PD-1 Ab Research Articles

Abstract PR10: Small-molecule inhibitors of CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumors

Abstract Introduction: Intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, requires the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to ADO). For this reason, various anti-CD73 antibodies are being advanced into clinical trials; however, there are few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Enzymatic assays: Ecto-nucleotidase activity was calculated based on the amount of inorganic phosphate (malachite green assay; absorbance at 620 nm) produced after 50-min incubation with 25μM AMP or ATP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; mCD73/E771 cells. Stable over-expression: hCD73/CHO; hCD39/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. CD8+ T cell functional assays: Human (enriched from buffy coats or leukopaks) and mouse (isolated from spleen of C57BL/6 mice) CD8+ T cells were pretreated with CD73 inhibitor or buffer control. One hour later, cells were stimulated (anti-CD3/CD28; +/- 50 μM AMP) in the presence of ADO deaminase inhibitor (10 μM). 3 days after stimulation, cell activation (CD25), proliferation (CFSE) and effector function (IFN-γ and granzyme B) were quantified by flow cytometry. Tumor model: CT26 cells were subcutaneously implanted into Balb/c mice. When tumor volumes were ∼100 mm3, mice were enrolled into cohorts (1: vehicle control; 2: A000830; 3: PD-1 Ab; 4: A000830 + PD-1 Ab) of ≥13 mice each. Interim immune phenotyping was performed when Group 1 tumor volumes were 800-1,000 mm3. Single-cell suspensions were generated from tumors and spleens (gentleMACS). 106 cells were blocked using purified CD16/32 antibodies (clone 2.4G2) and stained with the appropriate antibody cocktails. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 (IC50 against human and mouse CD73 of 1.0 nM and 3 nM, respectively). A000830 blocked ADO generation from AMP by human ovarian cancer cells (SKOV-3) with IC50: 0.2 nM and >10,000-fold selectivity relative to other ecto-nucleotidases (CD39, NTPDase2, NTPDase3, NTPDase8) and a large panel of unrelated enzymes, receptors and ion channels. In in vitro models of ADO-driven inhibition of human and mouse CD8+ T-cell activation, A000830 showed robust rescue of proliferation, CD25 expression, and IFNγ and granzyme B production. A000830 was easily administered (and well tolerated) to mice, resulting in sustained plasma concentrations at least 1,000 times higher than its potency against mouse CD73. In the spleen of CT26 tumor-bearing Balb/c mice, CD39 expression was found mainly on natural killer (NK) cells and myeloid-lineage cells, while in the tumor CD39 was highly expressed on myeloid cells (myeloid-derived suppressor cells (MDSC) and dendritic cells (DC)) as well as CD4+ and CD8+ T cells. CD73 expression was limited (spleen and tumor) to NK cells and CD4+ and CD8+ T cells. Therapeutic dosing of A000830 to these mice (tumors ∼100 mm3 in size) in combination with anti-PD1 antibody produced robust tumor growth inhibition, greater than either treatment alone. This effect was associated with increased CD8:Treg ratios in tumors (but not spleens). Conclusions: We describe a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human and mouse T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Juan C. Jaen, Jay P. Powers, Ada Chen, Manmohan R. Leleti, Jarek Kalisiak, Ulrike Schindler, Joanne Bl Tan, Steve Young, Shin-Heng Chiou, Laurent Debien. Small-molecule inhibitors of CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumors [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 PR10.

Abstract LB-218: Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics

Abstract Purpose: Recent clinical studies have demonstrated that immunotherapeutics, such as checkpoint inhibitors are a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still required. Axl and Mer, member of the TAM receptor tyrosine kinase family are known to be over-expressed in various types of hematological and solid tumor cancers and have been reported as poor prognostic factors. ONO-9330547 is a highly potent and selective oral Axl/Mer inhibitor. Previous study with ONO-9330547 demonstrated both increase proportion of CD8+ T cell and alternation of immunosuppressive microenvironment in syngeneic tumor graft model (Tanaka AACR 2015). Thus, we next went on to examine whether combining ONO-9330547 with anti-PD1 antibody has the potential to improve efficacy in a mouse colon cancer MC38 syngeneic model. Methods: MC38 tumor cells, a mouse colon cancer cell line, were implanted into female C57/B6 mice. Randomization of mice occurred when mean tumor volume reached 150 mm3. ONO-9330547 was fed in a diet containing 0.0013 or 0.013% ONO-9330547 (equivalent to 2 or 20 mg/kg/day). Anti-mouse PD-1 Ab (4H2) was administered intraperitoneally at a dose of 10 mg/kg every 6 days for 28 days. Tumor volumes were determined using the formula volume ( = width2 x length)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3. QPCR was used to determine the TAM ligands, Gas6 and Pros1. Results: Treatment with ONO-9330547 combined with 4H2 resulted in a significantly improved inhibition of tumor growth in the MC38 syngeneic model. In contrast, treatment of 4H2 monotherapy showed inferior anti-tumor activity compared with combination therapy. In particular, ONO-9330547 at 2 or 20 mg/kg/day combined with 4H2 resulted in tumor remission in 3/9 or 6/9 animals at day 28, respectively, whereas each monotherapy resulted in tumor remission in only 1/9 animals. Interestingly, the treatment with 4H2 significantly increased TAM ligands mRNA levels, Gas6 and Pros1 in tumor. Conclusion: Our results demonstrate that ONO-9330547, a highly potent dual Axl/Mer inhibitor, shows synergistic anti-tumor effect when combined with anti-PD-1 antibody which induces the expression of TAM ligands. These data support the concept that the immunomudulatory effect of ONO-9330547 is further enhanced when ONO-9330547 is combined with immunotherapy. Rational combination with immunotherapeutics is worth further investigation in the clinical setting. Citation Format: Toshio Yoshizawa, Kohei Tanaka, Tomoko Yasuhiro, Ryu Fujikawa, Suiho Ri, Kazuhito Kawabata. Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics. [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-218.

Synergistic Combination of PD-1 and EGFR Antibodies: A Proposed Phase 1 Clinical Trial Using Nivolumab and Cetuximab

Cetuximab induces head and neck cancer cell death through ADCC mediated by NK cells. NK cells when activated by the Fc exposed region of cetuximab bound to EGFR increase production of IFN-gamma. The high levels of IFN-gamma produced dendritic cell maturation and also increase CD8 T cell expression of PD1. The combination of an EGFR mab with a checkpoint (PD1) inhibitor may be synergistic through an enhancement of CD8 T cell activation and dendritic cell engagement which will increase cancer cytolysis. Based on this preclinical data, we proposed a phase 1 clinical trial exploring the combination of cetuximab plus nivolumab in recurrent head and neck cancer patients. The goal of the trial is to identify the maximal tolerated dose (MTD) and the recommended phase 2 dose (RP2D). Secondary endpoints would be the assessment of response rate, progression-free survival (PFS), and overall survival (OS). Patients with recurrent head and neck cancers will be eligible. We recommended a phase 1 clinical trial with a 3+3 dose escalation design as described here: (1) dose Level 1: cetuximab 200 mg/m2 IV loading dose followed by weekly cetuximab 125 mg/m2 IV nivolumab 1.5 mg/kg IV every 14 days; (2) dose Level 2: cetuximab 300 mg/m2 IV loading dose followed by weekly cetuximab 187.5 mg/m2 IV nivolumab 2.25 mg/kg IV every 14 days; and (3) dose Level 3: cetuximab 400 mg/m2 IV loading dose followed by weekly 250 mg/m2 IV nivolumab 3 mg/kg IV every 14 days. Since cetuximab as single agent has a response rate of 10% to 20% as a single agent in metastatic head and neck cancer, we anticipate a response rate of 30% with the proposed combination therapy. Since the agents do not have overlap toxicity, we do not anticipate major toxicities. PD-1 and EGFR Ab therapy may be synergistic, and further exploration in a clinical setting is proposed on this abstract.

Tumor immunologic heterogeneity influences response to radiation and combination immunotherapy

Clinical trials of CTLA-4 and PD-1/PD-L1 antibodies (Ab) have shown responses limited to 10-30% of patients. Increasing evidence suggests radiation (RT) can enhance responses to checkpoint therapies. However, understanding of tumor-derived factors that influence response is limited, and many preclinical models are designed to be immunogenic. We sought to develop a model of immunologic heterogeneity to study factors that influence responses to RT and immunotherapy. Two tumor clones Py117 and Py8119 derived from the PyMT mammary carcinoma in C57Bl/6 mice engraft efficiently and upon RT the Py117 but not Py8119 tumors regressed. There was no significant difference detected in radiosenstivity by clonogenic survival, hypoxia by pimonidazole staining, or angiogenesis through Meca32 staining. Tumor dissociation 10 days after 12 Gy treatment revealed a significant increase in CD45+ leukocytes, CD8+ T cells and decreased CD4+ T cells, myeloid derived suppressor cells, and macrophages in Py117 tumors but minimal changes in Py8119 tumors. Secondly, there was elevated MHCI and induced PD-L1 expression in Py117 compared to Py8119 tumors suggesting a greater response may be obtained with immunotherapy. Mice treated with 12 Gy, 12Gy + PD-1 Ab, and a single dose of CTLA-4 Ab 3 days prior to RT and PD-1 therapy revealed prolonged response with RT + PD-1 and RT + PD-1 + CTLA-4 groups, significant by repeated measures analysis. Notably 2/8 tumors treated with RT + PD-1 and 6/8 mice treated with RT + PD-1 +CTLA-4 were undetectable at day 81. Implantation of tumors with different ratios of Py117 and Py8119 cells revealed that 50% inoculation of Py117 cells leads to an immune response against both tumor clones characterized by greater tumor MHCI expression, CD8+ T cell infiltration and decreased macrophage infiltration. We hypothesized that treating the unresponsive Py8119 tumors with CD40 agonist would replicate the findings of the mixing experiment. CD40 Ab in combination with 20 Gy RT resulted in a greater antitumor response with significant decrease in tumor size not seen in either treatment alone. Evaluation of the tumor microenvironment revealed MHCI expression was enhanced, macrophage infiltration was decreased and CD8+ T cells were increased. These results support that tumor immunologic heterogeneity can influence immune responses after radiation and targeting suppressive factors in the tumor microenvironment can enhance responses. This model of heterogeneity without genetic manipulation is ideal to study tumor-derived factors that enhance or suppress the immune response after RT and could inform clinical approaches to radiation and immunotherapy combinations.

Anti-PD-1 mAb pre-radiotherapy (RT) loading dose and fractionated RT induce better tumor-specific immunity and tumor shrinkage than sequential administration in an HPV+ head and neck cancer model

Radiotherapy (RT) is a standard therapeutic strategy in the treatment in head and neck cancer (HNC), but many patients still experience recurrence and metastasis. Interestingly, radiotherapy (RT) may also induce immunomodulatory effects. Given the recent, exciting responses seen using anti-PD-1 (programmed death-1) checkpoint blockade immunotherapy in recurrent/metastatic disease, including HNC, we evaluated the combination of RT with anti-PD-1 therapy in a pre-clinical mouse model of locally advanced, untreated HPV-positive HNC. We compared utilizing PD-1 blockade before, during or after RT, as well as whether a single large faction (12Gy) or multiple smaller RT doses (2 Gy X 10 fractions) confers optimal antitumor immune responses and tumor shrinkage. We observed that fractionated doses of RT induced the highest PD-L1 (programmed death-ligand 1) expression on HNC cells in vitro and in treated mice. A loading dose of anti-PD-1 mAb therapy prior to RT appeared to be important for best therapeutic outcome, with greatest tumor response and tumor-specific immunity using PD-1 Ab loading dose than sequential administration of anti-PD-1 mAb after fractionated RT (p < 0.0001). Expression and intensity of PD-1 receptor expression on circulating T cells differentially impacted the T cell phenotype and anti-tumor outcome, with loss of PD-1(high) exhausted T cells during the best tumor response (p < 0.05). The combination of fractionated RT + anti-PD-1 Ab optimally upregulated the frequency of HPV E7 tumor antigen-specific T cells (p < 0.05). This study may facilitate strategies required for the combination of RT and immune checkpoint inhibitor in clinical trials, enabling more effective clinical activity and biomarker evaluation.

Abstract 266: Antitumor activity of REGN2810, a fully human anti-PD-1 monoclonal antibody, against MC38.Ova tumors grown in immune-competent humanized PD-1 mice

Abstract Programmed Cell Death Protein 1 (PD-1), is an inhibitory checkpoint receptor, expressed in an activation-induced manner on T cells. PD-1 signals upon binding to its ligands PD-L1 and PD-L2, expressed on APCs and some types of cancer cells, and delivers inhibitory signals to T cells, promoting T-cells functional exhaustion in a tumor microenvironment. Clinically, antibodies (Abs) that block the interaction between PD-1 and PD-L1 can release the cytotoxic function of tumor-specific T cells, yielding durable objective responses in multiple cancer types. REGN2810 is a fully human hinge-stabilized IgG4P monoclonal Ab that binds to the extracellular domain of human PD-1 with high affinity and specificity. REGN2810 was selected using a combination of binding studies (Surface Plasmon Resonance [SPR]-Biacore), enzyme-linked immunosorbent assays (ELISA), novel engineered cell-based bioassays, and preclinical studies in mouse models. REGN2810 was investigated in PD-1 humanized mice, an approach that allows direct testing of clinical PD-1 Ab that does not cross-react with mouse PD-1. Humanized PD-1 mice were created by Velocigene technology to express a humanized hybrid PD-1 protein comprising the extracellular portion of human PD-1 and the transmembrane and intracellular portion of mouse PD-1. Humanized PD-1 mice were validated for inducible cell surface expression of hybrid PD-1 protein on activated T-cells, intact PD-1/PD-L1 signaling and immune responses. A weakly immunogenic murine colorectal cancer cell line MC38 was engineered to express ovalbumin (MC38.Ova) in order to increase tumor immunogenicity and allow monitoring of T-cell immune responses to well-defined antigenic ovalbumin peptides. Prophylactic treatment with REGN2810 PD-1 Ab significantly suppressed growth of subcutaneous syngeneic MC38.Ova tumors in humanized PD-1 mice in a dose dependent manner, and improved mouse survival. Therapeutic treatment with REGN2810 also promoted dose dependent regression of established MC38.Ova tumors in humanized PD-1 mice. Treatment with REGN2810 induced CD8 T cells proliferation and cytokine production in spleens of tumor bearing huPD-1 mice. The mechanism of action and robust anti-tumor efficacy of REGN2810 support its clinical development for the treatment of human cancers. Citation Format: Elena Burova, Omaira Allbritton, William Poueymirou, Venus Lai, Janelle White, Dimitris Skokos, Nicholas Papadopoulos, Drew Murphy, Israel Lowy, Ella Ioffe, Gavin Thurston. Antitumor activity of REGN2810, a fully human anti-PD-1 monoclonal antibody, against MC38.Ova tumors grown in immune-competent humanized PD-1 mice. [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 266. doi:10.1158/1538-7445.AM2015-266

35 The role of toll-like receptor (TLR) agonists in combinational immunotherapy for head and neck cancer

Because of their stimulating effect on manifold components of the innate immune system, TLR-agonists are in the focus of the search for potent drugs in combinational anticancer therapy. They induce the maturation and cross-priming of Dendritic Cells (DC), and have been shown to enhance Natural Killer (NK) cell dependent lysis of tumor cells in combination with monoclonal antibodies (mAb) such as anti-epidermal growth factor receptor (EGFR) cetuximab. One of the reasons that may have limited the full potential of TLR-agonists so far is their sequential induction of suppressive factors as part of the immune system’s balancing component. Peripheral blood mononuclear cells (PBMC), NK, DC and monocytes were isolated and stimulated with different TLR agonists: poly I:C (TLR-3), LPS (TLR-4), VTX-2337 (TLR-8) and CpG (TLR-9). Analysis was performed using flow cytometry, cytokine ELISA, qPCR, T cell stimulation assays with VTX in the presence or absence of anti PD-1 Ab. Furthermore, Tumor-infiltrating Lymphocytes (TIL) of the pre- and post-treatment patient samples of trial patients that undergo a neoadjuvant combinational therapy with the TLR-8 agonist VTX-2337 and anti-EGFR mAb cetuximab were analyzed. TLR-agonists and combinational drugs show an additive effect. TLR8-stimulation enhanced monocyte maturation markers CD80, CD83, and CD86 alone and in co-culture with cetuximab-treated HNC cells. A skewing to the M1 phenotype of monocytes was evidenced by significantly increased secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005). Immature myeloid-derived suppressor cells (MDSC) that were treated with VTX-2337 showed upregulation of maturation markers. Furthermore, the treatment of monocytes with TLR-agonists such as VTX-2337 induced a strong upregulation of the inhibitory marker PD-L1. Our work focuses on the maturational effect of TLR agonists on monocytes, their skewing to an M1 phenotype and on the induction of suppressive pathways through surface molecules such as PD-L1. TLR-agonists contain a strong potential in combinational therapies, including drugs that inhibit the sequential induction of checkpoint receptors (e.g. PD-1 blockade, nivolumab) or show additive immune-enhancing effects (e.g. cetuximab).

MP35-08 TYROSINE-KINASE INHIBITORS TREATMENT INDUCES IL-6 SECRETION ON RENAL CARCINOMA CELLS

then tumor infiltrating lymphocyte was analyzed by immunohistochemistry. RESULTS: All three treatment groups ((1), (2) and (3)) showed significant decrease in tumor size compared with vehicle group. In particular, combination of PD-1 Ab and CTLA-4 Ab exhibited the most significant anti-tumor effect. The percentage of PCNA+ tumor cells were very similar amongst all therapies, but the percentage of Ki-67+ tumor cells were significantly decreased with combination of PD-1 Ab and CTLA-4 Ab. Combination of PD-1 Ab and CTLA-4 Ab demonstrated the most potent effect to trigger the infiltration of both CD4+ lymphocyte and CD8+ lymphocytes. These in vivo results indicate that combination of PD-1 Ab and CTLA-4 Ab may have a significant immunostimulatory effect to exert the anti-tumor effect to renal cell carcinoma. CONCLUSIONS: Combination therapy of anti-CTLA-4 antibody and anti-PD-1 antibody can be a potential therapeutic option for advanced kidney cancer patients.

High PD-1 expression and suppressed cytokine signaling distinguish T cells infiltrating follicular lymphoma tumors from peripheral T cells

AbstractDefects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4–, IL-10–, and IL-21–induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein–specific flow cytometry with several T-cell markers, we identified that CD4+CD45RO+CD62L− FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4+PD-1hi FL TILs, containing TFH and non-TFH cells, had lost their cytokine responsiveness, whereas PD-1− TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1+ histiocytes were found within the T cell–rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1hi subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.