CXCL12 retargeting of an adenovirus vector to cancer cells using a bispecific adapter

Figures & data

Figure 1 Flow cytometry analysis of CXCR4, CXCR7, hCAR, and integrin α_vβ₃ and α_vβ₅ cell surface expression.

Notes: Evaluation of antigen expression using an anti-CXCR4 antibody (A), an anti-CXCR7 antibody (B), an anti-hCAR antibody (C), and anti-integrin α_vβ₃ and α_vβ₅ antibodies (D) in human cancer cell lines, MDA-MB-435S, ZR-75-1, BT-20, and MCF-7, and in the immortalized non-tumorigenic breast epithelial cell line MCF-12A.
Abbreviation: hCAR, human Coxsackie and adenovirus receptor.

Table 1 Summary of surface antigen expression in cancer cell lines

Antibody	Cell line
	MDA-MB-435S	ZR-75-1	BT-20	MCF-7	MCF-12A
Unstained	1.95	2.56	3.46	3.60	3.03
Isotype (IgG2a κ)	1.91	2.65	3.46	3.57	3.03
Anti-CXCR4	6.41	6.48	11.27	11.17	3.78
Unstained	3.24	2.65	8.72	3.14	4.25
Isotype (IgG2a κ)	3.00	2.98	10.20	2.93	5.70
Anti-CXCR7	3.03	4.19	10.68	4.99	4.86
Unstained	2.38	2.02	2.23	1.80	3.13
Isotype (IgG1 κ)	3.71	3.26	2.73	2.66	3.64
Anti-hCAR	3.98	2.52	2.63	2.81	9.80
Unstained	1.05	1.31	3.10	1.26	2.18
Isotype (IgG1 κ)	1.34	2.18	3.28	1.51	3.24
Anti-anb3	4.38	2.42	3.56	2.09	5.48
Anti-anb5	3.64	6.22	17.30	4.00	4.36

Notes: Each cancer cell line was analyzed for the cell surface expression of CXCR4, CXCR7, hCAR, integrin anb3, and integrin anb5. The data are represented as MFI of gated unstained cells and cells stained with antigen-specific antibodies or isotype control antibodies.

Abbreviations: hCAR, human Coxsackie and adenovirus receptor; MFI, mean fluorescence intensity.

Figure 2 Expression and purification of the bispecific adapter (sCAR-CXCL12).

Notes: The structure of the sCAR-CXCL12 recombinant molecule was composed of an ectodomain portion of the human Coxsackie and Ad receptor followed by a 5-amino-acid peptide linker, a 6-His tag, fused to the mature human chemokine CXCL12 amino acid sequence (A). SDS-PAGE analysis and Coomassie staining of the sCAR-CXCL12 recombinant adapter protein purified by Ni column chromatography (B) and Western blot analysis using an anti-His tag antibody (C). Comparison of hCAR, CXCL12, and His tag antigen detection by Western blot analysis in recombinant sCAR-CXCL12 protein (D). M, molecular weight marker; numbers indicate molecular weight in kDa.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; hCAR, human Coxsackie and adenovirus receptor; His, histidine; PAGE, polyacrylamide gel electrophoresis; sCAR, soluble ectodomain form of the native Ad5 receptor; SDS, sodium dodecyl sulfate.

Figure 3 Binding specificity of the bispecific adapter (sCAR-CXCL12).

Notes: Purified sCAR-CXCL12 was tested in ELISA against recombinant Ad5 fiber knob (Ad5 knob) and recombinant Ad3 fiber knob (Ad3 knob). Each antigen was absorbed at 300 ng/well. The wells were incubated with dilutions of the adapter protein. The binding was detected by treating the wells with an anti-His tag antibody followed by an anti-rabbit IgG antibody conjugated with HRP and color reaction with TMB substrate. The color intensity was measured as optical density at 450 nm. Numbers indicate the mean ± SEM of absorbance from three replicate dishes. The concentration results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05.
Abbreviation: Ad, adenovirus; Ad3, Ad serotype 3; Ad5, Ad serotype 5; ELISA, enzyme-linked immunosorbent assay; His, histidine; HRP, horseradish peroxidase; sCAR, soluble ectodomain form of the native Ad5 receptor; SEM, standard error of the mean; TMB, 3,3′,5,5′-tetramethylbenzidine.

Figure 4 Infection of a cancer cell line by an Ad pre-complexed with a bispecific adapter, sCAR-CXCL12, is concentration and time dependent.

Notes: After an initial 4 h infection with 100 ifu/cell Ad5-CMV-GFP-luc alone or pre-complexed with increasing concentrations of sCAR-CXCL12, the percent of MDA-MB-435S cells positive for GFP expression was quantified at 48 h by flow cytometry analysis (A). After infection with 100 ifu/cell Ad5-CMV-GFP-luc alone or pre-complexed with 100 ng/10⁸ ifu of sCAR-CXCL12 for 5, 15, 30, 60, 120, and 240 min, the percent of MDA-MB-435S cells positive for GFP expression was quantified by flow cytometry analysis (B). Numbers indicate the mean ± SEM of percent GFP-positive cells from three replicate dishes.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; ifu, infectious unit; luc, luciferase; sCAR, soluble ectodomain form of the native Ad5 receptor; SEM, standard error of the mean.

Figure 5 An anti-CXCR4 antibody blocks sCAR-CXCL12-mediated binding of Ad to CXCR4-positive cells.

Notes: Prior to the Ad infection, MDA-MB-435S cells were incubated with increasing concentrations of an anti-CXCR4 antibody for 30 min at 4°C. Subsequently, the cells were infected for 1 h at 4°C with 100 ifu/cell Ad5-CMV-GFP-luc pre-complexed with 100 ng/10⁸ ifu of sCAR-CXCL12, and the amount of bound Ad5-CMV-GFP-luc DNA per dish was determined using a real-time PCR assay for the Ad5 E4 gene. Mean E4 copy number ± SEM from three replicate dishes is shown. The concentration results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; ifu, infectious unit; luc, luciferase; PCR, polymerase chain reaction; sCAR, soluble ectodomain form of the native Ad5 receptor; SEM, standard error of the mean.

Figure 6 Ablation of native liver tropism of Ad using a bispecific adapter (sCAR-CXCL12).

Notes: Luc assay at 48 h after infection of primary human liver slices with Ad5-CMV-GFP-luc in the absence or presence of 100 ng/10⁸ ifu sCAR-CXCL12. (A) Mean RLU/μg protein ± SEM of three replicate dishes. Real-time PCR assay for the Ad5 E4 gene in extracts from human primary liver slices with Ad5-WT in the absence or presence of 100 ng/10⁸ ifu sCAR-CXCL12 at 2 and 4 days after infection. (B) Mean E4 copy number ± SEM in extracts from three replicate dishes. The treatment results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; ifu, infectious unit; luc, luciferase; PCR, polymerase chain reaction; sCAR, soluble ectodomain form of the native Ad5 receptor; SEM, standard error of the mean; WT, wild type; RLU, relative light unit.

Figure 7 Ad targeted to CXCR4 by sCAR-CXCL12 enhances the efficacy of gene transfer to MDA-MB-435S cells.

Notes: MDA-MB-435S cells were plated at 1×10⁵ cells/well in a 24-well plate. The indicated amounts of Ad5-CMV-GFP-luc pre-complexed in the absence or presence of sCAR-CXCL12 (100 ng/well) were incubated for 4 h at 37°C followed by a medium change. (A) At 48 h post infection, GFP expression was examined by fluorescence microscopy (40 × magnification) in cells infected with increasing titers of Ad: (i and vi); 0 ifu/cell (ii and vii) 1 ifu/cell; (iii and viii) 10 ifu/cell; (iv and ix) 100 ifu/cell; (v and x) 1000 ifu/cell. (B) The percentage of GFP-positive cells was determined by flow cytometry. Numbers indicate the mean ± SEM of percent GFP-positive cells from three replicate dishes. The treatment results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; ifu, infectious unit; luc, luciferase; MOI, multiplicity of infection; sCAR, soluble ectodomain form of the native Ad5 receptor; SEM, standard error of the mean.

Figure 8 Determination of Ad targeting to CXCR4 by sCAR-CXCL12 in tumorigenic and immortalized non-tumorigenic cells.

Notes: Tumorigenic BT-20 (A), MCF-7 (B), and ZR-75-1 (C) cancer cells or non-tumorigenic MCF-12A epithelial cells (D) were plated at 1 × 10⁵ cells/well in a 24-well plate. Indicated amounts of Ad5-CMV-GFP-luc pre-complexed in the absence or presence of sCAR-CXCL12 (100 ng/well) were incubated for 4 h at 37°C followed by a medium change. At 48 h post infection, the percentage of GFP-positive cells was determined by flow cytometry. Numbers indicate the mean ± SEM of percent GFP-positive cells from three replicate dishes. The treatment results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; luc, luciferase; MOI, multiplicity of infection; sCAR, soluble ectodomain form of the native Ad5 receptor; SEM, standard error of the mean.

Figure 9 Liver untargeting of Ad infection using a bispecific adapter, sCAR-CXCL12, in SCID-bg mice in vivo.

Notes: Ad5-CMV-GFP-luc (1 × 10⁸ ifu/mouse) pre-incubated in the absence or presence of sCAR-CXCL12 (5 μg) was injected intravenously into SCID-bg mice. Ad-directed luc expression was monitored in living animals by bioluminescence imaging at 3 days after injection. (A) Examples of an untreated mouse, a mouse injected with Ad5-CMV-GFP-luc, and a mouse injected with Ad5-CMV-GFP-luc + sCAR-CXCL12. Luc expression was quantified from ROI of the liver regions. (B) Mean count ± SEM from ten animals. After bioluminescent imaging, the mice were sacrificed, liver extracts were prepared, and Ad was quantified by real-time PCR. (C) Mean E4 copy number ± SEM in livers from ten animals. The treatment results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05. Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; ifu, infectious unit; luc, luciferase; PCR, polymerase chain reaction; ROI, regions of interest; sCAR, soluble ectodomain form of the native Ad5 receptor; SCID-bg, severe combined immunodeficiency-beige; SEM, standard error of the mean.

Figure 10 Enhancement of Ad infection in xenograft tumors in vivo using a bispecific adapter (sCAR-CXCL12).

Notes: S.c. tumors were injected with Ad5-CMV-GFP-luc (1 × 10⁸ ifu/mouse) pre-incubated in the absence or presence of sCAR-CXCL12 (5 μg). Ad-directed luc expression was monitored in living animals by bioluminescence imaging at 3, 6, and 9 days after injection. (A) Examples of an untreated mouse, a mouse injected with Ad5-CMV-GFP-luc, and a mouse injected with Ad5-CMV-GFP-luc + sCAR-CXCL12. Luc expression was quantified from ROI of the tumor regions. (B) Mean count ± SEM from ten animals. The treatment results were compared using a Student’s t-test; the differences were considered statistically significant (*) if p<0.05.
Abbreviations: Ad, adenovirus; Ad5, Ad serotype 5; GFP, green fluorescent protein; ifu, infectious unit; luc, luciferase; ROI, regions of interest; sCAR, soluble ectodomain form of the native Ad5 receptor; s.c., subcutaneous; SEM, standard error of the mean.