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Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings., Graphical abstract, Weiner and colleagues report on a synthetic DNA vaccine targeting 40 neoantigens. The immunogenicity of individual epitopes was unaffected by their positioning on long vaccine strings or the size of vaccine constructs. These vaccines generated robust, long-term T cell immunity and controlled growth of heterogeneous multi-focal tumors.

Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumors in adults with a five-year survival rate less than 5%. In recent years, immunotherapeutic approaches that target a tumor-associated antigen (TAA), including EGFRvIII-targeted cancer vaccine and adoptive cell transfer, have been examined in clinical studies, which so far have shown limited efficacy in GBM patients. GBM is highly heterogeneous for targeting antigen expression. An immunotherapeutic agent that harnesses multiple TAAs simultaneously may mitigate tumor antigen escape and improve efficacy in GBM patients. Here, we describe a DNA-encoded tri-specific T cell engager (DTriTE) that targets CD3 and two TAAs, EGFRvIII and IL13Ra2, which are expressed in up to 30% and 75% of GBM cases, respectively. We designed EGFRvIII-IL13Ra2-DTriTE to include single-chain variable fragments targeting IL13Ra2, CD3, and EGFRvIII in a series, fused with glycine-serine linkers. EGFRvIII-IL13Ra2-DTriTE exhibited robust binding activities to both EGFRvIII+ and IL13Ra2+ tumor cells and primary T cells, inducing robust T cell activation and anti-tumor cytotoxicity. EGFRvIII-IL13Ra2-DTriTE showed durable in vivo expression of over 10 weeks after a single IM injection in NSG mice. We developed an orthotopic challenge of a heterogeneous GBM model, wherein a mixture of EGFRvIII+ tumor cells and IL13Ra2+ tumor cells were planted in the brains of NSG mice and primary human T cells were peripherally delivered. A single IM administration of EGFRvIII-IL13Ra2-DTriTE resulted in complete tumor clearance. An administration of DNA-encoded bispecific T cell engager (DBTE) targeting EGFRvIII alone induced tumor clearance in 3 of 10 (30%) and limited tumor control in 5 of 10 (50%) animals. Similarly, mono-targeting of IL13Ra2 alone induced tumor clearance in 1 of 9 (11%) and limited tumor control in 4 of 9 (44%) animals. EGFRvIII-IL13Ra2-DTriTE treatment exhibited significantly improved survival with 8 of 8 (100%) animals survived, while treatments of single EGFRvIII-DBTE, IL13Ra2-DBTE or a vehicle control resulted in 4 of 10 (40%), 4 of 9 (44%), and 0 of 10 (0%) animals survived, respectively. This study shows that dual tumor-targeting approach using DTriTE is highly effective at treating antigenically heterogeneous GBM tumors. Further study of DTriTE multi-antigen approaches for GBM and other heterogeneous tumors appears important. Citation Format: Daniel Hongil Park, Pratik Bhojnagarwala, Kevin Liaw, Devivasha Bordoloi, Ebony N. Gary, David B. Weiner. A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2976.

Ovarian cancer (OC) represents the deadliest gynecologic malignancy. Despite important advances in the field of OC therapy, recurrent OC still has very poor prognosis with a median survival of 1 year. Due to the close interaction between the ovarian cancer cells and tumor microenvironment, development of treatment strategies which not only target the tumor cells but also the components of the tumor microenvironment hold significance. Notably, a prime obstacle in the development of therapies is to identify targets with specific expression limited to the tumor surface and not the healthy tissues. The follicle-stimulating hormone receptor (FSHR) is one such target with selective expression in ovarian granulosa cells and thus, a potentially important therapeutic target in OC. Therefore, we developed monoclonal antibodies against FSHR and focused on studies of the most potent of these reagents. Anti-FSHR antibody bound to diverse FSHR expressing ovarian serous and clear cell adenocarcinoma cells suggesting these antibodies could be used to specifically target OC. We then designed a novel DNA encoded bispecific T cell engager targeting FSHR (FSHRxCD3) and evaluated this bispecific in therapeutic models for the treatment of OC. FSHRxCD3 bispecific in the presence of human PBMCs was highly specific in killing FSHR positive ovarian tumor lines. However, T cell approaches alone may not be fully effective in treating OC, referred as Immunologically “Cold” Tumors. Hence, we hypothesized that engaging the other components of immune system, would provide better tumor control. Increasing lines of evidence suggest that OC is receptive to Natural killer (NK) cell attack. We designed antibodies against human Siglec-7, an inhibitory receptor present on human NK cells and showed they could bind to NK cells. We then used these to create a novel class of bispecific NK engager (NKE) that simultaneously targets both Siglec-7 and FSHR (Siglec-7xFSHR). This NKE was potent at killing FSHR positive OC targets in both in vitro and in vivo assays. Multiple Ovarian tumors including BRCA mutated and PARPi resistant ovarian cancer cells could be targeted by this immune therapy. Our data demonstrate the therapeutic potential of these two novel bispecific molecules and initial studies suggest that their combination may be valuable in patients with OC. Citation Format: Devivasha Bordoloi, Pratik Bhojnagarwala, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, Alfredo Perales-Puchalt, Ryan P. O’Connell, Xizhou Zhu, Elizabeth M. Parzych, Rugang Zhang, Mohamed Abdel-Mohsen, David B. Weiner. Immunotherapy of ovarian cancer targeting FSHR by innate and adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4262.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease., Graphical abstract, Patel et al. demonstrate that immunization with a DNA vaccine encoding the SARS-CoV-2 spike antigen, INO-4800, induces durable immune responses in rhesus macaques and is associated with reduced viral loads after challenge.

SummaryCoronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. Here, we assess immunogenicity and anamnestic protective efficacy in rhesus macaques of the intradermal (ID)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800. INO-4800 is an ID-delivered DNA vaccine currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and neutralizing antibody responses against both the D614 and G614 SARS-CoV-2 spike proteins. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T and B cell responses. These responses were associated with lower viral loads in the lung and with faster nasal clearance of virus. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against COVID-19 disease.

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study., There is currently no licensed SARS-CoV-2 vaccine. Here, the authors generate an optimized DNA vaccine candidate encoding the SARS-CoV-2 spike antigen, demonstrating induction of specific T cells and neutralizing antibody responses in mice and guinea pigs. These initial results support further development of this vaccine candidate.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor clinical prognosis and there is significant need for new, improved therapies. Immunotherapy is a growing therapeutic research area for patients with advanced brain cancers. DNA encoded Tumor Attacking Bridges (dTABs) are bispecific antibodies that redirect T cell activity against Tumor Associated Antigens (TAAs) of interest. Here, we describe the design and development of in vivo generated dTABs targeting IL13Rα2, which is expressed in tumors of approximately 75% of GBM patients. Four dTABs targeting 2 different epitopes of IL13Rα2 were designed to compare alternate orientations of heavy and light chains in the dTABs. All dTABs bound to U87 cells expressing target as well as CD3 on T cells. The dTABs induced T cell activation, cytokine production, and tumor killing in the presence of target expressing tumor cells in a dose dependent manner. An important issue in the field of antibody guided therapies is on target - off tumor T cell activity which can cause serious side effects in patients. In order to minimize such toxicities, we identified one arrangement of dTAB which induced high T cell activation with potent tumor killing in the presence of target expressing tumor cells, but not when co-cultured with tumors that do not express IL13Rα2. We were able to detect expression of this lead dTAB for up to 19 days in the serum of NSG mice after a single DNA injection followed by electroporation. Serum from mice injected with this dTAB activated T cells which potently killed target expressing U87 GBM cells. In a subcutaneous xenograft mouse model of human GBM, this in vivo produced dTAB demonstrated significant tumor control and improved survival of mice. This study illustrates the potential of dTABs targeting IL13Rα2 as a novel approach for immunotherapy of this difficult to treat brain cancer. Citation Format: Pratik Sanjivkumar Bhojnagarwala, Ryan O'Connell, Daniel Park, Ebony Gary, Xizhou Zhu, David Weiner. Novel DNA encoded tumor attacking bridges (dTABs) targeting IL13Rα2 display potent tumor control in an animal model of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1822.

We report that human conventional CD15+ neutrophils can be isolated in the peripheral blood mononuclear cell (PBMC) layer during Ficoll gradient separation, and that they can impair T cell proliferation in vitro without concomitant neutrophil activation and killing. This effect was observed in a total of 92 patients with organ transplants, lung cancer or anxiety/depression, and in 18 healthy donors. Although such features are typically associated in the literature with the presence of certain myeloid-derived suppressor cell (PMN-MDSC) populations, we found that commercial centrifuge tubes that contained membranes or gels for PBMC isolation led to up to 70% PBMC contamination by CD15+ neutrophils, with subsequent suppressive effects in certain cellular assays. In particular, the suppressive activity of human MDSC should not be evaluated using lectin or microbead stimulation, whereas assays involving soluble or plate-bound antibodies or MLR are unaffected. We conclude that CD15+ neutrophil contamination, and associated effects on suppressor assays, can lead to significant artefacts in studies of human PMN-MDSC.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

Despite recent advances in the development of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The lack a high throughput, reproducible syngeneic animal model that replicates human disease is partly responsible for the paucity of novel therapeutic approaches. In this report, we present the first successful syngeneic, orthotopic model for esophageal cancer. This model was used to test an established adenoviral-based tumor vaccine. We utilized a murine esophageal cancer cell line established from the EDL2-cyclin D1;p53−/− mouse that was transduced to express a viral tumor antigen, the Human Papilloma Virus (HPV) E7 protein. The tumor was established in its natural microenvironment at the gastroesophageal (GE) junction. Tumor growth was consistent and reproducible. An adenoviral vaccine to E7 (Ad.E7) induced an E7-specific population of functionally active CD8+ T cells which trafficked into the tumors and retained cytotoxicity. Ad.E7 vaccination reduced local tumor growth and prolonged overall survival. These findings suggest that orthotopic tumor growth is a reasonable preclinical model to validate novel therapies.

Summary Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b + CD15 hi CD10 − CD16 low immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor T cell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer.

Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9,10-epoxide pathway (P450/epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. On the basis of B[a]P-1,6 and 3,6-dione formation, approximately 4 μM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 μM to 10 μM B[a]P with no significant increase seen with further escalation to 50 μM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7-8-dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones.

Recent studies suggest that immunotherapy may offer a promising treatment strategy for early-stage malignant pleural mesothelioma (MPM), but advanced tumor burden may limit the efficacy of immunotherapy. Therefore, we hypothesized that surgical cytoreduction could restore the efficacy of vaccine-based immunotherapy for MPM. We developed a murine model of MPM through transduction of a mesothelioma cell line with mesothelin. We used this model to evaluate the efficacy of a Listeria monocytogenes vaccine expressing mesothelin. Tumor growth was significantly inhibited at four weeks in animals vaccinated two weeks prior to tumor cell inoculation as compared to those given an empty vector control (1371 ± 420 mm(3) versus 405 ± 139 mm(3); p0.01). Mice vaccinated one week prior to tumor challenge also displayed significant reduction in tumor volume (1227 ± 406 mm(3) versus 309 ± 173 mm(3); p0.01). The vaccine had no effect when administered concurrently with tumor challenge, or after tumors were established. Flow cytometry showed reduced mesothelin expression in large tumors, as well as tumor-associated immunosuppression due to increased myeloid derived suppressor cells (MDSCs). These factors may have limited vaccine efficacy for advanced disease. Surgical cytoreduction of established tumors restored the antitumor potency of the therapeutic vaccine, with significantly reduced tumor burden at post-operative day 18 (397 ± 103 mm(3) versus 1047 ± 258 mm(3); p0.01). We found that surgery reduced MDSCs to levels comparable to those in tumor-naïve mice. This study demonstrates that cytoreduction surgery restores the efficacy of cancer vaccines for MPM by reducing tumor-related immunosuppression that impairs immunotherapy.

To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of “canonical” TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter. Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) stimulate antigen non-specific autologous T cell responses 2) directly stimulate antigen-specific autologous memory T cell responses, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction Citation Format: Jason Stadanlick, Abhishek Rao, Michael Annunziata, Edmund Moon, Shaun O'brien, Pratik Bhojnagarwala, Michael Feldman, Wayne Hancock, Jose Conejo-Garcia, Sunil Singhal, Stephen Albelda, Evgeniy Eruslanov. Tumor-associated neutrophils with antigen-presenting features in early-stage human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2017-3707

Careful preparation of human tissues is the cornerstone of obtaining accurate data in immunologic studies. Despite the essential importance of tissue processing in tumor immunology and clinical medicine, current methods of tissue disaggregation have not been rigorously tested for data fidelity. Thus, we critically evaluated the current techniques available in the literature that are used to prepare human lung tumors for immunologic studies. We discovered that these approaches are successful at digesting cellular attachments and ECMs; however, these methods frequently alter the immune cell composition and/or expression of surface molecules. We thus developed a novel approach to prepare human lung tumors for immunologic studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This enzymatic digestion cocktail optimized cell yield and cell viability, retrieved all major tumor-associated cell populations, and maintained the expression of cell-surface markers for lineage definition and in vivo effector functions. To our knowledge, we present the first rigorously tested disaggregation method designed for human lung tumors.

Introduction Defining tumor from non-tumor tissue is one of the major challenges of cancer surgery. Surgeons depend on visual and tactile clues to select which tissues should be removed from a patient. Recently, we and others have hypothesized near-infrared (NIR) imaging can be used during surgery to differentiate tumors from normal tissue. Methods We enrolled 8 canines and 5 humans undergoing cancer surgery for NIR imaging. The patients were injected with indocyanine green (ICG), an FDA approved non-receptor specific NIR dye that accumulates in hyperpermeable tissues, 16–24 hours prior to surgery. During surgery, NIR imaging was used to discriminate the tumor from non-tumor tissue. Results NIR imaging identified all tumors with a mean signal-to-background ratio of 6.7. Optical images were useful during surgery in discriminating normal tissue from cancer. In 3 canine cases and 1 human case, the tissue surrounding the tumor was inflamed due to obstruction of the vascular supply due to mass effect. In these instances, NIR imaging could not distinguish tumor tissue from tissue that was congested, edematous and did not contain cancer. Conclusions This study shows that NIR imaging can identify tumors from normal tissues, provides excellent tissue contrast, and it facilitates the resection of tumors. However, in situations where there is significant peritumoral inflammation, NIR imaging with ICG is not helpful. This suggests that non-targeted NIR dyes that accumulate in hyperpermeable tissues will have significant limitations in the future, and receptor-specific NIR dyes may be necessary to overcome this problem.

To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer. To address this question, freshly isolated tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of canonical TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). This subset of TANs was found in lung tumor tissue but not in adjacent uninvolved lung. We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter. Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. By contrast, mature CD11b+CD15+CD10+CD16hi neutrophils did not acquire the phenotype of hybrid TANs, when cultured under these conditions. In addition, we determined that IFN-γ and GM-CSF synergistically exerted APC promoting effects on immature neutrophils via the downregulation of the transcription factor, Ikaros. However, the development of APC-like hybrid neutrophils was profoundly inhibited under hypoxic conditions. Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) produce APC cytokines such as TNF and IL-12 after stimulation, 2) stimulate antigen non-specific autologous T cell responses induced by plate-bound anti-CD3 antibodies 3) directly stimulate antigen-specific autologous memory T cell responses to virus-derived antigens, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid TANs had enhanced ability to trigger and support T cell responses in direct cell-cell interactions. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Shaun O'Brien, Edmund Moon, Tom Stephen, Abhishek Rao, Alfred Garfall, Wayne Hancock, Jose Conejo-Garcia, Charuhas Deshpande, Michael Feldman, Sunil Singhal, Steven Albelda. The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A02.

Tumor-recruited myeloid cells represent a significant portion of inflammatory cells within the tumor microenvironment and influence nearly all steps of tumor progression. Although tumor-associated neutrophils (TANs) are present in large numbers, the role of TANs in cancer progression remains unclear and has only been recently investigated in murine models. The goal of this study was to provide a phenotypic and functional characterization of TANs in freshly harvested surgically resected lung cancer samples. Tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. Importantly, fresh lung tumors from patients were processed within 20 minutes of removal from the patient. We developed an approach to prepare human lung tumors for immunological studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This approach retrieved all major tumor-associated cell populations with high cell yield and maintained the expression of cell surface markers and the functions of isolated cells. In multicolor tracings, TANs were defined as CD15hi/CD66b+/CD11b+/MPO+/Arg1+/IL-5Ra-/ cells. We found that TANs comprised 5—25% of cells isolated from all digested human lung tumors (n=42). Compared to blood neutrophils, TANs displayed an activated phenotype (CD62Llo/CD54hi) and a novel repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. They also produced substantial quantities of the pro-inflammatory factors MCP-1, IL-8, MIP-1a, and IL-6, as well as the anti-inflammatory IL-1R antagonist. TANs were not hypofunctional cells, since they were able to phagocytize bacteria and produce reactive oxygen species. Understanding the role of TANs in regulating T cell responses in cancer patients is particularly important because cytotoxic T lymphocytes are the chief effector cells mediating antigen-driven anti-tumor immunity. We found that TANs were able to stimulate antigen non-specific T cell proliferation and IFN-γ release. Crosstalk between TANs and activated T cells led to substantial up-regulation of CD54, CD86, OX40L, and 4-1BBL co-stimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive feedback loop. This stimulatory activity of TANs appeared to be related to tumor size, as TANs from the majority of the smaller tumors (< 3cm) were more stimulatory than those from larger tumors. We also identified a novel subset of TANs with a hybrid “composite” phenotype of neutrophils and antigen-presenting cells (MPO+CD66b+CD14+HLA-DR+CCR7+CD86+). We found that this hybrid subset had the ability to: 1) dramatically stimulate antigen non-specific T cell proliferation, 2) induce the proliferation of allogeneic T cells in mixed lymphocyte reaction and 3) trigger antigen-specific memory T cell response to a mixture of T cell epitopes from human CMV, Epstein-Barr, flu viruses, as well as tetanus toxoid from all the common HLA types. Thus, our data suggest that in patients with early-stage lung cancer, TANs do not significantly contribute to inhibition of T cell responses. Rather, the majority of neutrophils recruited into the tumor microenvironment undergo phenotypic and functional changes that result in the formation of cells that resemble the murine anti-tumor “N1 TANs” and could potentially augment and support T cells responses. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Tom Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne Hancock, Jose Conejo-Garcia, Michael Feldman, Sunil Singhal, Steven Albelda. Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A66.

Aim: Advances in immunotherapy require a detailed understanding of host immunity. Data from murine models indicate solid tumors of various types can recruit Tregs and/or promote iTreg development, and thereby curtail antitumor responses. However, clinical data have been restricted to either evaluation of Treg numbers in tumors, or studies of PBMC Treg in patients with cancers. Surprisingly, no studies of Treg function have ever been reported using Tregs isolated from tumors themselves. Methods: Using our recently reported technique, we isolated Tregs within 20 mins of resection from 13 lung cancer patients, stages Ia-IIIa, and 2 control non-cancer patients (diaphragmatic hernia and lung granuloma). The mean age of the cancer patients (10 males, 3 females) was 65.4±2.2 years; 7 patients had adenocarcinoma, 3 had squamous cell carcinoma, and the remainder had large cell neuroendocrine carcinoma, colon adenocarcinoma and high-grade carcinoma. The mean size of their tumors was 4.0±0.8 cm, and 1 of the 13 patients developed metastases during the follow-up period. We compared the suppressive function of Tregs isolated from each patient's freshly resected tumor, LN and blood, using cryopreserved healthy donor PBMC aliquots as responders, and area-under-curve (AUC) analysis. We controlled for comparable CD4+ and FOXP3+ purity in all isolated Treg samples, and cell viability, excluding from further analysis all data from non-comparable FOXP3+ subsets. Thus, we were able to avoid artifacts arising from different cell conditions (e.g. use of autologous responder cells) or from unequal FOXP3+ content in Treg isolates. Results: Tumor-infiltrating Tregs had significantly enhanced suppressive function when compared with Tregs isolated from other locations in the same patient. Thus, tumor Tregs were significantly more suppressive than lung LN Tregs (p = 0.0003), and significantly more suppressive than blood Tregs (p = 0.03). Tregs isolated from LN in close proximity to tumors had comparable suppressive function to Tregs isolated from distant LN (p>0.05), and blood Tregs had comparable suppressive function to that of LN Tregs (p>0.05). Additional analyses showed that the enhanced suppressive function of tumor Treg was not due to Treg-induced killing/apoptosis of responder cells, cytokine and/or growth factor deprivation, or soluble factors produced by tumor Treg cells. Conclusions: We demonstrate for the first time that tumor-infiltrating human lung cancer Treg have significantly increased suppressive function. This finding is important for the development of effective anti-tumor therapy. While we are continuing to study the basis for such differences, our data suggest that studies of peripheral blood Tregs in cancer patients may be inadequate and inefficient approaches for understanding the fundamental mechanisms of limited host antitumor immune responses. Citation Format: Tatiana Akimova, Evgeniy B. Eruslanov, Pratik S. Bhojnagarwala, Jon G. Quatromoni, Jacqueline Morgen, Sunil Singhal, Steven M. Albelda, Wayne W. Hancock. Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells. [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 1291. doi:10.1158/1538-7445.AM2015-1291

Background Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation. Methods BALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells. Results Inhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p