J Immunother. 2015 Nov-Dec;38(9):357-70.
The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer-Cell Cytotoxicity Against Melanoma Cell Lines in vitro
Maria Luisa Dupuis1,Valentina Fiori2*, Alessandra Soriani3§, Biancamaria Ricci3, Sabrina Dominici4 , Diego Moricoli2, Alessandro Ascione1, Angela Santoni3, Mauro Magnani4 and Maurizio Cianfriglia1
1Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy
2Diatheva s.r.l, Fano (PU), Italy
3Department of Molecular Medicine, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy
4Department of Biomolecular Science, University of Urbino ‘‘Carlo Bo’’, Urbino (PU), Italy
- L. D., V. F. and A. S§. contributed equally to the work.
*Corresponding Author: Valentina Fiori, Diatheva srl, viale Piceno 137/F, 61032, Fano (PU) Italy. E-mail: email@example.com Tel.: +39 0721830605. Fax: +39 0721837154
Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin.
Conventional therapies for solid tumors and malignant melanoma are aimed at killing the maximum number of tumor cells by multi-modal treatments which may include different chemotherapy regimens or combination of chemical and biological drugs . While this strategy often achieves a significant reduction in tumor burden, most patients with disseminated metastatic cancers ultimately die because tumor cells evolve resistance. The limited successes of the reported anti cancer treatments have underscored the need to develop new and more effective curative strategies designed on the basis of novel immunotherapeutic modalities. In this context harnessing the immune system to fight cancer it has revealed an exciting powerful mean in the anti-tumor therapy in the past few years. However, despite the relevant results observed in melanoma and lung cancer in terms of objective and long lasting response, an important limit of immunotherapeutic interventions is the low rate of responder patients . For this reason the scientific community is spending efforts in the search of better immunotherapeutic target or novel combinations strategies that are able to increase the response rate and therapeutic efficacy possibly without affecting the overall toxicity profile. Even though these studies have permitted the production of several anti tumor protocols showing durable remission results from experimental animal models, the reported anti-tumor efficacy it is frequently affected, once translated in the clinical setting, by not tolerable adverse events.
A branch of immunotherapeutic research is actively focused on the identification of monoclonal antibodies (mAbs) that by targeting specific immune activating or inhibiting molecules expressed on the cell surface of immune effector and cancer cells act as biological mediators for curative processes.
In this context, CEACAM1 is an attractive target for cancer immunotherapeutic strategies because its expression profile, the role in the development of aggressive melanomas and carcinomas, and the expression in cancer metastasis.[4;5] Furthermore a large body of evidences have confirmed that CEACAM1 is a novel immune checkpoint inhibitor protein. It has been shown that tumors over-expressing CEACAM1 on their cell surface can evade the immune system surveillance by virtue of CEACAM1-CEACAM1 homophilic interactions with the same antigen expressed by activated T lymphocytes and NK cells [6-8]. These interactions produce an inhibitory effect on effector cells mediated by the cytoplasmatic domain of CEACAM1 and the recruitment of inhibitory molecules such as Src homology domain 2 containing phosphatase 1 and 2 (SHP-1 and SHP-2) .
As a consequence, mAbs against CEACAM1 able to interrupt the homophilic interactions, can restore the ability of T and NK lymphocytes to eliminate cancer cells (Fig.1). Moreover CEACAM1 is also able to interact with other antigens on the surface of T and NK cells. For example it has been recently shown that CEACAM1 regulates T-cell immunoglobulin domain and mucin domain-3 (TIM-3)-mediated tolerance and exhaustion of T lymphocytes  and that a direct interaction of CEACAM1 with NKG2D on NK cells leads to an inhibition of downstream signaling involved in the cytolysis of CEACAM1-bearing target cells . Anti-CEACAM1 mAbs have been proved to be effective when are used as single biological agents to render tumor cells de novo susceptible to T and NK cells [8;10]. However the anti-tumor efficacy it is much improved when it is combined with other immune checkpoint inhibitors such as anti-PD1 and anti-TIM3 mAbs in mice models .
In this context, we have used for the first time a fully human antibody in single chain fragment variable (scFv) format, named DIATHIS1, specific for CEACAM1 and highly reactive with melanoma and lung carcinoma [13;14]. In this work we demonstrated its efficacy in enhancing the NK cells citotoxicity against melanoma cells in vitro. The co-incubation of melanoma cells expressing CEACAM1 on their surface with different NK cell types (NK-92, NKL cell lines and activated NK cells from healthy donors) significantly enhances the activation of effector cells and the cell-mediated cytotoxicity against cancer cells without interfering with general cellular processes such as apoptosis and proliferation. Moreover we found a possible correlation between the expression of CEACAM1-3 splicing isoforms and a better response to DIATHIS1 treatment.
Antibody fragment in scFv format are composed by VH and VL chains that are joined via a flexible linker and represent the smallest functional domain of an antibody necessary for binding to the antigen with an affinity similar to that of the parent mAb. Currently human antibodies in scFv format are usually isolated from phage antibody libraries originated by different methodologies according with the finality of the scFvs utilization in the biomedical field . ScFvs with their small size (about 25 kDa) are more efficient than whole mAbs (150 kDa) in terms of tumor penetration and represent versatile molecules for numerous genetic engineering modifications aimed at improve their intrinsic features (as affinity, specificity, valency and pharmacokinetics) or to give rise to other therapeutic molecules such as antibody drug conjugates (ADCs) or chimeric antigen receptors (CARs)[16;17]. Moreover, thanks to their small size and fast clearance they are associated with limited toxicity and limited accumulation in off-target sites.
The human scFv DIATHIS1 in comparison with mAbs to human CEACAM1 originated by classical hybridoma technology, shows unique structural and functional properties. Its small size and affinity should allow good target selectivity and a homogeneous tumor penetration in comparison with whole mAbs. Moreover, the scFv DIATHIS1 does not bind to not-activated freshly isolated NK cells and shows no evident cross-reactivity with different human normal tissues.
Taken in aggregate our findings suggest that scFv DIATHIS1 represents a unique reagent for targeting CEACAM1 expressing tumors supporting its medical relevance in immunotherapeutic interventions. DIATHIS1 represents a potential platform for developing completely human anticancer therapeutics and may be used to complement the curative armamentarium of immunotherapeutic interventions against melanoma and to design effective models of cancer immune therapy mediated by new combination strategies with limited toxicity.
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