The dermis as a portal for dendritic cell-targeted immunotherapy of cutaneous melanoma.

Complete surgical excision at an early stage remains the only curative treatment for cutaneous melanoma with few available adjuvant therapy options. Nevertheless, melanoma is a relatively immunogenic tumor type and particularly amenable to immunotherapeutic approaches. A dense network of cutaneous dendritic cells (DC) may account for the reported efficacy of vaccination through the skin and provide an attractive target for the immunotherapy of melanoma. Several phenotypically distinct DC subsets are discernable in the skin, among others, epidermal Langerhans cells and dermal DC. Upon appropriate activation both subsets can efficiently migrate to melanoma-draining lymph nodes (LN) to prime T cell-mediated responses. Unfortunately, from an early stage, melanoma development is characterized by strong immune suppression, facilitating unchecked tumor growth and spread. Particularly the primary tumor site and the first-line tumor-draining LN, the so-called sentinel LN, bear the brunt of this melanoma-induced immune suppression-and these are exactly the sites where anti-melanoma effector T cell responses should be primed by DC in order to prevent early metastasis. Through local immunopotentiation or through DC-targeted vaccination, the dermis may be utilized as a portal to activate DC and kick-start or boost effective T cell-mediated anti-melanoma immunity, even in the face of this immune suppression.

Gene-directed enzyme prodrug therapy with carboxylesterase enhances the anticancer efficacy of the conditionally replicating adenovirus AdDelta24.

Conditionally replicating adenoviruses (CRAds) selectively replicate in and thereby kill cancer cells. The CRAd AdDelta24 with pRb-binding-deficient E1A kills cancer cells efficiently. Arming CRAds with genes encoding prodrug-converting enzymes could allow for enhanced anticancer efficacy by the combined effects of oncolytic replication and local prodrug activation. Here, we investigated combination treatment of human colon cancer cell lines with AdDelta24-type CRAds and gene-directed enzyme prodrug therapy (GDEPT) using two different enzyme/prodrug systems, that is, thymidine kinase/ganciclovir (TK/GCV) and carboxylesterase (CE)/CPT-11. On all three cell lines tested, GDEPT with TK/GCV made CRAd treatment less efficacious. In contrast, expression of a secreted form of CE (sCE2) combined with CPT-11 treatment markedly enhanced the efficacy of AdDelta24 virotherapy. Based on this observation, we constructed an AdDelta24 variant expressing sCE2. In the absence of CPT-11, this new CRAd Ad5-Delta24.E3-sCE2 was similarly effective as its parent in killing human colon cancer cells. Low concentrations of CPT-11 inhibited Ad5-Delta24.E3-sCE2 propagation. Nevertheless, CPT-11 specifically augmented the cytotoxicity of Ad5-Delta24.E3-sCE2 against all three-colon cancer cell lines. Hence, the positive contribution of sCE2/CPT-11 GDEPT to colon cancer cytotoxicity outweighed its negative influence on CRAd propagation. Therefore, CRAd-sCE2/CPT-11 combination therapy appears useful for more effective treatment of colon cancer.

Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11.

CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into SN-38 (7-ethyl-10-hydroxycamptothecin) by the enzyme carboxylesterase (CE). In humans, only a minority of CPT-11 is converted to SN-38. To increase the antitumour effect of CPT-11 by gene-directed enzyme prodrug therapy, we constructed a replication-deficient adenoviral vector Ad.C28-sCE2 containing a fusion gene encoding a secreted form of human liver CE2 targeted to the surface antigen epithelial cell adhesion molecule (EpCAM) that is highly expressed on most colon carcinoma cells. By targeting CE2 to EpCAM, the enzyme should accumulate specifically in tumours and leakage into the circulation should be minimised. Ad.C28-sCE2-transduced colon carcinoma cells expressed and secreted active CE that bound specifically to EpCAM-expressing cells. In sections of three-dimensional colon carcinoma spheroids transduced with Ad.C28-sCE2, it was shown that C28-sCE2 was capable of binding untransduced cells. Most importantly, treatment of these spheroids with nontoxic concentrations of CPT-11 resulted in growth inhibition comparable to treatment with SN-38. Therefore, Ad.C28-sCE2 holds promise in gene therapy approaches for the treatment of colon carcinoma.

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug.

Secreted and tumour targeted human carboxylesterase for activation of irinotecan.

Irinotecan (CPT-11) is an anticancer agent for the treatment of colon cancer. CPT-11 can be considered as a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. An approach to achieve tumour specific activation of CPT-11 is to transduce the cDNA encoding carboxylesterase into tumour cells. A secreted form of carboxylesterase may diffuse through a tumour mass and may activate CPT-11 extracellularly. This could enhance the antitumour efficacy by exerting a bystander effect on untransduced cells. In addition a secreted tumour-targeted form of carboxylesterase should prevent leakage of the enzyme from the site of the tumour into the circulation. We have constructed a secreted form of human liver carboxylesterase-2 by deletion of the cellular retention signal and by cloning the cDNA downstream of an Ig kappa leader sequence. The protein was secreted by transfected cells and showed both enzyme activity and efficient CPT-11 activation. To obtain a secreted, tumour-targeted form of carboxylesterase-2 the cDNA encoding the human scFv antibody C28 directed against the epithelial cell adhesion molecule EpCAM, was inserted between the leader sequence and carboxylesterase-2. This fusion protein showed CPT-11 activation and specific binding to EpCAM expressing cells. Importantly, in combination with CPT-11 both recombinant carboxylesterase proteins exerted strong antiproliferative effects on human colon cancer cells. They are, therefore, promising new tools for gene directed enzyme prodrug therapy approaches for the treatment of colon carcinoma with CPT-11.