Small-molecule CD73 Inhibitor Research Articles

Abstract LB131: CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization

Abstract Background: CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Immunosupressive adenosine contributes to immune evasion in solid tumors by accumulating within the tumor microenvironment and thereby inhibiting immune effector cells (e.g., CD8+ T cells). CD73 expression levels correlate with a worse prognosis in specific cancer types, including breast cancer. Adenosine production via CD73 can be therapeutically inhibited by two independent mechanisms: (1) enzyme inhibition, and (2) receptor internalization and subsequent degradation. CBO421, a drug Fc-conjugate (DFC), comprised of a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, achieves both mechanisms, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) with potential best-in-class efficacy. Methods: The activities of CBO421, a commercially sourced CD73 small molecule inhibitor, quemliclustat (AB680), and biosimilar anti-CD73 mAb, oleclumab (MEDI9447), were investigated. Enzymatic inhibition of soluble CD73 and membrane-bound CD73 on MDA-MB-231 cells was determined. Receptor-mediated internalization of CD73 was assessed using the human triple-negative breast cancer cell line (TNBC), MDA-MB-231, by flow cytometry. Cells were treated with CBO421 or oleclumab. CD73 internalization was measured as the decrease in CD73 surface receptor signal over a 6 h time-course using an anti-human CD73 detection antibody with non-overlapping epitopes to CBO421 or oleclumab. Results: CD73 inhibition by CBO421 of soluble CD73 or CD73 expressed on the surface of the MDA-MB-231 cell line was comparable or superior to AB680 or oleclumab. Receptor internalization activities of CBO421 and oleclumab were compared. CBO421 and oleclumab internalization percentages were determined at 1 h (52.3% vs 26.7%), 4 h (80.8% vs 38.0%), and 6 h (84.8% vs 42.9%) respectively, with CBO421 demonstrating superior maximal receptor internalization. The unconjugated hIgG1 Fc negative control did not exhibit any receptor-mediated internalization of CD73, as expected. IC50 values were also calculated for CBO421 and oleclumab at 1 h (0.20 nM vs 0.88 nM), 4 h (0.07 nM vs 0.27 nM), and 6 h (0.08 nM vs 0.14 nM), respectively. Conclusions: CBO421 demonstrated potent and complete CD73 enzyme inhibition and robust CD73 receptor internalization in CD73+ cancer cells that was superior to oleclumab, thereby further differentiating CBO421 from existing CD73-targeting therapeutics currently in clinical development. Based on these compelling results and additional emerging data, CBO421 is progressing as a potential best-in-class clinical development candidate for the treatment of solid cancers. Citation Format: Elizabeth Abelovski, Nicholas Dedeic, Simon Döhrmann, James Levin, Amanda Almaguer, Doug Zuill, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB131.

Abstract 2728: Discovery of a multispecific CD73/PD-1 targeting drug Fc-conjugate (DFC), which improves tumor reduction compared to PD-1 monotherapy in a humanized mouse model

Abstract Background: The approval of immune checkpoint therapies has revolutionized cancer treatment and established immuno-therapy as an additional treatment option. However, while anti-PD-(L)1 therapies have demonstrated durable responses, only a small subset of patients respond. One mechanism of tumor escape is via the release of immunosuppressive metabolites such as adenosine. CD73, an extracellular enzyme expressed on immune cells and some tumors, catalyzes the conversion of AMP to adenosine, the rate limiting step in adenosine production. As a strategy to improve response rates we developed a first in class CD73/PD-1 targeting DFC, comprised of a multivalent conjugate of a small molecule CD73 inhibitor stably linked to a proprietary human IgG1 Fc-fusion with a PD-1 inhibitor peptide. This multispecific DFC has the potential for differentiation and increased therapeutic potency compared to approved PD-(L)1 inhibitors. Methods: Binding of CD73/PD-1-001 to biotinylated hPD-1 was determined using ELISA and to human CD8+ T cells by flow cytometry. CD73 inhibition was measured in cell-free and cell-based assays. Efficacy was determined in a humanized mouse model optimized for PD-1 activity against a transgenic MC-38 colon cell line expressing hPD-L1 (Genoway, France). Tumor volumes were recorded and statistical analysis was conducted by t-test (Mann-Whitney) or two-way ANOVA. Results: In vitro, CD73/PD-1-001 demonstrated potent activity against both checkpoint targets. CD73/PD-1-001 bound to hPD-1 with an IC50 of < 1 nM and demonstrated functional inhibition of CD73 with an EC50 of <10 nM, which is on par with respective single agent molecules. Importantly, addition of the CD73 targeting moiety resulted in no significant loss in PD-1 binding relative to PD-1-001, a DFC containing only the PD-l inhibitor peptide. Efficacy was determined in a humanized mouse model against a transgenic MC-38 cell line. Both CD73/PD-1-001 and PD-1-001 DFCs were highly potent in this model with tumor growth inhibition (TGI) of >70% at dose concentrations of 3 mg/kg or greater (p≤0.01). However, at the lowest test concentration of 1 mg/kg, only CD73/PD-1-001 retained significant potency with a TGI of 56.0 % (p ≤ 0.05). Tumor reduction in the 1 mg/kg PD-1-001 group did not reach statistical significance (36.5%, p > 0.05). Conclusions: This work demonstrates that a multispecific CD73/PD-1 targeting DFC, is a potent inhibitor of tumor growth in a humanized mouse model. Furthermore, CD73/PD-1-001 was more potent than PD-1 monotherapy, demonstrating the therapeutic benefit of targeting two important checkpoints with a single agent molecule. Collectively, preclinical testing of a CD73/PD-1 DFC has validated the benefits of targeting the PD-(L)1 axis in combination with adenosine suppression, resulting in improved anti-tumor effects. Citation Format: James Levin, Simon Döhrmann, Nicholas Dedeic, Amanda Almaguer, Doug Zuill, Elizabeth Abelovski, Jason N. Cole, Jeffrey B. Locke, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Leslie W. Tari. Discovery of a multispecific CD73/PD-1 targeting drug Fc-conjugate (DFC), which improves tumor reduction compared to PD-1 monotherapy in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2728.

Abstract 2668: CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway

Abstract Background CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Adenosine contributes to immune evasion by solid tumors by concentrating immunosuppressive adenosine in the tumor microenvironment. CBO421, a Drug Fc-Conjugate (DFC), comprises a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) for potential best-in-class efficacy. Methods CBO421’s binding affinity to human CD73 and Fc receptors was assessed by surface plasmon resonance. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a commercial kit. Immunophenotyping and binding to cancer cells was measured by flow cytometry. Inhibition of soluble and membrane-bound CD73 on human PBMCs and mouse cancer cells was determined by measuring free phosphate levels. Functional activity was measured using PBMC rescue assays. In vivo efficacy of CBO421 monotherapy was evaluated in a syngeneic mouse model. Results CBO421 exhibited a binding affinity to human CD73 (KD = 0.8 nM) that was comparable to or greater to the affinity observed with small molecule inhibitors (AB680, OP-5244) and CD73-targeting mAbs (oleclumab, mupadolimab biosimilars). As expected, human FcRn binding of CBO421 was comparable to the unconjugated Fc and the full-length wild-type IgG1 control mAb. CBO421 binding to human Fc gamma receptors and ADCC activity was significantly reduced or abolished compared to the control mAb, such that it poses a minimal risk for undesired autoimmune activity. CBO421 potently inhibited soluble CD73 and CD73 expressed on human MDA-MB-231 and mouse EMT6 cancer cells. Immunophenotyping revealed that CD73 expression was primarily observed on CD8+ T and B cells from human PBMCs. In functional PBMC rescue assays, CBO421 demonstrated potent reactivation of AMP- or ATP-suppressed CD4+ or CD8+ T cells at levels comparable to small molecule inhibitors and greater than anti-CD73 mAb comparators. CBO421 monotherapy demonstrated a dose-dependent reduction in tumor volume in a syngeneic mouse model using MC38, a colorectal cancer cell line. Conclusions CBO421 exhibited strong binding and potent inhibition of both soluble and membrane-bound CD73, differentiating it from mAb CD73 inhibitors. The proprietary immune-silent Fc of CBO421 did not affect FcRn binding but substantially reduced Fc gamma receptor binding, leading to the elimination of ADCC activity to prevent undesired immune cell depletion in host tissues. CBO421 demonstrated high potency in functional cell-based assays and robust antitumor efficacy in a syngeneic mouse model. Currently, CBO421 is being advanced as a clinical development candidate for the treatment of solid cancers. Citation Format: Amanda Almaguer, Doug Zuill, Simon Döhrmann, James Levin, Nicholas Dedeic, Elizabeth Abelovski, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2668.

ARC-8: Phase 1/1b randomized study of quemliclustat + gemcitabine/nab-paclitaxel ± zimberelimab in patients with treatment-naive metastatic pancreatic adenocarcinoma.

665 Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) remains a challenging cancer to treat, with median survival of <1 year. In PDAC, extracellular adenosine (eADO) produced from ATP is released from tumor cells and suppresses antitumor immune responses. Quemliclustat (Q) is a potent and selective small-molecule inhibitor of soluble and cell-bound CD73, a key enzyme involved in the production of eADO within the tumor microenvironment. Elevated CD73 expression is found in 40-60% of PDAC and correlates with poor clinical outcome. ARC-8 evaluated Q ± anti-PD-1 antibody zimberelimab (Z) in combination with gemcitabine/nab-paclitaxel (G/nP). Methods: ARC-8 (NCT04104672) is a phase 1b dose escalation and expansion study in patients (pts) with treatment-naive mPDAC. Q 100mg was previously determined as the recommended dose for expansion (Manji et al., 2021). Q 100 mg is administered with standard doses of G/nP with Z (240 mg IV Q2W) in the dose expansion (Cohort A; QZ+G/nP) and in randomized (2:1) cohorts either with Z (Cohort A1; QZ+G/nP) or without Z (Cohort A2; Q+G/nP). Adverse events (AEs) are recorded and graded per NCI CTCAE 5.0. Clinical activity is assessed every 8 weeks per RECIST v1.1. Endpoints include measures of safety and median overall survival (OS). Results: As of June 19, 2023, 122 patients with untreated mPDAC were treated with Q 100mg, including 93 pts treated with QZ+G/nP (dose escalation, n=6; cohort A, n=26; and randomized cohort A1, n=61) and 29 pts treated with Q+G/nP (Cohort A2). Median age was 66 years, 66% ECOG of 1, and 65% had liver metastasis at baseline. Median survival follow-up was 21 months (mos). Efficacy is summarized in Table 1. All patients reported AE, 85% Grade 3+ AE, and 23% discontinued study treatment due to AE. Most common Grade 3+ AE was neutrophil count decreased (31%) and anemia (25%). Conclusions: Results from ARC-8 demonstrate the addition of Q 100 mg ± Z to G/nP was safe and tolerable, with no significant added toxicity to GnP. Modulation of eADO with Q may confer benefit beyond radiographic measures of disease. The OS is promising and supports further development of Q in mPDAC. Clinical trial information: NCT04104672 . [Table: see text]

Preliminary Results of the Oral CD73 Inhibitor, Oric-533, in Relapsed/Refractory Multiple Myeloma (RRMM)

Background: Although recently available immune-based approaches in multiple myeloma produce high response rates, curative outcomes have not yet been achieved, which highlights the need for new agents that further activate the immune system without increasing toxicity. ORIC-533 is a highly potent and selective, orally bioavailable, small molecule inhibitor of CD73, an enzyme expressed by various cell types, including immune cells and which catalyzes the generation of adenosine from adenosine monophosphate (AMP) (Allard 2019). Adenosine itself is highly immunosuppressive, diminishing activity of a range of immune cell types, including T, NK, and dendritic cells (Young 2014). By blocking adenosine production from AMP via inhibition of CD73, ORIC-533 has demonstrated the ability to restore and enhance immune function. Multiple myeloma (MM) patient bone marrow (BM) contains multiple cell types that also express CD73, including immune cells, cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells (Allard 2019). Moreover, elevated adenosine levels in the MM BM niche correlate with disease progression (Horenstein 2016; Yang 2020). We have previously observed that CD73-mediated adenosine activity suppresses the cytolytic antitumor immune function in the MM BM milieu (Ray 2022). ORIC-533 treatment reduced adenosine production, overcame this immune suppression with evidence of restored DC-mediated priming and activated cytotoxic CD8 + T-cell function, and significantly enhanced immune cell cytolytic behavior against RRMM tumor cells in autologous patient ex vivo experiments (Junttila 2022), making ORIC-533 a potential immunotherapy for patients with MM. Methods: ORIC-533-01 (NCT05227144) is an ongoing i3+3 Phase 1b dose escalation study in patients with RRMM. The primary objectives are to determine single agent safety/tolerability and pharmacokinetics (PK) of ORIC-533 to select the RP2D. Pharmacodynamic assessments include measurement of CD73 functional activity in serum and BM, as well as enumeration of immune cell subsets and activation state in blood and BM. Patients are also evaluated for clinical efficacy using IMWG criteria. Results: As of 23 June 2023, 17 patients received ORIC-533 once daily across 4 dose levels (400 mg to 1600 mg). All patients were triple-class refractory, 88% were penta-refractory, and 59% also received prior anti-BCMA/CD3 bispecific therapy or anti-BCMA CAR-T therapy. Overall, ORIC-533 was very well tolerated, with 5 patients experiencing a total of 10 treatment-related adverse events (TRAEs). Fatigue was the only G3 AE and the only TRAE seen in more than 1 patient (1 event each of G3 and G2); all other events seen in 1 patient each, were G1 or G2 in severity, and of no specific system organ class. No dose limiting toxicities, no Grade ≥4 TRAEs, and no treatment-related serious adverse events have been observed. PK showed increased exposure with dose with a plasma half-life of ~24 hrs. Soluble CD73 enzymatic activity was nearly completely inhibited by C1D15 in serum of all patients across all dose levels, while the largest relative suppression in surface CD73 activity in BM cells was observed at the highest dose level. There was also evidence for increases in the abundance of circulating NK and CD8+ T cells after 1 treatment cycle, with preliminary evidence of enhanced CD8+ T-cell activation at the highest dose levels tested in both peripheral blood and BM. In addition, early evidence of single agent clinical activity was observed. Conclusions: ORIC-533 has demonstrated an acceptable safety profile and preliminary evidence of immune activation in a heavily pretreated RRMM patient population. Enrollment is ongoing and updated data will be presented.

Development of a Concise Process for the Synthesis of the Azaindazole Core of the CD73 Inhibitor AB680

AB680 is a highly potent small-molecule CD73 inhibitor discovered and developed by Arcus Biosciences, currently in clinical trials for the treatment of pancreatic cancer. Herein, we report a concise synthesis of 4,6-dichloro-1H-pyrazolo[3,4-b]pyridine 3, which is the central azaindazole core of AB680. The process consists of four synthetic operations and three isolations, including a PMB-protected pyrazole formation, a telescoped two-step cyclization and aromatization sequence, and finally a one-pot PMB deprotection and chlorination to furnish 3. This chemistry was successfully scaled up to provide >200 g of 3 in 44% overall yield and 97.6% HPLC purity. Overall, the route developed toward 3 represents an efficient construction of an azaindazole core, which is a synthetically challenging yet prevalent structural motif.

Abstract 504: BPI-472372: a potent and orally bioavailable small molecule inhibitor of CD73 for cancer immunotherapy

Abstract CD73, also known as ecto-5'-nucleotidase, plays a major role in converting extracellular adenosine monophosphate (AMP) to immunosuppressive adenosine. It is frequently overexpressed across a variety of cancers. High adenosine level in the tumor microenvironment, predominantly signaling through the A2a/A2b receptors, suppresses innate and adaptive immune responses and mediates tumor immune evasion. CD73 overexpression is correlated with poor prognosis in several tumor types. Inhibition of CD73 to reduce adenosine production is a promising therapeutic approach for the treatment of cancer. Both monoclonal antibodies and small molecule inhibitors are being explored to target CD73. Small molecule CD73 inhibitor with good balance of activity and ADME properties are rare. Here we present BPI-472372, a potent, selective, and orally bioavailable small molecule CD73 inhibitor. It strongly inhibits both soluble and cell-bound CD73, with no off-target activity on other related nucleotidases (NTPDases 1/2/3/5), nor on a large panel of enzymes, receptors, and ion channels. BPI-472372 reversed AMP-induced CD4+ T cell suppression in a dose-dependent manner. In vivo, BPI-472372 exhibited anti-tumor effect in MC38 syngeneic mouse model with no visible adverse effect. Moreover, enhanced tumor growth inhibition activity was observed when BPI-472372 was combined with durvalumab (anti-PD-L1) in humanized PD-L1 syngeneic MC38 model. BPI-472372 exhibited desirable drug-like properties including high permeability, low hepatocytes clearance, and adequate oral exposure across multiple pre-clinical species. GLP toxicity study indicated that BPI-472372 was well tolerated at high dose following 4 weeks repeated administration in both mouse and dog. Taken together, BPI-472372 is a potent and orally bioavailable small molecule CD73 inhibitor with single agent anti-tumor efficacy, favorable ADME properties, and a wide therapeutic index. Citation Format: Han Han, Xiaofeng Yang, Ying Zhao, Jin Wang, Xiang Yang, Xuepeng Ju, Jiayu Zhao, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Jing Guo, Cheng Yang, Hong Lan, Hao Wu, Lieming Ding, Jiabing Wang. BPI-472372: a potent and orally bioavailable small molecule inhibitor of CD73 for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 504.

Abstract 5258: Preclinical testing of CD73 inhibitor AB680 for pancreatic cancer immunoprevention

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a profoundly immunosuppressive microenvironment. Innovative therapeutic strategies are urgently needed to stop the progression of precancerous lesions into aggressive PDAC, which remains a lethal malignancy. The goal of this research project is to test immunopreventive strategies by targeting the 5’ ectonucleotidase enzyme, CD73, one of the gatekeeper enzymes responsible for conversion of adenosine monophosphate (AMP), to an immunosuppressive metabolite, adenosine, in the tumor microenvironment (TME). We hypothesize that inhibition of CD73 will prevent pancreatic intraepithelial neoplasia (PanIN) formation and progression to PDAC by reversing adenosine directed immunosuppression. This research explores immunopreventive strategies aimed to restore tumor immune surveillance to prevent cancer initiation or progression. Materials and methods: We used two models: a syngeneic model of PDAC using cells derived from KrasG12D;Trp53R172H/+;PdxCre (KPC) mice and a KrasG12D;PdxCre (KC) genetically engineered mouse model (GEM) of PDAC. Oral gavage of AB680 (small molecule CD73 inhibitor) was given three days/week at 10mg/kg starting the day after KPC injections and tumor sizes were measured weekly. In the GEM model, the same treatment regimen began when the mice were between 6 and 9 weeks old and were euthanized either between 15 and 20 weeks of age or around 27 weeks of age, and pancreas tissue was harvested. Histology was analyzed and 6 fields per mouse were quantified using ImageJ. Results: As we have described (Singh, et al, bioRxiv), in the syngeneic model, there was a significant reduction in tumor growth and significant increase in activated CD8-positive T cells, dendritic cells, and macrophages from AB680 treated mice. The intratumoral adenosine levels were significantly decreased in AB680 treated mice compared to vehicle treated mice. In the KC GEM model, we quantified significantly fewer early PanIN lesions (p=0.0328), a trend in decreased advanced PanIN (p=0.0641), and significant decrease in PDAC (p=0.0058) in the AB680 treated mice when compared to the vehicle treated mice. We quantified abundance of collagen deposition as a marker of fibrosis and observed significantly decreased collagen (p<0.0001) in AB680 treated KC mice. In addition, we quantified abundance of CK19+ lesions and observed a significant decrease in CK19+ lesions in AB680 treated mice (p=0.0061) compared to vehicle treated mice. Conclusion: Inhibiting CD73 restructures TME and reduces PanIN incidence and progression to PDAC. CD73 inhibition may be a candidate immunoprevention strategy in pancreatic cancer. [Supported by NCI 75N91019D00021/75N91020F00002] Citation Format: Lincoln N. Strickland, Erika Y. Faraoni, Nicolette R. Mardik, Lana Vornik, Michelle I. Savage, Shizuko Sei, Mark S. Miller, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitor AB680 for pancreatic cancer immunoprevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5258.

Discovery of a Series of Potent, Selective, and Orally Bioavailable Nucleoside Inhibitors of CD73 That Demonstrates In Vivo Antitumor Activity.

CD73 (ecto-5'-nucleotidase) has emerged as an attractive target for cancer immunotherapy of many cancers. CD73 catalyzes the hydrolysis of adenosine monophosphate (AMP) into highly immunosuppressive adenosine that plays a critical role in tumor progression. Herein, we report our efforts in developing orally bioavailable and highly potent small-molecule CD73 inhibitors from the reported hit molecule 2 to lead molecule 20 and then finally to compound 49. Compound 49 was able to reverse AMP-mediated suppression of CD8+ T cells and completely inhibited CD73 activity in serum samples from various cancer patients. In preclinical in vivo studies, orally administered 49 showed a robust dose-dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship that correlated with efficacy. Compound 49 also demonstrated the expected immune-mediated antitumor mechanism of action and was efficacious upon oral administration not only as a single agent but also in combination with either chemotherapeutics or checkpoint inhibitor in the mouse tumor model.

Abstract IA013: Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention

Abstract Introduction: The all stages combined five-year survival rate for pancreatic adenocarcinoma (PDA) is 11%; however, the five-year survival rate for localized PDA is 42%. These statistics highlight the importance of early prevention strategies to prevent disease progression and metastatic dissemination. Through the NCI PREVENT program, this research program explores immunoprevention strategies for PDA by targeting CD73, a gatekeeper ectoenzyme responsible for production of extracellular adenosine. We have recently shown aggressive subtypes of pancreatic intraepithelial neoplasia (PanIN) and PDA arising in ductal pancreatic epithelium have elevated CD73 and intrapancreatic adenosine indicating adenosine generation may be an early trigger of immunosuppression. We hypothesize inhibition of CD73 and adenosine generation will promote a more robust anti-tumor immune response and prevent PanIN and PDA progression. Methods: We tested three small molecule CD73 inhibitors (APCP, OP-5244, and AB680) in a syngeneic PDA mouse model by injecting 100-200k murine PDA cells derived from KrasG12D;Trp53R172H/+;Pdx:Cre (KPC) mice in the flanks of C57BL/6 female mice. Tumor sizes were measured weekly and tumor volume and mass were recorded at time of death. Dosage: APCP oral gavage (3x/week at 20mg/kg) and intraperitoneal (IP) (3x/week at 20 mg/kg). OP-5244 oral (3x/week at 25mg/kg and 10mg/kg). AB680 oral gavage (3x/week at 10mg/kg). HPLC analysis was performed for each inhibitor to quantify adenosine levels. Results: IP delivery of APCP significantly reduced tumor growth and intratumoral adenosine levels; however oral gavage delivery did not reduce tumor growth. Similarly, oral gavage delivery of OP-5244 did not reduce tumor growth. AB680 significantly reduced tumor volume and intratumoral adenosine levels and CyTOF immunoprofiling showed activated CD8+ T cells, dendritic cells, and macrophages were significantly increased in the tumors from AB680 treated mice. Conclusion: APCP IP delivery is more effective than oral gavage delivery and OP-5244 oral gavage delivery does not significantly decrease tumor growth. AB680 oral gavage delivery significantly decreases tumor growth and tumor adenosine concentrations. We observed a significant increase in infiltration of activated CD8+ T cells. AB680 shows high translational potential for preclinical testing in spontaneous GEM models. Citation Format: Lincoln Ballew, Kanchan Singh, Vidhi Chandra, Tingting Mills, Erika Y. Faraoni, Victoria Mota, Trent Clark, Lana Vornik, Michelle I. Savage, Shizuko Sei, Altaf Mohammed, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA013.

Application of a fluorine strategy in the lead optimization of betulinic acid to the discovery of potent CD73 inhibitors

The ecto-5′-nucleotidase (CD73) is an important enzyme in the adenosine pathway and catalyzes the extracellular hydrolysis of adenosine monophosphate (AMP) yielding adenosine which is involved in the inflammation and immunosuppression. Inhibitors of CD73 have potential as novel immunotherapy agents for the treatment of cancer and infection. In this study, we discovered a series of fluorinated betulinic acid derivatives as potent CD73 inhibitors by a fluorine scanning strategy. Among these, three compounds ZM522, ZM553 and ZM557 exhibited inhibitory activity with IC50 values of 0.56 uM, 0.74 uM and 0.47 uM, respectively. In addition, these compounds showed a 7-fold, 5-fold and 8-fold increase in activity compared to the positive control drug α, β-methylene adenosine diphosphate (APCP) against the human CD73 enzyme. Two of these (ZM522 and ZM553) also exhibited effective interferon gamma (INF-γ) elevation and indicated the regulation of rescued T cell activation. Therefore, our study provides both a lead optimization strategy and potential compounds for further development of small molecule CD73 inhibitors.

Abstract 2074: ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with multiple myeloma

Abstract Adenosine accumulation within the tumor microenvironment can severely limit antitumor immunity by promoting the expansion of immunosuppressive cell types and impairing immune cell function, including T cells, natural killer cells and dendritic cells. The generation of adenosine from adenosine monophosphate (AMP) requires the activity of a cell surface ecto-5’-nucleotidase, CD73, expressed on the surface of various cell types including immune cells. CD73 overexpression is observed in many tumor types and correlates with unfavorable clinical outcomes. Given the essential role of CD73 in generating adenosine, inhibition of CD73 is a promising therapeutic strategy. Recent clinical proof of concept data demonstrated a significant improvement in progression free survival for non-small cell lung cancer patients upon targeting CD73 with the oleclumab anti-CD73 antibody in combination with anti-PDL1, relative to anti-PDL1 checkpoint inhibitor treatment alone.We developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73, that is highly selective for CD73 and exhibits picomolar potency in biochemical assays, completely blocking adenosine production from AMP. In ex vivo assays, ORIC-533 displayed greater potency in restoring immunosuppressed CD8+ T cell proliferation and activation compared to oleclumab, as well as greater potency versus small molecule inhibitors of CD73 and the adenosine receptor. In multiple myeloma (MM) patients, elevated adenosine levels in the bone marrow (BM) niche correlate with progression of multiple myeloma through a CD73-mediated pathway. Moreover, we recently reported a functional role of CD73 signaling in BM of MM patients, indicating that CD73 inhibition may represent a unique vulnerability and treatment strategy for MM (Ray et al., abstract #2675, ASH 2021). We tested the impact of CD73 inhibition in bone marrow aspirates from patients with relapsed or refractory MM, using an autologous ex vivo assay system comprised of the multiple myeloma BM microenvironment. CD73 inhibition stimulated the activation of plasmacytoid dendritic cells and T cell activation in the context of the MM BM milieu. Moreover, ORIC CD73 inhibitor as a single agent overcame immune suppression and triggered significant lysis and cell death of multiple myeloma cells by autologous T-cells in the bone marrow microenvironment. Taken together, these results demonstrate that the ORIC CD73 inhibitor potently inhibits the adenosine pathway, which restores anti-tumor immunity and therefore holds potential for patients with MM. Based upon these data, ORIC-533 is being studied as a single agent in a Phase 1 clinical trial in patients with relapsed or refractory multiple myeloma. Citation Format: Melissa R. Junttila, Arghya Ray, Robert Warne, Xi Chen, Ting Du, Dena Sutimantanapi, Jae H. Chang, Brian Blank, Jared Moore, Chudi O. Ndubaku, Tatiana Zavorotinskaya, Pratik Multani, Omar Nadeem, Dharminder Chauhan, Kenneth C. Anderson, Lori S. Friedman. ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with multiple myeloma [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 2074.

Abstract 2576: ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies

Abstract Background: CD73 is a cell surface enzyme which is highly expressed in the tumor microenvironment (TME). Degradation of adenosine monophosphate (AMP) into adenosine by CD73 results in the generation of an immunosuppressed and pro-angiogenic niche within the TME that promotes the onset and progression of cancer. Targeting CD73 has resulted in favourable anti-tumor effects in preclinical models and in clinic. One of the key mechanisms of therapeutic activity by CD73 inhibition is restoring the normal function of effector T cells from adenosine inhibition. ATG-037 is a highly potent and selective oral small molecule inhibitor of CD73. This study compared the ability of ATG-037 and clinical CD73 antibodies in reverting immunosuppression in higher-AMP environments in vitro. Methods: The activity of ATG-037 and CD73 antibodies (Hu101-28, MEDI9447) in inhibiting enzyme function of cell surface CD73 was evaluated by measuring the ATP-dependent oxidation of luciferin, which is inhibited by AMP, using Cell Titer Glo assay. Reversal of AMP/adenosine-mediated immune suppression of human T cells by CD73 inhibitors was determined by measuring T cell function in the presence of different concentrations of exogenous AMP. Markers for T cell proliferation, activation and cytotoxicity were assessed by flow cytometry, and the cytokine levels were measured by ELISA. Results: ATG-037, Hu101-28 and MEDI9447 inhibited the enzyme activity of cell surface-expressed CD73 on human A375 cells under 100 µM AMP with a half-maximal inhibitory concentration (IC50) of 0.36 nM,20.94 nM and 3.46 nM, respectively. ATG-037 demonstrated complete inhibition of CD73 activity, whereas MEDI9447 did not reach complete CD73 inhibition. T cell proliferation and activation induced by CD3/CD28 dynabeads were suppressed by 100 µM of extracellular AMP and the suppression was relieved by the addition of ATG-037 with half-maximal effective concentration (EC50) of 21.6 nM for CD8+ T cell proliferation, 27 nM for T cell activation, 2 nM for granzyme B expression, and 6.5 nM for T cell IFN-γ production. While Hu101-28 and MEDI9447 fail to restore the T cell function. In a mixed lymphocyte reaction assay, ATG-037, but not Hu101-28 or MEDI9447 reversed AMP-dependent T cell suppression. In addition, ATG-037 rescued immune cell activation in high AMP (500 µM, 1000 µM) environments. Conclusion: ATG-037 demonstrated potent and complete CD73 enzyme inhibition and stronger ability in restoring T cell function from high-level AMP mediated suppression, compared with clinical anti-CD73 antibodies. These data implicate the potential therapeutic advantages of small molecule CD73 inhibitors over blocking antibodies. Citation Format: Gang Bian, Jiahui Hua, Linjie Tian, Kevin Lynch, Jay Mei, Bo Shan, Bing Hou. ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies [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 2576.

Abstract 719: Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a profoundly immunosuppressive microenvironment. Innovative therapeutic strategies are urgently needed to stop the progression of precancerous lesions into aggressive PDAC, which remains a lethal malignancy. The goal of this research project is to test the immunopreventive strategies by targeting the ectonucleotidase CD73, one of the gatekeeper enzymes responsible for adenosine production and the formation of immunosuppressive tumor microenvironment in this malignancy. We hypothesize that inhibition of CD73 will prevent pancreatic intraepithelial neoplasia (PanIN) formation and progression to PDAC by reversing immunosuppression. Materials and methods: A syngeneic PDAC mouse model was employed by injecting 100k murine pancreatic cancer (KPC) cells in flanks of C57BL/6 female mice. Oral gavage of AB680 (small molecule CD73 inhibitor) was given three days/week at 10mg/kg starting the day after KPC injections and tumor sizes were measured weekly. Toxicity was analyzed by serum ALT analysis. At the time of death 6 weeks post-KPC inoculation, tumor volume and mass were recorded. In another experiment, AB680 was administered 3 days/week at 10mg/kg for 5 weeks. CyTOF immunoprofiling of digested tumors from control and AB680 treated mice and HPLC analysis on serum from the time of death were performed. Results: In experiment 1, by week three of the study, there was a significant difference in tumor volume between the control and AB680 treated groups (P=0.02). However, in weeks 4-5 AB680 reduced tumor growth compared to vehicle controls, but the grouped statistical analysis was not significant. When observing individual tumors, there was a reduction in tumor size in 30% of the AB680 treated mice between weeks 3-5, but the difference was not statistically different. ALT analysis determined AB680 does not induce liver toxicity. For experiment 2 (3x/week treatment with AB680), there was a significant reduction in tumor growth. Activated CD8-positive T cells, dendritic cells, and macrophages were significantly increased in the syngeneic tumors from AB680 treated mice. The intratumoral adenosine levels were significantly decreased in AB680 treated mice compared to vehicle treated mice. Conclusion: We conclude oral gavage delivery of CD73 inhibitor AB680 at 10mg/kg (6x/week) reduces tumor growth in KPC syngeneic tumor bearing mice. Treatment with AB680 at 10mg/kg 3x/week significantly increases tumor doubling time, significantly alters intratumoral immune cell populations, and results in a significant decrease in intratumoral adenosine levels. In addition, we observed a significant increase in infiltration of activated CD8-positive T cells indicating oral gavage delivery using AB680 reverses immunosuppression in vivo. [Supported by NCI 75N91019D00021/75N91020F00002] Citation Format: Kanchan Singh, Vidhi Chandra, Lincoln Ballew, Tingting Mills, Erika Y. Faraoni, Trent Clark, Lana A. Vornik, Michelle I. Savage, Holger K. Eltzschig, Altaf Mohammed, Shizuko Sei, Powel H. Brown, Florencia McAllister, Jennifer Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [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 719.

Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity.

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes.

Abstract LB163: Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments

Abstract Adenosine is recognized for its anti-inflammatory and immunosuppressive properties. Signaling through adenosine receptors expressed on immune cells initiates a cascade that diminishes activation and proliferation of cytotoxic T-cells, impairs activity of natural killer cells and CD4+ effector T-cells, and promotes the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, converts AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many solid tumors and may correlate with unfavorable clinical outcomes; therefore, reducing the level of adenosine via inhibition of CD73 is a strategy for enhancing antitumor immune responses and overcoming therapeutic resistance. We have developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73. ORIC-533 is highly selective for CD73 and exhibits picomolar potency in biochemical and cellular assays, blocking adenosine production from AMP. Expression of CD73 on immune cells is sufficient to drive immunosuppression in vitro when AMP is present. We have previously shown that ORIC-533 can rescue activation of CD8+ T- cells exposed to AMP and restore proliferation, cytokine secretion and CD25 expression at low nanomolar concentrations. To understand the role of CD73 in dendritic cell (DC) priming of helper CD4+ T-cells, we utilized co-culture of allogeneic immature DC and CD4+ T-cells, where AMP to adenosine conversion severely suppresses activation of helper CD4+ T-cells. Treatment with ORIC-533 completely restored activation of CD4+ T-cells in this setting. To better define the biologically relevant AMP concentrations likely to be encountered in tumors, we determined the concentration of AMP in a panel of primary patient tumors. We discovered that intratumoral AMP levels can reach up to 500 micromoles, emphasizing the requirement for CD73 inhibitors to be efficacious in a high AMP environment. We previously demonstrated that nanomolar concentrations of ORIC-533, unlike other CD73 inhibitors tested, can efficiently rescue cytotoxic T-cell function in the presence of millimolar AMP concentrations. Surprisingly, in these studies we found that inhibitors of adenosine receptors, A2A or A2A/B, can only rescue CD8+ T-cell function in the context of low micromolar AMP or adenosine and thus are likely to be ineffective in patient tumors with high AMP. Taken together, these preclinical results indicate the CD73 inhibitor ORIC-533 has best-in-class properties in reversing immunosuppression in tumors. ORIC-533 is currently undergoing GLP studies to support an IND filing in the first half of 2021. Citation Format: Dena Sutimantanapi, Chelsea Chen, Bob Warne, Alex Pankov, Jae Chang, Lori Friedman, Melissa Junttila, Tatiana Zavorotinskaya. Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB163.

Abstract 1713: Novel, potent and orally bioavailable small molecule CD73 inhibitors for cancer immunotherapy

Abstract CD73 is a cell surface ectoenzyme, which is overexpressed in many types of human and mouse cancers. CD73 is the primary enzymatic producer of immunosuppressive adenosine in the tumor microenvironment, and high CD73 expression is associated with significantly poorer prognosis in several tumor types. Inhibition of either adenosine generation or signaling by inhibiting CD73 have been shown to be effective therapeutic approaches. Here we sought to discover and develop novel and orally bioavailable small molecule inhibitors that target CD73. Aurigene's non-nucleotide small molecule exhibit potent inhibition of CD73 in respective biochemical and cellular assays. High potency translated into rescue of AMP induced repression of IFN-Υ and IL-2 in human PBMCs. Lead compounds exhibited desirable drug-like properties including solubility, Caco2 permeability, lack of CYP inhibition and excellent oral pharmacokinetic exposure. In summary, we have identified small molecule inhibitors with good drug like properties, which showed good in vitro potency and excellent PK. Evaluation of these lead compounds in syngeneic tumor model along with in vivo toxicity studies are currently under way. Citation Format: Chandregowda Venkateshappa, Kishore Narayanan, Prasath Kothandaraman, Garima Priyadarshani, Rashmi Nair, Megha Goyal, Vijay Kamal Ahuja, Sagar Dadhania, AB Aravind, DS Samiulla, Girish Daginakkatte, Thomas Antony, Kavitha Nellore, Sunil Panigrahi, Dinesh Chikkanna, Susanta Samajdar, Murali Ramachandra. Novel, potent and orally bioavailable small molecule CD73 inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1713.

ARC-8: Phase I/Ib study to evaluate safety and tolerability of AB680 + chemotherapy + zimberelimab (AB122) in patients with treatment-naive metastatic pancreatic adenocarcinoma (mPDAC).

404 Background: AB680, a potent, selective small-molecule inhibitor of soluble and membrane-bound CD73, targets a major pathway of extracellular adenosine production with the aim of eliminating adenosine-mediated immunosuppression within the tumor microenvironment. In mPDAC, programmed cell death protein-1 (PD-1) axis inhibitors have limited clinical activity as monotherapies or combined with standard-of-care (SOC) chemotherapy. KRAS mutations, present in >90% of invasive PDACs, are associated with significantly elevated CD73 expression and poor clinical outcomes. Thus, mPDAC may be particularly sensitive to CD73 inhibition combined with SOC chemotherapy + anti–PD-1 antibody (zimberelimab [Zim]) treatment. Methods: ARC-8 (NCT04104672) is an ongoing phase 1b, dose escalation and expansion study in patients (pts) with treatment-naive mPDAC. In the dose escalation, AB680 (25, 50, 75, or 100 mg) is administered intravenously once every 2 weeks (Q2W) with standard doses of nab-paclitaxel/gemcitabine (NP/Gem) + Zim (240 mg) in a 3+3 design to determine the recommended dose for expansion (RDE). In the dose expansion, AB680 will be administered at the RDE with NP/Gem + Zim. Adverse events (AEs) and dose-limiting toxicities (DLTs) are recorded and graded per NCI CTCAE 5.0. AB680 pharmacokinetics, pharmacodynamics, and biomarker evaluations are also being performed throughout the study. Clinical activity is assessed every 8 weeks per RECIST v1.1. Results: As of 04SEPT2020, enrolled patients include 4 in Cohort 1 (25 mg AB680), 6 in Cohort 2 (50 mg AB680), and 3 in Cohort 3 (75 mg AB680). Initial AE profiles were similar to those observed for NP/Gem. The most common treatment-related AEs were fatigue (n=6, 43%), anemia (n=4, 29%), and neutrophil count decrease (n=4, 29%); anemia (n=2, 14%) was the most common treatment-related grade 3/4 AE. Initial pharmacodynamic data (50 mg dose) indicated excellent peripheral target coverage at AB680 trough concentrations. Best overall responses for 9 evaluable pts were 3 with partial response (1 in Cohort 1; 2 in Cohort 2), including a complete response of a target lesion, and 5 with stable disease (1 in Cohort 1; 4 in Cohort 2). One pt in Cohort 1 discontinued the study due to progressive disease. As of the cutoff date, 1 DLT (Grade 2 autoimmune hepatitis) occurred in Cohort 2; the event completely resolved with steroids and the pt was able to resume study treatment. No DLTs were reported in Cohort 3. Conclusions: Preliminary results from ARC-8 indicate that AB680, the first clinical-stage small-molecule CD73 inhibitor, in combination with SOC chemotherapy + Zim has a manageable safety profile consistent with that expected for each agent alone and demonstrates early signals of clinical activity. Dose escalation to 100 mg AB680 (Cohort 4) is ongoing to inform RDE selection. Clinical trial information: NCT04104672.

Discovery of natural product ellagic acid as a potent CD73 and CD39 dual inhibitor

The ATP-adenosine pathway has been recently identified as an attractive immune-oncology target and several drug candidates have been entered clinic trials. Inspired by the report of the first small-molecule CD73inhibitor AB680, we describe the discovery of natural product ellagic acid as a dual CD73 and CD39 inhibitor with an IC50 value of 1.85 ± 0.21 μM and 0.50 ± 0.22 μM, respectively. The result of cytotoxicity assays indicated that ellagic acid is a valuable lead compound with low cytotoxicity effect for immune therapy.

Orally Bioavailable Small-Molecule CD73 Inhibitor (OP-5244) Reverses Immunosuppression through Blockade of Adenosine Production.

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.