Mach Jean-Pierre, Professor Emeritus

Taking advantage of our experience in tumor targeting with antibodies and in T lymphocyte cytotoxicity, we designed a new type of conjugates, consisting of antibody fragments coupled to Major Histocompatibility Complexes (MHC) containing antigenic viral peptides. We demonstrated that Fab fragments from different anti-tumor marker antibodies, conjugated to MHC/viral peptides, can target these antigens on the surface of cancer cells and induce their efficient lysis by virus specific cytotoxic T lymphocytes. We developed several in vivo syngeneic tumor models in viral infected mice and demonstrated the feasibility of this new immunotherapy strategy. In view of these promising results, we are presently testing in parallel similar approaches applied to non-polymorphic MHC related molecules, such as CD1d or MICA, to attract at the tumor site effector cells from the innate immune system such as NKT and NK cells.

Antibody-MHC/viral peptide conjugates for cancer cells targeting

Antibody-based cancer immunotherapy exploits the cell surface expression by cancer cells of tumour-associated antigens (TAA). The generation and engineering of high affinity anti-TAA monoclonal antibodies (mAbs) as native proteins, or as carriers for targeting radioactivity, toxins or cytokines to tumor cells have made important progresses and antibody-based cancer therapy is now effective for lymphoma, ovarian and breast cancer (for review, Mach 2002). Despite these very encouraging clinical results, however, one should remain aware that the injection of anti-tumor mAbs, such as anti-HER2 (herceptin) or anti-CD20 (Rituximab), when used as a single modality therapy, usually leads to partial tumor regression and mAb treatment needs to be combined with chemotherapy.

A second important strategy is to exploit the cellular immune response to cancer cells and T cell based cancer immunotherapy essentially focuses on the development of cancer vaccines to optimize the potent cytotoxicity of T lymphocytes against tumor derived antigens. Although a number of clinical trials are being conducted, this approach is still facing problems of tumor escape, like absence of costimulatory molecules or downregulation of MHC Class I expression on tumor cells.

Recently, we, and others, have developed an alternative strategy whereby anti-TAA mAbs are used to target on tumor cells recombinant MHC class I/viral peptide complexes in order to induce the killing of target cells by viral specific CD8 T lymphocytes. This strategy exploits the specific tumor localization of high affinity anti-TAA antibodies, which allows the coating of the cancer cells with class I molecules filled with a highly antigenic peptide. Altogether, this attractive therapeutic approach has clear advantages over the vaccination with poorly antigenic autologous tumor antigens. First, it will not be affected by the loss of endogenous MHC Class I expression by the tumour. Second, viral antigens are generally more potent than tumor antigens to generate cytotoxic T cells. Third, the lack of accessory molecules on the cancer cells, often reported as the cause of tolerance/anergy of T cells specific for TAA, will not affect the present strategy since the preexisting anti-viral memory T cell pool can be fully activated by a challenge with the appropriate vaccine at the time of treatment.

We have demonstrated for the first time in an entirely in vivo models of syngeneic carcinoma that this new tumor immunotherapy strategy can function in immunocompetent mice, using two different viral models, the Lymphochoriomeningitis virus (LCMV) and the Influenza virus (Flu), in mice grafted with subcutaneous tumor or lung metastasis, respectively. We have improved the quality of the conjugates by UV crosslinking the antigenic peptide into the groove of the MHC molecule, in collaboration with I. Luescher from the Ludwig Institute, so that the stability of the complex is greatly increased in vivo. We showed that systemic injection of such conjugates can efficiently induce tumor cell killing and tumor growth inhibition by the specific CD8+ CTLs generated by the viral infection (Cesson et al., 2006).

Antibody-CD1d fusion bifunctional molecule for targeting innate immunity to cancer cells

The aim of the present project is to activate at the tumor site effector cells of the innate immune system, such as CD1d-restricted NKT cells, by an antitumor antibody-mediated delivery of the CD1d non-polymorphic MHC class I related molecule loaded with the ligand a-galactosylceramide (α-GalCer) or analogs. Practically, this strategy is based on the development of a bifunctional molecule consisting of the CD1d-α-GalCer complex as the activating part, genetically fused to the tumor targeting part, such as single chain antibody fragments with high affinity for a tumor associated antigen (TAA). This antitumor antibody-CD1d molecule will recruit and activate at the tumor site CD1d-restricted NKT cells known for their cytoxicity and potent ability to activate NK cells,as shown in the schematic diagram of figure 2.

Fig. 2: NKT cells as transactivators of the innate and adaptive immune response. The glycolipid ligand α-GalCer presented by CD1d expressed on DC will activate NKT cells. In turn, activated NKT cells will rapidly secrete IFNγ and upregulate CD40L. These events will lead to NK activation, DC maturation and subsequent development of antigen-specific T lymphocytes, possibly against an existing tumor.

This strategy is similar to the one described above for tumor targeting of classical MHC class I-antigen and induction of specific cytotoxic T lymphocytes (CTL). However, replacing MHC Class I molecule with the MHC related CD1d has additional advantages. First, the CD1d protein is a monomorphic antigen presenting molecule, which would allow, in future clinical applications, the development of a single conjugate to treat a majority of patients in contrast to the polymorphic MHC Class I molecules. Second, even though the natural ligands of CD1d and the physiological role of NKT cells is still a matter of debate, an important antimetastatic activity of CD1d-NKT cells has been demonstrated in vitro and in vivo in the presence of the glycosphingolipid α-GalCer. Third, the fast activation of CD1d-NKT cells rapidly leads to the activation and proliferation of NK cells, which altogether greatly enhance cytotoxicity. Importantly, the tumor targeting part of the antibody-CD1d bifunctional molecule will restrict the recruitment of these cytotoxic effector cells at the tumor site and hence will increase efficacy, while limiting the known toxicity of an untargeted NKT and NK cell activation.

We have developed a genetic fusion of mouse β2 microglobulin - CD1d - anti-HER2 scFv, which is well produced by human embryonic kidney cells HEK293 in a transient transfection system. The fusion is well refolded as shown by binding to tumor cells expressing the HER2 antigen and by recognition with anti-CD1d antibody. The CD1d fusion is functional since it is able to activate NKT hybridoma, as demonstrated by the release of IL-2.

Our first original in vivo observation was that when αGalCer was loaded on the recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections led to a sustained iNKT and NK cell activation associated with interferon γ secretion as well as with DC maturation. In contrast, it is known that a single injection of the free form of αGalCer leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use.

Most importantly, when the αGalCer/sCD1d fused to the anti-HER2 scFv antibody fragment was injected, potent inhibition of experimental lung metastasis (Fig.3) and established subcutaneous tumors was observed even when systemic treatment was started 2 to 7 days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. Furthermore, we demonstrated that the anti-tumor activity of the CD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site (Stirnemann et al. 2008).

Our results strongly suggest that targeting iNKT cells to the tumor site can activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

Fig. 3: In vivo anti-tumor activity - Systemic Treatment. (A) Mice were grafted i.v. with 700'000 B16-HER2 cells and i.v. treatment was started 48 hours later. Mice were injected five times i.v. every 3 to 4 days (arrows) with either PBS (control), or equimolar amounts of αGalCer (0.4 μg), or αGalCer-loaded sCD1d-anti-HER2 fusion (40 μg). Mice were analyzed after 3 weeks and results are shown as pictures of tumors-invaded lungs (1 representative lung per group) and in the graph below expressed as percent of lung surface invaded by melanin-loaded tumor nodules. Results represent the mean ± SD of 5 mice per group of two independent experiments. **P < 0.005 versus control, *P < 0.04 versus αGalCer. (B) Mice were grafted as above and treatment was started 6 days after with the same protocol as in (A) including treatment with sCD1d (25 µg). Lung nodules were analyzed after 2 weeks. Results represent the mean ± SD of 6 mice per group of two independent experiments. ***P = 0.0006 versus control and **P < 0.004 versus αGalCer, *P < 0.02 versus sCD1d (Stirnemann et al. 2008).

Future development of the project by Dr. Alena Donda : In order to get closer to a clinical application, we will focus on three aspects: 1) Development of the human homologue of recombinant soluble CD1d and genetic fusion to different antibody fragments, in order to extend the targeting of CD1d to different type of cancer (anti-HER2 targeting breast cancer; anti-CEA targeting colon carcinoma). 2) Further analysis of the molecular mechanism allowing the sustained iNKT cell activation by αGalCer loaded on soluble CD1d molecules, as compared to the short lived activation induced by free αGalCer. 3) Development and exploitation of the effect of sustained iNKT cell activation on the adaptive immune response. In particular, the influence of the tumor-targeted CD1d treatment on T cell crosspriming by tumor antigens will be evaluated, taking as examples the xenoantigens HER2 and CEA, as well as different human TAA already used in active T cell immunotherapy of cancer patients. In parallel, the expected synergic antitumor effect of the continuous activation of iNKT and NK cells and an active antitumor immunization will be investigated in two models.

• Stirnemann, K., Romero, J.F., Robert, B., Cesson, V., Besra, G.S., Zauderer, M., Wurm, F., Corradin, G., Mach, J.P., MacDonald, H.R., Donda, A. (2008). Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2 scFv fusion protein induce antitumor effects in mice. J. Clin. Invest. 118, 994-1005. PubMed
• Larbouret, C., Robert, B., Navarro-Teulon, I., Thèzenas, S., Ladjemi, M.Z., Morisseau, S., Campigna, E., Bibeau, F., Mach, J.P., Pèlegrin, A., Azria, D. (2007). In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas. Clin. Cancer Res. 13, 3356-3362. PubMed
• Heer, A.K., Shamshiev, A., Donda, A., Uematsu, S., Akira, S., Kopf, M., Marsland, B.J. (2007). TLR signaling fine-tunes anti-influenza B cell responses without regulating effector T cell responses. J. Immunol. 178, 2182-2191. PubMed
• Buchegger, F., Antonescu, C., Delaloye, A.B., Helg, C., Kovacsovics, T., Kosinski, M., Mach, J.P., Ketterer, N. (2006). Long-term complete responses after 131I-tositumomab therapy for relapsed or refractory indolent non-Hodgkin's lymphoma. Br. J. Cancer 94, 1770-1776. PubMed
• Cesson, V., Stirnemann, K., Robert, B., Luescher, I., Filleron, T., Corradin, G., Mach, J.P., Donda, A. (2006). Active antiviral T-lymphocyte response can be redirected against tumor cells by antitumor antibody x MHC/viral peptide conjugates. Clin. Cancer Res. 12, 7422-7430. PubMed
• Fattah, O. M., Cloutier, S. M., Kundig, C., Felber, L. M., Gygi, C. M., Jichlinski, P., Leisinger, H. J., Gauthier, E. R., Mach, J. P., Deperthes, D. (2006). Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells. Int. J. Cancer 119, 2455-2463. PubMed
• Gorczynski, R.M., Alexander, C., Bessler, W., Fournier, K., Hoffmann, P., Mach, J.P., Rietschel, E.Th., Song, L., Waelli, Th., Westphal, O., Zahringer, U., Khatri, I. (2005). Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro. Immunol. Lett. 100, 120-129. PubMed
• Germain, C., Larbouret, C., Cesson, V., Donda, A., Held, W., Mach, J.P., Pelegrin, A. and Robert, B. (2005). MHC class I-related chain a conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells. Clin. Cancer Res. 11 (20): 7516-22. PubMed
• Muller, S., Hoffmann, P., Esche, U.V., Mach, J.P., Gorczynski, R.M., Waelli, T., Alexander, C., Zahringer, U., Rietschel, E.T., Bessler; W.G., Westphal, O. (2005). A fetal sheep liver extract containing immunostimulatory substances including LPS acts as leukocyte activator in cells of LPS responder and non responder mice. Int. Immunopharmacol. 5, 1809-1819. PubMed
• Buchegger, F., Adamer, F., Schaffland, A.O., Kosinski, M., Grannavel, C., Dupertuis, Y.M., de Tribolet, N., Mach, J.P. and Delaloye, A.B. (2004). Highly efficient DNA incorporation of intratumourally injected [125I]iododeoxyuridine under thymidine synthesis blocking in human glioblastoma xenografts. Int. J. Cancer 110 (1): 145-9. PubMed
• Donda, A., Cesson, V., Mach, J.P., Corradin, G., Primus, F.J., Robert, B. (2003). In vivo targeting of an anti-tumor antibody coupled to antigenic MHC class I complexes induces specific growth inhibition and regression of established syngeneic tumor grafts. Cancer Immunity 3, 11. PubMed