anti-PD1 Antibody Research Articles

Abstract 6115: Development of functionally differentiating anti-CD73 antibodies for cancer therapy

Abstract Background: Although the discovery and development of first-generation of immune checkpoint inhibitors (towards PD1 and CTLA-4) was a major milestone for cancer therapy, current clinical response rates are still considered very limited. Combination treatments are predicted to improve on these current immunotherapies including the targeting of the adenosine pathway (CD39, CD73 or A2AR), which has shown a lot of promise in preclinical and early clinical studies. CD73 is an ecto-5′-nucleotidase which transforms adenosine monophosphate (AMP) to adenosine. Adenosine is a soluble immunosuppressive metabolite that can suppress natural killer cells and cytotoxic CD8+ T cells. Blockade of CD73-mediated conversion of AMP to adenosine may therefore recover anti-tumor immunity through preventing the enrichment of adenosine in the tumor microenvironment. Method: In this campaign, two humanized and Fc-silenced IgG antibodies were generated named 7002-01 and 7002-04. The target binding epitopes of these candidates were revealed by binning experiments through bio-layer interferometry. Cell binding experiments were tested on human and cynomolgus CD73 overexpression CHO cell lines by flow cytometry. Cellular CD73 enzyme inhibition experiments were tested using A375, MDA-MB-231, H2030 and BT549 tumor cell lines via the CellTiter-Glo method. Soluble CD73 enzymatic tests were carried out on patient sera or recombinant CD73 protein using a similar method. T cell proliferation assays were performed using PBMC. In vivo efficacy studies were tested in B-NDG B2M-KO mice that were injected subcutaneously with A375 tumor cells and human PBMC. Results: Two candidates, 7002-01 and 7002-04, were selected based on their functional activity, that recognize different non-overlapping binding epitopes on CD73. Both candidates selectively bind to and can inhibit the activities of both membrane-bound and soluble human CD73 to high levels and seem to maintain inhibition at high dose-ranges without a hook effect. Both candidates can potently rescue adenosine-mediated T cell regulation. Additionally, 7002-01 and 7002-04 have combination synergy or additive effects for CD73 inhibition on soluble CD73, tumor cell lines that express CD73, and PBMC. 7002-01 and 7002-04 have single-agent anti-tumor efficacy and combination synergy with anti-PD1 antibodies in mice. 7002-01 has a typical antibody-like PK profile when rhesus monkeys were administered with a single intravenous dose at 25 or 50 mg/kg. No drug-related toxicities have been observed in GLP toxicity studies with dosages at 50, 250, and 500 mg/kg (QW, 4 weeks). Conclusion: Highly differentiating anti-CD73 antibodies were discovered that show maximal inhibition of both membranous and soluble CD73 without hook effects at high concentrations. 7002-01 was chosen as the lead molecule for its better overall activity profile and should be entering clinical trials by early 2022. Citation Format: Zhenqing Zhang, Yunli Jia, Xiaoniu Miao, Weifeng Huang, Chao Wang, Zhijun Yuan, Wenchao Jiang, Zhiyuan Li, Liandi Chen, Andy Tsun. Development of functionally differentiating anti-CD73 antibodies for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6115.

Abstract 2325: Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers

Abstract We discovered a crucial change common to all platinum resistant (CR) NSCLC cells, wherein a high level of reactive oxygen species (ROS) modulates tumor metabolism. CR cells activate the kynurenine (KYN) pathway (KP) during excessive oxidative stress to maintain high ROS for proliferation by catabolizing tryptophan (TRP) to KYN via indoleamine 2,3-dioxygenase-1 (IDO1); and by expressing (3-5x) higher levels of programmed death-ligand 1 (PD-L1) compared to platinum sensitive cells (p<0.02; n=8). We detected significant increases in IDO1 activity (3-4x), identified by the KYN/TRP ratio, in CR cells’ culture media (p<0.05; n=8). Extracellular KYN is known to program naïve T-cells to the immune suppressive regulatory T-cell (T-reg) phenotype. Using a human peripheral blood mononuclear cell (PBMC) co-culture system, we found a higher frequency of regulatory T-cells (Tregs; CD4+CD25+Foxp3+) and a lower frequency of effector T-cells (Teffs CD3+CD8+) and NK cell (CD3-CD314+CD335+) activity in co-culture with CR cells compared to cisplatin sensitive cancer cells. Importantly, inhibition of IDO1 and Tryptophan 2,3-Dioxygenase (TDO2), the enzymes involved in the rate limiting step in the KP, with the dual inhibitor AT-0174 at 20µM (48h) suppressed Tregs, but enhanced Teffs and NK cell populations in CR co-cultures. Next, we used a syngeneic mouse model of LLC (Lewis lung cancer) vs cisplatin resistant LLC-CR, to examine the effects of AT-0174 (170 mg/kg; P.O. once daily) in combination with anti-PD1 antibody (10 mg/ml; I.P. once every 3 days). Consistent with the in vitro model, analysis of mice with LLC-CR tumors found higher intratumoral T-reg and lower NK cell populations. Treatment with AT-0174 resulted in higher tumor infiltrating lymphocytes of Teffs and NK and lower Treg frequency in LLC-CR tumors than treatment with a selective IDO1 inhibitor (epacadostat). More importantly, the combination of AT-0174+antiPD1 reversed the immunosuppressive tumor microenvironments with further enhancement in Teffs and NK frequencies and diminished tumor growth in CR allografts with a significant reduction of total tumor weight compared with LLC mouse tumors (n=10, P<0.01). Our new findings support the potential therapeutic efficacy of the dual IDO/TDO inhibitor AT-0174 as an immuno-therapeutic treatment that enhances tumor immune surveillance and blocks tumor metabolism related survival mechanisms in platinum resistant NSCLC tumors. Support by the Dept. of Veterans Affairs. Citation Format: Sydney Spector, Chunjing Wu, Dan Nguyen, George Theodore, Ashley Garcia, Lynn Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2325.

Abstract 2646: Novel, isoform selective PAD4 inhibitors for the treatment of cancer

Abstract Background: Peptidyl Arginine Deiminase 4 (PAD4/PADI4) is an enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hyper citrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed Neutrophil Extracellular Traps (NETs). NETs have been shown to be associated with several pathological processes including, fibrosis, ischemic stroke, preeclampsia, thrombosis, cancers, and other autoimmune diseases. In the context of cancer, tumors secrete chemokines such as CXCL1 and GCSF to recruit and prime neutrophils to form NETs, which in turn, facilitate cancer metastasis by forming Circulating Tumor Cell (CTC) clusters and potentially increasing tumor cell extravasation. Therefore, cancer cell endogenous PAD4 seems to play a significant role in metastasis, and inhibition of PAD4 could be a novel strategy to inhibit cancer progression and metastasis. Here, we describe a series of novel small molecule PAD4 inhibitors that are selective and orally bioavailable for the treatment of cancer. Methods: Potency and selectivity were evaluated in ammonia release assay using recombinant PAD enzymes. Cellular potency was evaluated by assessing the modulation of citrullination in a human neutrophil-based assay. ADME surrogates and PK studies were conducted using appropriate preclinical models. In vivo efficacy studies were conducted in syngeneic mice tumor models. Results: Multiple leads with strong in vitro potency on PAD4 (< 0.200 µM) were identified. One of the leads (JTxPAD4i) showed an IC50 of 0.102 µM against recombinant human PAD4 in the biochemical assay and a similar potency on mouse PAD4. It was selective against PAD1, PAD2, and PAD3 enzymes (IC50 > 10 µM). In the human neutrophil-based assay, JTxPAD4i inhibited H3-Histone citrullination with an IC50 of 0.125 µM. It showed an oral bioavailability of greater than 50% across species, including mouse, rat, and dog. Oral dosing with JTxPAD4i at 50 mg/kg, BID, in 4T1 mice syngeneic breast cancer model resulted in ~50% tumor growth inhibition (TGI) as compared to ~35% TGI with anti-PD1 antibody and was well tolerated. TGI was associated with strong inhibition of histone citrullination and myeloperoxidase (MPO) that are markers for NET formation. There was also a strong and significant inhibition in inflammatory cytokines, including IL-6, IL-17, and IL-1β in the tumor. Similar TGI was observed in additional breast and lung cancer syngeneic models with more than one PAD4 inhibitor from our lead series. Further studies with our lead molecules are underway to elucidate the mechanism of action of these PAD4 inhibitors. Conclusion: These studies clearly emphasize the potential of PAD4 inhibition in the treatment of cancer and our lead PAD4 inhibitors are being profiled in advanced toxicology studies to be developed as a clinical candidate. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Chandru G, Mohd Zainuddin, Naveen Sadhu, Ramachandraiah Gosu, Luca Rastelli, Amir Siddiqui, Sharad Singh, Rudresh G, Sadanand Mullurwar, Saravanan Kandan, Santosh Vishwakarma, Gurulingappa Hallur, Ravi Trivedi, Jeyaraj D A. Novel, isoform selective PAD4 inhibitors for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2646.

Abstract 3882: Translating genotype to immunophenotype in HRAS mutated head and neck squamous cell carcinoma (HNSCC) to identify effective Tipifarnib partners for optimal patient outcomes

Abstract Background: Tipifarnib is a highly selective farnesyltransferase inhibitor that has shown promising activity in HRAS mutant recurrent/metastatic HNSCC. The incidence of HRAS mutations in HNSCC is 4-10%. Since HRAS mutated HNSCC can be successfully targeted with Tipifarnib, we sought to analyze the immune and genomic landscape of these tumors to guide combinatorial strategies. Methods: We identified thirteen patients with HRAS mutant tumors. We included in the analysis 50 patients with HRAS wild-type tumors. Genomic DNA from baseline biopsies of tumors was purified and subjected to targeted sequencing (TruSightTM Oncology 500 panel). PD-L1 expression in formalin-fixed tumor samples was assessed using the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies, Carpinteria, CA, USA) and characterized by the combined positive score (CPS). Assessment of PD-L1+ cells inside and at the periphery of the tumor was performed. HRAS mutational status was correlated with PD-L1 status. The genomic data derived from the targeted gene panel of HRAS mutant tumors were compared to those with wild-type HRAS. Results: Thirty-seven patients had both genomic and PD-L1 expression data available. The presence of HRAS mutations was associated with higher TPS PD-L1 scores (26% vs 40% vs 100% in TPS <1%, 1-49% and >50% respectively, p=0.043). Similar results were obtained for CPS (28% vs 80% in CPS <10% and =>10%, p=0.047). HRAS mutations were more commonly found with TP53 mutations (16% in WT TP53 and 55.6% in mutated TP53, p=0.073). Higher PD-L1 TPS showed increased survival without reaching significance when measuring either in the margins (64.1 vs 35.8, p=0.066) or the center of the tumor (p=0.097) due to the small sample size. Conclusions: HRAS mutant HNSCC are associated with high PD-L1 expression and co-occurrence of p53 mutations. Combinations of tipifarnib with an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA-4 antibody warrant clinical investigation in this rare subset of HNSCC. Citation Format: Amanda Psyrri, Miriam Anastasiou, Aris Spathis, Stergios Doumas, Panagiota Economopoulou, Ioannis Kotsantis, Anastasios Pantazopoulos, Anastasios Kyriazoglou, George Kavourakis, Theodoros Rampias, Periklis Foukas. Translating genotype to immunophenotype in HRAS mutated head and neck squamous cell carcinoma (HNSCC) to identify effective Tipifarnib partners for optimal patient outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3882.

Abstract 4261: CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses

Abstract CD27 is a member of the TNF-receptor superfamily, highly expressed on CD4+ and CD8+ T cells as well as on NK and NKT cells. It plays a key role on T cell proliferation and differentiation after stimulation with its ligand CD70. The co-stimulatory signal of CD27 on T cell is mediated via the NFκB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B. CD27/CD70 co-stimulation has the potential to boost immunity by T-cell activation, clonal expansion and enhanced differentiation into antigen specific cytotoxic and memory T cells. CD27/CD70 also influences the innate immune response via a direct activation of the NK cells and a subsequent secretion of interferon-gamma (IFN-γ). Therefore, CD27 signaling promotes cytotoxic T cell based anti-tumor immunity. With its central role in an immunological response, CD27 is a promising target for antitumor therapy. Previous works have demonstrated the efficacy of an agonistic CD27 antibody in controlling tumor growth and metastasis in different mice models including melanoma, renal cell carcinoma, breast cancer and lymphomas. This anti-tumor effect is mediated in part by an effective recruitment of IFN-γ producing CD8+ T cells within the tumor. Moreover, CD27 stimulation of Tumor Infiltrating Lymphocytes (TILs) can lower their threshold of activation and provide a broader repertoire of Ag-reactive T cells within the tumor. We have selected a lead therapeutic antibody from our library of 147 fully human anti-CD27 monoclonal antibodies generated in the Trianni mice. After confirming its binding potency and selectivity as well as its cross-reactivity with Non-Human Primate (NHP)-CD27 but not with the mouse-CD27, this lead candidate demonstrated strong agonistic proprieties. This was shown by its ability to induce a strong NFκB signal as well as to induce T cell proliferation and activation with secretion of pro-inflammatory cytokines. This antibody demonstrated agonistic proprieties without cross-linking confirming its potency. T cell activation observed after treatment with our anti-CD27 antibody only occurs in the presence of TCR engagement, preventing the risk of spontaneous activation of naïve T cells in vivo. The ability of our CD27 monoclonal antibody to increase an immune response was confirmed in a Mixed Lymphocyte Reaction assay with multiple donors. The role played by the NK cells and their activation via CD27 antibody was also demonstrated. To evaluate the anti-tumor functions of our lead antibody as a single agent or in combination with other immuno-therapies in vivo we have used human CD27 transgenic mice. We have demonstrated in the MB49 bladder tumor model as well as the EG7 thymoma model that our lead antibody induces a strong single agent anti-tumor activity and these tumors were totally controlled in combination with an anti-PD1 antibody. We are now analyzing the pharmacokinetic and pharmacodynamic of our antibody as well as its safety in a NHP model Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Shaarwari Sridhar, David Peckham, Jessica Cross, Nathan Eyde, Emily Frazier, Neda Kabi, Remington Lance, Kurt Lustig, Mei Xu, Tarcha Eric, Shawn Iadonato. CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4261.

Abstract 3505: CLN-617 is an IL-2/IL-12 fusion protein with a collagen-anchoring domain that induces potent systemic anti-tumor immunity upon intra-tumoral administration

Abstract Despite strong anti-tumor immune responses in the clinic, cytokine therapy for cancer treatment, has remained limited in use due to its narrow therapeutic window. Moreover, anti-tumor immunity is typically driven through multiple cytokines acting in concert in a local tumor microenvironment (TME) via autocrine or paracrine signaling. Yet most cytokine therapies focus on systemic delivery of individual cytokines causing adverse events without achieving therapeutic doses at the tumor site. Recently, intratumoral (IT) delivery of cytokines such as IL-2 and IL-12 anchored to collagen in the TME has resulted in promising anti-tumor efficacy with minimal toxicity in multiple murine tumor models, and remarkably also in spontaneously occurring large canine tumors (unpublished data). Cullinan Amber is developing CLN-617, a fusion protein that uniquely combines IL-2 and IL-12 with albumin and a collagen-binding domain in a single polypeptide. CLN-617 aims to increase cytokine persistence and retention in the TME upon IT administration and thus improve tolerability. Data presented in this abstract is generated using a murine surrogate, mCLN-617. mCLN-617 retains bioactivity of both IL-2 and IL-12 in the presence of collagen binding. Upon IT injection into B16F10 tumor-bearing mice, systemic drug levels of mCLN-617 were &lt 5% compared to mice dosed via intravenous (IV) administration. When IL-2/IL-12 fusion proteins lacking a collagen-binding domain were injected IT in B16F10-bearing mice, 60% of mice were euthanized due to severe body weight loss, whereas mCLN-617-treated mice maintained their body weight. In checkpoint-refractory B16F10 and MC38 models, mCLN-617 demonstrated 95% tumor growth inhibition and 100% CRs, respectively. All mice cured of their primary MC38 tumors were either protected from re-challenge or showed delayed tumor growth kinetics. Notably, 70% CRs were observed in the MC38 model after a single-dose treatment of mCLN-617. In mice bearing dual-flank MC38 tumors, only one of which was treated IT, 90% of treated tumors and 100% of distal untreated tumors were eliminated when mCLN-617 was combined with an anti-PD1 antibody, demonstrating a curative abscopal response. Of note, mCLN-617 monotherapy in the treated tumor also significantly delayed the growth of distal untreated tumors. Our human lead candidate CLN-617 is produced at high titers in CHO cells and is currently in IND enabling studies. We have demonstrated that the use of collagen-binding domains for tumor retention enables safe and effective delivery of a combination of IL-2 and IL-12 in a single multifunctional molecule. The strong anti-tumor efficacy of mCLN-617 as a monotherapy and significant deepening of responses in combination with checkpoint blockade, together with a favorable tolerability profile, supports future clinical development of CLN-617. Citation Format: Naveen K. Mehta, Bochong Li, Kavya Rakhra, K Dane Wittrup, Patrick A. Baeuerle, Jennifer S. Michaelson. CLN-617 is an IL-2/IL-12 fusion protein with a collagen-anchoring domain that induces potent systemic anti-tumor immunity upon intra-tumoral administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3505.

Abstract 612: Microbial determinants of immune checkpoint inhibition response in a murine model of obesity and metastatic basal-like breast cancer

Abstract Introduction: Breast cancer (BC) is the second leading cause of cancer-related mortality among American women and is exacerbated by obesity. The basal-like (BL) subtype accounts for 10-15% of all BCs and is a particularly aggressive disease due to its extreme intrinsic diversity compared with other subtypes. Paradoxically, obesity promotes immune checkpoint inhibition (ICI) response in some cancers. Further, obesity promotes altered intestinal microbial composition, and recent clinical studies of fecal microbiota transplants suggest causal links between the microbiome and immunotherapy responses. Hence, we evaluated the correlation of tumoral and cecal microbiota with anti-Programmed Cell Death Protein 1 (PD1) immunotherapy response in a murine model of BLBC and obesity. Methods: In an initial study, normoweight female C57BL/6J mice (n=8/group) received orthotopic injection of one of two novel C3TAg BLBC cell lines (1.02 and 2.51) syngeneic to C57BL/6J derived from a spontaneous tumor excised from B6-C3TAg transgenic mice. Gene expression profiling (Affymetrix arrays) with Gene Set Enrichment Analysis (GSEA) was performed on the resulting transplanted tumors. In a second study, female C57BL/6J mice were divided into a diet-induced obesity (DIO) group (n=30), promoted by high-fat diet feeding, and a lean control group (n=30). After 24 weeks, 2.51 cells were orthotopically injected into the fourth mammary fat pad of all mice, followed by treatment with anti-PD1 or IgG control antibodies (n=15/diet group). Tumor and cecal DNA was extracted and subjected to 16S rRNA amplicon sequencing. Results: GSEA from the initial study revealed that orthotopically transplanted line 2.51 tumors, relative to line 1.02 tumors, had lower levels of stromal remodeling as indicated by significant enrichment of gene sets related to extracellular matrix proteoglycans, collagen chain trimerization, and collagen biosynthesis and modification. Gene sets related to insulin and IGF-1 signaling were also lower in line 2.51 tumors than line 1.02 tumors. The in-life portion of the second study revealed a heterogeneous response to ICI therapies among lean and obese mice. To understand this diversity of response, 16S rRNA sequencing analysis of tumoral and cecal microbiota in response to DIO and ICI therapy is underway. Discussion: These data reveal striking differences in tumor microenvironment and nutrient sensing as being potentially important determinants of differential tumor growth in BLBC. Ultimately, this research may contribute to the development of precision nutrition approaches using pro-/pre-biotics to promote microbial communities, targeting the tumor microenvironment, which may improve immunotherapy response and disrupt the obesity-associated exacerbation of breast cancer. Financial Support: This work was supported by R35CA197627 to SDH. Citation Format: Tori L. McFarlane, Meredith S. Carson, Elaine M. Glenny, Violet A. Kiesel, Ashlee Taylor, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Michael F. Coleman, Stephen D. Hursting. Microbial determinants of immune checkpoint inhibition response in a murine model of obesity and metastatic basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 612.

Abstract 6219: WM-S1, the novel small molecule inhibitor of mutant RTK/receptor tyrosine kinase, for the treatment of cancer

Abstract As the representative targeted anticancer drug for colon cancer patients, cetuximab is the EGFR targeted therapeutic antibody and used for treatment of KRAS wild type cancers. Even some patient with KRAS wt gene did not respond cetuximab. However, there is no treatment available for cetuximab-resistant patient group, which is almost 50% of KRAS WT gene holders. Recently, our team identified cetuximab primary resistant related proteins named as mtRTK (mutant receptor tyrosine kinase) by array analysis based cetuximab responder or non-responder colon cancer patient tissues. We investigated mtRTK’s oncogenic potential as a novel anti-cancer target. A large proportion of colon cancer patients (36.2% Caucasian, 56.9% Korean) expressed the mtRTK was identified, using the sequencing analysis of patient samples. Based on these results, our efforts have led to the discovery of WM-S1, mtRTK inhibitor, which is the first mtRTK inhibitor in clinical development. The potent enzyme inhibitor showed a high anticancer activity confirmed in Patient-Derived Cells (PDC) and Patient-Derived Xenograft (PDX) animal models expressing the mutation. In preclinical studies demonstrate that WM-S1 is well tolerated in rats and dogs. Furthermore, WM-S1 has potent anticancer activities for various solid tumor (NSCLC, cholangiocarcinoma, etc.) including activated mtKRAS colon cancer expressing the mtRTK. Currently we are investigating WM-S1 in a phase 1a trial in AUS, which is the first mtRTK inhibitor in clinical development. Meanwhile, the mtRTK inhibitor WM-S1 drives antitumor immunity (with anti-PD-L1) in NSCLC. Combinational approaches with immunotherapy showed that synergistic effect of WM-S1 and anti-PD1 monoclonal antibody, suppressing tumor growth by 75% in anti-PD1 resistance NSCLC-derived humanized mouse model. A phase 1b trial is expected to develop WM-S1 through not only indication expansion but also combination therapy with immuno-checkpoint inhibitors in the USA, AUS and KOR from Q2 2022. In conclusion, mtRTK is a potential oncogenic driver mutation in various solid tumor. A first-in-class anticancer agent WM-S1 targeting mtRTK can be promising therapeutic agents for cetuximab-resistant colon cancer patients regardless of KRAS mutation status and other cancers. Citation Format: Joseph Kim, Jai-Hee Moon, Kyung-Mi Lee, Hyun Ryu, Eun Hye Park, Sang Hee Kim, Jeong Seok Kim, Young Ok Ko, Yong Seok Kim, Hyo Jin Kim, Tae Young Kim Kim, Moon Seong Yoo, Soll Jin, Seongrak Kim, Yoon Sun Park, Min Ki Lee, Mi So Lee, Ji Hyun Go, Yu Geun Ji, Jun Hyung Lee, Haneul Lee, Min Hwa Kim, Eun Hee Ko, Yeo Jin Lee, Seung-Mi Kim, Joon-yee Jeong, Yeon-seoung Choi, Seung-geon Bae, Jinwoo Lee, Won Jun Lee, Min-Kyeong Kim, Ji min Shin, Dong-in Koh, Sun-Chul Hur, Chun-Ho Park, Hyun Ho Lee, Dong-Hoon Jin. WM-S1, the novel small molecule inhibitor of mutant RTK/receptor tyrosine kinase, for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6219.

Abstract 654: CD28 shedding is a novel resistance mechanism to anti PD-1 therapy

Abstract Background: Resistance mechanisms to anti-PD1 therapy remain poorly understood. Studies have implicated that efficient anti-PD1 therapy relies specifically on intact CD28/B7 signaling. We have previously shown that membranal CD28 is actively shed by MMP-2 and MMP-13 upon T cell stimulation in humans, and that soluble CD28 can counteract the efficacy of anti-PD1 blocking antibodies in-vitro. Here we describe that CD28-shedding process is a potential novel resistance mechanism to PD-1 therapies in cancer patients, by using several ex-vivo and in-vivo methodologies. The prominent unfavorable effect of CD28-shedding on PD-1 blockade prompted the generation of BND-67, an agent that can selectively and efficiently block CD28 shedding. Methods: Soluble CD28 detection in plasma of cancer patients and in the culture media of stimulated T cells was achieved using a proprietary in-house ELISA. Cancer patient processed tumors and PBMC were used ex-vivo to identify membrane/soluble CD28 by ELISA and flow-cytometry. MC-38 challenged human CD28 transgenic mice (CD28-shedding permissive model), and WT mice (non-permissive CD28-shedding model) were compared for efficacy to anti-PD1 blocking antibody. BND-67 was isolated by screening a VHH library for binding to the CD28-shedding prone region and blocking of CD28 shedding. The lead clone was further affinity-matured and reformatted into a half-life extended construct. Results: CD28 receptor density was found to be lower on tumor-resident T cells then their matched counterparts in the periphery. Evaluation of plasma samples from patients undergoing anti-PD-1 therapy demonstrated an association of diseases relapse with elevated plasma levels of soluble CD28. Correspondingly, relapses from response to anti-PD-1 therapy were significantly higher in CD28-shedding permissive mice vs non-permissive mice. To overcome this novel regulatory mechanism, we generated BND-67, which is an affinity-matured VHH that can effectively bind CD28, block CD28 shedding and has no agonistic activity. In addition, an optimal approach for increasing the half-life of the agent was identified by fusing the VHH to human Fc. Conclusions: We report here the discovery of a potential novel resistance mechanism to anti-PD1 blockade involving the shedding of the CD28 receptor. This process occurs in the setting of cancer and was further reinforced by results observed in transgenic mice. Biond has generated BND-67, a therapeutic drug candidate for the inhibition of this novel regulatory mechanism. Citation Format: Motti Hakim, Anna Fridman Dror, Tal Shahar Gabay, Dror Alishekevitz, Edna Meilin, Ilana Mandel, Yair Sapir, Avidor Shulman, Tehila Ben Moshe. CD28 shedding is a novel resistance mechanism to anti PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 654.

Abstract 5528: A highly potent anti-CD39 biparatopic antibody and bispecific for cancer therapy

Abstract Background: There is an increasing demand for effective combinatory agents to improve upon PD-1/PD-L1-based therapeutics. One combinatory target axis is the adenosine metabolism pathway that consists of three major players, including CD39, CD73 and A2AR. Inhibition of any of these targets have shown enhanced preclinical efficacy in combination with PD-1/PD-L1 inhibitors. CD39 is an ectonucleotidase which degrades extracellular ATP to adenosine monophosphate (AMP). This is considered a rate-limiting step for the further degradation to adenosine by CD73. Adenosine is an immunosuppressive metabolite that can suppress NK and T cells. Blockade of CD39-mediated degradation of ATP to AMP may therefore recover anti-tumor immunity through preventing the enrichment of adenosine in the tumor microenvironment. Method: Two anti-CD39 VHH molecules were generated, named Ye-37 and Ye-46, which bind to two different epitopes on CD39. Binding experiments were carried out by bio-layer interferometry. Cell binding experiments were tested on CD39-overexpression cell lines by flow cytometry. Cellular CD39 enzymatic inhibition experiments were tested using an MOLP-8 cell line and PBMC via a luminescence-based assay. Soluble CD39 enzymatic tests were carried out on recombinant CD39 protein using a similar method. T cell proliferation assays were performed and observed on CD4+ or CD8+ T cell populations. In vivo efficacy studies were tested in B-NDG B2M-KO mice that were injected subcutaneously with A375-CD39+ tumor cells and human PBMC. An anti-PD1 x CD39 bispecific antibody was then generated by fusing the anti-CD39 biparatopic antibody to the C-terminus of an anti-PD1 IgG and tested using similar methods. Results: Two candidates, Ye-37 and Ye-46, were selected for their functional activity that recognize non-overlapping epitopes on CD39. The combination of Ye-37 and Ye-46 shows high potency in cell-based and soluble CD39 assays in blocking CD39 activity. Two biparatopic molecules were generated by fusing the biparatopic unit to the N- or C-termini of Fc (46-37-Fc and Fc-37-46) and showed similar activity to the combination. In vivo, we showed single-agent control of tumor growth and potentiation of tumor-growth inhibition when combined with anti-CD73 antibodies. An anti-PD1 x CD39 bispecific was generated and showed potent inhibition of PD-1/PD-L1 interactions by cell-based assays. Potent anti-tumor efficacy was shown, which was as effective as the combination of anti-PD1 plus anti-CD39 antibodies. Conclusion: Potent anti-CD39 and anti-PD1 x CD39 therapeutic candidates have been generated with promising activity as a combinatory or single agent, respectively. As such, we plan to file for clinical trial authorization of these programs by 2022. Citation Format: Zhenqing Zhang, Yunli Jia, Xiaoniu Miao, Weifeng Huang, Chao Wang, Zhijun Yuan, Wenchao Jiang, Liandi Chen, Zhiyuan Li, Andy Tsun. A highly potent anti-CD39 biparatopic antibody and bispecific for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5528.

Abstract 3366: Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer

Abstract The increased cancer incidence and associated mortality call for the development of novel treatment modalities to address needs of cancer patients, which do not respond to existing therapies. Oncolytic viruses emerge as a new modality in cancer therapy due to their natural ability to infect and kill cancer cells, and to stimulate patient's own immune system to eliminate cancer cells throughout the body. For this study, we used novel engineered oncolytic adenovirus, Ad5/35-3M (AVID-317), which demonstrated greatly-improved safety profile after systemic virus administration due to a set of targeted mutations in the adenovirus capsid (Atasheva et al, Science Translational Medicine, 2020, eabc6659). Systemic administration of AVID-317 to NCr/Nude mice with disseminated lung adenocarcinoma slowed tumor progression and significantly extended survival, compared to buffer-treated control tumor-bearing mice. In AVID-317 treated group, about 25% of mice demonstrated complete rejection of disseminated tumors and the rest of mice showed significantly extended survival. To understand the mechanism of AVID-317-mediated tumor suppression and to design more effective combination therapies, we analyzed disseminated tumors from the lungs in NCr/Nude mice using single cell RNA-seq and Mass Cytometry (Cy-TOF) after virotherapy. Our scRNA-seq analysis showed that in a subset of mice responding poorly to AVID-317 therapy, tumor-associated CSF1R-expressing myeloid cells expressed higher levels of MHC-II, Ctsb, and Arg1. In addition, CD3e+ T cells expressed functional exhaustion marker, PD-1, exclusively in mice that responded poorly to virotherapy. Mass Cytometry analysis with 37 metal isotope-labeled antibodies confirmed elevated expression of activation markers on myeloid cells after virotherapy. Next, we analyzed the efficacy of a combination therapy where in addition to AVID-317 administration, tumor-bearing mice were treated with anti PD-1 antibody to block immune-suppressive signaling in T cells, anti-CSF1R antibody to block CSF1R signaling, and anti-Asialo GM1 antibody to deplete anti-viral NK cells. Our analyses showed that combination therapy regimen significantly improved therapeutic outcomes in mice with disseminated lung cancer, by extending median survival to 95 days from 55-56 days observed in mice treated with either virus alone or combination drugs without the virus. Taken together, our study demonstrated feasibility of using AVID-317 oncolytic adenovirus for systemic therapy of disseminated lung cancer. Our study also showed that a combination of virotherapy with a defined select of immunotherapeutic drugs allows for the significant improvement of therapeutic efficacy in pre-clinical model of disseminated lung cancer. Citation Format: Jia Yao, Svetlana Atasheva, Dmitry Shayakhmetov. Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3366.

Abstract 1358: The selective HDAC6 inhibitor ITF3756 stimulates an antitumor immune response and leads to tumor regression in combination with anti CTLA-4 antibody in a colon carcinoma murine model

Abstract Stimulation of tumor immune response with antibodies blocking the repressive activity of immune checkpoints gives impressive results with long-lasting tumor regression that is achieved regrettably only in a fraction of patients. To overcome these suboptimal results, different targets and combination therapies are the aim of active non-clinical and clinical research. Importantly, clinical trials of combination therapies have demonstrated better efficacy but also an increase of adverse events. HDAC6 is a member of the Zn-dependent histone deacetylase family of enzymes with peculiar structure and functions. In contrast to what is observed with many other HDAC family members, HDAC6 KO mice are viable and have no peculiar phenotype indicating that selective HDAC6 inhibitors should be well tolerated. Since HDAC6 is an obligate regulator of cytokine-induced PD-L1 expression, HDAC6 inhibitors might have a role in modulating tumor immune responses. We here report the in vivo efficacy of ITF3756, a potent and selective HDAC6 inhibitor that reduces in vitro the expression of PD-L1 on human monocytes and on CD8 T cells, counters immune exhaustion in CD8 T cells but shows no direct cytotoxic effects on a panel of murine tumor cell lines. In syngeneic tumor models ITF3756 showed anti-tumor activity that was comparable to the efficacy of an anti PD1 antibody and that went along with increased immune cell infiltration and the generation of tumor-reactive CD8 and CD4 cells. ITF3756 was inactive in immune-deficient animals and selective depletion of either CD8 or CD4 cells severely blunted the antitumor activity of ITF3756. In combination with an anti CTLA-4 antibody, ITF3756 lead to a complete tumor eradication in 50% of the animals. Re-challenge of these animals did not result in tumor growth indicating that the combination therapy had elicited tumor immunity. Blocking the PD-1/PD-L1 axis either alone or in combination with inhibition of CTLA4 in NOD mice strongly accelerated the development of auto-immune diabetes that was lethal in some treatment groups. In contrast neither ITF3756 alone nor the combination with anti CTLA4 lead to any significant induction of diabetes despite the activity of ITF3756 on the PD-1/PD-L1 axis. We conclude that ITF3756 induces an in vivo antitumor immune response and a durable antitumor activity when combined with an anti CTLA-4 antibody. Neither ITF3756 monotherapy nor the combination with anti CTLA4 accelerated the induction of autoimmune diabetes in NOD mice suggesting a favorable safety profile that was confirmed in preclinical GLP toxicology studies. Phase I clinical trials with ITF3756 will be initiated this year. Citation Format: Gianluca Fossati, Chiara Ripamonti, Gianluca Caprini, Pietro Pozzi, Elisabetta Galbiati, Paola Cordella, Mattia Marchini, Barbara Vergani, Giovanni Sandrone, Andrea Stevenazzi, Christian Steinkuhler. The selective HDAC6 inhibitor ITF3756 stimulates an antitumor immune response and leads to tumor regression in combination with anti CTLA-4 antibody in a colon carcinoma murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1358.

Abstract 5600: Glycopeptides promote anti-cancer immune response against solid tumors

Abstract Only a fraction of patients benefits from immune checkpoint inhibitors (ICI). Tumor cells exhibit a distinct glycosylation pattern and supplementation of glycans has the potential to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a subcutaneous tumor mouse model, we investigated whether two glycopeptide-based test items reduce tumor burden when orally administered alone or in combination with ICI. C57/BL6 mice were supplemented with 3% GNU-201 (n=10) or 3% GNU-101 (n=10) in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice (n=10) received normal drinking water. On day 0, all mice were injected subcutaneously with YUMM1.7 melanoma cells in each flank. On days 6, 9 and 12, mice in each group received anti-PD1 antibody or an isotype control. Tumor size was measured every 3rd day. On day 15, the mice were sacrificed to analyze tumor weight and tumor infiltrating immune cells. 16S rDNA sequencing of fecal samples was performed on days -14, 0 and 14. GNU-201 and GNU-101 supplementation was well tolerated, no difference in water consumption and no abnormal behavior were detected that would indicate adverse effects of the products. While anti-PD1 on its own had no effect on tumor growth, we observed reduced tumor growth in the GNU-201+anti-PD1, GNU-101 and GNU-101+anti-PD1-treated groups, resulting in a significantly reduced tumor volume in these three groups on day 15. Administration of the two test items had a clear effect on anti-tumor T cell responses. In combination with anti-PD1, GNU-201 promoted the overall abundance of T cells, and among those the proportion of CD8+ T cells and IFNγ producing CD4+ T cells. Even in absence of anti-PD1, GNU-201 promoted the number of IFNγ+ CD8+ T cells, indicating that GNU-201 did not only promote tumor infiltration but also activation of CD8+ T cells, which are known to directly kill tumor cells. Finally, GNU-201+anti-PD1 resulted in significantly increased levels of Perforin and TNFα producing CD8+ T cells. With GNU-101 there was a trend towards elevated numbers of T cells and TNFα+ CD4 T cells. In mice that received GNU-101 with anti-PD1, we observed significantly elevated levels of TNFα+ CD8+ T cells and a trend towards higher numbers of IFNγ+ CD8+ T cells. GNU-201 and GNU-101 induced a significant modulation of the gut microbiota. In particular, the products promoted the growth of Akkermansia spp., a genus previously associated with anti-tumor immunity. In conclusion, GNU-201 and GNU-101 are both able to boost an anti-tumor immune response upon PD1 inhibition and GNU-101 shows moderate anti-tumor effects on its own. The response is linked with tumor-infiltration and activation of T cells and seems to be associated with changes in the gut microbiome. This study highlights a potential therapeutic role for glycopeptides in boosting anti-tumor immunity in response to ICI therapy. Citation Format: Marianne R. Spalinger, Romain Wyss, Sara Vidal, Yong Miao, Michael Scharl, Yemi Adesokan. Glycopeptides promote anti-cancer immune response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5600.

Abstract 4173: Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR

Abstract Cancer immunotherapy, especially those targeting the immune checkpoint PD-1 and PD-L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)-mediated anti-tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA-binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs. In this study, we show the direct binding of HuR protein to PD-L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP-IP) and further validated by RNA pulldown assay. We also show that PD-L1 mRNA decay is greatly expedited in HuR-knockout cells, which underlines the stabilization function of HuR on PD-L1 transcripts. Immunoblotting results demonstrate that PD-L1 protein expression level is decreased in HuR-knockout cells while up-regulated in HuR-proficient cells in comparison with control. These data together clarify HuR is engaged in PD-L1 post-transcription regulation. Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti-PD1 antibodies to overcome the limitation by regulating the tumor immune microenvironment. KH-200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4-week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. A similar combination effect was further observed in Lewis lung syngeneic lung cancer model. In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows effectiveness in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy. Citation Format: Qi Zhang, Yang Zhe, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4173.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV

SummaryBackgroundDysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated.MethodsWe studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles.FindingsHIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1− cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells.InterpretationTogether, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines.FundingNIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

Regorafenib in refractory metastatic colorectal cancer: A multi-center retrospective study.

e15580 Background: Regorafenib improves progression-free survival (PFS) and overall survival (OS) in patients with refractory metastatic colorectal cancer (mCRC). Here, we report the treatment patterns of regorafenib in the third- or late-line setting for mCRC in four centers in China. Methods: Patients with refractory mCRC in four centers in China administered regorafenib from February 1, 2018 to June 31, 2021 were enrolled. Patients were grouped into 3 cohorts, including the monotherapy (regorafenib alone), chemo (regorafenib plus chemotherapy) and immune (regorafenib plus anti-PD1 (programmed cell death 1) antibodies) groups. Demographic, clinical, survival and safety data were retrospectively analyzed. Results: A total of 177 patients were included in this study. Of them, 116 (65.5%) were treated with regorafenib alone, while 28 (15.9%) and 33 (18.6%) were administered regorafenib plus chemotherapy and anti-PD1 antibodies, respectively. The median followedup time was 9.2 months. The disease control rate (DCR) was 40.7%. The median PFS (mPFS) was 2.43 months and the median OS (mOS) was 12.2 months. The immune group had longer median PFS (3.5 m vs. 2.2 m, p = 0.043) compared with the monotherapy group. Patients administered regorafenib plus chemotherapy had longer median OS (15.9 m vs. 8.4 m, p = 0.032) compared with the monotherapy group. Patients who began regorafenib treatment at 120 mg had longer median PFS and OS compared with those who began at 80 mg (PFS: 3.7 m vs. 2.0 m; p < 0.001; OS: 13.4 m vs. 10.2 m; p = 0.005). Patients with a final dose of 120 mg had longer median PFS and OS compared with the 80 mg or less group (PFS: 5.0 m vs. 2.3 m; p = 0.045; OS: UR (unreach) vs. 10.9 m; p = 0.003). There were 87.0% (154/177) patients experience AEs. Three groups had similar rates of AEs (86.2% vs. 89.3% vs. 87.9%; p = 0.89). Conclusions: Patients administered regorafenib alone or regorafenib in combination with other agents were relieved to some extent, with a disease control rate of 40.7%. Regorafenib plus anti-PD1 antibodies showed better PFS, while regorafenib plus chemotherapy had the most benefit in OS. There was no significant difference among three groups in terms of AEs.

Efficacy of ipilimumab 3mg/kg following progression on low dose ipilimumab in metastatic melanoma.

e21533 Background: Anti-cytotoxic T lymphocyte-associated antigen 4 inhibitors, such as ipilimumab (IPI), are commonly used in melanoma management. Whilst a clear dose-toxicity relationship exists, and a dose-response relationship is seen with monotherapy in people with anti PD-1 antibody-naïve melanoma, there is no data on the outcomes of patients who progress following low dose IPI and are subsequently treated with standard dose IPI. We conducted a multicentre, retrospective review to assess the efficacy of this strategy. Methods: Patients with resected stage III, unresectable stage III or IV melanoma who received low dose IPI (< 3mg/kg) with an anti-PD1 antibody in the neoadjuvant, adjuvant or metastatic setting with recurrence (neo/adjuvant) or progressive disease (metastatic) as best response, who then received standard dose IPI (3mg/kg, IPI3) alone or in combination with an anti-PD1 antibody were eligible. IPI dose, frequency and best investigator-assessed RECIST response were collected. Progression free survival (PFS) and overall survival (OS) were analysed using the Kaplan Meier method. Results: 36 patients from 8 centres; 27 (75%) male, BRAF V600 mutant (18, 50%). All patients received low dose IPI with an anti-PD1 antibody, 18 (50%) in the neo/adjuvant and 18 (50%) in the metastatic setting; 15 (42%) received IPI 1mg/kg every 6 weeks, 13 (36%) IPI 1mg/kg every 3 weeks, 3 (8%) IPI 50mg every 6 weeks or 100mg every 12 weeks respectively, and 2 (6%) IPI 1mg/kg every 8 weeks. 23 (64%) received >1 intervening line of systemic therapy prior to IPI3. The most common intervening therapy was BRAF/MEK inhibitors; 14/23 (61%) as 2nd line treatment, 4/8 (50%) as 3rd line treatment, and all progressed. All patients received standard dose IPI3 for unresectable stage III or stage IV melanoma; median age 60 (29-78), 18 (50%) had M1d disease, 32 (89%) ECOG 0-1, 18 (50%) normal lactate dehydrogenase. 35 (97%) received IPI3 with nivolumab and 1 received single agent IPI3. 15 (42%) received 4 cycles of IPI3. The most common reason for IPI3 cessation was progressive disease (16, 44%). The response rate to IPI3 following progression on low dose IPI was 9/36 (25%); the disease control rate was 12/36 (33%). After a median follow up of 22 months (95% CI: 15-27 months), the median PFS and OS were not reached in patients who responded;1-year PFS and OS were 73% and 100% respectively. 13/36 (36%) had grade 3-4 immune-related adverse events (irAE). In 3/36 (8%), the same irAE was observed at low dose IPI and IPI3, and 1 patient had the same irAE (rash) at a higher grade with IPI3 compared to low dose IPI. Conclusions: Standard dose IPI3 following progression on low dose IPI has clinical activity.This is a reasonable treatment strategy for patients who progress on low dose IPI and provides further evidence of a dose-response relationship for IPI. Further research is needed to manage resistance to IPI with anti-PD1.

Trial in progress: A randomized phase II study of pembrolizumab with or without defactinib, a focal adhesion kinase inhibitor, following chemotherapy as a neoadjuvant and adjuvant treatment for resectable pancreatic ductal adenocarcinoma (PDAC).

TPS4192 Background: PDAC is an aggressive cancer. It remains refractory to checkpoint inhibition because of its significant desmoplastic and immunosuppressive tumor microenvironment (TME). Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is involved in tumor progression in many cancers and appears to be a targetable master regulator of the TME in PDAC. FAK inhibition combined with anti-PD1 antibody has been shown to modulate pancreatic stellate cells and decrease immunosuppressive myeloid and T-reg cells, leading to increased CD8 infiltration and improved survival in PDAC mouse models. A recent single arm phase I study of defactinib, a FAK inhibitor, combined with pembrolizumab, an anti-PD1 antibody, and chemotherapy was shown to be safe, and two confirmed partial responses were observed in patients with microsatellite-stable disease. Furthermore, increased CD8 T cell infiltration was observed in metastatic biopsies. Given the promising preclinical data and efficacy signals, as well as safety of the phase I clinical trial, our current study aims to assess the translational and clinical effects of sequentially combined defactinib and pembrolizumab following neoadjuvant and adjuvant chemotherapy in patients with high-risk resectable PDAC. Methods: This study is a multi-center, two-arm, randomized, open label, phase II clinical trial of neoadjuvant and adjuvant immunotherapy with defactinib and pembrolizumab, following neoadjuvant standard of care (SOC) gemcitabine and nab-paclitaxel in subjects with high-risk resectable PDAC. The primary objectives aim to assess changes in CD8 T cell intratumoral infiltration utilizing multiplex IHC and the pathologic complete response rate with defactinib and pembrolizumab or pembrolizumab alone, following neoadjuvant chemotherapy. Secondary objectives include assessment of disease-free survival, overall survival, and safety. Translational exploratory objectives include evaluating stromal and immune signatures among treatment groups via multiplex IHC and RNA/DNA sequencing. 36 subjects will be randomly assigned to receive 400 mg defactinib PO BID and 200 mg pembrolizumab IV every 3 weeks (Arm A) or 200 mg pembrolizumab IV alone every 3 weeks (Arm B). After enrollment, subjects will undergo 2 cycles (̃2 months) of standard neoadjuvant therapy of gemcitabine and nab-paclitaxel, followed by 2 cycles (6 weeks) of investigational treatment (Arm A or B) before surgical resection. Following surgery, subjects will receive SOC adjuvant chemotherapy followed by investigational treatment (Arm A or B) for 8 cycles (̃24 weeks). Key inclusion criteria include: resectable PDAC, CA 19-9>200, no prior systemic treatment for PDAC, and ECOG PS ≤1. As of February 2022, 14 patients have been enrolled. Clinical trial information: NCT03727880.

Anti–PD-1 antibody (sintilimab) plus decitabine as first-line treatment for patients with higher-risk myelodysplastic syndrome (MDS): Preliminary results from a single-arm, open-label, phase II study.

7052 Background: Hypomethylating agents (HMAs) are the preferred treatment for untreated patients (pts) with higher-risk MDS, but the survival of pts after HMAs treatment is poor. It has been demonstrated that the PD-1/PD-L1 expression was upregulated by HMAs in MDS pts, providing a strong rationale for combining HMAs with PD-1 antibody for MDS treatment. Therefore, this single-arm, open-label, clinical trial was performed to evaluate safety and efficacy of Sintilimab plus decitabine for pts with higher-risk MDS (ChiCTR2100044393). Methods: Adult pts with higher-risk MDS by the IPSS-R were enrolled. Patients received decitabine 20mg/m2 intravenously daily for 5 days and sintilimab 200mg IV starting on the first and 22nd day of a cycle every 42 days until unacceptable toxicity, relapse, or progression, for a maximum of 8 cycles. The primary endpoint was overall response rate (CR+PR+mCR). Simon’s optimal two-stage design was employed.If five or more pts in stage I (13 pts) achieved ORR, the study would enroll 34 additional pts in stage II (47 pts). Secondary endpoints included safety, and survival outcomes. The relationship between expression levels of immune-checkpoint and efficacy of the combination therapy, as well as other potential biomarkers were also explored via genomic profiling. Results: At data cut-off (January 31, 2022), 21 pts were enrolled with a median follow-up time of 5.8 months. The median age of pts was 64 years (range 30-83), and the other characteristics are summarised in Table.The ORR was 62%,with six pts reaching complete response (CR),four pts reaching marrow CR and three pts achieved marrow CR+ hematologic improvement (HI). In addition,four pts reaching HI. The most common grade 3 TEAEs( > 10%) were febrile neutropenia (76.2%) and pulmonary infection (38.1%). No grade 4 TEAEs and treatment-related deaths occurred. A total of 12 pts （57.1%）experienced immune-related adverse event, including rash (28.6%), pneumonia (9.5%), hypothyroidism (9.5%), elevated serum bilirubin (4.8%) and transpeptidase (4.8%), which were all resolved by glucocorticoid. In these 21 pts, the most frequently mutated genes are ASXL1（28.6%）, RUNX1 (14.3%) and TET2(14.3%). Updated biomarker data will be presented during the ASCO meeting. Conclusions: To the best of our knowledge, this is the first study to evaluate the efficacy and safety of sintilimab plus decitabine in pts with untreated higher-risk MDS. The preliminary results demonstrate that the combination therapy is relatively safe with anti-tumor activity. Clinical trial information: ChiCTR2100044393. [Table: see text]

Datopotamab deruxtecan (Dato-DXd) plus pembrolizumab in treatment-naive advanced/metastatic (adv/met) non–small cell lung cancer (NSCLC) with PD-L1 ≥ 50% and without actionable genomic alterations.

TPS3162 Background: First-line treatment with immunotherapy has significantly improved survival in patients with adv/met NSCLC. Pembrolizumab (pembro) as monotherapy has shown superior efficacy compared with chemotherapy in treatment-naive patients with advanced NSCLC and PD-L1 expression ≥50%, but most patients will ultimately experience progression. Dato-DXd is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody attached to a topoisomerase I inhibitor payload via a stable tetrapeptide-based cleavable linker. In the ongoing phase 1 TROPION-PanTumor01 study (NCT03401385; DS1062-A-J101), Dato-DXd 6 mg/kg monotherapy demonstrated an objective response rate (ORR) of 28% and a manageable safety profile in pretreated patients with NSCLC. In addition, preclinical studies showed that DXd ADCs combined with an anti–PD-1 antibody was more effective than monotherapy with either agent alone. The tolerability of Dato-DXd 6 mg/kg in combination with pembrolizumab was confirmed in the phase 1b TROPION-Lung02 trial (NCT04526691; DS1062-A-U102). Here we describe the phase 3 TROPION-Lung08 trial evaluating Dato-DXd combined with pembro in treatment-naive patients with adv/met NSCLC. Methods: TROPION-Lung08 (NCT05215340; DS1062-A-U304) is a global, randomized, open-label, phase 3 trial of Dato-DXd plus pembro vs pembro alone in treatment-naive patients with adv/met non-actionable oncogenic driven NSCLC with PD-L1 ≥50% (as determined by PD-L1 IHC 22C3 pharmDx assay). Approximately 740 patients will be randomized to receive pembro 200 mg plus Dato-DXd 6 mg/kg or pembro 200 mg alone every 21 days until discontinuation or 35 cycles of pembro. Randomization will be stratified by Eastern Cooperative Oncology Group performance status (0 vs 1), histology (squamous vs nonsquamous), geographic region (East Asia vs rest of world), and smoking status (former/current vs never). Patients must have stage IIIB/IIIC NSCLC ineligible for curative treatment or stage IV disease. Patients must not have received prior systemic therapy; if patients received neoadjuvant/adjuvant systemic therapy without immune checkpoint inhibitors, it must have been given ≥6 months before the diagnosis of adv/met disease. The primary endpoints are progression-free survival (assessed by blinded independent central review per Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1) and overall survival, with target hazard ratios of 0.65 and 0.75, respectively. Secondary endpoints include ORR, duration of response, time to response, disease control rate, safety, and antidrug antibody prevalence. Pharmacokinetic parameters, biomarkers, and patient-reported outcomes will also be explored. Clinical trial information: NCT05215340.