Wild-type interleukin (IL)-2 induces anti-tumor immunity and toxicity, predominated by vascular leak syndrome (VLS) leading to edema, hypotension, organ toxicity, and regulatory T cell (Treg) expansion. Efforts to uncouple IL-2 toxicity from its potency have failed in the clinic. We hypothesize that IL-2 toxicity is driven by cytokine release syndrome (CRS) followed by VLS and that coupling IL-2 with IL-10 will ameliorate toxicity. Our data, generated using human primary cells, mouse models, and non-human primates, suggest that coupling of these cytokines prevents toxicity while retaining cytotoxic T cell activation and limiting Treg expansion. In syngeneic murine tumor models, DK210 epidermal growth factor receptor (EGFR), an IL-2/IL-10 fusion molecule targeted to EGFR via an anti-EGFR single-chain variable fragment (scFV), potently activates T cells and natural killer (NK) cells and elicits interferon (IFN)γ-dependent anti-tumor function without peripheral inflammatory toxicity or Treg accumulation. Therefore, combining IL-2 with IL-10 uncouples toxicity from immune activation, leading to a balanced and pleiotropic anti-tumor immune response.

2589 Background: IL-2 induces anti-tumor immunity and toxicity, predominantly vascular leak syndrome (VLS), leading to edema, hypotension, organ toxicity, and therapy-inhibiting regulatory T cell (Treg) accumulation. DK2 10 (EGFR) couples wild-type IL-2 to a high affinity variant of EBV IL-10 via an scFv that binds to epidermal growth factor receptors. The IL-10 component was designed to block IL-2 mediated cytokine release syndrome (CRS) and VLS while retaining T cell activation and proliferation and limiting Treg expansion. We report the clinical and pharmacodynamic results from the dose escalation in the DEKA-1 phase 1 (NCT05704985) study. Methods: Eligible patients (pts) had advanced/metastatic tumors known to express EGFR, progressive disease on ≥1 lines of systemic treatment, and ECOG ≤1. DK2 10 (EGFR) (2-16 mg; 0.025-0.5 mg/kg for an 80 kg subject) was self-administered subcutaneously 3 times per week in 21-day cycles following a BOIN design. Adverse events (AEs) including serious (SAEs) were evaluated using CTCAE version 5.0. Cytokines and anti-drug antibodies were monitored during the first cycle and every 3 cycles thereafter. RECIST 1.1 tumor responses were evaluated every 9 weeks. Results: 35 pts (14 RCC, 6 NSCLC, 9 CRC, 5 PDAC, 1 SCC) were enrolled. Median age was 63 yrs (range 35-80). Treatment-related AEs (TRAEs; any grade) in ≥ 10% pts were injection site reactions (63%), fever (40%), fatigue (31%), nausea (23%), anemia (17%), chills (17%), eosinophilia (14%), CRS (14%), diarrhea (11%); the majority were G1-2. G3 TRAEs included fatigue (n = 4), anemia (n = 3), syncope (n = 3) and single events of acute kidney injury, cellulitis, hypoalbuminemia, and lymphopenia. No DLTs were observed. While MTD was not exceeded, a dose proportional induction of IFNγ was observed through 8 mg but not at 16 mg. Therefore, higher doses were not explored, and a 12 mg dose level was introduced for dose optimization. No appreciable increase in IL-6, TNF-a, or IL-1β was seen other than 1 subject at 4 mg with G2 CRS and elevated IL-6. IFNγ and IL-5 were concomitantly induced proportional to dose level and reached saturation between levels 3 and 4, consistent with pK exposure of DK2 10 (EGFR). Sustained IL-5 associated eosinophilia was observed and correlated with drug concentration but did not require intervention. IL-2 and IL-10 induction led to sustained elevation of IL-2Ra and IL-18, respectively. 33% of evaluable patients had a best ORR of SD. Conclusions: DK210 (EGFR) demonstrates strong IL-2 activity shown by IL-5 driven eosinophilia, shed IL-2Ra, T and NK cell proliferation and expansion but not Treg accumulation. These effects have been decoupled from IL-2 driven toxicity, confirming the hypothesis of the balancing effect of IL-10. These data support further evaluation in combination with T cell engagers, T cell therapeutics, and kinase inhibitors. Clinical trial information: NCT05704985 .

2584 Background: DK210 (EGFR) couples wild-type IL-2 to a high affinity variant of Epstein Barr Viral (EBV) IL-10 via a scaffold (scFv) that binds to epidermal growth factor receptors (EGFR). We report the dose escalation data from the DEKA-1 phase 1 (NCT05704985) study. Methods: Eligible patients (pts) had advanced/metastatic tumors known to express EGFR, progressive disease on ≥1 lines of systemic treatment, and ECOG ≤1. DK210 (EGFR) (2-16 mg; 0.025-0.5 mg/kg for an 80 kg subject) was self-administered subcutaneously (SC) three times per week (TIW) as an outpatient in 21-day cycles following a BOIN design. Adverse events (AEs) including serious (SAEs) were evaluated using CTCAE version 5.0. Cytokines and anti-drug antibodies were monitored during the first cycle, and then every 3 cycles thereafter. RECIST 1.1 tumor responses were evaluated every 9 weeks. Results: 17 pts (8 CRC, 5 PDAC, 4 NSCLC) were enrolled at 4 dose levels (2-16 mg TIW). Median age was 70 yrs (range 46-75) and 5 pts (29%) received previous immunotherapy. Median time on treatment was 9 wks (range: 1-30 wks). No DLTs were observed. A maximum tolerated dose has not yet been identified. Treatment-related AEs (TRAEs; any grade) in ≥ 10% pts were injection site reactions (53%), fatigue (41%), fever (35%), nausea (29%), diarrhea (18%); the majority of TRAEs were G1-2. Only 2 G3 TRAEs were reported: syncope and fatigue. No patients exhibited vascular leak or cytokine release syndrome associated with high dose IL-2, nor IL-10-associated toxicity. Dose modifications due to G2 or G3 fatigue occurred in 2 pts. Eosinophilia was observed and correlated with drug concentration but did not require intervention. An EC90 trough of 1 ng/mL was maintained for ~100 hrs at 4 mg and ~150 hrs at 8 mg dosing. At 8 mg dose, ranges of Cmax 5.42 – 13.2 ng/mL and trough 0.453 – 7.56 ng/mL were achieved. Fifteen pts were evaluable for response. Best overall response of stable disease was reported in 4 patients (1 mPDAC, 2 mCRC, 1 mNSCLC). In pts with stable disease systemic immune activation was characterized by the expansion of ~40 – 350 unique T cell clones starting at Day 5, expected wtIL-2 driven eosinophilia, up to 200-fold dose related increased plasma IFNɣ [median 417 pg/ml (range 10-1,000)] but without other pro-inflammatory cytokines associated with CRS. Treatment also induced IL-18, IL-18BP, IL-5, IL-2Ra, soluble PD-L1, LAG3 and TIGIT. Conclusions: DK210 (EGFR) was well tolerated and associated with therapeutically relevant on-target biomarker signals consisting of effector T cell expansion and elevated IFNɣ. These data suggest the EGFR targeted balanced combination of IL-2 with IL-10 improves safety and increases potency of anti-tumor function by dissociating CRS from T cell activation. Further exploration of DK210 (EGFR) in RCC and NSCLC as monotherapy and in combination is planned. Clinical trial information: NCT05704985 .

Background Both interleukin-2 (IL-2) and interleukin-10 (IL-10) have been extensively studied for their stimulatory function on T cells and their potential to obtain sustainable tumor control in renal, melanoma, lung, and pancreatic cancers as monotherapy. While approved, IL-2 exhibits significant toxicity in a high percentage of the patient population, limiting its widespread use. The significant efforts undertaken to uncouple IL-2 toxicity from its anti-tumor function have been unsuccessful and early phase clinical safety observed with PEGylated IL-10 was not met in a blinded Phase 3 trial. Deka Biosciences has engineered a novel molecule coupling wild-type IL-2 to a high affinity variant of Epstein Barr Viral (EBV) IL-10 via a scaffold that binds to epidermal growth factor receptors (EGFR). This patented molecule, named DK210 (EGFR), is targeted to EGFR expressing cells and demonstrated to be retained at high levels within the tumor microenvironment days after dosing. In addition to overlapping and non-redundant anti-tumor function, IL-10 reduces IL-2 mediated cytokine release syndrome (CRS) risks and inhibits IL-2 mediated T regulatory cell proliferation. Methods DK210 (EGFR) is being evaluated in an open-label, dose-escalation (Phase 1) study (BOIN design) with five (0.025–0.3 mg/kg) monotherapy dose levels. DK210 (EGFR) is home-administered via subcutaneous injection three times a week. The objectives of this study include evaluating the safety, CRS occurrence, pharmacokinetics, pharmacodynamic and predictive biomarkers, presence of anti-drug antibodies, and antitumor activity. Results As of June 22, 2023, three patients were enrolled, and are continuing DK210 (EGFR) treatment. Subjects improved clinically and no drug related toxicities, nor CRS were observed in any of the patients. One subject with pancreatic cancer achieved stable disease by RECIST 1.1. Conclusions DK210 (EGFR) couples wild-type IL-2 with a high affinity IL-10 and targets cytokines directly to the tumor microenvironment significantly changing Il-2s therapeutic window. Preliminary human data shows an encouraging safety and efficiency profile. The dose-escalation study is expected to be completed by the end of this year (NCT05704985). Acknowledgements The authors would like to thank all patients who participated in this study and their families, as well as all the investigators and site staff who made the study possible. Trial Registration Trial Registration www.clinicaltrials.gov; NCT05704985 Ethics Approval The study was approved by NEXT Oncology, Salus IRB, approval number NXVIR22.60, on 14-Feb-2023 and by Mary Crowley Medical Research Center Institutional Review Board, approval number 23–08, on 21-Apr-2023. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

PDF file - 206K, Suppl. Figure 1. PEG-IL-10 increases intratumoral CD8 T cells and induces CD8 T cell dependent tumor rejection. Suppl. Figure 2. Anti-tumor efficacy is abolished in IL-10R-/- mice. Suppl Figure 3. FTY720 treatment neither impairs IL-10 induced activation of tumor-resident CD8+ T cells nor rejection of CT26 tumors

Background: Immune therapies rely on successful activation of systemic immunity as well as expansion of the T-cell clones for tumor regression and successful therapeutic outcomes. PEGylated IL-10 (pegilodecakin or AM0010) monotherapy has been reported to achieve 25% objective tumor responses (ORR) in intermediate to poor risk renal cell cancer (RCC) in median 4th line of treatment (LOT) (range 1-8) (Naing A et al, JCO 2016). Here we report the immunological underpinnings of pegilodecakin induced tumor responses alone and in combination with anti-PD-1. Methods: Samples were collected post written consent from patients enrolled in a multi-basket trial (NCT0200944) and were analyzed in accordance with the IRB. Patients on AM0010 alone administered daily self-injection of pegilodecakin at 20 µg/kg, SC. Patients in the pembrolizumab + pegilodecakin received in addition pembrolizumab at 2mg/kg IV, Q3W. Normal healthy volunteers (NCT03267732) received a single (day 1) and multiple doses (days 4-9) of 5 µg/kg or 10 µg/kg AM0010 SC. Systemic and cellular antitumor immune responses were assessed by serum cytokine analysis (luminex) and by PBMC flow cytometry respectively. Additionally, immune fluorescence (IF) or immunohistochemistry was performed on formaldehyde fixed archival, pretreatment biopsies and on treatment biopsies CD8, granzyme B, phospho-STAT-3 or LAG-3, T-bet/CD3 and HLA-A. T-cell clones were quantified by TCR deep sequencing (Adaptive biotechnology) from DNA isolated using EDTA blood samples. Results: Pegilodecakin treatment induced a systemic anti-tumor immune cytokine response biased towards Th1 & Th2 cytokines (IFNg, IL-18, TNFa, IL-3, IL-4) along with IL-7. Tuppressive (TGFb) and Th17 cytokines (IL-23, IL-17) were reduced. Cytotoxic effector molecules (Granzyme B, FasL, lymphotoxinB) were increased in the serum. PBMC analysis by flow cytometry in pre- and post-treatment samples show invigoration of the exhausted, T-cells, with increased proliferative index among CD8+ T-cells expressing LAG3 and PD1 throughout pegilodecakin treatment. The increase of PD-1+ Lag-3+ Ki-67+ CD8+ T-cells correlates with objective response to AM0010. On-treatment biopsies showed that pegilodecakin increased GzmB+, Phospho-Stat3+, Lag-3+ CD8+ T-cells in the tumor. T-cell clonal analysis by TCR sequencing on PBMCs from patients during pegilodecakin treatment showed expansion of several hundred previously undetected T-cell clones per patient. Expansion of these T-cell clones in the blood correlated with tumor response, with patients with objective response showing increased number of novel clones as compared to patients with progressive disease. Conclusion: We report that pegilodecakin treatment induced a systemic Th1 immune activation with reduction of Th17 related cytokines. We further report the hallmarks of CD8+ T-cell immunity in these cancer patients, including the systemic elevation of IFNg and GranzymeB levels, expansion and activation of CD8+ TILs, and the proliferation and invigoration and expansion of PD-1+/Lag-3+ CD8+ T-cell sub-set. In addition, pegilodecakin treatment led to the expansion of T-cell clones that were undetectable pretreatment. Clinically, expansion of these novel T-cell clones during pegilodecakin treatment correlated with achievement of objective response. Citation Format: Martin Oft, Aung Naing, Jeffrey R. Infante, Kyriakos P. Papadopoulos, Ivan H. Chan, Cong Shen, Navneet P. Ratti, Karen A. Autio, Deborah J. Wong, Manish R. Patel, Patrick A. Ott, Gerald S. Falchook, Shubham Pant, Annie Hung, John B. Mumm, Matthew Adamow, Scott McCauley, Rakesh Verma, Phillip Wong, Peter VanVlasselaer, Joseph Leveque, Nizar M. Tannir. PEGylated IL-10 (pegilodecakin) induces systemic immune activation, CD8+ T-cell invigoration and polyclonal T-cell expansion in cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A016.

Tumor-reactive T cell exhaustion prevents the success of immune therapies. Pegilodecakin activates intratumoral CD8(+ )T cells in mice and induces objective tumor responses in patients. Here we report that pegilodecakin induces hallmarks of CD8(+ ) T cell immunity in cancer patients, including elevation of interferon-y and GranzymeB, expansion and activation of intratumoral CD8(+ ) T cells, and proliferation and expansion of LAG-3(+) PD-1(+) CD8(+ ) T cells. On pegilodecakin, newly expanded T cell clones, undetectable at baseline, become 1 %-10% of the total T cell repertoire in the blood. Elevation of interleukin-18, expansion of LAG-3(+) PD-1(+) T cells and novel T cell clones each correlated with objective tumor responses. Combined pegilodecakin with anti-PD-1 increased the expansion of LAG-3(+) PD-1(+) CD8(+ ) T cells.

Abstract Purpose: IL-10 inhibits inflammation but stimulates cytotoxic CD8 T cells. In preclinical models, PEGylated IL-10 induces rejection of tumors and establishes CD8 T cell memory. PEG-IL-10 (AM0010) activates the anti-apoptotic STAT3 in tumor infiltrating activated CD8 T cells. This leads to the expansion of tumor reactive memory CD8 T cells both in the tumor and the blood. The primary objective of this phase 1 study is to establish tolerability and anti-tumor activity of AM0010 alone and in combination with chemotherapy and anti PD-1 immunotherapy. Here we report the results of AM0010 in the monotherapy dose escalation and one expansion cohort in renal cell cancer (RCC). Procedures: 33 patients (pts) with advanced cancers including melanoma, RCC, non-small cell lung, colorectal, ovarian, prostate and pancreatic cancer were enrolled in cohorts of 3-6 pts each, followed by disease specific expansion cohorts at the recommended phase 2 dose (RP2D). AM0010 was self-administrated daily subcutaneously for the duration of treatment and responses were monitored following immune related response criteria (irRC). Immune responses were monitored through analysis of serum cytokines, activation of blood derived T cells, immunosequencing for peripheral T cell clonality and immunohistochemistry for the infiltration of tumors by CD8 T cells. Results: 33 pts were enrolled in dose escalation cohorts with AM0010 monotherapy at doses from 1 to 40 μg/kg. MTD was not established. The RP2D (20μg/kg) was determined based on tolerability, single agent anti-tumor activity and immune activation. Patients were heavily pretreated with a median of 5 prior treatments. Most treatment related adverse events (TrAE) were low grade and included anemia, thrombocytopenia, rash, injection site reaction, fatigue, increased lipase, dyslipidemia and transaminitis. G3/4 non-hematopoietic TrAEs were observed in 11 of 51 pts. G3/4 anemia or thrombocytopenia TrAEs were observed in 13 pts. Most TrAEs were transient with only one patient discontinuing treatment due to a TrAE. Immune related TrAEs such as colitis, pneumonitis and endocrine disruption were not observed. AM0010 induced a dose dependent Th1 cytokine signature (IL-18, IFNγ, IL-7) and a reduction of TGFβ in the serum of pts. AM0010 increased PD-1 positive activated CD8 T cells in the blood and the tumor and decreased proliferation of FoxP3 Tregs in the blood. AM0010 lead to an oligoclonal expansion of T cell clones in the blood without affecting the overall number of lymphocytes. Many of the expanded T cell clones were not detectable before treatment. Partial responses (PR) were observed in pts with RCC and uveal melanoma. Four of 15 RCC pts treated at RP2D (27%) had an objective response. Prolonged stable disease of 6 or more months was observed in several indications including colon cancer. Conclusion: AM0010 is well-tolerated and leads to sustained and systemic immune stimulation. The pharmacodynamics and clinical activity observed support the further exploration of AM0010 in monotherapy and in combination regimens with checkpoint inhibitors and with cytotoxic chemotherapies. Trial registration: www.clinicaltrials.gov NCT02009449 Citation Format: Jeffrey R. Infante, Aung Naing, Kyriakos P. Papadopoulos, Karen A. Autio, Patrick A. Ott, Deborah J. Wong, Gerald S. Falchook, Manish Patel, Shubham Pant, Melinda Whiteside, John B. Mumm, Ivan H. Chan, Johanna C. Bendell, Todd M. Bauer, Filip Janku, Milind Javle, Rivka R. Colen, Nizar M. Tannir, Martin Oft. Anti-tumor activity of PEGylated human IL-10 (AM0010) in patients with advanced solid tumors. [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 CT098.

IL-10 has been classically defined as a broad-spectrum immunosuppressant and is thought to facilitate the development of regulatory CD4(+) T cells. IL-10 is believed to represent one of the major suppressive factors secreted by IDO(+)FoxP3(+)CD4(+) Tregs. Contrary to this view, we have previously reported that PEGylated recombinant IL-10 (PEG-rIL-10) treatment of mice induces potent IFNγ and CD8(+) T-cell-dependent antitumor immunity. This hypothesis is currently being tested in clinical trials and we have reported that treatment of cancer patients with PEG-rHuIL-10 results in inhibition and regression of tumor growth as well as increased serum IFNγ. We have continued to assess PEG-rIL-10's pleiotropic effects and report that treatment of tumor-bearing mice and humans with PEG-rIL-10 increases intratumoral indoleamine 2, 3-dioxygenase (IDO) in an IFNγ-dependent manner. This should result in an increase in Tregs, but paradoxically our data illustrate that PEG-rIL-10 treatment of mice reduces intratumoral FoxP3(+)CD4(+) T cells in an IDO-independent manner. Additional investigation indicates that PEG-rIL-10 inhibits TGFβ/IL-2-dependent in vitro polarization of FoxP3(+)CD4(+) Tregs and potentiates IFNγ(+)T-bet(+)CD4(+) T cells. These data suggest that rather than acting as an immunosuppressant, PEG-rIL-10 may counteract the FoxP3(+)CD4(+) Treg suppressive milieu in tumor-bearing mice and humans, thereby further facilitating PEG-rIL-10's potent antitumor immunity.

### P189 Rational combinations of intratumoral T cell and myeloid agonists mobilize abscopal responses in prostate cancer #### Casey Ager1, Matthew Reilley2, Courtney Nicholas1, Todd Bartkowiak1, Ashvin Jaiswal1, Michael Curran1 ##### 1Department of Immunology, University of Texas MD Anderson

Purpose Interleukin-10 (IL-10) stimulates the expansion and cytotoxicity of tumor-infiltrating CD8+ T cells and inhibits inflammatory CD4+ T cells. Pegylation prolongs the serum concentration of IL-10 without changing the immunologic profile. This phase I study sought to determine the safety and antitumor activity of AM0010. Patients and Methods Patients with selected advanced solid tumors were treated with AM0010 in a dose-escalation study, which was followed by a renal cell cancer (RCC) dose-expansion cohort. AM0010 was self-administered subcutaneously at doses of 1 to 40 μg/kg once per day. Primary end points were safety and tolerability; clinical activity and immune activation were secondary end points. Results In the dose-escalation and -expansion cohorts, 33 and 18 patients, respectively, were treated with daily subcutaneous injection of AM0010. AM0010 was tolerated in a heavily pretreated patient population. Treatment-related adverse events (AEs) included anemia, fatigue, thrombocytopenia, fever, and injection site reactions. Grade 3 to 4 nonhematopoietic treatment-related AEs, including rash (n = 2) and transaminitis (n = 1), were observed in five of 33 patients. Grade 3 to 4 anemia or thrombocytopenia was observed in five patients. Most treatment-related AEs were transient or reversible. AM0010 led to systemic immune activation with elevated immune-stimulatory cytokines and reduced transforming growth factor beta in the serum. Partial responses were observed in one patient with uveal melanoma and four of 15 evaluable patients with RCC treated at 20 μg/kg (overall response rate, 27%). Prolonged stable disease of at least 4 months was observed in four patients, including one with colorectal cancer with disease stabilization for 20 months. Conclusion AM0010 has an acceptable toxicity profile with early evidence of antitumor activity, particularly in RCC. These data support the further evaluation of AM0010 both alone and in combination with other immune therapies and chemotherapies.

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10's dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10's increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10's cholesterol regulating biology is consistent between mice and humans.

3018 Background: IL-10 has anti-inflammatory properties but stimulates the cytotoxicity and proliferation of CD8 T cells. The observation that T cell receptor mediated activation induces the expression of IL-10 receptors and PD1 on CD8 T cells, provides the mechanistic rationale for combining AM0010 and anti-PD1 in the clinic. Tolerability and anti-tumor activity of AM0010 alone was established in the ongoing phase 1 study. Objective responses were observed in pts with uveal melanoma, cutaneous T cell lymphoma and in 4 of 15 pts with RCC. Methods: Pts with advanced melanoma, RCC or NSCLC were treated with AM0010 (daily SC) and Pembrolizumab (q3wk IV). 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. Results: In 19 pts, AM0010 10 mg/kg (n=13) or 20 mg/kg (n=6) in combination with anti-PD1 (2mg/kg) was well tolerated (observation period 10-15 months). All TrAEs were transient and DLTs or TrAEs leading to study discontinuation were not observed. There was no colitis, pneumonitis, or endocrine disruptions. G3/4 TrAEs were observed in 7 of 19 pts and included pruritus (1), anemia (3), thrombocytopenia (3) and malaise (1). Objective responses (PR/CR) were observed 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). AM0010 / anti-PD1 increased Th1 cytokines (IL-18, IFNg, IL-7) and the number and proliferation of PD1+ activated CD8 T cells while decreasing the proliferation of FoxP3+ Tregs and TGFb in the blood. AM0010 / anti-PD1 induced de-novo oligoclonal expansion of T cell clones in the blood without affecting total lymphocyte counts. AM0010 / anti-PD1 increased the number of tumor infiltrating Granzyme+ PD1+ CD8+ T cells in tumor biopsies of treated patients. Conclusions: AM0010 in combination with anti-PD1 is well-tolerated. The clinical activity and the observed CD8 T cell activation encourages the continued exploration of AM0010 in combination with anti-PD1. Clinical trial information: NCT02009449.Best Response SD PR CR CBR (SD, PR, CR: > 24 weeks) RCC (n=8) 4 3 1 5 NSCLC (n=5) 3 2 - 4 Melanoma (n=6) 2 2 - 2 (NR)

3082 Background: IL-10 induces activation of STAT3 in CD8 T cells leads to increased survival, proliferation and cytotoxicity of CD8 T cells. In preclinical studies, PEG-IL-10 induces CD8 mediated tumor rejection and synergizes with cytotoxic chemotherapies. Anti-tumor activity of AM0010 alone was established in the ongoing phase 1 basket trial. Objective responses were observed in pts with renal cell cancer (RCC; 4 of 15pts), uveal melanoma and cutaneous T cell lymphoma. Methods: Pts with advanced pancreatic cancer (PDAC) were treated daily with AM0010 alone (n = 24) or in combination with FOLFOX (5), capecitabine (5) or gemcitabine/nab-paclitaxel (6). Pts with colorectal cancer (n = 16) were treated with AM0010 alone. Tumor responses were monitored following irRC. Immune responses were monitored through analysis of serum cytokines, activation of blood derived T cells, immunosequencing for peripheral T cell clonality and immunohistochemistry for the infiltration of tumors by CD8 T cells. Results: In 24 PDAC pts and 16 CRC pts treated with AM0010 monotherapy (1-20 mg/kg), G3/4 TrAEs were observed in 16 pts, including anemia (5), thrombocytopenia (9), fatigue (2) rash (3) and transaminitis (1). Most TrAEs were transient, only one patient discontinued treatment due to anemia. Colitis, pneumonitis or endocrine abnormalities were not observed. In 16 pts with PDAC treated with AM0010 plus chemotherapy, TrAEs were observed in 11 pts, including thrombocytopenia (10). At 8 weeks, 8 of 17 pts with PDAC and in 4 of 15 pts with CRC on AM0010 alone had stable disease (SD). 11 of 15 PDAC pts had a reduction of CA 19-9. 4 patients (3 PDAC, 1 CRC) had prolonged PFS > 6 months. The median overall survival of PDAC was 5.1 months (n = 20), and CRC was 15.4 months (n = 13; observation > 15mo). 13 of 15 PDAC pts treated with AM0010 and chemotherapy had SD, 2 had a PR. AM0010 increased PD-1+ activated CD8 T cells and the de-novo oligoclonal expansion of T cell clones in the blood. AM0010 increased tumor infiltrating Phospho STAT3+, Granzyme+ and PD1+ CD8+ T cells in tumors. Conclusions: The preliminary clinical activity and the mechanistic data indicating enhanced immune stimulation encourages continued exploration of AM0010 in “immune resistant” cancers. Clinical trial information: NCT02009449.

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.

Recent advances in immunoncology have dramatically changed the treatment options available to cancer patients. However, the fundamental challenges with this therapeutic modality are not new and still persist with the current wave of immunoncology compounds. These challenges are centered on the activation and expansion, induction of intratumoral infiltration and persistence of highly activated, cytotoxic, tumor antigen specific CD8+ T cells. We have investigated the anti-tumor mechanism of action of pegylated recombinant interleukin-10, (PEG-rIL-10) both pre-clinically with murine (PEG-rMuIL-10) and now clinically (AM0010) with human pegylated interleukin-10. The preponderance of data suggest that IL-10's engagement of its receptor on CD8+ T cells enhances their activation status leading to antigen specific expansion. Quantitation of CD8+ T cell tumor infiltration reveals that treatment of both humans and mice with pegylated rIL-10 results in 3-4 fold increases of intratumoral, cytotoxic, CD8+ T cells. In addition, mice cured of their tumors with PEG-rMuIL-10 exhibit long term immunological protection from tumor re-challenge and long term treatment of cancer patients with AM0010 results in the persistence of highly activated CD8+ T cells. Cumulatively, these data suggest the IL-10 represents an emerging therapeutic that specifically addresses the fundamental challenges of the current wave of immunoncology assets.