Search

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs., Su Chul Jang et al. develop exoSTING, consisting of an engineered extracellular vesicle loaded with a potent cyclic dinucleotide (CDN) agonist of the STING pathway. They find that exoSTING shows more than 100-fold increased potency in in vivo tumor models and has increased tumor retention and lower levels of systemic inflammatory cytokine production as compared to free CDN.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

Background Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens. Results Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b+ and CD15+, HLA-DRdim and CD14− phenotype, were undetectable in frozen samples. Conclusion These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens. Electronic supplementary material The online version of this article (doi:10.1186/s12865-017-0192-1) contains supplementary material, which is available to authorized users.

Background: The Stimulator of Interferon Genes (STING) pathway is an attractive target in immuno-oncology. Selective activation of the STING pathway in antigen presenting cells (APCs) is essential for eliciting a potent and specific anti-tumor immune response. STING is ubiquitously expressed in normal cells including T cells and endothelial cells. Direct intra-tumoral administration of a free STING agonist results in activation of the STING pathway in all cells, resulting in loss of viability of immune cells, tissue damage, and systemic immune activation due to vascular leakage. Exosomes are an efficient natural messenger system that delivers macromolecules between cells. Leveraging exosome biology, we have developed a novel engineered exosome therapeutic, exoSTING, to selectively target the STING pathway in tumor resident APCs. Results: exoSTING is composed of exosomes engineered to express high levels of Protein X (PrX), a transmembrane glycoprotein naturally occurring on exosomes, and loaded with a STING agonist. Following intra-tumoral injection in checkpoint refractory B16F10 tumors, exoSTING selectively activates STING in APCs, leading to anti-tumor immunity with greater than 100-fold improvement in potency compared to free STING agonist. The activity of exoSTING is substantially diminished in the PrX knock out exosomes, highlighting the importance of this surface glycoprotein for preferential activation of APCs. In contrast to the free STING agonist, exoSTING is retained within the injected tumor, minimizing systemic exposure. Furthermore, exoSTING administration preserves the viability of T cells and APCs, reduces collateral tissue damage, and does not induce systemic cytokine production, resulting in a broader therapeutic window in contrast to free STING agonist. exoSTING produced an increased systemic tumor-specific T cell response as demonstrated by elimination of non-injected abscopal tumors. The specificity of exoSTING activity was demonstrated using a STING knockout mouse (Tmem173gt/J). T cells and myeloid cells play a critical role in exoSTING mediated anti-tumor immunity, in contrast to NK cells which are not required. exoSTING treatment results in significant induction of PD-L1 expression (P Conclusion: exoSTING is an engineered exosome therapeutic that leverages exosome biology and specifically targets the STING pathway in APCs, resulting in greater potency with preserved viability of T cells and APCs, greater systemic tumor antigen specific immune response, reduced systemic cytokine production, and enhanced efficacy. Citation Format: Su Chul Jang, Raymond J. Moniz, Chang Ling Sia, Rane A. Harrison, Damian Houde, Nikki Ross, Ke Xu, Nuruddeen Lewis, Raymond Bourdeau, Christine McCoy, Tong Zi, Agata Villiger-Oberbek, Scott Estes, Jorge Sanchez-Salazar, Kevin Dooley, William K. Dahlberg, Sriram Sathyanarayanan, Kyriakos D. Economides. exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen-presenting cells resulting in improved tumor antigen-specific adaptive immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 944.

Recent advances in single cell genomic technologies have enabled unbiased quantification of cellular states within complex tissues, mechanisms of differentiation, biomarker presentation in cellular subsets; yet their applications in evaluating the response to therapeutic interventions are still underexplored. In our study, we apply single cell RNA sequencing (scRNA-seq) to uncover mechanisms and drug targets for Multiple Myeloma(MM), a malignancy of plasma cells with estimates of 30,770 new cases and 12,770 deaths in 2018 within US alone. Using the 10x Genomics scRNA-seq platform, we profiled ten clinical MM patients, pre- and post-treatment with four standard of care chemotherapy regimens (VAD, VCD, VMP, MP; A=Adriamycin; C=Cyclophosphamide; D=Dexamethasone; M=Melphalan; P=Prednisone; V=Velcade). We sequenced a total of 106,000 cells and determined differential gene expression upon treatment at single-cell resolution comparing 37,926 pre- vs 44,842 post-treatment cells. We identify activation of immune cells with increased expression of IL1B, CD14, CD52, CCL4, IFITM3, PSMA4; and repression of translational machinery with reduced expression of PABPC1, PABPC4, FUS, POLR2A in post-treatment (abs(log2 FC >0.25) and P-value < 10-5). To understand the functional implication of gene expression changes, we identified that the upregulated genes are enriched for ontologies regulating protein ubiquitination pathway, communication between innate and adaptive immune cells, TREM1 signaling, dendritic cell maturation (P-value 3.16x10-17, 3.47x10-7, 3.09 x10-5, 4.07x10-5 respectively). Downregulated genes are enriched for EIF2 signaling, regulation of eIF4 and p70S6K signaling, mTOR signaling (P-value 3.16x10-13, 1.86x10-8, 2.24x10-6 respectively). Additionally, we performed a comparative analysis of differentially expressed genes in response to each drug regimen. We identified genes upregulated upon MP drug regimen are comparatively enriched for IL-8 signaling, while treatment with VMP enhances B-cell receptor signaling (P-value 5.5x10-12 and 9.0x10-9 respectively). Further, we have used our unbiased transcriptomic interrogation of MM clinical patients to identify a panel of 100+ biomarkers that are being evaluated at a proteomics level using our recently developed technique, RNA Expression and Protein sequencing (REAP-seq). This method enables us to obtain readout for whole transcriptome and proteins (>100) using oligo conjugated antibodies while retaining single cell resolution. Transcriptome and/or proteome analysis of bulk tumor often fails to detect signature of rare cell populations. Our study here highlights the use of single cell readouts for identifying novel oncology mechanisms, drug targets and drug combinations for more efficacious immunotherapies. Citation Format: Namit Kumar, Vanessa M. Peterson, Kelvin X. Zhang, Lixia Li, Philip W. Garrett-Engele, Raymond J. Moniz, Joel A. Klappenbach. Single cell RNA-sequencing of multiple myeloma clinical patients for identifying novel immuno-oncology drug targets [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 LB-012.

Given their immune-modulating capacity, regulatory T cells (Treg) cells may be important players in the induction of the protective T-cell response (Th1) to genital chlamydial infection. Recent work has demonstrated that plasmacytoid dendritic cells (pDC) respond to genital chlamydial infection, and that pDC may be uniquely positioned for the induction of Treg cells during this infection. Here, we present the first data demonstrating that Treg influx into the draining lymph node and the site of infection during genital chlamydial infection. We found that pDC depletion altered the numbers of Treg and nonprotective inflammatory cells [interferongamma-(IFNgamma)-producing CD8+ T and IFNgamma-producing natural killer T cells] in the spleens of mice genitally infected with Chlamydia muridarum. Furthermore, pDC depletion did not alter Th1 cell numbers, indicating that pDC modulate cells that could inhibit and promote nonprotective inflammation during genital chlamydial infection. Finally, we demonstrate that depletion of pDC results in less severe dilation and collagen deposition in the oviduct following resolution of infection, implicating pDC activity in the formation of sequelae following genital C. muridarum infection.

New methods are needed to eradicate or prevent Chlamydia trachomatis infections. Blockade of epithelial membrane protein 2 (EMP2) by genetic silencing or neutralizing polyclonal antibody reduced chlamydial infectivity in vitro. This study tests the prediction that recombinant anti-EMP2 diabody could reduce early chlamydial infection of the genital tract in vivo. In a murine infection model, pretreatment with anti-EMP2 diabody, as compared with control diabody, significantly reduced bacterial load, tissue production of inflammatory cytokines, recruitment of polymorphonuclear leukocytes, and local tissue inflammation. These findings support EMP2 as a potential preventative and therapeutic target for genital chlamydial infection.

Background Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. Methods The role of human anti-CAIX mAbs on CAIX+ RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX+ RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. Results Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ−/− mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX+ tumor growth. Conclusions Our findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX+RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users.

As a naturally occurring nanocapsule abundantly expressed in nearly all-eukaryotic cells, the barrel-shaped vault particle is perhaps an ideal structure to engineer for targeting to specific cell types. Recombinant vault particles self-assemble from 96 copies of the major vault protein (MVP), have dimensions of 72.5 × 41 nm, and have a hollow interior large enough to encapsulate hundreds of proteins. In this study, three different tags were engineered onto the C-terminus of MVP: an 11 amino acid epitope tag, a 33 amino acid IgG-binding peptide, and the 55 amino acid epidermal growth factor (EGF). These modified vaults were produced using a baculovirus expression system. Our studies demonstrate that recombinant vaults assembled from MVPs containing C-terminal peptide extensions, display these tags at the top and bottom of the vault on the outside of the particle, and can be used to specifically bind the modified vaults to epithelial cancer cells (A431) via the epidermal growth factor receptor (EGFR), either directly (EGF modified vaults) or as mediated by a monoclonal antibody (anti-EGFR) bound to recombinant vaults containing the IgG-binding peptide. The ability to target vaults to specific cells represents an essential advance towards using recombinant vaults as delivery vehicles.

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum. Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naive T cells in vivo. In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro. However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro. Our findings show that these two DC subsets likely have different functions in vivo. cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. To further support the clinical development of pembrolizumab and to aid the mechanistic understanding of anti-PD-1 immunotherapy, we generated a surrogate PD-1-blocking antibody (muDX400). We have used muDX400 to determine the antitumor activity, pharmacokinetics, and pharmacodynamics of PD-1 inhibition in multiple preclinical syngeneic tumor model systems. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive (ie, complete and durable tumor regressions were observed), partially responsive (ie, tumor growth inhibition was observed), and intrinsically resistant to therapy. Gene and protein expression signatures revealed that the more responsive models expressed higher levels of both PD-1 ligand (PD-L1) and tumor-infiltrating lymphocytes compared with nonresponsive models. To further evaluate mechanisms that could potentially enhance the antitumor activity of anti-PD-1 in these tumor models, muDX400 was combined with a number of different chemotherapies, targeted therapies, and other immunotherapies. Because immune suppression is a common side effect associated with many standard-of-care therapies, we evaluated the potential abrogation of muDX400-mediated antitumor activity when combined with approved therapies by scheduling the dosing regimen to examine concurrent and sequential administration of these agents. In the models in which enhanced antitumor activity was evident, we evaluated the immune landscape of blood, tumors, and draining lymph nodes by immuno-phenotyping and molecular profiling. These data provide preclinical support to expand the clinical development of pembrolizumab into additional cancer types as both a single agent and in combination with other approved anticancer therapies. Additional studies with muDX400 are ongoing to further elucidate the mechanism of action of PD-1 blockade and to better understand the antitumor responses observed in clinical trials of pembrolizumab. Citation Format: Elaine M. Pinheiro, Ruban Mangadu, Uyen T. Phan, Mingmei Cai, Yanhong Ma, Heather A. Hirsch, Terrill K. McClanahan, Raymond J. Moniz, Ali-Samer Al-Assaad, Samik Basu, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Brian J. Long. Evaluation of the antitumor activity of anti-PD-1 immunotherapy as a single agent and in combination with approved agents in preclinical tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 269. doi:10.1158/1538-7445.AM2015-269