<p>Included are additional material and methods, biochemical and functional data demonstrating target specificity and engagement, flow cytometry gating strategies, histograms, and analyses, and TME cytokine data.</p>
E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin–dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin–dependent adhesive interactions that direct homing of human HPC to BM.
Abstract Intratumoral (IT) delivery of plasmid IL-12 (tavokinogene telseplasmid; tavo) via electroporation (EP), collectively referred as IT-tavo-EP, generates immunologically-relevant levels of localized IL-12, triggering regression of both treated and distant tumors with minimal toxicity in preclinical and clinical studies. Our previous clinical trial data from melanoma patients treated with IT-tavo-EP identified a treatment-related increase of infiltrating T cells and transcripts related to immune activation, as well as a significant increase in the IFN-γ score of patients with a clinical benefit, suggesting that CD3+ tumor-infiltrating lymphocytes (TIL) may be critical in maximizing the anti-tumor effects of IT-tavo-EP. Furthermore, in-house biomarker data have identified an abundance of non-tumor reactive TIL that, if mobilized could additionally contribute to a clinical response. Accordingly, a plasmid-encoded membrane-bound polyclonal T cell-stimulating anti-CD3 (αCD3) hybrid antibody (scFv) was developed and used in combination with tavo (IT-tavo-αCD3-EP) to broaden the scope and depth of the T cell response. We previously demonstrated that membrane expression of αCD3 on neoplastic and stromal cells could activate CD3+ TIL, driving enhanced proliferation and cytotoxicity in a B16-OVA murine model. Here, using immune profiling of the tumor microenvironment (TME), we have demonstrated that this membrane-bound αCD3 therapeutic can significantly upregulate frequencies of CXCR3+CD8+ T cells and short-lived effector T cells, while reducing PD-1 expression on CD8+ T cells in vivo. Critically, naïve T cells, Treg cells, and exhausted T cells (subsets not typically associated with strong anti-tumor responses) displayed enhanced effector function (IFN-γ and granzyme B release) with engagement of membrane-bound αCD3 and IL-12. Furthermore, we found that this therapeutic approach could equally enhance proliferation of T cells regardless of the affinity for their cognate peptide:MHC, suggesting a TCR independent mechanism. Collectively, these observations demonstrate that IT-tavo-αCD3-EP can mobilize broad subsets of T cells beyond dominant anti-tumor effectors demonstrated. Thus, while enhanced cytolytic function is associated with this therapy, inclusion of additional atypical anti-tumor T cell subsets may also promote reshaping of the TME by production of effector cytokines upon engagement of surface-bound αCD3. Moreover, functional restoration of TIL isolated from a melanoma patient with active clinical progression on anti-PD-1 therapy, was possible with engagement of membrane-bound αCD3 in the presence of IL-12. Collectively, these data continue to support the utility of IT-tavo-αCD3-EP as a promising therapeutic approach for patients with melanoma and other accessible solid tumors. Citation Format: Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Erica Browning, Jon Salazar, Reneta Hermiz, Lauren Svenson, Chris Baker, Daniel O'Connor, Kellie Malloy, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of plasmid-encoded IL-12 and membrane-bound anti-CD3 increases tumor immunogenicity and augments the function of T cell subsets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-390.
<div>Abstract<p>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<sup>+</sup> 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.</p>Significance:<p>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.</p></div>
<div>Abstract<p>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<sup>+</sup> 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.</p>Significance:<p>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.</p></div>
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)