Bispecific Antibody Engineering for AML RIT

描述(由申请人提供)： 目前，尽管接受联合化疗和异基因干细胞移植(SCT)治疗，急性髓系白血病(AML)仍导致大多数患者死亡。放射性标记的抗CD45单抗可改善SCT中AML的预后，但毒性仍然很高，治愈率也不理想。这项研究提案的目的是使用一种新的方法来最大化AML的治愈率，该方法使用基因工程双特异性抗体的前靶向放射免疫治疗(PRIT)。在目标1中，我们将设计、表达和纯化与放射性金属配体共价且不可逆地结合的抗CD45 x抗配体双特异性抗体(scFv2-iFabs)。在目标2中，我们将评估抗CD45 x抗配体双特异性抗体(scFv2-iFabs)与CD45抗原和放射性金属配体螯合物的体外结合特性。在目标3中，我们将比较和对比使用新型分子工程抗CD45 x抗配体双特异性抗体(scFv2-iFabs)和我们目前的金标准方法PRIT使用链霉亲和素-生物素预靶向方法进行预靶向放射免疫治疗的药代动力学和生物分布。将在AML异种移植模型和AML播散性模型中进行比较。在目标4中，我们将比较和对比使用链霉亲和素-生物素方法的预靶向RIT和新型双特异性抗CD45 x抗配体双特异性抗体方法在异种移植和播散性AML模型中的治疗效果。在目的5中，我们将研究抗CD45双特异性抗体预靶向联合治疗在SCID小鼠人AML播散性模型中的毒性和疗效。我们假设，与传统的RIT和链霉亲和素-生物素PRIT相比，本提案中定义的新的双特异性“前靶向”RIT策略将放大向AML细胞传递的放射量，减少向肝、肺和其他正常器官传递的辐射，提高缓解和治愈率，延长生存期，并显著减轻毒性。我们预计这些临床前实验的结果将迅速转化为我们针对AML的临床RIT计划。公共卫生相关性：急性髓细胞白血病(AML)在13290名美国人中发生，尽管接受了化疗和干细胞移植治疗，但每年仍导致8820人死亡。在这个项目中，我们计划通过将放射性核素靶向AML上表达的CD45抗原来提高AML的治愈率，这种新方法被称为使用双特异性抗体进行“预靶向”放射免疫治疗。虽然这笔赠款专门用于AML，但正在开发的治疗方法也可以应用于其他表达CD45的恶性肿瘤，包括其他类型的白血病、骨髓发育不良和非霍奇金淋巴瘤，每年总共有超过12万美国人受到影响。我们预计，与目前可用的治疗方法相比，这种方法将治愈更多的患者，并导致更少的毒性反应。

DESCRIPTION (provided by applicant): Acute myelogenous leukemia (AML) currently kills the majority of afflicted patients despite treatment with combination chemotherapy and allogeneic stem cell transplantation (SCT). Radiolabeled anti-CD45 monoclonal antibodies (Ab) have been shown to improve outcomes for AML in the setting of SCT, but toxicity remains high and cure rates are suboptimal. The objective of this research proposal is to maximize the cure rate of AML using a novel approach employing pretargeted radioimmunotherapy (PRIT) with genetically engineered bispecific antibodies. In Aim 1, we will engineer, express, and purify anti-CD45 x anti-ligand bispecific Abs (scFv2-iFabs) that bind covalently and irreversibly to radiometal ligands. In Aim 2, we will assess the binding characteristics of anti-CD45 x anti-ligand bispecific Abs (scFv2-iFabs) for CD45 antigen and for radiometal ligand chelates in vitro. In Aim 3, we will compare and contrast the pharmacokinetics and biodistributions of pretargeted radioimmunotherapy using the novel molecularly engineered anti-CD45 x anti- ligand bispecific Abs (scFv2-iFabs) with our current gold standard method of PRIT using a streptavidin-biotin pretargeting method. Comparisons will be made in both an AML xenograft model and in a disseminated model of AML. In Aim 4, we will compare and contrast the therapeutic efficacy of pretargeted RIT using the streptavidin-biotin approach with the novel bispecific anti-CD45 x anti-ligand bispecific antibody approach in both xenograft and disseminated AML models. In Aim 5, we will investigate the toxicity and efficacy of combination therapy using anti-CD45 bispecific Ab pretargeting, with and without cytotoxic chemotherapy, in a disseminated model of human AML in SCID mice. We hypothesize that the novel bispecific "pretargeted" RIT strategy defined in this proposal will amplify the amount of radiation delivered to AML cells, decrease the radiation delivered to the liver, lungs, and other normal organs, improve remission and cure rates, prolong survival, and markedly attenuate toxicities compared to conventional RIT and to streptavidin-biotin PRIT. We anticipate rapid translation of the results of these preclinical experiments into our clinical RIT program for AML. PUBLIC HEALTH RELEVANCE: Acute myelogenous leukemia (AML) develops in 13,290 Americans and kills 8,820 of them each year despite treatment with chemotherapy and stem cell transplantation. In this project, we plan to improve the cure rate of AML by targeting radionuclides to the CD45 antigen expressed on AML using a new method called "pretargeted" radioimmunotherapy using bispecific antibodies. Although this grant is specifically focused on AML, the treatment being developed can also be applied to other CD45- expressing malignancies, including other types of leukemia, myelodysplasia, and non-Hodgkin's lymphoma, that affect a total of more than 120,000 Americans each year. We anticipate that this approach will cure more patients and cause fewer toxicities than currently available therapies.