Secreted production of assembled Norovirus virus-like particles from Pichia pastoris.

BACKGROUND: Norovirus virus-like particles (NoV VLPs) have recently been explored as potential vaccine platforms due to their ability to produce an effective immune response. Expression of the main structural protein, VP1, leads to formation of self-assembled particles with similar characteristics to the original virus. These NoV VLPs have been expressed in Escherichia coli, yeast and insect cells. Expression in E. coli and insect cells share downstream processing issues due to the presence of inclusion bodies or the need for numerous purification steps. NoV VLPs have also been produced in the yeast P. pastoris; however the protein was only expressed intracellularly. RESULTS: We have successfully expressed and secreted the VP1 protein in the novel P. pastoris strain, Bg11, using the methanol inducible pJ912 expression vector, containing the cDNA of NoV VP1. Expression of the VP1 protein in Bg11 was carried out in a 1.5 L bioreactor resulting in a total yield of NoV VLPs greater than 0.6 g/L. NoV VLPs obtained from the culture supernatant were purified via ion-exchange chromatography, resulting in particles with a purity over 90%. The average size of the particles after purification was 40 nm. Transmission electron microscopy was used to visualize the morphology of the particles and saliva-binding assay confirmed that the NoV VLPs bind to Histo-Blood Group Antigens (HBGA). CONCLUSIONS: In this study we describe the expression and characterization of fully assembled Norovirus virus-like particles obtained from P. pastoris. The particles are similar in size, morphology and binding capacity, as previously described, for the original NoV. Our results detail the successful expression and secretion of VLPs in P. pastoris, improving their candidacy as a vaccine platform.

Non-signaling Chimeric Antigen Receptors Enhance Antigen-Directed Killing by γδ T Cells in Contrast to αß T Cells.

Chimeric antigen receptor (CAR)-modified T cells have demonstrated efficacy against B cell leukemias/lymphomas. However, redirecting CAR T cells to malignant T cells is more challenging due to product-specific cis- and trans-activation causing fratricide. Other challenges include the potential for product contamination and T cell aplasia. We expressed non-signaling CARs (NSCARs) in γδ T cells since donor-derived γδ T cells can be used to prevent product contamination, and NSCARs lack signaling/activation domains, but retain antigen-specific tumor cell-targeting capability. As a result, NSCAR targeting requires an alternative cytotoxic mechanism, which can be achieved through utilization of γδ T cells that possess major histocompatibility complex (MHC)-independent cytotoxicity. We designed two distinct NSCARs and demonstrated that they do not enhance tumor-killing by αß T cells, as predicted. However, both CD5-NSCAR- and CD19-NSCAR-modified γδ T cells enhanced cytotoxicity against T and B cell acute lymphoblastic leukemia (T-ALL and B-ALL) cell lines, respectively. CD5-NSCAR expression in γδ T cells resulted in a 60% increase in cytotoxicity of CD5-expressing T-ALL cell lines. CD19-NSCAR-modified γδ T cells exhibited a 350% increase in cytotoxicity against a CD19-expressing B-ALL cell line compared to the cytotoxicity of naive cells. NSCARs may provide a mechanism to enhance antigen-directed anti-tumor cytotoxicity of γδ T cells through the introduction of a high-affinity interaction while avoiding self-activation.

Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions.

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating B cell malignancies in clinical trials; however, fewer studies have evaluated CAR T cell therapy for the treatment of T cell malignancies. There are many challenges in translating this therapy for T cell disease, including fratricide, T cell aplasia, and product contamination. To the best of our knowledge, no tumor-specific antigen has been identified with universal expression on cancerous T cells, hindering CAR T cell therapy for these malignancies. Numerous approaches have been assessed to address each of these challenges, such as (i) disrupting target antigen expression on CAR-modified T cells, (ii) targeting antigens with limited expression on T cells, and (iii) using third party donor cells that are either non-alloreactive or have been genome edited at the T cell receptor α constant (TRAC) locus. In this review, we discuss CAR approaches that have been explored both in preclinical and clinical studies targeting T cell antigens, as well as examine other potential strategies that can be used to successfully translate this therapy for T cell disease.

Ex vivo expanded patient-derived γδ T-cell immunotherapy enhances neuroblastoma tumor regression in a murine model.

An effective therapy regimen for relapsed/refractory high-risk neuroblastoma (NB) includes the anti-GD2 monoclonal antibody, dinutuximab, in combination with temozolomide and irinotecan, supporting a role for chemo-immunotherapy in NB. γδ T cells are an attractive anti-tumor immunotherapy because of their direct cytotoxic activity mediated through cell surface receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands whereas CD16 recognizes antibody bound to tumors and activates mechanisms of antibody-dependent cellular cytotoxicity (ADCC). This study demonstrates an efficient method for expanding and storing γδ T cells from NB patient-derived apheresis products at clinically relevant amounts. The expanded patient-derived γδ T cells were cytotoxic against the K562 cell line and multiple NB cell lines. Combining γδ T cells with dinutuximab led to a 30% increase in tumor cell lysis compared to γδ T cells alone. Furthermore, low-dose temozolomide in combination with expanded γδ T cells and dinutuximab resulted in increased IFNγ secretion and increased γδ T-cell surface expression of FasL and CD107a. IMR5 NB cell line xenografts established subcutaneously in NSG mice were treated with a regimen of dinutuximab, temozolomide, and γδ T cells. This combination caused targeted killing of NB xenografts in vivo, reducing tumor burden and prolonging survival. These data support the continued preclinical testing of dinutuximab and temozolomide in conjunction with γδ T-cell immunotherapy for patients with recurrent/refractory NB.

Development of chimeric antigen receptors targeting T-cell malignancies using two structurally different anti-CD5 antigen binding domains in NK and CRISPR-edited T cell lines.

Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors.

Chimeric antigen receptors (CARs) are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs). VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig)-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.