In Situ Regulation of DC Subsets and T Cells Mediates Tumor Regression in Mice

Cutaneous melanoma is a highly immunogenic disease, surgically curable at early stages, but life-threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis from precursor states to melanoma in situ to invasive tumor. Hallmarks of immunosuppression are detectable by the precursor stage, and when tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary. This environment is established by cytokine gradients that promote expression of MHC-II and IDO1 and by PDL1-expressing macrophages and dendritic cells engaging activated T cells. However, a few mm away, T cells synapse with melanoma cells in fields of tumor regression. Thus, invasion and immunoediting can co-exist within a few millimeters of each other in a single specimen.

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Improving efficacy of PD-1 blockade in unresponsive lymphoma tumors with in situ vaccination through induction of a highly efficient cross-presenting dendritic cell subset.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.76 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 76-76

Author(s):

Linda Hammerich ◽

Maxime Dhainaut ◽

Thomas A. Davis ◽

Tibor Keler ◽

Andres M. Salazar ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Cd8 T Cells ◽

Tumor Regression ◽

Low Grade ◽

Dendritic Cell Subset ◽

Term Survival ◽

Intratumoral Injections

76 Background: Low-grade non-Hodgkin’s B-cell lymphomas are generally incurable; preliminary results with anti-PD-1 therapy have yielded low response rates. Tumoral DC infiltration correlates with efficacy of checkpoint blockade and tumor-targeted vaccines represent promising, novel treatment strategies to induce anti-tumor T cells. Methods: A20 lymphoma-bearing mice were treated with a PD-1 blocking antibody with an in situ vaccine (ISV) consisting of intratumoral injections of FMS-like tyrosine kinase-3 ligand (Flt3L), local irradiation (XRT) of the tumor and intratumoral injections of the TLR3 agonist poly-ICLC (pIC). Results: Untreated lymphoma tumors contained very low numbers of DC and treatment with anti-PD1 alone did not induce tumor regression or increase survival. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC at the tumor site, the draining lymph node and the spleen. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on infiltrating TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by DC. Interestingly, tumor antigens were taken up mainly by CD103+ DC and not CD103- subtypes. CD103+ expression distinguishes a subset of migratory cross-presenting DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103- subsets. The combination of Flt3L with XRT and pIC induced tumor-reactive, IFNg-producing T cells, but delayed tumor growth and improved survival only in 40% of mice. ISV also increased expression of PD-L1 on tumor cells and tumor infiltrating DC. Consistently, combination of ISV with PD-1 blockade led to complete tumor regression and increased long-term survival in the majority of mice. PD-1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect. Conclusions: In situ vaccination can improve efficacy of anti-PD-1 in checkpoint-unresponsive lymphoma tumors through induction of a cross-presenting DC subset leading to long-term regression of established lymphoma tumors.

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598 Local radiation in combination with CpG and anti-OX40 induces enhanced T cell activation and proliferation

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.598 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A628-A628

Author(s):

Dan Spiegelman ◽

Alexander Pieper ◽

Luke Zangl ◽

Arika Feils ◽

Anna Hoefges ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Tumor Regression ◽

Tumor Model ◽

Cd8 T Cell ◽

External Beam ◽

In Situ Vaccine

BackgroundWe, and others, have previously shown that the in-situ vaccine of hypomethylated CG-enriched oligodeoxynucleotide (CpG) with agonist anti-OX40 antibody (OX40) is effective at curing mice in the A20 lymphoma model [1–4]. In separate preclinical models where CpG+OX40 fails to cause tumor regression, radiation therapy (RT) prior to the in-situ vaccine enhances the anti-tumor effect of CpG+OX40 [4]. We investigated the immune response, and specifically the activity of T cells, following treatment with RT+CpG+OX40 in the B78 melanoma model where CpG+OX40 typically fails to cause tumor regression.MethodsC57BL/6 mice were inoculated with 2x106 B78 melanoma cells on the right flank and allowed to grow until the average tumor size was ~150mm3. In two independent experiments, mice were randomized (n=4–5 per group per experiment) and treated with one of the following: 1) PBS, 2) CpG+OX40, 3) RT, 4) RT+CpG+OX40. 12 Gy external beam RT was dosed to the flank tumor on day 0 and intratumoral CpG (50µg)+OX40 (20 µg) were given on days 5, 7, and 9 after RT. Spleens and tumor draining lymph nodes (TDLNs) were harvested on day 12. T cell activation and proliferation were assessed via flow cytometry.ResultsCompared to all other groups in the study, mice treated with RT+CpG+OX40 demonstrated significantly elevated levels of CD4+ and CD8+ T cell activation in the TDLNs, as measured by interferon gamma expression. Similar trends of CD4+ and CD8+ T cell activation were measured in the spleens. Splenic CD8+ T cells from RT+CpG+OX40 treated mice demonstrated significantly elevated levels of proliferation over PBS and RT, as measured by Ki67.ConclusionsIn B78 melanoma, a weakly immunologic tumor model, combining RT with the in-situ vaccine CpG+OX40 enhances the activity of T cells, evidenced by significantly increased CD4+ and CD8+ T cell activation in the TDLN and spleen and elevated CD8+ T cell proliferation in the spleen.ReferencesHouot, R. and Levy, R. T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy. Blood, 2009. 113(15):3546–52.Marabelle, A., et al. Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. J Clin Invest, 2013. 123(6):2447–63.Sagiv-Barfi, I., et al. Eradication of spontaneous malignancy by local immunotherapy. Sci Transl Med, 2018. 10(426).Zangl, LM. Et al. External Beam Radiotherapy Required for Tumor Regression When Using CpG-Oligodeoxynucleotide and Anti-OX40 in an Immunologically Cold Tumor Model. Red Journal. 2019. 105:S88.

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Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models

Frontiers in Immunology ◽

10.3389/fimmu.2021.763888 ◽

2021 ◽

Vol 12 ◽

Author(s):

Alexander A. Pieper ◽

Luke M. Zangl ◽

Dan V. Speigelman ◽

Arika S. Feils ◽

Anna Hoefges ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cell Activation ◽

Tumor Response ◽

Tumor Regression ◽

Quantitative Polymerase Chain Reaction ◽

Response To Treatment ◽

Tumor Models ◽

Cpg Oligodeoxynucleotides

IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

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AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models

Cancer Cell International ◽

10.1186/s12935-019-1059-8 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Stephen M. Shaw ◽

Jenny Middleton ◽

Kim Wigglesworth ◽

Amber Charlemagne ◽

Oliver Schulz ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Tumor Cells ◽

Tumor Immunity ◽

Tumor Antigen ◽

Tumor Regression ◽

Antigen Uptake

Abstract Background Treatments that generate T cell-mediated immunity to a patient’s unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galβ1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity. Methods Various immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT−/−) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel–Cox test, C5a data by Mann–Whitney test, and single tumor regression by repeated measures analysis. Results In vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT−/− mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth. Conclusions We have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

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In situ immunization of a TLR9 agonist virus-like particle enhances anti-PD1 therapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000940 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000940

Author(s):

Yinwen Cheng ◽

Caitlin D Lemke-Miltner ◽

Wattawan Wongpattaraworakul ◽

Zhaoming Wang ◽

Carlos H F Chan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Activation ◽

Mononuclear Cells ◽

Immune Cell ◽

Tumor Regression ◽

Subcutaneous Administration ◽

Type I ◽

Peripheral Blood Mononuclear

BackgroundCMP-001 is a novel Toll-like receptor-9 agonist that consists of an unmethylated CpG-A motif-rich G10 oligodeoxynucleotide (ODN) encapsulated in virus-like particles. In situ vaccination of CMP-001 is believed to activate local tumor-associated plasmacytoid dendritic cells (pDCs) leading to type I interferon secretion and tumor antigen presentation to T cells and systemic antitumor T cell responses. This study is designed to investigate if CMP-001 would enhance head and neck squamous cell carcinoma (HNSCC) tumor response to anti-programmed cell death protein-1 (anti-PD-1) therapy in a human papilloma virus-positive (HPV+) tumor mouse model.MethodsImmune cell activation in response to CMP-001±anti-Qβ was performed using co-cultures of peripheral blood mononuclear cells and HPV+/HPV- HNSCC cells and then analyzed by flow cytometry. In situ vaccination with CMP-001 alone and in combination with anti-PD-1 was investigated in C57BL/6 mice-bearing mEERL HNSCC tumors and analyzed for anti-Qβ development, antitumor response, survival and immune cell recruitment. The role of antitumor immune response due to CMP-001+anti-PD-1 treatment was investigated by the depletion of natural killer (NK), CD4+ T, and CD8+ T cells.ResultsResults showed that the activity of CMP-001 on immune cell (pDCs, monocytes, CD4+/CD8+ T cells and NK cells) activation depends on the presence of anti-Qβ. A 2-week ‘priming’ period after subcutaneous administration of CMP-001 was required for robust anti-Qβ development in mice. In situ vaccination of CMP-001 was superior to unencapsulated G10 CpG-A ODN at suppressing both injected and uninjected (distant) tumors. In situ vaccination of CMP-001 in combination with anti-PD-1 therapy induced durable tumor regression at injected and distant tumors and significantly prolonged mouse survival compared with anti-PD-1 therapy alone. The antitumor effect of CMP-001+anti-PD-1 was accompanied by increased interferon gamma (IFNγ)+ CD4+/CD8+ T cells compared with control-treated mice. The therapeutic and abscopal effect of CMP-001+ anti-PD-1 therapy was completely abrogated by CD8+ T cell depletion.ConclusionsThese results demonstrate that in situ vaccination with CMP-001 can induce both local and abscopal antitumor immune responses. Additionally, the antitumor efficacy of CMP-001 combined with α-PD-1 therapy warrants further study as a novel immunotherapeutic strategy for the treatment of HNSCC.

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Combining In Situ Vaccination with Immune Checkpoint Blockade Induces Long-Term Regression of Lymphoma Tumors

Blood ◽

10.1182/blood.v128.22.465.465 ◽

2016 ◽

Vol 128 (22) ◽

pp. 465-465

Author(s):

Linda Hammerich ◽

Thomas A. Davis ◽

Tibor Keler ◽

Andres M Salazar ◽

Joshua Brody

Keyword(s):

T Cells ◽

Tumor Cells ◽

Research Funding ◽

Tumor Regression ◽

Low Grade ◽

Blocking Antibody ◽

Equity Ownership ◽

In Situ Vaccine

Abstract Background Low-grade non-Hodgkin's B-cell lymphomas are generally incurable, with standard therapies inducing only temporary remissions. Preliminary results with anti-PD-1 therapy have yielded low response rates, though tumor-targeted vaccines represent promising, novel treatment strategies. In a pre-clinical mouse model, we attempt to develop and optimize an in situ vaccine combining recruitment of dendritic cells (DC) and low-dose local radiotherapy (XRT) with intratumoral (i.t) administration of a toll-like receptor (TLR) agonist Methods A20 lymphoma-bearing mice were injected i.t. with FMS-like tyrosine kinase-3 ligand (Flt3L) daily for 9 days (30ug/mouse), followed by local XRT (9Gy) and i.t. injections of poly-ICLC (50ug/mouse) for 5 days. Leukocyte accumulation in tumors, lymph nodes, and spleens was analyzed by flow cytometry and animals were monitored for tumor growth and survival. To assess uptake of tumor antigens by DC, mCherry-expressing A20 cells were used. For assessment of systemic anti-tumor response tumors were inoculated on both flanks, but only one site was treated as described before. In some groups, anti-PD-1 blocking antibody was injected systemically during vaccination. Results Injection of Flt3L induced potent accumulation of DC at the tumor site, tumor-draining lymph node (TDLN) and the spleen, with intratumoral injection being superior to systemic injection in increasing intratumoral and TDLN DCs. Interestingly, Flt3L-treatment led to an 8-fold increase in TLR3+ DC in the tumor. Local XRT increased the amount of mCherry+ DC in the tumor, indicating enhanced uptake of dying tumor cells. XRT of A20 cells also induced activation of Flt3L-treated splenic DC in vitro. While combination of FLt3L and local XRT was not able to cure established tumors, the combination of Flt3L and XRT with poly-ICLC induced long-lasting tumor regression in 40% of mice as well as regression of untreated tumors. This was accompanied by induction of tumor-reactive, Interferon γ (IFN γ)-producing T cells. Of note, the combination of Flt3L and XRT increased expression of PD-1 and PD-L1 on tumor infiltrating T cells and tumor cells, respectively. Consistently, systemic treatment with a PD-1 blocking antibody significantly enhanced the efficacy of the Flt3L-primed in situ vaccine leading to complete tumor regression at the treated site and a significant survival benefit compared to the in situ vaccine without PD-1 blockade. PD-1 blockade also increased the number of tumor-reactive T cells. Conclusions In situ vaccination combining intratumoral Flt3L injection with local XRT, poly-ICLC and anti-PD-1 induces a potent anti-tumor immune response able to induce long-term regression of established lymphoma tumors. Disclosures Davis: Celldex Therapeutics: Employment. Keler:Celldex Therapeutics: Employment, Equity Ownership. Salazar:Oncovir Inc: Employment. Brody:Gilead: Honoraria, Other: Travel expenses, Speakers Bureau; Acerta Pharma: Research Funding; Immunogen: Equity Ownership; Pharmacyclics: Honoraria, Other: Travel expenses, Speakers Bureau; Novavax: Equity Ownership; Merck: Consultancy, Research Funding; Seattle Genetics: Consultancy; Synergy Pharmaceuticals: Equity Ownership.

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Faculty Opinions recommendation of In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727521981.793532240 ◽

2017 ◽

Author(s):

Surender Kharbanda

Keyword(s):

T Cells ◽

Synthetic Dna

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A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

Scientific Reports ◽

10.1038/s41598-021-90096-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Katherine E. Harris ◽

Kyle J. Lorentsen ◽

Harbani K. Malik-Chaudhry ◽

Kaitlyn Loughlin ◽

Harish Medlari Basappa ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Bispecific Antibody ◽

T Regulatory Cells ◽

Tumor Regression ◽

Interleukin 2 ◽

In Vivo Studies ◽

Preferential Binding

AbstractThe use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

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Alpha-Fetoprotein- and CD40Ligand-Expressing Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers13133375 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3375

Author(s):

Annabelle Vogt ◽

Farsaneh Sadeghlar ◽

Tiyasha H. Ayub ◽

Carlo Schneider ◽

Christian Möhring ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Dendritic Cells ◽

Tumor Regression ◽

Combined Therapy ◽

Term Survival ◽

Effector Cells ◽

Tumor Environment ◽

The Impact

Dendritic cells (DC) as professional antigen presenting cells are able to prime T-cells against the tumor-associated antigen α-fetoprotein (AFP) for immunotherapy of hepatocellular carcinoma (HCC). However, a strong immunosuppressive tumor environment limits their efficacy in patients. The co-stimulation with CD40Ligand (CD40L) is critical in the maturation of DC and T-cell priming. In this study, the impact of intratumoral (i.t.) CD40L-expressing DC to improve vaccination with murine (m)AFP-transduced DC (Ad-mAFP-DC) was analyzed in subcutaneous (s.c.) and orthotopic murine HCC. Murine DC were adenovirally transduced with Ad-mAFP or Ad-CD40L. Hepa129-mAFP-cells were injected into the right flank or the liver of C3H-mice to induce subcutaneous (s.c.) and orthotopic HCC. For treatments, 106 Ad-mAFP-transduced DC were inoculated s.c. followed by 106 CD40L-expressing DC injected intratumorally (i.t.). S.c. inoculation with Ad-mAFP-transduced DC, as vaccine, induced a delay of tumor-growth of AFP-positive HCC compared to controls. When s.c.-inoculation of Ad-mAFP-DC was combined with i.t.-application of Ad-CD40L-DC synergistic antitumoral effects were observed and complete remissions and long-term survival in 62% of tumor-bearing animals were achieved. Analysis of the tumor environment at different time points revealed that s.c.-vaccination with Ad-mAFP-DC seems to stimulate tumor-specific effector cells, allowing an earlier recruitment of effector T-cells and a Th1 shift within the tumors. After i.t. co-stimulation with Ad-CD40L-DC, production of Th1-cytokines was strongly increased and accompanied by a robust tumor infiltration of mature DC, activated CD4+-, CD8+-T-cells as well as reduction of regulatory T-cells. Moreover, Ad-CD40L-DC induced tumor cell apoptosis. Intratumoral co-stimulation with CD40L-expressing DC significantly improves vaccination with Ad-mAFP-DC in pre-established HCC in vivo. Combined therapy caused an early and strong Th1-shift in the tumor environment as well as higher tumor apoptosis, leading to synergistic tumor regression of HCC. Thus, CD40L co-stimulation represents a promising tool for improving DC-based immunotherapy of HCC.

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