anti-CD26 mAb Research Articles

IMMU-20. INNATE IMMUNE EVASION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cell surface proteome analysis of Group3-MB cell lines after direct MYC inhibition to identify changes in surface proteins and sensitivity to macrophage-mediated phagocytosis. METHODS We had previously demonstrated the preferential activity of HDACi (CI994) in MYC-driven medulloblastoma. CI-994, showed significant cell viability reduction mediated by reduction in the MYC (mRNA and protein), induction of apoptosis in MYC-driven medulloblastoma. In this study, we directly inhibited MYC using MYCi975 and OMO-MYC and carried out a cell surface proteomic analysis on Group3-MB cell lines to reveal CD24 and CD59 as potential immune evasion markers. While CD24 is a potential don’t eat me signal, CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that acts as an inhibitor to the membrane attack complex and protects cells from CDC. In certain tumors, CD59 expression is enhanced, posing a significant obstacle to treatment by hindering effective monoclonal antibody-induced CDC. We use inhibitors to CD24 and CD59 to confirm their role as innate immune evasion proteins in Group 3MB. RESULTS Meta-analysis of published datasets reveals CD24 and CD59 as strong prognostic indicators in Group3-MB as well as within the Group3g-subtype. Inhibition of MYC reduces surface levels of CD24, increasing macrophage-mediated phagocytosis. The use of a blocking anti-CD24 mAb significantly enhanced macrophage-mediated phagocytosis. Furthermore, a significant reduction in surface expression was observed in CD59. Blocking CD59 mAb significantly enhanced anti-CD47 mAb mediated phagocytosis. Combined treatment of anti-CD24 and anti-CD47 significantly enhanced the survival of Grp3-MB tumor-bearing mice.

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 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.

Feedback activation of CD73-Adenosine axis attenuates the antitumor immunity of STING pathway

The cGAS-STING pathway, a crucial component of innate immunity, has garnered attention as a potential therapeutic target for tumor treatment, but targeting this pathway is complicated by diverse feedback mechanisms of the cGAS-STING pathway. In this study, we demonstrated that STING activation enhanced the expression of CD73 and the subsequent production of adenosine in immune cells and cancer cells. Mechanistically, the feedback activation of CD73 depended on the type I IFN/IFNAR axis induced by STING activation. Furthermore, the combination of STING agonist and anti-CD73 mAb markedly blocked tumor growth in vivo by promoting the infiltration of CD8+ T cells and reducing the accumulation of Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment. Our work provides a rationale for the combination of STING agonists and CD73 inhibitors in cancer immunotherapy.

Humanized Monoclonal Antibody IMM47, Targeting CD24, Exhibits Exceptional Anti-Tumor Efficacy By Blocking the CD24/Siglec-10 Interaction and Can be Used As Monotherapy or in Combination with Anti-PD1 Antibodies for Cancer Immunotherapy

Background: To evaluate the anti-tumor mechanism of IMM47 by blocking the interaction of CD24/Siglec-10 through macrophage antigen presentation. Methods: IMM47 was constructed and produced using an in-house-developed CHO-K1 cell expression system. BLI, ELSIA, and flow cytometry methods were used to measure the EC50 of IMM47 binding, ADCC, ADCP, ADCT and CDC activities. hSiglec-10 Tg C57BL/6 mice congenitally transplanted with MC38-hCD24 were used to analyze the in vivo efficacy. IMM47 monotherapy or in combination with SIRP-Fc fusion protein (IMM01) or anti-PD-1 antibodies were used to test IMM47's anti-tumor efficacy in SCID mice with Jeko-1 or MCF-7 tumor cell xenotransplantation and hPD-1 Tg C57BL/6 mice with MC38-hCD24/hPD-L1 tumor cell homologous transplantation models. Results: The BLI assay results showed that the affinity between IMM47 and CD24 was 0.876 nM. ELISA results showed that the EC50s of IMM47 binding to CD24 were 0.2847 nM. The EC50 of IMM47 binding to REH, Hela, MCF-7, and HCC1954 cells were 14.09 nM, 7.14 nM, 7.54 nM and 54.89 nM, respectively. IMM47 can specifically bind to CD24-positive cells but not to CD24-negative cells. IMM47 binding to CD24 is independent of the glycosylation modification of the extracellular domain. IMM47 can only bind to CD24 in humans and chimpanzees but not in other species, including mice, rats, pigs, dogs, and cynomolgus. IMM47 does not bind to human erythrocytes but strongly binds to granulocytes. Mechanism of action study results showed that IMM47 blocks the binding of CD24 to Siglec-10, with ADCC EC50 values of 0.030 nM, 0.162 nM, 0.017 nM, and 0.202 nM on REH, Hela, MCF-7, and HCC1954, respectively. IMM47 has strong ADCT activity with an EC50 of 0.333 nM against REH. IMM47 has an ADCP EC50 of 0.067 nM and 0.099 nM on REH and MCF-7, respectively. The phagocytic activity of IMM47 was significantly stronger than that of IMM01, a SIRPα-Fc fusion protein. The CDC activity results showed that the EC50 of IMM47 on REH cells was 27.29 nM. With hSiglec-10 Tg C57BL/6 mice congenitally transplanted with MC38-hCD24 cells, in vivo efficacy assays showed that IMM47 has strong anti-tumor activity, with higher spleen M1 and M2 immune cells. After the IMM47 treatment, when tumor cells were reinoculated into mice, they did not form tumors, suggesting a memory immune response had been established. After the IMM47 treatment, tumors in 5 of 6 mice were completely cured at a dose of 3 mg/kg, with a dose-dependent relationship being observed. Pharmacodynamic in vivo study in the Jeko-1 tumor cell heterologous transplantation SCID mouse model, IMM47 exhibits strong anti-tumor activity and a dose-dependent relationship. The combination of IMM47 and IMM01 has significantly better efficacy than that of any single drug in the model of MCF-7 xenograft SCID mice. The combination of IMM47 and the PD-1 antibody Tislelizumab in the MC38-hCD24/hPD-L1 tumor cells in the homologously transplanted PD-1 transgenic C57BL/6 mouse model showed the best efficacy, and tumors in all mice were eliminated. The efficacy of IMM47 combined with either the PD-1 antibody Opdivo or Keytruda is better than that of monotherapy, and tumors in 5/6 and 6/6 mice were eliminated when combined with Opdivo and Keytruda, respectively. Four weeks after drug withdrawal, the re-inoculated cells could not form tumors and were eventually eliminated, while the re-inoculated cells in the control group grew normally and formed tumors. Conclusion: Our research showed that the humanized anti-CD24 mAb IMM47 had excellent anti-tumor effect. The extracellular domain's N-glycosylation alteration has no effect on IMM47's ability to bind to CD24. The in vitro assays revealed that IMM47 exhibits significant ADCC, ADCP, ADCT, and CDC activities. IMM47 displays strong anti-tumor activity in transgenic mouse models, and a memory immune response is generated after treatment. Additionally, an in vivo pharmacodynamics assay of IMM47 in combination with different PD-1 antibodies revealed that IMM47 exhibits synergistic therapeutic efficacy when combined with Tislelizumab, Opdivo, and Keytruda. This suggests that IMM47 has significant potential for use in the clinical setting for cancer immunotherapy, either alone or in combination with other immune checkpoint inhibitors. Finally, IMM47 avoids the adverse effects displayed by some anti-CD47 antibodies because it does not bind to human red blood cells and has a high safety profile.

Targeting CD24 “Don't Eat Me” Signal Restores Phagocytic Activity of Nurse-like Cells from Patients with Chronic Lymphocytic Leukemia

Introduction Chronic Lymphocytic Leukemia (CLL) is an indolent B-cell lymphoproliferative disease that carries the risk of evolving into a life-threatening aggressive histology, known as Richter's syndrome. Despite the advent of new agents such as ibrutinib and venetoclax that have dramatically changed the clinical fate of CLL, young patients still experience refractory and resistant disease, with few therapeutic options available over time. Therefore, the need for new strategies is pivotal for this chronic and insidious condition. Drug resistance might be triggered by the tumor microenvironment (TME), which provides survival signals to CLL tumor cells. Their interaction is mainly established with monocyte-derived nurse-like cells (NLCs). NLCs resemble the immunosuppressive M2-like tumor-associated macrophages (TAMs) and can create a profitable connection with CLL in terms of increasing proliferation and survival (Filip, Blood Cells Mol Dis. 2013). The possibility of targeting the TME by blocking the “Don't Eat Me” signal (DEMs) CD47 has provided restoration of phagocytosis within TME and interesting clinical results in heavily pretreated cases of lymphoma (Advani, NEJM. 2019). The use of agents targeting the DEMs within TME could be a promising approach in CLL as well. CD24 is another DEMs that was first validated in a preclinical model of solid cancer (Barkal, Nature. 2019). It was subsequently found to be upregulated in Mantle-cell lymphoma (MCL) and involved in DEMs as well, since CD24 blockade provided an increase in phagocytosis of MCL cells in vitro (Aroldi, Blood. 2021). This effect was particularly evident in association with anti-CD47 monoclonal antibody (mAb) and Rituximab (Aroldi, Blood. 2022). Although MCL has different pathogenesis compared to CLL, these two entities share some features, particularly regarding the B-cell of origin and the vital interaction with non-cancerous cells within TME. In this study, we explore the possibility of targeting CD24 in human primary samples of CLL (huCLL), redirecting the activity of NLCs against CLL cells using anti-CD24 mAb alone or in combination with other agents (anti-CD47, Rituximab). Methods We analyzed a microarray dataset of patients with CLL to check for any correlation between CD24 expression and survival (GSE22762). We also checked the CD24 antigen expression of huCLL, isolated and differentiated monocytes into M2-like TAMs in vitro, as well as conducted co-culture assays using flow cytometry. These methods were previously described (Aroldi, Blood. 2021; Aroldi, Blood. 2022). Unstimulated autologous CD14+ cell population - defined as precursors of NLCs - were isolated from patients with CLL and used for co-culture analysis without any previous cytokine stimulation for differentiation. Results In a cohort of CLL patients, higher CD24 levels correlated with a lower survival rate compared to lower expression ones (Figure 1). We also checked the CD24 surface in an extended cohort of huCLL and compared it to MEC-1 and PCL-12. huCLL showed higher CD24 surface levels than MEC-1 and PCL-12 and demonstrated better phagocytosis when co-cultured with M2-like TAMs and anti-CD24 mAb (Figure 2A). We then isolated precursors of circulating NLCs from 5 huCLL patients and found higher levels of Siglec-10 (CD24 ligand) than in healthy controls. In this setting, blocking CD24 redirected the phagocytic activity of NLCs against huCLL, showing higher phagocytic rate in cases of multiple combinations (anti-CD24, anti-CD47, anti-CD20, Figure 2B). Conclusions CLL could be another blood cancer subtype sensitive to CD24/Siglec-10 DEMs blocking agents, and CD24 expression negatively impacts survival. Anti-CD24 mAb, in particular, showed the ability to redirect patient-derived NLCs' phagocytic activity to cognate huCLL cancer cells, resulting in higher clearance in cases of multiple combination. Boosting the autologous innate immune system might, therefore, provide further therapeutic options in the setting of CD24 + CLL, particularly for those patients who have failed several lines of treatment and have limited options available.

IMM47, a humanized monoclonal antibody that targets CD24, exhibits exceptional anti-tumor efficacy by blocking the CD24/Siglec-10 interaction and can be used as monotherapy or in combination with anti-PD1 antibodies for cancer immunotherapy.

This study evaluates the anti-tumor mechanism of IMM47, a humanized anti-CD24 mAb. Biolayer interferometry, ELISA and flow cytometry methods were used to measure the IMM47 binding, affinity, ADCC, ADCP, ADCT and CDC activities. In vivo therapeutical efficacy was measured in transplanted mouse models. IMM47 significantly binds granulocytes but not human erythrocytes and blocks CD24's ability to bind to Siglec-10. IMM47 has strong ADCC, ADCT and ADCP activity against REH cells. IMM47's in vivo pharmacodynamics showed that IMM47 has strong anti-tumor effects in human siglec-10 transgenic mouse models with a memory immune response. IMM47 also has powerful synergistic therapeutic efficacy when combined with Tislelizumab, Opdivo and Keytruda, by blocking CD24/Siglec-10 interaction through macrophage antigen presentation with strong ADCC, ADCP, ADCT and CDC activities and with a safe profile. IMM47 binding to CD24 is independent of N-glycosylation modification of the extracellular domain.

Abstract 2923: JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73

Abstract Background: STING emerges as a key immune sensor and activated STING pathway plays a key role in innate and adaptive immune response. STING agonist has shown promising potency to stimulate anti-tumor immunity. Considering immune-related toxicity due to systemic administration, as well as difficulty in intra-tumor administration, targeted delivery of STING agonist by iADC is warranted. We developed CD73-STING iADC JAB-X1800, which uses anti-CD73 mAb for targeted delivery of payload JAB-27670, a highly potent non-cyclic dinucleotide STING agonist. Method: Binding assay and HEK293-STING-luciferase reporter assay were performed to test affinity of JAB-27670. Tumor cells with CD73 expression and THP-1 cells were co-cultured in the presences of iADC JAB-X1800 to assess the IFN-β production. The cell killing activity of iADC JAB-X1800 was evaluated when cancer cells co-cultured with human PBMCs. Furthermore, human and mouse plasma stability of iADC JAB-X1800 was assessed. Finally, the anti-tumor effects of iADC JAB-X1800 alone or in combination with anti-PD-1 were evaluated in animal models. Results: JAB-27670 is a highly potent STING agonist with sub-nanomolar EC50 in binding and luciferase reporter assays. In CD73-expressing cancer cells and THP-1 co-culture experiments, iADC JAB-X1800 induced IFN-β production with significant improvement compared with the free payload. JAB-X1800 also induced potent killing in vitro. JAB-X1800 is stable in human and mouse plasma. A single dose of JAB-X1800 induced tumor regression in MDA-MB-231 xenograft model and hCD73-MC38 syngeneic model. JAB-X1800 conferred immunologic memory when tumor free mice were re-challenged. JAB-X1800 showed synergistic anti-tumor activity with anti-PD-1 in hCD73-MC38 syngeneic model. Conclusions: The CD73-targeted STING agonist iADC JAB-X1800 demonstrated high stability in plasma, robust activity, and good tolerability in animal models, supporting clinical development of JAB-X1800 alone and in combination with anti-PD-1 for patients with CD73-expressing tumors. Citation Format: Yang Ye, Guiqun Yang, Hongling Wang, Xin Sun, Yanping Wang, Fashun Yan, Lei Wang, Wei Long. JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73 [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 2923.

190P Combination of IPH5201, a blocking antibody targeting the CD39 immunosuppressive pathway, with durvalumab and chemotherapies: Preclinical rationale

CD39 is an extracellular ectonucleotidase highly expressed in the tumor microenvironment (TME) that, sequentially with CD73, contributes to the production of adenosine (Ado), via hydrolysis of adenosine triphosphate (ATP). IPH5201 is a blocking anti-CD39 monoclonal antibody (mAb) that may promote antitumor immunity by accumulating immunostimulatory ATP released by necrotic cells and reducing immunosuppressive Ado levels in the TME. Targeting the Ado pathway has recently been reported to improve Durvalumab (D) efficacy in early-stage NSCLC patients, through the use of Oleclumab, an anti-CD73 mAb.

929MO Platform study of neoadjuvant durvalumab (D) alone or combined with novel agents in patients (pts) with resectable, early-stage non-small cell lung cancer (NSCLC): Pharmacodynamic correlates and circulating tumor DNA (ctDNA) dynamics in the NeoCOAST study

Neoadjuvant therapy with PD-(L)1 inhibitors, both as monotherapy and in combination, leads to pathological responses in resectable, early-stage NSCLC. NeoCOAST (NCT03794544) is a phase 2 study of the anti-PD-L1 monoclonal antibody (mAb) D alone or combined with the anti-CD73 mAb oleclumab (O), the anti-NKG2A mAb monalizumab (M), or the anti-STAT3 antisense oligonucleotide danvatirsen (Da) as neoadjuvant therapy.

Abstract 4259: PT199, a next generation anti-CD73 mAb that inhibits both membrane-bound and soluble CD73 activity to completion without “hook effect”

Abstract CD73, also known as ecto-5’-nucleotidase, is implicated in promoting immunosuppressive tumor microenvironment (TME) through production of adenosine. The first generation of anti-CD73 therapeutic antibodies currently in clinical studies have largely exhibited subdued clinical activities mainly due to several unfavorable characteristics such as incomplete enzyme inhibition and/or “hook effect”. These weaknesses are caused by the unfavorable target binding sites and enzyme inhibition mechanisms, which explains why these molecules do not inhibit soluble CD73 enzyme activity, or why the enzyme inhibition depends on certain stochiometric ratios of CD73 and the antibody and exhibits “hook effect” in dose-response curves. Here we report a next generation humanized monoclonal antibody against human CD73 (hCD73), PT199, which binds to a unique site on human CD73 that confers a different mechanism of enzyme inhibition and consequently does not have the flaws associated with the first generation of anti-CD73 mAbs currently in clinic. By directly binding to an epitope that is in or near the CD73 catalytic domain, PT199 potently inhibits both soluble shed CD73 and membrane-bound CD73 activity to completion without “hook effect”. Enzyme kinetic studies demonstrate that PT199 inhibits CD73 enzyme activity in a noncompetitive manner, allowing complete CD73 inhibition even at high AMP concentrations reported in TME. In a cell-based functional assay, PT199 inhibition of CD73 alleviates the immunosuppression effect of AMP on T cell proliferation. Compared with the first-generation molecules, PT199 completely inhibits CD73 activity in cancer patient serum. Furthermore, when administrated to hCD73 knock-in mice, PT199 greatly elevates endogenous AMP level, demonstrating a robust pharmacodynamic effect in blocking the conversion of AMP to adenosine. Comparative mechanistic studies indicate that PT199 binds to both the “open conformation” (inactive enzyme state) and the “closed conformation” (active enzyme state) with high affinity but with shifted binding curves, suggesting that PT199 may inhibit the CD73 enzyme activity by preventing the transition of the enzyme from the “open conformation” to the “closed conformation”. PT199 did not cause B cell reduction in either acute or repeated dosing studies in NHP. In NHP toxicology studies, PT199 exhibits no clinical observations at up to 300 mg/kg weekly repeated dosing and has an excellent PK profile typical of mAbs. Taken together, our data confirms PT199 as a potential best-in-class anti-hCD73 mAb. Phase 1 clinical trial is planned to start in the first quarter of 2022. Citation Format: Haiqun (John) Jia, Jack Li, Fen Pei, Lorraine Greenwood, Lauren Pejza, Yan Long, Kelan Chen, Jessica Perer, Ming Wang, Hui Zou. PT199, a next generation anti-CD73 mAb that inhibits both membrane-bound and soluble CD73 activity to completion without “hook effect” [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 4259.

Abstract CT011: NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC)

Abstract Background: Neoadjuvant anti-PD-(L)1 therapy may offer clinical benefit in resectable, early-stage NSCLC. NeoCOAST (NCT03794544) is a global, randomized phase 2 study of the anti-PD-L1 monoclonal antibody (mAb) durvalumab (D) alone or combined with the anti-CD73 mAb oleclumab (O), the anti-NKG2A mAb monalizumab (M), or the anti-STAT3 antisense oligonucleotide danvatirsen (Da) as neoadjuvant therapy. Methods: Pts with previously untreated, cytologically/histologically documented, resectable, Stage I [>2 cm]-IIIA NSCLC and ECOG PS 0-1 were randomized 1:1:1:1 (stratified by lymph node involvement) to receive D 1500 mg IV alone Q4W or combined with O 3000 mg IV Q2W, M 750 mg IV Q2W, or Da 200 mg IV QW, for one 28-day cycle, followed by surgery. Da monotherapy was also given on days 1, 3 and 5 of the week prior to combination treatment. The primary endpoint was investigator-assessed major pathological response (MPR; ≤10% residual viable tumor cells at tumor site and nodes, at surgery). Secondary endpoints were pathological complete response (pCR; no viable tumor cells), safety and tolerability, feasibility of surgery, PK and immunogenicity. Exploratory endpoints included tumor and microbiome biomarkers and blood mRNA signatures. Results: From March 2019 to Sept 2020, 84 pts were randomized and 83 received D (n=26), D+O (n=21), D+M (n=20), or D+Da (n=16). MPR occurred in 11.1% (95% CI, 2.4-29.2), 19.0% (95% CI, 5.4-41.9), 30.0% (95% CI, 11.9-54.3), and 31.3% (95% CI, 11.0-58.7%), respectively. pCR occurred in 3.7% (95% CI, 0.1-19.0), 9.5% (95% CI, 1.2-30.4), 10.0% (95% CI, 1.2-31.7%) and 12.5% (95% CI, 1.6-38.3), respectively. Rates of treatment-related AEs were 34.6% with D, 57.1% with D+O, 50.0% with D+M, and 43.8% with D+Da (grade ≥3 in 0%, 4.8%, 0% and 6.3%, respectively). Most pts (76/83; 91.6%) completed surgery with no significant delay; of the 7 pts unable to, 5 had progressive or stage IV disease. Across all arms, MPR was more common in pts with baseline tumor PD-L1 expression ≥1% vs those with <1%. With D, CD73 expression was correlated with greater residual viable tumor cells at surgery; however, with D+O, high CD73 (≥10% tumor cells) was associated with reduced viable tumor cells. Transcriptome analysis of post-treatment and baseline blood samples showed upregulation of genes involved in B cell activation and antigen presentation in pts with MPR in the D+O arm, and upregulation of genes associated with Tregs in pts with MPR in the D+M arm. Conclusions: One cycle of D + O, M or Da improved MPR and pCR rates vs D alone, with no new safety signals. Responses were associated with baseline tumor PD-L1 and CD73 expression levels. Pts with MPR receiving D+O or D+M had peripheral transcriptomic signatures related to immune cell function. These data warrant further investigation of these agents in resectable NSCLC. Citation Format: Tina Cascone, Rosario García-Campelo, Jonathan Spicer, Walter Weder, Davey Daniel, David Spigel, Maen Hussein, Julien Mazieres, Julio Oliveira, Edwin Yau, Alexander Spira, Raymond Mager, Oday Hamid, Lin-Yang Cheng, Ying Zheng, Jorge Blando, Lara McGrath, Italia Grenga, Yee Soo-Hoo, Rakesh Kumar, Patrick Forde. NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC) [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 CT011.

Abstract CT124: NeoCOAST-2: a randomized, open-label, phase 2 study of neoadjuvant durvalumab plus novel immunotherapies and chemotherapy (CT) followed by adjuvant durvalumab plus novel agents, in patients with resectable non-small-cell lung cancer (NSCLC)

Abstract Background: Neoadjuvant and adjuvant platinum-based CT improve survival rates in patients with resectable, early-stage NSCLC vs surgery alone. PD-1/PD-L1 inhibitors have shown clinical efficacy in patients with resectable, early-stage NSCLC, in both the neoadjuvant and adjuvant settings. The anti-PD-L1 monoclonal antibody (mAb) durvalumab, in combination with either the anti-CD73 mAb oleclumab or the anti-NKG2A mAb monalizumab, improved response rates and progression-free survival in patients with unresectable, Stage III NSCLC in the phase 2 COAST trial (NCT03822351). The NeoCOAST trial (NCT03794544) is currently evaluating the combination of durvalumab with oleclumab or monalizumab as neoadjuvant treatment in patients with resectable, early-stage NSCLC. NeoCOAST-2 (NCT05061550) is a multicenter study of neoadjuvant durvalumab combined with CT and either oleclumab or monalizumab, followed by surgery and adjuvant durvalumab plus oleclumab or monalizumab, in patients with resectable, Stage IIA-IIIA NSCLC. Methods: Eligible patients must have previously untreated, histologically/cytologically confirmed NSCLC, a WHO/ECOG performance status of 0 or 1, and life expectancy of ≥12 weeks. Patients must have tumor samples available for biomarker analyses, have planned surgery consisting of lobectomy, sleeve resection, or bilobectomy, and have a pre- or post-bronchodilator FEV1 of 1.0 L and DLCO >40% postoperative predictive value. Patients with sensitizing EGFR mutations or ALK translocations, history of allogeneic organ transplantation, active or prior documented autoimmune or inflammatory disorders, or uncontrolled intercurrent illness, are excluded. Approximately 140 patients will be randomized 1:1, stratified by PD-L1 expression (<1% vs ≥1%), to receive IV durvalumab + oleclumab or monalizumab + CT Q3W for 4 cycles prior to surgery, followed by durvalumab + oleclumab or monalizumab Q4W post surgery. Surgery must occur within 40 days of the last dose of neoadjuvant therapy. Patients will receive adjuvant therapy starting within 10 weeks after surgery, for 12 cycles or until disease progression (per RECIST v1.1). The primary endpoints are pathological complete response rate per blinded independent pathologist review, and safety and tolerability. Secondary endpoints include investigator-assessed event-free survival and disease-free survival, feasibility of planned surgery, major pathological response rate, changes in circulating tumor DNA, investigator-assessed objective response rate following neoadjuvant therapy, overall survival, pharmacokinetics and immunogenicity. Citation Format: Tina Cascone, Alexander Spira, Rosario G. Campelo, Dong-Wan Kim, Oday Hamid, Yee Soo-Hoo, Rakesh Kumar, Italia Grenga, Patrick Forde. NeoCOAST-2: a randomized, open-label, phase 2 study of neoadjuvant durvalumab plus novel immunotherapies and chemotherapy (CT) followed by adjuvant durvalumab plus novel agents, in patients with resectable non-small-cell lung cancer (NSCLC) [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 CT124.

LBA42 COAST: An open-label, randomised, phase II platform study of durvalumab alone or in combination with novel agents in patients with locally advanced, unresectable, stage III NSCLC

PACIFIC established consolidation durvalumab (D) as SoC for unresectable Stage III NSCLC with no progression after cCRT. Combination therapy may improve outcomes. CD73 is involved in RT resistance. RT induces NKG2A ligand and shows additive antitumour effects with PD-1 blockade preclinically. COAST (NCT03822351) is a global phase 2 study of D alone or combined with the anti-CD73 mAb oleclumab (O) or anti-NKG2A mAb monalizumab (M) as consolidation therapy.

Abstract 1877: IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models

Abstract CD73, an ecto-5'-nucleotidase, is frequently overexpressed in the tumor microenvironment and can be found on tumor, stromal cells and immune cells. Its function is to catalyze the conversion of adenosine monophosphate (AMP) to adenosine and phosphate, both at the cell surface and as a soluble factor. CD73 has gained interest as a therapeutic target in cancer due to the immunosuppressive role of adenosine in the tumor microenvironment. IOA-237 is a fully human, phage derived monoclonal antibody fully cross-reactive to mouse and human and with high potency against soluble and cell surface CD73. This novel antibody demonstrates in vitro and in vivo characteristics that suggest a best-in-class potential. We demonstrate that IOA-237 is able to significantly reduce tumor outgrowth in multiple mouse models of solid tumors. In the low CD73 expressing CT26 mouse colorectal cancer model, monotherapeutic application of IOA-237 showed tumor outgrowth reduction compared with isotype control. Best-in-class potential was confirmed with head-to-head comparison against a clinical stage anti-CD73 mAb, MEDI9447. Furthermore, inhibition of CD73 by IOA-237 was combined with inhibitors of novel immuno-oncology targets to study complementary modes of action in mouse models of solid tumors. These data indicate the potential of IOA-237 in monotherapeutic and combination therapeutic approaches and warrants further development. Additional in vitro and in vivo efficacy studies are ongoing and non-clinical safety studies are being planned. Citation Format: Marcel A. Deken, Alan M. Carruthers, Karolina Niewola-Staszkowska, Gráinne Gernon, Hilary Sandig, Pritom Shah, Michael M. Lahn, Zoë Johnson. IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models [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 1877.

CD73 expression is critical to therapeutic effects of human endometrial regenerative cells in inhibition of cardiac allograft rejection in mice.

The newly found mesenchymal‐like endometrial regenerative cells (ERCs) have been proved to induce immune tolerance in cardiac allograft transplantation. However, the therapeutic mechanism is not clear. The present study was undertaken to investigate whether ecto‐5′‐nucleotidase (CD73) expression on ERCs is critical to cardiac allograft protection. C57BL/6 mouse recipients receiving BALB/c mouse cardiac allografts were treated with unmodified ERCs or anti‐CD73 monoclonal antibodies (mAb) pretreated ERCs, respectively. It has been found that CD73 expression was critical to ERC‐induced attenuation of graft pathology. The blockage of CD73 expression on ERCs was related to the percentage decline of tolerogenic dendritic cells (Tol‐DCs), macrophages type 2 (M2), and regulatory T cells (Tregs). As compared with anti‐CD73 mAb pretreated ERCs group, CD73 expressing ERCs significantly increased the level of anti‐inflammatory cytokine IL‐10 but decreased levels of pro‐inflammatory cytokines including IFN‐γ and TNF‐α. In addition, CD73 expressing ERCs showed tissue protective function via the regulation of adenosine receptor expression which was related to the infiltration of CD4+ and CD8+ cells in the allografts. Furthermore, significant increase of A2B receptors in the cardiac allograft was also associated with CD73 expressing ERC‐induced prolongation of cardiac allograft survival.

Abstract 2258: CD73 targeting therapy enhances the anti-tumor effect of radiation therapy in rectal cancer

Abstract [Background and Purpose]Extracellular adenosine exerts strong immunosuppressive activity and is considered as an important metabolic immune-checkpoint molecule. CD73 (ecto-5'-nucleotidase) is the rate-limiting enzyme to produce adenosine, and high CD73 expression in tumor tissue is linked to poor prognosis. However, the role of CD73/adenosine signaling in the response against radiation therapy (RT) is less understood. Here, we examined the anti-tumor effects of the combination of CD73 blockade and RT. [Method]LuM1, a highly metastatic subtype of colon26 (murine colon cancer cell) were subcutaneously (sc) inoculated in Balb/c mouse. At day 12,14 and16, 4Gy RT was locally applied selectively to sc tumor with lead shielding of other body. Blocking CD73 mAb (200μg/body) was intraperitoneally administrated at day 12,14,16,19,22 and 25. The mice were sacrificed at day 28 and the growth of irradiated tumor and non-irradiated lung metastasis was evaluated. For evaluating immune function, tumor and spleen were excised at day 18. In addition, expression of CD73 in 64 human rectal cancer after chemoradiotherapy (CRT) was examined with immunohistochemistry and evaluated for the impact on patient outcome. [Result]Flowcytometric analysis showed that LuM1 positively expressed CD73 which was significantly enhanced by 10Gy RT in vitro. Local RT (4Gy x 2) also enhanced the expression of CD73 in LuM1 of sc tumor (MFI: Median=177, 120-234 vs 65.5, 24.3-72.3, n= 5, p=0.0079). Local RT caused significant delay in growth of sc tumor. However, the addition of anti-CD73 mAb further reduced the tumor weight as compared with isotype control (Μ=0.15g, 0.004-0.29g vs 0.34g, 0.1-0.73g, n=8, p=0.0379). Interestingly, the number of metastases in non-irradiated lung was also significantly decreased by anti-CD73mAb (Μ=1, 0-30 vs 12, 1-70, n=8, p=0.040).The percentage of IFNγ (+)/CD4 (+) cells and IFNγ (+)/CD8a (+) cells of splenocytes were increased in RT +Anti-CD73 groups compared to RT +Anti-Isotype group. (Μ=9.95%, 7.71-16.6% vs 3.64%, 1.01-11.7%, n=6, p=0.0022 and Μ=15.5%, 11.9-24.1% vs 5.98%, 1.51-16.6%, n=6, p=0.041).In addition, the percentage of IFNγ (+)/CD4 (+) cells of tumor infiltrating lymphocytes was increased in RT +Anti-CD73 groups compared to RT +Anti-Isotype group. (Μ=12.2%, 9.77-18.2% vs 7.09%, 1.55-9.41%, n=6, p=0.0022).Among the patients who received CRT, 13 patients with high expression level of CD73 both in remnant tumor cells and stroma showed significantly worse recurrence free survival and overall survival than other 51 patients (p=0.0059 and p=0.0077). [Conclusion]RT enhances the tissue expression of CD73, which can provide large amounts of adenosine in irradiated tumor tissue and may seriously suppress the local immune response and impair the response against RT. Our results suggest that functional blockade of CD73 can enhance not only direct but also abscopal effects of RT possibly through T cell activations, which may improve the outcome of the patients with advanced rectal cancer. Citation Format: Hidenori Tsukui, Hisanaga Horie, Akira Saito, Koji Koinuma, Hideyuki Ohsawa, Hironori Yamaguchi, Yasunaru Sakuma, Yoshinori Hosoya, Kotaro Yoshimura, Alan Lefor, Naohiro Sata, Joji Kitayama. CD73 targeting therapy enhances the anti-tumor effect of radiation therapy in rectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2258.

Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin \u03b13 in human pancreatic cancer

Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens and the development of targeting agents. Here, we report the development of two novel mAbs against CFPAC-1 human pancreatic cancer cells. Using ELISA, flow cytometry, immunoprecipitation, mass spectrometry, Western blot and immunohistochemistry, we found that the target antigens recognised by the two novel mAbs KU44.22B and KU44.13A, are integrin α3 and CD26 respectively, with high levels of expression in human pancreatic and other cancer cell lines and human pancreatic cancer tissue microarrays. Treatment with naked anti-CD26 mAb KU44.13A did not have any effect on the growth and migration of cancer cells nor did it induce receptor downregulation. In contrast, treatment with anti-integrin α3 mAb KU44.22B inhibited growth in vitro of Capan-2 cells, increased migration of BxPC-3 and CFPAC-1 cells and induced antibody internalisation. Both novel mAbs are capable of detecting their target antigens by immunohistochemistry but not by Western blot. These antibodies are excellent tools for studying the role of integrin α3 and CD26 in the complex biology of pancreatic cancer, their prognostic and predictive values and the therapeutic potential of their humanised and/or conjugated versions in patients whose tumours overexpress integrin α3 or CD26.

The chick embryo chorioallantoic membrane assay: In ovo model for personalized assessment and evaluation of the most effective therapeutic approach in cancer therapy.

e14634 Background: "Personalized medicine,” is the tailoring of medical treatment to a single person aiming to maximize efficacy and minimize toxicity. Currently there is no good prediction for response to therapy. The Chick Chorioallantoic Membrane (CAM) is naturally immuno-deficient and rich in vascularity therefore an ideal system, allowing generate 3D cancerous “organoids” in a very efficient, reproducible and cost-effective manner and translates basic research to the clinic. Aims: Generate a “personalized” HTP system for a quick, reliable and effective evaluation of different therapeutic options using 3D tumors in a "humanized egg" instead of mouse PDX model. Methods: Fertilized eggs were incubated until day 3 (37°C, 75-90% humidity). Then, 2ml of albumin was pulled out to separate the CAM from the eggshell and a small window in the eggshell has been made. To destroy the chicken immune system development, the eggs were irradiated at day 5 and human immune cells were then inoculated onto the CAM. On day 7, single cells suspension or tissues, derived from cancer patients, were transplanted onto the CAM and visible tumors were performed ("CAM-PDX"). Drugs, mAbs and chemotherapy, were applied via the yolk sac. Tumor growth was measured, weighted and monitored by caliper and IVIS fluorescent imaging platform. IHC was performed and confirmed the response of the particular specimens to the tested regiment. Results: Histology and IHC analysis confirmed that the established tumors retained their characteristics. Positive Ki-67 staining confirmed that cancer cells proliferate while the treated tumors showed reduced staining. Anti-CD24 mAb, FOLFOX, cetuximab, Foflorinox and Gemcitabine, given as single agent or combinations, successfully inhibited CR and pancreatic tumors (by 70-75%). Detection of active caspase 3 confirmed those results. Biopsies from human specimens, were successfully established and expanded by serial passages allows generation of bio-bank. The stimulated human PBMCs demonstrated enhanced proliferation in vitro and in ovo, even after 5 days in the egg. Irradiated eggs showed no functional immune system even after 2 weeks of development. Conclusions: The CAM is an ideal, effective, economical and powerful avatar-based precision medicine approach to predict the best protocol for cancer therapy.

A Novel Specific Anti-CD73 Antibody Inhibits Triple-Negative Breast Cancer Cell Motility by Regulating Autophagy.

Increasing researches have focused on cancer metastasis and development. The ectonucleotidase CD73 is one of the most common cell surface enzymes that are involved in immunosuppression. In this study, the recombinant plasmid pET28a-CD73 was constructed and the CD73 protein was overexpressed in E. coli as an inclusion body that was then subjected to refolding. The anti-CD73 monoclonal antibody (3F7) was obtained by hybridoma technology. The antibody subtype was identified as IgG2a with an affinity constant of 5.75 nM. This antibody could be applied to immunofluorescence and flow cytometry. The results showed that the CD73 protein was not only located in the cytoplasm but also distributed on the surface of triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468. Moreover, the level of CD73 protein was associated with the survival rate. Although the anti-CD73 antibody was not able to inhibit tumor cell growth, it could enhance the cytotoxic effect of Doxorubicin to triple-negative breast cancer cells. In vitro function assay results indicated that anti-CD73 mAb could inhibit cell migration and invasion in both human triple-negative breast cancer and mouse 4T1 cell lines. In this process, both the LC3I/LC3II ratio and p62 protein levels increased, which indicated that the blockage of CD73 could inhibit cell autophagy, and cell migration and invasion were restored by rapamycin. In vivo, anti-CD73 mAb could significantly inhibit lung metastasis of 4T1 cells in a mouse xenograft model. Taken together, this novel anti-CD73 antibody could be developed as an adjuvant drug for triple-negative breast cancer therapy and can be useful in tumor diagnosis.