Surgical Innovation &
Research

Novel CAR T Cell/NK Cell Based Therapies for Pancreatic and Ovarian Cancers

Pancreatic and ovarian cancers are relatively resistant to chemotherapy and typically diagnosed at an advanced stage, thereby precluding surgical resection and underscoring the need for novel therapies. One promising approach is the utilization of chimeric antigen receptor (CAR) T cell-based technology, which has ushered in a new era in cancer immunotherapy especially in hematological malignancies. CARs are genetically engineered simulated receptors expressed on T cells containing an extracellular single chain fragment variable (scFv) antibody fragment linked to intracellular T cell activation domain. 

Often known as a “living drug,” CAR T cells directly recognize tumor-specific antigen via scFv and kill tumor cells. Since this process circumvents the need for dendritic cell mediation in mounting immune responses, which often suffers from tumor-induced inhibition, it has revolutionized cancer immunotherapy. In other words, the extraordinary success of this therapy has been attributed to its ability to bypass tumor-imposed immune-inhibitory mechanisms. 

We have previously demonstrated in vitro killing of BxPC-3 pancreatic cancer cell-line and SKOV-3 ovarian cancer cell-line using autologous CAR T cells targeting tumor-specific antigen, mesothelin, generated via lentiviral transduction using conventional DNA. However, it has several important limitations as follows: 

1. Autologous T cell preparation is patient-specific, time consuming and labor intensive. 
2. Lentiviral vectors preferentially integrate into transcriptionally active chromosomal sites thus increasing the chances of insertional mutagenesis. 
3. Use of conventional DNA vector electroporation to avoid lentiviral vectors expresses unwanted antibiotic resistance genes and further triggers inflammatory responses due to the presence of bacterial unmethylated CpG. 
4. CAR T cell activity in pancreatic and ovarian cancers in clinical settings, in vivo, is inhibited by immunosuppressive tumor milieu. 

We developed two CAR related projects to address the above limitations. They are described below.

Project 1: “Off-the-shelf” Cellular Immuno-Therapy for Pancreatic and Ovarian Cancers

We have developed CAR therapy using bone marrow-derived primary natural killer (NK) cells as an alternative to autologous T cells which can be maintained as a continuously expandable off-the-shelf allogeneic therapy. We used sleeping beauty (SB) transposons, mobile genetic elements that are an alternative system for lentiviral transduction. SB transposons contain mesothelin scFv flanked by inverted terminal repeats enabling chromosomal integration. SB transposon chromosomal integration is without any preference for active transcriptional units, thus decreasing the risks of insertional mutagenesis. We further introduced novel minicircle (MC) plasmid technology in place of conventional DNA plasmids. MCs are devoid of redundant bacterial elements such as antibiotic resistant genes and unmethylated CpG. Being much smaller in size they are more efficient in entering cells. 

Thus, using modified natural killer cell-line (NK-92) to electroporate SB/MC mesothelin-CAR plasmid vector, we developed an allogeneic off-the-shelf therapy overcoming the limitations associated with lentiviral transduction. We demonstrated efficient killing of both pancreatic and ovarian cancer cells in vitro with an enhanced mesothelin-CAR engraftment. This has the potential to the improve the clinical safety profile of CAR T cell therapy by eliminating both viral and bacterial concerns while simultaneously increasing engraftment and cancer cell cytotoxicity.

Project 2: Blocking Inhibitory Cytokines in the Tumor Microenvironment of Pancreatic and Ovarian Cancers Potentiates Mesothelin-Chimeric Antigen Receptor NK-92MI Activity

We have previously demonstrated that mesothelin-CAR T cells kill both pancreatic and ovarian cancer cells in vitro. However, the efficacy of CAR T cell therapy for pancreatic and ovarian solid tumor treatment in vivo has not yet been successful. Solid tumors create an immune suppressive milieu in their tumor microenvironment (TME) consisting of inhibitory cytokines such as IL-10 and TGF-β secreted by regulator T cells (Tregs). IL-2 is known to protect the functional ability of T cells to overcome the negative effects of the TME. The NK-92MI cell line, which is derived from natural killer (NK) cells but genetically modified to secrete IL-2, can be utilized as part of a CAR NK cell treatment strategy to localizing IL-2 intratumorally. 

The pivotal challenge for the CAR T cell therapy against solid tumors such as pancreatic and ovarian cancers is the immunosuppressive TME that interferes with T cell activity. We posit that the blocking of IL-10 and TGF-β while producing immune-protective cytokines such as IL-2, may result in more effective CAR T and CAR NK cell therapy.

Using pancreatic (BKPC-3) and ovarian (SKOV-3) tumor-conditioned medium and patient-derived malignant ascites simulating the in vivo conditions, we can assess the effect of the TME and its reversal via depletion of IL10/TGF-β on mesothelin-CAR-NK cell cytotoxic  activity. Our approach has led to the establishment of an ex vivo system for testing various vaccine candidates against pancreatic and ovarian cancers. 

We have demonstrated a significant reversal of TME-mediated inhibition of mesothelin CAR-NK cell activity by antibody/siRNA mediated depleting IL10/TGF-β. This has been achieved with mesothelin-CAR T cells in autologous setting and more efficiently with an allogeneic off-the-shelf IL-2 secreting mesothelin-CAR-NK-92MI. This has potential for more effective clinical translation of mesothelin-engrafted CAR T cells or NK cells. 

We are in the process of deciphering various signaling pathways; if successful, our results will facilitate the development of therapeutic modalities that are non-overlapping and potentially synergistic with conventional treatment.