fc區M252Y、M428V、H435R (YVR) S354C T366W杵 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPGK 63 Fc區T366S、L368A、Y407V臼 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 64 Fc區H435R、T366S、L368A、Y407V臼 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 65 Fc區M252Y及M428V (YV) T366S、L368A、Y407V臼 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPGK 66 Fc區M252Y及M428L (YL) T366S、L368A、Y407V臼 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK 67 Fc區M252Y、M428L、H435R (YLR) T366S、L368A、Y407V臼 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSLSPGK 68 Fc區xELL P329G、H435R、T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 69 Fc區xELL P329G、S354C、T366W杵、ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 70 Fc區xELL S354C、T366W杵、ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 112 Fc區xELL H435R、T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 113 Fc區S354C T366W杵、ΔK447 DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG 114 Fc區H435R S354C T366W杵、ΔK447 DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPG 115 Fc區M252Y及M428L (YL) S354C T366W杵、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG 116 Fc區M252Y、M428L、H435R (YLR) S354C T366W杵、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSLSPG 117 Fc區M252Y、M428V、H435R (YVR) S354C T366W杵、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPG 118 Fc區T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 119 Fc區H435R、T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 120 Fc區M252Y及M428V (YV) T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPG 121 Fc區M252Y及M428L (YL) T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG 122 Fc區M252Y、M428L、H435R (YLR) T366S、L368A、Y407V臼、ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSLSPG 71 野生型人類IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 72 hzB7v15 VHH-Fc區2 IgG1 xELL S354C、T366W杵-IL-2-T3A-H16A-E61R-P65R-D84Y-C125S EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 73 hzB7v15 VHH-Fc區xELL H435R、T366S、L368A、Y407V臼 EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 74 hzB7v15 VHH-Fc區xELL P329G、S354C、T366W杵-IL-2-T3A-H16A-E61R-P65R-D84Y-C125S EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 75 hzB7v15 VHH- Fc區xELL P329G、H435R、T366S、L368A、Y407V臼 EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 76 hzB7v15 VHH-Fc區2 IgG1 xELL S354C、T366W杵-IL-2-T3A-H16A-P65R-D84S-C125S EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRSLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 78 IL-2-T3A-H16A-E61R-P65R-D84Y-C125S APASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 79 IL-2-T3A-H16A-P65R-D84S-C125S APASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRSLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 107 hzB7v31-Fc xELL- S354C、T366W杵-IL2-T3A H16A E61R P65R D84Y C125S QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 108 hzB7v31 Fc xELL H435R、T366S、L368A、Y407V臼 QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 110 hzB7v41-Fc xELL- S354C、T366W杵-IL2-T3A H16A E61R P65R D84Y C125S QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVRPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 111 hzB7v41 Fc xELL H435R、T366S、L368A、Y407V臼 QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVRPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 123 連接子1 GGGG 124 連接子2 GGSGGS 125 連接子3 GGSSGS The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. The foregoing embodiments are therefore intended in all respects to be illustrative and not restrictive of the invention. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and range of equivalencies to the claims are therefore intended to be included herein. table of certain sequences SEQ ID NO describe sequence 1 Human CD8a, precursor (signal sequence is amino acids 1-21) V ITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYV 77 CD8a-IgV LFc antigen for immunization (signal sequence is amino acids 1-21) MALPVTALLL PLALLLHAAR PSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKTGGS GGGGCPPCPA PELPGGPSVF VFPPKPKDVL SISGRPEVTC VVVDVGKEDP EVNFNWYIDG VEVRTANTKP KEEQFNSTYR VVSVLPIQHQ DWLTGKEFKC KVNNKALPAP IERTISKAKG QTREPQVYTL APHREELAKD TVSVTCLVKG FYPADINVEW QRNGQPESEG TYANTPPQLD NDGTYFLYSK LSVGKNTWQR GETLTCVVMH EALHNHYTQK SISQSLGK 2 B7VHH QVQLVQSGGGLVRPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMNGLKSEDTAVYVCAKGSPELQYDSWGQGTQVTVKPXX where each X series Gly may or may not exist 3 B7, hzB7v1, hzB7v2, hzB7v3, hzB7v4, hzB7v5, hzB7v6, hzB7v7, hzB7v8, hzB7v9, hzB7v10, hzB7v11, hzB7v12, hzB7v13, hzB7v14, hzB7v15, hzB7v16, hz B7v17, hzB7v18, hzB7v19, hzB7v20, hzB7v21, hzB7v25, hzB7v26, hzB7v27, CDR1 of hzB7v28, hzB7v29, hzB7v30, hzB7v31, hzB7v33, hzB7v34, hzB7v35, hzB7v36, hzB7v37, hzB7v39 and hzB7v40 GFTFDDYAMS 80 CDR1 of hzB7v23 GFTFDSYAMS 81 CDR1 of hzB7v24 GFTFSSYAMS 4 CDR2 of B7, hzB7v1, hzB7v2, hzB7v3, hzB7v4, hzB7v5, hzB7v8, hzB7v9, hzB7v13 and hzB7v14 TITWDGEGTD 12 CDR2 of hzB7v6 and hzB7v10 TITWEGEGTD 14 CDR2 of hzB7v7, hzB7v11, hzB7v15, hzB7v19, hzB7v20, hzB7v21, hzB7v23, hzB7v24 and hzB7v28 TITWDAEGTD twenty two CDR2 of hzB7v12 TITWSGEGTD 27 CDR2 of hzB7v16 TITWEGESTD 29 CDR2 of hzB7v17 TITWEGEGTY 31 CDR2 of hzB7v18 TITWEGESTY 82 CDR2 of hzB7v25 TITWSAEGTD 83 CDR2 of hzB7v26, hzB7v30 and hzB7v36 TITWDAGGTD 84 CDR2 of hzB7v27, hzB7v29 and hzB7v35 TITWDAEGTY 85 CDR2 of hzB7v31 and hzB7v37 TITWDAGGTY 86 CDR2 of hzB7v33 and hzB7v39 TITWSAEGTY 87 CDR2 of hzB7v34 and hzB7v40 TITTWSAGGTD 5 CDR3 of B7, hzB7v1, hzB7v2, hzB7v3, hzB7v4, hzB7v5, hzB7v6, hzB7v7, hzB7v10, hzB7v11, hzB7v12, hzB7v16, hzB7v17 and hzB7v18 GSPELQYDS 16 CDR3 of hzB7v8 and hzB7v13 GSPELQYES 18 hzB7v9, hzB7v14, hzB7v15, hzB7v19, hzB7v20, hzB7v21, hzB7v23, hzB7v24, hzB7v25, hzB7v26, hzB7v27, hzB7v28, hzB7v29, hzB7v30, hzB7v31, hzB7v 33. CDR3 of hzB7v34, hzB7v35, hzB7v36, hzB7v37, hzB7v39 and hzB7v40 GSPELQYDT 6 hzB7v1 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 7 hzB7v2 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 8 hzB7v3 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 9 hzB7v4 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 10 hzB7v5 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 11 hzB7v6 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 13 hzB7v7 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 15 hzB7v8 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYESWGQGTLVTVKPXX where each X series Gly or does not exist 17 hzB7v9 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESMKGRFTISRDNAKSTVYLQMSSLRAEDTAVYVCAKGSPELQYDTWGQGTLVTVKPXX where each X series Gly may or may not exist 19 hzB7v10 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 20 hzB7v11 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where Gly for each X series may or may not exist twenty one hzB7v12 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist twenty three hzB7v13 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYESWGQGTLVTVKPXX where each X series Gly may or may not exist twenty four hzB7v14 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDGEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPXX where Gly for each X series may or may not exist 25 hzB7v15 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPXX where Gly for each X series may or may not exist 26 hzB7v16 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGESTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where each X series Gly may or may not exist 28 hzB7v17 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGEGTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where Gly for each X series may or may not exist 30 hzB7v18 VHH EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWEGESTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDSWGQGTLVTVKPXX where Gly for each X series may or may not exist 88 hzB7v19 VHH QVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 89 hzB7v20 VHH EVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 90 hzB7v21 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 91 hzB7v23 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 92 hzB7v24 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 93 hzB7v25 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 94 hzB7v26 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 95 hzB7v27 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 96 hzB7v28 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 97 hzB7v29 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 98 hzB7v30 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 99 hzB7v31 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 100 hzB7v33 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 101 hzB7v34 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 102 hzB7v35 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 103 hzB7v36 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 104 hzB7v37 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 105 hzB7v39 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAEGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 106 hzB7v40 VHH QVQLVQSGGGEVKPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWSAGGTDYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKP 109 hzB7v41 VHH QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTV R P 32 Human IgG1 Fc region DKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGS FFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 33 Human IgG1 Fc xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 34 Fc area M252Y and M428V (YV) S354C T366W pestle DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLY SKL TVDKSRWQQG NVFSCSVVHE ALHNHYTQKS LSLSPGK 35 Fc area M252Y, M428V, H435R (YVR) T366S, L368A, Y407V mortar DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSK L TVDKSRWQQG NVFSCSVVHE ALHNRYTQKS LSLSPGK 36 Fc zone xELL H435R DKTHTC PPCPAPGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 37 Fc area xELL M252Y and M428V (YV) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 38 Fc area xELL M252Y and M428L (YL) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 39 Fc area xELL M252Y, M428L, H435R (YLR) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 40 Fc area xELL M252Y, M428V, H435R (YVR) DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 41 Fc area xELL S354C T366W pestle DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLY SK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 42 Fc area xELL H435R S354C T366W pestle DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLY SK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 43 Fc area xELL M252Y and M428V (YV) S354C T366W pestle DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVVHEALHNHYTQKSLSLSPGK 44 Fc area xELL M252Y and M428L (YL) S354C T366W pestle DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVLHEALHNHYTQKSLSLSPGK 45 Fc area xELL M252Y, M428L, H435R (YLR) S354C T366W pestle DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVLHEALHNRYTQKSLSLSPGK 46 Fc area xELL M252Y, M428V, H435R (YVR) S354C T366W pestle DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVVHEALHNRYTQKSLSLSPGK 47 Fc area xELL T366S, L368A, Y407V mortar DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 48 Fc area xELL H435R, T366S, L368A, Y407V mortar DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNRYTQKSLSLSPGK 49 Fc area xELL M252Y and M428V (YV) T366S, L368A, Y407V mortar DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVVHEALHNHYTQKSLSLSPGK 50 Fc area xELL M252Y and M428L (YL) T366S, L368A, Y407V mortar DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVLHEALHNHYTQKSLSLSPGK 51 Fc area xELL M252Y, M428L, H435R (YLR) T366S, L368A, Y407V mortar DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVLHEALHNRYTQKSLSLSPGK 52 Fc area xELL M252Y, M428V, H435R (YVR) T366S, L368A, Y407V mortar DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVVHEALHNRYTQKSLSLSPGK 53 Fc zone H435R DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 54 Fc area M252Y and M428V (YV) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 55 Fc area M252Y and M428L (YL) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 56 Fc area M252Y, M428L, H435R (YLR) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 57 Fc area M252Y, M428V, H435R (YVR) DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFF LYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 58 Fc area S354C T366W pestle DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLY SK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 59 Fc area H435R S354C T366W pestle DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLY SK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 60 Fc area M252Y and M428L (YL) S354C T366W pestle DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHNHYTQKSLSLSPGK 61 Fc area M252Y, M428L, H435R (YLR) S354C T366W pestle DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHNRYTQKSLSLSPGK 62 Fc area M252Y, M428V, H435R (YVR) S354C T366W pestle DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVVHEALHNRYTQKSLSLSPGK 63 Fc area T366S, L368A, Y407V mortar DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 64 Fc area H435R, T366S, L368A, Y407V mortar DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNRYTQKSLSLSPGK 65 Fc area M252Y and M428V (YV) T366S, L368A, Y407V mortar DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVVHEALHNHYTQKSLSLSPGK 66 Fc area M252Y and M428L (YL) T366S, L368A, Y407V mortar DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVLHEALHNHYTQKSLSLSPGK 67 Fc area M252Y, M428L, H435R (YLR) T366S, L368A, Y407V mortar DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVLHEALHNRYTQKSLSLSPGK 68 Fc area xELL P329G, H435R, T366S, L368A, Y407V mortar, ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNRYTQKSLSLSPG 69 Fc area xELL P329G, S354C, T366W pestle, ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 70 Fc area xELL S354C, T366W pestle, ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 112 Fc area xELL H435R, T366S, L368A, Y407V mortar, ΔK447 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SCSVMHEALHNRYTQKSLSLSPG 113 Fc area S354C T366W pestle, ΔK447 DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLY SK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG 114 Fc area H435R S354C T366W pestle, ΔK447 DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLY SK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPG 115 Fc area M252Y and M428L (YL) S354C T366W pestle, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHNHYTQKSLSLSPG 116 Fc area M252Y, M428L, H435R (YLR) S354C T366W, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHNRYTQKSLSLSPG 117 Fc area M252Y, M428V, H435R (YVR) S354C T366W pestle, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVVHEALHNRYTQKSLSLSPG 118 Fc area T366S, L368A, Y407V, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 119 Fc area H435R, T366S, L368A, Y407V, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNRYTQKSLSLSPG 120 Fc area M252Y and M428V (YV) T366S, L368A, Y407V, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVVHEALHNHYTQKSLSLSPG 121 Fc area M252Y and M428L (YL) T366S, L368A, Y407V, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVLHEALHNHYTQKSLSLSPG 122 Fc area M252Y, M428L, H435R (YLR) T366S, L368A, Y407V, ΔK447 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVLHEALHNRYTQKSLSLSPG 71 Wild-type human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 72 hzB7v15 VHH-Fc region 2 IgG1 xELL S354C, T366W杵-IL-2-T3A-H16A-E61R-P65R-D84Y-C125S EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLER ELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 73 hzB7v15 VHH-Fc area xELL H435R, T366S, L368A, Y407V mortar EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 74 hzB7v15 VHH-Fc area xELL P329G, S354C, T366W杵-IL-2-T3A-H16A-E61R-P65R-D84Y-C125S EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLER ELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 75 hzB7v15 VHH- Fc area xELL P329G, H435R, T366S, L368A, Y407V mortar EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 76 hzB7v15 VHH-Fc region 2 IgG1 xELL S354C, T366W杵-IL-2-T3A-H16A-P65R-D84S-C125S EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAEGTDYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE ELKRLEEVLNLAQSKNFHLRPRSLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 78 IL-2-T3A-H16A-E61R-P65R-D84Y-C125S APASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 79 IL-2-T3A-H16A-P65R-D84S-C125S APASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRSLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 107 hzB7v31-Fc xELL- S354C, T366W杵-IL2-T3A H16A E61R P65R D84Y C125S QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL ERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 108 hzB7v31 Fc xELL H435R, T366S, L368A, Y407V mortar QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVKPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 110 hzB7v41-Fc xELL- S354C, T366W杵-IL2-T3A H16A E61R P65R D84Y C125S QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVRPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGSAPASSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL ERELKRLEEVLNLAQSKNFHLRPRYLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 111 hzB7v41 Fc xELL H435R, T366S, L368A, Y407V mortar QVQLVQSGGGEVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSTITWDAGGTYYAAPVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAKGSPELQYDTWGQGTLVTVRPGGGGDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 123 Connector 1 GGGG 124 Connector 2 GGSGGS 125 Connector 3 GGSSGS

圖1A-1B展示如藉由流動式細胞測量術所評定的格式化為VHH-hIgG1-Fc融合蛋白之靶向CD8a之sdAb的結合。圖1A展示與經分離人類T細胞之結合。圖1B展示作為CD8a陰性對照與HEK293FS細胞之結合。Figures 1A-1B show binding of sdAb targeting CD8a formatted as VHH-hlgG1-Fc fusion protein as assessed by flow cytometry. Figure 1A shows binding to isolated human T cells. Figure 1B shows binding to HEK293FS cells as a negative control for CD8a.

圖2A-2B展示如藉由流動式細胞測量術所評定的格式化為VHH-hIgG1-Fc融合蛋白之靶向CD8a之sdAb的結合。圖2A展示與經分離人類T細胞之結合。圖2B展示作為CD8a陰性對照與HEK293FS細胞之結合。Figures 2A-2B show binding of sdAb targeting CD8a formatted as VHH-hlgG1-Fc fusion protein as assessed by flow cytometry. Figure 2A shows binding to isolated human T cells. Figure 2B shows binding to HEK293FS cells as a negative control for CD8a.

圖3A-3B展示如藉由流動式細胞測量術所評定的格式化為VHH-hIgG1-Fc融合蛋白之靶向CD8a之sdAb的結合。圖3A展示與人類CD8a-FL細胞之結合(表現全長CD8a)。圖3B展示食蟹獼猴CD8a-FL細胞上之結合。Figures 3A-3B show binding of sdAb targeting CD8a formatted as VHH-hlgG1-Fc fusion protein as assessed by flow cytometry. Figure 3A shows binding to human CD8a-FL cells (representing full length CD8a). Figure 3B shows binding on cynomolgus monkey CD8a-FL cells.

圖4A-4B展示如藉由流動式細胞測量術所評定的格式化為VHH-hIgG1-Fc融合蛋白之靶向CD8a之sdAb的結合。圖4A展示與經分離人類CD3+ CD4- T細胞之結合。圖4B展示與經分離食蟹獼猴CD3+ CD4-周邊血液單核細胞(PBMC)之結合。Figures 4A-4B show binding of sdAb targeting CD8a formatted as VHH-hlgG1-Fc fusion protein as assessed by flow cytometry. Figure 4A shows binding to isolated human CD3+ CD4- T cells. Figure 4B shows binding to isolated cynomolgus CD3+ CD4- peripheral blood mononuclear cells (PBMC).

圖5A-5B展示如藉由流動式細胞測量術評定的融合蛋白CD8a-hzB7v15 xELL-Fc之結合。圖5A展示與人類CD3+ CD4- Leuko 29 T細胞之結合。圖5B展示與食蟹獼猴CD3+ CD4- CD16- T細胞之結合。Figures 5A-5B show binding of the fusion protein CD8a-hzB7v15xELL-Fc as assessed by flow cytometry. Figure 5A shows binding to human CD3+ CD4- Leuko 29 T cells. Figure 5B shows binding to cynomolgus monkey CD3+ CD4- CD16- T cells.

圖6A-6C展示野生型IL-2及靶向CD8a之VHH-hIgG1-融合蛋白在IL-2報導細胞上之IL-2活性。圖6A展示野生型IL-2及融合蛋白在不表現CD8a之IL-2報導細胞上之活性，該融合蛋白包含有CD8a-hzB7v15及包含T3A、H16A、E61R、P65R、D84Y及C125S突變或T3A、H16A、P65R、D84S及C125S突變的IL-2。圖6B展示相同融合蛋白在表現CD8a之IL-2報導細胞上之IL-2活性。圖6C展示野生型IL-2、包含有CD8a-hzB7v31及包含T3A、H16A、P65R、D84S及C125S突變之IL-2的融合蛋白以及包含相同突變之非靶向IL-2變異體在表現CD8a之IL-2報導細胞上之活性。Figures 6A-6C show the IL-2 activity of wild-type IL-2 and VHH-hlgG1-fusion protein targeting CD8a on IL-2 reporter cells. Figure 6A shows the activity of wild-type IL-2 and fusion protein on IL-2 reporter cells that do not express CD8a. The fusion protein contains CD8a-hzB7v15 and contains T3A, H16A, E61R, P65R, D84Y and C125S mutations or T3A, H16A, P65R, D84S and C125S mutated IL-2. Figure 6B shows IL-2 activity of the same fusion protein on IL-2 reporter cells expressing CD8a. Figure 6C shows that wild-type IL-2, fusion proteins containing CD8a-hzB7v31 and IL-2 containing T3A, H16A, P65R, D84S and C125S mutations, and non-targeting IL-2 variants containing the same mutations express CD8a. IL-2 activity on reporter cells.

圖7展示在單次給藥包含有CD8a-hzB7v15及包含T3A、H16A、P65R、D84S及C125S突變之IL-2的融合蛋白之後食蟹獼猴之周邊血液中的細胞擴增。Figure 7 shows cell expansion in the peripheral blood of cynomolgus macaques after a single administration of a fusion protein containing CD8a-hzB7v15 and IL-2 containing T3A, H16A, P65R, D84S and C125S mutations.

圖8A-8B展示如藉由流動式細胞測量術評定的格式化為VHH均二聚體Fc融合蛋白或為包含減毒IL-2突變體之VHH杵臼Fc融合蛋白的靶向CD8a之sdAb的結合。圖8A展示與用全長人類CD8a (CD8a-FL)轉染之HEK 293F細胞的結合。圖8B展示與用全長人類CD8b (CD8b-FL)轉染之HEK 293F細胞的結合。Figures 8A-8B show binding of CD8a-targeting sdAb formatted as a VHH homodimeric Fc fusion protein or a VHH pestle Fc fusion protein containing an attenuated IL-2 mutant, as assessed by flow cytometry. . Figure 8A shows binding to HEK 293F cells transfected with full-length human CD8a (CD8a-FL). Figure 8B shows binding to HEK 293F cells transfected with full-length human CD8b (CD8b-FL).

圖9A-9B展示如藉由流動式細胞測量術評定的格式化為VHH均二聚體Fc融合蛋白或為包含減毒IL-2突變體之VHH杵臼Fc融合蛋白的靶向CD8a之sdAb的結合。圖9A展示與自人類全血中富集之泛T細胞(pan T cell)內之CD8 T細胞的結合。圖9B展示缺乏與自人類全血中富集之泛T細胞內之CD4 T細胞的結合。Figures 9A-9B show binding of CD8a-targeting sdAbs formatted as VHH homodimeric Fc fusion proteins or as VHH pestle Fc fusion proteins containing attenuated IL-2 mutants, as assessed by flow cytometry. . Figure 9A shows binding to CD8 T cells in pan T cells enriched from human whole blood. Figure 9B shows lack of binding to CD4 T cells within pan-T cells enriched from human whole blood.

圖10A-10H展示如藉由流動式細胞測量術所評定的格式化為VHH-hIgG1-Fc融合蛋白之靶向CD8a之sdAb的結合。圖10A-10B及圖10E-10F展示與自人類全血中富集之泛T細胞(圖10A及10B)或周邊血液單核細胞(PBMC) (圖10E及圖10F)內的CD8 T細胞(圖10A及圖10E)或CD4 T細胞(圖10B及圖10F)之結合。圖10C-10D及圖10F-10H展示與自食蟹獼猴全血分離之周邊血液單核細胞(PBMC)內的CD8 T細胞(圖10C及圖10G)或CD4 T細胞(圖10D及圖10H)之結合。Figures 10A-10H show binding of sdAb targeting CD8a formatted as VHH-hlgG1-Fc fusion protein as assessed by flow cytometry. Figures 10A-10B and Figures 10E-10F show CD8 T cells ( Figure 10A and Figure 10E) or CD4 T cells (Figure 10B and Figure 10F). Figures 10C-10D and Figures 10F-10H show CD8 T cells (Figure 10C and Figure 10G) or CD4 T cells (Figure 10D and Figure 10H) in peripheral blood mononuclear cells (PBMC) isolated from whole blood of cynomolgus monkeys. The combination.

圖11A-11D展示人類供體之周邊血液內的STAT5信號傳導細胞群。展示自人類全血中富集之泛T細胞(圖11A-11B)或PBMC (圖11C-11D)內的CD8 T細胞(圖11A及圖11C)或調節性T細胞(Treg，圖11B)或CD4 T細胞(圖11D)中的磷酸化STAT5 (pSTAT5)之含量(圖11A-11B)或表現pSTAT5之細胞百分比(圖11C-11D)。細胞用包含CD8a-hzB7v31或CD8a-B7v41、Fc區及突變減毒IL-2的融合蛋白；包含CD8a-hzB7v31及Fc區(無IL-2)的融合蛋白；包含非靶向VHH、Fc區及減毒IL-2的融合蛋白；或野生型IL-2處理。Figures 11A-11D show STAT5 signaling cell populations in peripheral blood of human donors. Display of CD8 T cells (Figure 11A and Figure 11C) or regulatory T cells (Treg, Figure 11B) from enriched pan-T cells (Figures 11A-11B) or PBMCs (Figures 11C-11D) in human whole blood or The amount of phosphorylated STAT5 (pSTAT5) in CD4 T cells (Figure 11D) (Figures 11A-11B) or the percentage of cells expressing pSTAT5 (Figures 11C-11D). Cells were treated with fusion proteins containing CD8a-hzB7v31 or CD8a-B7v41, Fc region and mutated attenuated IL-2; fusion proteins containing CD8a-hzB7v31 and Fc region (without IL-2); including non-targeting VHH, Fc region and Fusion proteins of attenuated IL-2; or wild-type IL-2 treatment.

圖12A-12C展示來自人類腫瘤樣品(兩個頭頸癌或腎癌病例及一個大腸癌病例，圖12A及圖12B)的經解離腫瘤細胞準備物或來自健康供體血液(圖12C)之PBMC內的CD8 T細胞(圖12A及圖12C)或CD4 T細胞(圖12B)之擴增，該等T細胞用包含CD8a-hzB7v31、Fc區及突變減毒IL-2的融合蛋白；包含CD8a-hzB7v31及Fc區(無IL-2)的融合蛋白；包含非靶向VHH、Fc區及減毒IL-2的融合蛋白；或野生型IL-2進行離體處理。Figures 12A-12C show dissociated tumor cell preparations from human tumor samples (two head and neck cancer or renal cancer cases and one colorectal cancer case, Figures 12A and 12B) or within PBMCs from healthy donor blood (Figure 12C) Expansion of CD8 T cells (Figure 12A and Figure 12C) or CD4 T cells (Figure 12B) using a fusion protein containing CD8a-hzB7v31, Fc region and mutated attenuated IL-2; containing CD8a-hzB7v31 and Fc region (without IL-2); fusion proteins containing non-targeting VHH, Fc region and attenuated IL-2; or wild-type IL-2 for ex vivo processing.

圖13A-13B展示食蟹獼猴中單一劑量(1 mg/kg)之包含CD8a-hzB7v15、Fc區及突變減毒IL-2之融合蛋白的活性。圖13A展示某些PBMC亞群之擴增，作為給藥後七天的細胞數相對於基線之變化倍數。圖13B展示在給藥前(基線)及給藥後七天此等亞群內Ki67+細胞的百分比。Figures 13A-13B show the activity of a single dose (1 mg/kg) of a fusion protein containing CD8a-hzB7v15, Fc region and mutant attenuated IL-2 in cynomolgus monkeys. Figure 13A shows the expansion of certain PBMC subpopulations as the fold change from baseline in cell number seven days post-dose. Figure 13B shows the percentage of Ki67+ cells within these subpopulations before dosing (baseline) and seven days after dosing.

圖14A-14B展示針對A431表皮樣癌細胞經富集之預刺激CD8 T細胞的細胞毒活性(圖14A)或PBMC之抗體依賴性細胞毒性(ADCC) (圖14B)。如所指示，用包含CD8a-hzB7v31、Fc區及減毒IL-2的融合蛋白；包含非靶向VHH、Fc區及減毒IL-2的融合蛋白；或野生型IL-2處理細胞。如所強調，以不同效應子與目標細胞比率(20:1、10:1或5:1)添加CD8 T細胞或PBMC。將EGFR特異性治療抗體西妥昔單抗添加至圖14B中之細胞培養物中。Figures 14A-14B show the cytotoxic activity of enriched pre-stimulated CD8 T cells against A431 epidermoid cancer cells (Figure 14A) or the antibody-dependent cytotoxicity (ADCC) of PBMC (Figure 14B). Cells were treated with fusion proteins containing CD8a-hzB7v31, Fc region, and attenuated IL-2; fusion proteins containing non-targeting VHH, Fc region, and attenuated IL-2; or wild-type IL-2, as indicated. As highlighted, CD8 T cells or PBMC were added at different effector to target cell ratios (20:1, 10:1 or 5:1). The EGFR-specific therapeutic antibody cetuximab was added to the cell culture in Figure 14B.

TW202309102A_111127072_SEQL.xmlTW202309102A_111127072_SEQL.xml

Claims (78)

一種多肽，其包含至少一個結合CD8之VHH域及經修飾IL-2，其中至少一個結合CD8之VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列，且其中該經修飾IL-2相對於包含SEQ ID NO: 71之胺基酸序列的野生型人類IL-2而言包含T3A、H16A、P65R、C125S突變及D84S或D84Y突變。A polypeptide comprising at least one CD8-binding VHH domain and modified IL-2, wherein at least one CD8-binding VHH domain comprises CDR1, which comprises the amino acid sequence of SEQ ID NO: 3, 80 or 81; CDR2, which An amino acid sequence comprising SEQ ID NO: 4, 12, 14, 22, 27, 29, 31, 82, 83, 84, 85, 86 or 87; and a CDR3 comprising SEQ ID NO: 5, 16 or 18 The amino acid sequence of SEQ ID NO: 71, and wherein the modified IL-2 includes T3A, H16A, P65R, C125S mutations and D84S or D84Y mutations relative to wild-type human IL-2 comprising the amino acid sequence of SEQ ID NO: 71 . 如請求項1之多肽，其中該經修飾IL-2包含T3A、H16A、P65R、C125S及D84S突變。The polypeptide of claim 1, wherein the modified IL-2 includes T3A, H16A, P65R, C125S and D84S mutations. 如請求項1之多肽，其中該經修飾IL-2包含T3A、H16A、E61R、P65R、C125S及D84Y突變。The polypeptide of claim 1, wherein the modified IL-2 includes T3A, H16A, E61R, P65R, C125S and D84Y mutations. 如請求項1或2之多肽，其中該經修飾IL-2包含SEQ ID NO: 79之胺基酸序列。The polypeptide of claim 1 or 2, wherein the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 79. 如請求項1或3之多肽，其中該經修飾IL-2包含SEQ ID NO: 78之胺基酸序列。The polypeptide of claim 1 or 3, wherein the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 78. 如請求項1至5中任一項之多肽，其中至少一個VHH域包含CDR1、CDR2及CDR3，其分別包含胺基酸序列SEQ ID NO: 3、4及5；3、12及5；3、14及5；3、4及16；3、4及18；3、22及5；3、14及18；3、27及5；3、29及5；3、31及5；80、14及18；81、14及18；3、82及18；3、83及18；3、84及18；3、85及18；3、86及18；或3、87及18。Such as the polypeptide of any one of claims 1 to 5, wherein at least one VHH domain includes CDR1, CDR2 and CDR3, which respectively include the amino acid sequences SEQ ID NO: 3, 4 and 5; 3, 12 and 5; 3, 14 and 5; 3, 4 and 16; 3, 4 and 18; 3, 22 and 5; 3, 14 and 18; 3, 27 and 5; 3, 29 and 5; 3, 31 and 5; 80, 14 and 18; 81, 14 and 18; 3, 82 and 18; 3, 83 and 18; 3, 84 and 18; 3, 85 and 18; 3, 86 and 18; or 3, 87 and 18. 如請求項1至6中任一項之多肽，其中至少一個VHH域包含CDR1，其包含SEQ ID NO: 3之胺基酸序列；CDR2，其包含SEQ ID NO: 85之胺基酸序列；及CDR3，其包含SEQ ID NO: 18之胺基酸序列。The polypeptide of any one of claims 1 to 6, wherein at least one VHH domain includes CDR1, which includes the amino acid sequence of SEQ ID NO: 3; CDR2, which includes the amino acid sequence of SEQ ID NO: 85; and CDR3, which includes the amino acid sequence of SEQ ID NO: 18. 如請求項1至7中任一項之多肽，其中至少一個VHH域或各VHH域經人類化。The polypeptide of any one of claims 1 to 7, wherein at least one VHH domain or each VHH domain is humanized. 如請求項1至8中任一項之多肽，其中至少一個VHH域包含與SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列至少85%、90%、95%或至少99%一致的胺基酸序列。The polypeptide of any one of claims 1 to 8, wherein at least one VHH domain includes SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23,24,25,26,28,30,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106 or The amino acid sequence of 109 is at least 85%, 90%, 95% or at least 99% identical to the amino acid sequence. 如請求項1至9中任一項之多肽，其中至少一個VHH域包含SEQ ID NO: 6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列。The polypeptide of any one of claims 1 to 9, wherein at least one VHH domain comprises SEQ ID NO: 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24 , 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 or 109 amines amino acid sequence. 如請求項1至10中任一項之多肽，其中至少一個VHH域包含SEQ ID NO: 99或109之胺基酸序列。The polypeptide of any one of claims 1 to 10, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99 or 109. 如請求項1至11中任一項之多肽，其包含兩個VHH域。The polypeptide of any one of claims 1 to 11, which contains two VHH domains. 如請求項1至11中任一項之多肽，其包含三個VHH域。The polypeptide of any one of claims 1 to 11, which contains three VHH domains. 如請求項1至13中任一項之多肽，其中該多肽包含Fc區。The polypeptide of any one of claims 1 to 13, wherein the polypeptide includes an Fc region. 如請求項14之多肽，其中該Fc區包含選自SEQ ID NO: 32-70及112-122之胺基酸序列。The polypeptide of claim 14, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 32-70 and 112-122. 如請求項14或請求項15之多肽，其中該經修飾IL-2融合至該Fc區之C端。The polypeptide of claim 14 or claim 15, wherein the modified IL-2 is fused to the C-terminus of the Fc region. 如請求項1至11、14及15中任一項之多肽，其中該多肽包含一個結合CD8之VHH域、Fc區及經修飾IL-2。The polypeptide of any one of claims 1 to 11, 14 and 15, wherein the polypeptide comprises a CD8-binding VHH domain, Fc region and modified IL-2. 如請求項1或請求項17之多肽，其中該多肽包含SEQ ID NO: 72、74、76、107或110之胺基酸序列。The polypeptide of claim 1 or claim 17, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 72, 74, 76, 107 or 110. 如請求項1至18中任一項之多肽，其中該多肽包含至少一個結合除CD8以外之抗原的抗原結合域。The polypeptide of any one of claims 1 to 18, wherein the polypeptide comprises at least one antigen-binding domain that binds an antigen other than CD8. 如請求項19之多肽，其包含至少一個結合以下之抗原結合域：TGFBR1、TGFBR2、Fas、TNFR2、1-92-LFA-3、5T4、α-4整合素、α-V整合素、α4β1整合素、α4β7整合素、AGR2、抗Lewis-Y、Apelin J受體、APRIL、B7-H3、B7-H4、B7-H6、BAFF、BCMA、BTLA、C5補體、C-242、CA9、CA19-9、(Lewis a)、碳酸酐酶9、CD2、CD3、CD6、CD9、CD11a、CD19、CD20、CD22、CD24、CD25、CD27、CD28、CD30、CD33、CD38、CD39、CD40、CD40L、CD41、CD44、CD44v6、CD47、CD51、CD52、CD56、CD64、CD70、CD71、CD73、CD74、CD80、CD81、CD86、CD95、CD117、CD123、CD125、CD132、(IL-2RG)、CD133、CD137、CD138、CD166、CD172A、CD248、CDH6、CEACAM5 (CEA)、CEACAM6 (NCA-90)、CLAUDIN-3、CLAUDIN-4、cMet、膠原蛋白、Cripto、CSFR、CSFR-1、CTLA4、CTGF、CXCL10、CXCL13、CXCR1、CXCR2、CXCR4、CYR61、DL44、DLK1、DLL3、DLL4、DPP-4、DSG1、EDA、EDB、EGFR、EGFRviii、內皮素B受體(ETBR)、ENPP3、EpCAM、EPHA2、EPHB2、ERBB3、RSV之F蛋白質、FAP、FcRH5、FGF-2、FGF8、FGFR1、FGFR2、FGFR3、FGFR4、FLT-3、葉酸受體α (FRα)、GAL3ST1、G-CSF、G-CSFR、GD2、GITR、GLUT1、GLUT4、GM-CSF、GM-CSFR、GP IIb/IIIa受體、Gp130、GPIIB/IIIA、GPNMB、GPRC5D、GRP78、HAVCAR1、HER2/neu、HER3、HER4、HGF、hGH、HVEM、玻尿酸酶、ICOS、IFNα、IFNβ、IFNγ、IgE、IgE受體(FceRI)、IGF、IGF1R、IL1B、IL1R、IL2、IL11、IL12、IL12p40、IL-12R、IL-12Rβ1、IL13、IL13R、IL15、IL17、IL18、IL21、IL23、IL23R、IL27/IL27R (wsx1)、IL29、IL-31R、IL31/IL31R、IL2R、IL4、IL4R、IL6、IL6R、胰島素受體、鋸齒狀配位體(Jagged Ligand)、鋸齒狀1、鋸齒狀2、KISS1-R、LAG-3、LIF-R、Lewis X、LIGHT、LRP4、LRRC26、Ly6G6D、LyPD1、MCSP、間皮素、MICA、MICB、MRP4、MUC1、黏蛋白-16 (MUC16、CA-125)、Na/K ATP酶、NGF、Nicastrin、Notch受體、Notch 1、Notch 2、Notch 3、Notch 4、NOV、OSM-R、OX-40、PAR2、PDGF-AA、PDGF-BB、PDGFRα、PDGFRβ、PD-1、PD-L1、PD-L2、磷脂醯絲胺酸、P1GF、PSCA、PSMA、PSGR、RAAG12、RAGE、SLC44A4、神經鞘胺醇1磷酸酯、STEAP1、STEAP2、TAG-72、TAPA1、TEM-8、TGFβ、TIGIT、TIM-3、TLR2、TLR4、TLR6、TLR7、TLR8、TLR9、TMEM31、TNFα、TNFR、TNFRS12A、TRAIL-R1、TRAIL-R2、運鐵蛋白、運鐵蛋白受體、TRK-A、TRK-B、TROP-2 uPAR、VAP1、VCAM-1、VEGF、VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGFR1、VEGFR2、VEGFR3、VISTA、WISP-1、WISP-2或WISP-3。Such as the polypeptide of claim 19, which includes at least one antigen-binding domain that binds to: TGFBR1, TGFBR2, Fas, TNFR2, 1-92-LFA-3, 5T4, α-4 integrin, α-V integrin, α4β1 integrin protein, α4β7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9 , (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD39, CD40, CD40L, CD41, CD44 , CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD73, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166 , CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F of RSV Protein, FAP, FcRH5, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptor, Gp130, GPIIB/IIIA, GPNMB, GPRC5D, GRP78, HAVCAR1, HER2/neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβ1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23 , IL23R, IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, insulin receptor, Jagged Ligand (Jagged Ligand), Jagged 1, Jagged 2. KISS1-R, LAG-3, LIF-R, Lewis 125), Na/K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα , PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72 , TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin Receptor, TRK-A, TRK-B, TROP-2 uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP- 1. WISP-2 or WISP-3. 如請求項19或20之多肽，其中至少一個結合除CD8以外之抗原的抗原結合域為VHH域。The polypeptide of claim 19 or 20, wherein at least one antigen-binding domain that binds an antigen other than CD8 is a VHH domain. 如請求項21之多肽，其中結合除CD8以外之抗原的各抗原結合域為VHH域。The polypeptide of claim 21, wherein each antigen-binding domain that binds an antigen other than CD8 is a VHH domain. 如請求項19至21中任一項之多肽，其中至少一個結合除CD8以外之抗原的抗原結合域包含重鏈可變區及輕鏈可變區。The polypeptide of any one of claims 19 to 21, wherein at least one antigen-binding domain that binds an antigen other than CD8 includes a heavy chain variable region and a light chain variable region. 如請求項23之多肽，其中結合除CD8以外之抗原的各抗原結合域包含重鏈可變區及輕鏈可變區。The polypeptide of claim 23, wherein each antigen-binding domain that binds to an antigen other than CD8 includes a heavy chain variable region and a light chain variable region. 一種包含第一多肽及第二多肽之複合物，其中該第一多肽為如請求項14至24中任一項之多肽，其中該第一多肽包含第一Fc區，且其中該第二多肽包含至少一個結合CD8之VHH域及第二Fc區，其中該等第一與第二Fc區相同或不同。A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is the polypeptide of any one of claims 14 to 24, wherein the first polypeptide comprises a first Fc region, and wherein the The second polypeptide includes at least one VHH domain that binds CD8 and a second Fc region, wherein the first and second Fc regions are the same or different. 一種包含第一多肽及第二多肽之複合物，其中該第一多肽包含至少一個結合CD8之VHH域及第一Fc區，且該第二多肽包含第二Fc區及經修飾IL-2，其中至少一個結合CD8之VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列；且其中該經修飾IL-2相對於包含SEQ ID NO: 71之胺基酸序列的野生型人類IL-2而言包含T3A或T3G、H16A、P65R、C125S及D84S或D84Y突變。A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least one CD8-binding VHH domain and a first Fc region, and the second polypeptide comprises a second Fc region and a modified IL -2, wherein at least one CD8-binding VHH domain comprises CDR1, which comprises the amino acid sequence of SEQ ID NO: 3, 80 or 81; CDR2, which comprises SEQ ID NO: 4, 12, 14, 22, 27, 29 , the amino acid sequence of SEQ ID NO: 5, 16 or 18; and wherein the modified IL-2 is relative to Wild-type human IL-2 comprising the amino acid sequence of SEQ ID NO: 71 contains T3A or T3G, H16A, P65R, C125S and D84S or D84Y mutations. 如請求項26之複合物，其中該CDR1包含SEQ ID NO: 3之胺基酸序列；該CDR2包含SEQ ID NO: 85之胺基酸序列；且該CDR3包含SEQ ID NO: 18之胺基酸序列。The complex of claim 26, wherein the CDR1 includes the amino acid sequence of SEQ ID NO: 3; the CDR2 includes the amino acid sequence of SEQ ID NO: 85; and the CDR3 includes the amino acid sequence of SEQ ID NO: 18 sequence. 如請求項26或請求項27之複合物，其中該經修飾IL-2包含T3A、H16A、P65R、C125S及D84S突變。The complex of claim 26 or claim 27, wherein the modified IL-2 includes T3A, H16A, P65R, C125S and D84S mutations. 如請求項26或請求項27之複合物，其中該經修飾IL-2包含T3A、H16A、E61R、P65R、C125S及D84Y突變。The complex of claim 26 or claim 27, wherein the modified IL-2 includes T3A, H16A, E61R, P65R, C125S and D84Y mutations. 如請求項26至29中任一項之複合物，其中該經修飾IL-2包含與SEQ ID NO: 78或79之胺基酸序列至少90%、至少95%、至少97%、至少98%、至少99%或100%一致的胺基酸序列。The complex of any one of claims 26 to 29, wherein the modified IL-2 comprises at least 90%, at least 95%, at least 97%, at least 98% of the amino acid sequence of SEQ ID NO: 78 or 79 , an amino acid sequence that is at least 99% or 100% identical. 如請求項26、27及29中任一項之複合物，其中該經修飾IL-2包含SEQ ID NO: 78之胺基酸序列。The complex of any one of claims 26, 27 and 29, wherein the modified IL-2 comprises the amino acid sequence of SEQ ID NO: 78. 如請求項26至31中任一項之複合物，其中至少一個VHH域或各VHH域經人類化。The complex of any one of claims 26 to 31, wherein at least one or each VHH domain is humanized. 如請求項26至32中任一項之複合物，其中至少一個VHH域包含與選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列至少85%、至少90%、至少95%或至少99%一致的胺基酸序列。The complex of any one of claims 26 to 32, wherein at least one VHH domain comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20 ,21,23,24,25,26,28,30,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105 , the amino acid sequence of 106 and 109 is at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence. 如請求項26至33中任一項之複合物，其中至少一個VHH域包含選自SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106及109之胺基酸序列。The complex of any one of claims 26 to 33, wherein at least one VHH domain comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, Amino acid sequences of 106 and 109. 如請求項26至34中任一項之複合物，其中至少一個VHH域包含SEQ ID NO: 99或109之胺基酸序列。The complex of any one of claims 26 to 34, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 99 or 109. 如請求項25至35中任一項之複合物，其中該第二多肽包含至少一個抗原結合域。The complex of any one of claims 25 to 35, wherein the second polypeptide comprises at least one antigen-binding domain. 如請求項36之複合物，其中該第二多肽之至少一個抗原結合域為VHH域。The complex of claim 36, wherein at least one antigen-binding domain of the second polypeptide is a VHH domain. 如請求項37之複合物，其中該第二多肽包含至少一個結合CD8之VHH域。The complex of claim 37, wherein the second polypeptide comprises at least one VHH domain that binds CD8. 如請求項25至38中任一項之複合物，其中該第二多肽包含至少一個結合CD8之VHH域，該VHH域包含CDR1，其包含SEQ ID NO: 3、80或81之胺基酸序列；CDR2，其包含SEQ ID NO: 4、12、14、22、27、29、31、82、83、84、85、86或87之胺基酸序列；及CDR3，其包含SEQ ID NO: 5、16或18之胺基酸序列。The complex of any one of claims 25 to 38, wherein the second polypeptide comprises at least one CD8-binding VHH domain, the VHH domain comprising CDR1 comprising the amino acid of SEQ ID NO: 3, 80 or 81 Sequence; CDR2, which includes the amino acid sequence of SEQ ID NO: 4, 12, 14, 22, 27, 29, 31, 82, 83, 84, 85, 86 or 87; and CDR3, which includes SEQ ID NO: 5, 16 or 18 amino acid sequences. 如請求項39之複合物，其中該第二多肽包含至少一個VHH域，該VHH域包含CDR1、CDR2及CDR3，其分別包含胺基酸序列SEQ ID NO: 3、4及5；3、12及5；3、14及5；3、4及16；3、4及18；3、22及5；3、14及18；3、27及5；3、29及5；3、31及5；80、14及18；81、14及18；3、82及18；3、83及18；3、84及18；3、85及18；3、86及18；或3、87及18。Such as the complex of claim 39, wherein the second polypeptide includes at least one VHH domain, and the VHH domain includes CDR1, CDR2 and CDR3, which respectively include the amino acid sequences SEQ ID NO: 3, 4 and 5; 3, 12 and 5; 3, 14 and 5; 3, 4 and 16; 3, 4 and 18; 3, 22 and 5; 3, 14 and 18; 3, 27 and 5; 3, 29 and 5; 3, 31 and 5 ; 80, 14 and 18; 81, 14 and 18; 3, 82 and 18; 3, 83 and 18; 3, 84 and 18; 3, 85 and 18; 3, 86 and 18; or 3, 87 and 18. 如請求項39或請求項40之複合物，其中該第二多肽包含至少一個VHH域，該VHH域包含CDR1，其包含SEQ ID NO: 3之胺基酸序列；CDR2，其包含SEQ ID NO: 85之胺基酸序列；及CDR3，其包含SEQ ID NO: 18之胺基酸序列。The complex of claim 39 or claim 40, wherein the second polypeptide includes at least one VHH domain, and the VHH domain includes CDR1, which includes the amino acid sequence of SEQ ID NO: 3; CDR2, which includes SEQ ID NO : The amino acid sequence of SEQ ID NO: 18; and CDR3, which includes the amino acid sequence of SEQ ID NO: 18. 如請求項39至41中任一項之複合物，其中該第二多肽之至少一個VHH域或各VHH域經人類化。The complex of any one of claims 39 to 41, wherein at least one or each VHH domain of the second polypeptide is humanized. 如請求項39至42中任一項之複合物，其中該第二多肽之至少一個VHH域包含與SEQ ID NO: 2、6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列至少85%、90%、95%或至少99%一致的胺基酸序列。The complex of any one of claims 39 to 42, wherein at least one VHH domain of the second polypeptide comprises SEQ ID NO: 2, 6, 7, 8, 9, 10, 11, 13, 15, 17 ,19,20,21,23,24,25,26,28,30,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103 , an amino acid sequence that is at least 85%, 90%, 95% or at least 99% identical to the amino acid sequence of 104, 105, 106 or 109. 如請求項39至43中任一項之複合物，其中該第二多肽之至少一個VHH域包含SEQ ID NO: 6、7、8、9、10、11、13、15、17、19、20、21、23、24、25、26、28、30、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106或109之胺基酸序列。The complex of any one of claims 39 to 43, wherein at least one VHH domain of the second polypeptide comprises SEQ ID NOs: 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 23, 24, 25, 26, 28, 30, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, Amino acid sequence 105, 106 or 109. 如請求項39至44中任一項之複合物，其中該第二多肽之至少一個VHH域包含SEQ ID NO: 99或109之胺基酸序列。The complex of any one of claims 39 to 44, wherein at least one VHH domain of the second polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 109. 如請求項25至45中任一項之複合物，其中該第二多肽包含一個結合CD8之VHH域。The complex of any one of claims 25 to 45, wherein the second polypeptide comprises a VHH domain that binds CD8. 如請求項25至46中任一項之複合物，其中該第一Fc區包含至少一個杵突變且該第二Fc區包含至少一個臼突變；或其中該第一Fc區包含至少一個臼突變且該第二Fc區包含至少一個杵突變。The complex of any one of claims 25 to 46, wherein the first Fc region comprises at least one hammer mutation and the second Fc region comprises at least one hammer mutation; or wherein the first Fc region comprises at least one hammer mutation and The second Fc region contains at least one clamp mutation. 如請求項47之複合物，其中該第一或第二Fc區包含T366W突變且該第一或第二Fc區中之另一者包含T366S、L368A及Y407V突變。The complex of claim 47, wherein the first or second Fc region comprises a T366W mutation and the other of the first or second Fc region comprises a T366S, L368A and Y407V mutation. 如請求項48之複合物，其中包含該等T366S、L368A及Y407V突變之該Fc區進一步包含H435R或H435K突變。For example, the complex of claim 48, wherein the Fc region including the T366S, L368A and Y407V mutations further includes an H435R or H435K mutation. 如請求項25至49中任一項之複合物，其中該第一多肽包含SEQ ID NO: 72、74、76、107或110之胺基酸序列。The complex of any one of claims 25 to 49, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 72, 74, 76, 107 or 110. 如請求項25至50中任一項之複合物，其中該第二多肽包含SEQ ID NO: 73、75、108或111之胺基酸序列。The complex of any one of claims 25 to 50, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 73, 75, 108 or 111. 如請求項51之複合物，其中該第一多肽包含SEQ ID NO: 72之胺基酸序列且該第二多肽包含SEQ ID NO: 73之胺基酸序列；該第一多肽包含SEQ ID NO: 74之胺基酸序列且該第二多肽包含SEQ ID NO:75之胺基酸序列；該第一多肽包含SEQ ID NO: 76之胺基酸序列且該第二多肽包含SEQ ID NO:75之胺基酸序列；該第一多肽包含SEQ ID NO: 107之胺基酸序列且該第二多肽包含SEQ ID NO:108之胺基酸序列；或該第一多肽包含SEQ ID NO: 110之胺基酸序列且該第二多肽包含SEQ ID NO:111之胺基酸序列。The complex of claim 51, wherein the first polypeptide includes the amino acid sequence of SEQ ID NO: 72 and the second polypeptide includes the amino acid sequence of SEQ ID NO: 73; the first polypeptide includes SEQ The amino acid sequence of ID NO: 74 and the second polypeptide includes the amino acid sequence of SEQ ID NO: 75; the first polypeptide includes the amino acid sequence of SEQ ID NO: 76 and the second polypeptide includes The amino acid sequence of SEQ ID NO:75; the first polypeptide includes the amino acid sequence of SEQ ID NO:107 and the second polypeptide includes the amino acid sequence of SEQ ID NO:108; or the first polypeptide includes the amino acid sequence of SEQ ID NO:107. The peptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 111. 如請求項25至52中任一項之複合物，其中該複合物在生理條件下形成。The complex of any one of claims 25 to 52, wherein the complex is formed under physiological conditions. 如請求項1至53中任一項之多肽或複合物，其中該CD8為人類CD8。The polypeptide or complex of any one of claims 1 to 53, wherein the CD8 is human CD8. 如請求項54之多肽或複合物，其中該人類CD8包含SEQ ID NO: 1之序列。The polypeptide or complex of claim 54, wherein the human CD8 comprises the sequence of SEQ ID NO: 1. 一種醫藥組合物，其包含如請求項1至55中任一項之多肽或複合物及醫藥學上可接受之載劑。A pharmaceutical composition comprising the polypeptide or complex of any one of claims 1 to 55 and a pharmaceutically acceptable carrier. 一種經分離核酸，其編碼如請求項1至55中任一項之多肽或複合物。An isolated nucleic acid encoding the polypeptide or complex of any one of claims 1 to 55. 一種載體，其包含如請求項57之核酸。A vector comprising the nucleic acid of claim 57. 一種宿主細胞，其包含如請求項57之核酸或如請求項58之載體。A host cell comprising the nucleic acid of claim 57 or the vector of claim 58. 一種宿主細胞，其表現如請求項1至55中任一項之多肽或複合物。A host cell expressing the polypeptide or complex of any one of claims 1 to 55. 一種產生如請求項1至55中任一項之多肽或複合物之方法，其包括在適合表現該多肽或複合物之條件下培育如請求項59或請求項60之宿主細胞。A method of producing a polypeptide or complex as claimed in any one of claims 1 to 55, comprising culturing a host cell as claimed in claim 59 or claim 60 under conditions suitable for expressing the polypeptide or complex. 如請求項61之方法，其進一步包含分離該多肽或複合物。The method of claim 61, further comprising isolating the polypeptide or complex. 一種增加CD8 +T細胞增殖之方法，其包含使T細胞與如請求項1至55中任一項之多肽或複合物接觸。 A method of increasing the proliferation of CD8 + T cells, comprising contacting the T cells with the polypeptide or complex of any one of claims 1 to 55. 如請求項63之方法，其中該等CD8 +T細胞係在活體外。 The method of claim 63, wherein the CD8 + T cells are in vitro. 如請求項63之方法，其中該等CD8 +T細胞係在活體內。 The method of claim 63, wherein the CD8 + T cells are in vivo. 一種治療癌症之方法，其包含向患有癌症之個體投與醫藥學上有效量之如請求項1至55中任一項之多肽或複合物或如請求項56之醫藥組合物。A method of treating cancer, comprising administering a pharmaceutically effective amount of the polypeptide or complex of any one of claims 1 to 55 or the pharmaceutical composition of claim 56 to an individual suffering from cancer. 如請求項66之方法，其中該癌症係選自基底細胞癌、膽道癌；膀胱癌；骨癌；腦及中樞神經系統癌；乳癌；腹膜癌；子宮頸癌；絨毛膜癌；大腸直腸癌；結締組織癌；消化系統癌；子宮內膜癌；食道癌；眼癌；頭頸癌；胃癌；胃腸癌；神經膠母細胞瘤；肝癌瘤；肝腫瘤；上皮內贅瘤；腎臟癌或腎癌；喉癌；肝癌；肺癌；小細胞肺癌；非小細胞肺癌；肺腺癌；鱗狀細胞肺癌(squamous carcinoma of the lung)；黑素瘤；骨髓瘤；神經母細胞瘤；口腔癌；卵巢癌；胰臟癌；前列腺癌；視網膜母細胞瘤；橫紋肌肉瘤；直腸癌；呼吸系統癌；唾液腺癌；肉瘤；皮膚癌；鱗狀細胞癌；胃臟癌；睪丸癌；甲狀腺癌；子宮或子宮內膜癌；泌尿系統癌；外陰癌；淋巴瘤；霍奇金氏淋巴瘤(Hodgkin's lymphoma)；非霍奇金氏淋巴瘤；B細胞淋巴瘤；低惡性度(low grade)/濾泡型非霍奇金氏淋巴瘤(NHL)；小淋巴球性(SL) NHL；中惡性度/濾泡型NHL；中惡性度彌漫性NHL；高惡性度免疫母細胞NHL；高惡性度淋巴母細胞NHL；高惡性度小型無裂隙細胞NHL；腫塊性病變NHL (bulky disease NHL)；套細胞淋巴瘤；AIDS相關淋巴瘤；瓦爾登斯特倫氏巨球蛋白血症(Waldenstrom's macroglobulinemia)；慢性淋巴球性白血病(CLL)；急性淋巴母細胞白血病(ALL)；毛細胞白血病；及慢性骨髓母細胞白血病。The method of claim 66, wherein the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer ; Connective tissue cancer; Digestive system cancer; Endometrial cancer; Esophageal cancer; Eye cancer; Head and neck cancer; Stomach cancer; Gastrointestinal cancer; Glioblastoma; Hepatocarcinoma; Liver tumor; Intraepithelial neoplasia; Kidney or kidney cancer ; Laryngeal cancer; Liver cancer; Lung cancer; Small cell lung cancer; Non-small cell lung cancer; Lung adenocarcinoma; Squamous cell carcinoma of the lung; Melanoma; Myeloma; Neuroblastoma; Oral cancer; Ovarian cancer ; Pancreatic cancer; Prostate cancer; Retinoblastoma; Rhabdomyosarcoma; Rectal cancer; Respiratory system cancer; Salivary gland cancer; Sarcoma; Skin cancer; Squamous cell carcinoma; Stomach cancer; Testicular cancer; Thyroid cancer; Uterine or intrauterine cancer. membrane cancer; urinary tract cancer; vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma Chikin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; High-grade small non-cleft cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; and chronic myeloblastic leukemia. 如請求項66或67之方法，其進一步包含投與額外治療劑。The method of claim 66 or 67, further comprising administering an additional therapeutic agent. 如請求項68之方法，其中該額外治療劑為抗癌劑。The method of claim 68, wherein the additional therapeutic agent is an anti-cancer agent. 如請求項69之方法，其中該抗癌劑係選自化學治療劑、抗癌生物製劑、放射線療法、CAR-T療法及溶瘤病毒。The method of claim 69, wherein the anti-cancer agent is selected from the group consisting of chemotherapeutic agents, anti-cancer biological agents, radiotherapy, CAR-T therapy and oncolytic viruses. 如請求項68之方法，其中該額外治療劑為抗癌生物製劑。The method of claim 68, wherein the additional therapeutic agent is an anti-cancer biologic. 如請求項71之方法，其中該抗癌生物製劑為抑制PD-1及/或PD-L1之藥劑。The method of claim 71, wherein the anti-cancer biological agent is an agent that inhibits PD-1 and/or PD-L1. 如請求項71之方法，其中該抗癌生物製劑為抑制VISTA、gpNMB、B7H3、B7H4、HHLA2、CTLA4或TIGIT之藥劑。The method of claim 71, wherein the anti-cancer biological agent is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4 or TIGIT. 如請求項69之方法，其中該抗癌劑為抗體。The method of claim 69, wherein the anti-cancer agent is an antibody. 如請求項71之方法，其中該抗癌生物製劑為細胞介素。The method of claim 71, wherein the anti-cancer biological agent is an interleukin. 如請求項69之方法，其中該抗癌劑為CAR-T療法。The method of claim 69, wherein the anti-cancer agent is CAR-T therapy. 如請求項69之方法，其中該抗癌劑為溶瘤病毒。The method of claim 69, wherein the anti-cancer agent is an oncolytic virus. 如請求項66至77中任一項之方法，其進一步包含腫瘤切除及/或放射線療法。The method of any one of claims 66 to 77, further comprising tumor resection and/or radiation therapy.
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