Immunotherapy for cancer treatment
State of the art in immunotherapy and T-cell adoptive transfer
Adoptive cell therapy (ACT) is a term used to describe the transfer of immune cells to patients for the treatment of cancer or infectious disease (ref1). With this approach, T cells can be modified to recognize tumors and elicit therapeutic effects upon transfer into the patient.
In early approaches, tumor infiltrating lymphocytes (TILs) were isolated from tumor specimens, expanded and adoptively transferred back into the patient (ref 2,3). To bypass the technical difficulties and labor intensity of TIL isolation, gene transfer–based strategies introducing antitumor receptors directed to specific tumor antigens (TA) into normal T cells have been developed (ref 1). There are two main types of antigen receptors used in genetic redirection (Figure 1). The first utilizes the native alpha and beta chains of a T Cell Receptor (TCR) specific for a tumor antigen. The second is termed a Chimeric Antigen Receptor (CAR), which is composed of a binding domain (i.e. specific antibody or receptor that binds a tumor antigen), linked to an intracellular signaling domain. Genes encoding these receptors are inserted into patients’ T cells, using viral or non-viral methods, to generate tumor-reactive T cells which will be infused to patients.
Figure 1: TCR and CAR-T cell therapies
Standard CARs are fusion genes comprised of a single-chain variable fragment (scFv) antibody recognizing the tumor antigen of interest, linked to intracellular signaling modules that mediate T-cell activation upon ligation of the CAR’s extracellular domain.
One of the major advantages versus the TCR-therapy is therefore that CARs recognize surface tumor antigens in a major histocompatibility complex (MHC)–independent fashion (no patient MHC-restriction) and would allow CAR-T cells to attack tumor cells in the immunosuppressive tumor microenvironment, where MHC-downregulation is one of the main immune evasion mechanism.
Progressively, CAR-T cells have become increasingly sophisticated and several generations of CARs have been developed. First generation CARs typically consist of an extracellular scFv of a monoclonal antibody, linked via a hinge and transmembrane region to an intracellular immunoreceptor tyrosine-based activation motif (ITAM) such as the CD3ζ-chain or less commonly the FcεRIγ. These CARs deliver the “primary activation" signal resulting in T-cell activation, target cell lysis and IL-2 secretion. However, when a co-stimulatory signal is lacking, this may result in T-cell anergy under physiological conditions. Therefore, second and third generation CARs have been designed to include intracellular costimulatory domains such as CD28, ICOS, 4-1BB (CD137) or OX40 either individually (2nd generation) or in combination (3rd generation), to mimic physiologic T-cell activation (Figure 2). CD28 was shown to confer greater effector memory functionality and 4-1BB CARs a greater persistence and enhanced survival to T cells (ref 4,5) but similar improvements in clinical outcomes were obtained in ALL patients with either costimulatory domains (ref 6-8). The optimal combination of costimulatory signals within a CAR is therefore still subject to debate as it depends on the clinical scenario, use of different scFvs, methods of transduction, and culture conditions
Figure 2: Classical CAR-T construct
Classical CAR-T therapeutic approach
The current CAR-T cell therapy is based on a "transplant procedure-like" approach, i.e., a single CAR-T cells infusion in lymphodepleted patient and for which persistence, expansion and survival of the transferred T cells is considered important (ref 3,10). This is usually achieved by administrating a specific conditioning chemotherapy regimen to patients prior to cell transfer and the use of specific ex-vivo stimulation and expansion procedure that promote the selection of a “T-memory stem cell” phenotype for the CAR-T infused (ref 11)
The proposed benefits of depleting immune cells before T cell transfer include
(i) in vivo expansion and long-term survival of transferred T cells through decreased competition for antigen-presenting cells and homeostatic cytokines,
(ii) expansion of immature dendritic cells that can present tumor antigens,
(iii) decrease in the number of immunosuppressive cells, such as myeloid-derived suppressor cells and regulatory T cells, which decrease the antitumor responses of transferred T cells.
The conditioning regimen administered prior to adoptive cell transfer must be sufficiently immunosuppressive to ensure engraftment and contributes to the anti-tumor impact of the procedure but might also enhance toxicity. A broad spectrum of regimens has been studied, varying in their intensity, whether high-dose or reduced intensity, and in the agents used.
Only a few preclinical studies targeting hematologic cancers with CAR-T cells were developed on immunocompetent, syngeneic animal models but they allowed to determine that for effective CD19 CAR-T cell function some form of lymphodepletion was required, presumably in part to decrease the antigen burden or possibly to deplete immunosuppressive regulatory T cells. In contrast, much more preclinical studies targeting solid cancers were performed without any lymphodepletion. It appears that CAR-T cells targeting tumor vasculature or affecting tumor microenvironment, eventually through co-transduction of a vector expressing IL-12, were the more effective treatments against solid tumors without requirement for lymphodepletion prior transfer.
The importance of lymphodepletion for clinical outcome was not clearly demonstrated, even for hematologic cancers, although it improves persistence of CAR T-Cells after infusion (ref 12), while acute toxicity was clearly associated to lymphodepletive conditioning (ref 13,14) but the cell dose injected, as well as type of CAR construct and combination with IL-2 therapy likely also increased this toxicity.
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