Improved Tumor Targeting of Salmonella VNP20009 via Ice-llama Antibody Guidance

描述(申请人提供)摘要虽然沙门氏菌vnp 20009(Vnp)已在动物模型中显示出在肿瘤靶向方面的前景，但很明显，肿瘤的再生长是结果，因为细菌优先生长在坏死的核心，无法到达 重要的外围设备。在转移性黑色素瘤患者的人体试验中，即使靶向本身也很差，没有观察到肿瘤消退。我们的目的是通过赋予VNP抗体介导的肿瘤靶向的成功识别潜力来提高VNP的肿瘤靶向性和肿瘤消退能力。我们之前曾试图使用LPP-OmpA表面展示平台和难溶的单链抗体来做到这一点，但后来发现，这两种抗体的结合导致细菌生长停滞，并限制了工程VNP的治疗潜力。我们推测，通过冰核蛋白(INP)展示小而高度可溶性的抗CEA单域抗体的VNP将在不抑制细菌侵袭或复制的情况下增强对CEA阳性肿瘤的靶向性，因此将能够比亲本VNP更有效地逆转肿瘤。我们的具体目标是：1、在不妨碍复制的情况下展示抗CEA sdAb的基因工程VNP，在体外能够结合固定化CEA的CEA；2、证明基因工程VNP能够内化到MCF-7细胞中，并且在三维球状细胞培养模型中具有同质靶向性；3、在小鼠乳腺癌模型中证明基因工程VNP具有更好的体内肿瘤靶向性，阐明肿瘤内的分布和肿瘤完全消退的可能性。VNP的多功能功能很好，包括移动性和携带大型有效载荷的能力 通过显示单个生物发光细胞的能力和通过抗生素轻松消除细菌的能力来平衡。因此，VNP作为一种高度可控且通用的癌症治疗方法的潜力是巨大的，只要我们能够提高靶向和后退能力。

DESCRIPTION (provided by applicant): ABSTRACT While Salmonella VNP 20009 (VNP) has shown promise in tumor targeting in animal models it is becoming apparent that regrowth of tumors is the outcome since the bacteria preferentially grow in the necrotic core and fail to reach the vital periphery. In human trials of metastatic melanoma patients, even targeting itself was very poor and no tumor regression was observed. We aim to improve the tumor targeting and tumor regressive ability of VNP by conferring upon it the successful recognition potential of antibody mediated tumor targeting. We had previously attempted to do this using the lpp-OmpA surface display platform and poorly soluble scFv antibodies but have since found out that the combination caused bacterial growth arrest and restricted the therapeutic potential of engineered VNP. We hypothesize that VNP displaying small and highly soluble anti- CEA single domain antibodies via the ice nucleation protein (INP) will show enhanced targeting of CEA positive tumors without inhibiting bacterial invasion or replication and will therefore be able to regress tumors more effectively than parental VNP. Our specific aims are to; 1, Engineer VNP to display anti-CEA sdAb without impeding replication, yet capable of binding immobilized CEA in vitro; 2, Demonstrate engineered VNP is capable of being internalized in MCF-7 cells and is also capable of homogenous targeting in three- dimensional spheroidal cell culture models; 3, Demonstrate improved in vivo tumor targeting of engineered VNP in a mouse model of breast cancer, elucidating intra-tumoral distribution and potential for complete tumor regression. The multifunctional capabilities of VNP including mobility and ability to carry large payloads are well balanced by the ability to visualize single bioluminescent cells and facile elimination of the bacteria by antibiotics. As such, the potential for VNP as a highly controllable yet versatile cancer therapeutic is enormous so long as we can improve targeting and regressive capacity.