義翹神州在線講座《免疫治療背景下：細(xì)胞因子的選擇及相關(guān)應(yīng)用》

免疫治療背景下：細(xì)胞因子的選擇及相關(guān)應(yīng)用SinoBiologicalInc.CONTENTSinoBiologicalInc.免疫治療中CAR-T細(xì)胞治療進(jìn)展概述1CAR-T細(xì)胞治療和抗體藥研發(fā)中細(xì)胞因子的應(yīng)用及相關(guān)產(chǎn)品的介紹2免疫治療中CAR-T細(xì)胞治療進(jìn)展概述01SinoBiologicalInc.

癌癥仍然是全球性的健康問(wèn)題。GLOBOCAN2020數(shù)據(jù)庫(kù)顯示，2020年全球新發(fā)癌癥大約在1千9百多萬(wàn)例，近千萬(wàn)例癌癥患者死亡(Figure1)[1]。

[1]HyunaSungetal.,“GlobalCancerStatistics2020:GLOBOCANEstimatesofIncidenceandMortalityWorldwidefor36Cancersin185Countries,”CA:ACancerJournalforCliniciansn/a,no.n/a,accessedFebruary7,2021,/10.3322/caac.21660.GlobalCancerStatistics2020什么是免疫治療？

目前治療癌癥的方法有許多，免疫治療就是其中之一。免疫治療主要是用免疫系統(tǒng)去對(duì)抗癌癥。干細(xì)胞移植免疫治療

手術(shù)激素治療靶向治療放療

化療癌癥治療

種類免疫治療的主要種類

單抗藥細(xì)胞因子CAR-T細(xì)胞治療…癌癥疫苗免疫檢查點(diǎn)抑制劑靶點(diǎn)FDA-approvedtherapeuticPD-1nivolumab(Opdivo?)pembrolizumab(Keytruda?)PD-L1atezolizumab(Tecentriq?)durvalumab(Imfinzi?)avelumab(Bavencio?)CTLA-4Ipilimumab(YERVOY?)靶點(diǎn)FDA-approvedtherapeuticCD20Rituximab(RITUXAN?)VEGFBevacizumab(Avastin?)HER2Trastuzumab(Herceptin?)義翹神州CAR-T細(xì)胞治療

靶點(diǎn)FDA-approvedtherapeuticIndicationFirstapproveddataCD19tisagenlecleucel(Kymriah?

)3-25歲患者復(fù)發(fā)性或難治性B細(xì)胞前體急性淋巴細(xì)胞白血?。怀赡昊颊邚?fù)發(fā)或難治性大B細(xì)胞淋巴瘤2017.8axicabtageneciloleucel(Yescarta?)成年患者復(fù)發(fā)性或難治性大B細(xì)胞淋巴瘤2017.10brexucabtageneautoleucel(Tecartus?)成年患者復(fù)發(fā)性或難治性套細(xì)胞淋巴瘤；成人復(fù)發(fā)性或難治性B細(xì)胞前體急性淋巴細(xì)胞白血病2020.7lisocabtagenemaraleucel(Breyanzi?)成年患者復(fù)發(fā)或難治性大B細(xì)胞淋巴瘤2021.2BCMA

idecabtagenevicleucel(Abecma?)成年患者復(fù)發(fā)或難治性多發(fā)性骨髓瘤2021.3ciltacabtageneautoleucel(CARVYKTITM)成年患者復(fù)發(fā)或難治性多發(fā)性骨髓瘤2022.2[1]“BridgingTCellstoTumors:CARsvsBsAb,”Immuno-OncologyResearchTrends,accessedMay30,2022,/home/2018/7/21/cancer-re-direction-and-t-cell-therapies-cars-vs-bites-or-tcr-and-immtacs.[2]NicholasTokarewetal.,“TeachinganOldDogNewTricks:Next-GenerationCARTCells,”BritishJournalofCancer120,no.1(January2019):26–37,/10.1038/s41416-018-0325-1.[3]VitaGolubovskayaandLijunWu,“DifferentSubsetsofTCells,Memory,EffectorFunctions,andCAR-TImmunotherapy,”Cancers8,no.3(March15,2016):36,/10.3390/cancers8030036.CAR細(xì)胞治療的簡(jiǎn)要發(fā)展時(shí)間線[1]FDA批準(zhǔn)的CAR-T

細(xì)胞治療[3]1-5代CAR的結(jié)構(gòu)[2]義翹神州CAR-T細(xì)胞治療和抗體藥研發(fā)中細(xì)胞因子的應(yīng)用及相關(guān)產(chǎn)品的介紹02義翹神州CAR-T細(xì)胞治療

[1]StacieIttershagenetal.,“Industry’sGiantLeapIntoCellularTherapy:CatalyzingChimericAntigenReceptorTCell(CAR-T)Immunotherapy,”CurrentHematologicMalignancyReports14,no.1(February1,2019):47–55,/10.1007/s11899-019-0498-6.

Tisagenlecleucel治療的主要步驟[1]采集患者血樣T細(xì)胞分離T細(xì)胞活化轉(zhuǎn)染CAR組件T細(xì)胞增殖和配制質(zhì)控和放行患者回輸(antiCD3antibody,antiCD28antibody)義翹神州CAR-T細(xì)胞治療

Tcell活化EffectofhumanCD28antibodyonstimulatingIL-2secretionofCD4Tcellsco-activatedwithCD3antibody.

EffectofHumanCD3antibodyontheproliferationandIFN-gammasecretionofCD4+Tcell.

antiCD3antibody

antiCD28antibody義翹神州CAR-T細(xì)胞治療

[1]StacieIttershagenetal.,“Industry’sGiantLeapIntoCellularTherapy:CatalyzingChimericAntigenReceptorTCell(CAR-T)Immunotherapy,”CurrentHematologicMalignancyReports14,no.1(February1,2019):47–55,/10.1007/s11899-019-0498-6.

Tisagenlecleucel治療的主要步驟[1]采集患者血樣T細(xì)胞分離T細(xì)胞活化轉(zhuǎn)染CAR組件T細(xì)胞增殖和配制質(zhì)控和放行患者回輸(Cytokines:IL2,IL21,IL7,IL15)(antiCD3antibody,antiCD28antibody)義翹神州[1]BarbaraSavoldoetal.,“CD28CostimulationImprovesExpansionandPersistenceofChimericAntigenReceptor–ModifiedTCellsinLymphomaPatients,”TheJournalofClinicalInvestigation121,no.5(May2,2011):1822–26,/10.1172/JCI46110.[2]HarjeetSinghetal.,“ManufactureofClinical-GradeCD19-SpecificTCellsStablyExpressingChimericAntigenReceptorUsingSleepingBeautySystemandArtificialAntigenPresentingCells,”P(pán)LoSONE8,no.5(May31,2013):e64138,/10.1371/journal.pone.0064138.[3]CorH.J.Lamersetal.,“TCellReceptor-EngineeredTCellstoTreatSolidTumors:TCellProcessingTowardOptimalTCellFitness,”HumanGeneTherapyMethods25,no.6(December1,2014):345–57,/10.1089/hgtb.2014.051.[4]YangXuetal.,“CloselyRelatedT-MemoryStemCellsCorrelatewithinVivoExpansionofCAR.CD19-TCellsandArePreservedbyIL-7andIL-15,”Blood123,no.24(June12,2014):3750–59,/10.1182/blood-2014-01-552174.GenerationandtransductionofactivatedTcells[1]收集到的PBMCs用OKT3antibody和rhIL-2(100U/ml)激活轉(zhuǎn)染轉(zhuǎn)染后的Tcelllines的

培養(yǎng)基中一周加入2次

rhIL-2(50–100U/ml)ManufactureofCAR+Tcells[2]按周期在過(guò)程中加入IL-21(30ng/mL)和50U/mLIL-2CAR-T細(xì)胞制備

Tcellactivation,transduction,andexpansionconditions[3]PMBC激活轉(zhuǎn)染增殖：加入IL-2(360?IU/ml)加入IL7+IL15at10+10?ng/ml加入IL15+IL21at10+10?ng/mlCelllinesandCAR-Tcellgeneration[4]PMBC激活轉(zhuǎn)染增殖：加入IL7+IL15at10+5?ng/ml義翹神州CAR-T細(xì)胞治療——

Tcell增殖

InducedInterferongammasecretionbyhumannaturalkillerlymphomaNK-92cells.

HumanIL21

HumanIL7Cellproliferationassayusinganti-CD3antibodyactivatedhumanPBMC.Thespecificactivityis>1x105units/μg,whichiscalibratedagainstthehumanIL-7referencestandard(NIBSCcode:90/530).≥95%asdeterminedbySEC-HPLC

HumanIL2CellproliferationassayusingCTLL2.ThespecificactivityofrecombinanthumanIL-2isapproximately20,000IU/μg.≥95%asdeterminedbySEC-HPLC

25℃加速48d凍融5次批間一致性MeasuredinacellproliferationassayusingMO7ehumanmegakaryocyticleukemiccells.

HumanIL15義翹神州質(zhì)量管理體系質(zhì)量保證管理質(zhì)量控制產(chǎn)品特點(diǎn)產(chǎn)品安全性符合《藥品生產(chǎn)質(zhì)量管理規(guī)范》體系供應(yīng)商管理體系和原材料管理體系符合《中華人民共和國(guó)藥典》2020年版三部通則：生物制品生產(chǎn)用原材料及輔料質(zhì)量控制真核&原核表達(dá)平臺(tái)細(xì)菌內(nèi)毒素＜5-10EU/mgISO9001:2015和ISO13485：2016質(zhì)量管理體系文件體系和記錄管理完全滿足GMP規(guī)范要求符合USP<1043>AncillaryMaterialsForCell,Gene,andTissue-engineeredProducts高純度宿主蛋白殘留檢測(cè)＜0.5μg/mg（500PPm）FDADMF備案中完整的批生產(chǎn)記錄、批檢驗(yàn)記錄、分裝記錄等生物活性檢測(cè)宿主DNA殘留檢測(cè)＜100pg/mg質(zhì)量文件/記錄經(jīng)QA審核和批準(zhǔn)支原體檢測(cè)建立和運(yùn)行設(shè)備維護(hù)和校準(zhǔn)程序無(wú)菌檢測(cè)專人負(fù)責(zé)管理人員培訓(xùn)體更高品質(zhì)細(xì)胞因子

義翹神州免疫治療的主要種類

單抗藥細(xì)胞因子CAR-T細(xì)胞治療…癌癥疫苗免疫檢查點(diǎn)抑制劑靶點(diǎn)FDA-approvedtherapeuticPD-1nivolumab(Opdivo?)pembrolizumab(Keytruda?)PD-L1atezolizumab(Tecentriq?)durvalumab(Imfinzi?)avelumab(Bavencio?)CTLA-4Ipilimumab(YERVOY?)靶點(diǎn)FDA-approvedtherapeuticCD20Rituximab(RITUXAN?)VEGFBevacizumab(Avastin?)HER2Trastuzumab(Herceptin?)義翹神州InduceInterferongammasecretionbyhumannaturalkillerlymphomaNK-92cells.IL18IL12CellproliferationassayusingMO7ehumanmegakaryocyticleukemiccells.免疫檢查點(diǎn)抑制劑有可能會(huì)在細(xì)胞上驗(yàn)證其活性。比如TIGIT會(huì)表達(dá)在具有抗腫瘤作用的T細(xì)胞和NK細(xì)胞上。TIGIT的表達(dá)會(huì)抑制NK細(xì)胞對(duì)于腫瘤細(xì)胞的殺傷。然而當(dāng)TIGIT被抑制后NK細(xì)胞和T細(xì)胞會(huì)重新恢復(fù)功能[1]。所以當(dāng)用NK細(xì)胞去研究TIGIT抑制劑功能的時(shí)候，細(xì)胞因子IL12[1]或者細(xì)胞因子的組合IL-12,IL-15andIL-18[2]會(huì)被加入其中用來(lái)刺激NK細(xì)胞。免疫檢查點(diǎn)抑制劑[1]FanqiaoMengetal.,“OverexpressionofTIGITinNKandTCellsContributestoTumorImmuneEscapeinMyelodysplasticSyndromes,”FrontiersinOncology10(2020),https://doi.org/10.3389/fonc.2020.01595.[2]XiaowanYinetal.,“ExpressionoftheInhibitoryReceptorTIGITIsUp-RegulatedSpecificallyonNKCellsWithCD226ActivatingReceptorFromHIV-InfectedIndividuals,”FrontiersinImmunology9(2018),/10.3389/fimmu.2018.02341.InduceIFN-gammasecretionbyKG?1humanacutemyelogenousleukemiacellsinthepresenceofTNF-alpha.IL15義翹神州免疫檢查點(diǎn)抑制劑[1]XinChenetal.,“AnFc-CompetentAnti-HumanTIGITBlockingAntibodyOciperlimab(BGB-A1217)ElicitsStrongImmuneResponsesandPotentAnti-TumorEfficacyinPre-ClinicalModels,”FrontiersinImmunology13(2022),/article/10.3389/fimmu.2022.828319.比如在免疫檢查點(diǎn)抑制劑(antiTIGITmab：BGB-A1217)Fc部分對(duì)于該免疫檢查點(diǎn)抑制劑抗腫瘤活性中的作用的研究中。作者設(shè)計(jì)了內(nèi)吞(trogocytosis)實(shí)驗(yàn)，這個(gè)實(shí)驗(yàn)中DC細(xì)胞被選用做其中一種acceptorcells.用來(lái)誘導(dǎo)DC細(xì)胞的培養(yǎng)基中加入了100ng/mLIL-4[1].CellproliferationassayusingTF-1humanerythroleukemiccells.ThespecificactivityofrecombinanthumanIL-4is>10,000IU/μgwhichiscalibratedagainstthehumanIL-4WHOInternationalStandard(NIBSCcode:88/656).IL4義翹神州≥95%asdeterminedbySEC-HPLC[1]Joe-MarcChauvinandHassaneM.Zarour,“TIGITinCancerImmunotherapy,”JournalforImmunoTherapyofCancer8,no.2(September1,2020):e000957,/10.1136/jitc-2020-000957.[2]MarianneWeulersseetal.,“Eomes-DependentLossoftheCo-ActivatingReceptorCD226RestrainsCD8+TCellAnti-TumorFunctionsandLimitstheEfficacyofCancerImmunotherapy,”Immunity53,no.4(October13,2020):824-839.e10,/10.1016/j.immuni.2020.09.006.TheTIGIT/CD226/CD96/CD112Raxis[1]免疫檢查點(diǎn)抑制劑在激活受體CD226的喪失會(huì)抑制CD8+T細(xì)胞功能和ICB功效的研究中，單核細(xì)胞衍生的成熟樹(shù)突細(xì)胞被用作CD226–CD8+T細(xì)胞增殖的刺激物[2]。Monocyte-DerivedMatureDendriticCells的制備從PBMCs中用C