In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin
In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin
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DOI：
10.1074/jbc.m409783200
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发表时间：
2005-01-07
影响因子：
4.8
通讯作者：
Pastan, I
中科院分区：
文献类型：
--
作者：
Ho, M;Kreitman, RJ;Pastan, I
重组免疫毒素BL22含有抗CD22抗体的Fv部分，在大多数耐药毛细胞白血病患者中产生完全缓解，但在CD22低表达的白血病患者中活性较低。互补决定区（CDR）诱变用于增加抗体亲和力，但很难成功执行。我们之前表明，通过在称为热点的特定DNA残基上定向突变，可以获得亲和力增强的抗体和活性增强的免疫毒素。由于热点既可以由体细胞突变产生，也可以存在于种系中，因此我们研究了哪种类型的热点更适合增加抗体亲和力。最初，针对抗体轻链CDR1内种系热点(Ser（30)-Asn(31)）的第二代抗体噬菌体展示文库发生突变。用精氨酸替代丝氨酸30或天门汀31产生的免疫毒素突变体的亲和力（0.8 nM）比BL22 （5.8 nM）增加了7倍，比第一代突变体HA22 （2.3 nM）增加了3倍。更重要的是，在包括每个细胞仅含有5500个CD22位点的WSU-CLL在内的各种B淋巴瘤细胞系中，观察到其活性比BL22增加10倍，比HA22增加2-3倍。为了进行比较，生成了两个靶向重链CDR1和CDR3非种系热点的噬菌体展示文库，但没有产生亲和性增加的Fv。我们的结果表明，种系热点而非非种系热点对体外抗体亲和成熟有效。
Recombinant immunotoxin BL22, containing the Fv portion of an anti-CD22 antibody, produced complete remissions in most patients with drug-resistant hairy cell leukemia but had less activity in leukemias with low CD22 expression. Complementarity-determining region (CDR) mutagenesis is used to increase antibody affinity but can be difficult to perform successfully. We previously showed that antibodies with increased affinity and immunotoxins with increased activity could be obtained by directing mutations at specific DNA residues called hot spots. Because hot spots can arise either by somatic mutation or be present in the germline, we examined which type of hot spot is preferred for increasing antibody affinity. Initially, a second generation antibody phage-display library targeting a germline hot spot (Ser(30)-Asn(31)) within CDR1 of the antibody light chain was mutated. Substitution of serine 30 or asparagine 31 with arginine produced mutant immunotoxins with an affinity (0.8 nM) increased 7-fold over BL22 (5.8 nM) and 3-fold over the first generation mutant HA22 (2.3 nM). More importantly, a 10-fold increase in activity over BL22 and a 2-3-fold increase over HA22 were observed in various B lymphoma cell lines including WSU-CLL that contains only 5500 CD22 sites per cell. For comparison, two phage-display libraries targeting non-germline hot spots in heavy chain CDR1 and CDR3 were generated but did not produce Fv with increased affinity. Our results demonstrate that germline hot spots but not non-germline hot spots are effective for in vitro antibody affinity maturation.