EGFR-Targeted Antibody–Drug Conjugate to Different Aminobisphosphonates:Direct and Indirect Antitumor Effects on Colorectal Carcinoma Cells

Antibody––drug conjugates (ADCs) are a promising delivery system that involves linking a monoclonal antibody (mAb) to a specific drug, such as a cytotoxic agent, to target tumor cells. This new class of antitumor therapy acts as a “biological missile” that can destroy tumor cells while increasing the therapeutic index and decreasing toxicity. One of the most critical factors in ADC design is selecting a target antigen that is highly expressed on the surface of cancer cells. In this study, we conjugated Cetuximab (Cet), a monoclonal antibody that targets the epidermal growth factor receptor (EGFR), to aminobisphosphonates (N-BPs) such as ibandronate (IBA) or risedronate (RIS) or zoledronate (ZA). Cetuximab is administered to patients with metastatic colorectal carcinoma (mCRC) with a wild-type (WT) EGFR transduction pathway. Also, it is well established that N-BPs can trigger the antitumor activity of Vδ2 T cells in both in vitro and in vivo experimental models. The resulting ADCs were added in co-culture to assess the effect on CRC cell line proliferation and sensitivity to Vδ2 T antitumor lymphocytes in comparison with the native antibody. These assays have been performed both in conventional and 3D spheroid cultures. We found that all three ADCs can increase the inhibitory effect on cell proliferation of the WT-EGFR cell line Caco-2 while only Cet-RIS and Cet-ZA can increase the cytotoxicity mediated by Vδ2 T cells against both WT and EGFR-mutated CRC cell lines (Caco-2, DLD-1, and HCT-116). Also, the ADCs can trigger the cell proliferation of Vδ2 T cells present in peripheral blood and tumor specimens. Our findings indicate that anti-EGFR antibodies bound to N-BPs can improve the antitumor effects of the native antibody possibly increasing the therapeutic effect.</p

EGFR-Targeted Antibody–Drug Conjugate to Different Aminobisphosphonates:Direct and Indirect Antitumor Effects on Colorectal Carcinoma Cells

Antibody––drug conjugates (ADCs) are a promising delivery system that involves linking a monoclonal antibody (mAb) to a specific drug, such as a cytotoxic agent, to target tumor cells. This new class of antitumor therapy acts as a “biological missile” that can destroy tumor cells while increasing the therapeutic index and decreasing toxicity. One of the most critical factors in ADC design is selecting a target antigen that is highly expressed on the surface of cancer cells. In this study, we conjugated Cetuximab (Cet), a monoclonal antibody that targets the epidermal growth factor receptor (EGFR), to aminobisphosphonates (N-BPs) such as ibandronate (IBA) or risedronate (RIS) or zoledronate (ZA). Cetuximab is administered to patients with metastatic colorectal carcinoma (mCRC) with a wild-type (WT) EGFR transduction pathway. Also, it is well established that N-BPs can trigger the antitumor activity of Vδ2 T cells in both in vitro and in vivo experimental models. The resulting ADCs were added in co-culture to assess the effect on CRC cell line proliferation and sensitivity to Vδ2 T antitumor lymphocytes in comparison with the native antibody. These assays have been performed both in conventional and 3D spheroid cultures. We found that all three ADCs can increase the inhibitory effect on cell proliferation of the WT-EGFR cell line Caco-2 while only Cet-RIS and Cet-ZA can increase the cytotoxicity mediated by Vδ2 T cells against both WT and EGFR-mutated CRC cell lines (Caco-2, DLD-1, and HCT-116). Also, the ADCs can trigger the cell proliferation of Vδ2 T cells present in peripheral blood and tumor specimens. Our findings indicate that anti-EGFR antibodies bound to N-BPs can improve the antitumor effects of the native antibody possibly increasing the therapeutic effect.</p

Clonal heterogeneity in chronic lymphocytic leukemia cells: superior response to surface IgM cross-linking in CD38, ZAP-70-positive cells.

Background Patients with chronic lymphocytic leukemia whose cells express CD38 and ZAP-70 and utilize unmutated Ig VH region genes have a very poor prognosis. We studied whether cells expressing CD38 and ZAP-70 are more susceptible to stimulation through B-cell receptors than are cells that do not express CD38 and ZAP-70.Design and Methods CD38-positive and CD38-negative leukemic cells were separated from single cases and compared for their response to B-cell receptor cross-linking and ZAP-70 expression. Cohort studies were also carried out by measuring the apoptotic response to surface immunoglobulin M (IgM) cross-linking in 82 patients with chronic lymphocytic leukemia and the protein tyrosine phosphorylation induced by surface IgM in 21 patients.Results CD38-positive cells, isolated from cases of chronic lymphocytic leukemia classified as CD38-positive or CD38-negative, expressed more ZAP-70 than the corresponding CD38-negative cells, exhibited more robust protein tyrosine phosphorylation and had a greater tendency to apoptosis upon B-cell receptor cross-linking. In the cohort studies, surface IgM-induced protein tyrosine phosphorylation correlated significantly with CD38 and ZAP-70 expression and with the absence of Ig VH gene mutations. Apoptosis induced by surface IgM cross-linking correlated significantly only with the proportion of CD38-positive cells. Difficulties in finding more definitive correlations were probably related to imprecision in the in vitro test system and in the definition of cases as positive or negative.Conclusions Collectively, these data indicate that CD38-positive, ZAP-70-positive cells have a greater capacity for signaling through the B-cell receptor and suggest a function for B-cell receptor signaling in promoting chronic lymphocytic leukemia cell expansion, especially within the CD38-positive fraction of the leukemic clone

A non-invasive approach to monitor chronic lymphocytic leukemia engraftment in a xenograft mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI).

This work was supported by: Associazione Italiana Ricerca sul Cancro (AIRC) [Grant 5 x mille n.9980, (to M.F., F.M. and A. N.)]; AIRC I.G. [n. 14,326 (to M.F.)], [n.10136 and 16,722 (A.N.)], [n.15426 (to F.F.)]. AIRC and Fondazione CaRiCal co-financed Multi Unit Regional Grant 2014 [n.16695 (to F.M.)]. Italian Ministry of Health 5 × 1000 funds (to F.F). A.G R. was supported by Associazione Italiana contro le Leucemie-Linfomi-Mielomi (AIL) Cosenza - Fondazione Amelia Scorza (FAS). S.M. C.M., F.V., L. E., S. B., were supported by AIRC.Peer reviewedPostprin

Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells

Microenvironment regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence

The development and progression of Chronic Lymphocytic Leukemia (CLL) require co-operation of both microenvironment and cytokines. Investigating the IL-23R/IL-23 axis we found that circulating cells of early-stage CLL patients with shorter time-to-treatment (but not of those with a more benign course) expressed a defective form of the IL-23R complex lacking the IL-12Rß1 chain. However, the cells from both patient groups expressed the com-plete IL-23R complex in tissue infiltrates and could be induced to express it when co-cultured with activated T cells or other CD40L-bearing cells. IL-23 production by CLL cells activated in vitro in this fashion and in lymphoid tissues was observed suggesting the exist-ence of an autocrine/paracrine loop causing CLL cell proliferation. Culture of CLL cells with stromal cells, nurse like cells and stimulation with anti IgM antibodies and IL-4 failed to activate this loop. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 anti-body proved effective in controlling disease onset/expansion in xenografted mice, suggest-ing potential therapeutic strategies

Unexpected effects of biphosphonates in in vitro models of activated CLL cells

Recent studies suggest that the commonly prescribed anti-osteoporosis drugs bisphosphonates (BPs) might also exhibit antitumor activity. We investigated a possible anticancer effect of BPs on B-chronic lymphocytic leukemia (CLL) cells obtained from peripheral blood of 26 CLL patients. Zoledronate, etidronate and clodronate were administered in vitro simultaneously to following activation stimuli: i) CD40L-expressing fibroblasts, ii) soluble recombinant CD40L produced in our laboratory +IL-4, iii) CpG ODN 2006+IL-15 with or without bone marrow stromal cells (BMSC). CLL cell viability, activation/proliferation were monitored by flow cytometry. We unexpectedly observed that BPs generated a protective effect from spontaneous apoptosis in 11/26 (42%) patients (viability + 18%-392%) and an augmentation in CLL cell activation/proliferation in 61% of the samples (S+G2M phase: +100%±25). Interestingly, protection from spontaneous apoptosis or increment of cell activation, required the presence of either fibroblasts, BMSC or autologous Nurse Like Cells (NLC). We thus hypothesized that supportive cells are involved in the BPs effects either through cell-cell interactions with leukemic cells or T cells, or through soluble factors release in the medium. Functional experiments with transwells suggest that stromal cells, in presence of Clodronate, release soluble factors in the medium that may probably concur to the unexpected Clodronate-mediated enhancement of CLL cell activation/proliferation. This work is in progress and several critical questions on the mechanisms are still unanswered. Nevertheless, the phenomenological data argue that caution should be taken when administering BPs against osteoporosis in elderly persons, who could have Monoclonal B Lymphocytosis or CLL

Relevance of Stereotyped B-Cell Receptors in the Context of the Molecular, Cytogenetic and Clinical Features of Chronic Lymphocytic Leukemia

Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome

The Pleiotropic Effects of Gut Microbiota in Colorectal Cancer Progression: How to Turn Foes into Friends

Colorectal Cancer (CRC) is one of most frequent malignant cancers, showing high lethality worldwide [...