CAR T Cell Therapy: A Game Changer in Cancer Treatment.

The development of novel targeted therapies with acceptable safety profiles is critical to successful cancer outcomes with better survival rates. Immunotherapy offers promising opportunities with the potential to induce sustained remissions in patients with refractory disease. Recent dramatic clinical responses in trials with gene modified T cells expressing chimeric antigen receptors (CARs) in B-cell malignancies have generated great enthusiasm. This therapy might pave the way for a potential paradigm shift in the way we treat refractory or relapsed cancers. CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation.

Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement.

The manufacture of clinical grade cellular products for adoptive immunotherapy requires ex vivo culture and expansion of human T cells. One of the key components in manufacturing of T cell therapies is human serum (HS) or fetal bovine serum (FBS), which can potentially expose immunotherapy recipient to adventitious infectious pathogens and are thus considered as non-cGMP compliant for adoptive therapy. Here we describe a novel xeno-free serum replacement (SR) with defined components that can be reproducibly used for the production of clinical grade T-cell therapies in combination with several different cell culture media. Dynabeads CD3/CD28 Cell Therapy System (CTS)-activated or antigen-specific T cells expanded using the xeno-free SR, CTS Immune Cell SR, showed comparable growth kinetics observed with cell culture media supplemented with HS or FBS. Importantly the xeno-free SR supplemented medium supported the optimal expansion of T cells specific for subdominant tumour-associated antigens and promoted expansion of T cells with central memory T-cell phenotype, which is favourable for in vivo survival and persistence following adoptive transfer. Furthermore, T cells expanded using xeno-free SR medium were highly amenable to lentivirus-mediated gene transduction for potential application for gene-modified T cells. Taken together, the CTS Immune Cell SR provides a novel platform strategy for the manufacture of clinical grade adoptive cellular therapies.

Ex vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy.

Adoptive T cell therapy is a promising treatment strategy for patients with different types of cancer. The methods used for generation of high numbers of tumor specific T cells usually require long-term ex vivo culture, which frequently lead to generation of terminally differentiated effector cells, demonstrating low persistence in vivo. Therefore, optimization of protocols for generation of T cells for adoptive cell therapy is warranted. The aim of this work was to develop a protocol for expansion of antigen-specific T cells using Dynabeads CD3/CD28 to obtain T cells expressing markers important for in vivo persistence and survival. To achieve high numbers of antigen-specific T cells following expansion, we have tested the effect of depleting regulatory T cells using Dynabeads CD25 and including a pre-stimulation step with peptide prior to the non-specific expansion with Dynabeads. Our data demonstrate that virus- and tumor specific T cells can be expanded to high numbers using Dynabeads CD3/CD28 following optimization of the culture conditions. The expansion protocol presented here results in enrichment of antigen-specific CD8(+) T cells with an early/intermediate memory phenotype. This is observed even when the antigen-specific CD8(+) T cells demonstrated a terminal effector phenotype prior to expansion. This protocol thus results in expanded T cells with a phenotypic profile which may increase the chance of retaining long-term persistence following adoptive transfer. Based on these data we have developed a cGMP protocol for expansion of tumor specific T cells for adoptive T cell therapy.

Cell isolation and expansion using Dynabeads.

This chapter describes the use of Dynabeads for cell isolation and expansion. Dynabeads are uniform polystyrene spherical beads that have been made magnetisable and superparamagnetic, meaning they are only magnetic in a magnetic field. Due to this property, the beads can easily be resuspended when the magnetic field is removed. The invention of Dynabeads made, by Professor John Ugelstad, has revolutionized the separation of many biological materials. For example, the attachment of target-specific antibodies to the surface of the beads allows capture and isolation of intact cells directly from a complex suspension such as blood. This is all accomplished under the influence of a simple magnetic field without the need for column separation techniques or centrifugation. In general, magnetic beads coated with specific antibodies can be used either for isolation or depletion of various cell types. Positive or negative cell isolation can be performed depending on the nature of the starting sample, the cell surface markers and the downstream application in question. Positive cell isolation is the method of choice for unprocessed samples, such as whole blood, and for downstream molecular applications. Positive cell isolation can also be used for any downstream application after detachment and removal of the beads. Negative cell isolation is the method of choice when it is critical that cells of interest remain untouched, i.e., no antibodies have been bound to any cell surface markers on the cells of interest. Some cell populations can only be defined by multiple cell surface markers. Such populations of cells can be isolated by the combination of negative and positive cell isolation. By coupling Dynabeads with antibodies directed against cell surface activation molecules, the beads can be used both for isolation and expansion of the cells. Dynabeads are currently used in two major clinical applications: 1) In the Isolex 300i Magnetic Cell Selection System for CD34 Stem Cell Isolation--2) For ex vivo T cell isolation and expansion using Dynabeads ClinExVivo CD3/CD28 for clinical trials in novel adoptive immunotherapy.

A mouse C kappa-specific T cell clone indicates that DC-SIGN is an efficient target for antibody-mediated delivery of T cell epitopes for MHC class II presentation.

In vaccine development, a major objective is to induce strong, specific T cell responses. This might be obtained by targeting antigen to cell surface molecules that efficiently channel the antigen into endocytic compartments for loading of MHC molecules. Antibodies have been used to deliver antigen; however, it is important to define optimal targets on antigen-presenting cells (APC) for efficient delivery. For this purpose, we have established a T cell readout that can be used to screen large numbers of different mAb for their ability to load MHC class II molecules. The novel human CD4+ T cell clone is specific for mouse Ig C kappa (40-48) and restricted by HLA-DR4 (DRA1,B1*0401). DR4 apparently presents both mouse and human C kappa 40-48, but there is no cross-reaction at the T cell level. B cells from DR4 transgenic mice spontaneously process and present the mouse C kappa peptide. The mouse C kappa -specific T cell readout was used to demonstrate that mouse mAb specific for human dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN), a novel DC-specific molecule, were 10- to 1000-fold more potent at inducing kappa-specific human CD4+ T cell proliferation compared to control mAb. Consistent with this finding, DC-SIGN-specific mAb were rapidly internalized upon binding and found in intracellular vesicles. These results strongly argue that DC-SIGN-specific mAb are channeled into the MHC class II presentation pathway. Thus, DC-SIGN could be an efficient target for antibody-mediated delivery of T cell epitopes in vaccine development.