Combination Of anti-PD1 Research Articles

Abstract B67: Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo

Abstract Recent success of T cell based cancer immunotherapy approaches like chimeric antigen receptor T cells (CAR-Ts) and especially bispecific T cell engagers (BiTEs) has suggested great potential for cancer therapy. However, these technologies have several drawbacks like high risks for side-effects, such as autoimmunity, as well as inappropriate T cell activation due to a non-natural synapse. Hence, we sought to use antibody-mediated delivery of viral peptides to tumor cells in order to mimic viral infection and induce tumor cell lysis by virus-specific cytotoxic CD8+ T cells. Here, we generated immunoconjugates called antibody-targeted pathogen-derived peptides (ATPPs), where mature virus-derived MHC class I peptides from Influenza A or Epstein-Barr virus were conjugated to a tumor-associated antigen-specific antibody via a disulfide bond. Using optical imaging in combination with fluorescence resonance energy transfer, we revealed that after ATPP binding to the target antigen and subsequent internalization, the peptides are released in endosomes and loaded onto recycling MHC class I complexes. These peptide-MHC complexes are subsequently presented on the cell surface and mediate activation of human peptide-specific CD8+ T cells as shown by Interferon-γ release using enzyme-linked immunosorbent assay. Moreover, ATPP-loaded cancer cells from different tumor types were efficiently killed in vitro by human CD8+ T cells in a dose-dependent manner, reaching up to 100% killing after 24h at 0,13nM with an effector-to-target ratio of 3:1. Tumor cell death was assessed by the xCELLigence system as well as lactate dehydrogenase quantification in the supernatant. Interestingly, ATPPs were about 1000-times more potent than free peptide, while antibody alone, a non-cleavable ATPP construct, as well as peptides conjugated to a non-binding antibody exhibited no effects. Importantly, all used antibodies carried the P329G/LALA mutation for abolished Fc-receptor binding. In addition, we performed an experimental subcutaneous xenograft in vivo study with PDL1-expressing MDA-MB231 breast cancer cells in NOG mice in combination with anti-PD1 treatment. We observed that ATPPs successfully recruited adoptively transferred human peptide-specific CD8+ T cells into the tumor and induced 60% tumor growth inhibition of an established tumor after 3 weeks in the anti-PD1 combination setting. Altogether, we generated first in class immunoconjugates for antibody-targeted loading of cancer cells with foreign MHC-I restricted peptide antigens. Our results demonstrate potent ATPP-mediated antitumor efficacy in vitro and in vivo, by loading tumor cells with viral peptide antigens and redirecting virus-specific cytotoxic T cells against cancer. Citation Format: Julian Peter Sefrin, Lars Hillringhaus, Valeria Lifke, Alexander Lifke. Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B67.

Abstract PR08: Antitumor activity and immune correlates of PEGylated human IL-10 (AM0010) alone or in combination with anti-PD-1

Abstract Background: The success of and the durability of immune therapy of cancer is thought to depend on the activation and expansion of tumor reactive and infiltrating CD8+ T cells. The response to immune checkpoint blockade, depends on a pre-existing, CD8 T cell-rich tumor microenvironment. IL-10 stimulates the antigen mediated cytotoxicity, survival and proliferation of intra-tumoral CD8+ T cells and simultaneously dampens chronic inflammation. T cell receptor mediated activation of CD8 T cells induces the expression of IL-10 receptors on these cells. IL-10 activates with STAT3 an essential survival and proliferation signal in antigen activated CD8 T cells. This also provides a mechanistic rationale for combining AM0010 and anti-PD1 in the clinic. To evaluate the clinical activity, tolerability and anti-tumor activity of AM0010 alone or in combination with chemotherapy or immune checkpoint inhibitors a multi-basket phase 1 study was conducted. Additional disease specific expansion cohorts for the combination of AM0010 with FOLFOX in pancreatic cancer or with nivolumab in RCC or NSCLC are currently evaluated Results: Tolerability and anti-tumor activity of AM0010 alone or in combination with chemotherapy or immune checkpoint inhibitors was established in this multi-basket phase 1 study. In monotherapy, objective responses were observed in pts with uveal melanoma, cutaneous T cell lymphoma and in 4 of 15 pts with RCC. Patients with advanced melanoma, RCC or NSCLC were also treated with AM0010 (daily SC) in combination with anti-PD-1 immune checkpoint blockade. Tumor responses were monitored following irRC. Immune responses were measured by analysis of serum cytokines, activation of blood derived T cells, peripheral T cell clonality and immunohistochemistry of tumor infiltrating CD8 T cells. In 19 pts, AMO010 10 μg/kg (n = 13) or 20 μg/kg (n = 6) in combination with anti-PD1 - pembrolizumab (2mg/kg) was well tolerated (observation period 10-15 months). Immune related TrAE occured in the frequency and severity as expected from pembrolizumab montherapy. The combination of AM0010 with pembrolizumab achieved objective responses (PR/CR) in 4 of 8 RCC pts, 2 of 5 NSCLC pts and 2 of 6 melanoma pts. 2 additional melanoma pts had tumor increase followed by decrease (pseudoprogression). Independent of the combination with either chemotherapy or anti-PD-1, AM0010 increased Th1 cytokines (IL-18, IFNγ, IL-7) in a dose dependent fashion. FasL and lymphotoxin beta - products of cytotoxic T cells - were also increased in the serum of AM0010 treated patients. In contrast, mediators of chronic inflammation, such as IL-23 and IL-17 and the immune suppressive cytokine TGFbeta were reduced in the serum of patients. AM0010 increased the number and proliferation of PD1+ activated CD8 T cells while decreasing the proliferation of FoxP3+ Tregs and TGFβ in the blood. AM0010 induced de-novo oligoclonal expansion of T cell clones in the blood of patients without affecting total lymphocyte counts. This clonal expansion appeared enhanced and accelerated in patients treated with a AM0010 anti PD-1 combination, but was also seen in patients which received a AM0010 - chemotherapy combination. AM0010 also increased the number of tumor infiltrating Phospho-STAT3+ CD8 T cells in tumors and the number of Granzyme+ PD1+ CD8+ T cells in tumor biopsies of treated patients. Conclusion: AM0010 alone or in combination with anti-PD1 is well-tolerated. The clinical activity and the observed CD8 T cell activation encourages the phase 2/3 studies of AM0010 in combination with anti-PD1 planned for later in 2016. Trial registration: www.clinicaltrials.gov NCT02009449. Citation Format: Aung Naing, Jeffrey R. Infante, Kyriakos P. Papadopoulos, Deborah J. Wong, Karen A. Autio, Gerald S. Falchook, Manish Patel, Shubham Pant, Amita Patnaik, Melinda Whiteside, Johanna C. Bendell, John Mumm, Ivan H. Chan, Gail L. Brown, Peter VanVlasselaer, J. R. Hecht, David S. Hong, Nizar M. Tannir, Martin Oft. Antitumor activity and immune correlates of PEGylated human IL-10 (AM0010) alone or in combination with anti-PD-1 [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR08.

Interferon-γ Production by Peripheral Lymphocytes Predicts Survival of Tumor-Bearing Mice Receiving Dual PD-1/CTLA-4 Blockade

Immune checkpoint inhibitors are transforming the way cancer is treated. However, these therapies do not benefit all patients and frequently cause significant immune-related adverse events. Biomarkers that identify patients with a favorable early response to therapy are essential for guiding treatment decisions and improving patient outcomes. In this report of our study, we present evidence that shortly after administration of dual PD-1/CTLA-4 blockade, the proinflammatory capacity of peripheral lymphocytes is predictive of tumor progression and survival outcomes in multiple murine models. Specifically, we observed that the quantity of interferon-γ (IFNγ) produced by peripheral lymphocytes in response to CD3/CD28 stimulation was robustly correlated with subsequent survival outcomes. In the tumor models and early time points assessed in this study, this relationship was considerably more predictive than a host of other potential biomarkers, several of which have been previously reported. Overall, these findings suggest that measuring the capacity of peripheral lymphocytes to produce IFNγ may help identify which patients are benefitting from combination anti-PD-1/anti-CTLA-4 immunotherapy. Cancer Immunol Res; 4(8); 650-7. ©2016 AACR.

Abstract LB-052: Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model

Abstract We demonstrate for the first time that blocking of the programmed death-1 (PD-1) pathway in conjunction with immunogenic cell death induced by CpG-ODN and activatable nanodelivery of doxorubicin can generate curative responses in both primary and contralateral tumors. Activatable nanotherapeutics are attractive since the toxicity of chemotherapeutics can be constrained to a small region; combining such a strategy with immunotherapy is the goal of this study. We have previously shown that administration of CpG-ODN as an adjuvant, together with local release of doxorubicin from temperature sensitive liposomes (TSL) resulted in regression of directly-treated tumors, suppressed growth of contralateral tumors and reduced chemotherapeutic-mediated toxicity in a murine breast cancer model.1 Increases in cytotoxic CD8+ T lymphocytes and a reduction in regulatory T cells and myeloid-derived suppressor cells were observed in both directly treated and contralateral tumors. This combinatorial approach was curative for directly-treated tumors and overall survival was significantly extended, however, the contralateral tumor returned in all treated mice. The following is the protocol explored for the addition of anti-PD1: immune intact FVB/n mice with bilateral invasive neu deletion syngeneic transplanted tumors were treated with a combination of anti-PD1 (aPD-1, 200 μg, i.p.) and intratumoral administration of CpG-ODN (100 μg, i.t.) on days 0, 7, 14 and 0, 3, 7, 10, 17 and 24, respectively. Doxorubicin TSL were prepared from DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-drug/mg-lipid and administrated i.v. at 6 mg doxorubicin/kg body weight on days 10, 17, 24. The formation of a complex between doxorubicin and copper was created to enhance the circulation and stability of TSL and to reduce systemic toxicity. To trigger drug release, hyperthermia was induced in the primary tumor with ultrasound (peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz) at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Immediately afterwards, 100 μg of CpG-ODN 1826 was administered intratumorally to the insonified tumor. Upon treatment with this combination of locally-released doxorubicin, local administration of CpG-ODN and systemic aPD-1, 100% of treated and contralateral tumors regressed by at least 80%; further, all of the directly-treated tumors and 50% of the contralateral tumors were eliminated without recurrence. Thus, a 50% complete response rate was achieved, with tumor regression observed immediately after the incorporation of the doxorubicin treatment. By contrast, administration of CpG-ODN and systemic aPD-1 alone resulted in regression of 66% of treated and contralateral tumors. *MS and AK contributed equally to this work. 1. J Control Release (2015); 220: 253-264. Citation Format: Matthew T. Silvestrini, Azadeh Kheirolomoom, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Josquin Foiret, Samantha Tucci, Neil E. Hubbard, Alexander D. Borowsky, Katherine W. Ferrara. Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-052.

Imprime PGG, a yeast β-glucan PAMP elicits a coordinated immune response in combination with anti-PD1 antibody

Abstract Imprime PGG (Imprime), a b glucan PAMP is currently in clinical development in combination with tumor-targeting antibodies, anti-angiogenic antibodies and immune checkpoint inhibitors. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in terms of both objective tumor response and survival. As a PAMP, Imprime is readily recognized by innate immune cells, triggering immune responses including repolarization of M2 macrophages and dendritic cell (DC) maturation. Earlier results have shown that both the M2 macrophages and DC derived from Imprime-treated monocytes have higher expression of PD-L1 and CD86. Since PD-L1 expression, a marker for adaptive immune resistance, and CD86, a co-stimulatory marker for T cell activation could potentiate the activity of anti-PD1 antibody, we hypothesized that the combination will show synergistic T cell stimulatory activity. Ex- vivo treatment of T cells with anti-PD1 antibody, nivolumab showed increased proliferation in response to CD3/CD28 stimulation. Coculture of these T cells with Imprime-treated M2s or DCs drove further T cell expansion and increased production of several cytokines including IFN-γ, IL-2, TNF-α, GM-CSF, and IP-10. We next evaluated the anti-tumor efficacy of Imprime and murine anti-PD1 in a syngeneic tumor model. In the CT-26 syngeneic model, the median tumor volume in the anti-PD1 alone treatment group was 936 mm3, while it was only 172 mm3 in the Imprime and anti-PD1 combination group. Collectively, these data suggest that the combination of Imprime and checkpoint inhibitors holds a lot of promise for cancer immunotherapy.

Successful Immunotherapy against a Transplantable Mouse Squamous Lung Carcinoma with Anti–PD-1 and Anti-CD137 Monoclonal Antibodies

IntroductionAnti–programmed cell death 1 (anti–PD-1) and anti–programmed cell death ligand 1 (PD-L1) antagonist monoclonal antibodies (mAbs) against metastatic non–small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. MethodsWe generated a transplantable squamous cell carcinoma cell line (UN-SCC680AJ) from a lung tumor induced by chronic N-nitroso-tris-chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti-CD137 and/or anti–PD-1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. ResultsIn syngeneic mice, large 12-day–established tumors derived from the transplantable cell line variant UN-SCC680AJ were amenable to curative treatment with anti–PD-1, anti–PD-L1, or anti-CD137 immunostimulatory mAbs. Single-agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti–PD-1 plus anti-CD137 achieved complete rejections. Tumor cells expressed weak baseline PD-L1 on the plasma membrane, but this could be readily induced by interferon-γ. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co-expressing CD137 and PD-1 were prominent. ConclusionsThese promising results advocate the use of combined anti–PD-1/PD-L1 plus anti-CD137 mAb immunotherapy for the treatment of squamous non–small cell lung cancer in the clinical setting.

Abstract 255: Dual targeting of Delta-like ligand 4 (DLL4) and programmed death 1(PD1) inhibits tumor growth and generates enhanced long-term immunological memory

Abstract Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cells (CSC) population. We are currently testing an anti-DLL4 antibody, demcizumab, in Phase1B trials in NSCLC, pancreatic, and ovarian cancer. DLL4 is also known to modulate immune responses. In the current study we examine the impact of anti-DLL4 on anti-tumor immune responses as a single agent and in combination with the key immune checkpoint inhibitor Programmed Cell Death Protein 1 (PD1). While the recent clinical success of PD1 inhibitors represents a new and promising cancer immunotherapeutic approach, high initial response rates are often associated by a lack of long-term, durable effects in a significant number of patients. Therefore, we hypothesized that dual blockade of DLL4 and PD1 might further impact tumor growth by further enhancing anti-tumor immune immunity. Our data demonstrates that dual blockade of DLL4 and PD1 using antibodies not only reduces tumor growth, but also led to tumor rejection in ∼50% in CT26WT tumor-bearing mice, similar to those treated with anti-PD1 alone (no tumor rejection was observed with anti-DLL4 alone). Anti-PD1 increased specific CD8+ T cell-mediated IFN-γ production while decreasing IL6. Anti-DLL4 treatment reduced IL17 production. Interestingly, only the dual blockage led to increased production of IL2 by splenocytes. Since IL2 is required for secondary population expansion of CD8+ memory T cells, increased IL2 in the combination group suggests potential for increased T cell activation, maintenance and memory T cell function, as compared to single agent anti-DLL4 and anti-PD1. While anti-PD1 reduced inhibition of CD4+ T cell proliferation by Tregs, the dual blockade significantly reduced Treg-mediated CD8+ T cell suppression. Furthermore, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population. Interestingly, anti-PD1 decreased granulocytic MDSCs, while anti-DLL4 reduced monocytic MDSCs. Mice cured with single-agent anti-PD1 and anti-DLL4/anti-PD1 combination treatments were protected from series of re-challenge with tumor cells, suggesting the existence of immunologic memory. Interestingly, more mice were protected from tumor re-challenge when both DLL4 and PD1 were blocked, as compared to PD1 alone. Surprisingly, mice previously treated with the anti-DLL4/anti-PD1 combination produced more IL2, clearly indicating the role of DLL4 blockade in enhancing anti-tumor immunity. Therefore, these results show that dual targeting of DLL4 and PD1 may be an effective and durable cancer therapy by increasing anti-tumor immune response and promoting long-term immunological memory. Citation Format: Minu Srivastava, Christopher L. Murriel, Julie Roda, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Rui Yun, Erin Mayes, Trevor Bentley, Belinda Cancilla, Raymond Tam, Tracy Tang, Ann Kapoun, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Dual targeting of Delta-like ligand 4 (DLL4) and programmed death 1(PD1) inhibits tumor growth and generates enhanced long-term immunological memory. [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 255. doi:10.1158/1538-7445.AM2015-255

Phase 1 study of pembrolizumab (pembro; MK-3475) plus ipilimumab (IPI) as second-line therapy for advanced non-small cell lung cancer (NSCLC): KEYNOTE-021 cohort D.

8011 Background: Pembro is a potent anti–PD-1 monoclonal antibody. IPI, an anti–CTLA-4 antibody, has shown activity in advanced NSCLC. In melanoma, combined anti–PD-1 and anti–CTLA-4 treatment has shown robust efficacy and manageable toxicity. We report interim results from a phase 1 study evaluating pembro + IPI in patients (pts) with recurrent NSCLC. Methods: Pts with stage IIIB/IV NSCLC that recurred after ≤ 2 prior regimens received pembro + IPI every 3 wk for 4 cycles followed by maintenance pembro. Based on emerging data from the nivolumab + IPI advanced NSCLC study, doses were reduced from 10 mg/kg to 2 mg/kg for pembro and from 3 mg/kg to 1 mg/kg for IPI). Primary end point was safety and incidence of dose-limiting toxicities (DLTs) in the first 3 wk of dosing. Response was assessed every 6 wk per RECIST 1.1 by investigator review. Results: As of Dec 2014,17 pts were enrolled: 3 at pembro 10 mg/kg + IPI 3 mg/kg, 3 at pembro 10 mg/kg + IPI 1 mg/kg, and 11 at pembro 2 mg/kg + IPI 1 mg/kg. No DLTs or...