A facile method for producing bispecific antibodies from full-length IgG

描述（由申请人提供）：双特异性抗体已成为一种有前途的癌症治疗方法，具有越来越多的令人鼓舞的临床结果。例如，Blinatumomab（一种鼠抗人CD 3 x抗人CD 19双特异性抗体）在前体B细胞急性淋巴细胞白血病（B-ALL）患者中产生了临床缓解，其剂量比常规抗体治疗（如利妥昔单抗（抗人CD 20））低数千倍。[1]这些发现激发了该领域的大量兴趣和增长，特别关注开发新的方法来产生双特异性抗体（例如Triomab、BiTE、Dock和Lock等）。产率和纯度高。尽管不断进步，但所有目前的双特异性抗体技术仍然需要大量的抗体工程改造和前期克隆以产生甚至单一的功能产物，这可能是耗时且具有挑战性的。利用scFv的技术还面临着对功能性、溶解度、稳定性、亲合力和药代动力学的关注。生产双特异性抗体的挑战之一是对其作用模式的不完全理解。例如，并非所有的抗CD 3抗体都同样有效地触发T细胞活化。考虑到这些不确定性以及生产所需的高成本和时间，允许快速生产双特异性抗体而不需要抗体工程化和克隆的方法将是成本有效的，显著增加通量，并最终导致对导致改善的治疗功效的潜在生物学机制的更深入理解。因此，我们提出开发一种简便的方法，用于从全长未修饰的IgG有效生产双特异性抗体，而不需要抗体工程化、克隆或修饰。双特异性抗体将在短短一天内以高纯度产生。使用完整IgG工作的几个益处是它们是稳定的，可以高产率产生，提供高亲合力二价结合，并且预期维持Fc效应子功能，包括抗体依赖性细胞介导的细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）。该提案的具体目的是（1）优化从全长未修饰IgG制备双特异性抗体的程序;（2）体外评价抗CD 20 x抗CD 3双特异性抗体的功能和功效;（3） 确定体内CD 20 x CD 3双特异性抗体的药代动力学和功效。

DESCRIPTION (provided by applicant): Bispecific antibodies have emerged as a promising cancer treatment, with a growing list of encouraging clinical results. For example, blinatumomab, a murine anti-human CD3 x anti-human CD19 bispecific antibody has produced clinical remission in precursor B cell acute lymphoblastic leukemia (B-ALL) patients at thousands times lower dosage than conventional antibody therapies, such as rituximab (anti-human CD20).[1] These findings have spurred a great deal of interest and growth in the field, with particular attention being focused on developing new methodologies to generate bispecific antibodies (e.g. Triomabs, BiTEs, Dock and Lock, etc.) in high yields and purity. Despite continual progress, all current bispecific antibody technologies still require a tremendous amount of antibody engineering and cloning upfront to generate even a single functional product, which can be time consuming and challenging. Technologies utilzing scFv's are also faced with concerns over functionality, solubility, stability, avidity, and pharmacokinetics. Addin to the challenges in producing bispecific antibodies is an incomplete understanding of their modes of action. For example, not all anti-CD3 antibodies work equally well to trigger T-cell activation. Given these uncertainties and the high cost and time required for production, a methodology that allows bispecific antibodies to be rapidly produced without the need for antibody engineering and cloning would be cost-effective, significantly increase throughput, and ultimately lead to a deeper understanding of the underlying biological mechanisms that lead to improved therapeutic efficacy. We therefore propose to develop a facile method for the efficient production of bispecific antibodies from full- length unmodified IgG, without the need for antibody engineering, cloning, or modifications. The bispecific antibodies will be produced with high purity in as little as one day. Several benefits of working with intact IgG are that they are stable, can be produced in high yield, offer high-avidity bivalent binding, and are expected to maintain Fc-effector functions, including antibody dependent cell-mediated cytoxicity (ADCC) and complement- dependent cytotoxicity (CDC). The specific aims for the proposal are (1) Optimize procedure for preparing bispecific antibodies from full-length, unmodified IgG; (2) Evaluate the functionality and efficacy of anti-CD20 x anti-CD3 bispecific antibodies in vitro; (3) Determine the pharmacokinetics and efficacy of CD20 x CD3 bispecific antibody in vivo.