Table_1_A strategy for high antibody expression with low anti-drug antibodies using AAV9 vectors.pdf

IntroductionUse of adeno-associated virus (AAV) vectors is complicated by host immune responses that can limit transgene expression. Recent clinical trials using AAV vectors to deliver HIV broadly neutralizing antibodies (bNAbs) by intramuscular administration resulted in poor expression with anti-drug antibodies (ADA) responses against the bNAb.
MethodsHere we compared the expression of, and ADA responses against, an anti-SIV antibody ITS01 when delivered by five different AAV capsids. We first evaluated ITS01 expression from AAV vectors three different 2A peptides. Rhesus macaques were selected for the study based on preexisiting neutralizing antibodies by evaluating serum samples in a neutralization assay against the five capsids used in the study. Macaques were intramuscularly administered AAV vectors at a 2.5x10^12 vg/kg over eight administration sites. ITS01 concentrations and anti-drug antibodies (ADA) were measured by ELISA and a neutralization assay was conducted to confirm ex vivo antibody potency.
ResultsWe observed that ITS01 expressed three-fold more efficiently in mice from AAV vectors in which heavy and light-chain genes were separated by a P2A ribosomal skipping peptide, compared with those bearing F2A or T2A peptides. We then measured the preexisting neutralizing antibody responses against three traditional AAV capsids in 360 rhesus macaques and observed that 8%, 16%, and 42% were seronegative for AAV1, AAV8, and AAV9, respectively. Finally, we compared ITS01 expression in seronegative macaques intramuscularly transduced with AAV1, AAV8, or AAV9, or with the synthetic capsids AAV-NP22 or AAV-KP1. We observed at 30 weeks after administration that AAV9- and AAV1-delivered vectors expressed the highest concentrations of ITS01 (224 µg/mL, n=5, and 216 µg/mL, n=3, respectively). The remaining groups expressed an average of 35-73 µg/mL. Notably, ADA responses against ITS01 were observed in six of the 19 animals. Lastly, we demonstrated that the expressed ITS01 retained its neutralizing activity with nearly the same potency of purified recombinant protein.
DiscussionOverall, these data suggest that the AAV9 capsid is a suitable choice for intramuscular expression of antibodies in nonhuman primates.

引言
腺相关病毒（adeno-associated virus, AAV）载体的应用受到宿主免疫反应的限制，此类免疫反应会抑制转基因表达。近期有临床试验采用AAV载体经肌内给药递送HIV广谱中和抗体（broadly neutralizing antibodies, bNAbs），结果显示转基因表达效果不佳，且受试者产生了针对该bNAb的抗药物抗体（anti-drug antibodies, ADA）应答。

方法
本研究针对5种不同的AAV衣壳（capsids）递送的抗SIV抗体ITS01的表达水平及其ADA应答进行了比较。首先，我们采用3种不同的2A肽（2A peptides）构建AAV载体，评估其介导的ITS01表达效果。本研究依据预存中和抗体水平筛选恒河猴：通过针对本研究所用5种衣壳的中和试验检测血清样本以完成筛选。随后以2.5×10^12 vg/kg的剂量，经8个给药位点对恒河猴实施肌内AAV载体接种。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测ITS01浓度与ADA水平，并通过中和试验验证体外（ex vivo）抗体效价。

结果
我们观察到，与携带F2A或T2A肽的载体相比，采用P2A核糖体跳过肽分隔重链与轻链基因的AAV载体，在小鼠中介导的ITS01表达效率提升了3倍。随后我们检测了360只恒河猴针对3种经典AAV衣壳的预存中和抗体应答，发现分别有8%、16%与42%的个体对AAV1、AAV8与AAV9呈血清阴性。最后，我们对血清阴性的恒河猴分别肌内转导AAV1、AAV8、AAV9，或合成衣壳AAV-NP22、AAV-KP1并检测ITS01的表达水平。给药后30周时，AAV9与AAV1载体介导的ITS01表达浓度最高，分别为224 μg/mL（n=5）与216 μg/mL（n=3）；其余各组的平均表达浓度为35~73 μg/mL。值得注意的是，19只受试动物中有6只产生了针对ITS01的ADA应答。最终我们证实，表达得到的ITS01保留了中和活性，其效价与纯化重组蛋白几乎一致。

讨论
综上，本研究数据表明，AAV9衣壳是非人灵长类动物肌内递送抗体的适宜选择。