Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the ...first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse–human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody–drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host’s immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.
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.
Relevant advances have been made in the management of relapsed/refractory (r/r) Hodgkin Lymphomas (HL) with the use of the anti-CD30 antibody-drug conjugate (ADC) brentuximab-vedotin (Bre-Ved). ...Unfortunately, most patients eventually progress despite the excellent response rates and tolerability. In this report, we describe an ADC composed of the aminobisphosphonate zoledronic acid (ZA) conjugated to Bre-Ved by binding the free amino groups of this antibody with the phosphoric group of ZA. Liquid chromatography-mass spectrometry, inductively coupled plasma-mass spectrometry, and matrix-assisted laser desorption ionization-mass spectrometry analyses 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 Bre-Ved. 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 Bre-Ved. Eventually, Bre-Ved-ZA ADC, in contrast with the native antibody, can trigger the proliferation and activation of cytolytic activity of effector-memory Vδ2 T-lymphocytes against HL-cell lines. These findings may support the potential use of this ADC in the management of r/r HL.
B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of ...patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5
and CD5
B cells from the spleen and peripheral blood (PB).
Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5
and CD5
cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution.
CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5
B (0.57%) cells compared to CD5
B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family.
CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.
Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the ...mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.
Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene ...alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus del(17p) were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations accompanied by del(17p) in 9 cases, 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5
+
B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell ...receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5
+
B cells, nor in specific B-cell subpopulations or the CD5
+
cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.
PDF
