anti-CD3 Monoclonal Antibody Research Articles

#4570 RELATION OF KIDNEY BIOPSY FINDINGS TO CLINICAL OUTCOMES IN PATIENTS WITH LUPUS NEPHRITIS TREATED WITH AN ANTAGONISTIC ANTI-CD40 ANTIBODY (BI 655064)

Abstract Background and Aims Lupus nephritis (LN) is the most serious complication of systemic lupus erythematosus with a high risk of end-stage kidney disease and a high need of better treatment. A recent randomized, double-blind, placebo-controlled, phase II trial tested doses of 120, 180 and 240 mg of the anti-CD40 monoclonal antibody BI 655064 as add-on to standard of care (MMF + glucocorticoids), in patients with active LN (proteinuria > 1 g/day) for 52 weeks. Although the trial did not demonstrate a dose-response relationship for the primary endpoint determined by complete renal response, a potential efficacy benefit of BI 655064 180 mg and 240 mg was found in a post-hoc analysis. We investigated whether a treatment effect was related to findings in the pre-treatment renal biopsy. Method Biopsies from 101 patients with LN class III or IV, confirmed in a central review conducted according to a previously established protocol, were included. Protein/creatinine ratio (UPCR, based on spot urine) and eGFR were determined at every visit. For each, the last available UPCR and the last available eGFR values, linear regressions (based on the respective baselines) of all patients irrespective of therapy were calculated, and subjects were classified according to their individual performance, “Better” or “Worse” than the average. The modified activity and chronicity indices as well as all biopsy parameters, which are not already contained in the indices, were compared for differences between the “Better” and “Worse”. Parameters with p< 0.1 in the univariate analyses were entered into a multivariate logistic regression model, and a routine model selection procedure (with p< 0.2) was used to identify parameters predictive for clinical outcome. As monocytes express CD40, which is the target of BI 655064, a further question was whether high dose treatment with BI 655064 (180/240 mg) was beneficial when monocytes were present in the biopsy, using a model which adjusted for all predictors identified in the multivariate model. Results The results of the univariate and the multivariate analysis of biopsy parameters are shown in Table 1. Improvement in UPCR was better if the modified chronicity index was low. In addition, LN class IV subjects had a marginally better prognosis than class III. The effect of high-dose treatment with BI 655064 was better when monocytes were present. eGFR outcome was better if the modified chronicity index was low and, conversely, the modified activity index was high. Further, the presence of synechia/adhesions was a negative predictor for positive outcome. There was no indication for an effect of high-dose treatment with 655064 on eGFR when monocytes were present. Conclusion This post-hoc analysis of the BI 655064 trial used a novel approach which investigated whether specific histological lesions in renal biopsies were related to treatment outcome. Although the number of patients was limited, our results suggest that treatment with higher dose anti-CD40 may improve the reduction of proteinuria when monocytes are present in the biopsy. This indicates that specific information from renal biopsies could ultimately improve the choice of treatment for individual LN patients.

Targeting CD137 (4-1BB) towards improved safety and efficacy for cancer immunotherapy.

T cells play a critical role in antitumor immunity, where T cell activation is regulated by both inhibitory and costimulatory receptor signaling that fine-tune T cell activity during different stages of T cell immune responses. Currently, cancer immunotherapy by targeting inhibitory receptors such as CTLA-4 and PD-1/L1, and their combination by antagonist antibodies, has been well established. However, developing agonist antibodies that target costimulatory receptors such as CD28 and CD137/4-1BB has faced considerable challenges, including highly publicized adverse events. Intracellular costimulatory domains of CD28 and/or CD137/4-1BB are essential for the clinical benefits of FDA-approved chimeric antigen receptor T cell (CAR-T) therapies. The major challenge is how to decouple efficacy from toxicity by systemic immune activation. This review focuses on anti-CD137 agonist monoclonal antibodies with different IgG isotypes in clinical development. It discusses CD137 biology in the context of anti-CD137 agonist drug discovery, including the binding epitope selected for anti-CD137 agonist antibody in competition or not with CD137 ligand (CD137L), the IgG isotype of antibodies selected with an impact on crosslinking by Fc gamma receptors, and the conditional activation of anti-CD137 antibodies for safe and potent engagement with CD137 in the tumor microenvironment (TME). We discuss and compare the potential mechanisms/effects of different CD137 targeting strategies and agents under development and how rational combinations could enhance antitumor activities without amplifying the toxicity of these agonist antibodies.

Early safety and efficacy from a phase I clinical study of TWP-101, a novel CD137 agonist antibody, in patients with advanced melanoma and urothelial carcinoma.

9530 Background: CD137 plays the roles as a potent co-stimulator of both adaptive and innate immune cells, being an attractive target for cancer immunotherapy. TWP-101 is a fully humanized agonistic anti-CD137 monoclonal IgG4 antibody, targeting a novel epitope of CD137 with a unique mechanism of actions as a CD137 agonist but not CD137 ligand antagonist. The phase I study of TWP-101 in patient (pt)s with advanced melanoma and urothelial carcinoma was initiated (NCT04871334). Methods: Enrolled pts were advanced melanoma refractory to standard therapy. Dose-escalation includes accelerated titration (0.01 and 0.03mg/kg) and conventional Fibonacci 3+3 dose levels (0.1, 0.3, 1.0 and 3.0 mg/kg). TWP-101 was administered intravenously every Q2W until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. The primary objectives were to define the safety profile, to determine the maximum tolerated dose and RP2D of TWP-101. Secondary objectives were to evaluate pharmacokinetics (PK), immunogenicity and preliminary clinical efficacy. Exploratory objectives were to determine pharmacodynamics (PD) biomarkers. Results: From Feb 2021 to Mar 2022,13 melanoma pts (median age 54 years, range 39-73; 6 men, 7 women; median 2 prior lines of therapy, range 1-6; 12 with prior immunotherapy) were treated. The following five dose levels had been evaluated from 0.01 to 1.0 mg/kg. 3mg/kg dose escalation is ongoing, no MTD has been reached. The median treatment time was 16 wks. (range 2-59). On cutoff date of October 31, 2022, 13 pts discontinued treatment due to progression disease (n = 12) and protocol deviation (n = 1). No DLTs were observed. 9 pts (69.2%) experienced treatment-related adverse events (TRAE). The most common TRAEs (≥10%) were neutropenia (23.1%), leukopenia (15.4%), hypertriglyceridemia (15.4%), anemia (15.4%), hyponatremia (15.4%). 1 pt experienced grade 3-4 TRAEs: hyponatremia, Asthenia and reduced appetite. In all TRAEs, one grade 1 decreased free triiodothyronine and one grade 2 pyrexia were possibly related, and all other TRAEs, including 2 treatment-related SAEs (1 grade 4 hyponatremia and 1 grade 2 subarachnoid hemorrhage), were identified as possibly unrelated. Deaths were due to progression disease (n = 3). Preliminary PK analysis showed dose-proportional kinetics. For 12 evaluable pts, 2 pts achieved PR (16.7%), 5 pts was SD. DCR was 58.3%. Median PFS was 16 wks. (range 16-60), PFS of 2 PR pts was 24 and 60 wks. Conclusions: TWP-101 demonstrated a good safety profile without hepatotoxicity frequently observed with other studied CD137 antibodies. Both favorable tolerability and preliminary antitumor activity warrant further evaluation in pts with advanced melanoma and urothelial carcinoma. Clinical trial information: NCT04871334 .

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Advances in Immunogenic Cell Death for Cancer Immunotherapy

RELT is overexpressed in human inflammatory bowel disease and breast cancer.

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR member that activates NF-kB and is implicated in several cancers. We therefore sought to characterize the association of RELT with inflammatory bowel disease (IBD) and breast cancer (BC). Additionally, we further characterized the association of RELT with Filamin A (FLNA), an actin-binding protein that can promote tumorigenesis, or counteract tumors, depending on its cellular location. Female CB-17 SCID mice were injected with anti-CD40 monoclonal antibody to induce colitis and euthanized seven days later. Immunohistochemistry (IHC) was utilized to compare RELT expression in the mouse colitis model with uninjected control mice. IHC was utilized to compare the expression of RELT in biopsies of human IBD and non-inflamed tissue, as well as human BC versus non-malignant tissue. Luciferase assays and an X-gal morphology assay were utilized to examine cell death in the BC cell line MDA-MB-231 (231). RELT immunostaining is significantly increased in both mouse colitis and human IBD biopsies in comparison to that of species-matched non-inflamed tissue. An apparent association between colonic histological damage and RELT immunostaining was observed in both mice and human specimens. RELT staining was also increased in human BC biopsies versus patient-matched non-malignant tissue. RELT overexpression induced death in 231 cells, an effect that was not abrogated by a RELT mutation in the binding site for the OXSR1 kinase. However, co-expression of a S2152A mutant of FLNA did abrogate the ability of RELT to induce death in 231 cells. Collectively, these results implicate RELT as being important for human disease and further our understanding of the interaction between RELT and FLNA.

Mutation of the TGN1412 anti-CD28 monoclonal antibody lower hinge confers specific FcγRIIb binding and retention of super-agonist activity.

The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγRs and agonistic action was consequentially lost, confirming the FcγR dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilizing mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies retained their super-agonistic ability, demonstrating that CD28- and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signaling.

Modulating CNS neuroinflammation in animal models of intracerebral hemorrhage by nasal anti-CD3 (S4.010)

<h3>Objective:</h3> Investigate the therapeutic benefit of nasal anti-CD3 treatment in animal model of ICH. <h3>Background:</h3> Intracerebral hemorrhage (ICH) is the most devastating type of stroke, with a disproportionately high mortality approaching 40%. Current investigations in ICH treatment have focused on limiting hematoma expansion which unfortunately, have not led to an effective treatment. Neuroinflammation play a critical role in secondary injury post-ICH. Exposure of brain parenchyma to blood products initiates a complex inflammatory cascade start with activation of resident microglia. Resulting cytokine release recruits circulating monocytes and lymphocytes, further enhancing inflammation, and contributing to secondary injury. The cellular and molecular mechanisms leading to neurological deficits following ICH are poorly understood, and there is no effective therapy to modulate the mitigate CNS injury and promote recovery. The mucosal immune system is a unique tolerogenic organ that provides a physiological approach for the induction of regulatory T cells (Tregs). We found that nasal anti-CD3 monoclonal antibody induces IL-10-secreting Tregs that migrate to the brain and suppresses microglial inflammation in a mouse model of multiple sclerosis. The therapeutic potential of the Tregs/IL-10 axis in ICH is largely unexplored. <h3>Design/Methods:</h3> We employed the collagenases model of ICH in mice and administered nasal anti-CD3 treatment within 24 hours of ICH which was continued up to 1-month post-bleed. We performed Flow cytometry, histopathology and behavioral analyses to characterize the cells and assess the effects of treatment on the behavioral outcomes at 1-month post-ICH. <h3>Results:</h3> Nasal anti-CD3 increased FoxP3+ Tregs and IL10 producing FoxP3+ Tregs in the brain. It also reduced microglial activation and lesion volume in the ipsilateral hemisphere post-ICH. Treatment improved behavioral outcomes, including motor, spatial learning, and hippocampal-dependent working memory functions at 1-month post-ICH. <h3>Conclusions:</h3> Our findings suggest that the nasal anti-CD3 may represent a novel therapeutic approach for treating ICH and potentially other types of acute brain injury. <b>Disclosure:</b> Dr. Izzy has received publishing royalties from a publication relating to health care. Taha Yahya has nothing to disclose. Dr. Cao has nothing to disclose. Dr. Waed has nothing to disclose. Dr. Moreira has nothing to disclose. Dr. Rezende has nothing to disclose. Dr. Weiner has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Genentech. Dr. Weiner has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Medday Pharmaceuticals. Dr. Weiner has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for vTv Therapeutics. Dr. Weiner has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Tiziana Life Sciences. Dr. Weiner has received personal compensation in the range of $10,000-$49,999 for serving on a Scientific Advisory or Data Safety Monitoring board for vTv Therapeutics. Dr. Weiner has received personal compensation in the range of $10,000-$49,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Medday Pharmaceuticals. Dr. Weiner has received personal compensation in the range of $10,000-$49,999 for serving as an officer or member of the Board of Directors for vTv Therapeutics. Dr. Weiner has received stock or an ownership interest from vTv Therapeutics. The institution of Dr. Weiner has received research support from National Institute of Health. The institution of Dr. Weiner has received research support from National MS Society. The institution of Dr. Weiner has received research support from Genzyme Corp. The institution of Dr. Weiner has received research support from Genentech, Inc. . The institution of Dr. Weiner has received research support from Verily Life Sciences LLC. The institution of Dr. Weiner has received research support from EMD Serono, Inc..

Photonic Hyperthermia Synergizes with Immune-Activators to Augment Tumor-Localized Immunotherapy.

Photothermal immunotherapy, the combination of photothermal hyperthermia and immunotherapy, is a noninvasive and desirable therapeutic strategy to address the deficiency of traditional photothermal ablation for tumor treatment. However, insufficient T-cell activation following photothermal treatment is a bottleneck to achieve satisfactory therapeutic effectiveness. In this work, a multifunctional nanoplatform is rationally designed and engineered on the basis of polypyrrole-based magnetic nanomedicine modified by T-cell activators of anti-CD3 and anti-CD28 monoclonal antibodies, which have achieved robust near infrared laser-triggered photothermal ablation and long-lasting T-cell activation, realizing diagnostic imaging-guided immunosuppressive tumor microenvironment regulation following photothermal hyperthermia by reinvigorating tumor-infiltrating lymphocytes. By virtue of high-efficient immunogenic cell death and dendritic cell maturation combined with T-cell activation, this nanosystem markedly restrains primary and abscopal tumors as well as metastatic tumors with negligible side effects in vivo, exerting the specific function for suppressing tumor recurrence and metastasis by establishing a long-term memory immune response.

TNX-1500, a crystallizable fragment–modified anti-CD154 antibody, prolongs nonhuman primate cardiac allograft survival

Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to supplement or replace current clinical immunosuppression in solid organ transplantation. We evaluated the tolerability and activity of a novel humanized anti-CD154 monoclonal antibody, TNX-1500 (TNX), in a nonhuman primate heterotopic cardiac allogeneic (allo) transplant model. TNX-1500 contains a rupluzimab fragment antigen-binding region and an immunoglobin G4 crystallizable fragment region engineered to reduce binding to the crystallizable fragment gamma receptor IIa and associated risks of thrombosis. Recipients were treated for 6 months with standard-dose TNX (sTNX) monotherapy, low-dose TNX monotherapy (loTNX), or loTNX with mycophenolate mofetil (MMF) (loTNX + MMF). Results were compared with historical data using chimeric humanized 5c8 monotherapy dosed as for loTNX but discontinued at 3 months. Median survival time was similar for humanized 5c8 and both loTNX groups, but significantly longer with sTNX (>265 days) than with loTNX (99 days) or loTNX + MMF (88 days) (P < 0.05 for both comparisons against sTNX). Standard-dose TNX prevented antidonor alloantibody elaboration, inhibited chronic rejection, and was associated with a significantly reduced effector T cells/regulatory T cells ratio relative to loTNX with MMF. No thrombotic complications were observed. This study demonstrated that TNX was well tolerated, prolongs allograft survival, and prevents alloantibody production and cardiac allograft vasculopathy in a stringent preclinical nonhuman primate heart allotransplant model.

TNX-1500, a crystallizable fragment–modified anti-CD154 antibody, prolongs nonhuman primate renal allograft survival

The blockade of the CD154-CD40 pathway with anti-CD154 monoclonal antibody has been a promising immunomodulatory approach to prevent allograft rejection. However, clinical trials of immunoglobulin G1 antibodies targeting this pathway revealed thrombogenic properties, which were subsequently shown to be mediated by crystallizable fragment (Fc)-gamma receptor IIa–dependent platelet activation. To prevent thromboembolic complications, an immunoglobulin G4 anti-CD154 monoclonal antibody, TNX-1500, which retains the fragment antigen binding region of ruplizumab (humanized 5c8, BG9588), was modified by protein engineering to decrease Fc binding to Fc-gamma receptor IIa while retaining certain other effector functions and pharmacokinetics comparable with natural antibodies. Here, we report that TNX-1500 treatment is not associated with platelet activation in vitro and consistently inhibits kidney allograft rejection in vivo without clinical or histologic evidence of prothrombotic phenomena. We conclude that TNX-1500 retains efficacy similar to that of 5c8 to prevent kidney allograft rejection while avoiding previously identified pathway-associated thromboembolic complications.

Abstract 1880: A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile

Abstract Background: CD137 (TNFRSF9, 4-1BB) is a member of the tumor necrosis factor receptor superfamily that functions as a costimulatory molecule of immunocytes and is mainly expressed on the surface of immune cells including activated T and natural killer (NK) cells. Agonists against CD137 have shown promising therapeutic activity in mouse tumor models but have suffered setback in the clinic due to notably hepatotoxicity, or suboptimal agonistic potency. Here, we developed a fully human agonistic anti-CD137 monoclonal IgG4 antibody, ZG033, with an exclusive epitope, to achieve a better efficacy and safety profile for immunotherapy. Methods: The antigen binding specificity and affinity of ZG033 were determined by ELISA, surface plasmon resonance (SPR). X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms. The activities were determined using cell-based and reporter gene assays. In vivo antitumor activities were evaluated in human 4-1BB transgenic mice bearing with syngeneic tumors, and possible toxicity was evaluated in humanized mice and cynomolgus monkeys. Cytokine release assay was conducted by incubating ZG033 with PBMCs from healthy donors. The pharmacokinetic (PK) behavior of ZG033 were characterized in cynomolgus monkeys. A phase 1 study was initiated to evaluate the safety and preliminary clinical activities of ZG033, as well as the immunogenicity and pharmacokinetics. Results: ZG033 showed high specificity for human CD137 without cross-species reactivity across mouse, rat and exhibited potent CD137 agonist activity dependent on FcγR crosslinking. The binding affinity of ZG033 determined by surface plasmon resonance (SPR) was moderate (KD=10 nM). X-ray crystallography revealed that the Fab of ZG033 binds CD137 at an epitope overlap with the ligand binding interface. ZG033 increased CD137-driven NFκB reporter gene activation and the production of IFN-γ by human T cells in a FcγR-dependent manner. In human CD137 transgenic mice, ZG033 showed robust antitumor activity and induced durable antigen-specific immunological memory that prevented the tumor formation in the rechallenged mice. There were no signs of toxicity in human cytokine release assay which confirmed that ZG033 did not induce pro-inflammatory cytokines in the absence of TCR stimulation. Toxicology data in mice showed that ZG033 was well-tolerated with normal ALT and AST levels. ZG033 was further confirmed to well tolerated up to 180 mg/kg (MTD≥180mg/kg) dose without systemic toxicity in cynomolgus monkeys. Patients with advanced tumors, who are refractory after exhausting available therapies, were enrolled for ZG033 treatment. We will report the results from early dose escalation part of the study. Conclusion: These data demonstrate that ZG033 is an FcγR crosslinking-dependent CD137 agonist that displays a favorable safety profile and has potential in either mono- or combinational immunotherapies. Citation Format: Yang Huang, Liangwei Li, Wenting Liu, Dayan Zhang, Pengfei Zhou, Qing Fang, Weiming Zhou, Fengrong Wang, Guodong Shen, Liansheng Cheng. A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile [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 1880.

Abstract 1546: ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models

Abstract Regulatory T (Treg) cells are a distinctive lineage of CD4+ T cells as characterized by expression of FoxP3 and CD25 markers. Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Accumulation of Treg cells in the tumor microenvironment (TME) is often associated with an adverse prognosis in multiple cancers and also has implication for predicting response to immunotherapy. Treg cell depletion strategy has been explored in preclinical and clinical settings to induce effective antitumor immune responses in TME. CD25, a high-affinity binding subunit alpha of the IL-2 receptor, is constitutively and highly expressed on Treg cells but also transiently upregulated on effector T cells. The high level of CD25 expression on Treg cells could deprive of IL-2 for effector T cells and inhibit their proliferation. Thus, an effective approach to selectively suppress Treg cell function is to deplete Treg cells while avoid blocking IL-2 binding to CD25 for allowing T effect cell proliferation and activation. Using our immune tolerance breaking antibody platform, we generated a humanized IgG1 subtype anti-CD25 monoclonal antibody (TST010). TST010 bound to human CD25 with high affinity and selectivity but did not block IL-2 binding to CD25. No blocking activity to IL-2 signaling on human PBMC was further confirmed by the phosphorylation of Stat5 using FACS analysis. By reducing fucosylation during cell culture process, TST010 mAb gained the enhanced ADCC activity. In an ADCC reporter assay, TST010 showed its sub-nanomolar ADCC activity against CD25-overexpression SU-DHL-1 cell line and human induced Treg cells, but much less to human activated CD8+ T effect cells due to lower CD25 expression. Similarly, in a NK cytotoxicity assay, TST010 selectively killed human induced Treg cells in contrast to activated CD8+ T cells. In vivo antitumor activity of TST010 was evaluated in the syngeneic mouse tumor models. The mouse colon cancer MC38 model was developed on the human CD25 knock-in mice. In the MC38 tumor model, TST010 markedly reduced the Treg (FoxP3+CD4+) population in both peripheral blood and TME. Importantly, the CD8+T/Treg ratio in tumor was increased starting at 2 weeks post TST010 treatment, and it resulted in a significant single agent tumor growth inhibition at a 10 mg/kg dose. Furthermore, in the LLC tumor model which does not respond well to checkpoint inhibitors, the anti-CD25 mAb showed a good combination effect with a PD-L1 inhibitor. In summary, we have discovered a novel therapeutic anti-CD25 mAb that can deplete Treg cells without blocking IL-2 signaling. As demonstrated in our preclinical tumor models, it has a good potential to induce effective antitumor immune responses in TME and tumor growth inhibition especially in combination with PD-1/PD-L1 treatment. Citation Format: Di Sun, Huanhuan Guo, Hongjun Li, Xinlai Yao, Yiqing Xu, Shenjie Zhang, Shijie Zhou, Xiaoli Zi, Lisa Zheng, Yi Gu, Xueming Qian. ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models [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 1546.

Abstract 1531: Human anti-CD40 agonistic antibodies with enhanced FcgR engagement activate immune cell and promote anti-tumor efficacy

Abstract Checkpoint inhibitors represent a major advance in cancer immunotherapy, and combinatorial immunotherapies with secondary drivers of anti-tumor immunity provide beneficial effects for patients that do not show a strong endogenous immune response. CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing B cell and dendritic cell activity and fostering anti-tumor immune responses. Engagement of the inhibitory Fcg-receptor (FcgR) IIb is an absolute requirement for in vivo antitumor activity of agonistic anti-CD40 monoclonal antibodies, and Fc region mutant designs of anti-CD40 antibodies had been discovered to enhance FcgRIIb engagement. With Twist’s precision DNA writing technologies, we have created phage display VHH and scFv libraries with diversity greater than 1 × 1010 for optimal discovery. In this study, we performed high affinity binding of the antibodies by SPR and cell surface binding. The leads are reformatted on human IgG1, IgG4, and IgG1 mutant. The in vitro properties of the CD40 agonistic antibodies demonstrate enhanced FcgRIIb engagement by NFkB activation. B cell activation is also detected by upregulation of CD86 and IL6 secretion. In humanized hCD40 mice model, we observed the engineered anti-CD40 agonists enhance anti-tumor immune function in vivo. These studies suggest that our antibodies can be potential drug for cancer immunotherapy. Citation Format: Linya Wang, Tom Z. Yuan, Mouna Villalta, Zhen Han, Hansika Wadhwa, Kara Y. Chan, Marisa Yang, Tammy Htoy, Paul VanDyke, Carson Holliday, Hector Franco, Hoa Giang, Fumiko Axelrod, Aaron Sato. Human anti-CD40 agonistic antibodies with enhanced FcgR engagement activate immune cell and promote anti-tumor efficacy [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 1531.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [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 1807.

Nano-adjuvants and immune agonists promote antitumor immunity of peptide amphiphiles.

Immunostimulatory cues play an important role in priming antitumor immunity and promoting the efficacy of subunit cancer vaccines. However, the clinical use of many immunostimulatory agents is often hampered by their inefficient in vivo delivery which may decrease immune response to the vaccination. To promote vaccine efficacy, we develop vaccine formulations which integrate three key elements: (1) a nano-adjuvant formulated by conjugating an agonistic anti-CD40 monoclonal antibody (αCD40) to the surface of a polyIC-loaded lipid nanoparticle, (2) a peptide amphiphile containing an optimized CD8+ T-cell epitope that derived from a melanoma antigen gp100, (3) an agonistic anti-4-1BB monoclonal antibody (α4-1BB) that boosts the efficacy of vaccinations. In a syngeneic mouse model of melanoma, the vaccine formulations enhanced innate immunity and activated multiple innate immune signaling pathways within draining lymph nodes, as well as promoted antigen-specific immune responses and reduced immunosuppression in the tumor microenvironment, leading to profound tumor growth inhibition and prolonged survival. Thus, our vaccine formulations represent an attractive strategy to stimulate antitumor immunity and control tumor progression. STATEMENT OF SIGNIFICANCE: The clinical use of many immunostimulatory agents is often hampered by their inefficient in vivo delivery which may decrease immune response to the vaccination. To promote the antitumor immunity of subunit vaccines, we develop novel vaccine formulations that integrate multifunctional modalities including (1) a nano-adjuvant containing anti-CD40 monoclonal antibody (αCD40) and TLR3 agonist which activate innate immunity through diverse signaling pathways, (2) a peptide amphiphile containing an optimized CD8+ T-cell epitope from tumor antigen, (3) an anti-4-1BB monoclonal antibody (α4-1BB) that boosts the efficacy of vaccinations. In this study, our vaccine formulations stimulate superior antitumor immunity and control tumor progression. The above nano-engineered platform and immunogenic biomacromolecules can be further applied to other T-cell-inducing vaccines.

Failure of Costimulatory Blockade-induced Regulatory T Cells to Sustain Long-term Survival of High Ischemic Allografts.

Costimulatory blockade-induced allograft tolerance has been achieved in rodent models, but these strategies do not translate well to nonhuman primate and clinical transplants. One confounder that may underlie this discrepancy is the greater ischemic inflammation imposed on the transplants. In mice, cardiac allografts subjected to prolonged cold ischemic storage (CIS) before transplant have increased ischemia-reperfusion injury, which amplifies infiltrating endogenous memory CD8 T-cell activation within hours after transplantation to mediate acute graft inflammation and cytotoxic lymphocyte-associated molecule-4 immunoglobulin-resistant rejection. This study tested strategies inhibiting memory CD8 T-cell activation within such high ischemic allografts to achieve long-term survival. A/J (H-2 a ) hearts subjected to 0.5 or 8 h of CIS were transplanted to C57BL/6 (H-2 b ) recipients and treatment with peritransplant costimulatory blockade. At 60 d posttransplant, regulatory T cells (Treg) were depleted in recipients of high ischemic allografts with anti-CD25 monoclonal antibody (mAb) or diphtheria toxin. Whereas peritransplant (days 0 and +1) anti-lymphocyte function-associated antigen-1 mAb and anti-CD154 mAb prolonged survival of >60% allografts subjected to minimal CIS for >100 d, only 20% of allografts subjected to prolonged CIS survived beyond day 80 posttransplant and rejection was accompanied by high titers of donor-specific antibody. Peritransplant anti-lymphocyte function-associated antigen-1, anti-tumor necrosis factor-α, and anti-CD154 mAb plus additional anti-CD154 mAb on days 14 and 16 obviated this donor-specific antibody and promoted Treg-mediated tolerance and survival of 60% of high ischemic allografts beyond day 100 posttransplant, but all allografts failed by day 120. These studies indicate a strategy inducing prolonged high ischemic allograft survival through Treg-mediated tolerance that is not sustained indefinitely.

Cannabinoid Receptor 1 Agonist ACEA and Cannabinoid Receptor 2 Agonist GW833972A Attenuates Cell-Mediated Immunity by Different Biological Mechanisms

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) are components in the endocannabinoid system that play significant roles in regulating immune responses. There are many agonists for the cannabinoid receptors; however, their effects on T cell regulation have not been elucidated. In the present study, we determined the effects of the CB1 selective agonist ACEA and the CB2 selective agonist GW833972A on T cell responses. It was found that both agonists impaired anti-CD3 monoclonal antibody induced T cell proliferation. However, ACEA and GW833972A agonists down-regulated the expression of activation markers on CD4+ and CD8+ T cells and co-stimulatory molecules on B cells and monocytes in different manners. Moreover, only GW833972A suppressed the cytotoxic activities of CD8+ T cells without interfering in the cytotoxic activities of CD4+ T cells and NK cells. In addition, the CB2 agonist, but not CB1 agonist, caused the reduction of Th1 cytokine production. Our results demonstrated that the CB1 agonist ACEA and CB2 agonist GW833972A attenuated cell-mediated immunity in different mechanisms. These agonists may be able to be used as therapeutic agents for inducing T cell hypofunction in inflammatory and autoimmune diseases.

Nasal administration of anti-CD3 mAb (Foralumab) downregulates NKG7 and increases TGFB1 and GIMAP7 expression in T cells in subjects with COVID-19

T cells are present in early stages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and play a major role in disease outcome and long-lasting immunity. Nasal administration of a fully human anti-CD3 monoclonal antibody (Foralumab) reduced lung inflammation as well as serum IL-6 and C-reactive protein in moderate cases of COVID-19. Using serum proteomics and RNA-sequencing, we investigated the immune changes in patients treated with nasal Foralumab. In a randomized trial, mild to moderate COVID-19 outpatients received nasal Foralumab (100 μg/d) given for 10 consecutive days and were compared to patients that did not receive Foralumab. We found that naïve-like T cells were increased in Foralumab-treated subjects and NGK7+ effector T cells were reduced. CCL5, IL32, CST7, GZMH, GZMB, GZMA, PRF1, and CCL4 gene expression were downregulated in T cells and CASP1 was downregulated in T cells, monocytes, and B cells in subjects treated with Foralumab. In addition to the downregulation of effector features, an increase in TGFB1 gene expression in cell types with known effector function was observed in Foralumab-treated subjects. We also found increased expression of GTP-binding gene GIMAP7 in subjects treated with Foralumab. Rho/ROCK1, a downstream pathway of GTPases signaling was downregulated in Foralumab-treated individuals. TGFB1, GIMAP7, and NKG7 transcriptomic changes observed in Foralumab-treated COVID-19 subjects were also observed in healthy volunteers, MS subjects, and mice treated with nasal anti-CD3. Our findings demonstrate that nasal Foralumab modulates the inflammatory response in COVID-19 and provides a novel avenue to treat the disease.

Synthesis of Bispecific Antibody Conjugates Using Functionalized Poly-ADP-ribose Polymers.

Poly-ADP-ribose (PAR) is a natural type of polymer derived from enzymatic reactions catalyzed by cellular poly(ADP-ribose) polymerases (PARPs). Given its notable solubility and biocompatibility, the PAR polymer may function as effective carriers for therapeutics in addition to modulating biomolecular interactions in cells. To explore its therapeutic potential, we herein developed a PAR polymer-based bispecific antibody targeting both human epidermal growth factor receptor 2 (HER2) and T-cell CD3 antigens. This was accomplished by conjugating anti-HER2 and anti-CD3 monoclonal antibodies to azido-functionalized PAR polymers through click chemistry. The generated PAR polymer-anti-HER2/anti-CD3 antibody conjugate could not only bind specifically to both HER2- and CD3-expressing target cells but also display potent cytotoxicity against HER2-positive breast cancer cells in the presence of non-activated human peripheral blood mononuclear cells (PBMCs). Functionalized PAR polymers provide a new strategy for synthesizing bispecific antibodies and may enable generation of PAR polymer-based conjugates with unique pharmacological activities for biomedical applications.

IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo.

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA-NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ-STAT1 cascade augmented NK2R expression in CD8+ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8+ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8+ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8+ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8+ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.