Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, United States of America.
The Jesse Brown VA Medical Center, Chicago, Illinois, United States of America.
Sci Rep. 2017 Aug 31;7(1):10224. doi: 10.1038/s41598-017-10758-4.
L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar K for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar K for asparagine. We recently identified the low K guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low K chimeras we developed a suitable bacterial selection system (E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low K clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63-h and 65-h, share respectively 85.7% and 87.1% identity with the hASNase1 but have a K similar to gpASNase1. These clones possess 100-140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential.
天冬酰胺酶是一种用于治疗急性淋巴细胞白血病 (ALL) 的化疗药物。天冬酰胺酶临床疗效的主要前提条件是对天冬酰胺的微摩尔 K 值，以允许血液中天冬酰胺完全耗尽。由于目前批准的天冬酰胺酶来源于细菌，因此免疫原性是一个挑战，如果是人类酶则可以减轻这种挑战。然而，所有人类天冬酰胺酶对天冬酰胺的 K 值都在毫摩尔范围内。我们最近鉴定了对 K 值要求低的豚鼠天冬酰胺酶（gpASNase1）。由于 gpASNase1 和人类天冬酰胺酶 1（hASNase1）共享约 70%的氨基酸同一性，因此我们决定通过 DNA 改组生成与 hASNase1 的嵌合体来对 gpASNase1 进行人源化。为了鉴定低 K 值嵌合体，我们开发了一个合适的细菌选择系统（E. coli 菌株 BW5Δ）。用改组文库转化 BW5Δ，可鉴定出几个低 K 值克隆。为了进一步人源化这些克隆，将 gpASNase1 的 C 末端结构域替换为 hASNase1 的 C 末端结构域。在鉴定的两个克隆 63-h 和 65-h 中，它们与 hASNase1 的同源性分别为 85.7%和 87.1%，但 K 值与 gpASNase1 相似。与 hASNase1 相比，这两个克隆的催化效率分别提高了 100-140 倍。值得注意的是，我们还表明这些高度类似人类的天冬酰胺酶保持了它们体外杀死 ALL 的潜力。
