A Synthetic Circuit Empowering Reprogrammed B Cells for Therapeutic Proteins Expression Regulated by Tumor Detection.

Cancer remains a leading cause of death worldwide, but immunotherapies hold promises to cure it by awaking the patient's immune system to provide long-term protection. Cell therapies, involving the infusion of immune cells, either directly or genetically modified, are being developed to recognize and destroy cancer cells. Here, we explored the potential of a new synthetic circuit to reprogram B cells to cure cancers. This circuit consists in a sensor (a membrane-anchored IgG1), a transducer (a fragment of the NR4A1 promoter) and an effector molecule. Upon recognition of its target, this sensor triggers signaling pathways leading to the activation of the transducer and to effector expression (here, a reporter molecule). We showed that this circuit could discriminate tumors expressing the target antigen from those that did not, in a dose dependent manner in vitro. Going further, we replaced the original membrane-anchored sensor by an immunoglobulin expression cassette that can not only be membrane-anchored but also be secreted depending on B-cell maturation status. This allowed concomitant activation of the circuit and secretion of transgenic antibodies directed against the targeted antigen. Of note, these antibodies could correctly bind their target and were recognized by FcR expressed at the surface of immune cells, which should synergically amplify the action of the effector. The potential of reprogrammed B cells remains to be assessed in vivo by implementing a therapeutic effector. In the future, B-cell reprogramming platforms should allow personalized cancer treatment by adapting both the sensor and the therapeutic effectors to patients.

Development of NK cell-based cancer immunotherapies through receptor engineering.

Natural killer (NK) cell-based immunotherapies are attracting increasing interest in the field of cancer treatment. Early clinical trials have shown promising outcomes, alongside satisfactory product efficacy and safety. Recent developments have greatly increased the therapeutic potential of NK cells by endowing them with enhanced recognition and cytotoxic capacities. This review focuses on surface receptor engineering in NK cell therapy and discusses its impact, challenges, and future directions.Most approaches are based on engineering with chimeric antigen receptors to allow NK cells to target specific tumor antigens independent of human leukocyte antigen restriction. This approach has increased the precision and potency of NK-mediated recognition and elimination of cancer cells. In addition, engineering NK cells with T-cell receptors also mediates the recognition of intracellular epitopes, which broadens the range of target peptides. Indirect tumor peptide recognition by NK cells has also been improved by optimizing immunoglobulin constant fragment receptor expression and signaling. Indeed, engineered NK cells have an improved ability to recognize and destroy target cells coated with specific antibodies, thereby increasing their antibody-dependent cellular cytotoxicity. The ability of NK cell receptor engineering to promote the expansion, persistence, and infiltration of transferred cells in the tumor microenvironment has also been explored. Receptor-based strategies for sustained NK cell functionality within the tumor environment have also been discussed, and these strategies providing perspectives to counteract tumor-induced immunosuppression.Overall, receptor engineering has led to significant advances in NK cell-based cancer immunotherapies. As technical challenges are addressed, these innovative treatments will likely reshape cancer immunotherapy.

Efficient adoptive transfer of autologous modified B cells: a new humanized platform mouse model for testing B cells reprogramming therapies.

Here, we report a novel experimental setup to perform adoptive transfer of gene-edited B cells using humanized immune system mice by infusing autologous HIS mouse-derived human B cells "educated" in a murine context and thus rendered tolerant to the host. The present approach presents two advantages over the conventional humanized PBMC mouse models: (i) it circumvents the risk of xenogeneic graft-versus-host reaction and (ii) it mimics more closely human immune responses, thus favoring clinical translation. We show that the frequencies and numbers of transduced B cells in recipient's spleens one week post-transfer are within the range of the size of the pre-immune B cell population specific for a given protein antigen in the mouse. They are also compatible with the B cell numbers required to elicit a sizeable immune response upon immunization. Altogether, our findings pave the way for future studies aiming at assessing therapeutic interventions involving B cell reprogramming for instance by an antibody transgene in a "humanized" hematopoietic setting.

Exploiting B Cell Transfer for Cancer Therapy: Engineered B Cells to Eradicate Tumors.

Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient's immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.

Towards Physiologically and Tightly Regulated Vectored Antibody Therapies.

Cancers represent highly significant health issues and the options for their treatment are often not efficient to cure the disease. Immunotherapy strategies have been developed to modulate the patient's immune system in order to eradicate cancerous cells. For instance, passive immunization consists in the administration at high doses of exogenously produced monoclonal antibodies directed either against tumor antigen or against immune checkpoint inhibitors. Its main advantage is that it provides immediate immunity, though during a relatively short period, which consequently requires frequent injections. To circumvent this limitation, several approaches, reviewed here, have emerged to induce in vivo antibody secretion at physiological doses. Gene delivery vectors, such as adenoviral vectors or adeno-associated vectors, have been designed to induce antibody secretion in vivo after in situ cell modification, and have driven significant improvements in several cancer models. However, anti-idiotypic antibodies and escape mutants have been detected, probably because of both the continuous expression of antibodies and their expression by unspecialized cell types. To overcome these hurdles, adoptive transfer of genetically modified B cells that secrete antibodies either constitutively or in a regulated manner have been developed by ex vivo transgene insertion with viral vectors. Recently, with the emergence of gene editing technologies, the endogenous B cell receptor loci of B cells have been modified with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas-9) system to change their specificity in order to target a given antigen. The expression of the modified BCR gene hence follows the endogenous regulation mechanisms, which may prevent or at least reduce side effects. Although these approaches seem promising for cancer treatments, major questions, such as the persistence and the re-activation potential of these engineered cells, remain to be addressed in clinically relevant animal models before translation to humans.

VP24 is a molecular determinant of Ebola virus virulence in guinea pigs.

In sharp contrast to human and nonhuman primates, guinea pigs and some other mammals resist Ebola virus (EBOV) replication and do not develop illness upon virus inoculation. However, serial passaging of EBOV in guinea pigs results in a selection of variants with high pathogenicity. In this report, using a reverse genetics approach, we demonstrate that this dramatic increase in EBOV pathogenicity is associated with amino acid substitutions in the structural protein VP24. We show that although replication of recombinant EBOV carrying wild-type VP24 is impaired in primary peritoneal guinea pig macrophages and in the liver of infected animals, the substitutions in VP24 allow EBOV to replicate in guinea pig macrophages and spread in the liver of infected animals. Furthermore, we demonstrate that both VP24/wild type and the guinea pig-adapted VP24/8mc are similar in their ability to block expression of interferon-induced host genes, suggesting that the increase in EBOV virulence for guinea pigs is not associated with VP24 interferon antagonist function. This study sheds light on the mechanism of resistance to EBOV infection and highlights the critical role of VP24 in EBOV pathogenesis.