αv integrins (initial binding target)
Neuropilin-1 (NRP-1, secondary binding target after proteolytic cleavage) [1]

Three processes are involved in iRGD peptide-mediated tumor penetration: first, it binds to αv-integrins on tumor vasculature or tumor cells; second, it exposes a C-terminal motif that binds to neuropilin-1 (NRP-1); and third, it internalizes the cell. The insertion of an iRGD peptide into the C terminus of the ICOVIR15K fiber only improves binding and internalization in MCF7 cells that express integrins and NRP-1. Viral infection and replication are unaffected by iRGD insertion[1]. When coupled with 5-FU, iRGD peptide (0.3 μmol/mL) increases the chemotherapeutic efficacy of 5-FU on gastric cancer cells through NRP1[2]. However, iRGD peptide alone has no discernible effect on gastric cancer cells.

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The insertion of the iRGD peptide into the C-terminus of the oncolytic adenovirus ICOVIR15K fiber protein resulted in a modified virus (ICOVIR15K-iRGD). In MCF7 cells (which express αvβ5 integrins and NRP-1, but low levels of the adenovirus primary receptor CAR), the iRGD-modified virus showed enhanced binding and internalization compared to the control virus (ICOVIR15K). However, this enhanced binding/internalization did not lead to increased transduction (gene expression) or cytotoxicity in the tested cell lines (A549, MIA PaCa-2), suggesting that iRGD-mediated internalization may not facilitate the virus's natural entry and replication pathway. [1]
As a functional control, a virus modified with the RPARPAR peptide (a CendR prototype NRP-1-binding peptide) also showed enhanced binding and internalization in cell lines expressing high/moderate levels of NRP-1 (HUVEC, A549, MCF7). [1]

When iRGD is introduced into the oncolytic adenovirus ICOVIR15K, the result is an increased anticancer effect in mice as well as improved early viral diffusion into the tumor mass[1]. When 5-FU is combined with iRGD (4 mmol/kg, iv), it considerably slows down the formation of tumors in nude mice that are harboring human stomach cancer cells[2].

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When the iRGD peptide was inserted into the capsid of a tumor-retargeted adenoviral vector (AdGLK-iRGD) and administered intravenously to mice bearing MIA PaCa-2 pancreatic xenograft tumors, it significantly enhanced tumor transduction (approximately 2-fold higher luciferase signal) compared to the vector without iRGD (AdGLK). This enhancement was specific to the tumor, as transduction of other organs (e.g., liver, spleen) remained unchanged. [1]
Insertion of the iRGD peptide into the oncolytic adenovirus ICOVIR15K (ICOVIR15K-iRGD) enhanced early viral dissemination within the tumor mass. At day 11 post intravenous administration in A549 lung carcinoma xenograft models, the E1A-positive area (indicating viral replication foci) in tumors treated with ICOVIR15K-iRGD was approximately 4-fold larger than in those treated with the control virus, suggesting improved intratumoral penetration. [1]
The iRGD peptide insertion significantly improved the antitumor efficacy of the oncolytic adenovirus. In both A549 (high NRP-1) and MIA PaCa-2 (low NRP-1) subcutaneous xenograft models, a single intravenous dose of ICOVIR15K-iRGD led to greater tumor growth inhibition and prolonged animal survival compared to the control virus ICOVIR15K and PBS groups. [1]

Adenovirus Binding and Internalization Assay: Cells (HUVEC, MCF7, A549, MIA PaCa-2) were seeded in 96-well plates. After 24 hours, cells were infected with purified viruses (e.g., ICOVIR15K, ICOVIR15K-iRGD) at a dose of 10,000 viral genomes per cell in cold media and incubated for 90 minutes at 4°C with agitation to allow binding. For binding measurements, cells were washed with cold PBS and lysed. For internalization assays, after binding, cells were washed, fresh media was added, and they were incubated for 60 minutes at 37°C. Cells were then washed, trypsinized, and pelleted for lysis. Viral genomes in the lysates were quantified by TaqMan-based qPCR. [1]
Transduction Assay: Cells (MCF7, 293, A549, MIA PaCa-2) were seeded in 96-well plates and infected the next day with adenoviral vectors (e.g., AdGLK, AdGLK-iRGD) at specific viral particle per cell ratios. At 36 hours post-infection, cells were lysed, and luciferase expression was quantified using a luciferase assay system. [1]
In vitro Cytotoxicity Assay: MIA PaCa-2 or A549 cells were infected in suspension with serial dilutions of oncolytic viruses (e.g., ICOVIR15K, ICOVIR15K-iRGD). At 5-6 days post-infection, total cellular protein was quantified. The IC₅₀ (half-maximal inhibitory concentration) values were calculated using non-linear regression analysis. [1]

Biodistribution Study: Female BALB/C nu/nu mice bearing subcutaneous MIA PaCa-2 pancreatic adenocarcinoma xenografts (approx. 200 mm³) were randomized into groups. Mice received a single intravenous injection via the tail vein of either PBS, or 4 x 10¹⁰ viral particles of AdGLK or AdGLK-iRGD. Three days post-injection, mice were injected intraperitoneally with D-luciferin, euthanized after 15 minutes, and organs were harvested for ex vivo bioluminescence imaging to quantify luciferase expression. [1]
Antitumor Efficacy Study: Female BALB/C nu/nu mice bearing subcutaneous A549 lung adenocarcinoma or MIA PaCa-2 pancreatic adenocarcinoma xenografts (reaching specified volumes) were randomized. Mice received a single intravenous injection via the tail vein of PBS, or 4 x 10¹⁰ viral particles of ICOVIR15K or ICOVIR15K-iRGD. Tumor dimensions and body weight were measured twice weekly. Tumor volume was calculated. Animals were euthanized when tumor volume exceeded 500 mm³. [1]
Analysis of Viral Dissemination: Mice bearing A549 tumors received a single intravenous dose of PBS or viruses (ICOVIR15K, ICOVIR15K-iRGD, ICOVIR15K-RPARPAR). At day 11 post-injection, tumors were harvested, embedded, and sectioned. Viral replication within the tumor was assessed by immunofluorescence staining for adenovirus E1A protein, and the E1A-positive area was quantified. [1]

[1]. iRGD tumor-penetrating peptide-modified oncolytic adenovirus shows enhanced tumor transduction, intratumoral dissemination and antitumor efficacy. Gene Ther. 2014 Aug;21(8):767-74.

[2]. Combination of NRP1-mediated iRGD with 5-fluorouracil suppresses proliferation, migration and invasion of gastric cancer cells. Biomed Pharmacother. 2017 Sep;93:1136-1143.

Internalized arginylglycyl aspartate cyclic peptide (iRGD) is a 9-amino acid cyclic tumor-specific targeting peptide with a structure based on the arginine-glycine-aspartate (RGD) sequence (CRGDKRGPDC) and tumor-penetrating activity. iRGD contains both an RGD motif and a C-terminal binding (CendR) motif, the latter enhancing its internalization effect. After administration, the RGD motif of the iRGD peptide specifically targets the tumor by binding to αvβ3/αvβ5 integrins on tumor endothelial cells. Subsequently, iRGD is cleaved by a tumor-specific protease, exposing the positively charged CendR motif. This motif binds to neuropilin-1 (NRP-1), an overexpressed receptor on certain tumor cells. This increases tumor vascular permeability, promoting tumor penetration. Compared to other RGD peptides, this drug improves delivery efficiency and increases the accumulation of concomitant or coupled chemotherapeutic drugs in the tumor.

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iRGD peptide (circular sequence: CRGDKGPDC) is a tumor-penetrating peptide identified by in vivo phage display technology. Its mechanism of action includes three steps: 1) binding to αν integrin overexpressed on tumor blood vessels or tumor cells; 2) being hydrolyzed by tumor-associated proteases to expose the C-terminal motif; 3) the exposed CendR motif binds to neurociliary protein-1 (NRP-1), mediating tissue penetration and internalization. This mechanism endows the drug with tumor selectivity. [1] In this study, the iRGD peptide was genetically inserted into the C-terminus of adenovirus fibrin along with the flexible (GGGGS)₃ linker to enhance the virus's ability to infiltrate from tumor blood vessels and penetrate deep into the tumor parenchyma. The effect of this peptide has an additive effect with the existing RGDK fiber axon modification of the basic virus (ICOVIR15K). [1]
The in vivo antitumor efficacy of iRGD-modified virus was observed in tumor models with high NRP-1 expression (A549) and low NRP-1 expression (MIA PaCa-2) in tumor cells, indicating that interaction with NRP-1 on tumor blood vessels and/or matrix plays a key role in its enhanced permeability. [1]

C35H57N13O14S2

948.035784482956

947.358

1392278-76-0

134611625

White to off-white solid powder

-11.3

14

19

13

64

1750

7

S1CC(C(=O)O)NC(C(CC(=O)O)NC(C2CCCN2C(CNC(C(CCCCN)NC(C(CC(=O)O)NC(CNC(C(CCC/N=C(\N)/N)NC(C(CS1)N)=O)=O)=O)=O)=O)=O)=O)=O

YHTTWXCDIRTOQX-FQJIPJFPSA-N

InChI=1S/C35H57N13O14S2/c36-8-2-1-5-18-30(57)42-14-25(50)48-10-4-7-23(48)33(60)46-21(12-27(53)54)32(59)47-22(34(61)62)16-64-63-15-17(37)28(55)44-19(6-3-9-40-35(38)39)29(56)41-13-24(49)43-20(11-26(51)52)31(58)45-18/h17-23H,1-16,36-37H2,(H,41,56)(H,42,57)(H,43,49)(H,44,55)(H,45,58)(H,46,60)(H,47,59)(H,51,52)(H,53,54)(H,61,62)(H4,38,39,40)/t17-,18-,19-,20-,21-,22-,23-/m0/s1

(6S,9S,15S,18R,23R,26S,29S)-18-amino-6-(4-aminobutyl)-9,26-bis(carboxymethyl)-15-[3-(diaminomethylideneamino)propyl]-2,5,8,11,14,17,25,28-octaoxo-20,21-dithia-1,4,7,10,13,16,24,27-octazabicyclo[27.3.0]dotriacontane-23-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 50 mg/mL (~52.74 mM)

Solubility in Formulation 1: 100 mg/mL (105.48 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)