SAR442085, a novel anti-CD38 antibody with enhanced antitumor activity against multiple myeloma.

Anti-CD38 monoclonal antibodies (mAbs) represent a breakthrough in the treatment of multiple myeloma (MM), yet some patients fail to respond or progress quickly with this therapy, highlighting the need for novel approaches. In this study we compared the preclinical efficacy of SAR442085, a next-generation anti-CD38 mAb with enhanced affinity for activating Fcγ receptors (FcγR), with first-generation anti-CD38 mAb daratumumab and isatuximab. In surface plasmon resonance and cellular binding assays, we found that SAR442085 had higher binding affinity than daratumumab and isatuximab for FcγRIIa (CD32a) and FcγRIIIa (CD16a). SAR442085 also exhibited better in vitro antibody-dependent cellular cytotoxicity (ADCC) against a panel of MM cells expressing variable CD38 receptor densities including MM patients' primary plasma cells. The enhanced ADCC of SAR442085 was confirmed using NK-92 cells bearing low and high affinity FcγRIIIa (CD16a)-158F/V variants. Using MM patients' primary bone marrow cells, we confirmed that SAR442085 had an increased ability to engage FcγRIIIa, resulting in higher natural killer (NK) cell activation and degranulation against primary plasma cells than preexisting Fc wild-type anti-CD38 mAbs. Finally, using huFcgR transgenic mice that express human Fcγ receptors under the control of their human regulatory elements, we demonstrated that SAR442085 had higher NK cell-dependent in vivo antitumor efficacy and better survival than daratumumab and isatuximab against EL4 thymoma or VK*MYC myeloma cells overexpressing human CD38. These results highlight the preclinical efficacy of SAR442085 and support the current evaluation of this next-generation anti-CD38 antibody in phase I clinical development in patients with relapsed/refractory MM.

Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.

A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

HSP110 T17 simplifies and improves the microsatellite instability testing in patients with colorectal cancer.

BACKGROUND: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex). METHODS: The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins. RESULTS: HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS. CONCLUSIONS: HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC.

Small bowel adenocarcinomas complicating Crohn's disease are associated with dysplasia: a pathological and molecular study.

BACKGROUND: Crohn's disease (CD) is associated with an increased risk of small bowel adenocarcinoma (SBA). However, there are no guidelines for the screening and early diagnosis of SBA. Colorectal cancer associated with chronic colitis arises from dysplasia. High-risk patients benefit from surveillance colonoscopies aimed to detect dysplasia. The dysplasia-carcinoma sequence remains poorly documented in CD-associated SBA. Moreover, molecular data about SBA complicating CD and associated dysplasia are very limited. We therefore assessed dysplasia and several key molecular markers of carcinogenesis in SBA and dysplasia developed in patients with CD. METHODS: Forty-five SBA complicating CD and 4 specimens with dysplasia without SBA were screened. In SBA, we looked for dysplasia and determined their pathological characteristics (type, grade, distribution). We also stained for mismatch repair proteins (MLH1, MSH2, MSH6, PMS2), p53, ß-catenin, and p16 and looked for KRAS, BRAF and PIK3CA mutations. RESULTS: All neoplastic lesions, except 1 lesion, were found in inflamed mucosal areas. Dysplasia was found in 20 of 41 patients with SBA (49%). Dysplasia was flat or raised, low grade or high grade, and adjacent or distant to concomitant SBA. Molecular markers of SBA carcinogenesis complicating CD were similar to those observed in chronic colitis-related colorectal cancer (KRAS, BRAF, p53, MSI), although differences were observed for ß-catenin and p16. No PIK3CA mutations were observed. CONCLUSIONS: These results suggest that there is an inflammation-dysplasia-adenocarcinoma sequence in at least half of CD-related SBA, similar to what is observed in chronic colitis-related colorectal cancer and may have implications for the prevention and treatment of this cancer.

NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi.

Congenital melanocytic nevus (CMN) is a particular melanocytic in utero proliferation characterized by an increased risk of melanoma transformation during infancy or adulthood. NRAS and BRAF mutations have consistently been reported in CMN samples, but until recently results have been contradictory. We therefore studied a series of large and giant CMNs and compared them with small and medium CMNs using Sanger sequencing, pyrosequencing, high-resolution melting analysis, and mutation enrichment by an enhanced version of ice-COLD-PCR. Large-giant CMNs displayed NRAS mutations in 94.7% of cases (18/19). At that point, the role of additional mutations in CMN pathogenesis had to be investigated. We therefore performed exome sequencing on five specimens of large-giant nevi. The results showed that NRAS mutation was the sole recurrent somatic event found in such melanocytic proliferations. The genetic profile of small-medium CMNs was significantly different, with 70% of cases bearing NRAS mutations and 30% showing BRAF mutations. These findings strongly suggest that NRAS mutations are sufficient to drive melanocytic benign proliferations in utero.

Overexpression of microRNAs-155 and 21 targeting mismatch repair proteins in inflammatory bowel diseases.

Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency is reported in 5-10% of colorectal cancers (CRCs) complicating inflammatory bowel diseases (IBD). The molecular mechanisms underlying MMR deficiency may be different in IBD CRCs, and in sporadic and hereditary MSI tumors. Here, we hypothesize that overexpression of miR-155 and miR-21, two inflammation-related microRNAs that target core MMR proteins, may constitute a pre-neoplastic event for the development of MSI IBD CRCs. We studied miR-155 and miR-21 expression using real-time quantitative PCR in MSI (n = 10) and microsatellite stable (n = 10) IBD CRCs, and in MSI (n = 32) and microsatellite stable (n = 30) non-IBD CRCs. We also screened colonic samples from IBD patients without cancer (n = 18) and used healthy colonic mucosa as controls (n = 20). MiR-155 and miR-21 appeared significantly overexpressed not only in the colonic mucosa of IBD subjects without CRC but also in neoplastic tissues of IBD patients compared with non-IBD controls (P < 0.001). Importantly, in patients with IBD CRCs, miR-155 and miR-21 overexpression extended to the distant non-neoplastic mucosa (P < 0.001). Ratios of expressions in tumors versus matched distant mucosa revealed a nearly significant association between miR-155 overexpression and MSI in IBDs (P = 0.057). These results show a strong deregulation of both MMR-targeting microRNAs in IBD subjects with or without cancer. MiR-155 overexpression being particularly associated to MSI IBD CRCs and extending to distant non-neoplastic mucosa, strongly suggests that a pre-neoplastic miR-155 field defect may promote MSI-driven transformation of the colonic mucosa. The detection and monitoring of miR-155 field defect may, therefore, have implications for the prevention and treatment of MSI IBD CRCs.

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures.

The "RNA world" hypothesis proposes that--early in the evolution of life--RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg(2+), which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg(2+) on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na(+) or K(+)), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

MiRNA genes constitute new targets for microsatellite instability in colorectal cancer.

Mismatch repair-deficient colorectal cancers (CRC) display widespread instability at DNA microsatellite sequences (MSI). Although MSI has been reported to commonly occur at coding repeats, leading to alterations in the function of a number of genes encoding cancer-related proteins, nothing is known about the putative impact of this process on non-coding microRNAs. In miRbase V15, we identified very few human microRNA genes with mono- or di-nucleotide repeats (n = 27). A mutational analysis of these sequences in a large series of MSI CRC cell lines and primary tumors underscored instability in 15 of the 24 microRNA genes successfully studied at variable frequencies ranging from 2.5% to 100%. Following a maximum likelihood statistical method, microRNA genes were separated into two groups that differed significantly in their mutation frequencies and in their tendency to represent mutations that may or may not be under selective pressures during MSI tumoral progression. The first group included 21 genes that displayed no or few mutations in CRC. The second group contained three genes, i.e., hsa-mir-1273c, hsa-mir-1303 and hsa-mir-567, with frequent (≥ 80%) and sometimes bi-allelic mutations in MSI tumors. For the only one expressed in colonic tissues, hsa-mir-1303, no direct link was found between the presence or not of mono- or bi-allelic alterations and the levels of mature miR expression in MSI cell lines, as determined by sequencing and quantitative PCR respectively. Overall, our results provide evidence that DNA repeats contained in human miRNA genes are relatively rare and preserved from mutations due to MSI in MMR-deficient cancer cells. Functional studies are now required to conclude whether mutated miRNAs, and especially the miR-1303, might have a role in MSI tumorigenesis.