靶向突变型p53：新型抗p53R175H单克隆抗体作为诊断工具的评价-动脉网

Targeting mutant p53: Evaluation of novel anti-p53R175H monoclonal antibodies as diagnostic tools

Nature 等信源发布 2025-01-06 23:50

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Abstract

摘要

About 50% of all cancers carry a mutation in p53 that impairs its tumor suppressor function. The p53 missense mutation p53

大约50%的癌症携带p53突变，从而损害其抑癌功能。p53错义突变p53

R175H

(p53

（p53

R172H

in mice) is a hotspot mutation in various cancer types. Therefore, monoclonal antibodies selectively targeting clinically relevant mutations like p53

在小鼠中）是各种癌症类型的热点突变。因此，单克隆抗体选择性靶向p53等临床相关突变

R175H

could prove immensely value. We aimed to evaluate the in vitro and in vivo binding properties of two novel anti-p53

可以证明它具有巨大的价值。我们旨在评估两种新型抗p53的体外和体内结合特性

R175H

monoclonal antibodies and to assess their performance as agents for molecular imaging. In vitro,

单克隆抗体，并评估其作为分子成像剂的性能。在体外，

125

I-4H5 and

I-4H5和

125

I-7B9 demonstrated long shelf life and antigen-specific binding. Our in vivo study design allowed head-to-head comparison of the antibodies in a double tumor model using repeated SPECT/CT imaging, followed by biodistribution and autoradiography. Both tracers performed similarly, with marginally faster blood clearance for .

。我们的体内研究设计允许使用重复的SPECT/CT成像，然后进行生物分布和放射自显影，在双肿瘤模型中对抗体进行头对头比较。两种示踪剂的表现相似，血液清除率略快。

125

I-7B9. Repeated molecular imaging demonstrated suitable imaging characteristics for both antibodies, with the best contrast images occurring at 48 h post-injection. Significantly higher uptake was detected in the mut-p53-expressing tumors, confirmed by ex vivo autoradiography. We conclude that molecular imaging with an anti-p53.

I-7B9。重复的分子成像显示了两种抗体的合适成像特征，最佳对比度图像发生在注射后48小时。通过离体放射自显影证实，在表达mut-p53的肿瘤中检测到明显更高的摄取。我们得出结论，使用抗p53进行分子成像。

R175H

tracer could be a promising approach for cancer diagnostics and could be further applied for patient stratification and treatment response monitoring of mutant p53-targeted therapeutics.

示踪剂可能是一种有前途的癌症诊断方法，并可进一步应用于突变型p53靶向治疗剂的患者分层和治疗反应监测。

Introduction

简介

p53 is the most mutated gene across all cancer types, and p53 mutations are heavily involved in carcinogenesis and the response to therapy

p53是所有癌症类型中突变最多的基因，p53突变与癌变和治疗反应密切相关

. About 50% of all cancers carry a mutation in p53 that impairs its tumor suppressor function, making p53 a rare, almost universal, cancer marker in an otherwise very heterogeneous landscape of oncogenes and tumor suppressor genes. Among the p53 mutations, several hotspot mutations occur frequently

。大约50%的癌症携带p53突变，该突变会损害其抑癌功能，使p53在癌基因和抑癌基因的其他非常异质的情况下成为罕见的，几乎普遍的癌症标志物。在p53突变中，几个热点突变频繁发生

. These include R175(4.8%), G245(3.12%), R248 (6.79%), R249 (2.59%), R273 (6.55%) and R282 (2.59%)

。其中包括R175（4.8%），G245（3.12%），R248（6.79%），R249（2.59%），R273（6.55%）和R282（2.59%）

. The location of these mutations within the DNA-binding domain suggests their importance in disrupting the normal function of the p53 protein. There are generally two types of p53 mutants based on their effects on the protein structure and function. The first type, known as contact mutants, includes mutations like R248W and R273H.

这些突变在DNA结合域中的位置表明它们在破坏p53蛋白正常功能中的重要性。根据它们对蛋白质结构和功能的影响，通常有两种类型的p53突变体。第一种类型称为接触突变体，包括R248W和R273H等突变。

. These mutations specifically affect the ability of the mutant p53 protein to bind to DNA. As a result, mutant p53 loses its ability to carry out its tumor-suppressor functions, which include regulating cell growth, inducing cell death, and preventing the formation of cancerous cells. The second type of p53 mutants are conformational mutants, such as R175H and G245S.

这些突变特别影响突变p53蛋白与DNA结合的能力。结果，突变型p53失去了执行其肿瘤抑制功能的能力，包括调节细胞生长，诱导细胞死亡和防止癌细胞形成。第二类p53突变体是构象突变体，例如R175H和G245S。

. These mutations alter the three-dimensional structure of the DNA-binding domain, rendering it incapable of properly interacting with DNA. Overall, both conformational and contact mutants of p53 result in defective DNA binding and the loss of p53’s tumor-suppressor functions. This disruption in p53 function allows cancer cells to proliferate uncontrollably and evade normal cellular mechanisms that would normally prevent tumor formation..

这些突变改变了DNA结合域的三维结构，使其无法与DNA正确相互作用。总体而言，p53的构象和接触突变体均导致DNA结合缺陷和p53抑癌功能丧失。p53功能的这种破坏使癌细胞无法控制地增殖，并逃避通常会阻止肿瘤形成的正常细胞机制。。

Furthermore, p53 mutations are involved in about 70% of Li-Fraumeni syndrome (LFS) patients, an inherited genetic disorder characterized by early onset of various types of sarcomas and carcinomas

此外，约70%的Li-Fraumeni综合征（LFS）患者涉及p53突变，这是一种遗传性遗传疾病，其特征是各种类型的肉瘤和癌症的早期发作

. The p53 missense mutation (p53

p53错义突变（p53

R175H

) is one of the most common hotspot mutations of p53, and is also encountered in the germline of LFS patients. Gaining a deeper understanding of the entire spectrum of p53 mutations and their role in various cancer types remains a promising but difficult prospect, and understanding the biology of mutant p53 is fundamental to unveiling its role in carcinogenesis and response to therapy.

)是p53最常见的热点突变之一，在LFS患者的种系中也会遇到。深入了解p53突变的整个范围及其在各种癌症类型中的作用仍然是一个有希望但困难的前景，了解突变型p53的生物学对于揭示其在致癌作用和治疗反应中的作用至关重要。

Such understanding can also open the possibility of using specific single-amino-acid p53 variants as biomarkers for disease diagnostics, treatment follow-up, and targets for novel therapeutic solutions. One limiting factor has been the capacity for raising antibodies specific enough for mutant p53, since antibodies against single-amino-acid changes may be highly cross-reactive with wildtype p53..

这种理解还可以为使用特定的单氨基酸p53变体作为疾病诊断，治疗随访和新型治疗方案靶点的生物标志物提供可能性。一个限制因素是产生对突变型p53足够特异的抗体的能力，因为针对单个氨基酸变化的抗体可能与野生型p53高度交叉反应。。

However, recent advances in monoclonal antibody development have led to the generation of novel antibodies specific to p53 missense mutations, engineered to possess high binding affinity and selectivity for specific mutant p53 proteins

然而，单克隆抗体开发的最新进展导致产生了针对p53错义突变的新型抗体，这些抗体被设计为对特定突变p53蛋白具有高结合亲和力和选择性

. The high specificity of monoclonal antibodies (mAbs) allows for the targeted binding to specific mutant p53 proteins and can help distinguish cancer cells from normal cells. These monoclonal antibodies have the potential to be used as vector molecules for therapeutic purposes but also molecular imaging techniques.

单克隆抗体（mAb）的高特异性允许靶向结合特定的突变p53蛋白，并有助于区分癌细胞和正常细胞。这些单克隆抗体有可能用作治疗目的的载体分子，但也可以用作分子成像技术。

By conjugating these antibodies with specific imaging probes such as radionuclides, they can be used as molecular imaging agents for the detection and visualization of mutant p53 in vivo by positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Mutant p53 has been shown to have a half-life of several hours, compared to wild-type p53 of approximately 20 min.

。与野生型p53约20分钟相比，突变型p53的半衰期为数小时。

. As a result, these mutant proteins can accumulate in large quantities specifically in tumor cells, making them an even more attractive target for molecular imaging

因此，这些突变蛋白可以在肿瘤细胞中大量积累，使其成为分子成像的更有吸引力的靶标

. Targeting intracellular proteins with antibodies does however pose unique challenges since antibodies typically have limited access to the intracellular environment. Nonetheless, intracellular proteins can act as targets for therapy and imaging using specific antibodies if the antigen becomes accessible through cell permeability events occurring in tumor tissues.

然而，用抗体靶向细胞内蛋白质确实带来了独特的挑战，因为抗体通常对细胞内环境的访问有限。尽管如此，如果通过肿瘤组织中发生的细胞通透性事件可以获得抗原，则细胞内蛋白质可以作为使用特异性抗体进行治疗和成像的靶标。

Furthermore, recent advancements in antibody engineering and delivery methods have opened up new possibilities for intracellular protein targeting, including protein-transduction domain fusion and nanoparticle-driven delivery.

此外，抗体工程和递送方法的最新进展为细胞内蛋白质靶向开辟了新的可能性，包括蛋白质转导结构域融合和纳米粒子驱动的递送。

Consequently, if successful, molecular imaging offers the advantage of non-invasively visualizing and quantifying mutant p53 in cancer patients, allowing for early detection and precise characterization of cancerous lesions, guiding treatment decisions, and monitoring treatment response.

因此，如果成功，分子成像将提供无创可视化和定量癌症患者突变型p53的优势，从而可以早期发现和精确表征癌症病变，指导治疗决策并监测治疗反应。

The present study aimed to for the first time to radiolabel two new and highly selective mAbs raised against p53

本研究旨在首次放射性标记两种针对p53的新型高选择性单克隆抗体

R175H

(p53

（p53

R172H

in mice) and to evaluate the in vitro and in vivo binding properties. The overall goal was to demonstrate the potential of these antibodies for cancer diagnostics, patient stratification, and treatment response monitoring in cases involving mutant p53.

在小鼠中），并评估体外和体内结合特性。总体目标是证明这些抗体在涉及突变型p53的病例中用于癌症诊断，患者分层和治疗反应监测的潜力。

Results

结果

Ab selection and in vitro characterization of the mAbs 4H5 and 7B9

单克隆抗体4H5和7B9的Ab选择和体外表征

Initial antibody generation and characterization of the two R175H-specific antibody clones (7B9 and 4H5) has been previously described

先前已经描述了两个R175H特异性抗体克隆（7B9和4H5）的初始抗体产生和表征

. In brief, immunization with a fusion protein bearing multiple repeats of the p53 sequence focused around the R175H mutation elicited a mutant-specific antibody response, resulting in the generation of several hybridoma clones. The monoclonal antibodies (mAbs) only recognized the R175H mutant protein and not the wild-type p53 or other mutant p53 proteins with different amino acid substitutions.

简而言之，用带有围绕R175H突变的p53序列多个重复序列的融合蛋白进行免疫，引发了突变特异性抗体反应，从而产生了几个杂交瘤克隆。单克隆抗体（mAb）仅识别R175H突变蛋白，而不识别野生型p53或其他具有不同氨基酸取代的突变p53蛋白。

Immunoblotting using the 4H5, 7B9 antibody confirmed the specificity for the R175H mutant without any cross-reactivity to wild-type p53 (Fig. .

使用4H5、7B9抗体进行的免疫印迹证实了R175H突变体的特异性，而对野生型p53没有任何交叉反应（图）。

a, Supplementary Fig. 2a). Similar results were obtained by immunofluorescence staining using both the 7B9 and 4H5 clones (Fig.

a、补充图2a）。使用7B9和4H5克隆通过免疫荧光染色获得了类似的结果（图）。

b). A quantitative comparison of the antibodies is provided in Supplementary Fig. 2b, showing the percentage of positively stained cells. Although no significant difference was detected between the performance of 4H5 and 7B9, the D01 antibody demonstrated higher staining levels. This discrepancy is likely due to differences in affinity of the mAbs or variability in transfection efficiency, as the p53-null cells were transfected with the p53-R175H mutant, potentially leading to inconsistent mutant p53 expression..

b）。补充图2b提供了抗体的定量比较，显示了阳性染色细胞的百分比。尽管在4H5和7B9的性能之间未检测到显着差异，但D01抗体显示出更高的染色水平。这种差异可能是由于单克隆抗体亲和力的差异或转染效率的差异所致，因为p53无效细胞被p53-R175H突变体转染，可能导致突变体p53表达不一致。。

Fig. 1

图1

Characterization of anti-p53

抗p53的表征

R175H

antibodies, by immunoblotting, immunofluorescent staining, and immunohistochemistry staining. (

抗体，通过免疫印迹，免疫荧光染色和免疫组织化学染色。（笑声）(

一

) H1299 cells were transfected with indicated plasmids and equal amounts of lysates were used for immunoblotting with the 7B9, 4H5, DO1 (anti-human p53), and GAPDH antibodies. (

)用指定的质粒转染H1299细胞，并将等量的裂解物用于7B9、4H5、DO1（抗人p53）和GAPDH抗体的免疫印迹。（笑声）(

b类

) Immunofluorescent detection of p53

)p53的免疫荧光检测

R175H

protein in p53

p53中的蛋白质

R175H

transfected H1299 cells using 4H5, 7B9, 9D9 (anti-GST) and D01 mAbs. After p53

使用4H5、7B9、9D9（抗GST）和D01 mAb转染H1299细胞。p53之后

R175H

transfection, cells were fixed in 4% paraformaldehyde and then permeabilized for mAb immunological fluorescent staining. (

。（笑声）(

c级

) Detection of endogenous mouse p53

)内源性小鼠p53的检测

R172H

protein in p53

p53中的蛋白质

R172H/R172H

mouse tumor tissue section using Digoxigenin conjugated 4H5, 7B9, anti-GST control 9D9, or IC12 (against mouse and human p53) mAbs. Both 4H5 and 7B9 mAb specifically recognized and stained cell nuclei expressing endogenous mouse p53

使用洋地黄毒苷缀合的4H5、7B9，抗GST对照9D9或IC12（针对小鼠和人p53）mAb的小鼠肿瘤组织切片。4H5和7B9单克隆抗体均能特异性识别和染色表达内源性小鼠p53的细胞核

R172H

protein in tumor derived from p53

p53衍生的肿瘤中的蛋白质

R172H/R172H

mutant mice. Representative images are shown.

突变小鼠。显示了代表性的图像。

Full size image

全尺寸图像

Immunohistochemistry results further confirmed the specificity of both 4H5 and 7B9 clones, upon conjugation with DIG, which eliminates non-specific-binding caused by secondary anti-mouse Ig, on mouse p53

免疫组织化学结果进一步证实了4H5和7B9克隆与DIG结合后的特异性，DIG消除了继发性抗小鼠Ig对小鼠p53的非特异性结合

R172H

tumors (Fig.

肿瘤（图）。

c). Thus, both the 7B9 and 4H5 clones were pursued for subsequent radio-labelling studies.

c）。因此，7B9和4H5克隆都被用于随后的放射性标记研究。

In vitro characterization of radioiodinated 4H5 and 7B9

放射性碘标记4H5和7B9的体外表征

Radioiodination of the mAbs 4H5 and 7B9 with Iodine-125 (

用碘-125对单克隆抗体4H5和7B9进行放射性碘标记(

125

I) using the Iodogen method resulted in high yield and purity (> 98%). In vitro characterizations of

一）使用碘原法可获得高产率和纯度（>98%）

125

I-4H5 and

I-4H5和

125

I-7B9 demonstrated stability and long shelf life of the radio-conjugates (Fig.

I-7B9证明了放射性共轭物的稳定性和较长的保质期（图）。

a). > 90% of both conjugates were still intact after 48 h at room temperature in PBS. ELISA assays with

a）。>室温下在PBS中48小时后，两种缀合物的90%仍然完整

125

I-4H5 and

I-4H5和

125

I-7B9 against the p53

I-7B9对抗p53

R175H

protein, other different p53 mutations, as well as unrelated control proteins proved specificity for the R175H mutation for both antibodies as well as intact antigen-specific binding after the radiolabeling procedure (Fig.

。

b).

b）。

Fig. 2

图2

Stability and ELISA measurements of radioiodinated anti-p53R175H mAbs 4H5 and 7B9. (

放射性碘标记的抗p53R175H mAb 4H5和7B9的稳定性和ELISA测量。（笑声）(

一

) Stability measurement of

)稳定性测量

125

I-4H5 and

I-4H5和

125

I-7B9 after 24 and 48 h post labelling (in PBS at room temperature). (

标记后24和48小时后的I-7B9（在室温下在PBS中）。（笑声）(

b类

) In vitro binding properties of the radioiodinated anti-p53R175H mAbs 4H5 and 7B9 (10 nM) against mutated p53 forms and unrelated negative control proteins.

)放射性碘标记的抗p53R175H mAb 4H5和7B9（10 nM）对突变的p53形式和不相关的阴性对照蛋白的体外结合特性。

125

I-4H5 and

I-4H5和

125

I-7B9 bound specifically to the mut p53

I-7B9与mut p53特异性结合

R175H

protein. One-way ANOVA and Tukey’s multiple comparison posttest. **

蛋白质。单因素方差分析和Tukey的多重比较后测**

≤ 0.01 and **** for

≤ 0.0001. Data are presented as the means ± standard deviation (SD),

≤0.0001。数据表示为平均值±标准偏差（SD），

= 3.

Full size image

全尺寸图像

In vivo characterization of the mAbs 4H5 and 7B9

单克隆抗体4H5和7B9的体内表征

In vivo, a cross-reactivity analysis of the radiolabeled mAb

在体内，放射性标记的单克隆抗体的交叉反应性分析

125

I-4H5 and

I-4H5和

125

I-7B9 (50 µg, 200 kBq) was first performed in a negative control mouse model that does not express any p53 protein with mouse melanoma B16 p53 knockout tumors (B16-KO) (Fig.

I-7B9（50μg，200 kBq）首先在阴性对照小鼠模型中进行，该模型不表达任何p53蛋白与小鼠黑色素瘤B16 p53敲除肿瘤（B16-KO）（图）。

). Biodistribution data collected 48 h and 72 h after tracer injection confirmed no non-specific accumulation of the tracers in any organ (Fig.

)。示踪剂注射后48小时和72小时收集的生物分布数据证实示踪剂在任何器官中都没有非特异性积累（图）。

a).

a）。

125

I-4H5 and

I-4H5和

125

I-7B9 followed a similar distribution pattern. Furthermore, uptake of both radioconjugates in the p53 B16-KO tumors was comparable to tracer uptake in the other normal organs, except for blood (tumor-to-organ ratios of 0.44–0.49), probably due to the relatively long circulation time of full-size antibodies in the body (Fig. .

I-7B9遵循类似的分布模式。。

b).

b）。

Fig. 3

图3

Biodistribution analysis. In vivo cross-reactivity analysis of the iodinated mAbs 4H5 and 7B9 in a mouse model with B16-KO tumors (8 mice/group). (

生物分布分析。在具有B16-KO肿瘤的小鼠模型（8只小鼠/组）中碘化mAb 4H5和7B9的体内交叉反应性分析。（笑声）(

一

) % injected dose/gram tissue (%ID/g) and (

)%注射剂量/克组织（%ID/g）和(

b类

) tumor-to-organ ratios of

)肿瘤与器官的比例

125

I-4H5 and

I-4H5和

125

I-7B9 48 h and 72 h p.i. Data are presented as means ± standard deviation (SD).

I-7B9 48小时和72小时p.I.数据表示为平均值±标准偏差（SD）。

Full size image

全尺寸图像

Next, the biodistribution of

接下来，生物分布

125

I-4H5 and

I-4H5和

125

I-7B9 (50 µg, 200 kBq) was evaluated in a dual tumor mouse model, with one mouse primary tumor (Mut-T-B6) expressing the mutp53 protein (p53

在双肿瘤小鼠模型中评估了I-7B9（50µg，200 kBq），其中一个小鼠原发肿瘤（Mut-T-B6）表达mutp53蛋白（p53

R172H/R172H

) on the right posterior flank and one p53 lacking mouse primary tumor B16-KO on the left (Fig.

)在右后侧和左侧一个缺乏p53的小鼠原发性肿瘤B16-KO（图）。

Fig. 4

图4

Biodistribution analysis. In vivo specificity of the iodinated mAbs 4H5 and 7B9 in a mouse model with Mut-T-B6 and B16-KO tumors. (

生物分布分析。。（笑声）(

一

) % injected dose/gram tissue (%ID/g) 48 h (8 mice/group) and (

)%注射剂量/克组织（%ID/g）48小时（8只小鼠/组）和(

b类

) 72 h (3 mice/group) p.i. of

)72小时（3只小鼠/组）

125

I-4H5 and

I-4H5和

125

I-7B9 for normal organs and tumors. (

I-7B9用于正常器官和肿瘤。（笑声）(

c级

) % injected dose/gram tissue for positive and negative tumors 48 h and 72 p.i. of

)阳性和阴性肿瘤48小时和72 p.i.的%注射剂量/克组织

125

I-4H5 and

I-4H5和

125

I-7B9. (

I-7B9。（笑声）(

) Tumor/blood ratios for positive and negative tumors 48 h and 72 h p.i. of

)阳性和阴性肿瘤48小时和72小时的肿瘤/血液比率

125

I-4H5 and

I-4H5和

125

I-7B9. One-way ANOVA and Tukey’s multiple comparison posttest. *

I-7B9。单因素方差分析和Tukey的多重比较后测*

≤ 0.05 and **

≤0.05和**

≤ 0.01. Data are presented as means ± SD.

≤0.01。数据表示为平均值±SD。

Full size image

全尺寸图像

Similar to the previous cross-reactivity in vivo biodistribution experiment

类似于先前的体内交叉反应生物分布实验

125

I-4H5 and

I-4H5和

125

I-7B9 showed comparable results (Fig.

I-7B9显示出可比的结果（图）。

), high activity in blood and blood-rich organs with slightly faster blood clearance for

)，血液和富含血液的器官中的高活性，血液清除速度略快

125

I-7B9 compared to

I-7B9与

125

I-4H5 at the 72 h time point (from 4.8% ID/g at 48 h to 2.7% ID/g and 1.7% ID/g for

125

I-4H5 and

I-4H5和

125

I-7B9, respectively). There was a significant difference in size between KO and Mut-T-B6 tumors (Supplementary Fig. 3a and b). However both

分别为I-7B9）。KO和Mut-T-B6肿瘤之间的大小存在显着差异（补充图3a和b）。然而，两者

125

I-4H5 and

I-4H5和

125

I-7B9 uptake (CPM and %ID/g) was significantly higher in the p53

p53中的I-7B9摄取（CPM和ID/g百分比）显着更高

R172H

positive tumors compared to KO tumors 48 h p.i. This difference became less pronounced 72 h p.i. (Fig.

与KO肿瘤相比，阳性肿瘤在48小时p.i.这种差异在72小时p.i.变得不那么明显。（图）。

a-c, Supplementary Fig. 3c and d). As a consequence of the remaining activity in the blood, tumor-to-blood ratios were < 1, however a significant difference between positive and negative tumor was observed for both investigated endpoints (Fig.

a-c，补充图3c和d）。由于血液中剩余的活性，肿瘤与血液的比率小于1，然而，对于两个研究终点，观察到阳性和阴性肿瘤之间的显着差异（图）。

d).

d）。

We further tested the CF750-conjugated 4H5 mAb in p53

我们进一步测试了CF750结合的4H5单克隆抗体在p53中的表达

R172H/R172H

mutant B6 mice bearing spontaneous tumors. Mice were sacrificed 3 days post-injection for organ and tumor imaging using the IVIS Spectrum. As shown in Supplementary Fig. 4, we observed a strong signal in the tumors, along with some hotspots in normal tissues (carrying the R172H mutation), particularly in blood-rich organs like the kidneys and lungs.

携带自发性肿瘤的突变B6小鼠。注射后3天处死小鼠，使用IVIS光谱进行器官和肿瘤成像。如补充图4所示，我们在肿瘤中观察到了强烈的信号，以及正常组织（携带R172H突变）中的一些热点，特别是在肾脏和肺部等富含血液的器官中。

This could reflect either antibody uptake in these normal organs or elevated levels of circulating mAbs still present in the blood..

这可能反映了这些正常器官中的抗体摄取或血液中仍然存在的循环单克隆抗体水平升高。。

Molecular imaging of

分子成像

125

I-4H5 and

I-4H5和

125

I-7B9 using repeated SPECT/CT scans

I-7B9使用重复SPECT/CT扫描

To study the in vivo imaging performance of

研究体内成像性能

125

I-4H5 and

I-4H5和

125

I-7B9 we used the same dual tumor model with p53 knockout and p53

I-7B9我们使用了相同的双重肿瘤模型，其中p53基因敲除和p53

R172H

expressing mouse primary tumors, derived from p53 null and p53

表达源自p53-null和p53的小鼠原发性肿瘤

R172H/R172H

mice, as described in the biodistribution study.

小鼠，如生物分布研究中所述。

Repeated molecular imaging of both tracers in 2 subjects scanned simultaneously with a SPECT/CT camera at 24 h, 48 h, and 6 d post-injection allowed head-to-head comparison of both iodinated antibodies in the same subjects over time. Both

在注射后24小时，48小时和6天用SPECT/CT相机同时扫描的2名受试者中两种示踪剂的重复分子成像允许随着时间的推移对同一受试者中的两种碘化抗体进行头对头比较。两者

125

I-4H5 and

I-4H5和

125

I-7B9 demonstrated suitable imaging characteristics binding to the mutp53

I-7B9表现出与mutp53结合的合适成像特征

R172H

expressing tumors.

表达肿瘤。

Figure

图

a-c displays the SPECT/CT scans over time of four mice (2 for

a-c显示四只小鼠随时间的SPECT/CT扫描（2只

125

I-4H5 and 2 for

I-4H5和2

125

I-7B9). Thyroid uptake was visible due to free

I-7B9）。由于游离，甲状腺摄取可见

125

I, while liver, lungs, and heart were visible most likely due to the slow blood clearance and slow secretion of the antibodies. The highest accumulation of

一、而肝脏，肺和心脏很可能是由于血液清除缓慢和抗体分泌缓慢所致。最高的积累

125

I-4H5 and

I-4H5和

125

I-7B9 was detected in the mutp53 expressing tumors at all time points. Interestingly, some activity was already detected in the KO tumors at the time of 24-hour imaging, which remained at a similar level for 48-hour imaging, while activity in the positive tumors increased significantly from 24 to 48 h.

在所有时间点，在表达mutp53的肿瘤中均检测到I-7B9。有趣的是，在24小时成像时已经在KO肿瘤中检测到一些活性，其在48小时成像中保持相似的水平，而阳性肿瘤中的活性从24小时显着增加至48小时。

This likely explains why the scans conducted at the 48-hour post-injection time poind yield images with the most pronounced contrast. Six days post injection, tracers remained detectable in both blood and tumors. Because imaging was conducted longitudinally, tumors had grown considerably larger by the 6-day time point compared to the earlier time points, influencing the biodistribution data where the injected activity is divided by tumor weight.

这可能解释了为什么在注射后48小时进行的扫描会产生对比度最明显的图像。注射后六天，示踪剂在血液和肿瘤中均可检测到。由于成像是纵向进行的，因此与较早的时间点相比，肿瘤在6天的时间点生长得更大，从而影响了注射活性除以肿瘤重量的生物分布数据。

SPECT images are presented in relative SUV scale (SUVR = 10) with respect to muscle..

SPECT图像以相对于肌肉的相对SUV比例（SUVR=10）呈现。。

Fig. 5

图5

Repeated molecular imaging and analysis. Repeated SPECT/CT imaging of iodinated mAbs 4H5 and 7B9 in a mouse model with Mut-T-B6 and B16-KO tumors. 2 subjects were scanned simultaneously at (

重复分子成像和分析。。同时对2名受试者进行扫描(

一

) 24 h, (

)24小时(

b类

) 48 h and (

)48小时和(

c级

) 6 d post-injection. The upper and lower rows represent the experimental replicates. SPECT images are presented in RGB and CT in gray color scale, respectively. (

)注射后6天。上面和下面的行代表实验重复。SPECT图像分别以RGB和CT的灰度表示。（笑声）(

) Ex vivo Autoradiography of iodinated mAbs 4H5 and 7B9 in Mut-T-B6 and B16-KO tumors 72 h p.i. (6–8 animals/group). One-way ANOVA and Tukey’s multiple comparison posttest. *

)Mut-T-B6和B16-KO肿瘤中碘化单克隆抗体4H5和7B9的离体放射自显影72小时（6-8只动物/组）。单因素方差分析和Tukey的多重比较后测*

≤ 0.05. Data are presented as means ± SD. (

≤0.05。数据表示为平均值±SD(

) Representative images of ex vivo Autoradiography of iodinated mAbs 4H5 and 7B9 in Mut-T-B6 and B16-KO tumors 72 h p.i.

)在Mut-T-B6和B16-KO肿瘤中72小时碘化mAb 4H5和7B9的离体放射自显影的代表性图像。

Full size image

全尺寸图像

Autoradiography of

放射自显影

125

I-4H5 and

I-4H5和

125

I-7B9 in KO and mutp53 tumors

I-7B9在KO和mutp53肿瘤中的表达

Ex vivo autoradiography results confirmed the specific binding of both tracers to the p53

离体放射自显影结果证实了两种示踪剂与p53的特异性结合

R172H

-positive tumors, significantly higher than the negative control tumors (Fig.

-阳性肿瘤，显着高于阴性对照肿瘤（图）。

d). However, uptake in the p53 null tumors (as also seen in the SPECT scans) was higher than expected. In both tumor models, the highest activity was measured at the rim of the tumor and in the narrow periphery of the blood vessels (Fig.

d）。然而，p53无效肿瘤的摄取（如SPECT扫描所示）高于预期。在两种肿瘤模型中，在肿瘤边缘和血管狭窄周围测量到最高活性（图）。

e).

e）。

Discussion and conclusion

讨论和结论

Several hotspots in the p53 genetic sequence exist, where mutations are primarily encountered. Mutated p53 proteins encoded by these hotspot mutations almost always lose the functions of wild-type p53 and may instead guide functions associated with cancer development, such as promotion of proliferation, migration, initiation, invasion, angiogenesis, disruption of tissue architecture, and resistance to anticancer drugs.

p53基因序列中存在几个热点，主要遇到突变。由这些热点突变编码的突变p53蛋白几乎总是失去野生型p53的功能，而是可能指导与癌症发展相关的功能，例如促进增殖，迁移，起始，侵袭，血管生成，破坏组织结构和抗癌药物的耐药性。

. The addiction of cancer cells to mutp53 makes it an attractive target for developing new diagnostic and therapeutic tools

. Furthermore, understanding the specific mutations and their functions can provide valuable insights for novel approaches to restore the tumor-suppressive properties of p53 in cancer treatment.

此外，了解特定突变及其功能可以为恢复p53在癌症治疗中的肿瘤抑制特性的新方法提供有价值的见解。

While techniques like next-generation sequencing (NGS) are crucial for identifying genetic mutations across the genome, they often require invasive biopsies and may not capture the full spectrum of genetic variation in all tumor lesions. Additionally, NGS provides a static snapshot of the tumor’s genetics and does not reflect real-time functional activity.

虽然下一代测序（NGS）等技术对于鉴定整个基因组的基因突变至关重要，但它们通常需要进行侵入性活检，并且可能无法捕获所有肿瘤病变中的全部遗传变异。。

In contrast, molecular imaging with specific tracers offers a non-invasive, real-time view of tumors, providing dynamic insights into the behavior of specific mutant proteins, such as R175H p53, within living organisms. This approach allows for the monitoring of changes in protein expression and tumor activity over time, providing functional data that complements the genetic information obtained from whole-genome sequencing..

相反，使用特定示踪剂进行分子成像可以提供肿瘤的非侵入性实时视图，从而动态了解生物体内特定突变蛋白（例如R175H p53）的行为。这种方法可以监测蛋白质表达和肿瘤活性随时间的变化，提供功能数据，补充从全基因组测序获得的遗传信息。。

In the present study, we evaluated the in vitro and in vivo binding properties of two monoclonal antibodies, 4H5 and 7B9, previously described in

在本研究中，我们评估了先前在

, designed to target the p53R175H mutation, one of the most frequent p53 alterations in human cancers. The specificity of these antibodies was demonstrated through immunoblotting, immunofluorescence as well as ELISA techniques, showing recognition of the R175H mutant without cross-reactivity to wild-type or other mutant forms of p53.

，旨在靶向p53R175H突变，这是人类癌症中最常见的p53改变之一。通过免疫印迹，免疫荧光以及ELISA技术证明了这些抗体的特异性，显示出对R175H突变体的识别，而对野生型或其他突变形式的p53没有交叉反应。

Radioiodinated .

放射性碘。

125

I-4H5 and

I-4H5和

125

I-7B9 demonstrated long shelf life and antigen-specific binding. Our findings confirm the potential of these antibodies as agents for molecular imaging.

。我们的发现证实了这些抗体作为分子成像剂的潜力。

Several efforts have been undertaken, but mutant p53 is still difficult to drug due to a lack of thermostability and suitable binding pockets for small molecule drugs. However, generating mutation-specific reagents, like the monoclonal antibodies explored here, has significant potential for broad applications in biomedical research, supporting both basic studies and translational diagnostic and therapeutic efforts..

。然而，产生突变特异性试剂（如此处探索的单克隆抗体）在生物医学研究中具有广泛应用的巨大潜力，可支持基础研究以及转化诊断和治疗工作。。

Most progress has been made targeting the Y220C mutation where both the pocket formed and reactive cysteine have been explored. For the Y220C mutation for example, an allele-specific reactivator drug rezatapopt (PC14586, mutant converted to WT activity) is currently in phase I and II clinical trials (NCT04585750).

针对Y220C突变已经取得了大多数进展，其中已经探索了口袋形成和反应性半胱氨酸。例如，对于Y220C突变，等位基因特异性再激活剂药物rezatapopt（PC14586，突变转化为WT活性）目前正在I期和II期临床试验中（NCT04585750）。

. In preclinical studies, treatment with the small molecule rezatapopt induced complete regression of tumors in mice and potentiated checkpoint therapy. These experiments demonstrated that changing mutant p53 protein and restoring its wildtype function not only resulted in p53-mediated cell death but also improved the recognition and killing of the tumors by the immune system in small animal models.

在临床前研究中，用小分子rezatapopt治疗可诱导小鼠肿瘤完全消退，并增强检查点治疗。这些实验表明，改变突变型p53蛋白并恢复其野生型功能不仅导致p53介导的细胞死亡，而且还改善了小动物模型中免疫系统对肿瘤的识别和杀伤。

This included increased infiltration and activation of lymphocytes and other immune cells, similar to what was recently described for p53 activation in wildtype tumors using the stapled peptide Sulanemadlin.

这包括淋巴细胞和其他免疫细胞的浸润和活化增加，类似于最近使用钉合肽Sulanemadlin在野生型肿瘤中描述的p53活化。

It is generally believed that antibodies typically require the target to be available extracellularly (e.g. on the outside of cell membranes or as a free molecule in the blood), with a concern that that antibodies targeting intracellular antigens may be hindered by the cell membrane. However, recent studies indicate that the integrity of cell membranes may be compromised within the tumor environment, thereby facilitating antibody access.

通常认为，抗体通常需要靶标在细胞外可用（例如在细胞膜的外部或作为血液中的游离分子），担心靶向细胞内抗原的抗体可能会受到细胞膜的阻碍。然而，最近的研究表明，细胞膜的完整性可能在肿瘤环境中受到损害，从而促进抗体的获取。

For example, Dadachova et al. showed that a monoclonal antibody against melanin can deliver effective therapeutic doses of 188-rhenium to mouse melanoma tumor cells.

例如，Dadachova等人表明，抗黑色素的单克隆抗体可以向小鼠黑色素瘤肿瘤细胞提供有效治疗剂量的188铼。

. While the antibody showed an apparent preference for tumor cell melanin presumably because of accessibility caused by tumor cell membrane damage it is a concern that the antibody is not intrinsically tumor-specific. Here, we overcome this difficulty by using antibodies that are specific to tumor mutations in p53..

虽然该抗体显示出对肿瘤细胞黑色素的明显偏好，可能是因为肿瘤细胞膜损伤引起的可及性，但令人担忧的是该抗体本质上不是肿瘤特异性的。在这里，我们通过使用特异于p53肿瘤突变的抗体来克服这一困难。。

Complicating antibody recognition, specific p53 mutations may be masked intracellular by interactions with other proteins, such as p73

使抗体识别复杂化的是，特定的p53突变可能被细胞内与其他蛋白质（如p73）的相互作用所掩盖

, potentially obscuring the R175H epitope. While the exact structure of the p53-p73 complex is unknown, studies suggest that p73 binds at the tetramerization domain (OD), distant from the DNA-binding domain (DBD) where R175H is located, indicating that the R175H epitope remains accessible

，可能会掩盖R175H表位。虽然p53-p73复合物的确切结构尚不清楚，但研究表明p73结合在远离R175H所在的DNA结合域（DBD）的四聚结构域（OD），表明R175H表位仍然可以接近

. Additionally, phosphorylated wild-type p53 is thought to bind p73 without blocking the DBD, suggesting that mutant p53 may behave similarly. Thus, antibodies like 4H5 and 7B9 should still effectively recognize and bind to mutant p53.

此外，磷酸化的野生型p53被认为可以结合p73而不会阻断DBD，这表明突变型p53的行为可能相似。因此，4H5和7B9等抗体仍应有效识别并结合突变型p53。

Another major challenge of antibody-based imaging techniques is the conjugation of the mAb with the radionuclide. Numerous direct and indirect labeling techniques can be used, however, only a fraction of these can be used without compromising the functionality and orientation of the mAb. Direct radioiodine labeling with Iodine-125, as performed in the current study, requires the presence of an aromatic moiety such as tyrosine or histidine.

基于抗体的成像技术的另一个主要挑战是单克隆抗体与放射性核素的结合。可以使用许多直接和间接标记技术，但是，在不损害mAb的功能和方向的情况下，只能使用其中的一小部分。如在当前研究中所进行的，用碘-125直接放射性碘标记需要存在芳香族部分，例如酪氨酸或组氨酸。

. Tyrosine is the primary site of iodine addition, but if the pH is above 8.5, the secondary site on the histidine ring is preferred. Since tyrosine is likely to be present in the binding region, it is important to evaluate the immunoreactivity and high molar activity of the binder after labeling and purification.

酪氨酸是碘添加的主要位点，但如果pH值高于8.5，则组氨酸环上的次要位点是首选。由于酪氨酸可能存在于结合区中，因此在标记和纯化后评估粘合剂的免疫反应性和高摩尔活性很重要。

. We were able to radio-iodinate 4H5 and 7B9 with a high yield and long shelf life. Moreover, the two radio-iodinated mAbs,

我们能够以高产率和较长的保质期放射性碘化4H5和7B9。此外，两种放射性碘化单克隆抗体，

125

I-4H5 and

I-4H5和

125

I-7B9, demonstrated selective in vitro binding properties after radiolabeling.

。

While the use of

而使用

125

I as an imaging agent in preclinical studies has many advantages for preclinical work, particularly practical ones, such as availability and long half-life, there are also limitations. Due to the long half-time of 59.5 days and the relatively low gamma emission,

I作为临床前研究中的显像剂，对于临床前工作具有许多优势，特别是实用的，如可用性和半衰期长，也存在局限性。，

125

I is rather impractical as an imaging agent in the clinical setting. However,

我在临床环境中作为成像剂是相当不切实际的。但是，

125

I can be easily replaced by other halogen radioisotopes without changing the radiochemical labeling method, including

在不改变放射化学标记方法的情况下，我可以很容易地被其他卤素放射性同位素取代，包括

124

I, which is suitable for PET imaging, and

一、适用于PET成像，以及

123

I, which is suitable for human SPECT imaging.

一、适用于人体SPECT成像。

In this study, we could exclude in two separate small animal models any off-target accumulation in normal organs for both tracers. We observed a long circulation time in blood, which is often preferred in a therapeutic setting but is not optimal for diagnostic imaging.

。我们观察到血液循环时间长，这在治疗环境中通常是首选，但对于诊断成像来说并不是最佳选择。

Nevertheless, a significantly higher uptake of both

尽管如此，两者的吸收率都要高得多

125

I-4H5 and

I-4H5和

125

I-7B9 was observed in the p53

在p53中观察到I-7B9

R172H

expressing tumors at the tested time points.

在测试的时间点表达肿瘤。

In the in vivo study, a significant size difference between p53 KO and mutant p53 tumors was observed at 6 days post-injection, which complicates direct comparisons between the two models. To gain more insight into the specific impact of different p53 mutations on tumor behavior, further studies using tumors from the same cancer type with more similar growth rates would be beneficial.

在体内研究中，在注射后6天观察到p53 KO和突变型p53肿瘤之间存在显着的大小差异，这使两种模型之间的直接比较变得复杂。为了更深入地了解不同p53突变对肿瘤行为的具体影响，使用生长速度更相似的相同癌症类型的肿瘤进行进一步研究将是有益的。

This could include tumors with other p53 hotspot mutations, such as R273H or G245S, to better understand how these mutations influence tumor growth and tracer uptake. Additionally, including immunocompetent mice in future studies would provide a more comprehensive evaluation of the immune response in these models.

这可能包括具有其他p53热点突变的肿瘤，例如R273H或G245S，以更好地了解这些突变如何影响肿瘤生长和示踪剂摄取。此外，在未来的研究中包括免疫活性小鼠将为这些模型中的免疫反应提供更全面的评估。

Together, these approaches would allow for more precise comparisons and help clarify the contribution of p53 mutation status to therapeutic outcomes and imaging biomarker efficacy..

总之，这些方法将允许更精确的比较，并有助于阐明p53突变状态对治疗结果和成像生物标志物功效的贡献。。

Although the double tumor model used in this study is not ideal, using %ID/g accounts for size differences and shows higher tracer uptake in Mut-T-B6 tumors, not just due to their larger size. Importantly, the increased growth of the positive tumors does not lead to overestimation of uptake per gram, as smaller tumors usually show higher uptake.

尽管本研究中使用的双肿瘤模型并不理想，但使用%ID/g可以解释大小差异，并且在Mut-T-B6肿瘤中显示出更高的示踪剂摄取，而不仅仅是由于其较大的大小。。

Larger tumors generally exhibit lower uptake per gram due to reduced antibody penetration.

由于抗体渗透减少，较大的肿瘤通常表现出较低的每克摄取量。

. Therefore, the significant differences observed in tracer uptake between p53 KO and mutant tumors are even more remarkable, as the larger tumor size of the mutant group would have been expected to mask, rather than exaggerate, the differences.

因此，p53 KO和突变肿瘤之间在示踪剂摄取方面观察到的显着差异甚至更为显着，因为突变组较大的肿瘤大小有望掩盖而不是夸大差异。

In addition, elevated activity was observed in the thyroid gland, suggesting that in vivo deiodination may occur. Therefore, blocking the thyroid gland with e.g. perchlorate before imaging would be beneficial

此外，在甲状腺中观察到活性升高，表明可能发生体内脱碘。因此，在成像前用高氯酸盐等阻断甲状腺将是有益的

. It is also important to note that SPECT analysis alone cannot differentiate between various cell populations within a tumor. Previous research has identified mutant p53 secreted into the tumor microenvironment via extracellular vesicles suggesting that macrophages could uptake the R175H mutant from this environment.

。同样重要的是要注意，单独的SPECT分析不能区分肿瘤内的各种细胞群。先前的研究已经确定了通过细胞外囊泡分泌到肿瘤微环境中的突变型p53，这表明巨噬细胞可以从这种环境中摄取R175H突变体。

. However, our prior analyses of paraffin-embedded tumors stained with 4H5 and 7B9 did not show any cross-reactivity with wild-type p53, other mutp53 hotspot mutants, or immune cells, such as macrophages

然而，我们先前对4H5和7B9染色的石蜡包埋肿瘤的分析未显示与野生型p53，其他mutp53热点突变体或免疫细胞（例如巨噬细胞）有任何交叉反应

Despite the high activity in the blood pool, we were able to visualize the p53

尽管血池中的活性很高，但我们能够看到p53

R172H

mutation by repeated SPECT/CT imaging with the best contrast at 48 h. In further studies, imaging performance could be improved by developing smaller tracer variants. In contrast to whole-size mAbs, engineered affibody molecules and antibody fragments such as minibodies, diabodies, single chain variable region fragments (scFvs), and nanobodies are much smaller but retain the essential specificities and affinities.

通过重复SPECT/CT成像突变，在48小时时对比度最佳。在进一步的研究中，可以通过开发较小的示踪剂变体来提高成像性能。与全尺寸mAb相比，工程化的抗体分子和抗体片段（例如小体，双抗体，单链可变区片段（scFv）和纳米体）要小得多，但保留了基本的特异性和亲和力。

Advantages of smaller tracers include shorter blood circulation times (hours rather than weeks) improving the signal-to-noise ratio, deeper tissue penetration, and enabling same-day imaging.

较小示踪剂的优点包括缩短血液循环时间（数小时而不是数周），提高信噪比，加深组织穿透，并实现当天成像。

. In addition, the lack of the Fc region also lowers the nonspecific binding of the fragment and therefore may improve image quality. Imaging tracers smaller than 60 kDa are preferably excreted via the renal system and are not metabolized or retained by the liver. However, compared to full-size antibodies, smaller molecules may have poorer affinities and lower overall tumor uptake.

此外，缺乏Fc区也会降低片段的非特异性结合，因此可以提高图像质量。。然而，与全尺寸抗体相比，较小的分子可能具有较差的亲和力和较低的总体肿瘤摄取。

Autoradiography of the negative and positive tumors revealed an accumulation of tracers in the narrow periphery of blood vessels and at the edge around the tumor, a phenomenon that also deserves further investigation in follow-up studies. While mAbs have the ability to bind specifically to tumor cells, their initial diffusion from the vasculature into tumor tissue is similar to that of non-targeting molecules and relies on the enhanced permeability and retention (EPR) effect, a unique property of tumors that affects diffusion of macromolecules through leaky blood vessels in the tumor area.

阴性和阳性肿瘤的放射自显影显示示踪剂在狭窄的血管周围和肿瘤周围的边缘积累，这种现象也值得在后续研究中进一步研究。虽然单克隆抗体具有特异性结合肿瘤细胞的能力，但它们从脉管系统到肿瘤组织的初始扩散与非靶向分子相似，并且依赖于增强的渗透性和保留（EPR）效应，这是肿瘤的独特性质，影响大分子通过肿瘤区域的渗漏血管扩散。

. Due to the rapid growth of tumors, the surrounding blood vessels can have a defective architecture, which is exacerbated by the production of various permeability factors. Molecules larger than 40 kDa benefit from the EPR effect, promoting the accumulation in the tumor. However, the efficacy varies and is influenced by multiple factors including vascular permeability, endothelial receptors, vascular maturation, extracellular matrix, hypoxia, interstitial fluid pressure, and tumor cell density.

由于肿瘤的快速生长，周围的血管可能具有缺陷的结构，这由于各种渗透因子的产生而加剧。大于40 kDa的分子受益于EPR效应，促进肿瘤中的积累。然而，疗效各不相同，并受多种因素的影响，包括血管通透性，内皮受体，血管成熟，细胞外基质，缺氧，间质液压力和肿瘤细胞密度。

. Strategies that promote EPR and release mAbs accumulated at the periphery of blood vessels could improve the access and availability of the mAb to the target. In agreement with this, we hypothesize that the uptake observed in the p53 negative tumors is due to the EPR effect may be reduced by using molecules smaller than 40 kDa.

。促进EPR和释放在血管周围积累的单克隆抗体的策略可以改善单克隆抗体对靶标的获取和可用性。与此一致，我们假设在p53阴性肿瘤中观察到的摄取是由于EPR效应所致，可以通过使用小于40 kDa的分子来减少。

Here, for example, the use of Fab fragments could potentially achieve even better contrast between positive and negative tumors and could instead be better suited for diagnostic imaging..

例如，在这里，Fab片段的使用可能会在阳性和阴性肿瘤之间实现更好的对比，并且可能更适合于诊断成像。。

While our study highlights the R175H mutation linked to LFS, we recognize that antibodies may not be suitable for treating LFS patients due to the presence of mutant p53 in healthy tissues. Our preliminary optical imaging results in R172H mutant mice using the CF750-conjugated 4H5 mAb showed greater uptake in spontaneous tumors, but also some uptake was also observed in normal organs (carrying the R172H mutation).

虽然我们的研究强调了与LFS相关的R175H突变，但我们认识到，由于健康组织中存在突变型p53，抗体可能不适合治疗LFS患者。我们使用CF750偶联的4H5 mAb在R172H突变小鼠中进行的初步光学成像结果显示，自发性肿瘤的摄取更大，但在正常器官（携带R172H突变）中也观察到了一些摄取。

Nonetheless, mutation specific antibodies could still be valuable for ex vivo applications, such as IHC, to detect and assess mutant p53 in LFS biopsy samples, thereby aiding diagnosis and research into the molecular landscape of LFS tumors..

尽管如此，突变特异性抗体对于离体应用（例如IHC）仍然很有价值，可以检测和评估LFS活检样本中的突变p53，从而有助于诊断和研究LFS肿瘤的分子格局。。

This proof-of-concept study shows encouraging initial results and we can conclude that antibody targeting of intracellular proteins holds great potential for precision medicine and therapeutics. We believe that molecular imaging with anti-p53

这项概念验证研究显示了令人鼓舞的初步结果，我们可以得出结论，针对细胞内蛋白质的抗体在精准医学和治疗方面具有巨大潜力。我们认为抗p53的分子成像

R175H

tracer could be a promising approach for cancer diagnostics and could be further applied for patient stratification and treatment response monitoring of mut-p53-targeted therapeutics as companion diagnostics. Further research and development in antibody engineering, delivery systems, and optimization of intracellular protein targeting approaches will be key to unlocking the full potential of this emerging field..

示踪剂可能是一种很有前途的癌症诊断方法，可以进一步应用于mut-p53靶向治疗的患者分层和治疗反应监测，作为伴随诊断。抗体工程，递送系统和细胞内蛋白质靶向方法优化的进一步研究和开发将是释放这一新兴领域全部潜力的关键。。

Methods

方法

p53

R175H

targeting antibodies

靶向抗体

Construction and production of the mAb clones 4H5 and 7B9 specific for the p53

p53特异性单克隆抗体4H5和7B9的构建和生产

R175H

mutant protein was previously described in

突变蛋白先前在

. The hybridoma clones were adapted to serum-free media by serial passage. The supernatant media was harvested and purified by protein A chromatography. PAGE analysis of the purified antibody showed heavy and light chain bands and no contaminants. The heavy chain and light chain sequences of the antibodies have been cloned.

杂交瘤克隆通过连续传代适应无血清培养基。收获上清液培养基并通过蛋白A色谱纯化。纯化抗体的PAGE分析显示重链和轻链条带，没有污染物。已经克隆了抗体的重链和轻链序列。

The recombinant expression of these clones gave the same specificities as the parent hybridoma. Thus, the antibodies are sequence verified..

这些克隆的重组表达具有与亲本杂交瘤相同的特异性。因此，抗体被序列验证。。

Iodination of the mAbs 4H5 and 7B9

单克隆抗体4H5和7B9的碘化

Reaction vessels were coated with 20 µg Iodogen (1,3,4,6-Tetrachloro-3α,6α-diphenylglycouril) dissolved in dichloromethane at 0.1 mg/mL. After evaporation,

反应容器涂有20µg碘原（1,3,4,6-四氯-3α，6α-二苯乙酰基），以0.1 mg/mL的浓度溶解在二氯甲烷中。蒸发后，

125

I and 4H5 or 7B9 (150 µg in PBS) were mixed and added in an equivalent volume as sodium phosphate buffer, pH 7.4. The mixture was incubated at room temperature for seven minutes and gently shaken every 30 s. The reaction mixture was then transferred to a new tube with 1 mL 0.05 M sodium phosphate and 5 M NaCl, pH 7.4.

将I和4H5或7B9（PBS中150µg）混合，并以等体积添加为磷酸钠缓冲液（pH 7.4）。将混合物在室温下孵育7分钟，并每30秒轻轻摇动一次。然后将反应混合物转移到含有1 mL 0.05 M磷酸钠和5 M NaCl（pH 7.4）的新管中。

buffer. After 10 min, one mL 0.05 M sodium phosphate, 5% potassium iodide, and 0.5% BSA (w/v) buffer was added and the sample was mixed thoroughly. Labeled conjugates were separated from unreacted radionuclide and low molecular weight reaction components using NAP-5 columns pre-equilibrated with PBS.

缓冲区。10分钟后，加入1 mL 0.05 M磷酸钠，5%碘化钾和0.5%BSA（w/v）缓冲液，并将样品充分混合。使用用PBS预平衡的NAP-5柱将标记的缀合物与未反应的放射性核素和低分子量反应组分分离。

Labelling yield and stability of the mAbs were evaluated by instant thin-layer chromatography (ITLC, Biodex Medical Systems) with subsequent quantification in a phosphoimager (BAS-1800II, Fujifilm)..

通过即时薄层色谱（ITLC，Biodex Medical Systems）评估单克隆抗体的标记产率和稳定性，随后在phosphoimager（BAS-1800II，Fujifilm）中进行定量。。

ELISA assays

ELISA分析

R175H-Trx, Y220C-Trx, and Trx proteins were coated on Nunc 96-stripwell plates at the concentration of 0.5 µg/mL. Plates were left overnight at 4 °C. The following day, plates were washed with 0.05% PBS-Tween 20 (PBS-T) and blocked with 5% FBS in PBS with 0.05% (w/v) Tween-20 (blocking buffer) for an hour at 37 °C and washed three times.

将R175H-Trx，Y220C-Trx和Trx蛋白以0.5µg/mL的浓度包被在Nunc 96 stripwell平板上。将平板在4°C下放置过夜。第二天，将板用0.05%PBS-Tween 20（PBS-T）洗涤，并用含有0.05%（w/v）Tween-20（封闭缓冲液）的PBS中的5%FBS在37℃下封闭1小时并洗涤三次。

125

I-4H5 and

I-4H5和

125

I-7B9 were diluted in PBS-T (1nM, 10nM, 100nM) and added to the wells for one hour at 37 °C. After three washes, individual wells were separated and the associated activity was counted in a gamma counter (1480 Wizard 3′, Wallace).

将I-7B9在PBS-T（1nM，10nM，100nM）中稀释，并在37℃下加入孔中1小时。洗涤三次后，分离单个孔，并在伽马计数器（1480 Wizard 3'，Wallace）中计数相关活性。

Cell lines

细胞系

The murine melanoma cell line (H-2b background) B16-F10 was purchased from ATCC. B16-KO was generated by CRISPR-targeting (Supplementary Fig. 1a). The sgRNA target sequences used for targeting Exon 1 of murine p53 are CACCGTGACACCCTGCTGGGAAGG and AAACCCTTCCCAGCAGGGTGTCAC was cloned into PX458 plasmid with spCas9.

鼠黑素瘤细胞系（H-2b背景）B16-F10购自ATCC。B16-KO是通过CRISPR靶向产生的（补充图1a）。用于靶向鼠p53外显子1的sgRNA靶序列是CACCGTGACACCCTGGGAAGG，并且用spCas9将AAACCCTTCCCAGCAGGTGTCAC克隆到PX458质粒中。

p53 knockout B16-F10 cell lines were generated by transfecting 1 µg of PX458_mmp53KO plasmid with ~ 1 × 10.

通过用〜1×10转染1μgpx458μmmp53KO质粒产生p53敲除B16-F10细胞系。

B16-F10 cells as per the manufacturer’s instruction (Lipofectamine 3000). After 48 h post-transfection, B16 cells were dissociated into single cells and FACS sorted (MoFlo XDP 4) into single cells, in wells containing DMEM media complete with 20% FCS. To screen the population for CRISPR knockout efficiency, genomic DNA from B16 cells was extracted using QuickExtract DNA solution (Epicenter) and primers flanking the targeted region were used in PCR to obtain amplicons that are subsequently subcloned into pCR-BluntII-TOPO vector (Life Technologies).

B16-F10细胞按照制造商的说明（Lipofectamine 3000）。。为了筛选CRISPR敲除效率的群体，使用QuickExtract DNA溶液（Epicenter）提取来自B16细胞的基因组DNA，并在PCR中使用靶向区域侧翼的引物以获得扩增子，随后将其亚克隆到PCR BluntII TOPO载体（Life Technologies）中。

The amplicons were checked by sequencing (using the primer pairs: Mmp53_Ex1_F: GTTCTGTAGCTTCAGTTCATTGG and Mmp53_Ex1_R: CCGAGAGGTCTCGTCACGCTC) to assess the efficiency of sgRNA targeting p53. Clonal cells possessing p53 knockout were checked by sequencing and confirmed via western blot and immunohistochemistry with the corresponding antibodies (Supplementary Fig. 1b)..

通过测序（使用引物对：Mmp53\u Ex1\u F:GTTCTGTAGCTTCAGTTCATTGG和Mmp53\u Ex1\u R:CCGAGAGGTCTCGTCACGCTC）检查扩增子，以评估靶向p53的sgRNA的效率。通过测序检查具有p53敲除的克隆细胞，并通过蛋白质印迹和免疫组织化学用相应的抗体进行确认（补充图1b）。。

The primary mouse p53

原代小鼠p53

R172H/R172H

cell line, referred to as Mut-T-B6, was established from a spontaneous primary sarcoma-like tumor in a p53

细胞系，称为Mut-T-B6，是由p53中的自发性原发性肉瘤样肿瘤建立的

R172H/R172H

mutant B6 mouse with H-2b background. These cells have been characterized, with data of the morphology and p53 expression status determined by Western blotting shown in Supplementary Fig. 1c and d.

具有H-2b背景的突变B6小鼠。已经表征了这些细胞，补充图1c和d中显示了通过蛋白质印迹确定的形态和p53表达状态的数据。

All cell lines were grown in DMEM supplemented with 10% FBS and a mixture of streptomycin/penicillin, Mut-T-B6, and B16-KO with an additional 5% sodium pyruvate.

所有细胞系均在补充有10%FBS和链霉素/青霉素，Mut-T-B6和B16-KO与另外5%丙酮酸钠的混合物的DMEM中生长。

Immunoblotting

免疫印迹

Cell lysates were prepared by sonication in 0.1% Triton X PBS (PBST) with protease inhibitors. Protein concentration in cell lysates was determined using the QuickStart Bradford assay. 20g of cell lysates were mixed with NuPAGE LDS buffer, heated, and loaded onto 4–12% Mini-PROTEAN gels. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes.

通过在含有蛋白酶抑制剂的0.1%Triton X PBS（PBST）中超声处理来制备细胞裂解物。使用QuickStart Bradford测定法测定细胞裂解物中的蛋白质浓度。将20g细胞裂解液与NuPAGE LDS缓冲液混合，加热，并加载到4-12%的Mini-PROTEAN凝胶上。通过电泳分离蛋白质并转移到硝酸纤维素膜上。

Membranes were blocked in 5% milk in PBST. 7B9, 4H5, or other indicated antibodies were used as primary antibodies and detected with goat anti-mouse IgG (H + L) (1:3000, Jackson Laboratories). Chemiluminescence detection was performed using SuperSignal West Dura Substrate. Imaging was done with Licor Odyssey Fc and Image Studio software.

将膜在PBST中的5%牛奶中封闭。将7B9、4H5或其他指定的抗体用作一抗，并用山羊抗小鼠IgG（H + L）（1:3000，Jackson Laboratories）检测。使用SuperSignal West Dura底物进行化学发光检测。成像是使用Licor Odyssey Fc和Image Studio软件完成的。

For uncropped membranes see Supplementary Fig. 2..

对于未裁剪的膜，请参见补充图2。。

Immunofluorescence (IF) staining and immunohistochemistry (IHC) staining

免疫荧光（IF）染色和免疫组织化学（IHC）染色

Cells or frozen tumor tissue sections were fixed with 4% paraformaldehyde, permeabilized, and blocked non-specific binding with 5% BSA (for IF) or 5% normal goat serum (for IHC) in PBS. Apply the indicated primary antibody or Digoxigenin (DIG) conjugated antibody to the sections and incubate overnight.

将细胞或冷冻的肿瘤组织切片用4%多聚甲醛固定，透化，并用PBS中的5%BSA（用于IF）或5%正常山羊血清（用于IHC）阻断非特异性结合。将指定的一抗或洋地黄毒苷（DIG）缀合的抗体应用于切片并孵育过夜。

After rinsing samples to remove the unbound primary antibody, Alexa Fluor 488-conjugated -anti-mouse Ig (1:500, Invitrogen, California, USA) secondary antibodies were used for IF. After 1 h incubation, samples were rinsed and stained nuclei with DAPI before mounting the coverslip onto the slides using an anti-fade mounting medium.

冲洗样品以除去未结合的一抗后，将Alexa Fluor 488偶联的抗小鼠Ig（1:500，Invitrogen，California，USA）二抗用于IF。孵育1小时后，冲洗样品并用DAPI染色细胞核，然后使用抗褪色固定介质将盖玻片固定在载玻片上。

IF staining images were observed using an Olympus FV1000 upright confocal microscope. Quantification was performed using NIH ImageJ software, according to.

使用Olympus FV1000立式共聚焦显微镜观察IF染色图像。根据NIH ImageJ软件进行定量。

. First, image contrast was enhanced (Image > Adjust > Brightness/Contrast) with images converted to 16-bit format. We applied thresholding (Image > Adjust > Threshold, “Dark Background”) to isolate cells, followed by watershed segmentation (Process > Binary > Watershed) to define cell edges. Cells were counted using Analyze > Analyze Particles.

首先，图像对比度增强（图像>调整>亮度/对比度），图像转换为16位格式。我们应用阈值（图像>调整>阈值，“暗背景”）来分离细胞，然后进行分水岭分割（过程>二进制>分水岭）来定义细胞边缘。使用Analyze>Analyze颗粒对细胞进行计数。

Total cell number (A) was obtained from DAPI-stained images of p53R175H-transfected H1299 cells, and mAb-positive cells (B) were quantified from corresponding FITC-stained images. The percentage of positive cells was calculated as: % Positive = (B / A) × 100..

从p53R175H转染的H1299细胞的DAPI染色图像获得总细胞数（A），并从相应的FITC染色图像定量mAb阳性细胞（B）。阳性细胞的百分比计算为：%阳性=（B/A）×100。。

For IHC, HRP conjugated -anti-DIG secondary antibody (1:50, Jackson ImmunoResearch Lab) was added. After 1 h incubation, samples were washed and DAB solution, with hydrogen peroxide, was added. HRP zcatalyzes DAB oxidation, forming a brown precipitate at the antigen sites for visualizing. IHC Images were captured with a Zeiss AxioImager upright microscope..

对于IHC，添加了HRP偶联的抗DIG二抗（1:50，Jackson ImmunoResearch Lab）。温育1小时后，洗涤样品并加入含有过氧化氢的DAB溶液。HRP Z催化DAB氧化，在抗原位点形成棕色沉淀物以进行可视化。IHC图像是用蔡司AxioImager立式显微镜拍摄的。。

Small animal studies

小动物研究

Female nu/nu Balb/c mice (

雌性无/无小balb/c(

= 58 total, age = 6 weeks, obtained from Janvier-Labs) were housed under standard laboratory conditions and were given ad libitum access to a standard laboratory diet and water. All animal studies were carried out in accordance with relevant guidelines, regulations and ARRIVE guidelines. The methods and protocols utilized in the animal study complied with Swedish law and were approved by the Uppsala Committee of Animal Research Ethics, Uppsala, Sweden (permit C33/16).

总共58岁，年龄6周，从Janvier实验室获得）在标准实验室条件下饲养，并随意获得标准实验室饮食和水。所有动物研究均按照相关指南，法规和ARRIVE指南进行。动物研究中使用的方法和方案符合瑞典法律，并得到了瑞典乌普萨拉乌普萨拉动物研究伦理委员会的批准（许可证C33/16）。

All animals were randomized to the treatment groups. At endpoint all animals were euthanized with a mixture of ketamine (250 mg/kg) and xylazine (25 mg/kg) solution followed by heart puncture..

将所有动物随机分配到治疗组。终点时，用氯胺酮（250 mg/kg）和甲苯噻嗪（25 mg/kg）溶液的混合物对所有动物实施安乐死，然后进行心脏穿刺。。

Biodistribution in mice carrying p53 negative B16-KO tumors.

。

32 mice were used to analyze the in vivo cross-reactivity of 4H5 and 7B9 to xenografts carrying p53-negative B16-KO tumors. 1 × 10

32只小鼠用于分析4H5和7B9与携带p53阴性B16-KO肿瘤的异种移植物的体内交叉反应性。1×10

B16-KO cells were injected subcutaneously into the right posterior leg.

将B16-KO细胞皮下注射到右后腿。

Biodistribution in mice carrying Mut-T-B6 and B16-KO tumors.

携带Mut-T-B6和B16-KO肿瘤的小鼠的生物分布。

26 mice were used to analyze binding of 4H5 and 7B9 in mice carrying Mut-T-B6 and B16-KO tumors. 1 × 10

26只小鼠用于分析携带Mut-T-B6和B16-KO肿瘤的小鼠中4H5和7B9的结合。1×10

Mut-T-B6 cells and 2 × 10

Mut-T-B6细胞和2×10

B16-KO cells were injected subcutaneously in the lower left and right flank respectively.

将B16-KO细胞分别皮下注射到左下侧和右下侧。

50 µL

50µL

125

I-4H5 or

I-4H5或

125

I-7B9 (both 200 kBq, 50 µg) was given intravenously. At 48 and 72 h post-injection (p.i.) blood, tumors heart, liver, kidneys, spleen, colon, stomach, GI, skin, bone, brain, and muscle were collected, weighed, and measured in a gamma well counter.

静脉注射I-7B9（均为200 kBq，50µg）。在注射后48和72小时（p.i.），收集血液，肿瘤心脏，肝脏，肾脏，脾脏，结肠，胃，胃肠道，皮肤，骨骼，大脑和肌肉，称重并在伽玛井计数器中测量。

In vivo optical imaging of p53

p53的体内光学成像

R172H/R172H

mutant B6 mice.

。

4H5 antibody was labeled with XenoLight CF750 using a Fluorescent Rapid Antibody Labeling Kit (Caliper Life Science, Hopkinton, MA, USA) according to the manufacturer’s instructions. The mutant p53

根据制造商的说明，使用荧光快速抗体标记试剂盒（Caliper Life Science，Hopkinton，MA，USA），用XenoLight CF750标记4H5抗体。突变型p53

R172H/R172H

mice were generated as previously described

如前所述产生小鼠

. Two 6- to 8-month-old male mice bearing spontaneous tumors were used for tumor and organ imaging. The mice were intravenously injected with 50 ug XenoLight CF750- labelled 4H5 mAb in 100 µL PBS per mouse. Three days after the CF750-4H5 mAb injection, the mice were euthanatized by cervical dislocation which was done when the animal was fully anaesthetised with 2.5% Avertin.

两只携带自发性肿瘤的6至8个月大的雄性小鼠用于肿瘤和器官成像。每只小鼠在100µL PBS中静脉注射50 ug XenoLight CF750标记的4H5 mAb。注射CF750-4H5 mAb三天后，将小鼠通过颈椎脱位安乐死，这是在用2.5%阿弗丁完全麻醉动物时完成的。

The injected mice were sacrificed 3 days after the injection, and the tumor, heart, lung, liver, spleen, kidney, intestine, colon and fat were scanned by Caliper IVIS image system (Perkin Elmer). The experiments were approved by the University Animal Care and Use Committee of A*STAR Biological Resource Center (Singapore), and were carried out in Biological Resource Center (Singapore) animal facility..

注射后3天处死注射的小鼠，并通过Caliper IVIS图像系统（Perkin Elmer）扫描肿瘤，心脏，肺，肝，脾，肾，肠，结肠和脂肪。。。

SPECT/CT imaging

SPECT/CT成像

Mice carrying both Mut-T-B6 and B16-KO tumors (as described above) received an intravenous injection via the tail vein with 50 µl

同时携带Mut-T-B6和B16-KO肿瘤的小鼠（如上所述）通过尾静脉注射50l

125

I-4H5 (7 MBq, 50 µg), or

I-4H5（7 MBq，50[UNK]µg），或

125

I-7B9 (200 kBq, 50 µg). At 24 h, 48 h, and 6 days h p.i. two animals (one for each tracer) were anaesthetized using isoflurane and imaged simultaneously with a static whole-body tomographical scan in the NanoScan SPECT/CT (Mediso Medical Imaging Systems Ltd., Hungary). First, a whole-body CT scan was acquired with the following parameters.

I-7B9（200 kBq，50µg）。在第24小时，48小时和6天，使用异氟烷麻醉两只动物（每种示踪剂一只），并在NanoScan SPECT/CT（Mediso Medical Imaging Systems Ltd.，匈牙利）中与静态全身断层扫描同时成像。首先，使用以下参数获取全身CT扫描。

Scan method: Semi circle FOV; projections 480; X-ray, 50 kVp and 600 µA; binning, 1:4; acquisition time, 2 min 47 s. SPECT scan was performed on the same scan range as CT, for 10 min with the following parameters. Frame time, 8 s; acquisition over 28,4 keV. SPECT raw data were reconstructed in Nucline software (3.04.015.000) using the TeraTomo 3D algorithm with 3 subsets, 48 iterations, and corrected for scattering and attenuation artifacts.

扫描方式：半圆视野；投影480；X射线，50 kVp和600µA；binning，1:4；采集时间，2分钟47秒。SPECT扫描在与CT相同的扫描范围内进行，持续10分钟，参数如下。帧时间，8秒；收购超过284keV。。

The CT raw files were reconstructed using Filter Back Projection. SPECT and CT Dicom files were fused and analyzed using PMOD v 4.105 (PMOD Technologies Ltd., Switzerland)..

使用滤波反投影重建CT原始文件。使用PMOD v 4.105（瑞士PMOD Technologies Ltd.）对SPECT和CT Dicom文件进行融合和分析。。

Autoradiography

放射自显影

72 h p.i., Mut-T-B6 and B16-KO tumors were collected from the animals included in the SPECT/CT imaging and embedded in an O.C.T medium (VWR, Belgium). and sectioned with a microtome (20-µm sections). The tumors were then sliced into 20-µm sections using a microtome, and the distribution of remaining radioactivity was captured with a phosphorimager (Fujifilm BAS-1800 II, Japan).

从SPECT/CT成像中包括的动物中收集72小时p.i.，Mut-T-B6和B16-KO肿瘤，并将其包埋在O.C.T培养基（VWR，比利时）中。并用切片机（20µm切片）切片。然后使用切片机将肿瘤切成20µm的切片，并用phosphorimager（Fujifilm BAS-1800 II，日本）捕获剩余放射性的分布。

ImageJ for Mac OSX version 1.48v (NIH, Bethesda, MD, USA) was employed to measure the activity distribution within the tumor Sect.

。

. Activity was quantified as pixel intensity per tumor area in the phosphorimager output file, using an arbitrary scale and normalized to control.

使用任意比例将活性量化为phosphorimager输出文件中每个肿瘤区域的像素强度，并进行标准化以进行控制。

Statistical analysis

统计分析

Data are presented as the means ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism 7 for Mac (GraphPad Software, CA, USA). Data was analyzed using an unpaired Students-Test for comparison of 2 groups, otherwise using One-way ANOVA and Tukey’s multiple comparison posttest.

数据表示为平均值±标准偏差（SD）。使用GraphPad Prism 7 for Mac（GraphPad Software，CA，USA）进行统计分析。使用不成对的学生测试对数据进行分析，以比较两组，否则使用单因素方差分析和Tukey的多重比较后测。

p-values of less than 0.05 were considered statistically significant. Asterisks indicate significance levels at * for .

小于0.05的p值被认为具有统计学意义。星号表示*的显着性水平。

≤ 0.05, ** for

≤0.05，**表示

≤ 0.01, *** for

≤0.01，***表示

≤ 0.001, and **** for

≤0.001，和****用于

≤ 0.0001.

≤ 0,0001.

Data availability

数据可用性

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

在当前研究期间生成和/或分析的数据集可根据合理要求从通讯作者处获得。

References

参考文献

Sabapathy, K. & Lane, D. P. Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others.

Sabapathy，K。＆Lane，D.P。p53的治疗靶向：所有突变体都是相等的，但一些突变体比其他突变体更相等。

Nat. Rev. Clin. Oncol.

国家修订临床。Oncol公司。

(1), 13–30 (2018).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

Rivlin, N. et al. Mutations in the p53 tumor suppressor gene: important milestones at the various steps of Tumorigenesis.

Rivlin，N。等人，《p53肿瘤抑制基因突变：肿瘤发生各个阶段的重要里程碑》。

Genes Cancer

基因癌症

(4), 466–474 (2011).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Tang, Q. et al. Mutant p53 on the path to Metastasis.

Tang，Q。等人。突变型p53在转移途径中的作用。

Trends Cancer

趋势癌症

(1), 62–73 (2020).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学者

Liu, D. P., Song, H. & Xu, Y. A common gain of function of p53 cancer mutants in inducing genetic instability.

Liu，D.P.，Song，H。＆Xu，Y。p53癌症突变体在诱导遗传不稳定性方面的共同功能获得。

Oncogene

癌基因

(7), 949–956 (2010).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Schneider, K. et al.

Schneider，K。等人。

Li-Fraumeni Syndrome

Li-Fraumeni综合征

(University of Washington, Seattle, Seattle (WA), 1993).

（华盛顿大学，西雅图，西雅图（华盛顿州），1993年）。

Hwang, L. A. et al. Monoclonal antibodies against specific p53 hotspot mutants as potential tools for Precision Medicine.

Hwang，L.A。等人。针对特定p53热点突变体的单克隆抗体作为精准医学的潜在工具。

Cell. Rep.

细胞。代表。

(1), 299–312 (2018).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

SoussiT The p53 tumor suppressor gene: from molecular biology to clinical investigation.

SoussiT p53肿瘤抑制基因：从分子生物学到临床研究。

Ann. N Y Acad. Sci.

安。N和ACAD。科学。

910

, 121–137 (2000).

Article

文章

ADS

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Ozaki, T. & Nakagawara, A. Role of p53 in cell death and human cancers.

Ozaki，T。＆Nakagawara，A。p53在细胞死亡和人类癌症中的作用。

Cancers (Basel)

癌症（巴塞尔）

(1), 994–1013 (2011).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Slastnikova, T. A. et al.

Slastnikova，T.A.等人。

Targeted intracellular delivery of antibodies: the state of the art

靶向细胞内递送抗体：最新技术

Front. Pharmacol.

正面。药理学。

9. (2018).

Muller, P. A. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities.

Muller，P.A。＆Vousden，K.H。癌症中的突变型p53：新功能和治疗机会。

Cancer Cell.

癌细胞。

(3), 304–317 (2014).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Vikhanskaya, F. et al. Cancer-derived p53 mutants suppress p53-target gene expression–potential mechanism for gain of function of mutant p53.

Vikhanskaya，F。等人。癌症衍生的p53突变体抑制p53靶基因表达-突变体p53功能获得的潜在机制。

Nucleic Acids Res.

核酸研究。

(6), 2093–2104 (2007).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Bossi, G. et al. Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression.

Bossi，G。等人。突变型p53功能获得：通过消除突变型p53表达来降低人类癌细胞系的肿瘤恶性程度。

Oncogene

癌基因

(2), 304–309 (2006).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Levine, A. J. Targeting the P53 protein for Cancer therapies: the translational impact of P53 Research.

Levine，A.J。针对癌症治疗的P53蛋白：P53研究的转化影响。

Cancer Res.

癌症研究。

(3), 362–364 (2022).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Dumbrava, E. E. et al. First-in-human study of PC14586, a small molecule structural corrector of Y220C mutant p53, in patients with advanced solid tumors harboring a TP53 Y220C mutation.

Dumbrava，E.E.等人首次对携带TP53 Y220C突变的晚期实体瘤患者进行了PC14586的人体研究，PC14586是Y220C突变型p53的小分子结构校正剂。

J. Clin. Oncol.

J、克林。肿瘤。

(16_suppl), 3003–3003 (2022).

（16_suppl），3003–3003（2022）。

Article

文章

MATH

数学

Google Scholar

谷歌学者

Carter, B. Z. et al. Selective targeting of TP53-Y220C mutant AML by PC14586 results in TP53 wild-type conformation and Synergistical apoptosis induction by concomitant inhibition of Xpo-1, MDM2, or Bcl-2.

Carter，B.Z.等人。通过PC14586选择性靶向TP53-Y220C突变体AML导致TP53野生型构象和协同凋亡诱导，同时抑制Xpo-1，MDM2或Bcl-2。

Blood

血液

142

(Supplement 1), 2261–2261 (2023).

（补编1），2261-2261（2023）。

Article

文章

Google Scholar

谷歌学者

Ingelshed, K. et al. MDM2/MDMX inhibition by Sulanemadlin synergizes with anti-programmed death 1 immunotherapy in wild-type p53 tumors.

Sulanemadlin对MDM2/MDMX的抑制作用与野生型p53肿瘤中的抗程序性死亡1免疫疗法协同作用。

iScience

(6), 109862 (2024).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Dadachova, E. et al. Dead cells in melanoma tumors provide abundant antigen for targeted delivery of ionizing radiation by a mAb to melanin.

Dadachova，E。等人。黑色素瘤肿瘤中的死细胞为单克隆抗体将电离辐射靶向递送至黑色素提供了丰富的抗原。

Proc. Natl. Acad. Sci. U S A

程序。纳特尔。阿卡德。科学。美国

101

(41), 14865–14870 (2004).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Wolf, E. R. et al.

Wolf，E.R。等人。

Mutant and wild-type p53 form complexes with p73 upon phosphorylation by the kinase JNK.

突变型和野生型p53在被激酶JNK磷酸化后与p73形成复合物。

Sci Signal, 11(524). (2018).

Sci信号，11（524）。（2018年）。

Di Como, C. J., Gaiddon, C. & Prives, C. 73 function is inhibited by tumor-derived p53 mutants in mammalian cells.

Di Como，C.J.，Gaiddon，C。＆Prives，C。73功能被哺乳动物细胞中肿瘤衍生的p53突变体抑制。

Mol. Cell. Biol.

摩尔电池。生物。

(2), 1438–1449 (1999).

Article

文章

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Kumar, K. & Woolum, K.

Kumar，K.和Woolum，K。

A Novel reagent for Radioiodine labeling of New Chemical entities (NCEs) and Biomolecules

一种新的化学实体和生物分子放射性碘标记试剂

Molecules

分子

(14) (2021).

Hagan, P. L. et al. Tumor size: effect on monoclonal antibody uptake in tumor models.

Hagan，P.L。等。肿瘤大小：对肿瘤模型中单克隆抗体摄取的影响。

J. Nucl. Med.

J.核。医学。

(3), 422–427 (1986).

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Wolff, J. Perchlorate and the thyroid gland.

Wolff，J。高氯酸盐和甲状腺。

Pharmacol. Rev.

药理学。版次：。

(1), 89–105 (1998).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Bhatta, B. et al.

Bhatta，B.等人。

Cancer cells Shuttle Extracellular vesicles containing oncogenic mutant p53 proteins to the Tumor Microenvironment

Cancers (Basel)

癌症（巴塞尔）

. 13(12) (2021).

Fu, R. et al. Antibody fragment and Affibody ImmunoPET Imaging agents: Radiolabelling Strategies and Applications.

Fu，R。等。抗体片段和Affibody ImmunoPET显像剂：放射性标记策略和应用。

ChemMedChem

(23), 2466–2478 (2018).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

MATH

数学

Google Scholar

谷歌学者

Dammes, N. & Peer, D. Monoclonal antibody-based molecular imaging strategies and theranostic opportunities.

Dammes，N。＆Peer，D。基于单克隆抗体的分子成像策略和治疗诊断机会。

Theranostics

治疗诊断科技

(2), 938–955 (2020).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Fang, J., Nakamura, H. & Maeda, H. The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect.

Fang，J.，Nakamura，H。＆Maeda，H。EPR效应：用于药物递送的肿瘤血管的独特特征，涉及的因素以及效应的限制和增强。

Adv. Drug Deliv Rev.

Adv.Drug Deliv修订版。

(3), 136–151 (2011).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Handala, L. et al.

Handala，L.等人。

QuantIF: an ImageJ Macro to automatically determine the percentage of infected cells after Immunofluorescence

QuantIF:ImageJ宏，用于自动确定免疫荧光后感染细胞的百分比

Viruses

病毒

(2) (2019).

Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome.

Olive，K.P.等人。突变型p53在两种Li-Fraumeni综合征小鼠模型中的功能获得。

Cell

细胞

119

(6), 847–860 (2004).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis.

Schneider，C.A.，Rasband，W.S。和Eliceiri，K.W。NIH Image to ImageJ：25年的图像分析。

Nat. Methods

自然方法

(7), 671–675 (2012).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

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Acknowledgements

致谢

We thank Prof Borek Vojtesek for producing the purified p53R175H mAbs for this study. The SPECT/CT imaging work in this publication was performed at the Preclinical PET-MRI Platform (PPP).

我们感谢Borek Vojtesek教授为本研究生产纯化的p53R175H单克隆抗体。本出版物中的SPECT/CT成像工作是在临床前PET-MRI平台（PPP）上进行的。

Funding

资金

Open access funding provided by Uppsala University. The research was supported by grants from Swedish Cancer society (21 0371 FE, 24 3787 Pj, 24 3485 Pj), The Swedish Childhood Cancer Fund (FT2023-0023, PR2023-0111), Swedish Research Council (Vetenskapsrådet, 2024-03447) and the National Medical Research Council Singapore and NCC Cancer Fund..

。这项研究得到了瑞典癌症协会（21 0371 FE，24 3787 Pj，24 3485 Pj），瑞典儿童癌症基金会（FT2023-0023，PR2023-0111），瑞典研究委员会（Vetenskapsrådet，2024-03447）以及新加坡国家医学研究委员会和NCC癌症基金会的资助。。

Author information

作者信息

Authors and Affiliations

作者和隶属关系

Department of Immunology, Genetics, Pathology, Uppsala University, Uppsala, Sweden

瑞典乌普萨拉乌普萨拉大学免疫学、遗传学、病理学系

Diana Spiegelberg & Marika Nestor

戴安娜·斯皮格尔伯格和玛丽卡·内斯特

Department of Surgical Sciences, Uppsala University, Uppsala, Sweden

瑞典乌普萨拉乌普萨拉大学外科

Diana Spiegelberg

戴安娜·斯皮尔伯格

Divisions of Cellular & Molecular Research, National Cancer Centre Singapore, Singapore, 168583, Singapore

新加坡国家癌症中心细胞与分子研究部，新加坡168583

Le-Ann Hwang & Sashwini Chandra Kumar

黄乐安&萨希文尼·钱德拉·库马尔

School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore

南洋理工大学生物科学学院，新加坡637551

Le-Ann Hwang, Sashwini Chandra Kumar & Kanaga Sabapathy

黄乐安[UNK]沙士威尼·钱德拉·库马尔[UNK]&[UNK][Kanaga Sabathy

Institute of Molecular and Cellular Biology, ASTAR, Singapore, 138673, Singapore

新加坡阿斯塔分子与细胞生物学研究所，新加坡138673

Khian Hong Pua, Xin Yu Koh & Xiao Hui Koh

Khian Hong Pua、Xin Yu Koh和Xiao Hui Koh

Preclinical PET-MRI Platform, Part of Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden

Ram Kumar Selvaraju

拉姆·库马尔·塞尔瓦拉朱

Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden

瑞典斯德哥尔摩卡罗琳学院生命科学实验室微生物学、肿瘤和细胞生物学系

David Lane

大卫·莱恩

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D.S. initiated and designed the study, contributed to experimental studies with focus on ELISA and in vivo biodistribution and SPECT/CT studies, contributed to data analysis and interpretation, and drafted and revised the manuscript. L-A H. initiated and designed the study, contributed to experimental studies with focus on antibody characterization, cell line generation and optical imaging, contributed to data analysis and interpretation, and revised the manuscript.

D、美国发起并设计了这项研究，为实验研究做出了贡献，重点是ELISA和体内生物分布以及SPECT/CT研究，为数据分析和解释做出了贡献，并起草和修订了手稿。L-A H.发起并设计了这项研究，为实验研究做出了贡献，重点是抗体表征，细胞系生成和光学成像，为数据分析和解释做出了贡献，并修订了手稿。

K.H.P. contributed to design, generation and characterization of the p53 knock out cell line, B16 KO.S.C.K. contributed to WB and data analysis.X.Y.K. contributed to IHC and data analysis.X.H.K. contributed to IF and data analysis.R.K.S. contributed to experimental studies with focus on SPECT/CT, contributed to data analysis and interpretation, and revised the manuscript.

K、 H.P.为p53敲除细胞系B16KO的设计，生成和表征做出了贡献。S、 C.K.为WB和数据分析做出了贡献。十、 Y.K.为IHC和数据分析做出了贡献。十、 H.K.为IF和数据分析做出了贡献。R、 K.S.为实验研究做出了贡献，重点是SPECT/CT，为数据分析和解释做出了贡献，并修订了手稿。

K.S. designed part of the study and contributed to the data analysis and interpretation, and revised the manuscript.M.N. initiated and designed the study, contributed to data analysis and interpretation, and revised the manuscript. D.P.L. initiated and designed the study, provided the mABs, contributed to data analysis and interpretation, and revised the manuscript..

K、。M、 N.发起并设计了这项研究，为数据分析和解释做出了贡献，并修订了手稿。D。P、 L.发起并设计了这项研究，提供了单克隆抗体，为数据分析和解释做出了贡献，并修订了手稿。。

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Diana Spiegelberg

戴安娜·斯皮尔伯格

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Spiegelberg, D., Hwang, LA., Pua, K.H.

Spiegelberg，D.，黄，洛杉矶，蒲，K.H。

et al.