Following are public and technical abstracts for the Personalized Cellular Immunotherapy project funded by the Department of Defense Kidney Cancer Research Program (KCRP) for 2017.
Principal Investigator: Haviz Ahmed
Institution: GlycoMantra
Funding Mechanism: Concept Award
Award Amount: $103,013
Public Abstract
There is no efficient therapy for aggressive kidney cancer. About 30% kidney cancer patients do not respond to the most commonly used drug, sunitinib. The remaining 70% of kidney cancer patients respond initially to common drugs but develop resistance to those drugs in 6 to 15 months and die. The inefficiency of these therapies is largely due to the fact that the tumor is heterogeneous in nature; also, the level of tumor antigens expressed in the different stages of cancer varies. Therefore, innovative strategies for the treatment of kidney cancer are urgently needed.
Recently, chimeric antigen receptor (CAR) T cell immunotherapy has shown improved antitumor efficacy in some forms of blood cancers. However, the therapeutic potential of CAR T cells in solid tumors, such as kidney cancer, is very limited due to their functional heterogeneity and associated toxicity. In this respect, natural killer T (NKT) cells are more efficient than T cells in cytotoxicity and trafficking to solid tumors.
A novel concept of personalized cellular immunotherapy for kidney cancer is proposed here. The idea is to screen biopsy specimens first from individual patients for a panel of known overexpressed tumor antigens and then label two or three of the most abundant antigens with a tag. Once the tumor is marked with the tag, immunotherapy with CAR-NKT cells targeting the tag begins. The engineered NKT cells will recognize and specifically kill kidney cancer cells. Thus, the proposed CAR-NKT is a universal cellular therapy for individualized kidney cancer.
The use of CAR-NKT as proposed here for the treatment of kidney is highly innovative in many ways. First, the proposed CAR-NKT cell immunotherapy is the first-ever tool that weaponizes NKT cells to targeted killing of any type of kidney cancer (personalized). The CAR-NKT will also target galectin in the tumor microenvironment promoting the patient’s antitumor immune response. Particularly relevant to the US military and Veterans, expression of galectin is increased in the presence of polycyclic aromatic hydrocarbons, common hazardous chemicals associated with war. So, the proposed CAR-NKT is a universal cellular therapy designed to specifically recognize and kill any type of kidney cancer regardless of tumor heterogeneity (personalized based on the abundance of tumor antigens) not only from the civilian population, but also from Veterans and military personnel.
Technical Abstract
Background and Rationale: There is no efficient therapy for metastatic renal cell carcinoma (mRCC). Thirty percent (30%) of mRCC patients do not respond to the most commonly used drug, sunitinib, and the rest, who respond initially, develop drug resistance in 6 to 15 months. Therefore, alternative strategies for the treatment of mRCC are urgently needed. Recently, the engineered expression of chimeric antigen receptors (CARs) on the surface of T cells has shown improved antitumor efficacy in some forms of blood cancers. However, therapeutic potential of CAR T cells in solid tumors is very limited due to their functional heterogeneity and associated toxicity. In this respect, natural killer T (NKT) cells are more efficient than CD8+T cells in cytotoxicity, production of desired cytokines, and trafficking to solid tumors. Moreover, NKT cells’ toxicity is less in the autologous or allogeneic settings as NKT cognate receptor signaling gene; CD1d is monomorphic and expressed by only a few cell types.
A novel concept of personalized cellular immunotherapy for kidney cancer is proposed here. The idea is to screen a biopsy specimen first from an individual patient for a panel of known inducive tumor antigens and then treat the patient with FITC conjugated antibodies against two or three of the most abundant antigens obtained from the screen. Once the tumor is marked with antibody-FITC, cellular therapy with CAR+-NKT cell (CAR-NKT) expressing FITC scFv begins. Thus, the engineered NKT cells will recognize and specifically kill kidney cancer cells. The FITC-targeting CAR is equipped with CD28, 4-1BB, and CD3-zeta for cell activation, IL-12 for immune regulation, and an inhibitory peptide for targeting galectin involved in the progression of kidney cancer. Thus, the proposed CAR-NKT is a universal adaptive cellular immunotherapy for individualized kidney cancer.
Objectives: The efficacy of this novel concept will be demonstrated in vitro using carbonic anhydrase IX (CAIX) expressing CD1d- mRCC cells (Caki-1) as an example after treating with anti-CAIX mAb-FITC conjugated antibodies. Specific objectives of this concept proposal are as follows: (1) Phenotypic characterization and cytokine-switching of CAR+ NKT cells. (2) Evaluate dual cytotoxicity of CAR-NKT against anti-CAIX mAb-FITC treated Caki-1 cells and M2 macrophages. (3) Evaluate the efficacy of the inhibitory peptide of galectin on angiogenesis and tumor-endothelial cell interactions.
Study Design: Lentiviral CAR_FITC construct and various control constructs and their virus packaging will be made. For NKT cell isolation, stimulation, expansion, transduction, and re-expansion, NKTs from PBMC (buffy coat) will be purified by anti-iNKT microbeads. Isolated NKTs will be expanded after stimulation with irradiated autologous negative PBMC, recombinant IL-2, and alpha-galactosylceramide (alphaGalCer). Phenotypic characterization of expanded NKT cells will be determined using relevant mAbs. For CAR-NKT mediated cytokine profiling, Th1-Th2 polarization including IL-12 will be measured using a Human Cytokine/Chemokine Immunoassay kit in a co-culture of parental and engineered NKTs stimulated with irradiated, anti-CAIX mAb-FITC treated Caki-1 cells. The cytotoxicity of parental and CAR-NKTs against antibody-pulsed Caki-1 cells and CD1d+ M2 macrophages (isolated from PBMC) will be evaluated using a standard 4-hour 51Cr-release assay. To evaluate the efficacy of the inhibitory peptide of galectin, secretory products from transduced NKTs will be applied on angiogenesis and tumor-endothelial cell assays using calcein-labeled Caki-1 cells on HUVEC.
Innovation: The use of CAR-NKT as proposed here for the treatment of mRCC is highly innovative in many ways. First, the proposed CAR-NKT construct for immunotherapy is the first-ever multi-pronged construct that weaponizes NKT cells to targeted killing of any type of kidney cancer (personalized). Second, the use of an inhibitory peptide of galectin in the CAR construct to block tumor progression is utterly original. The CAR-NKT is expected to be Th1-biased. Third, the use of NKT cells in kidney cancer is the first. Fourth, scFv FITC has never been used on NKT cells for kidney or any other cancer. Fifth, the proposed CAR-NKT along with the antigen-probed targeted cells in the tumor microenvironment will also kill CD1d signaling tumor-associated pro-tumorigenic M2 macrophage (TAM-M2) in kidney cancer.
Impact: There is no effective therapy for mRCC. As the tumor microenvironment (TME) of RCC is heterogeneous, monotherapy most likely will not work. So, therapy based on the presence of induced signature tumor antigens in a patient may be the ideal solution. The proposed project aims to develop a personalized cellular immunotherapy for kidney cancer. Tumor antigens will be identified first from the biopsy sample and the patient will then be treated with FITC conjugated antibodies against a few of the most abundant antigens found from the biopsy screen. Once the tumor is marked with antibody-FITC, cellular therapy with CAR-NKT expressing FITC scFv will be initiated. Thus, the proposed CAR-NKT is a universal cellular therapy designed to specifically recognize and kill any type of kidney cancer regardless of intratumor heterogeneity (personalized based on the abundance of tumor antigens). Moreover, the proposed CAR-NKT is functionalized with an inhibitory peptide of galectin that acts not only against tumor cells, but also in the TME, resulting in tumor suppression.