Monfardini C, Kieber-Emmons T, VonFeldt J M, O'Malley B, Rosenbaum H, Godillot A P, Kaushansky K, Brown C B, Voet D, McCallus D E
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia.
J Biol Chem. 1995 Mar 24;270(12):6628-38. doi: 10.1074/jbc.270.12.6628.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody. Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDABL vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones isolated. One clone (23.2) which inhibited 126.213 binding to GM-CSF was sequenced revealing a murine kappa light chain of subgroup III. Comparison of the 23.2 sequence with the human GM-CSF sequence revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.
粒细胞-巨噬细胞集落刺激因子（GM-CSF）在许多免疫和炎症过程中都很重要。GM-CSF与属于最近描述的一个超基因家族的特定细胞受体结合。这些受体是药物设计的潜在靶点，而这种设计依赖于对配体-受体相互作用的分子理解。剖析关键分子间相互作用的一种方法是开发潜在重要的特定相互作用位点的类似物。在先前的研究中，单克隆抗体已被用于这些目的。在此，我们展示了重组抗体技术在开发与一种中和性抗GM-CSF单克隆抗体结合的GM-CSF上一个位点的类似物中的应用。在BALB/c小鼠中制备了具有高滴度中和活性的抗人GM-CSF多克隆抗血清。制备纯化的免疫球蛋白并用于免疫同基因小鼠。开发出了抗抗GM-CSF，其表现出针对GM-CSF依赖性细胞增殖的生物拮抗活性。从小鼠脾脏细胞中提取RNA，合成cDNA，并使用针对鼠κ轻链V区的特异性引物进行聚合酶链反应。将聚合酶链反应产物克隆到pDABL载体中并构建表达文库。用抗GM-CSF中和单克隆抗体126.213对其进行筛选，并分离出几个结合克隆。对一个抑制126.213与GM-CSF结合的克隆（23.2）进行测序，揭示其为III亚组的鼠κ轻链。将23.2序列与人类GM-CSF序列进行比较，发现特定互补决定区（CDR）与人类GM-CSF仅有微弱的序列相似性。结构分析显示23.2的CDR I、CDR II和FR3区域中的特定氨基酸与GM-CSF的B和C螺旋上的残基存在潜在的模拟。CDR I的一种合成肽类似物能与126.213结合，特异性拮抗GM-CSF与细胞的结合并阻断GM-CSF的生物活性。这些研究表明使用重组抗体文库作为相互作用位点类似物来源的可行性。
