Raedun Clarke

Mouse models of non-Hodgkin lymphoma reveal Syk as an important therapeutic target

We have generated mouse models of non-Hodgkin lymphoma (NHL) that rely on the cooperation between MYC overexpression and B-cell antigen receptor (BCR) signaling for the initiation and maintenance of B-cell lymphomas. Using these mouse models of NHL, we have focused on the identification of BCR-derived signal effectors that are important for the maintenance of NHL tumors. In the present study, we concentrate on Spleen tyrosine kinase (Syk), a nonreceptor tyrosine kinase required to transduce BCR-dependent signals. Using a genetic approach, we showed that Syk expression is required for the survival of murine NHL-like tumors in vitro and that tumor cells deficient in Syk fail to expand in vivo. In addition, a pharmacologic inhibitor of Syk was able to induce apoptosis of transformed B cells in vitro and led to tumor regression in vivo. Finally, we show that genetic or pharmacologic inhibition of Syk activity in human NHL cell lines are generally consistent with results found in the mouse models, suggesting that targeting Syk may be a viable therapeutic strategy.

A new function for LAT and CD8 during CD8-mediated apoptosis that is independent of TCR signal transduction.

The majority (>95%) of thymocytes undergo apoptosis during selection in the thymus. Several mechanisms have been proposed to explain how apoptosis of thymocytes that are not positively selected occurs; however, it is unknown whether thymocytes die purely by “neglect” or whether signaling through a cell‐surface receptor initiates an apoptotic pathway. We have previously demonstrated that on double positive thymocytes the ligation of CD8 in the absence of TCR engagement results in apoptosis and have postulated this is a mechanism to remove thymocytes that have failed positive selection. On mature single positive T cells CD8 acts as a co‐receptor to augment signaling through the TCR that is dependent on the phosphorylation of the adaptor protein, linker for activation of T cells (LAT). Here, we show that during CD8‐mediated apoptosis of double positive thymocytes there is an increase in the association of CD8 with LAT and an increase in LAT tyrosine phosphorylation. Decreasing LAT expression and mutation of tyrosine residues of LAT reduced apoptosis upon crosslinking of CD8. Our results identify novel functions for both CD8 and LAT that are independent of TCR signal transduction and suggest a mechanism for signal transduction leading to apoptosis upon CD8 crosslinking.

Abstract 3576: FT500, an off-the-shelf NK cell cancer immunotherapy derived from a master pluripotent cell line, enhances T-cell activation and recruitment to overcome checkpoint blockade resistance

The development of PD1/PDL1 targeting checkpoint inhibitors (CI) has transformed the oncology landscape, providing long term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. Novel therapeutic approaches with the ability to overcome CI resistance are needed, and there is significant opportunity for therapies capable of additively or synergistically enhancing T-cell activation and recruitment when combined with CI. Adoptive transfer of NK cells from healthy donors has the potential to recruit T cells to the tumor microenvironment and augment T-cell activation at the tumor site. NK cells have both direct anti-tumor activity and the capacity to secrete inflammatory cytokines and chemokines upon activation, enabling the cells to play a unique and critical role in regulating anti-tumor T cell activity. We sought to determine whether FT500, an off-the-shelf NK cell product derived from a clonal master pluripotent cell line, could synergize with CI to relieve local immunosuppression and enhance T-cell activation and recruitment to the tumor site. FT500 is universally negative for cell surface PD1, and expression of PDL1 on tumor lines had no discernable effect on FT500 cytotoxicity. Similarly, addition of PDL1 blocking antibody had no effect on FT500 cytotoxicity or degranulation, suggesting that FT500 is inherently resistant to PDL1-PD1 mediated inhibition. Additionally, activation of FT500 induced the secretion of soluble factors capable of enhancing T-cell activation, as evidenced by increased upregulation of CD69. We hypothesized that FT500 might also enhance CI by promoting recruitment of T cells to the tumor site. Using conventional in vitro transwell migration assays, we found that FT500 produced soluble factors that promoted the migration of activated T cells. Additional profiling confirmed FT500 production of a range of chemokines, including CCL3, CCL4, CXCL10 and CCL22. Furthermore, using an in vivo recruitment model, FT500 was able to recruit T cells out of the circulation and into the peritoneal cavity. Similarly, utilizing a three-dimensional tumor spheroid model in vitro, infiltration of T cells into tumor spheroids was significantly enhanced when combined with FT500, suggesting that FT500 can enhance tumor infiltration of T cells. Our data suggest that FT500 is a potent producer of chemokines and can facilitate the recruitment of T cells to the tumor site. In addition to its direct cytotoxic potential, FT500 is also able to enhance T-cell activation, suggesting an ability to synergize with CI to reduce tumor burden. Together, our data provide evidence supporting the combination of FT500, an off-the-shelf NK cell cancer immunotherapy, with CI to overcome checkpoint blockade resistance. Citation Format: Ryan Bjordahl, Sajid Mahmood, Svetlana Gaidarova, Ramzey Abujarour, Raedun Clarke, Laurel Stokely, Paul Rogers, Moyar Ge, Megan Robinson, Betsy Rezner, Tom Tong Lee, Bahram Valamehr. FT500, an off-the-shelf NK cell cancer immunotherapy derived from a master pluripotent cell line, enhances T-cell activation and recruitment to overcome checkpoint blockade resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3576.

Abstract 609: Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy

Using epitope scanning of 272 short, synthetic peptides representing the amino acid sequence of the CB-11 peptide of type II collagen, we have shown that five strains of rat, immunized with type II collagen, produce antibodies to a region 37-45 amino acids from the amino end of CB-11 peptide. Antibodies to this region always gave the highest binding values suggesting that it is an immunodominant region. Wistar rats immunized with a synthetic peptide representing this region, coupled to keyhole limpet haemocyanin, produced antibodies to this peptide which could still be detected at 1:4000 to 1:8000 dilution but none developed clinical arthritis. All sera also showed binding of antibodies to denatured bovine type II collagen but not to native type II collagen, keyhole limpet haemocyanin or to bovine serum albumin by ELISA. Sera from peptide-immunized rats were examined for antibody binding to the 272 short peptides of the CB-11 peptide and to the synthetic peptides representing shortened forms of the immunodominant region and forms of it with substituted amino acids. These results showed that the antibodies in the peptide-immunized rats were not identical to those produced to that peptide by rats immunized with type II collagen but may represent subpopulations of them. These findings suggest caution in interpreting the role of antibodies to individual peptides in arthritis induction without knowledge of their fine specificity.

Abstract 3755: Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy

The unique attributes of a combinatorial tumor recognition system, diminished off-tumor cytotoxicity, and multifaceted effector function make natural killer (NK) cells a prime candidate for a universal approach to cancer immunotherapy. In addition, NK cells are the principal mediator of antibody-directed cellular cytotoxicity (ADCC). However, NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system and the therapeutic efficacy of monoclonal antibodies. To address the need for advanced and combinatorial cancer therapies, we developed a unique and effective strategy to create a renewable source of engineered “off-the-shelf” NK cells with augmented function, including enhanced ADCC and persistence. Key challenges associated with genetic editing, limited expansion, persistence and variability of peripheral blood (PB)-derived NK cells were overcome by utilizing our induced pluripotent stem cell (iPSC) technology as the unlimited starting material for the reproducible and consistent derivation of engineered NK cells. Through targeted transgene integration, we produced a clonal iPSC master cell line to continuously produce NK cells engineered to uniformly express a high affinity, non-cleavable version of CD16 (hnCD16-NK). In directed differentiation, the hnCD16-NK cells displayed homogeneous expression of CD16 (>95%) and a mature CD56+ NK cell phenotype, as exhibited by expression of KIR, NCRs, DNAM-1, and NKG2D. In contrast to endogenous CD16 expression, the engineered hnCD16 molecule was shown to be cleavage resistant upon NK cell activation (>95% CD16+ hnCD16-NK vs. Citation Format: Ryan Bjordahl, Frank Cichocki, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, Greg Bonello, Tom Lee, Weijie Lan, Matthieu Bauer, Dave Robbins, Betsy Rezner, Sarah Cooley, Bruce Walcheck, Stewart Abbot, Bruce Blazar, Scott Wolchko, Daniel Shoemaker, Jeffrey S. Miller, Bahram Valamehr. Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2017-3755