Search

Personalized neoantigen vaccines have shown strong immunogenicity in clinical trial, but still face various challenges in facilitating an efficient antitumour immune response. Here we prepared a personalized neoantigen nanovaccine (PNVAC) platform for adjuvant cancer immunotherapy. PNVAC triggered superior protective efficacy against tumor recurrence and promoted longer survival than free neoantigens, especially when combined with anti-PD-1 treatment in a murine tumor model. A phase I clinical trial (ChiCTR1800017319) was initiated to study the safety, immunogenicity, and prophylactic effect of PNVAC on preventing tumor recurrence in patients with high-risk gastric/gastroesophageal junction cancer after adjuvant chemotherapy post-surgical resection. The 1- and 2-year disease-free survival rates were significantly higher than historical control. PNVAC induced both CD4+ and CD8+ T cell responses as well as antigen-experienced memory T cell phenotype. Furthermore, the immune response was persistent and remained evident one year after the vaccination. Our work provides a safe and feasible strategy for development of neoantigen vaccines to delay gastric cancer recurrence after surgery.

Poor infiltration of activated lymphocytes into tumors represents a fundamental factor limiting the therapeutic effect of adoptive cell immunotherapy. A tumor-penetrating peptide, iRGD, has been widely used to deliver drugs into tumor tissues. In this study, we demonstrate for the first time that iRGD could also facilitate the infiltration of lymphocytes in both 3D tumor spheroids and several xenograft mouse models. In addition, combining iRGD modification with PD-1 knockout lymphocytes reveals a superior anti-tumor efficiency. Mechanistic studies demonstrate that the binding of iRGD to neuropilin-1 results in tyrosine phosphorylation of the endothelial barrier regulator VE-cadherin, which plays a role in the opening of endothelial cell contacts and the promotion of transendothelial lymphocyte migration. In summary, these results demonstrate that iRGD modification could promote tumor-specific lymphocyte infiltration, and thereby overcome the bottleneck associated with adoptive immune cell therapy in solid tumors., The therapeutic efficacy of adoptive T cell transfer is limited by their ability to infiltrate solid tumours. Here, the authors show that loading the tumor penetrating cyclic peptide iRGD on the cell surface of T cells enhances their ability to penetrate the tumour, resulting in enhanced efficacy in a mouse model of gastric cancer.

BackgroundPoor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.MethodsT-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.ResultsWe generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.ConclusionAltogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.

Background: The mortality of pancreatic cancer is increasing year by year, and the prognosis of pancreatic cancer with liver metastasis is worse. There is still no recognized effective second-line treatment strategy. In this trial, the efficacy and safety of the triple-drug regimen by S-1 chemotherapy combined with anti-PD-1 McAb (Sintilimab) and anti-angiogenic drug (Anlotinib) were evaluated in pancreatic cancer patients with liver metastasis. Methods: Pancreatic cancer patients with liver metastases and ECOG 0~2 were recruited. These patients had never been given 5-FU, anti-PD-1 McAb and/or anti-angiogenic drugs in first-line treatment. S-1 was administered orally at 25 mg/m2 bid from day 1 to day 14, Sintilimab was administered intravenously at 200 mg on day 1, and Anlotinib was administered orally 12mg qd from day 1 to day 14. This method were repeated every 21 days till disease progression. The therapeutic effect was evaluated every 3 cycles. Progression-free survival (PFS) and overall survival (OS) were estimated via the K-M method. Some patients’ tumor tissues have done high-throughput sequencing and the relationship between with effect has been analyzed. Results: From March 2020 to June 2021, a total of 23 patients were enrolled in this study and 19 patients had objective efficacy evaluation. Two patient (10.5%) had confirmed partial response and eight patients (42.1%) had stable disease. Median PFS and OS were 3.53 months and 7.15 months. Grade 3 adverse events were 26.3%, and there were no grade 4 or above adverse events. The tumor tissues of 16 patients have done high-throughput sequencing, and genetic test results show that 6 patients were evaluated as HRD-High and 10 patients as HRD-Low. Patients with HRD-H had shorter PFS than those with HRD-L (2.43 vs. 5.45 months; p = 0.043), However, OS was not significantly different between these two groups (4.43vs.9.35 months; P = 0.11). Conclusions: This study suggests that S-1 chemotherapy combined with anti-PD-1 McAb (Sintilimab) and anti-angiogenic drug (Anlotinib) is efficacy and safety as a second-line treatment for pancreatic cancer patients with liver metastasis. Subgroup analysis showed that patients with HRD-L had longer PFS. HRD-H were found to be related to worsen PFS, which may indicate that other therapies should be explored regarding to these patients. Citation Format: Juan Du, Xin Qiu, Changchang Lu, Huizi Sha, Yahui Zhu, Weiwei Kong, Xing Li, Xiaoping Qian, Baorui Liu. Efficacy and safety of second-line therapy by S-1 combined with Sintilimab and Anlotinib in pancreatic cancer patients with liver metastasis: A single-arm, phase II clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5192.

9550 Background: Mucosal melanoma is a rare subtype in white populations, but the second most common subtype in Asian populations. This subtype is a more aggressive malignancy, with high risk of metastasis and death. Immune checkpoint inhibitor combined with anti-angiogenic agent has been investigated in many solid tumors, including some preliminary evidence (NCT03086174 and NCT04091217) in Chinese patients with advanced mucosal melanoma. This study investigated the efficacy and safety of camrelizumab plus apatinib in patients with advanced mucosal melanoma. Methods: In this prospective, single-arm study (ChiCTR1900023277), patients with inoperable stage III-IV or recurrent/metastatic mucosal melanoma and Eastern Cooperative Oncology Group performance status of 0-1 were enrolled. Patients received camrelizumab 200 mg once every 2 weeks and apatinib 500 mg once daily until disease progression or intolerable toxicity. The primary endpoint was objective response rate (ORR) according to the Response Evaluation Criteria In Solid Tumors, version 1.1. Results: Between April 2019 and January 2022, a total of 30 patients were enrolled (Table). As of January 2022, 21 patients had at least one efficacy assessment, and the median follow-up duration was 8.1 months. The ORR was 42.9%, including one (4.8%) patient with confirmed complete response, six (28.6%) with confirmed partial response (PR), and two (9.5%) with unconfirmed PR. The disease control rate (DCR) was 81.0%. The median progression-free survival was 7.2 months (95%CI, 5.8-not reached [NR]) in 21 patients, 7.7 months (95%CI, 5.8-NR) in 19 patients with first-line camrelizumab plus apatinib treatment, and 9.8 months (95%CI, 4.2-NR) in 11 patients without prior chemotherapy. The most common treatment-related adverse events in 27 patients with available safety data were fatigue (17 [63.0%]), hypertension (15 [55.6%]), and elevated transaminase (14 [51.9%]). No treatment-related deaths occurred. Exploratory analysis found a tendency that patients with high T cell receptor diversity had better prognosis. Higher frequencies of Vβ-Jβ (including Vβ5-8Jβ2-7, Vβ28J2-4 and Vβ12-5Jβ1-1) indicate better survival, and Vβ12-5Jβ1-1 is an independent factor after multivariate adjustment. Conclusions: Camrelizumab plus apatinib showed favorable ORR and DCR in patients with advanced mucosal melanoma, with an acceptable safety profile. Follow-up for survival outcomes is ongoing. Clinical trial information: ChiCTR1900023277. [Table: see text]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells and augments chemotherapeutics in vivo. Here, we developed sTRAIL-iRGD, a recombinant protein consisting of sTRAIL fused to CRGDKGPDC, a C-terminal end binding peptide with an integrin-binding arginine-glycine-aspartic acid (iRGD) motif. CRGDKGPDC is a tumor-homing peptide with high penetration into tumor tissue and cells. We found that sTRAIL-iRGD internalized into cultured gastric cancer tumor cells and localized to both the tumor mass in vivo and three-dimensional multicellular spheroids in vitro. sTRAIL-iRGD had an antitumor effect in tumor cell lines, multicellular spheroids and nude mice with tumors. Repeated treatment with sTRAIL-iRGD reduced tumor growth and volume in vivo. Mice treated with sTRAIL-iRGD and paclitaxel (PTX) in combination showed no sign of sTRAIL-iRGD-related liver toxicity. Our data suggest that sTRAIL-iRGD is a promising anti-gastric cancer agent with high selectivity and limited systemic toxicity.

Adoptive T cell transfer is one of the most promising ways to combat solid tumors. However, the weak infiltration of T cells into tumor sites has restricted their antitumor efficacy. To overcome this obstacle, we used the lipophilic protein painting strategy to improve tumor targeting and penetrating capacity of lymphocytes for the first time. We synthesized the lipid anchor consisting of a bispecific recombinant protein iRGD-antiEGFR and DSPE-PEG derivates, then successfully inserted it into the membranes of T cells. This surface modification was non-invasive and could efficiently improve the infiltration ability of T cells into multicellular spheroids and tumor masses. The surface modified T cells also displayed superior antitumor activities in EGFR-positive tumor xenografts via systematic infusion. Moreover, the permeability and antitumor efficacy of these surface painted T cells could be remarkably enhanced when used in combination with local low-dose irradiation.

Xinyu Bian,* Puyuan Wu,* Huizi Sha, Hanqing Qian, Qing Wang, Lei Cheng, Yang Yang, Mi Yang, Baorui LiuComprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: In this study, we report a novel kind of targeting with paclitaxel (PTX)-loaded silk fibroin nanoparticles conjugated with iRGD–EGFR nanobody recombinant protein (anti-EGFR-iRGD). The new nanoparticles (called A-PTX-SF-NPs) were prepared using the carbodiimide-mediated coupling procedure and their characteristics were evaluated. The cellular cytotoxicity and cellular uptake of A-PTX-SF-NPs were also investigated. The results in vivo suggested that NPs conjugated with the recombinant protein exhibited more targeting and anti-neoplastic property in cells with high EGFR expression. In the in vivo antitumor efficacy assay, the A-PTX-SF-NPs group showed slower tumor growth and smaller tumor volumes than PTX-SF-NPs in a HeLa xenograft mouse model. A real-time near-infrared fluorescence imaging study showed that A-PTX-SF-NPs could target the tumor more effectively. These results suggest that the anticancer activity and tumor targeting of A-PTX-SF-NPs were superior to those of PTX-SF-NPs and may have the potential to be used for targeted delivery for tumor therapies. Keywords: EGFR, nanobody, iRGD, recombinant protein, targeting drug carriers, antitumor efficiency

Recombinant anti‑epidermal growth factor receptor‑internalizing arginine‑glycine‑aspartic acid (anti‑EGFR single‑domain antibody fused with iRGD peptide) protein efficiently targets the EGFR extracellular domain and integrin αvβ/β5, and shows a high penetration into cells. Thus, this protein may improve penetration of conjugated drugs into the deep zone of gastric cancer multicellular 3D spheroids. In the present study, a novel tumor‑targeting contrast agent for magnetic resonance imaging (MRI) was developed, by coupling gadolinium‑diethylene triamine pentaacetate (Gd‑DTPA) with the bispecific recombinant anti‑EGFR‑iRGD protein. The anti‑EGFR‑iRGD protein was extracted from Escherichia coli and Gd was loaded onto the recombinant protein by chelation using DTPA anhydride. Single‑targeting agent anti‑EGFR‑DTPA‑Gd, which served as the control, was also prepared. The results of the present study showed that anti‑EGFR‑iRGD‑DTPA‑Gd exhibited no significant cyto-toxicity to human gastric carcinoma cells (BGC‑823) under the experimental conditions used. Compared with a conventional contrast agent (Magnevist), anti‑EGFR‑iRGD‑DTPA‑Gd showed higher T1 relaxivity (10.157/mM/sec at 3T) and better tumor‑targeting ability. In addition, the signal intensity and the area under curve for the enhanced signal time in tumor, in vivo, were stronger than Gd‑DTPA alone or the anti‑EGFR‑Gd control. Thus, Gd‑labelled anti‑EGFR‑iRGD has potential as a tumor‑targeting contrast agent for improved MRI.

436 Background: Pancreatic carcinoma is one of the most lethal malignancies worldwide and is characterized by extremely poor prognosis. Gemcitabine plus Nab-paclitaxel (GNp) has been recommended by international guidelines for first-line treatment of locally advanced or metastatic pancreatic carcinoma. However, exploration into molecular biomarkers of GNp treatment is still limited. This study aims to observe and evaluate the efficacy and safety of first-line GNp treatment of pancreatic carcinoma, and to explore potential predictive and prognostic biomarkers to screen advantageous population. Methods: From December 20, 2018 to July 6, 2020, patients with metastatic pancreatic carcinoma, who received first-line GNp treatment, were enrolled. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were overall survival (OS), overall response rate (ORR), disease control rate (DCR), and adverse events (AE). Patients were collected formalin fixed paraffin embedded (FFPE) samples and sequenced by a next-generation sequencing (NGS)-based 450 gene panel carried out by OrigiMed, a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Results: In this study, tissue samples from 101 patients were assessed by NGS, including 58 (57.4%) primary tissues and 43 (42.6%) metastasis tissues. So far, 72 patients were available for efficacy analysis, the ORR and DCR were 38.2% and 77.9%, respectively, and were similar between patients sampled from primary lesions and metastasis lesions. The PFS and OS were 176 days and 308 days, respectively. The most frequently mutated genes were KRAS (91.9%), TP53 (76.8%), CDKN2A (32.3%) and SMAD4 (26.3%). Germline pathogenic BRCA2 mutations were detected in 3 (3.0%) patients, while somatic mutations of BRCA2 were found in 2 (2.0%) patients. For KRAS, missense mutations resulted in single amino acid substitutions primarily at G12 (88.1%), and lower frequencies at Q61 (4.0%) and V14 (1.0%). At G12, five different amino acid substitutions have been identified, with G12D (37.6%) the predominant mutation, G12V (32.7%), G12R (11.9%), G12C (3.0%) and G12A (1.0%). The ORR to AG treatment was significantly higher in TP53-alteration (-alt, 47.1%) than in TP53-wild-type (-WT, 11.1%) subgroups (p=0.01). Median OS times in CDKN2B-alt and CDKN2B-WT patients were 180 days and 323 days (p=0.029), respectively. Conclusions: Current evidence indicates that GNp is a valuable option for the first-line treatment of metastatic pancreatic cancer. TP53 may serve as a novel response-predictive gene for GNp treatment in pancreatic carcinoma. Overall survival was significantly prolonged in CDKN2B-WT patients compared with CDKN2B-alt patients. This study provided insights in identifying patients who might potentially benefit from GNp therapy. Clinical trial information: NCT03768687.

384 Background: To date, chemotherapy remains the only effective treatment of unresectable pancreatic adenocarcinoma. In the last few years, the interest in the use of immunological anticancer strategies is greatly increased. AGIG is a novel chemo-immunotherapy regimen that combines nab-paclitaxel + gemcitabine chemotherapy with sequential recombinant interleukin-2 and granulocyte-macrophage colony stimulating factor (GM-CSF) therapy. We conducted a single-arm prospective phase II study to determine the efficacy and safety of the first-line treatment of advanced pancreatic cancer with AGIG regimen. Methods: Nab-paclitaxel (120 mg/m2) and gemcitabine (1000 mg/m2) were administered intravenously to all patients on days 1 and 8 triweekly, interleukin-2 and GM-CSF (100 µg) were administered subcutaneously on days 3-5 after chemotherapy. The primary end point was ORR by the Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary end points included safety profile, progression-free survival (PFS), overall survival (OS). Patients’ conditions and the efficacy and safety were assessed every 4 cycles. Results: Between 11/2018 and 01/2020, 64 patients were enrolled. In the 64 evaluable patients, the ORR and DCR were 43.75% and 76.6%, respectively. The median follow-up time was 12.1 (range 7.1–22.4) months, the median PFS was 5.7 (range 1.63–15.8) months, and the median OS was 14.2 (range 2.9–22.0) months. The most common adverse event was fever (75%). The incidence of grade 3/4 neutropenia was 4.69%. In subgroup analyses, we found that eosinophil count in the blood elevated three times higher than baseline level predicted a longer survival. Conclusions: The AGIG Chemo-immunotherapy regimen has presented encouraging ORR, OS, and manageable toxicities as first-line therapy for advanced pancreatic cancer. This regimen may be a novel reliable therapeutic option for patients with preserved performance status. The improvement of treatment efficiency may be related to the activation of non-specific immune response. Clinical trial information: NCT0376867. Research Sponsor: The Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School. [Table: see text]

Background: T-cell targeting of mutation-derived epitopes (neoantigens) has been demonstrated to drive anti-tumor responses. Nanotechnology has been reported to enhance immune responses of vaccines. Moreover, immunizing patients against such neoantigens, in combination with checkpoint inhibitor (CPI) and/or antiangiogenic drugs may elicit greater anti-tumor responses. Methods: Patient-specific mutation-containing neoantigens were selected on the basis of tumour-specific mutations whole-exome sequencing (WES) and RNA sequencing. Cancer testis antigens were obtained according to immunohistochemical staining and HLA-binding affinity prediction. Personalized neoantigen/cancer testis antigen nanovaccine (PVAC) is an amphiphiles nanovaccine loaded with personalized vaccine encoding multiple neoantigens designed to induce neoantigen specific T cells responses. Patients will receive PVAC in combination with PD-1 monoclonal antibody and/or antiangiogenic drugs. Primary end points include safety and tolerability. Results: 9 microsatellite stability (MSS) patients, which had relapsed from standard treatments, are enrolled in this study. 4 patients (1 gastric cancer, 1 liver cancer, 1 cervical carcinoma, 1 soft tissue sarcoma) received PVAC in combination with PD-1 mAb, and another 5 patients (2 gastric cancer, 1 colon cancer, 1 NSCLC, 1 renal carcinoma) received PVAC in combination with PD-1 mAb and antiangiogenic therapy. No DLTs were reported. Five patients developed grade 1 subcutaneous indurations in the injection sites,which collected with nanovaccine. One patient had grade 2 rash caused by antiangiogenic drug. No drug related SAEs have been observed. There are 1 CR, 4 PR, 3 SD and 1 PD. Neoantigen specific T cell responses have been detected by IFN-γ Elispot from PBMCs. Conclusions: PVAC is safe and well tolerated. Clinical responses have been observed in combination with PD-1 mAb and antiangiogenic drugs and neoantigen-specific T cell response have been observed after vaccination. Clinical trial information: ChiCTR1900022986. Citation Format: Jia Wei, Qin Liu, Hanqing Qian, Jie Shao, Lijing Zhu, Lifeng Wang, Lixia Yu, Huizi Sha, Baorui Liu. Personalized neoantigen/cancer testis antigen nanovaccine (PVAC) in combination with PD-1 monoclonal antibody and/or antiangiogenic treatment in patients with metastatic solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1049.

Background: 35% of stage IIIB/C Gastric cancer patients will recurrent after D2 gastrectomy within one year. Mutation-derived epitopes (neoantigens) has been demonstrated to induce tumor cell specific immune responses controlling the tumor growth. Nanovaccine can increase antigen presentation efficiency and elicit potent antitumor T cell responses with robust therapeutic efficacy. We hypothesized that vaccination with neoantigens/cancer testis (CT) antigens could expand pre-existing and induce antigen-specific T-cells populations, favouring of tumor control enhancement. Here, we report the first-in-human application of this concept in gastric cancer. Methods: Patient-specific mutation-containing neoantigens were selected on the basis of tumour-specific mutations whole-exome sequencing (WES) and RNA sequencing. Cancer testis antigens were obtained according to immunohistochemical staining and HLA-binding affinity prediction. PVAC is an amphiphiles nanovaccine loaded with multiple personalized neoantigens/cancer testis antigens designed to induce antigen specific T cells and associated antitumor responses. PVAC will be administrated to stage IIIB/IIIC gastric carcinoma after six cycles of adjuvant chemotherapy (S-1/Oxaliplatin or S-1/docetaxel). Each patient received PVAC by subcutaneous injection on Days 1, 4, 8, 15, 22, 43, 64, 85, 169, administrated with the adjuvant montanide ISA 51 VG. Safety, immunogenicity and clinical efficacy will be evaluated. Results: 25 stage IIIB or IIIC gastric cancer patients were enrolled in this study. Mean age was 54.3 years old (range: 34-70), and ECOG performance scores were 0 or 1. Repeated dosing has been well tolerated with mild local discomfort and no DLTs. 10 of the 25 patients developed grade 1 local skin reactions with redness, swelling or subcutaneous indurations in the injection sites. Three patients were observed grade 2 local skin reactions in the injection sites. Four had grade 1 to 2 fever. No SAEs related to PVAC have been observed. Among median follow up time of 11.6 months (range: 7.5-24.0 months), only one patient had local recurrence at 10.5 months after surgery. The rest 24 patients remain disease free on study. Neoantigen specific T cell responses have been detected by IFN-γ Elispot from PBMCs. Conclusions: PVAC is a multiple neoantigens/CT antigens nanovaccine that personalizes tumor specific antigens and the individual patient's capacity to respond. Addition of PVAC may prolong progression-free survival (PFS) after the standard of care chemotherapy. Clinical trial information: ChiCTR1800017319. Citation Format: Qin Liu, Hanqing Qian, Jie Shao, Qiuping Xu, Huizi Sha, Yang Yang, Weifeng Wang, Lixia Yu, Baorui Liu, Jia Wei. A phase I study to assess the safety, tolerability, and immunogenicity of personalized neoantigen/cancer testis antigen nanovaccine (PVAC) for high-risk resected gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT237.

3134 Background: T-cell targeting of mutation-derived epitopes (neoantigens) has been demonstrated to drive anti-tumor responses. Nanotechnology has been reported to enhance immune responses of vaccines. Moreover, immunizing patients against such neoantigens, in combination with checkpoint inhibitor (CPI) and/or antiangiogenic drugs may elicit greater anti-tumor responses. Methods: Patient-specific mutation-containing neoantigens were selected on the basis of tumour-specific mutations whole-exome sequencing (WES) and RNA sequencing. Cancer testis antigens were obtained according to immunohistochemical staining and HLA-binding affinity prediction. Personalized neoantigen/cancer testis antigen nanovaccine (PVAC) is an amphiphiles nanovaccine loaded with personalized vaccine encoding multiple neoantigens designed to induce neoantigen specific T cells responses. Patients will receive PVAC in combination with PD-1 monoclonal antibody and/or antiangiogenic drugs. Primary end points include safety and tolerability. Results: 13 microsatellite stability (MSS) patients, which had relapsed from standard treatments, are enrolled in this study. 5 patients (1 gastric cancer, 1 liver cancer, 1 cervical carcinoma, 1 soft tissue sarcoma, 1 renal carcinoma) received PVAC in combination with PD-1 mAb, and another 8 patients (3 gastric cancer, 2 colon cancer, 1 NSCLC, 1 renal carcinoma) received PVAC in combination with PD-1 mAb and antiangiogenic therapy. No DLTs were reported. Five patients developed grade 1 and one patients developed grade 2 subcutaneous indurations in the injection sites, which collected with nanovaccine. One patient had grade 2 rash caused by antiangiogenic drug. No drug related SAEs have been observed. There are 1 CR, 6 PR, 4 SD and 2 PD. Neoantigen specific T cell responses have been detected by IFN-γ ELISpot from PBMCs. Conclusions: PVAC is safe and well tolerated. Clinical responses have been observed in combination with PD-1 mAb and antiangiogenic drugs and neoantigen-specific T cell response have been observed after vaccination. Clinical trial information: ChiCTR1900022986 .

4535 Background: 35% of stage IIIB/C Gastric cancer patients will recurrent after D2 gastrectomy within one year. Mutation-derived epitopes (neoantigens) has been demonstrated to induce tumor cell specific immune responses controlling the tumor growth. Nanovaccine can increase antigen presentation efficiency and elicit potent antitumor T cell responses with robust therapeutic efficacy. We hypothesized that vaccination with neoantigens/cancer testis (CT) antigens could expand pre-existing and induce antigen-specific T-cells populations, favouring of tumor control enhancement. Here, we report the first-in-human application of this concept in gastric cancer. Methods: Patient-specific mutation-containing neoantigens were selected on the basis of tumour-specific mutations whole-exome sequencing (WES) and RNA sequencing. Cancer testis antigens were obtained according to immunohistochemical staining and HLA-binding affinity prediction. PVAC is an amphiphiles nanovaccine loaded with multiple personalized neoantigens/cancer testis antigens designed to induce antigen specific T cells and associated antitumor responses. PVAC will be administrated to stage IIIB/IIIC gastric carcinoma after six cycles of adjuvant chemotherapy (S-1/Oxaliplatin or S-1/docetaxel). Each patient received PVAC by subcutaneous injection on Days 1, 4, 8, 15, 22, 43, 64, 85, 169, administrated with the adjuvant montanide ISA 51 VG. Safety, immunogenicity and clinical efficacy will be evaluated. Results: 25 stage IIIB or IIIC gastric cancer patients were enrolled in this study. Mean age was 54.3 years old (range: 34-70), and ECOG performance scores were 0 or 1. Repeated dosing has been well tolerated with mild local discomfort and no DLTs. Three patients were observed grade 2 local skin reactions in the injection sites. No SAEs related to PVAC have been observed. Among median follow up time of 12.6 months (range: 8.5-25.0 months), only two patients had local recurrence at 24.0 months and 10.5 months after surgery, respectivelt. The rest 23 patients remain disease free on study. Neoantigen specific T cell responses have been detected by IFN-γ Elispot from PBMCs. Conclusions: PVAC is a multiple neoantigen/CT antigens nanovaccine that personalizes tumor specific antigens and the individual patient’s capacity to respond. Addition of PVAC may prolong progression-free survival (PFS) after the standard of care chemotherapy. Clinical trial information: ChiCTR1800017319 .

e16773 Background: Pancreatic cancer is a kind of digestive tumor with low incidence but high degree of malignancy. It is characterized by difficult in early detection and invasive metastasis. Together with high recurrence and metastasis rate after resection, the prognosis of pancreatic cancer is extremely poor. To explore the role of BRCA1 mRNA expression in the chemotherapy of unresectable pancreatic cancer and the synergistic effect of chemotherapy and immunotherapy, our center has carried out a clinical trial which focuses on individualized chemotherapy combined with sequential immunotherapy according to BRCA1 mRNA expression in the first-line treatment of unresectable pancreatic cancer. Methods: The expression of BRCA1 mRNA in tumor tissues of patients with pancreatic cancer was detected. According to the expression level, gemcitabine combined with nab-paclitaxel-based or gemcitabine combined with oxaliplatin-based biweekly chemotherapy combined with sequential GM-CSF and IL-2 immunotherapy was applied. Patients’ conditions and the efficacy and safety were assessed every 4 cycles. Results: A total of 25 patients were enrolled in the study. All of them were observed for toxic side effects and 24 of them were evaluated for efficacy. The median overall survival and median progression-free survival were 11.9 months and 6.3 months. The disease control rate was 91.7%, of which 37.5% (9/24) patients achieved partial remission (PR), 54.2% (13/24) patients achieved stable disease (SD) and 8.3% (2/24) patients were assessed as progressive disease(PD). Of the 15 patients with medium or high expression in BRCA1 mRNA, 7 achieved PR and 8 achieved SD. Of the 9 patients with low BRCA1 mRNA expression, 2 achieved PR, 5 achieved SD and 2 had PD. The proportion of eosinophils in the blood of some patients with good therapeutic effects was significantly higher than that before treatment. Hematological and non-hematological toxicity during the treatment were mostly grade 1~2. The two most common grade 3~4 adverse events were fever and thrombocytopenia. Conclusions: Our results suggest that individualized selection of chemotherapy combined with sequential immunotherapy according to BRCA1 mRNA expression level in the treatment of unresectable pancreatic cancer have curative effect and controllable adverse reactions. The improvement of treatment efficiency may be related to the activation of non-specific immune response.

Paclitaxel is widely used as a radiosensitizer for various tumors, including esophageal cancer, but its therapeutic effect remains to be improved. In this study, we constructed a novel nano-radiosensitizer, anti-EGFR-iRGD-conjugated (iE)-PRNPs, by conjugating the recombinant protein anti-epidermal growth factor receptor (EGFR)-internalizing arginine-glycine-aspartic (iRGD) to the surface of paclitaxel-loaded red blood cell membrane nanoparticles (PRNPs). The iE-PRNPs were confirmed to possess tumor-targeting, high penetrability, and sustained release properties that free paclitaxel does not possess. Compared with that of paclitaxel, the sensitizer enhancement ratio of iE-PRNPs was significantly increased (1.32-fold and 1.25-fold) in esophageal cancer cells with high and low expression levels of EGFR, respectively. Additionally, compared with that of unmodified PRNPs, the sensitizer enhancement ratio of iE-PRNPs in EGFR-overexpressing esophageal cancer cells was significantly increased (1.27-fold), while that of PRNPs in esophageal cancer cells with a low EGFR expression level increased slightly (1.06-fold). The improved radiosensitization effect was associated with enhanced G2/M arrest, increased reactive oxygen species, and more effective induction of DNA double-strand breaks. In summary, iE-PRNPs appear to be a novel type of radiosensitizer with the potential to overcome the bottleneck of esophageal cancer radiotherapeutic efficacy.

Hong Chen,1–3,* Huizi Sha,2,* Lianru Zhang,2 Hanqing Qian,2 Fangjun Chen,2 Naiqin Ding,2 Liulian Ji,2 Anqing Zhu,2 Qiuping Xu,2 Fanyan Meng,2 Lixia Yu,2 Yan Zhou,3 Baorui Liu1,2 1The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, People’s Republic of China; 2The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, People’s Republic of China; 3Department of Oncology, Yixing People’s Hospital, Jiangsu, People’s Republic of China *These authors contributed equally to this work Background: There is currently much interest in cancer cell targeting and tumor penetrating for research and therapeutic purposes.Purpose: To improve targeting delivery of antitumor drugs to gastric cancer, in this study, a tumor-targeting biocompatible drug delivery system derived from erythrocyte membrane for delivering paclitaxel (PTX) was constructed.Methods: Erythrocyte membrane of human red blood cells (RBCs) were used for preparing of erythrocyte membrane-derived vesicles. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide[polyethylene glycol]-3400) (DSPE-PEG-MAL), a phospholipid derivative, was used to insert tumor-targeting molecular into erythrocyte membrane-derived vesicles. A lipid insertion method was used to functionalize these vesicles without the need for direct chemical conjugation. Furthermore, a tumor-penetrating bispecific recombinant protein named anti-EGFR-iRGD was used for the first time in this work to enable nanosystem to target and penetrate efficiently into the tumor site.Results: Paclitaxel (PTX)-loaded anti-EGFR-iRGD-modified erythrocyte membrane nanosystem (anti-EGFR-iRGD-RBCm-PTX, abbreviated to PRP) were manufactured. PRP was spheroid, uniformly size, about 171.7±4.7 nm in average, could be stable in vitro for 8 days, and released PTX in a biphasic pattern. PRP showed comparable cytotoxicity toward human gastric cancer cells in vitro. In vivo studies showed that, PRP accumulated in tumor site within 2h of administration, lasted longer than 48h, and the tumor volume was reduced 61% by PRP treatment in Balb/c nude mice, without causing severe side effects.Conclusion: PRP has potential applications in cancer treatment and as an adjunct for other anticancer strategies. Keywords: tumor-penetrating bispecific recombinant protein, erythrocyte membrane, biomimetic nanoparticle, drug delivery

Zhen Zhang,1,2 Hanqing Qian,1 Jie Huang,1 Huizi Sha,1 Hang Zhang,1 Lixia Yu,1 Baorui Liu,1 Dong Hua,3 Xiaoping Qian1 1Comprehensive Cancer Center, Nanjing Drum Tower Hospital, Clinical Cancer Institute of Nanjing University, Nanjing, Jiangsu, People’s Republic of China; 2Department of Integrated Traditional Chinese Medicine and Western Medicine Oncology, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, People’s Republic of China; 3Department of Medical Oncology, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, People’s Republic of China Background: Red blood cell membrane-coated nanoparticle (RBCm-NP) platform, which consist of natural RBCm and synthetic polymeric core, can extend circulation time in vivo with an improved biocompatibility and stability of this biomimetic nanocarrier. To achieve better bioavailability of antitumor drugs that were loaded in RBCm-NPs, the functionalization of coated RBCm with specific targeting ability is essential. Bispecific recombinant protein anti-EGFR-iRGD, containing both tumor penetrating peptide (internalizing RGD peptide) and EGFR single-domain antibody (sdAb), seems to be an optimal targeting ligand for RBCm-NPs in the treatment of multiple tumors, especially colorectal cancer with high EGFR expression. Materials and methods: We modified the anti-EGFR-iRGD recombinant protein on the surface of RBCm-NPs by lipid insertion method to construct iE–RBCm–PLGA NPs and confirmed the presentation of active tumor-targeting ability in colorectal cancer models with high EGFR expression when compared with RBCm–PLGA NPs. In addition, potential anti- tumor drug gambogic acid (GA) was loaded into the NPs to endow the antitumor efficiency of iE–RBCm–GA/PLGA NPs. It was simultaneously evaluated whether GA can reach better biocompatibility benefiting from the improved antitumor efficiency of iE–RBCm–GA/PLGA NPs in colorectal cancer models. Results: We successfully modified anti-EGFR-iRGD proteins on the surface of biomimetic NPs with integrated and stable “shell–core” structure. iE–RBCm–PLGA NPs showed its improved targeting ability in vitro (multicellular spheroids [MCS]) and in vivo (nude mice bearing tumors). Besides, no matter on short-term cell apoptosis at tumor site (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]) and long-term tumor inhibition, iE–RBCm–GA/PLGA NPs achieved better antitumor efficacy than free GA in spite of the similar effects of cytotoxicity and apoptosis to GA in vitro. Conclusion: We expect that the bispecific biomimetic nanocarrier can extend the clinical application of many other potential antitumor drugs similar to GA and become a novel drug carrier in the colorectal cancer treatment. Keywords: biomimetic nanocarrier, anti-EGFR-iRGD recombinant protein, gambogic acid, targeting ability, antitumor efficiency, colorectal cancer

Three-dimensional (3D) culture methods, building a bridge between the traditional two-dimensional (2D) monolayer cell culture and the in-vivo conditions, launch a new era in cell culture. A reliable and stable method for 3D cell culture is required for further cytology research or chemosensitivity tests, considering its high similarity with conditions in vivo . Here we present a novel 3D tumor cell culture model based on mulberry silk fibroin derived from Bombyx mori , which is easy to access in China. This is a biocompatible 3D silk fibroin scaffold, without adding any toxic organic solvents, surfactants or other toxic agents during the preparation process. We investigated its microstructure and confirmed that the gastric cancer cell lines were successfully seeded and grown on the scaffold. In conclusion, the silk fibroin scaffold, as a promising 3D cell culture model, has the potential for more accurate cytology research and clinical chemosensitivity tests in the future.

Radiotherapy has been the primary method for the local control of several types of unresectable tumor, including gastric cancer. Patients with gastric cancer frequently express high levels of epidermal growth factor receptor (EGFR), which have been found to increase following radiotherapy treatment. This provides a basis for the combination of antibodies targeting EGFR and radiotherapy. In our previous study, a protein (anti‑EGFR‑iRGD) with bispecific targets and high permeability was constructed, and its effects on inhibiting the proliferation of gastric cancer cells was investigated. In the present study, the capacity of anti‑EGFR‑iRGD to modulate a radiation response was investigated and the specific mechanisms underlying these interactions were evaluated in gastric cancer cell lines and xenografts exhibiting high levels of EGFR. The radioenhancement of anti‑EGFR‑iRGD was associated with inhibited radiation‑induced upregulation of EGFR, inhibited cell proliferation and promotion of cell apoptosis. In addition, anti‑EGFR‑iRGD appeared to permeate more into the tumor tissue following radiation. These findings indicated that the recombinant protein anti‑EGFR‑iRGD was a selective and effective radiosensitizer in EGFR‑overexpressing gastric cancer cells and xenografts. These results further suggested that anti‑EGFR‑iRGD is a potential superior EGFR‑targeted therapy combined with radiotherapy. Overall, the present study suggested that anti‑EGFR‑iRGD may be a promising candidate for preclinical and clinical use.

Radiotherapy has been the primary method for the local control of several types of unresectable tumor, including gastric cancer. Patients with gastric cancer frequently express high levels of epidermal growth factor receptor (EGFR), which have been found to increase following radiotherapy treatment. This provides a basis for the combination of antibodies targeting EGFR and radiotherapy. In our previous study, a protein (anti‑EGFR‑iRGD) with bispecific targets and high permeability was constructed, and its effects on inhibiting the proliferation of gastric cancer cells was investigated. In the present study, the capacity of anti‑EGFR‑iRGD to modulate a radiation response was investigated and the specific mechanisms underlying these interactions were evaluated in gastric cancer cell lines and xenografts exhibiting high levels of EGFR. The radioenhancement of anti‑EGFR‑iRGD was associated with inhibited radiation‑induced upregulation of EGFR, inhibited cell proliferation and promotion of cell apoptosis. In addition, anti‑EGFR‑iRGD appeared to permeate more into the tumor tissue following radiation. These findings indicated that the recombinant protein anti‑EGFR‑iRGD was a selective and effective radiosensitizer in EGFR‑overexpressing gastric cancer cells and xenografts. These results further suggested that anti‑EGFR‑iRGD is a potential superior EGFR‑targeted therapy combined with radiotherapy. Overall, the present study suggested that anti‑EGFR‑iRGD may be a promising candidate for preclinical and clinical use.

An integrated γ-cyclodextrin-gated mesoporous silica delivery system via dual dynamic covalent bonds was constructed with dual drug loading for simultaneous and cascade release in targeted combination drug therapy.

Paclitaxel (PTX) is a cytotoxic chemotherapy drug with encouraging activity in human malignancies. However, free PTX has a very low oral bioavailability due to its low aqueous solubility and the gastrointestinal drug barrier. In order to overcome this obstacle, we have designed erythrocyte membrane nanoparticles (EMNP) using sonication method. The permeability of PTX by EMNP was 3.5-fold (Papp = 0.425 nm/s) and 16.2-fold (Papp = 394.1 nm/s) higher than free PTX in MDCK-MDR1 cell monolayers and intestinal mucosal tissue, respectively. The in vivo pharmacokinetics indicated that the AUC0-t (μg/mL·h) and Cmax (μg/mL) of EMNP were 14.2-fold and 6.0-fold higher than that of free PTX, respectively. In summary, the EMNP appears to be a promising nanoformulation to enhance the oral bioavailability of insoluble and poorly permeable drugs.

Stimuli-responsive multidrug delivery systems need to be invented for clinical combination therapy by the controlled release behavior of each drug individually. A facile, effective, and universal platform was built for simultaneous and cascade release of two drugs from DNA-gated gold nanorod-embedded mesoporous silica nanoparticles (MSNs) functionalized with titanium(IV)-chelating phosphonates. Coordination chemistry is the first strategy for DNA capping through multivalent chelating interactions in drug delivery systems not only as a gatekeeper but also as a drug carrier. One drug was entrapped in the MSN pores, and the other drug intercalated within the capping duplex DNA. The two drugs were simultaneously released upon triggering of endonuclease degradation or photothermal dehybridization and were successively released upon first triggering of basic pH and subsequent triggering of photothermal heating. The designed native DNA-gated MSN delivery systems integrated a simultaneous and cascade release of two drugs into smart single nanovehicles for promising practical applications in targeted combination drug therapy.

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

KLA (sequence, KLAKLAKKLAKLAK) is a peptide which leads to programmed cell death by disrupting the mitochondrial membrane. However, low penetration in tumors greatly limits its application and efficacy. To develop a KLA-based cancer therapy, KLA-iRGD, a recombinant protein was constructed. It consists of the KLA peptide and iRGD (CRGDKGPDC), a tumor-homing peptide with high penetration into tumor tissue and cells. The conjugated KLA exhibits pro-apoptotic activity to prevent the growth of a tumor once it is inside the cell. Once KLA-iRGD is internalized in cultured tumor cells, via the activation of the receptor neuropilin-1, it spreads extensively throughout the mass of the tumor. The recombinant KLA-iRGD protein showed antitumor activity in vivo in mice and in vitro in tumor cell lines. Repeated treatment with KLA-iRGD greatly prevented tumor growth, resulting in a considerable reduction in tumor volume. According to our data, KLA-iRGD may serve as a potential anticancer agent with limited systemic toxicity and high selectivity for the treatment of MKN45 gastric cancer, which may lead to the enhancement of new targeted anticancer agents.

The objective of this study is to develop a new-type biodegradable, biocompatible curcumin-loaded nanoerythrosomes (Cur-RBC-NPs) by means of the sonication method. The size of Cur-RBC-NPs was optimized by varying drug loading parameters. The morphology, size distribution, stability, in vitro release pattern, cellular uptake of nanoparticles and in vitro anti-tumor effects were evaluated, respectively. The results showed the prepared Cur-RBC-NPs were nearly uniform spheres, with an average diameter of (245.7 ± 1.3) nm. Encapsulation efficiency (EE) and load efficiency (LE) of Cur-RBC-NPs were 50.65% ± 1.36% and 6.27% ± 0.29%. And the nanoparticles had a good sustained release property. According to the in vitro experiment, Cur-RBC-NPs were effectively taken in by tumor cells, and exhibited a significant anti-tumor effect. In conclusion, the method for preparing Cur-RBC-NPs is convenient, with a good sustained release behavior and anti-tumor efficacy, and so expected to be a new-type nano-drug delivery system in clinical practice.

e24324Background: Radiotherapy resistance of tumor cells remains a major therapeutic problem in tumor therapy, and hypoxia is one of the most major resistance mechanisms in various tumor types. A n...

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

It has been a major challenge for drug penetration in solid tumor tissues because of the complicated tumor microenvironment. We have previously constructed a protein of bispecific targets and high permeability named anti-EGFR-iRGD and investigated its inhibiting cell proliferation of gastric cancer. Paclitaxel (PTX) is widely used for treating various kinds of cancer. In this paper, we investigated the effects of anti-EGFR-iRGD in combination with chemotherapeutic drugs including PTX in epidermal growth factor receptor highly expressing gastric cancer. We demonstrated the therapeutic efficacy of PTX combined with anti-EGFR-iRGD on monolayer cells (2D), multicellular spheroids (3D) and tumor-bearing mice for the first time and investigated the mechanism of this synergy effect. Our results provide impetus for further studies to use anti-EGFR-iRGD with standard cytotoxic treatment regimens for enhancing therapy of gastric cancer patients.

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

e14076 Background: We report a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this platform, T-lymphocyte membranes were camouflaged to the surface of poly-lactic-co-glycolic acid nanoparticles, with local low-dose irradiation (LDI) as a chemoattractant. These carriers were further anchored with the recombinant protein anti-EGFR-iRGD, improving tumor accumulation, facilitating tumor uptake. Methods: The T-lymphocyte membrane coating was verified by dynamic light scattering, transmission electron microscopy and confocal laser scanning microscopy. The particle phagocytosis study was performed using a human phagocytic cell line. In vivo NIR fluorescence imaging was performed to monitor the route of nanoparticles. EGFR expression of tumor cells was tested before and after LDI. Results: This new platform reduced phagocytosis of macrophages by 23.99% (p = 0.002). iRGD-EGFR anchored T-lymphocyte membrane-encapsulated nanoparticles accumulated in tumor site more than unfunctionalized groups, while LDI significantly enhanced the targeting ability. LDI could up-regulate EGFR expression on tumor cells, which was important in the process of localization of iRGD-EGFR anchored T-lymphocyte membrane-encapsulated nanoparticles in tumors. Conclusions: This new platform included both the long circulation time and tumor sites accumulation ability while LDI could significantly enhance the tumor accumulation ability.

e14047 Background: There is a great interest in targeting and penetrating of cancer cells for research or therapeutic purposes. Red blood cells (RBCs) are readily available and fully biocompatible long-circulating intravascular carriers that are amenable to chemical modifications, drug loading and reinjection. The purpose of this study was to design a tumor-targeting biocompatible drug delivery system for delivery of antitumor drugs. Methods: DSPE-PEG-MAL, phospholipid derivatives was used to insert into erythrocyte membrane nanoparticles. To make nanoparticles active targeting to the tumor site, a tumor-penetrating bispecific recombinant protein named anti-EGFR-iRGD was used. The characterization, bio-distribution, tumor targeting ability and antitumor activity of paclitaxel loaded anti-EGFR-iRGD modified erythrocyte membrane nanoparticle were evaluated. Results: In this study, anti-EGFR-iRGD-RBC-PTX nanoparticles was successfully constructed with a size of around 100 nm. A lipid-insertion method is employed to functionalize these nanoparticles without the need for direct chemical conjugation. It showed signifcantly targeted skill and increased cytotoxic effect toward both nontargeted RBC-PTX and combination of anti-EGFR-iRGD and RBC-PTX. The tissue distribution and antitumor assays in mice bearing gastric cancer xenograft confrmed the superior penetration tumor effcacy and antitumor activity of anti-EGFR-iRGD-RBC-PTX. Conclusions: We designed and successfully prepared a novel anti-EGFR-iRGD decorated, erythrocyte membrane sourced nanoparticle for targeted drug delivery, with enhanced tumor targeting and anti-tumor effect. Anti-EGFR-iRGD-RBC-PTX represents a potential effective nanomedicine against gastric cancer.