Intratumoral injection of adenoviral vectors encoding tumor-targeted immunoconjugates for cancer immunotherapy

Abstract

The efficacy and safety of a new immunotherapy protocol for cancer were tested in a severe combined immunodeficient mouse model of human skin and metastatic lung melanoma. The protocol involves intratumoral injections of replication-incompetent adenoviral vectors encoding immunoconjugates composed of the Fc region of an IgG1 immunoglobulin conjugated to a tumor-targeting domain. One targeting domain is factor VII that binds to tissue factor expressed on endothelial cells lining the tumor neovasculature and on tumor cells; the active site of factor VII was mutated to inhibit the initiation of blood coagulation. Another targeting domain is a single-chain Fv antibody that binds to a cognate antigen expressed on human melanoma cells. The adenoviral vectors infect mainly the cells of the injected tumor, which synthesize and secrete the immunoconjugates. The bloodborne immunoconjugates induce a cytolytic immune response against the targeted neovasculature endothelial cells and tumor cells. The mouse model experiments showed that intratumoral delivery of the factor VII immunoconjugate, either alone or together with the single-chain Fv immunoconjugate, resulted in growth inhibition and regression of the injected tumor, and also of distant metastatic tumors, without evidence of damage to normal organs. There was extensive destruction of the tumor neovasculature, presumably mediated by the factor VII immunoconjugate bound to tissue factor on neovasculature endothelial cells. Because tissue factor is generally expressed on neovascular endothelial cells and tumor cells, a factor VII immunoconjugate could be used for immunotherapy against a broad range of human solid tumors.

Figure 1

Diagram of an immunoconjugate molecule. TD, targeting domain; H, hinge region of an IgG1 immunoglobulin with 2 disulfide bridges; CH2 and CH3, constant regions of an IgG1 immunoglobulin.

Figure 2

Distribution of an adenoviral vector in tumor and liver after intratumoral injection. The control vector encoding the GFP protein but not an immunoconjugate was injected into three sites of a human melanoma skin tumor growing in SCID mice. The total vector dose was 6 × 10⁹ IU. The tumor and liver were dissected 40 h after the injection and were examined intact under a dissecting microscope with fluorescence optics. The GFP signal was detected with 480-nm excitation and 630-nm emission, and the background signal was detected with 577-nm excitation and 630-nm emission. (A) Tumor GFP. (B) Tumor background. (C) Liver GFP. (D) Liver background. A bright fluorescent spot similar to the one in A also was detected at two other tumor sites, presumably corresponding to the injection sites. The photographs are focused at one level in the tissues. However, the GFP spot in the tumor also could be detected by focusing above and below that level, suggesting that the tumor cells adjacent to the path traversed by the injection needle are the only cells infected by the vector. (Bar = 50 μm.)

Figure 3

Dosage effect of intratumorally injected adenoviral vectors on the growth of a human melanoma tumor in SCID mice. The mice were first injected s.c. with the human melanoma cell line LXSN, and when a skin tumor had grown to the size indicated on day 0 the tumor was injected with a mixture of the two adenoviral vectors encoding the mfVIIasm and G71-1 immunoconjugates. The dose for the two vectors was as follows. ○, 6 × 10⁹ IU; ▵, 2 × 10⁹ IU; □, 7 × 10⁸ IU. The dose for the control vector that did not encode an immunoconjugate was as follows. ⋄, 6 × 10⁹ IU.

Figure 4

Effect of a high dose of intratumorally injected adenoviral vectors on the growth of a human melanoma tumor in SCID mice. The mice were first injected s.c. with the human melanoma line LXSN, and when a skin tumor had grown to the size indicated on day 0 the tumor was injected with 6 × 10⁹ IU of the following adenoviral vectors. ●, control vector (3 mice); ■, vector encoding the mfVIIasm immunoconjugate (6 mice); ▵, mixture of the two vectors encoding the mfVIIasm and G71-1 immunoconjugates (5 mice). Additional injections were done on days 2, 5, 7, 9, 11, 13, 15, and 17. The points on each curve are the average (±20%) of the measurements for all mice in the corresponding group.

Figure 5

Liver sections from SCID mice injected intravenously or intratumorally with the adenoviral vectors. The i.v. experiment was described previously (see Fig. 5 in ref. 11), and the intratumoral experiment is described in Fig. 4. The panels on the left were injected with the control vector, and the panels on the right were injected with a mixture of the two vectors encoding the mfVIIasm and G71-1 immunoconjugates. The livers were dissected 2 days after the last injection, were fixed in formaldehyde, and were embedded in paraffin. The sections were stained with hematoxylin and eosin and were photographed at a magnification of 100×.

Figure 6

Melanoma skin tumors from SCID mice injected intratumorally with the null adenooviral vector control (top row) or with the adenoviral vectors encoding the mfVIIasm and G71-1 immunoconjugates (bottom row). The experiment is described in Fig. 4. The tumors were dissected 2 days after the last injection and were photographed. The full scale at the top of the figure is 1 cm.

Figure 7

Effect of i.v. injections into immunocompetent mice of the adenoviral vector encoding the mfVIIasm immunoconjugate on the growth of a mouse skin melanoma. C57BL/6 mice were injected s.c. with the mouse melanoma line B16F10, and when a skin tumor had grown to the size indicated on day 0 the mice were injected intravenously with 3 × 10¹⁰ IP of the vector encoding the mfVIIasm immunoconjugate. The Fc domain of the immunoconjugate was derived from a mouse IgG1 immunoglobulin. The mice were injected intravenously again on day 5 with 1.5 × 10¹⁰ IP of the same vector.