(B) Cell area calculated by image analysis (CELLCYTE Studio software) on images of proliferating KMH2 cell cultures, without or with BreCVedCZA or BreCVed (2.0 g/mL or 0.2 g/mL) taken at 24, 48, 72 h, expressed as mm2. confirmed the covalent linkage between the antibody and ZA. The novel ADC has been tested for its reactivity with the HL/CD30+ lymphoblastoid cell lines (KMH2, L428, L540, HS445, and RPMI6666), showing a better titration than native BreCVed. Once the HL-cells are entered, the ADC co-localizes with the lysosomal LAMP1 in the intracellular vesicles. Also, this ADC exerted a stronger anti-proliferative and pro-apoptotic (about one log fold) effect on HL-cell proliferation compared to the native antibody BreCVed. Eventually, BreCVedCZA ADC, in Atazanavir contrast with the native antibody, can trigger the proliferation and activation of cytolytic activity of effector-memory V2 T-lymphocytes against HL-cell lines. These findings may support the potential use of this ADC MDS1-EVI1 in the management of r/r HL. Keywords: Hodgkin lymphoma(s), V2 T-cells, ADC, zoledronic acid, brentuximab, CD30 1. Introduction The involvement of T cells in the control of tumors, including leukemias and lymphomas, is confirmed [1,2,3]. In particular, T lymphocytes can recognize phosphoantigens (pAg) accumulated and presented by neoplastic cells [4,5,6,7]. Also, the antitumor activity of these lymphocytes, bearing at the cell surface the Fc receptor IIIa/CD16, can be triggered through the Fc portion of therapeutic antibody [7,8,9]. Different compounds, such as aminobisphosphonates (N-BPs), can stimulate the proliferation of the peripheral blood V2 T-cell subset, increasing the number of circulating Atazanavir T-lymphocytes able to destroy tumor cells [5,6,7,8]. Among N-BPs, zoledronic acid (ZA) is a chemically stable analog of inorganic pyrophosphate able to interfere with the farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) synthase of the cholesterol synthesis pathway. This inhibition induces the intracellular accumulation of isopentenyl pyrophosphate (IPP); in turn, Atazanavir the IPP is presented by the cell-membrane butyrophilins such as BTN3A1 and BTN2A1 to the V9V2 T-cell receptor eliciting their preferential growth and activation [7,8,9,10]. The two mechanisms, immunostimulation and direct anticancer action, are the rationale for the use of different N-BPs in clinical trials [11,12,13]. We have reported that ZA can stimulate the expansion of V2T-cells with anti-leukemic and lymphoma activity, possibly acting as a therapeutic tool to enhance conventional therapies [14,15]. In the specific case of Hodgkin lymphoma (HL), monoclonal antibodies (mAb) directed to the CD30 antigen expressed by tumor cells represent a key therapeutic tool, especially for treating relapsed/refractory (r/r) HL [16,17,18]. Indeed, the use of the anti-CD30 antibody brentuximab linked to the microtubule inhibitor vedotin leading to the antibodyCdrug conjugate (ADC) named brentuximabCvedotin (BreCVed) can evoke a strong anti-HL cytotoxic effect improving the chemotherapy [16,17,18]. Also, Atazanavir resistance is not rare, and in second-line treatments, survival advantage is often lower than expected and is reached only in combination with other antiblastic drugs [16,17,18]. The expression of CD30 on HL is finely regulated by the action of a disintegrin and metalloproteinase (ADAM) 10 activity, and BreCVed effects can be increased using specific ADAM10 inhibitors [15]. Thus, it is conceivable that the anti-CD30 ADC-mediated therapeutic effect could be enhanced by eliciting the generation of anti-lymphoma effector lymphocytes as well as using drug combinations [15]. It should be considered that although several ADCs have been synthesized for treating different types of cancers [19,20,21] exploiting the Atazanavir cytotoxic effect of a drug linked to an antibody, only a few are currently used due to incomplete or unsatisfactory clinical outcomes [19]. More recently, we have described a new type of ADC, based on the anti-EGFR therapeutic antibody Cetuximab, covalently linked to ZA exerting anticancer properties [22]. This ADC can trigger the expansion of V2 T-cells in patients suffering from colorectal carcinoma that, in turn, can kill patient-derived organoids. This effect was mediated by both V2 TCR triggering and antibody-dependent cellular cytotoxicity (ADCC) [22]. In this paper, we study the reactivity and functional properties of.