Cell-based Immunotherapy

Cell-based immunotherapy uses a cell type from the immune system as therapeutic agent. For this purpose, the cells are first removed from the body, then activated or modified, expanded and finally re-infused into the patient. 

These are some of the strategies currently being investigated:

• Adoptive T cell therapy focuses on increasing tumor specific T cells: Various sources and types of T cells have been used for adoptive therapy including tumor-infiltrating lymphocytes (TILs) expanded ex vivo or peripheral blood T cells genetically engineered ex vivo to either express a cancer-specific T cell receptor (TCR) or a chimeric antigen receptor (CAR; CAR-T cells) against a cancer epitope.
   
• Natural killer (NK) cell therapy aims to activate innate immune responses. Read more 
   
• Dendritic cell (DC) strategies use these antigen-presenting cells (APCs) to activate the immune system. To increase presentation of a tumor-specific antigen, DCs are pulsed ex vivo with tumor lysate or a specific cancer antigen, or genetically engineered to present the antigen. After re-infusion into the patient, the APCs activate the adaptive immune response via T lymphocytes.

While most cell-based approaches are still in the experimental phase compared to other cancer immunotherapy strategies, the first two adoptive T cell therapies have already been approved for the US, and partially approved for the EU market. Both products, Kymriah^® from Novartis and Yescarta^® from Gilead, are CAR-T cells targeting CD19 B-cell lymphomas.

In order to overcome immunosuppressive effects of cancer cells, researchers have genetically modified T cells ex vivo to re-target them towards cancer cells. Such re-targeting can be achieved by generating a T cell that expresses a cancer antigen specific T cell receptor (TCR) or a chimeric antigen receptor (CAR). CARs combine the signaling pathway of a TCR with the specificity and the binding properties of a monoclonal antibody. This enables the resulting T cells, the CAR-T cells, to recognize tumor antigens in their native conformation, independent of their presentation via the major histocompatibility complex (MHC) which might be mutated on cancer cells. This provides an advantage over TCR, because antigen recognition by TCR relies on MHC presentation of the cancer antigen.  Furthermore, the use of CAR expands the range of potential targets, as a CAR can bind not only to proteins but also to carbohydrate, lipids and heavily glycosylated proteins.

Because of these features, CAR-T cells are representing a promising addition to the cancer immunotherapy toolbox for the treatment of hematopoietic malignancies as well as solid tumors. 

For CAR-T cell therapies, T cells are isolated from a patient and then genetically modified ex vivo to express a CAR specific to an antigen of the patient’s cancer cells. Subsequent to characterization and expansion, the engineered CAR-T cells are re-infused into the patient to specifically target and destroy the tumor cells.

Researchers follow two different approaches when developing CAR-T cell immunotherapies. Most current strategies are autologous. For this patient-specific therapy, T cells are isolated from an individual patient, genetically engineered by introducing the CAR, expanded and then re-infused into the donating patient. With an allogeneic approach, T cells are isolated from a healthy donor, genetically engineered, expanded and then used to treat many patients. However, in this case the engineering process requires two steps: the introduction of the CAR and the knock-out of the TCR to minimize graft-versus-host disease. Researchers are also looking into deriving allogeneic T cells from induced pluripotent stem cells.

In the past years, research on CAR-T cells for cancer immunotherapy has gained a significant focus and resulted in over 100 ongoing clinical trials (Hay & Turtle 2017). CAR-T cells targeting the most investigated antigen CD19 which is up-regulated in many hematological malignancies, have experienced much clinical success, leading to approval of the first two CAR-T cell therapies (Kymriah^® from Novartis and Yescarta^® from Gilead).

The manufacturing of CAR-T cells involves an ex vivo modification step to transfer the CAR expressing transgene into T cells isolated from a patient. Such ex vivo delivery of the CAR transgene into human T cells and its insertion into the T cell genome can either be achieved by viral transduction or by non-viral transfection of plasmids in combination with genome editing tools. 

Viruses, like gamma-retrovirus, lentivirus, adeno or adeno-associated virus, are widely used because of their high transduction/insertion efficiency. However, depending on the virus type, there might be some limitations with regard to safety concerns (immunogenicity, insertional mutagenesis), production costs and lead times, and DNA insert size.

Alternatively, non-viral transfection methods are under exploration for transferring the CAR to overcome some of the limitations affiliated to viruses. Non-viral methods are supposed to be more cost- and time-effective and offer more flexibility in terms of insert size and with regards to the cargo type used. When aiming for stable insertion into the T cell genome, a CAR plasmid is typically transfected together with a transposition system (e.g. Sleeping Beauty or piggyBac^TM transposon/transposase).* Furthermore, CAR mRNA can be used if only a transient CAR expression and more dosage control is desired.

Primary T cells are considered being hard-to-transfect by non-viral methods, leading to low expression of the CAR gene, especially due to the need for co-transfection when using a transposition system. However, advanced electroporation technologies, like the Nucleofector^TM Technology, allow for efficient transfection of pre-stimulated, or even resting or human T cells and have been proven for more complex transfection scenarios where multiple or even different substrate types need to be co-delivered: for example, for iPSC generation, genome editing with CRISPR, ZFN or TALENs. 

Successful generation of CAR T cells has been shown for Nucleofection in combination with Sleeping Beauty or piggyBac^TM transposon-transposase systems (Kebriaei et al. 2016; Magnani et al. 2016; Saito et al. 2014; Singh et al. 2015).* As large amounts of DNA can be toxic for T cells, the use of minimalistic DNA vectors encoding transposon and transposase, so called mini-circles, might be a promising alternative. For the Sleeping Beauty system, researchers demonstrated that transfection of mini-circle CAR transposons with mini-circle- or mRNA-based transposase provided significantly higher transfection efficiency and less toxicity compared to a plasmid-based approach (Monjezi et al. 2017), while keeping functional effects comparable to virus. Such DNA toxicity as well as uncontrolled integration into the genome can also be overcome by transfecting CAR mRNA. Furthermore, using transiently expressed CAR can temporally limit the CAR-T activity and thus reduce off-tissue toxicity to normal tissue (Caruso et al. 2016). 

More recently, CRISPR/Cas9 has been used for targeted insertion of the CAR sequence into the T cell receptor (TCR) locus. This allowed for endogenous control of CAR expression with parallel knockout of the TCR (Eyquem et al. 2017). CRISPR/Cas9 has also been used to knockout the inhibitory checkpoint PD-1 receptor in T cells to potentially improve the efficiency of CAR-T cell-based therapeutics (Su et al. 2016).

*piggyBac is a registered trademark of Poseida Therapeutics. Sleeping Beauty is a registered trademark of Regents of the University of Minnesota.

References

Caruso et al. (2016) J Immunother 39(5):205-217
Hay & Turtle (2017) Drugs 77(3):237–245
Kebriaei et al. (2016) J Clin Invest 126(9):3363-3376
Magnani et al. (2016) Oncotarget 7(32):51581-51597
Monjezi et al. (2017) Leukemia 31(1):186-194
Saito et al. (2014) Cytotherapy 16(9):1257-1269
Singh et al. (2015) Cancer Gene Ther 22(2):95-100

Antibody therapy is currently the most established treatment that has been approved and successfully used in immunotherapy for a wide range of cancers. In this case, a recombinant monoclonal antibody is designed to bind a certain cancer antigen, often a membrane protein or receptor. Upon binding, it induces apoptosis, activates the complement system or prevents interaction of a receptor with its natural ligand.

More than a dozen antibodies have been approved for cancer treatment and many clinical trials are ongoing.

So called “immune checkpoints” are typically aimed as for self-control of the immune system. These “brakes” play a key role in regulating and maintaining the balance between T cell activation and immune tolerance. However, certain cancer types express immunosuppressive molecules on their cell surface, such as PD-L1, which is common for several human solid tumors including melanoma, lung, and ovarian tumors. PD-L1 can initiate that “brake” by acting as a ligand for T cell-specific PD-1 receptor. Upon binding of that ligand to the receptor, it prevents activation of the T cell. This undesirable suppression of the T cell by the cancer cell can be blocked by immune checkpoint inhibitors, helping to release the “brake”. For example, the PD-L1/PD-1 interaction can be inhibited by antibodies, that either bind and thus block the PD-L1 ligand of the tumor cell (anti PD-L1) or the PD-1 receptor of the T cell (anti PD-1).

Several immune checkpoint inhibitors have been approved for clinical use.

Cancer vaccines can be administered prophylactically against viruses that may cause cancer (for example HPV or HBV). Therapeutic cancer vaccines aim to treat already existing tumors by boosting the patient’s immune system. Vaccine types can be manifold comprising proteins/peptides, cell-based vaccines or genetic vaccines. A comprehensive overview is provided by Guo et al. (2013) Adv Cancer Res 119: 421–475.