Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of plasmid CXCL9 synergizes with plasmid IL-12 therapy to elicit robust anti-tumor immunity.

Clinical studies have demonstrated that local expression of the cytokine IL-12 drives interferon-gamma expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our late-stage melanoma trials identified a significant on-treatment increase of intratumoral CXCR3 transcripts that was restricted to responding patients, underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. In this study, we sought to understand if the addition of DNA-encodable CXCL9 could augment the anti-tumor immune responses driven by intratumoral IL-12. We show that localized IL-12 and CXCL9 treatment reshapes the tumor microenvironment to promote dendritic cell licensing and CD8+ T cell activation. Additionally, this combination treatment results in a significant abscopal anti-tumor response and provides a concomitant benefit to anti-PD-1 therapies. Collectively, these data demonstrate that a functional tumoral CXCR3/CXCL9 axis is critical for IL-12 anti-tumor efficacy. Furthermore, restoring or amplifying the CXCL9 gradient in the tumors via intratumoral electroporation of plasmid CXCL9 can not only result in efficient trafficking of cytotoxic CD8+ T cells into the tumor but can also reshape the microenvironment to promote systemic immune response.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

Intratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells. To encourage a more favorable on-treatment tumor microenvironment (TME), we explored combining this IL12 therapy with an intratumoral polyclonal T-cell stimulator membrane-anchored anti-CD3 to productively engage a diverse subset of lymphocytes including the nonreactive and suppressive T cells. This study highlighted that combined intratumoral electroporation of IL12 and membrane-anchored anti-CD3 plasmids can enhance cytokine production, T-cell cytotoxicity, and proliferation while limiting the suppressive capacity within the TME. These collective antitumor effects not only improve regression of treated tumors but drive systemic immunity with control of nontreated contralateral tumors in vivo. Moreover, combination of IL12 and anti-CD3 restored the function of TIL isolated from a patient with melanoma actively progressing on programmed cell death protein 1 (PD-1) checkpoint inhibitor therapy. IMPLICATIONS: This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic.

Immuno-oncological Efficacy of RXDX-106, a Novel TAM (TYRO3, AXL, MER) Family Small-Molecule Kinase Inhibitor.

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106-mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types. SIGNIFICANCE: The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.

The mechanisms of up-regulation of dendritic cell activity by oxidative stress.

Whereas DC have increasingly been recognized for their role in activating the inflammatory cascades during IRIs, the mechanisms by which oxidative stress enhances DC activation remain to be explored. We examined the role of oxidative stress on two important features of DC: T cell activation and trafficking. Bone marrow-derived OS-DC were compared with untreated DC. DC exposed to oxidative stress augmented allogeneic T cell proliferation and showed increased migration in a chemotaxis chamber. These results were confirmed by using hypoxanthine and xanthine oxidase as another inducer of oxidative stress. We used OT-II and OT-I mice to assess the effect of oxidative stress on DC activation of OVA-specific CD4(+) and CD8(+) T cells, respectively. Oxidative stress increased DC capacity to promote OVA-specific CD4(+) T cell activity, demonstrated by an increase in their proliferation and production of IFN-γ, IL-6, and IL-2 proinflammatory cytokines. Whereas oxidative stress increased the DC ability to stimulate IFN-γ production by OVA-specific CD8(+) T cells, cellular proliferation and cytotoxicity were not affected. Compared with untreated DC, oxidative stress significantly reduced the capacity of DC to generate T(regs), which were restored by using anti-IL-6. With regard to DC trafficking, whereas oxidative stress increased DC expression of p-Akt and p-NF-κB, targeting PI3Kγ and NF-κB pathways abrogated the observed increase in DC migration. Our data propose novel insights on the activation of DC by oxidative stress and provide rationales for targeted therapies, which can potentially attenuate IRI.

Dynamic alterations in chemokine gradients induce transendothelial shuttling of human T cells under physiologic shear conditions.

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies, yet the molecular effectors of this process are relatively uncharacterized. For extravasation, studies based predominantly on static transwell assays support a general model, whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However, this model of chemotaxis cannot readily reconcile how chemokines influence intravasation, as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus, to analyze how T cells integrate perivascular chemokine signals under physiologic flow, we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment, transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However, when exposedto abluminal CXCL12, transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent, augmented by apical flow, blocked by antibodies to integrins, and reduced by AMD3100 in a dose-dependent manner. Moreover, CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream.