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景的癌症治疗方法，具有越来越多令人鼓舞的临床结果。例如，blinatumomab，一种小鼠抗人CD3 x抗人CD19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，其剂量比常规抗体疗法（如利妥昔单抗（抗人CD20））低数千倍这些发现激发了该领域的极大兴趣和增长，特别关注的是开发新的方法来产生高产量和高纯度的双特异性抗体（例如Triomabs， BiTEs， Dock和Lock等）。尽管取得了不断的进步，但目前所有的双特异性抗体技术仍然需要大量的抗体工程和克隆，以产生一个单一的功能产品，这可能是耗时和具有挑战性的。利用scFv的技术也面临着功能性、溶解度、稳定性、亲和性和药代动力学方面的问题。在生产双特异性抗体的挑战之外，还有对其作用方式的不完全了解。例如，并不是所有的抗cd3抗体都能很好地激活t细胞。考虑到这些不确定性以及生产所需的高成本和时间，一种无需抗体工程和克隆就能快速生产双特异性抗体的方法将具有成本效益，显著提高吞吐量，并最终导致对潜在生物学机制的更深入了解，从而提高治疗效果。因此，我们建议开发一种简便的方法，从全长未修饰的IgG中高效地生产双特异性抗体，而无需进行抗体工程，克隆或修饰。高纯度的双特异性抗体将在一天内生产出来。使用完整IgG的几个好处是它们稳定，可以高产，提供高亲和力的二价结合，并有望保持fc效应功能，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）。该提案的具体目标是：(1)优化从全长未修饰的IgG制备双特异性抗体的程序；(2)体外评价抗cd20 x抗cd3双特异性抗体的功能和疗效；（3）

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.