Guenther Lametschwandtner

Phase I clinical trial of adoptive cellular immunotherapy with APN401 in patients with solid tumors

Meeting abstracts Casitas-B-lineage lymphoma protein-b (Cbl-b), an E3 ubiquitin ligase, has been identified as a key intracellular checkpoint limiting lymphocyte activation. Inhibiting Cbl-b has been shown to enhance T cell and natural killer cell mediated antitumor activity in mouse tumor models.

Development of a personalized cellular ex-vivo cbl-b silencing cancer immunotherapy

Background The E3 ubiquitin ligase cbl-b has been identified as an important gatekeeper limiting T cell activation. Concordantly, the immune system of cbl-b deficient mice can effectively fight tumors, thereby validating cbl-b as an excellent target to enhance anti-tumor immune activity. We have recently shown in proof-of-concept experiments that transfer of transiently cbl-b silenced murine T cells had efficacy to enhance the anti-tumor immune response in mouse models. Methods A design algorithm was used to screen for siRNAs that are highly effective to silence cbl-b, and the optimized siRNA was produced at a GMP manufacturer. PBMCs were isolated from healthy donors or cancer patients, transfected with siRNA by electroporation and immune cell phenotype and activation was determined by FACS and ELISA. For enhancement of DC vaccination responses, PBMCs were ex vivo silenced, and co-administrated with the DC preparations intranodally to the cancer patient. Results We have established a highly efficient transfection protocol using a commercial electroporation device enabling us to simultaneously transfect T, B, NK cells and monocytes with minimal cell damage. Using this protocol, we have identified a siRNA that was able to shut down cbl-b expression for more than 7 days in stimulated human T cells, resulting in strong enhancement of T cell activation, cytokine production and proliferation. Moreover, simultaneous silencing of cbl-b in all immune cells of the PBMCs yielded additional advantages, most notably enhancing NK cell reactivity against tumor cell and IL-2 stimulation. Silencing of cancer patient PBMCs yielded similar results ex vivo and intranodal transfer of autologous cbl-b silenced cells together with activated DCs to patients with advanced cancers was feasible and well tolerated. Conclusions To enable the clinical implementation of a cbl-b ex vivo silencing treatment, we have established and tested a protocol that can be easily performed on any clinical unit that applies adoptive cell therapies to patients. Based on these results, a Phase I trial for the systemic administration of cbl-b silenced PBMCs to patients with advanced cancers is presently being set up.

Cbl-b silenced human NK cells respond stronger to cytokine stimulation

The E3 ubiquitin ligase cbl-b has been identified as an important gatekeeper limiting T cell activation and more recently also NK cell activation. Hence, cbl-b deficient NK cells displayed stronger anti-tumor responses and this has been linked to a key role of cbl-b for modulation of TAM-receptors. However, in T cells cbl-b has been described to interact with different key regulators of T cell receptor and costimulatory signaling pathways. We have therefore investigated, whether abrogation of cbl-b function in human NK cells mediates increased responsiveness to cytokine stimulation. Primary human NK cells were isolated and cbl-b silenced by electroporation with siRNA directed against cbl-b. Cbl-b silenced NK cells reacted stronger to tumor cell contact and this response was synergistically enhanced by cytokine stimulation with IL-2 and IL-12. Moreover, stimulation of cbl-b silenced NK cells with cytokines in the absence of tumor cells, either with IL-2 and IL-12 or IL-2 alone led to enhanced activation of NK cells, resulting in increased secretion of effector cytokines like IFN-g and TNF-a and upregulation of the activation marker CD69. Similar results were obtained for stimulation of cbl-b silenced T cells with other key cytokines of innate immune responses, particularly type I Interferons like IFN-a and IFN-b. These data demonstrate that the proposed central role of cbl-b as a negative regulator of various signaling pathways, including Akt, also applies to NK cells. Together, these results show that interfering with cbl-b function in human NK cells enables synergistic responses to key cytokines of innate and adaptive immune responses. Thus, targeting cbl-b in the context of tumor-immune therapy should not be confined to the T cell compartment, but include NK cells as well. This can be achieved in the context of adoptive cell therapies, when autologous patient PBMCs are silenced for cbl-b ex vivo and retransferred afterwards. Such a protocol was established and is currently tested in a Phase I trial at Wake Forest University. The observed synergism of cbl-b silencing and NK cell stimulation by cytokines like IL-2 could be of particular relevance when tumor-reacting cbl-b silenced T cells infiltrate the tumor and secrete enhanced amounts of IL-2. In addition, it also provides a rationale for combinations of cbl-b targeting approaches with local application of cytokines at the tumor site (e.g. intratumoral injection of IL-2).

Treatment of a cancer patient by an adoptive cell therapy protocol combining DC vaccination with cbl-b ex vivo silencing

Meeting abstracts The E3 ubiquitin ligase cbl-b has been identified as a key intracellular checkpoint limiting T and NK cell activation. Concordantly, blockade of cbl-b function by genetic deletion strongly enhances anti-tumor immune responses, thereby validating cbl-b as target for immunotherapy.

Overcoming immune-cell unresponsiveness in cancer

Despite significant recent progress, cancer immunotherapy efficacies are still limited, since tumors can down-modulate and escape the immune system by various mechanisms. To overcome these limitations, we aimed to identify low molecular weight molecules that can enhance T cell reactivity in the context of otherwise insufficient or suppressive stimulation. In an HTS campaign, 80000 maximal diverse compounds were screened on anti-CD3/CD28 stimulated human PBMCs using enhancement of IL2 production as readout. The confirmed hit list contained 3 chemically independent series, which enhanced the production of inflammatory cytokines, especially IL-2, IFN-γ and TNF-α, from TCR-stimulated T cells, while lacking any agonist activity on unstimulated T cells. The compound-mediated enhancement of TCR-stimulation was also observed on the level of T cell activation markers like CD25, CD69, CD71 and CD40L. In line with these observations, compounds of these series enhanced the in vitro anti-tumor responsiveness of PBMCs against tumor cell lines, originating from melanoma (M21), neuroblastoma (LAN) and leukemia (K562). For MHC-positive tumor cells, refined analysis demonstrated that compounds enhanced CD8 T cell activation, resulting in selective tumor cell killing while compounds in the absence of immune cells did not affect tumor cell viability. In contrast, the PBMC-response against K562 cells, which is a commonly used NK cell target due to the lacking MHC-expression, was mainly mediated by NK cells. Moreover, compounds also enhanced IL-2 mediated NK cell proliferation and cytokine (IFN-γ, TNF-α) and effector molecule (Granzyme B, Perforine) secretion. The activities of these compounds were further confirmed in an antigen-specific ex vivo T cell stimulation assay of human PBMCs with Tetanus toxoid, which resulted in enhanced proliferation of T cells. In addition, enhanced IL-2 secretion by activated T cells led to consequently increased NK cell activation and proliferation in antigen-stimulated stimulated PBMCs. To enable future assessment of compound activities in murine tumor models in vivo, we have verified that compounds of all 3 series enhanced T cell activation and cytokine production of TCR-stimulated murine T cells, while they had no effect on unstimulated T cells. Together, we show here the identification of a novel class of low molecular weight compounds with drug-like properties that selectively enhance activation of T and NK cells in the context of antigen-specific and anti-tumor immunity with high potential for development of improved tumor immunotherapeutics.