Julian Kim

Development of Y-shaped peptide for constructing nanoparticle systems targeting tumor-associated macrophages in vitro and in vivo

Tumor-associated macrophage (TAM) is increasingly being viewed as a target of great interest in tumor microenvironment due to its important role in the progression and metastasis of cancers. It has been shown that TAM indeed overexpresses unique surface marker legumain. In this study, we designed and synthesized a Y-shaped legumain-targeting peptide (Y-Leg) with functional groups allowing for further conjugation with imaging and therapeutic moieties (vide infra). The in vitro cell experiments using FITC-conjugated Y-Leg revealed its specific and selective interaction with M2-polarized macrophages (i.e., TAMs) with preference to M1 macrophages, and that the interaction was not interfered with by conjugating FITC to its functional group. Further, we constructed a nanotube system by grafting Y-Leg onto oxidized carbon nanotubes (OCNTs) loaded with paramagnetic Fe3O4 nanoparticles. The intravenous injection of the resultant Y-Leg-OCNT/Fe3O4 nanotubes to 4T1 mammary tumor-bearing mouse led to the magnetic resonance imaging (MRI) of TAM-infiltrated tumor microenvironment, revealing the targeting specificity of Y-Leg-conjugated nanotubes in vivo. The Y shape of peptide and its functional groups containing amines and imidazole can protonate at different pHs, contributing to the in vitro and in vivo targeting specificity. This study represents the first development of novel peptide and peptide-grafted nanotube system targeting M2-polarized TAMs in vivo. The methodology developed in this study is applicable to the construction of various multifunctional nanoparticle systems for selectively targeting, imaging and manipulating of TAMs for the diagnosis and treatment of cancers and inflammatory diseases identified with macrophage-infiltrated disease tissue.

Modulating mammary tumor growth, metastasis and immunosuppression by siRNA-induced MIF reduction in tumor microenvironment

Macrophage migration inhibitory factor (MIF) has been identified as a major gene product upregulated in breast cancer cells–tissues upon the accumulation of macrophages. However, regulatory role of MIF in tumor microenvironment is not well understood. Previously, we have developed small interfering RNA (siRNA)-loaded nanoparticle system to effectively reduce MIF expression in both breast cancer cells and macrophages. Using this nanoparticle system, in this study we demonstrated that the siRNA-induced MIF reduction in murine mammary cancer line 4T1 and human breast cancer line MDA-MB-231 resulted in significant reduction of cell proliferation and increase of apoptosis; the siRNA-induced MIF reduction in tumor-associated macrophages resulted in a significant reduction of surface expression of CD74 and CD206 and a significant increase of surface expression of major histocompatibility complex II, as well as intracellular expression of tumor necrosis factor-α and interleukin-2. A direct injection of the MIF-siRNA-loaded nanoparticles into 4T1 tumor in mice resulted in effective reduction of intratumoral MIF. This led to a reduction of tumor growth and metastasis. This also resulted in a reduction of circulating myeloid-derived suppressive cells both in number and in suppressive function. CD4 T-cell infiltration to tumor was increased. More importantly, this not only slowed the growth of treated 4T1 tumor, but also delayed the growth and metastasis of a contralateral untreated 4T1-luc tumor, suggesting the development of systemic antitumor responses. This study demonstrates for the first time that the siRNA-mediated intratumoral MIF reduction can induce antitumoral immune response via reducing systemic immune suppression.

Systemic administration of β-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer

ABSTRACT Converting an immunosuppressive melanoma microenvironment into one that favors the induction of antitumor immunity is indispensable for effective cancer immunotherapy. In the current study we demonstrate that oat-derived β-(1-3)-(1-4)-glucan of 200 kDa molecular size (BG34-200) previously shown to mediate direct interaction with macrophages could alter the immune signature within melanoma microenvironment. Systemic administration of BG34-200 resulted in reversion of tolerant melanoma microenvironment to an immunogenic one that allows M1-type activation of macrophages, the induction of pro-inflammatory cytokines/chemokines including IFN-γ, TNF-α, CXCL9, and CXCL10, and enhanced IRF1 and PD-L1 expression. In turn, BG34-200 induced a superior antitumor response against primary and lung metastatic B16F10 melanoma compared to untreated controls. The enhanced tumor destruction was accompanied with significantly increased tumor infiltration of CD4+ and CD8+ T cells as well as elevated IFN-γ in the tumor sites. Systemic administration of BG34-200 also provoked systemic activation of tumor draining lymph node T cells that recognize antigens naturally expressing in melanoma (gp100/PMEL). Mechanistic studies using CD11b-knockout (KO), CD11 c-DTR transgenic mice and nude mice revealed that macrophages, DCs, T cells and NK cells were all required for the BG34-200-induced therapeutic benefit. Studies using IFN-γ-KO transgenic mice showed that IFN-γ was essential for the BG34-200-elicited antitumor response. Beyond melanoma, the therapeutic efficacy of BG34-200 and its immune stimulating activity were demonstrated in a mouse model of osteosarcoma. Together, BG34-200 is highly effective in modulating antitumor immunity. Our data support the potential therapeutic use of this novel immune modulator in the treatment of metastatic melanoma.

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes.

Our laboratory has previously demonstrated that melanoma draining lymph node (MDLN) samples from stage III patients contained both CD4+ and CD8+ T cells that can be readily expanded to mediate tumor cell apoptosis in vitro and improve survival in mice bearing human melanoma xenografts. In this study, we investigated whether MDLN T cells contain melanoma-reactive CD4+ T-cell compartment and what they are. To test this, we performed multiparametric (11-color and 6-color) fluorescence-activated cell sorting analyses to monitor phenotypic and functional property of CD4+ T cells in response to melanoma cell antigen reexposure. Our results have demonstrated that the antigen reexposure could result in a generation of CD4+CCR7+CD62L+CD27− T-cell subsets with various effector cell-like properties. Within the CD4+CCR7+CD62L+CD27− T-cell compartment, in response to antigen reexposure, some of the cells expressed significantly upregulated CD40L and/or CXCR5, and some of them expressed significantly upregulated interleukin-2 and/or tumor necrosis factor-&agr;. This may suggest the existence of melanoma-reactive CD4+ “effector-precursor” cells within the expanded MDLN cells and their differentiation into various effector lineages in response to antigen restimulation. Recent clinical trials have demonstrated that effective adoptive cellular immunotherapy maybe enhanced by antigen-specific CD4+ T cells. Therefore, results of this study may significantly benefit innovative design of +adoptive cellular immunotherapy that can potentially mediate enhanced and durable clinical responses.

Requirement of Innate Immunity in Tumor-Bearing Mice Cured by Adoptive Immunotherapy Using Tumor-Draining Lymph Nodes

Background. The purpose of this study was to determine the cellular effectors of both the adoptively transferred cells and the tumor-bearing host that participate in the antitumor response to adoptive immunotherapy using culture-activated tumor-draining lymph nodes (TDLNs). Methods. TDLNs harvested from mice with 4T1 carcinoma cells were fractionated to derive the L-selectinlow subpopulation and activated ex vivo prior to in vitro cytokine release assays and adoptive transfer into BALB/c mice bearing 3-day established subcutaneous tumors. Tumor-bearing recipients were SCID (lacking T, B, and NK cells), Rag2 deficient (lacking T and B cells), and wild-type BALB/c mice. Results. Culture-activated L-selectinlow 4T1 TDLN from BALB/c mice secreted significant levels of interferon-gamma in response to 4T1 but not control tumor cells in vitro. CD4 cells within the adoptively transferred effector cell population contributed significantly to the antitumor effect in vivo. Culture-activated L-selectinlow TDLNs from BALB/c wild-type mice were able to cure Rag2 deficient but not SCID mice bearing 4T1 subcutaneous tumors, suggesting a requirement of NK cells within the innate immune system of the tumor-bearing host during the antitumor response. Conclusions. These results identify the cellular effectors involved in tumor regression following adoptive transfer and demonstrate the requirement for intact innate immunity within the tumor-bearing host.

Use of high throughput T cell receptor (TCR) DNA sequencing to characterize T cell infusion products and track clonal expansion in vivo

Background During the process of T cell priming to peptide antigens, the T cell receptor (TCR) undergoes gene rearrangements that result in a peptide-binding region (complementarity determining region, CDR) which can act as a unique molecular fingerprint for similar T cell clonotypes. The purpose of this study was to assess high-throughput TCR DNA sequencing to analyze qualitative and quantitative measures of human T cell clonotype expansions both in vitro and in vivo. Methods Human lymph nodes derived from patients with melanoma (melanoma-draining lymph node cells, MDLN) were culture-activated using either anti-CD3/CD28 beads and IL-2 100 IU/ml alone or in combination with antiVEGF neutralizing antibody, for 14 days. The two resultant MDLN cultures were compared for overall T cell expansion, cell surface phenotype, intracellular cytokine staining and high-throughput TCR DNA sequencing (ImmunoSEQ, Adaptive Biotechnologies). In addition, both TDLN cell cultures were injected intravenously into SCID mice bearing A375 melanoma xenografts and bone marrow was harvested to assess for persistence of transferred human melanoma MDLN cells. Results Overall T cell expansion and cell surface phenotype of MDLN cell cultures was similar. MDLN cells cultured in the presence of anti-VEGF neutralizing antibody demonstrated higher baseline levels of intracellular interferon-g. TCR sequencing analysis comparing day 0 versus day 14 cultured MDLN cells demonstrated approximately 10% shared T cell clonotypes following culture activation with anti-CD3/CD28 beads and IL-2. As a point of reference, the proportion of shared T cell clonotypes between two different patient MDLN cells was approximately 0.6%. Addition of anti-VEGF antibody during culture of MDLN cells resulted in common T cell clonotypes of approximately 1% (day 0 versus day 14), confirming the expansion of molecularly distinct T cell clonotypes. Human MDLN cells infused into SCID mice bearing A375 melanoma xenografts demonstrated persistence of T cell clonotypes in the bone marrow several weeks after infusion. It is notable that none of the low frequency T cell clonotype expansion differences between samples could be readily identified using VDJ gene usage or CDR3 length analysis. Conclusions

Abstract 16: Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal associated with the growth and metastasis of tumors. In addition, VEGF has also been implicated in the suppression of immune cell function. The purpose of this study was to characterize VEGF secretion, receptor expression and effect of soluble VEGF neutralization in human lymph node cells both at rest and during activation. Methods: Human lymph nodes derived from patients with melanoma (IRB CASE 3610) containing T cells, B cells, monocyte/macrophages and dendritic cells were cultured with anti-CD3/CD28 beads and IL-2 in order to induce an inflammatory response in vitro. Culture supernatant was assayed for the presence of soluble VEGF on days 4, 7, 11 and 14 of culture. Multiparameter FACS analysis was performed to assess the cell surface expression of VEGF receptor subtypes 1 and 2 over the 14-day culture period. In addition, intracellular expression of cytokines within effector T cells was assessed at the end of the culture. Finally, a parallel culture containing a VEGF neutralizing antibody was performed to assess the effect of VEGF neutralization on effector T cell phenotype. High-throughput T cell receptor CDR3 sequencing was used to determine the molecular sequence of the variable region of the T cell receptors (ImmunoSEQ, Seattle, WA). Results: VEGF was measureable in the culture supernatant on days 4 and beyond. The proportion of cells expressing intracellular VEGF peaked at day 4 and subsequently decreased. CD11b+(monocyte/macrophage), CD11c+ (dendritic cells) and CD19+ (B) cells had the highest proportion of cells which expressed intracellular VEGF. VEGF R1 and R2 were not constitutively expressed on resting immune cells, but receptor expression was upregulated upon activation of the T cells in vitro. Day 14 T cells demonstrated intracellular IFN-γ, IL-2 and TNF-α. The addition of VEGF-blocking antibody in the culture of MDLN cells effectively neutralized the amount of soluble VEGF detected in the culture supernatant at all days. The addition of the VEGF-blocking antibody also increased the proportion of T cells expressing intracellular IFN-γ as compared T cells in the non-VEGF blocked culture and resulted in distinct molecularly defined T cell clones. Conclusion: These results confirm that soluble VEGF is produced by immune cells during activation, even in the absence of tumor. VEGF receptor expression on lymph node immune cell subsets is constitutive, but low, and then upregulated during in vitro activation of T cells, presumably due to secondary cytokine production. Neutralization of soluble VEGF resulted in qualitative differences in effector cytokine expression and T cell receptor sequences. Whether the effect on these T cells is direct via VEGF interaction with the cognate receptor or from an indirect effect on antigen-presenting cells is currently under investigation. Citation Format: Madeleine P. Strohl, Hallie Graor, Mei Zhang, Anthony Visioni, John Ammori, Isabelle Rivers-McCue, Julian Kim. Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2014-16