Neville D M, Srinivasachar K, Stone R, Scharff J
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
J Biol Chem. 1989 Sep 5;264(25):14653-61.
We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).
我们在构建抗体 - 白喉毒素偶联物时使用了一类新型的酸可裂解蛋白交联试剂（Srinivaschar, K.，和Neville, D. M., Jr.（1989年）《生物化学》28卷，2501 - 2509页）。通过抑制携带CD5的细胞（Jurkat细胞）上的蛋白质合成来测定的抗CD5偶联物的效力与交联剂的水解速率相关。与不可裂解的传统偶联物相比，可裂解偶联物效力的最大增幅为50倍，并且通过与过量抗CD5竞争判断，这种增幅对CD5摄取途径具有特异性。由白喉毒素和抗高分子量黑色素瘤相关抗原（HMW - MAA）制成的偶联物的效力通过可裂解交联剂提高了3 - 10倍。然而，转铁蛋白或抗CD3白喉毒素偶联物的效力仅略有提高（2 - 3倍）。先前显示与野生型相比结合亲和力表达不到1/100的突变型白喉毒素CRM103和CRM9被替换到这些偶联物中，作为可能的细胞内毒素 - 受体相互作用的探针进行研究。两种突变体对Jurkat靶细胞具有同等毒性，其效力为野生型的1/700。对于转铁蛋白和抗CD3介导的摄取，CRM9不可裂解偶联物与野生型偶联物具有同等效力，但对于抗CD5介导的摄取则不然，其毒性比野生型类似物降低了60倍。可裂解交联剂增强了抗CD5 - CRM103和抗CD5 - CRM9偶联物的毒性，但效力仅为类似野生型可裂解偶联物的1/10。这些数据与一个模型一致，在该模型中，可裂解交联剂增强抗CD5和抗高分子量黑色素瘤相关抗原偶联物的毒性是由于细胞内毒素 - 毒素受体相互作用增强，最终导致毒素向细胞质区室的转运增加。相比之下，这些数据表明抗CD3和转铁蛋白摄取系统不需要这种相互作用,这与之前的研究工作一致（Johnson, V.G., Wilson, D., Greenfield, L., 和Youle, R. J.（1988年）《生物化学杂志》263卷，1295 - 1300页）。
