The First Approved Gene Therapy Product for Cancer Ad- p53 (Gendicine): 12 Years in the Clinic

Abstract

Gendicine (recombinant human p53 adenovirus), developed by Shenzhen SiBiono GeneTech Co. Ltd., was approved in 2003 by the China Food and Drug Administration (CFDA) as a first-in-class gene therapy product to treat head and neck cancer, and entered the commercial market in 2004. Gendicine is a biological therapy that is delivered via minimally invasive intratumoral injection, as well as by intracavity or intravascular infusion. The wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumor suppressor that is activated by cellular stress, and mediates cell-cycle arrest and DNA repair, or induces apoptosis, senescence, and/or autophagy, depending upon cellular stress conditions. Based on 12 years of commercial use in >30,000 patients, and >30 published clinical studies, Gendicine has exhibited an exemplary safety record, and when combined with chemotherapy and radiotherapy has demonstrated significantly higher response rates than for standard therapies alone. In addition to head and neck cancer, Gendicine has been successfully applied to treat various other cancer types and different stages of disease. Thirteen published studies that include long-term survival data showed that Gendicine combination regimens yield progression-free survival times that are significantly longer than standard therapies alone. Although the p53 gene is mutated in >50% of all human cancers, p53 mutation status did not significantly influence efficacy outcomes and long-term survival rate for Ad-p53-treated patients. To date, Shenzhen SiBiono GeneTech has manufactured 41 batches of Gendicine in compliance with CFDA QC/QA requirements, and 169,571 vials (1.0 × 10¹² vector particles per vial) have been used to treat patients. No serious adverse events have been reported, except for vector-associated transient fever, which occurred in 50–60% of patients and persisted for only a few hours. The manufacturing accomplishments and clinical experience with Gendicine, as well as the understanding of its cellular mechanisms of action and implications, could provide valuable insights for the international gene therapy community and add valuable data to promote further developments and advancements in the gene therapy field.

Introduction

Gendicine (recombinant human p 53 adenovirus [Ad5RSV-p53]), developed by Shenzhen SiBiono GeneTech Co. Ltd., was approved by the China Food and Drug Administration (CFDA) in October 2003 for the treatment of head and neck squamous-cell carcinoma (HNSCC).^1
–3 Gendicine was the first-ever approved gene therapy drug, and also the first to enter the commercial market. In fact, the approval of Gendicine superseded its sister product, INGN 201 (Advexin, Ad5CMV-p53), which started clinical development 4 years earlier. In 2003, INGN 201 was still in the middle of Phase III trials in the United States and was just granted Orphan Drug Designation and Fast Track status for drug development by the U.S. FDA. Although Shenzhen SiBiono GeneTech has not conducted any formal clinical studies outside of China to support regulatory approval of Gendicine in other countries, the company is pursuing strategies to bring Gendicine to the United States for clinical trials in support of a Biologics License Application.

The approval of first-in-class Gendicine by the CFDA prompted varied reactions. On one side, there was renewed excitement for this novel class of therapeutics emerging from the international gene therapy community that had been clouded by unexpected clinical events in late 1999.^4,5 The safety, efficacy, and approval of Gendicine reignited other gene therapy Investigational New Drugs (INDs) and clinical trials. On the other side, there were concerns about the clinical trial data for Gendicine, particularly whether the methodology used to collect the data was comparable to U.S. FDA standards of Good Clinical Practice and whether Chinese scientists and clinicians could conduct unbiased clinical trials.⁶

Nevertheless, Shenzhen SiBiono Gene Tech and Gendicine persevered. Between approval in 2004 and 2013, Shenzhen SiBiono Gene Tech manufactured 41 batches of Gendicine with a total of 169,571 vials (1.0 × 10¹² vector particles [vp] per vial). All batches complied with CFDA QC/QA standards. Based upon an average usage rate of five Gendicine vials/patient, >30,000 patients, 10% of whom resided in one of >50 nations outside China, have been treated with Gendicine alone or in combination with chemo- and/or radiation therapies, thermotherapy, and various other regimens. Overall, Gendicine treatment has produced 30–40% complete response (CR) and 50–60% partial response (PR), with total response rates (CR + PR) ranging from 90% to 96% in various clinical applications or studies, consistent with the results of the Phase II and Phase III clinical trials on Gendicine that formed the basis for CFDA approval. To date, the most common adverse events reported for Gendicine treatment have been fever, arthralgia, and myalgia. Within 24 h of Gendicine administration, between 50% and 60% of treated patients experienced fever ranging from 37.5°C to 39.5°C that persisted for a few hours.

The clinical and commercial success of Gendicine has encouraged the international advancement of gene therapy.⁶ Development of a recombinant human endostatin adenovirus for cancer treatment began in 2004 and is now in Phase III clinical study in China.⁷ Meanwhile, other viral vector-based gene therapy products have also had expedited development. In July 2012, 9 years after Gendicine approval, the second commercial gene therapy product, Glybera (AAV-lipoprotein lipase, alipogene tiparvovec) was approved by the European Medicines Agency.^8,9 The third commercial gene therapy product, approved by the U.S. FDA on October 27, 2015,¹⁰ was Imlygic (talimogene laherparepvec), a recombinant herpes simplex virus type 1 carrying the effector gene of granulocyte-macrophage colony-stimulating factor for the treatment of melanoma.¹¹ On August 30, 2017, the U.S. FDA approved two chimeric antigen receptor (CAR) T-cell therapies (ex vivo gene therapy). The first of these was Kymriah (CTL019, tisagenlecleucel) for the treatment of relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia in pediatric and young adult patients.¹² The other, Yescarta (axicabtagene ciloleucel), was approved on October 18, 2017, for the treatment of adult patients with certain types of large B-cell lymphoma who did respond to or who relapsed after at least two other kinds of treatment.¹³ Most recently, on December 19, 2017, the U.S. FDA approved Luxturna (voretigene neparvovec), an adeno-associated virus (AAV)-based gene therapy for patients with vision loss due to confirmed biallelic, RPE65-mediated inherited retinal disease.^14,15 Based on this activity among the international medical field, it is apparent that gene therapy is entering a new era.

Gendicine exerts its therapeutic efficacy through a combination of the human wt p53 gene and adenovirus serotype-5 vector (Ad5). The p53 protein, discovered in the late 1970s, functions as a central mediator of genome stability.^16
–18 Cellular stress, such as hypoxia, DNA damage, and oncogenic stress, causes accumulation of p53 protein within normal cells. p53 then induces cell-cycle arrest, senescence, apoptosis, and/or autophagy in response to these stresses and prevents the accumulation of genetic mutations within the cell.^19
–21 p53 is also a transcription factor that regulates the expression of several target genes such as p21/WAF1, Bid, and DR5. Patients with Li–Fraumeni syndrome have loss of function (LOF) mutations in p53 and develop breast, brain, adrenal, and connective tissue cancers. Furthermore, the p53 gene is mutated in 60–80% of all human cancers. Loss of p53 function is critical for the progression of human cancers, and its activity is essential for the efficacy of chemotherapy and radiation.²² Restoring wt p53 function became a major therapeutic approach for the treatment of cancer due to the tumor suppressor function of p53. Recently, a gain of function (GOF) p53 gene mutation in the transcriptional activation domain 2 (TAD2) was shown to generate a “super tumor-suppressor”, which further emphasized the importance and application potential of p53 in cancer therapy or prevention.^23,24

Recombinant adenovirus (Ad) is the most widely used viral vector for gene therapy^25,26 and has the following favorable characteristics for gene therapy products: (1) high gene-transfer efficiency; (2) large gene-carrying capacity; (3) selective gene delivery; (4) mild cytotoxicity; (5) potential therapeutic immunogenicity; (6) ease of construction and manipulation; and (7) cost-effective commercial manufacture.

Among the 2,463 gene therapy clinical trials that have been conducted worldwide since 1989, 509 (21%) involved adenovirus vectors.²⁷ For efficient gene delivery and high-level expression of p53 in cancer cells, Ad is the ideal vector. The application potential of p53 gene delivery by Ad vector made Ad-p53 gene therapy for cancer an attractive IND candidate.^28

–34 There have been three Ad-p53 INDs: Advexin, SCH-58500, and Gendicine.^3,35,36 A number of preclinical and clinical studies have been conducted using these three Ad-p53 product candidates, and the study findings have been previously reviewed.³⁷ Among them, only Gendicine has obtained regulatory approval (CFDA), and in 2004 it became the first gene therapy product to enter the market.

Gendicine Chemistry, Manufacturing, and Controls

The Chemistry, Manufacturing, and Controls (CMC) for Gendicine were established to be fully compliant with IND and current Good Manufacturing Practice requirements for both the CFDA and the U.S. FDA.

Gendicine production system

Gendicine is generated through co-transfection of the p53 expression cassette shuttle vector and Ad5 genome recombinant vector in HEK 293 cells.^38,39 The p53 expression cassette is composed of the Rous sarcoma virus promoter, the wt human p53 gene, and a bovine poly-A signal. The genome structure of Gendicine was confirmed via DNA sequencing analysis. The recombinant human Ad-p53 adenovirus and HEK 293 production cell line underwent rigorous process development and optimization to develop the commercial manufacturing production system.

Manufacturing and quality controls

The preclinical studies and process/assay development of Gendicine followed the Points to Consider designated by the U.S. FDA. These points later evolved into the Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy IND Applications.^40,41 CMC for Gendicine in China was groundbreaking and contributed to the development of CFDA guidelines for human gene therapy product quality control.⁴²

Gendicine production

Gendicine production technology involves a proprietary large-scale perfusion-based bioreactor system. The producer cell line SBN-Cel, a subclone derived from the HEK 293 cell line, exhibits optimal characteristics for efficient and cost-effective manufacture, including: strong adhesion to the culture surface, robust growth (doubling time approximately 18 h faster than the parental 293 cells), and good virus productivity. Shenzhen SiBiono GeneTech established and qualified primary, master, and working cell banks for the production cell line and vector, which represent critical raw materials for the commercial production of Gendicine. Each Gendicine batch production begins by seeding the production cells in T-flasks. After expansion, the cells are inoculated into a perfusion-based bioreactor, where the cells are infected with Gendicine at an optimized multiplicity of infection. Infected cells are further grown for 5–8 days until vector propagation peaks prior to starting cell lysis, and then the vector-containing cells are harvested. The final-stage culture medium containing vector and cell debris, totaling 14 L per batch, is then collected.

Gendicine purification

The downstream process for Gendicine purification consists of membrane filtration, ultrafiltration, and chromatography. The bioreactor harvest is first subjected to tangential flow filtration for initial clarification followed by ultrafiltration for concentration and dialysis. The concentrate is further treated with nuclease to degrade large cellular DNA. The material is then subjected to additional purification by ion chromatography in an automated chromatography system (fast protein liquid chromatography; GE Healthcare). After this single-step purification, the purity of the Gendicine final product is >98%. Each 14 L single-batch bioreactor run generates 4–5 × 10¹⁵ vp of purified final product.

Gendicine QC/QA

The critical components, such as master cell and vector banks, working cell and vector banks, and other raw materials, were all analyzed according to robust assay panels. The major in-process testing items are vector infectivity and validity, vector quantity in the crude harvest, vector purity in the end process lot, and impurity analysis in the end process lot. The main lot release testing parameters are sterility, vector purity, concentration, vp measurement, infectivity (PFU), potency (p53 gene expression/bioactivity), replication-competent adenovirus, AAV, residuals (host cell DNA, protein, bovine serum albumin), endotoxin, and mycoplasma.

The Gendicine commercialization experience

From 2004 to 2013, Shenzhen SiBiono GeneTech produced 41 batches of Gendicine. All batches, totaling 169,571 vials (1.0 × 10¹² vp per vial) passed lot release testing in compliance with CFDA QC/QA standards. The standard Gendicine dose is a once a week injection with 10¹² vp per dose for 4 weeks. Regimens can vary by one to two courses, depending on treatment response. More than 30,000 patients have been treated with Gendicine either alone or in combination with chemo- and/or radiation therapies, thermotherapy, and various other regimens (based upon average use of five vials of Gendicine per patient).

Clinical Studies and Case Reports

Gendicine has been a useful tool for scientists and clinicians to collaborate on various clinical studies. In addition to the clinical application of Gendicine to treat HNSCC, application of Gendicine for other types of cancer has been carried out in various clinical studies.^43,44 Data for >30 clinical studies have been published, the majority of which include statistical analyses (Table 1). By analyzing the data and making side-by-side comparisons of standard therapies, Gendicine combined with chemo- and/or radiotherapy or other therapeutic regimens generates response rates that are generally significantly higher than those for standard therapy alone.

Table 1.

Summary on Gendicine clinical studies

No.	Year	Type of cancer	Group	Patient number	Regimen	CR (%)	PR (%)	CR + PR (%)	SD (%)	Effective rate (CR + PR + SD)	Long-term survival follow-up	Statistical analysis p-values ^*	Reference
1	2001 2002	HNSCC	GTRT	20	Gendicine + radiotherapy	59.0	35.0	94.0	6.0	100.0	NA	Efficacy GTRT > RT; p = 0.016	Zhang et al.⁵⁴
			RT	22	Radiotherapy alone	22.0	59.0	81.0	19.0	100.0
2	20012003	HNSCC	GTRT	36	Gendicine + radiotherapy	64.3	32.1	96.4	3.6	100.0	NA	Efficacy GTRT > RT; p = 0.003	Zhang et al.⁴⁶
			RT	33	Radiotherapy alone	19.4	5.6	25.0	16.7	41.7
3	NA	HNC	GTCT	7	Gendicine + chemotherapy	28.57	NA	NA	NA	NA	NA	Toxic syndromes and complications GTCT < CT; p < 0.05	Li et al.⁴⁵
			GT	6	Gendicine	16.67	NA	NA	NA	NA
			CT	10	Chemotherapy	10.0	NA	NA	NA	NA
4	20012003	NPC	GTRT	42	Gendicine + radiotherapy	66.7	31.0	97.7	2.3	10.0	5-year locoregional tumor control rate GTRT > RT (2.7% vs. 28.0%; p = 0.002)	CR rate GTRT > RT; p = 0.01	Pan et al.⁵⁰
			RT	40	Radiotherapy alone	22.4	61.0	83.4	14.6	98.0
5	20052006	NPC	GTRT + CT	15	Gendicine + radiotherapy + chemotherapy	6.67	93.33	100.0	0	100.0	NA	NA	Liu et al.⁴⁹
			RT + CT	15	Radiotherapy + chemotherapy	0	40.0	40.0	60.0	100.0
6	20072008	NPC	GTRTCT	14	Gendicine + radiotherapy + chemotherapy	86.0	7.0	93.0	7.0	100.0	Response rate GTRTCT > RTCT in 3 months; p = 0.037	Tumor shrink rate in 3 months GTRTCT > RTCT; p = 0.037	Si et al.⁵⁵
			RTCT	15	Radiotherapy + chemotherapy	33.0	33.0	66.0	34.0	100.0
7	20102012	NPC	GTCRT	54	Gendicine + chemoradiotherapy	33.33	26.93	62.96	22.22	85.16	PFS and 3-year survival rate GTCRT > CRT or GT; p < 0.05	Effective rate GTCRT > CRT or GT; p < 0.05	Ma et al.⁴⁸
			CRT	54	Chemoradiotherapy	16.7	24.07	40.77	38.89	79.66
			GT	54	Gendicine	14.81	25.93	40.74	40.74	81.48
8	20092010	NPC	GTRT	24	Gendicine + radiotherapy	37.5	54.2	91.7	8.3	100.0	8 weeks after the treatment effective rate GTRT > RT (92% vs. 62%); p < 0.05	Effective rate and survival rate GTRT > RT; p < 0.05 (Fisher's exact test)	Wang et al.⁵¹
			RT	26	Radiotherapy	11.5	50.0	61.5	26.9	88.4
9	NA	NPC	GTRT	24	Gendicine + radiotherapy	62.5	NA	NA	NA	NA	3-year overall survival, GTRT was 14.4% higher than that of GT	NA	Zhang et al.⁴⁷
			RT	25	Radiotherapy	26.9	NA	NA	NA	NA
10	20032007	OSCC	GTCT	35	Gendicine + chemotherapy	48.5	33.3	81.8	NA	NA	NA	Non-responder rate GTCT < GT + C or CT + C; p < 0.02 (Kruskal–Wallis test); survival rate GTCT > C + C; p = 0.019	Li et al.⁵⁶
			GT + C	33	Gendicine + placebo	16.7	36.7	53.4	NA	NA
			CT + C	31	Chemotherapy + placebo	17.2	34.5	51.7	NA	NA
11	20052011	OSCC	GTRT	57	Gendicine + radiotherapy	NA	NA	NA	NA	NA	Overall recurrent rate: EG GTRT < RT (7% vs. 32%) DFS GTRT > RT	NA	Liu et al.⁵⁷
			RT	50	Radiotherapy	NA	NA	NA	NA	NA
12	20042005	HCC	GT + TACE	68	Gendicine + TACE	0	67.6	67.6	22.0	89.6	GT + TACE had higher survival rate (89.71% vs. 68.15%; p < 0.01)	Effective rate GT + TACE > TACE; p < 0.05	Guan et al.⁵⁸
			TACE	82	TACE	0	51.2	51.2	32.9	84.1
13	20042007	HCC	GTCT + TACE	23	Gendicine +5-FU + TACE	0	21.7	21.7	52.2	73.9	Median OS: 12.8 months in treatment group vs.10.4 months in control group	No significant difference	Tian et al.⁵⁹
			TACE	23	TACE	0	21.7	21.7	47.8	69.5
14	20042007	HCC	GTRT	20	Gendicine + fSRT	35.0	50.0	85.0	15.0	100.0	The 1-year survival rates: GTRT (90%) vs. RT (70%); the 1-year DFS rates: GTRT (85%) vs. RT (65%)	NA	Yang et al.⁶⁰
			RT	20	fSRT	20.0	50.0	70.0	30.0	100.0
15	20072009	HCC	GTTACE(p53+)	30	Gendicine + TACE	0	73.3	73.3	13.3	86.6	NA	Effective rate in p53+ patients GTTACE > TACE; p < 0.05	Ou et al.⁶¹
			TACE(p53+)	30	TACE	0	46.7	46.7	26.7	73.4
			GTTACE(p53–)	30	Gendicine + TACE	0	66.7	66.7	20.0	86.4
			TACE(p53-)	30	TACE	0	60.0	60.0	23.3	83.3
16	20062009	HCC	GTCT	30	Gendicine + chemotherapy	0	13.3	13.3	70.0	83.3	6-month survival rate GTCT > CT (50% vs. 11.1%; p < 0.05)	Mutant p53 expression GTCT < CT (23.74% vs. 11.81%); p < 0.01	Chen et al.⁶²
			CT	18	Chemotherapy	0	0	0	55.5	55.5
17	NA	HCC	GTCTCTM	20	Gendicine + chemotherapy + CTM	NA	NA	NA	NA	NA	Liver function after treatment: GTCTCTM > GTCT (AST/ALT, GGT/ALT, Child–Pugh; p < 0.05)	Tumor number and size GTCTCTM < GTCT; p < 0.05	Shi et al.⁶³
			GTCT	20	Gendicine + chemotherapy	NA	NA	NA	NA	NA
18	20092010	HCC	GTCT	30	Gendicine + chemotherapy	NA	NA	70.0	NA	97.0	NA	Response rate GTCT > GT; p < 0.05	Shi et al.⁶⁴
			GT	30	Chemotherapy	NA	NA	40.0	NA	90.0
19	20082010	HCC	GTCT (intratumoral)	56	Gendicine (intratumoral) + chemotherapy	10.7	75	85.7	8.9	94.6	6- and 12-month survival rate: GTCT > CT; p < 0.05	Effective rate GTCT (intratumoral) or GTCT (arterial) > CT; p < 0.01	Feng et al.⁶⁵
			GTCT (arterial)	62	Gendicine (arterial perfusion) + chemotherapy	11.3	67.7	79.0	12.9	91.9
			CT	44	Chemotherapy	2.3	50.0	52.3	29.5	81.8
20	20102011	NSCLC	GTRTCT	31	Gendicine + radiotherapy + chemotherapy	9.67	61.3	70.97	16.13	87.10	1-year survival rate GTRTCT > RTCT (74.19% vs. 69.60%; no significant difference)	Effective rate GTRTCT > RTCT; p < 0.05	Wang et al.⁶⁶
			RTCT	33	Radiotherapy + chemotherapy	3.03	42.42	45.45	27.27	72.72
21	20042005	NSCLC	GTCT	19	Gendicine + chemotherapy	10.5	36.8	47.3	42.1	89.4	NA	Adverse events GTCT < CT; p < 0.05	Guan et al.⁶⁷
			CT	39	Chemotherapy	0	38.4	38.4	15.3	53.7
22	20112013	NSCLC	GTCT	50	Gendicine + chemotherapy	2	46.0	48.0	48.0	96.0	NA	Effective rate GTCT > CT; p = 0.039	Liu et al.⁶⁸
			CT	50	Chemotherapy	0	28.0	28.0	62.0	90.0
23	20052006	NSCLC	GTCT	19	Gendicine + chemotherapy	0	10.5	10.5	42.1	52.6	NA	No significant difference	Ning et al.⁶⁹
			CT	21	Chemotherapy	0	0	0	61.9	61.9
24	NA	Esophageal carcinoma	GTRT	22	Gendicine + radiotherapy	36.4	59.1	95.5	4.5	100.0	NA	Response rate and CR rate GTRT > RT; p < 0.05	Lu et al.⁷⁰
			RT	23	Radiotherapy	13.0	60.9	73.9	21.7	95.6
25	20072011	Ovarian carcinoma	GTCT	25	Gendicine + chemotherapy	NA	NA	64.0	28.0	92.0	CA-125 return to normal: GTCT > CT (68.0% vs. 33.3%); disease control rate GTCT > CT (92% vs. 87.5)	Rate of CA125 regression: GTCT > CT; p < 0.05	Cui et al.⁷¹
			CT	24	Chemotherapy	NA	NA	70.8	16.7	87.5
26	NA	Cervical cancer	GTRT	69	Gendicine + radiotherapy	66.7	33.3	100.0	0	100.0	5-year survival rate: GTRT > RT (74.2% vs. 56.7%; p = 0.047)		Su et al.⁷²
			RT	35	Radiotherapy	54.3	37.1	91.4	8.6	100.0
27	20122013	Cervical cancer	GTRT	10	Gendicine + radiotherapy	60.0	30.0	90.0	10.0	100.0	NA	Effective rate GTRT > RT; p = 0.020	Xu et al.⁷³
			RT	10	Radiotherapy	20.0	10.0	30.0	70.0	100.0
28	20042005	Advanced cancer	GTCT	13	Gendicine + chemotherapy	0	46.2	46.2	30.8	76.8	NA	NA	Qi et al.⁷⁴
			GT	10	Gendicine	0	30.0	30.0	40.0	70.0
29	20072008	Malignant pleural or peritoneal effusion	GTCT	27	Gendicine + chemotherapy	22.3	40.7	63.0	14.8	87.8	NA	Karnofsky score after treatment: GTCT > CT; p < 0.05	Dong et al.⁷⁵
			CT	21	Chemotherapy	19.1	23.8	42.9	38.1	82.0
30	NA	Malignant serosal cavity effusion	GTCT	30	Gendicine + chemotherapy	NA	NA	86.7	NA	NA	Quality of life improvement: GTCT > CT (70% vs. 30%)	Response rate: GTCT > CT; p = 0.01; quality of life improvement: GTCT > CT; p = 0.006	Wang et al.⁷⁶
			CT	30	Chemotherapy	NA	NA	60.0	NA	NA

Unless specified, all p-values are based on rank sum test (chi-square test).

CR, complete response; PR, partial response; SD, stable disease; GTRT, Gendicine + radiotherapy; GTCT, Gendicine + chemotherapy; GT, gene therapy; C, chemotherapy; RT, radiotherapy; HNSCC, head and neck squamous-cell carcinoma; HNC, head and neck cancer; NPC, nasopharyngeal cancer; OSCC, oral squamous-cell carcinoma; HCC, hepatocellular carcinoma; NSCLC, non-small-cell lung cancer; TACE, transcatheter arterial chemoembolization fSRT, fractionated stereotactic radiotherapy; NA, not available.

Long-term survival data for patients treated with Gendicine combination regimens versus standard regimens alone were reported in 13 published clinical studies (Table 2). Five of these studies presented statistical data demonstrating that patients treated with a Gendicine combination regimen have significantly longer post-treatment survival than those receiving standard regimens alone. Three other studies with statistical analyses have long-term survival rates for Gendicine combination arms that are numerically higher than those in the standard regimen alone arms, but the differences were not statistically significant. The remaining five studies had no statistical analysis, but Gendicine combination regimen groups had numerically higher long-term survival rates than the standard regimens alone. Overall, the data from these 13 studies indicate improved survival rates with Gendicine combination therapy.

Table 2.

Survival-rate comparison of Gendicine-treated cancer patients

				Survival rate (%)
No.	Group (regimen)	Type of cancer	Patient number	1 month	3 months	6 months	9 months	1 year	2 years	3 years	5 years	Statistical analysis p-values	Reference
1	GTCT: Gendicine (intratumoral) + chemo	HCC	56	—	—	80.4	—	72.7	23.6	—	—	6-month and 1-year survival rate: GTCT > CT; p < 0.05; 2-year survival rate: no significant difference	Feng et al.⁶⁵
	GTCT: Gendicine (arterial perfusion) + chemo		62	—	—	77.4	—	67.7	21.3	—	—
	CT: chemo		44	—	—	56.8	—	47.7	14.0	—	—
2	GTCT: Gendicine + chemo	HCC	30	96.6	83.3	50.0	23.3	6.7	—	—	—	p < 0.05	Chen et al.⁶²
	CT: chemo		18	94.4	55.6	11.1	0	0	—	—	—
3	GTCT: Gendicine + chemo (TACE)	HCC	68	—	89.71	76.13	—	43.3	—	—	—	p = 0.0002	Guan et al.⁵⁸
	CT: chemo (TACE)		82	—	68.15	36.98	—	24.02	—	—	—
4	GTRT: Gendicine + radiotherapy	NPC	42	—	—	—	—	97.6	—	78.6	66.7	No significant difference	Pan et al.⁵⁰
	RT: radiotherapy		40	—	—	—	—	95.0	—	65.0	59.2
5	GTRT: Gendicine + radiotherapy	Cervical cancer	69	—	—	—	—	97.1	—	83.9	74.2	No significant difference	Su et al.⁷²
	RT: radiotherapy		35	—	—	—	—	97.1	—	71.4	56.7
6	GTRT: Gendicine + ¹³¹I + surgery	Thyroid cancer	31	—	—	—	—	100	—	—	—	NA	Zhu et al.⁷⁷
	RT: ¹³¹I + surgery		29	—	—	—	—	86.2	—	—	—
7	GTRT: Gendicine + fSRT	HCC	20	—	—	—	—	90.0	—	—	—	NA	Yang et al.⁶⁰
	RT: fSRT		20	—	—	—	—	70.0	—	—	—
8	GTCT: Gendicine + chemo	OSCC	35	—	—	—	—	88.6	82.9	71.4	51.4	p = 0.015 (GTCT vs. CT)	Li et al.^56,a
	GT: Gendicine		33	—	—	—	—	75.8	48.5	45.5	33.3
	CT: chemo		31	—	—	—	—	71.0	41.9	38.7	29.0
9	GTRT: Gendicine + radiotherapy	Soft-tissue sarcoma	37	—	—	—	—	56.8	21.6	16.2	10.8	NA	Geng et al.^78,b
10	GTRT: Gendicine + radiotherapy	Cervical cancer	31	—	—	—	—	—	—	—	85.7	NA	Zhang^79,c
	RT: radiotherapy		140,000	—	—	—	—	—	—	—	55.3
11	Gendicine + surgery	Oral mucosa	29	—	—	—	—	—	39.6	—	—	NA	Li⁸⁰
	Surgery		28	—	—	—	—	—	16.7	—	—
12	GTCRT: Gendicine + chemoradiotherapy	NPC	54	—	—	—	—	—	—	75.9	—	p < 0.05 (GTCRT vs. CRT or GT)	Ma⁴⁸
	CRT: chemoradiotherapy		54	—	—	—	—	—	—	53.7	—
	GT: Gendicine		54	—	—	—	—	—	—	51.9	—
13	GTCRT: Gendicine + hemoradiotherapy	NSCLC	31	—	—	—	—	74.19	—	—	—	No significant difference	Wang⁶⁶
	CRT: Chemoradiotherapy		33	—	—	—	—	69.10	—	—	—

Results based on Kaplan–Meier survival curves.

No control group.

The survival rate of control group come from a study including 140,000 cases with stage I–IV of cervical cancer treated using radiotherapy alone.

¹³¹I, radioactive iodine.

Use of Gendicine to treat head and neck cancer

Head and neck cancer (HNC) is a group of cancers that arise within the mouth, nose, throat, larynx, sinuses, or salivary glands. Gendicine is the CFDA-approved indication for HNCs. In post-marketing studies, nasopharyngeal carcinoma (NPC), a subgroup of HNC, was the cancer most frequently treated with Gendicine. Most treatment courses also included radiation and/or chemotherapy. The outcomes of these studies reaffirmed the data and conclusions of the Phase II–III clinical trials for Gendicine that formed the basis for CFDA approval.^{45

–53} All of these studies demonstrated improvement compared to use of first-line standard therapy methods alone (chemotherapy and/or radiotherapy; Table 1). The total response rate, complete remission (CR) + partial remission (PR), for HNC treated with Gendicine was >90%, which is significantly higher than that for standard cancer treatments alone.⁴⁷ The reported 1-, 2-, 3-, and 5-year survival rates were also higher than those of the group that received conventional therapy alone.^50,51 In all studies that included statistical analysis, groups treated with Gendicine showed significant improvement in response compared to control groups (p < 0.05; Table 1).

Off-label Gendicine applications: other types of cancer

Lung cancer

Gendicine has been used to treat lung cancers, especially non-small-cell lung cancer in combination with chemo- or radiotherapy. According to clinical reports, Gendicine combined with chemotherapy and radiotherapy produced better results than Gendicine combined with chemotherapy alone. Although both methods showed improved results compared to the control group, the response rate for Gendicine combined with chemotherapy and radiotherapy was 76.9%, which was higher than that for Gendicine combined with chemotherapy alone (47.3%, 48.0%, and 10.5%).^66

–69 All reports concluded there was improved efficacy and quality of life for the patients.^66

–69,81

Breast cancer

A clinical study on use of Gendicine to treat triple-negative breast cancer has been conducted in China (manuscript in preparation). Among the 128 enrolled patients, 64 were treated with Gendicine combined with Xeloda, a 5-fluorouracil prodrug, while the other 64 were treated with Xeloda alone as a control. Gendicine (2.0 × 10¹² vp) was given via intratumoral injection every 3 days for a total of 10 injections. The response rate for the Gendicine group was 84.4%, which was significantly higher than the 71.8% for the control group (p < 0.05). The results suggested that Gendicine is a safe and efficacious treatment for triple-negative breast cancer.

Cancers of female reproductive organs

The p53 mutation rate in cervical and ovarian cancers is relatively high compared to other types of cancer. Gendicine has been used extensively to treat these two cancers.^72,73,82 For the treatment of cervical cancer, the response rate for Gendicine combined with radiotherapy treatment was three times that of radiotherapy alone (90% vs. 30%; p = 0.02).⁷³ In a separate clinical study, the 5-year overall survival (OS) rate and 5-year progression-free survival (PFS) rate of Gendicine combined with radiotherapy treatment were 17.5% and 17.1% higher, respectively, than that for radiotherapy alone (p = 0.047).⁷² The use of Gendicine to treat ovarian cancer was studied between May 2009 and October 2010. The response rate for ovarian cancer reached 90% (20 cases). Among the patients who had malignant pleural or peritoneal effusion condition (18 cases), 100% had resolution of the effusion.⁸³ The efficacy of Gendicine combined with chemotherapy for the treatment of uterine sarcoma was studied via a retrospective analysis of clinical data from 12 patients. The interval between the first operation and diagnosis of recurrence (first PFS, PFS1) was 1–18 months (median 3 months). All of the patients were treated with local administration of Gendicine followed by chemotherapy (local injection of bleomycin and intravenous infusion of cisplatin, epirubicin, and isocyclophosphamide). Efficacy was evaluated, and the CR + PR was calculated. In follow-up, second PFS (PFS2) and OS data were also obtained. The treatment resulted in one CR, seven PR, three cases of stable disease (SD), and one case of progressive disease (PD). The objective response rate (CR + PR) was 66.7%, and the total response rate (CR + PR + SD) was 91.7%. PFS2 ranged from 2 to 62 months, with a median of 13 months, which was 10 months longer than that of PFS1 and was statistically significant (p = 0.0038).⁸⁴

Liver and pancreatic cancers

Liver cancer is the second largest off-label application of Gendicine. Various controlled clinical studies have been reported concerning hepatocellular carcinoma (HCC) treated with Gendicine in combination with chemotherapy (Table 1). Gendicine combined with other regimens such as Traditional Chinese Medicine (TCM) and gelatin sponge microspheres were also reported. The average response rate for Gendicine combined with standard therapy was as high as 85%.⁶⁰ Some of the patients showed CR of the cancer and no recurrence.^65,85
–87 The survival rate of the combined therapy was also significantly higher than that of the control group (Table 1).^58,62,65 All reports also described an improvement in quality of life after treatment with Gendicine.^{58
–60,62

–65,85

–90} In pancreatic carcinoma, Gendicine combined with chemotherapy or radiotherapy was investigated for safety and efficacy. Results showed that the response rate was 80% and the 1-year PFS and OS were 40.0% and 51.1%, respectively. The combination of Gendicine for treatment of unresectable pancreatic carcinoma suggested modest benefit.⁹¹

Digestive tract cancers

In a clinical study, Gendicine was combined with radiotherapy for the treatment of esophageal carcinoma. The overall response rate of the treatment group was significantly higher than that of the control group, and the CR rate of the treatment group was threefold higher than that of the control group (p < 0.05). The clinical results showed that when combined with radiotherapy, Gendicine significantly improved treatment efficacy compared to that of radiotherapy alone, with only minor side effects.⁷⁰ Another clinical study was conducted on the safety and efficacy of Gendicine combined with FOLFOX4 to treat advanced colorectal cancer.⁹² The results demonstrated that the Karnofsky performance score of all patients increased significantly after the treatment. All patients were alive up to the follow-up date, indicating the effectiveness of Gendicine for treatment of advanced colorectal cancer.

Other cancers

Gendicine has also been used alone or in combination with chemotherapy, radiotherapy, and hyperthermia for the treatment of other types of cancer, such as soft-tissue sarcoma, schwannoma, and conditions caused by cancer such as pleural effusion or ascites.^75,93

–96 In a clinical study of 30 patients with advanced soft-tissue sarcoma, after treatment with Gendicine combined with hyperthermia or hyperthermia plus radiotherapy, the tumor size of 29 (96.6%) patients was reduced.⁹³ Reports of other clinical applications for the treatment of advanced soft-tissue sarcoma reproduced the effectiveness of Gendicine in terms of tumor shrinkage and improved quality of life.⁹⁴

Variation in route of application and regimen

Route of application

The CFDA-approved route of administration for Gendicine is intratumoral injection, and this is therefore the most widely applied method.^46,53,91,94 This approach ensures optimal locoregional vector concentration and transfection efficiency. However, for some cancers in which cancer cells are widespread, or for patients with metastases, direct intratumoral injection is not applicable. In clinical applications, various routes of application, including arterial injection, intraoperative, intra-tract (bronchial, esophageal, rectal, urethral, vaginal), intracranial, intra-cavity, or systemic infusion, have been applied according to the patient's condition in order to improve gene delivery efficiency.^{52,75,81,96
–98} The optimal application route should be selected for each patient to maximize the efficacy of Gendicine. For example, artery perfusion is widely used to treat liver cancer.

Dose and course

The dose and treatment course for Gendicine depends on several aspects, such as tumor size and location, cancer stage, and patient tolerance. Dose amount and frequency, as well as application route for Gendicine, are currently at the physician's discretion. In general, each injection will deliver 1–4 × 10¹² vp, and injections are administered once every 3–7 days over the course of 3–8 weeks. Patients may receive more injections, depending on the response to treatment.^{50,58,70,81,93,95}

Use of Gendicine alone as primary treatment

Although rare, Gendicine has been used alone as the primary treatment for some indications, particularly ovarian cancer, malignant pleural effusions, or peritoneal ascites. Some patients have declined standard therapies and requested Gendicine as the primary treatment. Reports show that Gendicine monotherapy could be efficacious against these cancers.^74,83,99

Combination with standard regimens

Gendicine was approved to treat HNC in combination with chemo- or radiotherapy. A number of clinical studies have shown that Gendicine in combination with chemotherapy increased chemotherapy efficacy.^53,67,85,97 In addition, Gendicine is also widely used in combination with radiotherapy to improve outcomes.^49,78,91 The combination of Gendicine with chemo- or radiotherapy often reduces the size of the tumor and can afford options for surgical removal of tumors. In most clinical applications, chemo- or radiotherapy are applied concurrently with Gendicine. However, because the Gendicine transfection and expression requires around 72 h to reach peak efficacy, chemo- or radiotherapy should be administered at least 48 h after Gendicine injection, which is supported by multiple case reports showing that chemo- or radiotherapy given 48–72 h after Gendicine injection was associated with improved outcomes.⁴⁵

Combination with thermotherapy

For some patients with sarcomas or malignant serosal cavity effusion, hyperthermia combined with Gendicine was effective in reducing tumor size and relieving symptoms while demonstrating good efficacy.^76,83,93 Clinical studies showed that Gendicine had enhanced efficacy for the treatment of advanced cancers when combined with hyperthermia.^100,101

Combination with immunotherapy

A clinical study of Gendicine combined with autologous immune reactive cell reinfusion for various types of cancers was presented in 2008.¹⁰² Although the detailed therapy regimen was not disclosed at this presentation, the total effective rate reached 76.9%. Since Gendicine itself could stimulate the immune system to fight cancer, combining it with immunotherapy could be a treatment option for advanced cancers.¹⁰² The experimental data for Ad-p53-dependent activation of lymphokine activated killer cells (LAK) and cytotoxic T lymphocytes (CTL) provide some insight into the mechanism by which Gendicine may enhance immunotherapy.^103,104

Combination with TCM

Gendicine has been combined with TCM for the treatment of cancer. Simultaneous injection of Aidi solution (a type of TCM) and Gendicine has been used for the treatment of HCC. Six months after treatment, the AFP/CEA level of the treatment group was reduced compared to the control group. One year after treatment, the aspartate transaminase/alanine transaminase (ALT), gamma-glutamyl transpeptidase/ALT, and the Child–Pugh score of the treatment group were also improved compared to the control group, indicating an improvement in liver function. The result was encouraging and provided an additional method of cancer treatment.⁶³ In another clinical study, Gendicine was combined with the TCM Elemene to prevent recurrence of small HCC.¹⁰⁵ Three injections of Gendicine were given intravenously every other day, and Elemene was given every day for 5 days. After the treatment, none of the five patients tested showed recurrence. This result demonstrated that Gendicine combined with TCM could be used for the prevention of tumor recurrence.

Pharmacokinetics and immune responses

Biodistribution and pharmacokinetics

Biodistribution and pharmacokinetics of Ad, including Ad-p53, have previously been extensively studied in both animal models and human clinical trials. For Gendicine, the major route of administration is intratumoral injection, which allows concentration of the viral vector in the tumor mass. In preclinical animal models, Gendicine transfected cancer cells within 1 h of intratumoral injection, and Ad-p53 protein expression was detected after 3 h. The p53 protein expression level rose to 47% of maximum at 12 h, reached peak expression on day 3, and then decreased gradually to 30% by day 5. Residual p53 expression remained detectable on day 14 but was undetectable 3 weeks after injection. A similar time course of p53 expression was observed in clinical studies.⁵⁸ In one clinical study, no virus was detected in serum, urine, and sputum samples following intra-arterial (external carotid artery) administration, suggesting that Gendicine did not distribute systemically.¹⁰⁶ In another study, detection of Ad-p53 DNA levels following repetitive intravenous infusion showed that the quantity and duration of detectable DNA was proportional to the administered dose.¹⁰⁷ At a dose of 1 × 10¹² vp, Ad-p53 DNA was detectable on day 14 and diminished at later time points. The half-life for the serum concentration of the Ad-p53 vector was about 7 min. Vector-specific DNA sequences remained for up to 2 weeks in various tissues but were generally undetectable 3 weeks after dosing.¹⁰⁸

Immune responses

Immune responses to adenoviral vectors delivered via intratumoral injection or systemically have been studied extensively. Although most patients have been previously exposed to adenovirus and thus have pre-existing neutralizing antibodies, the clinical efficacy of Ad5-based therapies (including Gendicine and other Ad-p53 vectors) appeared to be effective and was not suppressed by neutralizing antibodies.^37,107 Preliminary results demonstrated that Ad-p53 can induce immune responses, not only against the vector, but also invokes activities that boost anticancer immunity, such as activation of several cytokine genes, tumor antigen genes, and co-stimulatory molecule genes.¹⁰⁹ In other clinical trials using adenoviral vector, the majority of patients presented with neutralizing antibodies and almost all showed a significant increase in viral titer after the initial Ad vector injection.^110,111 In the clinic, Gendicine delivered systemically was reported to be safe and effective in enhancing responses to chemo- and/or radiotherapy. Therefore, the presence of pre-existing Ad immunity and the rapid development of Ad vector immunity did not completely block Gendicine function.^37,107 This was confirmed by a clinical study of Gendicine delivered via intra-external carotid artery infusion.⁵⁶ Anti-vector neutralizing antibody titers increased or became positive in all patients in Groups I and II after intra-arterial infusion with Gendicine (Group I: basal level 176 ± 103 to week 2 of treatment 2,129 ± 1,198; and group II: basal level 168 ± 101 to week 2 of treatment 2,137 ± 1,173). Antibody titers increased after administration of subsequent cycles of treatment. However, the antitumor activity of Gendicine was not affected by these neutralizing antibodies.⁵⁶ Nonetheless, improvements in Ad vector design and development of other methods that enhance the safety and efficacy of systemic delivery of Ad vectors are needed for Gendicine and other Ad vectors.^98,112

Adverse events

The CFDA has established a Center for Reporting Adverse Events and maintains open access to its database. No serious adverse event reports were received by the Shenzhen SiBiono GeneTech QC/QA department, or were found in the searchable Center for Reporting Adverse Events database, or other online databases such as China Knowledge Resource Integrated Database, Wanfang Med Online, or PaperPass.

The one adverse event that has been seen to occur within 24 h of Gendicine administration is vector-associated transient fever.^58,106 The majority of fevers ranged from 37.5°C to 38.3°C. In some cases, the fever rose to as high as 38.5–39.5°C.

A few patients also experienced influenza-like symptoms, muscle ache, or pain at the injection site. These symptoms were also self-limiting and resolved quickly.^68,70,99 No significant abnormal laboratory results have been reported in association with Gendicine treatment, including routine testing of urine or blood, or results for electrocardiogram or chest radiography.

Mechanisms of Action and Implications

One remarkable feature of Ad-p53 is that it enables highly efficient gene transfer and high-level nuclear expression of p53 protein in transfected cells.^29,113 The anticancer mechanisms of Gendicine are thus mainly attributed to overexpression of the exogenous wt p53 protein in the transfected cancer cells. Some effects may be caused by the Ad vector and some by the combination of Ad vector and p53 protein.

A large number of studies have revealed multiple potential mechanisms of p53 anticancer action through direct and indirect effects.²³ Most of the studies on the mechanisms of p53-mediated tumor suppression were done in cancer cell lines and animal models.¹¹⁴ It is beyond the scope of this review to discuss the many potential mechanisms of p53 action, but it is important to understand the known direct effects of the p53 protein (e.g., arresting cell growth, apoptosis, autophagy, and cellular senescence), especially as they pertain to anticancer activity.

Direct effects

Arresting cell growth

The p53 protein has pivotal functions in cell-cycle progression due its regulation of G1/S, S, and G2/M cell-cycle checkpoints. p53 upregulates expression of the CDK inhibitor p21 (CIP1/WAF1), which binds to and inhibits cyclin E/cdk2 and cyclin A/cdk2 complexes, halts cell-cycle progression and DNA replication, and allows for DNA repair. The p53–p21 signaling axis appears to be instrumental for maintaining S-phase checkpoints. p53 also impacts the G2/M checkpoint by transcriptionally modulating the expression of multiple, physically and functionally intertwined targets, including Cdc25C, 14-3-3σ, p21, and GADD45.¹¹⁵ A study on the human lung adenocarcinoma cell lines GLC-82 and A549 clearly showed that cells were arrested in the G0/G1 phase and that very few cells were in S phase following transfection with Gendicine.¹¹⁶ A different study on the A549 cell line confirmed this result wherein Gendicine added to cells in combination with cisplatin (DDP) showed higher numbers of cells arrested in the G0/G1 and G2/M phases, and fewer cells in S phase (9.63% vs. 24.44%) relative to untreated control cells.¹¹⁷ In a clinical study conducted between 2006 and 2007, 18 patients with dysplastic oral leukoplakia were treated with Gendicine. Biopsies taken from these patients after the final Gendicine injection showed significant elevations in p53 and p21 protein expression (100% and 89%, respectively) relative to pretreatment biopsies. Statistical analysis revealed positive correlations between p53 expression and that of p21.¹¹⁸ In a clinical study involving patients with recurrent glioma, p21 protein expression presumably induced by exogenous p53 was detected at the injection site.¹¹⁹

Inducing apoptosis

Multiple mechanisms of apoptosis induced by p53 have been previously discussed.¹²⁰ p53 acts via two major apoptotic pathways: the extrinsic, death receptor pathway that triggers activation of the caspase cascade, and the intrinsic, mitochondrial pathway, which shifts the balance in Bcl-2 family proteins toward the pro-apoptotic members and promotes apoptosome formation.^121,122 These two mechanisms ensure efficient, p53-dependent induction of apoptosis in a stage-, tissue-, and stress signal-specific manner. The effect of Gendicine on apoptosis studied in three different gastric cancer cell lines (SGC-7901, BGC-823, and HGC-27) showed inhibition of cell growth at clinical concentrations of Gendicine (10⁹ vp).¹²³ Immunohistochemistry demonstrated a decrease in Bcl-2 levels and an increase in that of Bax and caspase-3 after infection with Gendicine, suggesting promotion of apoptosis. A study of the effect of Gendicine on the pancreatic carcinoma cell line SW1990 showed that following Gendicine infection Bax expression was upregulated significantly (76.5% vs. 28.30%).¹²⁴ Studies performed in GLC-82, A549, HeLa, and HepG2 cell lines demonstrated both increased proliferation inhibition and apoptosis induction after Gendicine treatment combined with chemotherapy drugs compared to control cells treated with Gendicine or chemotherapy drug alone.^116,125,126 F-box and WD repeat domain-containing 7 (Fbxw7) is a p53-dependent suppressor gene that induces the degradation of the oncoproteins c-Myc, cyclin E, notch, and others.¹²⁷ A Fbxw7, c-Myc, and cyclin E expression test in human HCC cells LO2 and Hep3B treated with Gendicine showed that Gendicine infection significantly increased the expression of Fbxw7, decreased c-Myc and cyclin E levels, and induced apoptosis.¹²⁸ An in vivo study in a mouse model confirmed Fbxw7 upregulation and demonstrated tumor inhibition by Gendicine. Moreover, tumor size was reduced by half in the Gendicine-treated group compared to the control group.¹²⁸

Regulating autophagy

Autophagy is a major catabolic process that involves degradation and recycling of cytosolic components in autophagosomes, which fuse with lysosomes. Autophagy is a survival strategy induced in response to stress conditions and is also a means to remove harmful or damaged macromolecules.^129,130 Through autophagy, nutrient-starved cells can fulfill energy requirements and maintain metabolic states, thus facilitating their survival, especially in cancer pathogenesis. p53 plays dual roles in regulating autophagy, depending on its subcellular localization.¹³¹ Nuclear p53 facilitates autophagy by trans-activating its target genes, whereas cytoplasmic p53 mainly inhibits autophagy through extranuclear, transcription-independent mechanisms. Ad-p53 enables high-level nuclear expression of p53.¹¹³ p53-dependent activation of autophagy suggests that this process is part of the anticancer function of p53.^132,133 Furthermore, the p53-induced modulator of autophagy, DRAM, is critical for apoptosis.¹³⁴

Mediating cell senescence

Among cell-cycle molecules such as p53, pRB, p21, p16, mechanistic target of rapamycin (mTOR), and p27, mTOR is recognized as a key molecule in switching on/off senescence/quiescence. Although maximal p53 activation blocks mTOR and causes quiescence, partial p53 activation sustains mTOR activity, and subsequent senescence. In contrast to quiescence, senescence is permanent cell-cycle arrest and a degenerative process that precedes certain types of cell death.^135,136 p53 induces cellular senescence through transcriptional activation of target genes such as p21, PAI1, and PML in response to cell stressors such as oxidative stress or DNA damage.¹²⁰ Although no preclinical or clinical studies have been performed to examine the effect of Gendicine on cell senescence, many studies have demonstrated that Gendicine infection promotes the expression of p21, which is a key protein in cell senescence progression.^118,119 These results indicate that Gendicine could induce cell senescence in tumors. In addition, multiple experiments in animal models have demonstrated that p53-mediated tumor suppression correlates with cellular senescence.^137
–139

Indirect effects

Numerous potential indirect effects of p53 protein and the adenovirus vector used in Gendicine (Ad-p53) have been reported, including adenovirus-induced tumor neoantigen and antitumor immunity,^140

–143 inhibition of angiogenesis by p53 via downregulation of VEGF expression and inhibition of HIF-1 activity,^{50,101,144

–148} and bystander effects.^149,150 Restoration of p53 activities in transduced cells was also reported to promote modulation of the tumor microenvironment.^151,152 Inhibition of metastasis by Gendicine was previously studied.¹⁵³ The effect of interfering with cell adhesion and invasion may occur through suppression of the epithelial mesenchymal transition and stabilization of cell–cell junction proteins (e.g., E-cadherin).^154

–157

Molecular mechanisms

Generally, p53 functions through two networks: signaling and epigenetic.^114,120 Gendicine intratumoral injection causes the transduced cancer cells to express high levels of exogenous wt-p53,²⁹ which is shown to be concentrated in the nucleus.¹¹³ At a molecular level, Ad-p53 mechanisms of action are attributed mainly to p53-mediated regulation of extensive signaling networks. p53 binds to the enhancer/promoter elements of target genes and regulates their transcription, thus initiating cellular programs that account for most of its tumor-suppressor functions.^{19,120,133,158

–162} To transactivate pro-apoptotic genes and thereafter execute apoptosis, p53 levels must exceed and be maintained at a certain threshold.¹⁶³

The p53 protein not only enforces genome stability by preventing genetic alterations in cells, but also plays a role in regulating epigenetic changes that can occur in cells.¹⁶⁴ In response to cellular stresses, the interplay between p53-mediated methylation, demethylation, and other post-transcriptional modifications could fine-tune p53 activity ultimately to prevent tumor formation.¹⁶⁵

Enhancing other therapies

Various studies revealed that Ad-p53 enhances conventional therapies, such as chemotherapy, radiotherapy, and/or hyperthermia.^76,94 These enhancements are achieved primarily as a result of p53-mediated autophagy and apoptosis.^{74,119,120,123,124,128,166

–170}

In addition to synergistic effects on chemo- or radiotherapy, Gendicine could be used to augment immunotherapy. Ad-p53 gene therapy enhanced LAK immunotherapy in the treatment of HNC.¹⁰⁴ Combining a dendritic cell–based p53 vaccine (p53-DC) with rAd-p53 gene therapy can induce p53 overexpression and thus enhance the cytotoxicity of CTLs differentiated in response to the p53-DC vaccine in prostate cancer cells that do not express p53. This suggests that combination therapy with p53-DC vaccination and Ad-p53 gene therapy together may represent a new paradigm for the treatment of metastatic castration resistant prostate cancer.¹⁰³

The chemotherapy-augmenting effects of Gendicine may act by reversing multidrug resistance (MDR) through regulation of autophagy. p53 is thought to contribute to lung cancer cell radiosensitivity by regulating autophagy and apoptosis.¹⁷⁰ Another study suggested that regulatory effects of p53 on MDR may be determined by induction of autophagy in ovarian cancer cells.¹⁷¹

Significant efforts have been undertaken to reverse MDR. Autophagy could overcome drug resistance upon its activation by promoting cell death through role reversal from pro-survival to pro-death (i.e., apoptosis or necrosis). Likewise, autophagy inhibition could counteract MDR by sensitizing cells to anticancer drugs. Because the effects of p53 on MDR may depend upon p53 protein levels and location in cancer cells, enhancing chemotherapy efficacy may involve autophagy regulation by p53.^171,172

Regulating energy supply

Beyond its canonical functions described above, p53 also plays an important role in metabolism.¹³³ Previously, p53 was shown to regulate cellular energy supplies by suppressing gluconeogenesis, limiting glucose uptake via downregulation of glucose transporter GLUT1 and GLUT4 gene expression, and reduction of ATP generation by interfering with cytochrome bc1 complex function.^173
–175

Recently, in vitro and in vivo studies established a role for p53 in gluconeogenesis through a mechanism involving (1) direct activation of the gene encoding the NAD-dependent deacetylase sirtuin 6 (SIRT6), (2) SIRT6-dependent deacetylation and nuclear exclusion of forkhead box protein O1 (FoxO1), and (3) downregulation of FoxO1-activated genes (G6PC and PCK1) that are rate-limiting for gluconeogenesis. One study demonstrated that p53 cooperated with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion.¹⁷⁶ The results have implications for proposed tumor-suppressor functions of p53 through regulation of metabolic pathways.

Interestingly, the suppressive effect of p53 on glucose metabolism was observed in the clinic when Gendicine was given via systemic administration to cancer patients with insulin-dependent type 2 diabetes. Blood glucose levels decreased following Gendicine treatment, and these patients used insulin less frequently (manuscript in preparation). This effect persisted for >1 year after Gendicine treatment. Some preclinical and controlled clinical studies are underway to investigate this observation further.

p53 Mutation status and treatment response

Across all types of cancer, p53 has the most diversity in terms of cell functionality and the highest frequency of mutation (50–60%). The data for p53 in cancers were recently organized into a display of a “rainbow of mutants.” Based on the TP53 Database (27,852 somatic and 1,509 germ-line TP53 mutations in cancer) from the International Agency for Research of Cancer,¹⁷⁷ a novel, simplified classification system was proposed to be used to categorize the various functional classes of p53 mutants for use in clinical oncology.¹⁷⁸ These categories are predicated on the location of the mutation within the N-terminal, DNA-binding, or oligomerization domain, as well as the often context-dependent effects of the mutation on p53 function: complete or partial LOF, a dominant-negative (DN) effect, and/or GOF properties.

Optimal therapeutic targeting of these functionally variant categories of mutant p53 requires mutation-specific approaches, ranging from restoring wt activity to the mutant protein, to degradation of mutant p53.¹⁷⁸ Some strategies for developing more effective medical approaches include: (1) restoration of wt activity to mutant p53; (2) selectively increasing the activity of wt-p53; (3) eliciting residual activity of mutant p53; (4) selectively degrading mutant p53; (5) exploiting synthetic lethal vulnerabilities; (6) blocking co-factor proteins (e.g., PIN1 and PML); (7) suppressing mutant-p53-activated survival mechanisms; and (8) interfering with p53/MDM2/MDM4 interactions. Restoring wt activity of p53 is the most straightforward approach.¹⁷⁸

Ad-p53 gene therapy must overcome the challenges of the presence of mutant p53 expressed by target cancer cells.^179
–181 Either GOF mutation or high-level accumulation of mutant p53 proteins, particularly DN mutations, may antagonize or abrogate the tumor suppressive function afforded by the exogenous wt-p53.^178,182 However, at a molecular level, whether the restoration of wt-p53 functionality exerted by exogenous wt-p53 can override cancer progression driven by mutant p53 will likely be decided by the stoichiometry of wt-p53 and mutant p53 proteins. Early in vitro studies demonstrated that Ad-p53 infection rendered high-level exogenous wt-p53 expression in the nucleus of the infected cancer cells. p53 gene transduction efficiencies varied between cell lines. More than 90% growth inhibition occurred in seven out of eight cell lines after infection with Ad-p53 if a viral dose that resulted in infection of 50% of cells was used. Regardless of endogenous p53 status, apoptosis occurred in cells infected with Ad-p53.^{29,113,183,184} The results of cell culture studies were later confirmed by Ad-p53 in clinical applications (summarized in Tables 1 and 2).^116,129,185 Overall, restoration of wt-p53 by Gendicine, especially high-level nuclear expression of exogenous p53 in infected cells or in treated patients, can overcome the negative or competitive effects from endogenous mutant p53 to generate anticancer efficacy.

p53 as biomarker for gene therapy

p53 and its associated factors at genetic, epigenetic, and protein levels are not only therapeutic targets but also valuable clinically relevant biomarkers for diagnosis and prognosis. Early studies showed that diagnosis based on p53 mutation status is important and significant for cancer treatment and prognosis.^186,187 Based on the reported correlation between p53 and MDM2, monitoring the relative changes in both proteins may be more clinically meaningful.^188
–190

In an attempt to develop p53 further as a biomarker, a systematic search and collection of meta-analyses of p53-related alterations and cancers published in the past 5 years was conducted through appropriate queries in the PubMed database. Composed “gray-scale” tables demonstrate significant levels for each variant, and the potential heterogeneity was subsequently discussed. The data show that p53-related alterations are extremely complex biomarkers in terms of their clinical translation. Together with experimental studies on p53-related alterations, a gold-standard approach is still needed, as is additional evidence from clinical studies that include large, prospectively planned cohorts, to understand fully the potential of p53 as a cancer biomarker.¹⁹¹

p53 has been proposed to be a biomarker to predict Ad-p53 gene therapy efficacy in recurrent HNSCC.¹⁹² Tumor p53 biomarkers were evaluated in 116 patients, including 29 treated with methotrexate in a Phase III randomized controlled trial. Profiles that would be favorable for p53 gene therapy efficacy were hypothesized to have either normal p53 gene sequences or low-level p53 protein expression, whereas unfavorable p53 inhibitor profiles were predicted to have high-level expression of mutated p53 that can inhibit normal p53 protein function. A statistically significant increase in tumor responses was observed for patients with favorable p53 efficacy profiles compared to those with unfavorable p53 inhibitor profiles in Phase I/II trials. The subsequent Phase III trial showed statistically significant increases in time to progression (TTP) and survival following p53 gene therapy in patients with favorable p53 profiles compared to unfavorable p53 inhibitor profiles (median TTP: 2.7 months vs. 1.4 months, p = 0.0121; median survival: 7.2 months vs. 2.7 months, p < 0.0001). These results indicate that tumor p53 biomarker profiles may predict p53 gene therapy efficacy in recurrent HNSCC.¹⁹² A recent review of current information about p53 mutations available via The Cancer Genome Atlas–based analysis of HNSCC also suggested use of p53 mutation status as a potential evaluation or stratification biomarker for prognosis and a predictor of clinical response to radiotherapy and chemotherapy in HNSCC patients.¹⁹³

Based on Gendicine clinical data gathered over the past 12 years, the p53 mutation status did not significantly influence efficacy outcomes or long-term (3- to 5-year) survival rate of cancer patients given Ad-p53 gene therapy.

Discussion

Gendicine manufacturing experience

One of the remarkable accomplishments in Gendicine clinical development is that between its commercial launch in 2004 and 2013, 41 batches of Gendicine (totaling 169,571 vials, 1.0 × 10¹² vp per vial) were manufactured and applied in clinical oncology. All batches were compliant with CFDA QC/QA standards. The standard dose of Gendicine is one weekly injection with 10¹² vp per dose for 4 weeks. Treatment regimens can vary by one or two courses, depending on treatment response. To date, >30,000 patients have been treated with Gendicine alone or in combination with chemo- or radiotherapies and various other regimens. Approximately 3,000 international patients from >50 nations have been treated with Gendicine.

Safety, efficacy, and long-term benefit

Since Gendicine entered the market in 2004, numerous clinical oncology data and study reports have demonstrated its safety and efficacy.^3,37,43,107 The clinical data show that Gendicine combined with standard regimens resulted in CR rates 25–50% higher than those patients who received the standard regimen alone.^{45

–76} Among the >30,000 patients treated, the average response rate (CR + PR) reached 90%, and a large group of 5-year survivors was observed during treatment follow-up.^{50,56,72,78,79} A common adverse event observed was vector-associated transient fever, which occurred in 50–60% of patients treated.^3,37 The fever typically occurred within 24 h of Gendicine administration and persisted for 4–6 h. Other adverse events had a low frequency and were minor.

Spectrum of cancer treatment

Gendicine has been studied and found to be effective for treatment of HNC, NPC, and a variety of other types of cancer at various stages of disease, particularly in cases for which standard regimens failed and no other treatment options were available. The various controlled clinical studies presented in this review have demonstrated that Gendicine has been successfully used to treat several cancer types, including liver cancer, lung cancer, female reproductive cancer, digestive tract cancer, brain cancer, and soft-tissue cancer.^{45

–81,83

–97,99

–102} Gendicine intratumoral injection is minimally invasive. Successful use of Gendicine as a primary cancer treatment has also been reported.^74,83,99 Therefore, using Gendicine to eradicate early-stage cancer may be an alternative to surgical treatment to minimize the traumatic burden to patients, especially when the surgery is disfiguring or when no surgical option is available.

Enhancement of other treatment regimens

The p53 protein plays a central role in maintaining cell genome stability and cell growth regulation, and also functions as a chemo-radiotherapy sensitizer and anticancer therapy enhancer.^{49,53,67,78,85,91,97} In addition to its applicability to a wide spectrum of cancer types, Gendicine is also amenable to different routes of administration,^{50,58,70,81,93,95} and use in combination with other cancer regimens.^{63,76,83,93,102

–105} Gendicine is being further developed in the clinic as a general anticancer enhancer that can play various roles in different combinational cancer regimens at the physician's discretion, depending on clinical needs, disease status, and patient situation.

Further development and application potential

In numerous clinical studies, Gendicine has demonstrated effectiveness in cancer treatment via intratumoral injection and intra-cavity application. Gendicine also enhanced chemo-radiotherapy via systemic administration, intra-arterial injection, or intravenous infusion. However, adenoviruses induce strong innate and acquired immunity in vivo. As such, systemic Gendicine application may be further developed via modifications to the vector surface and/or temporarily attenuating immune responses. There are a variety of potential new approaches that have been reviewed and discussed recently that seek to overcome the limitations of Ad-vectors and enhance their efficacy in cancer gene therapy.^194
–196

Prevention of cancer is equally as important and as valuable as cancer treatment. Recent preclinical studies in an animal model of pancreatic cancer reported that a gain of function mutation in the TAD2 region of p53 resulted in a stronger suppression of pancreatic cancer.²⁴ High-efficiency gene delivery of p53 and high-level expression of p53 protein in cells may allow Gendicine to play a role in cancer prevention. Another area that is important for cancer prevention is vaccine development, especially an oral vaccine,^197
–199 which may be incorporated into Gendicine treatment regimens to enhance the prophylactic effects of cancer prevention. Controlled experiments and clinical studies could be carried out to investigate these potential opportunities further.

After more than a decade of commercial experience and treatment of >30,000 patients, Gendicine (Ad-p53) has demonstrated an exemplary safety profile. Gendicine has been effective in multiple tumor types and successfully deployed in combination with various standard anticancer therapies. Moreover, Gendicine has enabled the development of new cancer treatment algorithms to amplify treatment efficacy responses and expand medical applications.

Footnotes

Acknowledgments

We thank all the researchers, clinicians, nurses, regulatory personnel, and management who have worked with great dedication throughout the past years on Gendicine R&D, manufacture, and clinical applications. We are particularly grateful to Dr. Yuquan Wei for helpful discussion for this review and his continual promotion of gene therapy in China. We also acknowledge Drs. Arnold J. Levine, Chu-Tse Wu, Yixin Zeng, Yan Sun, Wenlin Huang, Xiangming Fang, Robert E. Sobol, Mr. Chris Reinhard, and many others who have contributed to advancing the development and application of Gendicine as well as the gene therapy field. We are grateful to the patients and their families who participated in Gendicine clinical trials and clinical applications.

Author Disclosure

W-W.Z. is a scientific advisory board member of Shenzhen SiBiono GeneTech Co. Ltd., but is not employed by the company. W.L., X.X., H.L., A.H., and W.X. are employees of Shenzhen SiBiono GeneTech Co. Ltd. The other co-authors have neither business interests nor operations involvement in the company and have no other competing financial interests.

References

Brief Report, First gene therapy medication approved. (Gendicine, developed by Shenzhen SiBiono Gene Technologies). Biopharm Int, 2003; 16–14. http://connection.ebscohost.com/c/articles/11716683/first-gene-therapy-medication-approved (last accessed February 5, 2018 ).

Wilson

. Gendicine: the first commercial gene therapy product. Hum Gene Ther, 2005; 16:1014–1015.

Peng

. Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers. Hum Gene Ther, 2005; 16:1016–1027.

Sibbald

. Death but one unintended consequence of gene-therapy trial. CMAJ, 2001; 164:1612.

Engel

, Kohn

, Podsakoff

. Update on gene therapy of inherited immune deficiencies. Curr Opin Mol Ther, 2003; 5:503–507.

Ginn

, Alexander

, Edelstein

, et al. Gene therapy clinical trials worldwide to 2012—an update. J Gene Med, 2013; 15:65–77.

, Huang

, Liu

, et al. Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery. World J Gastroenterol, 2004; 10:1867–1871.

European Medicines Agency (EMA) Committee for Medicinal Products for Human Use (CHMP). Summary of positive opinion for Glybera. July 19, 2012. EMA/CHMP/472167/2012.

Miller

. Glybera and the future of gene therapy in the European Union. Nat Rev Drug Discov, 2012; 11:419–419.

10.

Amgen Press Release. FDA approves IMLYGIC™ (Talimogene Laherparepvec) as first oncolytic viral therapy in the US, October 27, 2015. www.amgen.com/media/news-releases/2015/10/fda-approves-imlygic-talimogene-laherparepvec-as-first-oncolytic-viral-therapy-in-the-us/ (last accessed February 5, 2018 ).

11.

Harrington

, Puzanov

, Hecht

, et al. Clinical development of talimogene laherparepvec (T-VEC): a modified herpes simplex virus type-1–derived oncolytic immunotherapy. Expert Rev Anticancer Ther, 2015; 15:1389–1403.

12.

Bach

, Giralt

, Saltz

. FDA approval of Tisagenlecleucel: promise and complexities of a $475,000 cancer drug. JAMA, 2017; 318:1861–1862.

13.

Roberts

, Better

, Bot

, et al. Axicabtagene ciloleucel, a first-in-class CAR T cell therapy for aggressive NHL. Leuk Lymphoma, 2017; 1–12.

14.

Philippidis

. FDA Advisory Panel unanimously recommends approval of Spark Therapeutics' gene therapy Luxturna. GEN News Highlights, October 13, 2017. https://www.genengnews.com/gen-news-highlights/fda-advisory-panel-unanimously-recommends-approval-of-spark-therapeutics-gene-therapy-luxturna/81255043 (last accessed February 5, 2018 ).

15.

Russell

, Bennett

, Wellman

, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, Phase 3 trial. Lancet, 2017; 390:849–860.

16.

Linzer

, Levine

. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell, 1979; 17:43–52.

17.

Dippold

, Jay

, DeLeo

, et al. p53 transformation-related protein: detection by monoclonal antibody in mouse and human cells. Proc Natl Acad Sci U S A, 1981; 78:1695–1699.

18.

Levine

, Reich

, Thomas

. The regulation of a cellular protein, p53, in normal and transformed cells. Prog Clin Biol Res, 1982; 119:159–169.

19.

, Levine

. p53 and E2F-1 cooperate to mediate apoptosis. Proc Natl Acad Sci U S A, 1994; 91:3602–3606.

20.

Lowe

. Cancer therapy and p53. Curr Opin Oncol, 1995; 7:547–553.

21.

Robins

, Alexe

, Harris

, et al. The first twenty-five years of p53 research. In: Hainaut

, Wiman

, eds. 25 Years of P53 Research. Dordrecht, The Netherlands: Springer, 2005:1–25.

22.

, El-Deiry

. Targeting p53 for enhanced radio- and chemo-sensitivity. Apoptosis, 2009; 14:597.

23.

Bieging

, Mello

, Attardi

. Unravelling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer, 2014; 14:359–370.

24.

Mello

, Valente

, Raj

, et al. A p53 Super-tumor suppressor reveals a tumor suppressive p53-Ptpn14-Yap axis in pancreatic cancer. Cancer Cell, 2017; 32:460–473.

25.

Zhang

. Adenoviral vectors: development and application. Expert Opin Investig Drugs, 1997; 6:1419–1457.

26.

Zhang

. Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Ther, 1999; 6:113–138.

27.

J Gene Med. Gene therapy clinical trials worldwide—update online www.wiley.com//legacy/wileychi/genmed/clinical/Charts & Table/Vectors.

28.

Zhang

, Fang

, Branch

, et al. Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques, 1993; 15:868–872.

29.

Zhang

, Fang

, Mazur

, et al. High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther, 1994; 1:5–13.

30.

Zhang

W-W

, Fujiwara

, Grimm

, et al. Advances in cancer gene therapy. Adv Pharmacol, 1995; 32:289–341.

31.

Nielsen

, Lipari

, Dell

, et al. Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer. Clin Cancer Res, 1998; 4:835–846.

32.

Wills

, Maneval

, Menzel

, et al. Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther, 1994; 5:1079–1088.

33.

Barnard

. Technology evaluation: Sch-58500, Canji. Curr Opin Mol Ther, 2000; 2:586–592.

34.

INGN 201: Ad-p53, Ad5CMV-p53, adenoviral p53, p53 gene therapy—introgen, RPR/INGN 201. Drugs RD, 2007; 8:176–187.

35.

Tazawa

, Kagawa

, Fujiwara

. Advances in adenovirus-mediated p53 cancer gene therapy. Expert Opin Biol Ther, 2013; 13:1569–1583.

36.

Chen

, Zhang

, He

, et al. Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives. Onco Targets Ther, 2014; 7:1901–1909.

37.

Sobol

, Guan

, Li

, et al. Tp53 gene therapy for cancer treatment and prevention. In: Hainaut

, Olivier

, Wiman

, eds. p53 in the Clinics. New York: Springer, 2013:189–207.

38.

McGrory

, Bautista

, Graham

. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology, 1988; 163:614–617.

39.

Graham

, Smiley

, Russell

, et al. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol, 1977; 36:59–72.

40.

Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), April 15, 2008. https://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/UCM078694.pdf (last accessed February 5, 2018 ).

41.

Husain

, Han

, Au

, et al. Gene therapy for cancer: regulatory considerations for approval. Cancer Gene Ther, 2015; 22:554–563.

42.

Shenzhen Sibiono GeneTech, National Institute for the Control of Pharmaceutical and Biological Products. Points to consider for human gene therapy and product quality control State Food and Drug Administration of China. BioPharm Int, 2004; 17:73–76.

43.

, Li

, et al. Key points of basic theories and clinical practice in rAd-p53 (Gendicine™) gene therapy for solid malignant tumors. Expert Opin Biol Ther, 2015; 15:437–454.

44.

Lee

, Bishop

, Zhang

, et al. Adenovirus-mediated gene delivery: potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes Dis, 2017; 4:43–63.

45.

, Huang

, Wang

, et al. A combination therapy of selective intraarterial Gendicine infusion with chemotherapy for locally advanced head and neck carcinoma. Mol Ther, 2006; 13:S20.

46.

Zhang

, Xiao

, Liu

, et al. Recombinant adenovirus-p53 gene therapy combined with radiotherapy for head and neck squamous-cell carcinoma. Zhonghua Zhong Liu Za Zhi, 2005; 27:426–428.

47.

Zhang

, Xiao

, Sun

, et al. Clinical trial of recombinant adenovirus-p53 (Gendicine) combined with radiotherapy in nasopharyngeal carcinoma patients. Mol Ther, 2006; 13:S280.

48.

, Ma

, Xing

. Efficacy and safety of recombinant human adenovirus p53 combined with chemoradiotherapy in the treatment of recurrent nasopharyngeal carcinoma. Anticancer Drugs, 2017; 28:230–236.

49.

Liu

, Ji

, Chen

. Clinical effect of recombinant human p53 adv injection (Gendicine) in combination with radiotherapy in patients suffering from recurrent nasopharyngeal carcinoma. J Otolaryngol Ophthalmol Shandong Univ, 2010; 24:13–16.

50.

Pan

, Zhang

, Chen

, et al. Effect of recombinant adenovirus-p53 combined with radiotherapy on long-term prognosis of advanced nasopharyngeal carcinoma. J Clin Oncol, 2008; 27:799–804.

51.

Wang

, Wang

, Zhang

, et al. Clinical observations of recombinant adenovirus-p53 combined with radiotherapy on nasopharyngeal squamous carcinoma. Mod J Int Tradit Chin West Med, 2012; 21:924–934.

52.

, Xu

, Guo

, et al. Therapeutic p53 gene agent in the treatment of NPC cases: preliminary results. J Clin Oncol, 2015; 33:e13551–e13551.

53.

Chang

, Wu

, Xu

. The clinical observation of recombinant adenovirus p53 injection combined with chemotherapy in the treatment of neck metastatic carcinoma. Med Innov China, 2013; 17:006.

54.

Zhang

, Xiao

, Liu

, et al. Treatment of head and neck squamous cell carcinoma by recombinant adenovirus-p53 combined with radiotherapy: a Phase II clinical trial of 42 cases. Zhonghua Yi Xue Za Zhi, 2003; 83:2023–2028.

55.

, He

, Lan

, et al. Recombinant adenovirus p53 agent injection combined with radiotherapy and chemotherapy for intermediate and advanced stage nasopharyngeal carcinoma. Chin J Clin Oncol, 2009; 36:1031–1039.

56.

, Li

, Wang

, et al. Selective intra-arterial infusion of rAd-p53 with chemotherapy for advanced oral cancer: a randomized clinical trial. BMC Med, 2014; 12:16.

57.

Liu

, Chen

, Hu

, et al. Randomized, controlled Phase II study of post-surgery radiotherapy combined with recombinant adenoviral human p53 gene therapy in treatment of oral cancer. Cancer Gene Ther, 2013; 20:375–379.

58.

Guan

, Liu

, He

, et al. p53 gene therapy in combination with transcatheter arterial chemoembolization for HCC: one-year follow-up. World J Gastroenterol, 2011; 17:2143.

59.

Tian

, Liu

, Zhou

, et al. Multiple hepatic arterial injections of recombinant adenovirus p53 and 5-fluorouracil after transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma: a pilot Phase II trial. Anticancer Drugs, 2009; 20:389–395.

60.

Yang

, Wang

, et al. Clinical study of recombinant adenovirus-p53 combined with fractionated stereotactic radiotherapy for hepatocellular carcinoma. J Cancer Res Clin Oncol, 2010; 136:625–630.

61.

, Ma

, Kang

, et al. Recombinant adenovirus-p53 gene therapy combined with transcatheter arterial chemoembolization for p53-positive and p53-negative hepatocellular carcinoma. Chin J Interv Imaging Ther, 2010; 7:354–357.

62.

Chen

, Chen

, Xi

, et al. Clinical therapeutic effect and biological monitoring of p53 gene in advanced hepatocellular carcinoma. Am J Clin Oncol, 2014; 37:24–29.

63.

Shi

, Cui

, Hu

, et al. Observation of curative effect of combination P53 gene therapy drug with Traditional Chinese Medicine on primary hepatocellular carcinoma. Mod J Int Tradit Chin West Med, 2012; 7:693–694.

64.

Shi

, Dong

, Hu

, et al. Clinical study of P53 gene therapy on primary hepatocarcinoma. Mod J Int Tradit Chin West Med, 2012; 10:1036–1037.

65.

Feng

. Gendicine in interventional chemotherapy of primary hepatocarcinoma. World Chin J Digestol, 2013; 21:1437–1441.

66.

Wang

, Wang

, Yang

, et al. Treatment of local advanced non-small cell lung cancer with recombinant human p53adenovirus combined with radiochemotherapy. J Guiyang Med Col, 2014; 39:225–228.

67.

Guan

, Liu

, Zou

, et al. Adenovirus-mediated wild-type p53 gene transfer in combination with bronchial arterial infusion for treatment of advanced non-small-cell lung cancer, one year follow-up. J Zhejiang Univ Sci B, 2009; 10:331–340.

68.

Liu

. Safety and short-term efficacy of advanced malignant cancer by Gendicine combined with chemotherapy. J Qiqihar Univ Med, 2015; 36:16–18.

69.

Ning

, Sun

, Wang

, et al. Docetaxel plus trans-tracheal injection of adenoviral-mediated p53 versus docetaxel alone in patients with previously treated non-small-cell lung cancer. Cancer Gene Ther, 2011; 18:444–450.

70.

, Yang

, Huang

, et al. Antitumor activity of a combination of rAd2p53 adenoviral gene therapy and radiotherapy in esophageal carcinoma. Cell Biochem Biophys, 2011; 59:147–152.

71.

Cui

, Guan

, Liu

, et al. Outcome of patients with recurrent epithelial ovarian carcinoma following treatment with recombinant human adenovirus p53 combined with chemotherapy. Chinese J Cancer Biother, 2014; 21:450–454.

72.

, Chen

, Xiao

, et al. Effect and safety of recombinant adenovirus-p53 transfer combined with radiotherapy on long-term survival of locally advanced cervical cancer. Hum Gene Ther, 2016; 27:1008–1014.

73.

, Quan

, Jiang

. Clinical observations of intensity modulated radiation therapy combined with recombinant human p53 adenovirus injection on middle-late type giant piece of cervical carcinoma. Anhui Med J, 2015; 36:19–22.

74.

, Yang

, Han

, et al. The clinical effect of recombinant human Ad.p53 agent-Gendicine in advanced cancer patients. J Modern Oncol, 2006; 14:1295–1297.

75.

Dong

, Li

, Hong

, et al. Advanced malignant pleural or peritoneal effusion in patients treated with recombinant adenovirus p53 injection plus cisplatin. J Int Med Res, 2008; 36:1273–1287.

76.

Wang

, Zhao

, Zhang

. rAd-p53 combined with chemotherapy and local thermotherapy for patients with malignant serosal cavity effusion. China Cancer, 2012; 21:717–712.

77.

Zhu

. Preoperative recombinant adeno-viral human p53 gene (rAd-p53) therapy in treatment of locally advanced locally advanced papillary thyroid cancer (PTC) and folliculary thyroid cancer (FTC). J Clin Oncol, 2011; 29:5583–5583.

78.

Geng

, Xiao

, Zhang

, et al. Clinical effectiveness of recombinant adenovirus-p53 combined with radiotherapy in advanced soft tissue sarcoma: a report of 37 cases. J Clin Oncol, 2014; 32:e21514–e21514.

79.

Zhang

. Recombinant adenovirus-p53 (rAd-p53) in combination with radiotherapy for treating cervical cancer. J Clin Oncol, 2011; 29:5096.

80.

. Phase II study of recombinant adeno-viral human p53 (rAd-p53) gene therapy combined with surgery in treatment of melanomas of oral mucosa. J Clin Oncol, 2011; 29:8533–8533.

81.

Yang

, Wang

, Zheng

. A primary report of recombinant adeno-viral human p53 gene (rAd-p53) in combination with concurrent radio-chemotherapy in patients with T4N0-2M0 stage non-small cell lung cancer in elderly. Chin J Coal Ind Med, 2013; 16:1586–1589.

82.

Marks

, Davidoff

, Kerns

, et al. Overexpression and mutation of p53 in epithelial ovarian cancer. Cancer Res, 1991; 51:2979–2984.

83.

Wan

, Xu

R-L

, Zhu

, et al. Clinical observation on recombinant adenoviral human p53 gene therapy for advanced ovarian cancer. Chi J Clin Oncol Rehab, 2015; 22:782–785.

84.

Xia

, Du

, Wang

, et al. Treatment of uterine sarcoma with rAd-p53 (Gendicine^®) followed by chemotherapy—clinical study on TP 53 gene therapy. Hum Gene Ther, 2017 Dec 27 [Epub ahead of print]; DOI: 10.1089/hum.2017.206.

85.

Sheng

, Zheng

, Cui

, et al. Complete remission of multiple lung metastases after ablation of hepatocellular carcinoma by transarterial infusion with the p53 gene. Anticancer Drugs, 2015; 26:227–231.

86.

Guan

, Liu

, Sun

, et al. Successful management of postoperative recurrence of hepatocellular carcinoma with p53 gene therapy combining transcatheter arterial chemoembolization. World J Gastroenterol, 2005; 11:3803–3805.

87.

, Chen

, Zhang

. p53 gene therapy for pulmonary metastasis tumor from hepatocellular carcinoma. Anticancer Drugs, 2010; 21:882–884.

88.

Guan

, Liu

, Zhou

, et al. p53 gene (Gendicine) and embolisation overcame recurrent hepatocellular carcinoma. Gut, 2005; 54:1318–1319.

89.

Tian

, Liu

, Sui

. A patient with huge hepatocellular carcinoma who had a complete clinical response to p53 gene combined with chemotherapy and transcatheter arterial chemoembolization. Anticancer Drugs, 2009; 20:403–407.

90.

Zhou

, Zhang

, Zhao

, et al. The preliminary study of recombinant adenovirus p53 combined with transarterial embolization with particles for advanced hepatocellular carcinoma. Zhonghua Yi Xue Za Zhi, 2014; 94:660–663.

91.

J-L

, Cai

, Zhang

, et al. Combination of recombinant adenovirus-p53 with radiochemotherapy in unresectable pancreatic carcinoma. Chin J Cancer Res, 2011; 23:194–200.

92.

Zhang

. Recombinant adeno-viral human p53 gene combined with FOLFOX4 in treatment of advanced colorectal cancer. J Clin Oncol, 2011; 29:e14133–e14133.

93.

Gang

, Xiao

, Liu

, et al. Clinical effect of recombinant adenovirus-p53 combined with hyperthermia for advanced soft tissue sarcoma. Sci Tech Rev, 2013; 25:749–755.

94.

Xiao

, Liu

, Sun

. Clinical observation of the effect of recombinant adenovirus-p53 plus radio-thermotherapy on soft tissue sarcoma. Chin. J Clin Oncol, 2007; 34:65–67.

95.

Liu

, Zhao

, Jiang

, et al. A patient with a large intrathoracic malignant schwannoma who showed a complete clinical response to rAd-p53-combined with radiotherapy. Anticancer Drugs, 2015; 26:902–906.

96.

Zhang

, Hu

, Wang

, et al. Efficacy of recombinant adenoviral human p53 gene in treatment of malignant pleural or peritoneal effusions. Chin J Lung Cancer, 2013; 16:153–156.

97.

Rong

, Pan

, Gao

, et al. Evaluation of efficacy and safety for recombinant human adenovirus-p53 in the control of the malignant pleural effusions via thoracic perfusion. Sci Rep, 2016; 6:39355.

98.

Green

, Seymour

. Adenoviral vectors: systemic delivery and tumor targeting. Cancer Gene Ther, 2002; 9:1036–1042.

99.

Chen

, HU

, Zhang

. Effects of recombinant adenovirus p53 injection on malignant pleural effusion. Med J West China, 2010; 22:635–636.

100.

Zhang

, Xu

, Liu

, et al. Clinical study of recombinant adenovirus-p53 (Adp53) combined with hyperthermia in advanced cancer (a report of 15 cases). Intl J Hyperthermia, 2005; 21:631–636.

101.

, Xiao

, Li

, et al. Clinical antiangiogenic effect of recombinant adenovirus-p53 combined with hyperthermia for advanced cancer. Chin J Cancer Res, 2013; 25:749–755.

102.

Zhang

, Li

, Liu

, et al. Clinical observation on 21 cases of malignant tumor treated by Gendicine combined with comprehensive immunotherapy. In: 5th China Tumor Academic Conference and 7th Cross-Strait Tumor Conference, the International Conference of Cell and Gene Therapy on Tumor, 2nd China–Japan Tumor Interventional Therapy Conference, 2008:354–355. www.doc88.com/p-494332847864.html (last accessed February 5, 2018 ).

103.

Saito

, Kitagawa

, Yoneda

, et al. Combination of p53-DC vaccine and rAd-p53 gene therapy induced CTLs cytotoxic against p53-deleted human prostate cancer cells in vitro . Cancer Gene Ther, 2017; 24:289–296.

104.

Saito

, Ando

, Morishita

, et al. A combined lymphokine-activated killer (LAK) cell immunotherapy and adenovirus-p53 gene therapy for head and neck squamous cell carcinoma. Anticancer Res, 2014; 34:3365–3370.

105.

Xie

, Zhou

. Prevention of postoperative recurrence of small hepatocellular carcinoma by combination of elemene-5Fu-p53 gene via portal vein pathway. Zhonghua Yi Xue Hui, 2011; 163–164.

106.

, Li

, Zhang

, et al. In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia. Int J Oral Maxillofac Surg, 2009; 38:433–434.

107.

Tolcher

, Hao

, De

, et al. Phase I, pharmacokinetic, and pharmacodynamic study of intravenously administered Ad5CMV-p53, an adenoviral vector containing the wild-type p53 gene, in patients with advanced cancer. J Clin Oncol, 2006; 24:2052–2058.

108.

Morrissey

, Horvath

, Snyder

, et al. Rodent nonclinical safety evaluation studies of SCH 58500, an adenoviral vector for the p53 gene. Toxicol Sci, 2002; 65:266–275.

109.

Nemunaitis

, O'Brien

. Head and neck cancer: gene therapy approaches. Part II: genes delivered. Expert Opin Biol Ther, 2002; 2:311–324.

110.

Ganly

, Kirn

, Eckhardt

, et al. A Phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. Clin Cancer Res, 2000; 6:798–806.

111.

Mulvihill

, Warren

, Venook

, et al. Safety and feasibility of injection with an E1B-55 kDa gene-deleted, replication-selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a Phase I trial. Gene Ther, 2001; 8:308–315.

112.

Ahi

, Bangari

, Mittal

. Adenoviral vector immunity: its implications and circumvention strategies. Curr Gene Ther, 2011; 11:307–320.

113.

Zhang

, Alemany

, Wang

, et al. Safety evaluation of Ad5CMV-p53 in vitro and in vivo . Hum Gene Ther, 1995; 6:155–164.

114.

Kaiser

, Attardi

. Deconstructing networks of p53-mediated tumor suppression in vivo . Cell Death Differ, 2018; 25:93–103.

115.

Giono

, Manfredi

. The p53 tumor suppressor participates in multiple cell cycle checkpoints. J Cell Physiol, 2006; 209:13–20.

116.

Wang

, Lu

, Wang

, et al. Effect of recombinant adenovirus-p53 on growth and chemosensitivity of human lung adenocarcinoma cell lines. Zhongguo Fei Ai Za Zhi, 2006; 9:127–131.

117.

Wang

, Jiao

. Effect of combined use of rAd-p53 and DDP in different time sequence on tumoricidal activity against human lung adenocarcinoma cells in vitro . J Modern Oncol, 2008; 16:339–341.

118.

Zhang

, Zhang

, Li

, et al. Biologic analysis of recombinant human adenovirus-p53 injection in patients with oral leukoplakia. Hua Xi Kou Qiang Yi Xue Za Zhi, 2008; 26:670–672.

119.

Lang

, Bruner

, Fuller

, et al. Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results. J Clin Oncol, 2003; 21:2508–2518.

120.

Brady

, Attardi

. p53 at a glance. J Cell Sci, 2010; 123:2527–2532.

121.

Haupt

, Berger

, Goldberg

, et al. Apoptosis—the p53 network. J Cell Sci, 2003; 116:4077–4085.

122.

Stegh

. Targeting the p53 signaling pathway in cancer therapy—the promises, challenges and perils. Expert Opin Ther Targets, 2012; 16:67–83.

123.

Chen

, Zheng

, Liu

, et al. rAd-p53 enhances the sensitivity of human gastric cancer cells to chemotherapy. World J Gastroenterol, 2011; 17:4289–4297.

124.

, Pan

, Zhu

, et al. Recombinant adenovirus-p53 (Gendicine) sensitizes a pancreatic carcinoma cell line to radiation. Chin J Cancer Res, 2013; 25:715–721.

125.

Liu

, Zheng

, Liu

. The mechanism and inhibitory effect of recombinant human P53 adenovirus injection combined with paclitaxel on human cervical cancer cell HeLa. Eur Rev Med Pharmacol Sci, 2015; 19:1037–1042.

126.

Zhenchao

, Liting

, Jun

. Effects of rAd-p53 on growth of human hepatic carcinoma cells. Acta Universitatis Medicinalis Anhui, 2013; 48:353–356.

127.

Welcker

, Clurman

. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer, 2008; 8:83–93.

128.

, Zheng

, Zhou

, et al. Recombinant human adenovirus-p53 injection induced apoptosis in hepatocellular carcinoma cell lines mediated by p53-Fbxw7 pathway, which controls c-Myc and cyclin E. PLoS One, 2013; 8:e68574.

129.

Crighton

, Wilkinson

, Ryan

. DRAM links autophagy to p53 and programmed cell death. Autophagy, 2007; 3:72–74.

130.

Tasdemir

, Chiara

, Morselli

, et al. A dual role of p53 in the control of autophagy. Autophagy, 2008; 4:810–814.

131.

Tang

, Di

, Cao

, et al. p53-mediated autophagic regulation: a prospective strategy for cancer therapy. Cancer Lett, 2015; 363:101–107.

132.

White

. Autophagy and p53. Cold Spring Harb Perspect Med, 2016; 6:a026120.

133.

Berkers

, Maddocks

, Cheung

, et al. Metabolic regulation by p53 family members. Cell Metab, 2013; 18:617–633.

134.

Crighton

, Wilkinson

, O'Prey

, et al. DRAM, a p53-Induced modulator of autophagy, is critical for apoptosis. Cell, 2006; 126:121–134.

135.

Haq

, Brenton

, Takahashi

, et al. Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence. Cancer Res, 2002; 62:5076–5082.

136.

Terzi

, Izmirli

, Gogebakan

. The cell fate: senescence or quiescence. Mol Biol Rep, 2016; 43:1213–1220.

137.

Collado

, Gil

, Efeyan

, et al. Tumour biology: senescence in premalignant tumours. Nature, 2005; 436:642.

138.

Dankort

, Filenova

, Collado

, et al. A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors. Genes Dev, 2007; 21:379–384.

139.

Tyner

, Venkatachalam

, Choi

, et al. p53 mutant mice that display early ageing-associated phenotypes. Nature, 2002; 415:45–53.

140.

Miller

, Jasty

, Mancini

, et al. Hamster tumors induced by type I avian adenovirus (CELO). I. Histopathology and neoantigen of virus-induced and transplanted tumors. Arch Gesamte Virusforsch, 1970; 32:221–228.

141.

Avgousti

, Herrmann

, Kulej

, Pancholi

, et al. A core viral protein binds host nucleosomes to sequester immune danger signals. Nature, 2016; 535:173–177.

142.

Endo

, Sakai

, Ouchi

, et al. Virus-mediated oncolysis induces danger signal and stimulates cytotoxic T-lymphocyte activity via proteasome activator upregulation. Oncogene, 2008; 27:2375–2381.

143.

Yen

, Ioannides

, Xu

, et al. Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type. P53 (Ad-p53). Cancer Gene Ther, 2000; 7:530–536.

144.

Teodoro

, Evans

, Green

. Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome. J Mol Med (Berl), 2007; 85:1175–1186.

145.

Fujiwara

, Nishizaki

, Tanaka

. Recombinant adenovirus expressing wild-type p53 is antiagiogenic: implication for lung cancer gene therapy. Gan To Kagaku Ryoho, 2000; 27:1217–1224.

146.

Zhang

, Yu

, Hu

, et al. Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res, 2000; 60:3655–3661.

147.

Sano

, Minamino

, Toko

, et al. p53-induced inhibition of Hif-1 cause scardiac dysfunction during pressure overload. Nature, 2007; 446:444–448.

148.

Dai

, Chen

, Song

, et al. A natural small molecule harmine inhibits angiogenesis and suppresses tumor growth through activation of p53 in endothelial cells. PLoS One, 2012; 7:e52162.

149.

Nishizaki

, Fujiwara

, Tanida

, et al. Recombinant adenovirus expressing wild-type p53 is antiangiogenic: a proposed mechanism for by-stander effect. Clin Cancer Res, 1999; 5:1015–1023.

150.

Frank

, Frederick

, Liu

, et al. Bystander effect in the adenovirus-mediated wild-type p53 gene therapy model of human squamous cell carcinoma of the head and neck. Clin Cancer Res, 1998; 4:2521–2528.

151.

Guo

, Cui

. New perspective on targeting the tumor suppressor p53 pathway in the tumor microenvironment to enhance the efficacy of immunotherapy. J Immunother Cancer, 2015; 3:9.

152.

Cui

, Guo

. Immunomodulatory function of the tumor suppressor p53 in host immune response and the tumor microenvironment. Int J Mol Sci, 2016; 17:E1942.

153.

Wang

, Lan

, Si

, et al. Differential expression of KAI1 and CD44 in nasopharyngeal carcinoma before and after Gendicine combined with radiotherapy and chemotherapy treatment. Chin J Otorhinolaryngol, 2014; 20:96–100.

154.

Shiota

, Izumi

, Onitsuka

, et al. Twist and p53 reciprocally regulate target genes via direct interaction. Oncogene, 2008; 27:5543–5553.

155.

Bolós

, Peinado

, Pérez-Moreno

, et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci, 2003; 116:499–511.

156.

Shih

, Tsai

, Chang

, et al. Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res, 2005; 11:8070–8078.

157.

Muller

PAJ

, Vousden

, Norman

. p53 and its mutants in tumor cell migration and invasion. J Cell Biol, 2011; 192:209–218.

158.

Jin

, Levine

. The p53 functional circuit. J Cell Sci, 2001; 114:4139–4140.

159.

Qiu

, Wu

, Walsh

, et al. Retinoblastoma protein modulates gankyrin-MDM2 in regulation of p53 stability and chemosensitivity in cancer cells. Oncogene, 2008; 27:4034–4043.

160.

Fuchs

, Adler

, Buschmann

, et al. Mdm2 association with p53 targets its ubiquitination. Oncogene, 1998; 17:2543–2547.

161.

, Lozano

. NF-kappa B activation of p53. A potential mechanism for suppressing cell growth in response to stress. J Biol Chem, 1994; 269:20067–20074.

162.

, Kim

, Roeder

. Ordered cooperative functions of PRMT1, p300, and CARM1 in transcriptional activation by p53. Cell, 2004; 117:735–748.

163.

, Ye

, Tang

, et al. p53 dynamics orchestrates with binding affinity to target genes for cell fate decision. Cell Death Dis, 2017; 8:e3130.

164.

Levine

, Berger

. The interplay between epigenetic changes and the p53 protein in stem cells. Genes Dev, 2017; 31:1195–1201.

165.

Scoumanne

, Chen

. Protein methylation: a new mechanism of p53 tumor suppressor regulation. Histol Histopathol, 2008; 23:1143–1149.

166.

Lebedeva

, Bagdasarova

, Tyler

, et al. Tumor suppression and therapy sensitization of localized and metastatic breast cancer by adenovirus p53. Hum Gene Ther, 2001; 12:763–772.

167.

Wang

, Yang

, Zhang

, et al. The clinical observation of effect of recombinant human Ad-p53 combined with chemotherapy or radiotherapy in advanced cancer patients. Chin Clin Oncol, 2008; 13:896–900.

168.

Chen

, Tong

, Ma

. The recent clinical efficacy of recombinant human Ad-P53 agent–Gendicine combined chemotherapy in malignant tumor patients. J Clin Exp Med, 2014; 13:1326–1329.

169.

, Han

, Zhao

, et al. Synergistic cytotoxic effects of recombinant human adenovirus p53 and radiation at various time points in A549 lung adenocarcinoma cells. Oncol Lett, 2012; 4:529.

170.

Cheng

, Kong

, Hou

, et al. The tumor suppressor, p53, contributes to radiosensitivity of lung cancer cells by regulating autophagy and apoptosis. Cancer Biother Radiopharm, 2013; 28:153–159.

171.

Kong

, Ma

, Liang

, Yi

, et al. The different regulatory effects of p53 status on multidrug resistance are determined by autophagy in ovarian cancer cells. Biomed Pharmacother, 2012; 66:271–278.

172.

Hodorová

, Rybárová

, Vecanová

, et al. Relation between expression pattern of wild-type p53 and multidrug resistance proteins in human nephroblastomas. Acta Histochem, 2013; 115:273–278.

173.

Schwartzenberg-Bar-Yoseph

, Armoni

, Karnieli

. The tumor suppressor p53 down-regulates glucose transporters GLUT1 and GLUT4 gene expression. Cancer Res, 2004; 64:2627–2633.

174.

Brasseur

, Tron

, Dujardin

, et al. The nuclear ABC1 gene is essential for the correct conformation and functioning of the cytochrome bc1 complex and the neighbouring complexes II and IV in the mitochondrial respiratory chain. Eur J Biochem, 1997; 246:103–111.

175.

Iiizumi

, Arakawa

, Mori

, et al. Isolation of a novel gene, CABC1, encoding a mitochondrial protein that is highly homologous to yeast activity of bc1 complex. Cancer Res, 2002; 62:1246–1250.

176.

Zhang

, Tu

, Wang

, et al. Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion. Proc Natl Acad Sci U S A, 2014; 111:10684–10689.

177.

Bouaoun

, Sonkin

, Ardin

, et al. TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data. Hum Mutat, 2016; 37:865–876.

178.

Sabapathy

, Lane

. Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others. Nat Rev Clin Oncol, 2018; 15:13–30.

179.

Freed-Pastor

, Prives

. Mutant p53: one name, many proteins. Genes Dev, 2012; 26:1268–1286.

180.

Olotu

, Soliman

MES

. From mutational inactivation to aberrant gain-of-function: unraveling the structural basis of mutant p53 oncogenic transition. J Cell Biochem, 2017 Oct 23 [Epub ahead of print]; DOI: 10.1002/jcb.26430.

181.

Yue

, Zhao

, Xu

, et al. Mutant p53 in cancer: accumulation, gain-of-function, and therapy. J Mol Biol, 2017; 429:1595–1606.

182.

Zeimet

, Marth

. Why did p53 gene therapy fail in ovarian cancer?. Lancet Oncol, 2003; 4:415–422.

183.

Wolf

, Mills

, Bazzet

, et al. Adenovirus-mediated p53 growth inhibition of ovarian cancer cells is independent of endogenous p53 status. Gynecol Oncol, 1999; 75:261–266.

184.

Harris

, Sutjipto

, Wills

, et al. Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein. Cancer Gene Ther, 1996; 3:121–130.

185.

Buller

, Runnebaum

, Karlan

, et al. A Phase I/II trial of rAd/p53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer Gene Ther, 2002; 9:553–556.

186.

Eissa

, Zohny

, Zekri

, et al. Diagnostic value of fibronectin and mutant p53 in the urine of patients with bladder cancer: impact on clinicopathological features and disease recurrence. Med Oncol, 2010; 27:1286–1294.

187.

Guo

, Wang

. Significance of mutant p53, Ki-67, ER and PR in differential diagnosis of uterine endometrial carcinoma and uterine serous carcinoma. Cancer Res Clin, 2015; 27:406–408.

188.

Noon

, Polański

, El-Fert

, et al. Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy. BJU Int, 2012; 109:1250–1257.

189.

Deben

, Deschoolmeester

, Lardon

, et al. TP53 and MDM2 genetic alterations in non-small cell lung cancer: evaluating their prognostic and predictive value. Crit Rev Oncol Hematol, 2016; 99:63–73.

190.

Abolhasani

, Salarinejad

, Asgari

. P53 and MDM2 over-expression and five-year survival of kidney cancer patients undergoing radical nephrectomy—Iranian experience. Asian Pac J Cancer Prev, 2015; 16:5043–5047.

191.

Xiao

, Wang

, Chen

. The clinical translational potential of p53-related alterations as cancer biomarkers. Histol Histopathol, 2015; 30:1171–1183.

192.

Nemunaitis

, Clayman

, Agarwala

, et al. Biomarkers predict p53 gene therapy efficacy in recurrent squamous cell carcinoma of the head and neck. Clin Cancer Res, 2009; 15:7719–7725.

193.

Zhou

, Liu

, Myers

. TP53 mutations in head and neck squamous cell carcinoma and their impact on disease progression and treatment response. J Cell Biochem, 2016; 117:2682–2692.

194.

Wold

, Toth

. Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Curr Gene Ther, 2013; 13:421–433.

195.

Alonsopadilla

, Papp

, Kaján

, et al. Development of novel adenoviral vectors to overcome challenges observed with hAdV-5-based constructs. Mol Ther, 2015; 24:6–16.

196.

Yamamoto

, Nagasato

, Yoshida

, et al. Recent advances in genetic modification of adenovirus vectors for cancer treatment. Cancer Sci, 2017; 108:831–837.

197.

Mason

, Lam

, Arntzen

. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci U S A, 1992; 89:11745–11749.

198.

Lei

, Xu

, Chen

, et al. Immunoprotection against influenza H5N1 virus by oral administration of enteric-coated recombinant lactococcus lactis mini-capsules. Virology, 2010; 407:319–324.

199.

Tam

, Lam

, et al. Oral immunization and edible vaccines: a viable option or mirage?. Biotech in Hong Kong, 2015; 2:201–213.