Search

Genetic engineering of T cells with chimeric T-cell receptors (CARs) is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV-) specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34+ hematopoietic progenitors. Moreover, after intravenous administration into CD33+ human acutemyeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone. [ABSTRACT FROM AUTHOR]

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53R175H, was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells. [ABSTRACT FROM AUTHOR]

A major obstacle to the expansion of abnormal cells with significant proliferative potential is the induction of programmed cell death. Consequently, oncogene-driven hyperproliferation must be associated with apoptosis inhibition to allow malignant outgrowth. The oncogenic cooperation of N-Myc and Twist-1 in the development of neuroblastoma, the most common and deadly solid tumour of childhood, perfectly illustrates such a process. N-Myc promotes cell proliferation, whereas Twist-1 counteracts its pro-apoptotic properties by knocking-down the ARF/p53 pathway. On the basis of numerous recent studies reporting its overexpression in a variety of human cancers, we discuss in this review the role of Twist-1 as a potent inhibitor of the cell safety programs engaged in response to an abnormal mitogenic activity. [ABSTRACT FROM AUTHOR]

Abstract Background The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNΔ1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein. Methods Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCNΔ1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. Results Both are translated, but higher levels of protein were seen with MYCNΔ1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNΔ1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCNΔ1b mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with MYCN mRNA. Here, we showed that MYCNOT: MYCN Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of MYCNΔ1b mRNA. Conclusions Existence of upstream ORF in MYCN transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.

During the last few years Antibody-Drug Conjugates (ADCs) have become one of the most active and very promising therapeutic weapons. Lessons learned from the traditional chemical conjugations (via lysine or cysteine residues of the antibodies) and the clinical studies of the developed ADCs have recently paved the way to the improvement of the conjugation technologies. Use of site-specific conjugation is considered as the promising path for improving the design and development of homogeneous ADCs with controlled Drug-Antibody ratio (DAR). Moreover, some of these conjugations can be applied to antibody fragments such as Fab, scfv and VHH for which random and chemical conjugation showed significant limitations. In this study, we identified a novel small peptide substrate (Q-tag) with high affinity and specificity of bacterial transglutaminase which can be genetically fused to different formats of antibodies of interest for the development of enzymatic site-specific conjugation we named "CovIsolink" platform. We describe the synthesis of chemically defined drugs conjugation in which the site and stoichiometry of conjugation are controlled using a genetically encoded Q-tag peptide with specific amino acids which serves as a substrate of bacterial transglutaminase. This approach has enabled the generation of homogeneous conjugates with DAR 1,7 for full IgG and 0,8 drug ratio for Fab, scfv and VHH antibody fragments without the presence of significant amounts of unconjugated antibody and fragments. As a proof of concept, Q-tagged anti Her-2 (human IgG1 (Trastuzumab) and the corresponding fragments (Fab, scfv and VHH) were engineered and conjugated with different aminated-payloads. The corresponding Cov-ADCs were evaluated in series of in vitro and in vivo assays, demonstrating similar tumor cell killing potency as Trastuzumab emtansine (Kadcyla®) even with lower drug-to-antibody ratio (DAR)., (© 2024. The Author(s).)

Intratumoral delivery of viruses and virus-associated molecular patterns can achieve antitumor effects that are largely mediated by the elicitation or potentiation of immune responses against the malignancy. Attenuated vaccines are approved and marketed as good manufactiring practice (GMP)-manufactured agents whose administration might be able to induce such effects. Recent reports in mouse transplantable tumor models indicate that the rotavirus, influenza and yellow fever vaccines can be especially suitable to elicit powerful antitumor immunity against cancer following intratumoral administration. These results highlight that intratumoral anti-infectious vaccines can turn cold tumors into hot, and underscore the key role played by virus-induced type I interferon pathways to overcome resistance to immune checkpoint-targeted antibodies., Competing Interests: Competing interests: Consulting: BMS, AZ, Roche, F-star, Genmab, Alligator, Bioncotech, Numab, EMD. Grants: Roche, BMS, Alligator, Bioncotech., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Although immune checkpoint-targeted therapies are currently revolutionizing cancer care, only a minority of patients develop durable objective responses to anti-PD-1, PD-L1, and CTLA-4 therapy. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors and overcome the resistance to these immunotherapies. Oncolytic properties of common viruses can be exploited for the priming of antitumor immunity, and such oncolytic viruses are currently in active clinical development in combination with immune checkpoint-targeted therapies. However, the routine implementation of these therapies is limited by their manufacturing constraints, the risk of exposure of clinical staff, and the ongoing regulations on genetically modified organisms. We sought to determine whether anti-infectious disease vaccines could be used as a commercially available source of immunostimulatory agents for cancer immunotherapy. We found that rotavirus vaccines have both immunostimulatory and oncolytic properties. In vitro, they can directly kill cancer cells with features of immunogenic cell death. In vivo, intratumoral rotavirus therapy has antitumor effects that are dependent on the immune system. In several immunocompetent murine tumor models, intratumoral rotavirus overcomes resistance to and synergizes with immune checkpoint-targeted therapy. Heat- and UV-inactivated rotavirus lost their oncolytic activity but kept their synergy with immune checkpoint-targeted antibodies through the up-regulation of the double-stranded RNA receptor retinoic acid-induced gene 1 (RIG-I). Rotavirus vaccines are clinical-grade products used in pediatric and adult populations. Therefore, in situ immunization strategies with intratumoral-attenuated rotavirus could be implemented quickly in the clinic., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Therapeutic monoclonal antibodies targeting immune checkpoints (ICPs) have changed the treatment landscape of many tumors. However, response rate remains relatively low in most cases. A major factor involved in initial resistance to ICP inhibitors is the lack or paucity of tumor T cell infiltration, characterizing the so-called "cold tumors." In this review, we describe the main mechanisms involved in the absence of T cell infiltration, including lack of tumor antigens, defect in antigen presentation, absence of T cell activation and deficit of homing into the tumor bed. We discuss then the different therapeutic approaches that could turn cold into hot tumors. In this way, specific therapies are proposed according to their mechanism of action. In addition, ''supra-physiological'' therapies, such as T cell recruiting bispecific antibodies and Chimeric Antigen Receptor (CAR) T cells, may be active regardless of the mechanism involved, especially in MHC class I negative tumors. The determination of the main factors implicated in the lack of preexisting tumor T cell infiltration is crucial for the development of adapted algorithms of treatments for cold tumors.

Introduction: Trastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC., Methods: We set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model., Results: We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells' cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models., Conclusions: Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification., (Copyright © 2015, American Association for the Advancement of Science.)

In neuroblastoma, MYCN amplification is associated with a worse prognosis and is a criterion used in the clinic to provide intensive treatments to children even with localized disease. In correlation with MYCN amplification, upregulation of TWIST1, a transcription factor playing a crucial role in inhibition of apoptosis and differentiation, was previously reported. Clinical data set analysis of MYCN, MYC and TWIST1 expression permits us to confirm that TWIST1 expression is upregulated in MYCN amplified neuroblastoma but also in a subset of neuroblastoma harboring high expression of MYCN or MYC without gene amplification. In silico analyses reveal the presence of several MYC regulatory motifs (E-Boxes and INR) within the TWIST1 promoter. Using gel shift assay and reporter activity assays, we demonstrate that both N-Myc and c-Myc proteins can bind and activate the TWIST1 promoter. Therefore, we propose TWIST1 as a direct MYC transcriptional target., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92(CD16)) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92(CAR)). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92(CD16) was always inferior to that observed after direct recognition by NK-92(CAR). In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92(CD16) + trastuzumab but not of NK-92(CAR) induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

Stem cells are defined by their unique property to self-renew and starting from one single cell to generate all the different progenies required for tissue regeneration. In adults, stem cells are still present in the majority of tissues and organs where they are responsible for continuous organs and tissues homeostasis. Adult stem cells have been isolated in various tissues and all share common specific characteristics (localization in stem cell niches, drug transporter expression, adhesion, levels of apoptosis inhibitors, DNA methylation, -) involved in high levels of drug resistance of this specific cell subtype. Several studies have identified different populations of cancer cells, within the same tumor, some of them which present properties closely related to normal stem cells and raised the concept of cancer stem cells. Interestingly, the cell surface markers expressed by these particular cancer cells are the same as those expressed by normal stem cells, suggesting that cancer can arise in some cases from the malignant transformation of stem cells. The cancer stem cell (CaSC) model predicts that, even if "conventional" cancer cells can be killed by chemotherapy or radiotherapy, only the destruction of CaSC, considered responsible for relapse, will allow full recovery, thus demonstrating the importance of CaSC-targeting for patient outcome. Therapeutic innovations will emerge from a better understanding of the biology and environment of cancer stem cells. The tumor environment can create a niche favoring the survival and proliferation of CaSC. It also protects CaSC from chemotherapy-induced apoptosis. Clinically, it is crucial to get rid of quiescent and resistant cells and to adapt the therapeutic strategy to cancer stem cells sheltered in niches. Here, we review the major characteristics of cancer stem cells and their behavior in response to chemotherapy; we also highlight the main issues to be considered for efficient and specific cancer stem cell targeting.

Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.

A major obstacle to the expansion of abnormal cells with significant proliferative potential is the induction of either cellular senescence or programmed cell death. Consequently, oncogene-driven hyperproliferation must be associated with apoptosis inhibition to allow malignant outgrowth. The oncogenic cooperation of N-Myc and Twist 1 in the development of neuroblastoma, the most common and deadly solid tumour of childhood, perfectly illustrates such a process. N-Myc promotes cell proliferation whereas Twist 1 counteracts its pro-apoptotic properties by knocking-down the ARF/p53 pathway. This observation provides a mechanistic explanation for the rarity of p53 mutations in neuroblastomas. It also highlights the involvement of two crucial regulators of embryogenesis in human cancer development. In this review, we discuss the possible role of Twist 1 in tumour progression, based on the numerous recent studies reporting its overexpression in a variety of human cancers.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and the third most common pediatric cancer. Although numerous factors including patient age, disease stage and genetic abnormalities have been shown to be predictive of outcome, the mechanisms responsible for the highly variable clinical behavior of this tumor remain largely unknown. In order to gain new insights into the biology of this tumor, we performed microarray analysis and compared the gene expression patterns of NB detected by mass screening, characterized by highly probable spontaneous regression, versus stage 4 NB with poor prognosis. The bioinformatics analysis revealed a set of 19 discriminatory genes that may play a significant role in the natural progression of the disease. Validation of these results and further mechanistic studies would shed new light on the biology of tumor progression, and provide new tools to predict clinical outcome in children with NB.

N-Myc oncogene amplification is a frequent event in neuroblastoma and is strongly correlated with advanced disease stage and treatment failure. Similarly to c-Myc oncogenic activation, N-Myc deregulation promotes both cell proliferation and p53-dependent apoptosis by sensitizing cells to a variety of insults. Intriguingly, p53 mutations are uncommon in neuroblastomas, strongly suggesting that an alternative cooperating event circumvents this safeguard against oncogene-driven neoplasia. By performing a pangenomic cDNA microarray analysis, we demonstrate that human Twist is constantly overexpressed in N-Myc-amplified neuroblastomas. H-Twist overexpression is responsible for the inhibition of the ARF/p53 pathway involved in the Myc-dependent apoptotic response. This oncogenic cooperation of two key regulators of embryogenesis causes cell transformation and malignant outgrowth.

The p53-transcriptional target, BTG2(TIS21/PC3), was previously identified as an antiproliferative gene. However, the precise biological functions of the protein product remain to be elucidated. BTG2(TIS21/PC3) expression is induced in vivo during neurogenesis, and the gene is transiently expressed in vitro in rat pheochromocytoma PC12 cells after induction of neuronal differentiation by addition of nerve growth factor (NGF). These observations suggest that BTG2(TIS21/PC3) is functionally significant during the neuronal differentiation process. To test this hypothesis, a vector that expressed BTG2(TIS21/PC3) under the control of an inducible promoter was introduced into PC12 cells. Growth arrest and differentiation in response to NGF were greatly enhanced by BTG2(TIS21/PC3) overexpression. Furthermore, an antisense oligonucleotide complementary to BTG2(TIS21/PC3) mRNA, which was able to inhibit endogenous BTG2(TIS21/PC3) expression, triggered programmed cell death in differentiated PC12 cells. These observations confirm that BTG2(TIS21/PC3) expression promotes neuronal differentiation and that it is required for survival of terminally differentiated cells.

We have previously reported a set of Moloney murine leukemia virus derived envelopes retargeted to the Pit-2 phosphate transporter molecule, by insertion of the Pit-2 binding domain (BD) at the N terminus of the ecotropic retroviral envelope glycoproteins (S. Valsesia-Wittmann et al., J. Virol. 70:2059-2064, 1996). The resulting chimeric envelopes share two BDs: an additional N-terminal BD (Pit-2 BD) and the BD of the ecotropic envelope (mCAT-1 BD). By inserting a variety of different amino acid spacers between the two binding domains, we showed that retroviruses can potentially use the targeted cell surface receptor Pit-2, the ecotropic retroviral receptor mCAT-1, or both receptors cooperatively for entry into target cell (S. Valsesia-Wittmann et al., EMBO J 6:1214-1223, 1997). An extreme example of receptor cooperativity was encountered when envelopes with specific proline-rich interdomain spacers (PRO spacers) were tested: both receptors had to be coexpressed at the surface of the targeted cells to cooperatively allow infection. Here, we characterized the role of PRO spacer in the cooperation of receptors. We have shown that the particular organization of the PRO spacer-a beta-turn polyproline-was responsible for the cooperative effect. In the native configuration of the viruses, the structure masked the regions located downstream of the PRO spacer, thus the mCAT-1 BD. After interaction with the targeted Pit-2 receptor, the BD of the backbone envelope became accessible, and we demonstrated that interaction between the mCAT-1 BD and the mCAT-1 receptor is absolutely necessary. This interaction leads to natural fusion triggering and entry of viruses into targeted cells.

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a lacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.