Evaluation of 68Ga-labeled TPP-1: A Targeted PD-L1 Probe for PET Imaging of Colorectal Cancer

Colorectal cancer is a common clinical malignancy of the digestive system. In recent years, with the improvement of living standards and changes in dietary habits, the incidence and death rate of colorectal cancer has been on the rise [1]. At present, effective treatments for colorectal cancer include surgery, chemotherapy and radiotherapy, but most lesions have already metastasized at the time point of being detected, resulting in a high death rate. The development of immunotherapeutic agents targeting PD-1/PD-L1 has affected the treatment strategy for many digestive system tumors due to their high efficacy and low toxicity compared to conventional treatments [2,3]. Unfortunately, although immuno-blocking therapy (IBT) targeting the PD-1/PD-L1 pathway has shown impressive clinical results, only a small number of patients have been benefited [4].


Introduction
Colorectal cancer is a common clinical malignancy of the digestive system. In recent years, with the improvement of living standards and changes in dietary habits, the incidence and death rate of colorectal cancer has been on the rise [1]. At present, effective treatments for colorectal cancer include surgery, chemotherapy and radiotherapy, but most lesions have already metastasized at the time point of being detected, resulting in a high death rate. The development of immunotherapeutic agents targeting PD-1/PD-L1 has affected the treatment strategy for many digestive system tumors due to their high efficacy and low toxicity compared to conventional treatments [2,3]. Unfortunately, although immuno-blocking therapy (IBT) targeting the PD-1/PD-L1 pathway has shown impressive clinical results, only a small number of patients have been benefited [4].
Multiple studies have shown that PD-L1 antigen expression is a prognostic marker of response to PD-1/PD-L1 IBT. Recently, positron emission tomography (PET) imaging with radiolabeled anti-PD-L1 antibodies has shown vital advantages over immunohistochemistry in evaluating PD-L1 expression [5]. PD-1/PD-L1-targeted nuclear imaging enables the quantification of primary and metastatic lesions as well as systemic PD-1/PD-L1 expression by replacing invasive tests such as puncture, providing a more accurate screening method for patients on PD-1/PD-L1 immunotherapy. However, the disadvantage of nuclide-labeled intact monoclonal antibodies is of the need to use long half-life nuclear and prolong the time required for imaging. Herein, small molecule peptide probes are ideal as a PD-L1 PET imaging agent as they are more penetrating to tumors and can be rapidly up taken by tumors and cleared from the blood and background tissue, enabling the same-day imaging by one injection. TPP-1 is a PD-L1-targeting and clinically translatable peptide that has demonstrated its potential for rapid and highly specific binding to PD-L1 in several preclinical studies [6,7]. In this study, the tracer 68 Ga-TPP-1 showed good application in PD-L1 expression imaging in MC38 xenograft tumors and had promising applications in guiding immunotherapy.

Methods
For the synthesis of 68 Ga-TPP-1, TPP-1-DOTA (50μg, in 0.25M sodium acetate, 1mL) was mixed with fresh 68 Ga 3+ (in 0.05M HCl, 4mL). The reaction mixture was incubated at 100℃ for 10min and purified using a sep-pak C-18 cartridge with 50% ethanol as the eluent. For animal studies, the right upper limb of three combined immunodeficient mice (line name:

Results
The radio-HPLC results showed the radiochemical purity of 68 Ga-TPP-1 was approximately 99% after purification. 68 Ga-TPP-1 was stable (>95%) in 0.01M PBS at 37℃ for 6h, as well as displayed acceptable stability (> 90%) in 1% mouse serum for 4 hours. PET imaging showed that the tumor was of highly uptake of 68 Ga-TPP-1, and the physiological uptake in the kidney and bladder were detected. Tumor uptake of 68 Ga-TPP-1 was detected at 30 min post injection, and the SUV max of the tumor and contralateral muscles was 0.95±0.06 and 0.42±0.11 at 60min post-injection, respectively. The targeting uptake at the tumor increased gradually during the first-hour post injection, and the tumor can be clearly seen at 90min post injection. The tumor-to-muscle (T/M) uptake ratio peaked at 2.33±0.55 at 60min post injection. Uptake of 68 Ga-TPP-1 in tumor was significantly (p<0.05) blocked when compared to non-blocked group, where the SUV max of the tumor was 0.33 ( Figure  1). This revealed that, in addition to its physiological distribution in the kidney and bladder, 68 Ga-TPP-1 has better specific binding to PD-L1 high expression colorectal cancer. Based on the immunohistochemistry results of tumors tissues, PD-L1 antigens were expressed in MC38 tumor massively where the brown-yellow area stands for positive expression (Figure 1).

C.
Immunohistochemistry confirmed the high PD-L1 expression in tumor tissue (original magnification ×200).

Discussion
In contrast to traditional treatments for gastrointestinal tumors, immunotherapy focuses on improving the microenvironment in and around the tumor and enhancing the body's own immune function, resulting in the inhibition of tumor cell growth. However, the low efficiency and high cost of treatment restrict the application of IBT, and the current methods of screening patients for benefit, such as IHC and dMMR, do not accurately predict the efficacy of PD-1/ PD-L1 IBT, which remains a serious issue pending to be solved. The clinical significance of immune-related molecular imaging is demonstrated in guiding treatment and predicting therapeutic efficacy. Functional imaging in nuclear medicine has been shown to be useful in assessing PD-1/PD-L1 expression in a variety of tumor types. Currently, three PD-1/PD-L1 targeting probes have been successfully applied in translational clinical studies [5,9]. For example, Nature Medicine reported that it is possible to image non-small cell lung cancer, advanced bladder cancer and triple negative breast cancer with 89 Zr-Atezolizumab PET, demonstrating heterogeneous variation between lesions, patients and tumor types as well as laying the foundation for establishing clinical treatment options. In terms of PD-1/PD-L1 molecular imaging, the targeting ability and metabolic pathway of the probes play a key role. Objective issues such as poor penetration of intact antibodies in solid tumor tissue, low stability, restricted routes of drug delivery and difficulty to control pharmacokinetics limit their use in clinics [10].
In this situation, the advantages of small molecule PD-L1 inhibitors with a high degree of specificity are highlighted. Compared to antibodies, peptide-based PD-1/PD-L1 inhibitors have favorable biocompatibility and ease of synthesis, offering an advantage in avoiding accumulation in non-target organs. Therefore, the development of low molecular weight probes as 68 Ga-TPP-1 that specifically bind to PD-L1 with high affinity will be an important direction for future research. This will enable highly specific detection of PD-1/PD-L1 expression in patients while performing SPECT or PET imaging in a short time after probe injection and will provide a more convenient imaging method for the clinical application of tumor immunotherapy.