Volume 11, Issue 1, Pages 149-159 (January 2005) Clinical and local biological effects of an intratumoral injection of mda-7 (IL24; INGN 241) in patients with advanced carcinoma: a phase I study  C. Casey Cunningham, Sunil Chada, James A. Merritt, Alex Tong, Neil Senzer, Yuan Zhang, Abner Mhashilkar, Karen Parker, Sasha Vukelja, Don Richards, Jill Hood, Keith Coffee, John Nemunaitis  Molecular Therapy  Volume 11, Issue 1, Pages 149-159 (January 2005) DOI: 10.1016/j.ymthe.2004.09.019 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Study schema for clinical protocol. The dose levels of INGN 241 and biopsy schedules are indicated. bx, biopsy; rxn, resection. Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Excisional biopsy procedure. (1) INGN 241 vector admixed with Isosulfan blue allows identification of the site of injection. (2) Lesion resected 24 h after injection. (3) Postresection processing. The bisected lesion is serially sectioned and the left portion is fixed and evaluated by immunohistochemistry. The right half is sectioned and immediately flash-frozen for quantitative PCR analyses. Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Pharmacodynamics of INGN 241 vector and expression. (A) Dose response of INGN 241 vector DNA and RNA. Tumor sections were obtained from preinjected (Pre), proximal, and distal sections from cohort 1–3 patients and vector-specific signals evaluated by quantitative DNA- and RT-PCR. RNA was not available from cohort 2 tumor. (B) Dose response of INGN 241 transgenic MDA-7 expression and TUNEL reactivity. Tumor sections were obtained from preinjected (Pre), proximal, and distal sections from cohort 1–3 patients and vector-specific signals evaluated. (C) Spread of INGN 241 DNA and (D) RNA from injection site. Genomic DNA and RNA were isolated from tumor sections and analyzed using vector-specific primers for INGN 241. Signals were quantitated using real-time PCR. Data are plotted to indicate signals compared to distance from injection site. Correlation coefficient for DNA decay = 0.9 (P < 0.02) and for RNA decay = 0.82 (P < 0.05). (n > 40 samples were used in the analysis.) Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Intratumoral pharmacokinetics of vector DNA and mRNA. (A) Decay of INGN 241 vector at injection site. The median number of DNA copies at each time point postinjection is shown; number of patients per sample is indicated below. The number of DNA copies/μg genomic DNA was converted to illustrate the average number of vector DNA copies per cell—shown above graph. (B) Decay of INGN 241 vector RNA at injection site. The median number of RNA copies/μg at each time point is shown; number of patients per sample is indicated below. Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Spread of MDA-7 protein and biological effect. MDA-7 protein expression correlates with apoptosis. Serial sections from each tumor were evaluated for (A) MDA-7 expression and (B) TUNEL reactivity using immunohistochemistry. Data are plotted to indicate signals compared to distance from injection site. Both MDA-7 and TUNEL staining show strong correlation with distance; correlation coefficient for MDA-7 = 0.69 and for TUNEL = 0.77 (P < 0.02). (n > 30 samples were used in the analysis.) Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 MDA-7 transgene expression correlates with distribution of vector throughout the tumor. One-half of the tumor was analyzed for MDA-7 protein expression and the other half for vector-specific DNA and RNA levels. The number of DNA copies/μg genomic DNA and number of RNA copies/μg total RNA are shown for each tumor section. TUNEL reactivity was 50% in the central section and 30% at the periphery (Section 3). Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Objective clinical response to INGN 241 in a patient with metastatic melanoma. Cohort 8 patient with metastatic melanoma. Injected lesion was on right clavicle (dashed circle in (A)). (B) By day 4, region is inflamed. (C) At the end of cycle 1 (day 30), lesion has completely regressed. This patient is still alive >600 days after initiating treatment. Molecular Therapy 2005 11, 149-159DOI: (10.1016/j.ymthe.2004.09.019) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions