Targeting interference of CTLA-4 co-inhibition to tumour specific T cells to enhance activity and reduce toxicity

Blockade of Cytotoxic T lymphocyte antigen-4 (CTLA-4) can enhance anti-tumour responses in preclinical models. A monoclonal antibody targeting human CTLA-4 (αCTLA4 mAb), is FDA approved for clinical use against metastatic melanoma. Paradigms suggest that αCTLA4 mAb blocks CTLA-4 dually on CTLA-4hi Tregs (restricting regulatory activity) and CTLA-4int-hi CD4 Teff (enhanced expansion) resulting in intratumoural Teff dominance and a net gain in anti-tumour activity. More recently, antibody-dependent cellular cytotoxicity (ADCC)-mediated depletion of CTLA-4hi Tregs in the tumour has been flagged as a critical factor driving anti-tumour immune responses. We explored the ADCC hypothesis in an adoptive cell transfer model of murine melanoma. We compared intratumoural Treg numbers in groups receiving CTLA-4+/+ or CTLA-4-/- tumour-specific T cells with/without exogenous αCTLA4 mAb. Exogenous αCTLA4 mAb resulted in diminished intratumoural tumour-specific Treg numbers in groups receiving CTLA-4+/+ but not CTLA-4-/- T-cells. Endogenous CTLA-4+/+ Tregs were depleted intratumourally with αCTLA4 mAb, irrespective of the CTLA-4 status of the adoptive cells. This data supports the proposed ADCC mechanism of αCTLA4 mAb and its dependence upon target cell CTLA-4 expression. Furthermore, we engineered tumour-specific T-cells to secrete αCTLA-4 mAb on IgG2a isotype (high ADCC activity) or IgG1 isotype (low ADCC activity) for adoptive transfer into melanoma-bearing mice. We found that transduced primary murine T-cells could engraft, infiltrate tumours and secrete biologically active αCTLA4 mAb. Secreted IgG2a αCTLA-4 mAb was strongly associated with intratumoural depletion of CTLA-4hi Tregs, but unexpectedly CTLA-4-replete transduced Teff were also vulnerable to depletion, and this significantly compromised the anti-tumour potential of this therapy. Intratumoural depletion of CTLA-4hi T-cells was not associated with (secreted) IgG1 αCTLA-4 mAb or (secreted) isotype control mAb. We believe that depletion of intratumoural Tregs is an important area for cancer immunotherapy, but our experience of αCTLA-4 mAb underlines the importance of careful immunomodulatory target selection to avoid toxicitiy

Novel Cellular Therapies for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Most patients present with advanced disease, and current gold-standard management using tyrosine kinase inhibitors or immune checkpoint inhibitors (ICIs) offers modest clinical benefit. Cellular immune therapies targeting HCC are currently being tested in the laboratory and in clinical trials. Here, we review the landscape of cellular immunotherapy for HCC, defining antigenic targets, outlining the range of cell therapy products being applied in HCC (such as CAR-T and TCR-T), and exploring how advanced engineering solutions may further enhance this therapeutic approach

CAR-T cell manufacturing landscape—Lessons from the past decade and considerations for early clinical development

CAR-T cell therapies have consolidated their position over the last decade as an effective alternative to conventional chemotherapies for the treatment of a number of hematological malignancies. With an exponential increase in the number of commercial therapies and hundreds of phase 1 trials exploring CAR-T cell efficacy in different settings (including autoimmunity and solid tumors), demand for manufacturing capabilities in recent years has considerably increased. In this review, we explore the current landscape of CAR-T cell manufacturing and discuss some of the challenges limiting production capacity worldwide. We describe the latest technical developments in GMP production platform design to facilitate the delivery of a range of increasingly complex CAR-T cell products, and the challenges associated with translation of new scientific developments into clinical products for patients. We explore all aspects of the manufacturing process, namely early development, manufacturing technology, quality control, and the requirements for industrial scaling. Finally, we discuss the challenges faced as a small academic team, responsible for the delivery of a high number of innovative products to patients. We describe our experience in the setup of an effective bench-to-clinic pipeline, with a streamlined workflow, for implementation of a diverse portfolio of phase 1 trials

Cellular Therapeutic Approaches to Cytomegalovirus Infection Following Allogeneic Stem Cell Transplantation

INTRODUCTION Cytomegalovirus (CMV) infection following allogeneic hematopoietic stem cell transplantation (HSCT) is a major cause of morbidity and mortality. Early clinical trials demonstrate that adoptive transfer of donor-derived virus-specific T cells to restore virus-specific immunity is an effective strategy to control CMV infection after HSCT, conferring protection in 70–90% of patients (1). The field has evolved rapidly to develop solutions to some of the CMV cell therapy manufacturing challenges identified in early clinical studies and to define strategies to deliver CMV cell therapy to patients with virus-naive donors. This review discusses the seminal early studies and explores cutting-edge novel technologies that broaden the feasibility and the scope of virus-specific T cells for at risk patients

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format