Almog O, Benhar I, Vasmatzis G, Tordova M, Lee B, Pastan I, Gilliland G L
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
Proteins. 1998 May 1;31(2):128-38.
A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (BldsFv) crystallizes in space group P6(1)22 with the unit cell parameters a = b = 80.1 A, and c = 138.1 A. The crystal structure of the BldsFv has been determined at 2.1-A resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 A and in bond angle of 2.74 degrees. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv is a deep depression 10-A wide and 17-A long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab.
已制备出一种重组Fv构建体，其为识别人类癌细胞上LewisY相关碳水化合物表位的B1单克隆抗体。该Fv由独立表达并作为包涵体分离的VH和VL结构域的多肽链组成。通过将等摩尔量的经氯化胍溶解的包涵体进行合并和重折叠来制备Fv。Fv通过在VL100和VH44残基之间构建的链间二硫键来稳定。该构建体具有与单链构建体相似的结合亲和力（Benhar和Pastan，《临床癌症研究》1:1023 - 1029，1995年）。B1二硫键稳定化Fv（BldsFv）在空间群P6(1)22中结晶，晶胞参数a = b = 80.1 Å，c = 138.1 Å。已使用分子置换技术在2.1 Å分辨率下测定了BldsFv的晶体结构。最终结构的晶体学R值为0.187，键长的均方根偏差为0.014 Å，键角的均方根偏差为2.74度。将BldsFv结构与其他免疫球蛋白片段Fv区域的已知结构进行比较，结果显示二级和三级结构密切相关。BldsFv的抗原结合位点是一个宽10 Å、长17 Å的深凹陷，凹陷的壁由来自互补决定区L1、L3、H1、H2和H3的残基组成，其中许多是酪氨酸。模型构建研究表明，LewisY四糖Fuc - Gal - Nag - Fuc能够以与早期生化研究中预测的表位一致的方式容纳在抗原结合位点中（Pastan、Lovelace、Gallo、Rutherford、Magnani和Willingham，《癌症研究》51:3781 - 3787，1991年）。因此，构建的二硫键似乎对Fv结构几乎没有造成（如果有也是极小的）扭曲，使其成为B1 Fab的有效替代物。
