4-1BB Agonist Research Articles

Abstract 628: Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Abstract Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved Chimeric Antigen Receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and co-stimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-Mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression. Results: CD137 co-stimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic co-stimulatory activity elicited by agonist CD137-targeted agents. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [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 628.

Abstract 5558: Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation

Abstract Background Claudin 6 (CLDN6) is a tight junction transmembrane protein normally only expressed during embryonic development, but is aberrantly expressed in a variety of tumors, including ovarian cancer, testicular cancer, hepatocellular and lung adenocarcinoma. Here, we generated a CLDN6 X 4-1BB bispecific antibody (BsAb) which could conditionally activate T cells through 4-1BB stimulation upon CLDN6 engagement, positioning it as a potential novel immunotherapy for ovarian cancer and other CLDN6 positive tumors. Methods CLDN6 X 4-1BB BsAb was evaluated for its antigen binding through cell-based binding assay. In vitro function of the antibody was confirmed in a 4-1BB signaling reporter assay and primary human PBMC assay. In vivo efficacy of the antibody was further demonstrated in 4-1BB humanized syngeneic mouse model. Ex vivo analysis of tumor infiltrating and peripheral immune cells was conducted by FACS. Safety parameters such as liver enzymes and pathology upon antibody administration were also assessed. Results CLDN6 X 4-1BB BsAb showed strong binding to several ovarian cancer cell lines with different levels of CLDN6 expression. Functional evaluation of CLDN6X4-1BB BsAb indicated that the activation of 4-1BB signaling was dependent on CLDN6 expression on tumor cells and the activity was stronger than reference 4-1BB monoclonal antibody urelumab. In a humanized 4-1BB mouse model, CLDN6X4-1BB BsAb showed tumor growth inhibition superior to the combination of anti-CLDN6 and anti-4-1BB monospecific antibodies, and the treatment effect was associated with an increase in tumor infiltrating CD45 and CD8 cells as well as CD8/Treg ratio. From the safety perspective, there were no significant changes in liver enzymes or liver histopathology following repeated BsAb administration, suggesting little risks for liver toxicity commonly induced by other 4-1BB agonist antibodies. Conclusions We have successfully generated a novel CLDN6-targeted 4-1BB bispecific antibody which could induce potent 4-1BB stimulation and anti-tumor activity in a CLDN6-dependent manner while minimizing the risk of liver toxicity. Taken together, these data support further development of CLDN6 X 4-1BB bispecific antibody towards IND and clinical trial in 2022. Citation Format: Jian Li, Wenqing Jiang, Yan Liu, Zhengyi Wang, Xi Chen, Taylor B. Guo. Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation [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 5558.

Abstract 296: Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity

Abstract Background: Clinical progress of α4-1BB has been impeded by hepatotoxicity. Current research is focused on improving targeted delivery of α4-1BB to the tumor using tumor-specific markers. However, targeted delivery of 4-1BB agonists to tumors faces challenges due to a lack of tumor biomarkers and effector T cells within tumors. To overcome these challenges, we developed antigen-independent targeting approach based on unnatural sugar-mediated metabolic glycoengineering and bio-orthogonal click chemistry to deliver α4-1BB to tumors. We utilized nanoparticles that can deliver a sugar analogue containing click-chemistry moiety to the tumor microenvironment. Method: The Ac4ManNAz was loaded in mPEG-PLGA nanoparticles (MazNP), and the loading efficiency was determined by HPLC. DBCO-functionalized α4-1BB (DBCO-α4-1BB) was synthesized via amine-NHS coupling reaction. The tumor inhibition efficiency was assessed on mice bearing different tumor models. Mice were either inoculated subcutaneously on the left flank (75,000 B16F10 cells) or injected on the left fourth mammary fat pad (100,000 4T1 cells). Mice were given MazNPs (IV) on day 5, 6, 10, and 11, and DBCO-α4-1BB (IV) and αPD-1 (IP) on day 8 and 13. T lymphocytes were analyzed by fluorescent IF staining. Serum liver enzyme was examined to evaluate hepatotoxicity of treatments. Liver pathology was assessed by H&E and CD8+ IHC staining. Results: The αPD1 plus DBCO-α4-1BB with MazNPs showed a 36.4% cure rate, compared to 0% of αPD1 plus DBCO-α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz in B16F10 melanoma model. We then re-challenged the cured mice with 200,000 B16F10 cells and 50% of cured mice survived without any further treatment. In 4T1 breast cancer model, the survival data analyzed using the log-rank test showed that the median survival of αPD1 plus DBCO-α4-1BB with MazNP was increased by 71%, compared to the PBS, and 26% αPD1 plus α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz. We showed that the involvement of NK and CD8+ T cells in the antitumor efficacy of αPD1 plus DBCO-α4-1BB with MazNP. Notably, a massive CD8+ T cell infiltration in the liver was observed in both αPD1 plus α4-1BB (29.8 ± 18.2%) and αPD1 plus DBCO-α4-1BB with Ac4ManNAz (22.1 ± 10.9), significantly higher than PBS control (0.9 ± 0.6%), while the percentage of CD8+ T cells in the MazNP treatment (5.4 ± 5.8%) was five times lower. Hepatotoxicity was further confirmed by serum liver enzyme analysis that ALT and AST levels were substantially elevated by αPD1 plus α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz, as compared to PBS control group, while the MazNP-treated mice had normal serum ALT and AST levels except for one. Conclusion: Our findings demonstrated that antigen-independent targeted delivery of α4-1BB can improve anti-tumor immune responses while reducing hepatotoxicity. Citation Format: Hyesun Hyun, Bo Sun, Stephanie A. Montgomery, Teresa Griffin, Juanzhu Yan, Albert Wielgus, Yue Wang, Tian Zhang, Jianjun Cheng, Andrew Z. Wang. Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity [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 296.

Validation of a clinically relevant humanized mouse model for the safety assessment of 4-1BB agonists utomilumab and urelumab.

e14602 Background: Immunotherapy is proven to be powerful and effective in treating cancer. One of the strategies for the therapy is to boost a patient’s immune system by stimulating tumor necrosis factor receptors via the activation of 4-1BB (CD137) costimulatory molecule. Urelumab was the first 4-1BB agonist investigated in clinical trials and showed clinical promise but doses were limited by severe liver toxicity. Preclinical assessment of toxicity failed to predict the clinical safety outcome. Methods: To address the critical needs of in vivo evaluation of toxicity in a preclinical setting, we have developed an animal model using PBMC humanized mice to assess the safety of immunotherapy. In this model, agents targeting human immune system including monoclonal and bispecific antibodies can induce an acute response of cytokine release within hours of treatment and with some therapies a systemic response can manifest in tissue damage and lethality of mice days later. Results: The potential clinical relevance of the PBMC humanized mouse model was demonstrated using 4-1BB agonists urelumab and utomilumab. The antibody-treated PBMC humanized mice were evaluated daily for clinical score and assessed for clinical chemistry and liver histopathology at study terminus. Animals dosed with urelumab at 10 mg/kg exhibited body weight loss and underwent early euthanasia, while animals dosed with utomilumab survived to scheduled euthanasia with minimum body weight loss. Urelumab showed marked liver toxicity relative to utomilumab and controls with more necrosis in the tissue and higher mean ALT, AST and GLDH activities, and the tolerability of urelumab was increased by lowering the dose to 1 mg/kg. The differences captured by the humanized mouse model presented a safety profile similar to the findings about urelumab and utomilumab from the clinical trials. Utomilumab was well tolerated at the dose that caused severe toxicity for urelumab in patients, and the toxicity of urelumab could be reduced by lowering the dose. Conclusions: Our data suggest that the PBMC humanized mouse model could identify human 4-1BB agonists that caused potentially severe liver toxicity. Preclinical safety assessment using humanized mouse models as used for these studies could be an important step in the course of the development of novel immunotherapy for the safety of patients as well as mitigating drug development cost.

DuoBody-CD40x4-1BB induces dendritic-cell maturation and enhances T-cell activation through conditional CD40 and 4-1BB agonist activity

BackgroundDespite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational...

A phase 1/2 study of RTX-224, an engineered red blood cell expressing 4-1BB ligand and membrane-bound IL-12, for the treatment of patients with select advanced solid tumors.

TPS2680 Background: RTX-224 is a genetically engineered off-the-shelf, allogeneic red blood cell that expresses the costimulatory molecule 4-1BB ligand (4-1BBL) and cytokine interleukin-12 (IL-12) on the cell surface. The use of 4-1BB and IL-12 agonists for cancer immunotherapy has been limited due to systemic toxicities. Unlike agonist antibodies and recombinant cytokines, which are distributed systemically, Red Cell Therapeutics, such as RTX-224, are restricted to the vasculature and spleen, which may limit toxicities previously observed with agonist 4-1BB monoclonal antibodies and recombinant IL-12. RTX-224 is designed to be a broad immune agonist of both adaptive and innate responses that activates and expands effector and memory CD8+ and CD4+ T cells, cytotoxic natural killer (NK) cells and produces inflammatory cytokines and chemokines, leading to enhanced antigen presentation. In preclinical studies, the combined activation of both adaptive and innate immune responses by the murine surrogate of RTX-224 led to antitumor activity while the restricted biodistribution improved the safety profile. Methods: The RTX-224-01 study is a Phase 1/2, first-in-human, multi-center, dose-escalation and expansion study of RTX-224 in patients with relapsed or refractory urothelial cancer, squamous cell carcinoma of the head and neck, non-small cell lung cancer, triple negative breast cancer and cutaneous melanoma. Safety, tolerability, pharmacokinetics and pharmacodynamics and anti-tumor activity of RTX-224 will be assessed. Approximately 28 patients will be enrolled across dose level cohorts to identify the recommended Phase 2 dose (RP2D). The starting dose is 100 million (1x108) cells administered intravenously every 3 weeks (Q3W) and the dose will be escalated by half-log increments following a Bayesian logarithmic regression model (BLRM) with overdose control. Following RP2D selection, each of the 5 expansion cohorts will enroll approximately 20 patients. Pharmacodynamic and exploratory biomarker studies correlative to clinical response will be evaluated on peripheral blood and paired tumor biopsies. Multiple technologies will be employed to profile the innate and adaptive responses following RTX-224 treatment. The study is open and enrolling patients in Phase 1. Clinical trial information: NCT05219578.

Effect of immunotherapy and time-of-day infusion chronomodulation on survival in advanced cancers.

1588 Background: Emerging clinical studies report correlation of time-of-day infusion (TOI) to immunotherapy outcomes and the intricate interplay of the human circadian rhythm and cancer and immunotherapy exposure. Preclinical and clinical studies have shown cancer chronotherapy to play important role in transcriptional rhythmicity of oncogenic process. We evaluated the association of TOI and immunotherapy outcomes in a large cohort to determine impact of TOI on overall survival (OS) and progression free survival (PFS). Methods: We reviewed charts of patients with solid tumors who received immunotherapy, specifically anti programmed cell death protein 1 (PD1) and anti-programmed death-ligand 1 (PD-L1) agents at MD Anderson Cancer Center. Infusion times were divided into two hours cohorts from 8am-8pm and one overnight cohort from 8pm-8am. Accelerated failure time models were used to model OS (log-normal distribution) and PFS (Weibull distribution) with relation to TOI after adjusting for factors such as age, gender, tumor type and prednisone > 10mg daily use within 1 month of immunotherapy initiation to minimize confounding factors. Results: Of 6151 patients with advanced tumors, 4441 patients received immunotherapy therapy after adjusting for minimum 140 patients in each tumor cohort from 10/15/2016 until 10/15/2021. Tumor types included advanced lung cancer (31.4%), melanoma (28.7%), renal (14.4%), breast (7.6%), colon (6.2%), liver cancer (3.9%) and head and neck (H&N) cancers (2.8%). Median age was 63 (15-99) with 58% males and 42% female. 1894 (43%) patients received investigational agents and 2547 (57%) received standard of care therapies. Mechanism of action of various agents: anti PD1, anti-PDL1, TGF-βRII and anti-PDL1, anti-PD-1 with OX40+ T Cell activation, PD-1 / PD-L1 bispecific antibody, PD-L1 dependent 4-1BB agonist and anti PD-1 and CTLA-4 activity. Among all patients, median OS was 26.4 months while median PFS was 4.8 months. Preponderance of immunotherapy TOI occurred between 12-2pm and 2-4pm in all patients. Patients receiving overnight TOI with lung, renal and breast cancer demonstrated significantly (p < 0.05) poorer OS compared to TOI (10am-4pm) whereas in melanoma, significantly lower OS was seen with overnight, 8-10am and 6-8pm compared to 10am-4pm TOIs. Among H&N cancer patients, early TOI (8-10am) was associated with significantly lower OS compared to afternoon TOI (12pm-4pm). PFS trends were less distinct than for OS but showed a slighted inverted tendency towards lower PFS following daytime TOI. Conclusions: In this large cohort of patients treated with immunotherapy, clinically significant association of TOI with overall survival is seen. These intriguing findings warrant prospective validation in a future randomized trial to harness the role of chronomodulation of cancer therapies for improving outcomes.

Enhancing the antitumor response of CD8+ T cells by 4-1BB (CD137) co-stimulation with distinct inactivation of the type 2 adenosine receptors.

Abstract Activating the immune co-stimulatory receptor 4-1BB (CD137) with agonist antibody binding and crosslinking-inducing agents that elicit 4-1BB intracellular signaling potentiates the anti-tumor responses of CD8+ T cells. However, the underlying in-depth mechanisms remain to be defined. The type 2 adenosine receptors (primarily the high affinity receptor, A2AR) predominantly expressed on CD8+ T cells inhibit T cell activation and expansion by blocking TCR signaling in a cAMP-dependent manner. Here, we show that inactivation of the low affinity receptor A2BR rather than A2AR by continuous treatment of antagonists and/or genetic deletion induces superior survival advantage of effector CD8+ T cells with agonistic 4-1BB co-stimulation especially upon chronic TCR stimulation and/or long-term antigen exposure. Mechanistically, A2BR inactivation helps sustain the increased energy and biosynthetic requirements through the accumulation of intracellular glutathione (GSH) in response to agonistic 4-1BB co-stimulation. Importantly, A2BR inactivation in combination with agonistic 4-1BB co-stimulation displays a greater ability to modulate mitochondrial fitness for anti-tumor CD8+ T cell expansion while minimize T cell exhaustion. Thus, the A2BR pathway plays an unexpected role in metabolic reprogramming of GSH upon agonistic 4-1BB co-stimulation that allows the fine-tuning of the anti-tumor responses of CD8+ T cells. Supported by NCI CCSG P30 CA060553 R01CA258857

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

BackgroundOn the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane...

An Fc-inert PD-L1×4-1BB bispecific antibody mediates potent anti-tumor immunity in mice by combining checkpoint inhibition and conditional 4-1BB co-stimulation

ABSTRACT Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis have changed the treatment paradigm for advanced solid tumors; however, many patients experience treatment resistance. In preclinical models 4-1BB co-stimulation synergizes with ICI by activating cytotoxic T- and NK-cell-mediated anti-tumor immunity. Here we characterize the mechanism of action of a mouse-reactive Fc-inert PD-L1×4-1BB bispecific antibody (mbsAb-PD-L1×4-1BB) and provide proof-of-concept for enhanced anti-tumor activity. In reporter assays mbsAb-PD-L1×4-1BB exhibited conditional 4-1BB agonist activity that was dependent on simultaneous binding to PD-L1. mbsAb-PD-L1×4-1BB further blocked the PD-L1/PD-1 interaction independently of 4-1BB binding. By combining both mechanisms, mbsAb-PD-L1×4-1BB strongly enhanced T-cell proliferation, cytokine production and antigen-specific cytotoxicity using primary mouse cells in vitro. Furthermore, mbsAb-PD-L1×4-1BB exhibited potent anti-tumor activity in the CT26 and MC38 models in vivo, leading to the rejection of CT26 tumors that were unresponsive to PD-L1 blockade alone. Anti-tumor activity was associated with increased tumor-specific CD8+ T cells and reduced regulatory T cells within the tumor microenvironment and tumor-draining lymph nodes. In immunocompetent tumor-free mice, mbsAb-PD-L1×4-1BB treatment neither induced T-cell infiltration into the liver nor elevated liver enzymes in the blood. Dual targeting of PD-L1 and 4-1BB with a bispecific antibody may therefore address key limitations of first generation 4-1BB-agonistic antibodies, and may provide a novel approach to improve PD-1/PD-L1 checkpoint blockade.

The PD-L1/4-1BB Bispecific Antibody-Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner.

While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.

Abstract IAP0301: Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities

Abstract T cell-engaging bispecific antibody constructs (TCEs) have the potential to engage all cytotoxic T cells for redirected cancer cell lysis, including CD8, CD4, gamma/delta, NKT and regulatory T cells. TCEs transiently connect T cells with target cells leading to formation of a cytolytic synapse. Once activated by TCEs, T cells can adopt a serial lysis mode and increase troop size by their local proliferation. In numerous animal models, TCEs can eradicate large tumors as single-agent therapy. TCEs show a remarkable single-agent activity in hematological diseases, including ALL, NHL (DLBCL and FL) and AML. A CD19/CD3-bispecific TCE called blinatumomab (Blincyto) has been approved in 2014, and six other TCEs targeting CD20, BCMA and CD123 are in late-stage development. With complete response rates >50% and durability of responses, TCEs are in par with autologous CD19 CAR-T cells in NHL but are easier to manufacture and allow for a faster treatment start. Treatment of solid tumor indications with TCEs is not as successful as treatment of blood-borne cancers. This may be due to issues with tumor penetration, the hostile tumor microenvironment and the scarcity of T cells in cold tumors. A variety of TCEs explore their single-agent activity against solid tumors by targeting surface antigen like PSMA, GPC3, GD2, or HER-2. Enhancements in the form of combination therapy with checkpoint inhibitors, or 4-1BB and CD28 agonists are being explored as ways of augmenting solid tumor activity of TCEs. The first TCE showing robust anti-tumor activity in a solid tumor indication is targeting a gp100 peptide/MHC (developed by Immunocore). It is made from a soluble TCR fragment of very high affinity for the gp100 peptide HLA*0201 complex fused to an anti-CD3 scFv fragment. Although only few partial responses were observed in uveal melanoma patients, the single-agent treatment showed a very robust improvement in overall survival with a hazard ratio of 0.51 compared to investigators’ choice. This data will inspire the development of more TCEs that are specific for peptide/MHC complexes. This class of targets can also be addresses by TCR-mimetic antibodies. TCEs using such antibodies showed high anti-tumor activity in mouse models against MAGE-A4, p53 mutant or tyrosinase peptide/MHC complexes, all targets that are otherwise not amenable to classical TCEs. Another emerging opportunity for treating cancers are conditional TCEs. These are infused as inactive precursors. Inside tumors, the locally produced proteases convert the precursors into highly active TCEs. One example is the so called ProTriTACs, where a half-life extending albumin-binding domain masks the CD3-binding domain. The mask confers a 500-fold reduction in the cytolytic activity of the TCE. Inside tumors, the mask is cleaved off and a TCE is activated that has a very short serum half-life once it is released from the tumor. Conditional TCEs promise not only a wider therapeutic index but can also leverage as targets tumor-associated antigens that are widely expressed on tumors but also on healthy normal tissues, such as EGFR or EpCAM/TROP1. On-target toxicity of TCEs relates to the quality of target antigens. Finding more tumor-specific surface antigens, appropriate pMHC targets (e.g., mutant oncogene epitopes) and conditional T cell engagers are ways to address this challenge. Off-target toxicity of TCEs relates to T cell activation because of redirected target cell lysis. In the worst case, a cytokine release syndrome is induced. In the best case, a low-level cytokine release is contributing to the efficacy of TCEs. CRS can be managed by steroids, step dosing, anti-IL6 antibody and/or dasatinib. Another promising means is subcutaneous administration of TCEs. A dozen TCE programs currently explore this route, which allows reduction of Cmax and of the initial cytokine release. TCEs will soon become a major weapon in the arsenal of cancer therapy. They will not only work in blood-borne cancers but will soon conquer select solid tumor indications. Peptide/MHC-targeting and conditional TCEs are very promising approaches to achieve solid tumor activity of TCEs. Citation Format: Patrick A. Baeuerle. Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr IAP0301.

Augmenting Efficacy of T-Cell Bispecific Antibodies in AML through a Tumor Stroma-Targeted 4-1BB Agonist

Bispecific antibodies represent a promising treatment option for acute myeloid leukemia (AML). We have recently described a novel T-cell bispecific antibody (TCB) targeting the intracellular tumor antigen Wilms tumor 1 (WT1) in the context of HLA-A*02 (Augsberger et al. Blood 2021). Based on these findings a multicenter first-in-human clinical trial was initiated in relapse/refractory AML (NCT04580121). Possible immune escape mechanisms against T-cell based immunotherapy are provided by the tumor microenvironment (TME) of the bone marrow by co-inhibition of T cells or stromal cells shielding leukemic cells from immune effector cells. To overcome the immunosuppressive effect of the TME and to enhance T-cell responses, we evaluated the combination of the WT1-TCB with an antibody fusion protein that targets a stromal antigen (Fibroblast-activation protein; FAP) and provides a positive costimulatory signal (4-1BBL) to T cells. FAP is upregulated on cancer-associated fibroblasts after remodulation of the bone marrow niche by leukemic cells, and the FAP specificity of the molecule therefore provides T-cell co-stimulation tightly restricted to the tumor niche.Efficacy of the combination (WT1-TCB + FAP-4-1BBL antibody fusion protein) was evaluated in co-culture assays over 4 days with primary HLA-A*02 + AML cells, healthy donor (HD) T cells and three NIH-3T3 fibroblast cell lines. NIH-3T3 cell lines were genetically modified to express low and high levels of FAP, respectively. Wild-type NIH-3T3 cells were included as control. Additionally, a control (Ctrl)-TCB and a Ctrl-4-1BBL antibody fusion protein recognizing a non-tumor target derived from the human germline repertoire were included. Enhancement of T-cell mediated cytotoxicity by the FAP-4-1BBL antibody fusion protein was evaluated by (1) specific lysis of primary AML cells, (2) upregulation of the T-cell activation markers CD25 and 4-1BB, (3) T-cell expansion calculated as fold change compared to day 0, and (4) Granzyme B-expression which was evaluated by intracellular staining.After 4 days of co-culture, with an E:T ratio of 1:2, we observed a mean specific lysis of 55.1±8.2% (±SEM; n=4) of primary AML cells mediated by HD T cells and WT1-TCB. Notably, this was reduced to 19.4±5.9% (±SEM; n=4) in the presence of NIH-3T3 cells. However, AML cell lysis was restored by the addition of the FAP-4-1BBL antibody fusion protein in the presence of high FAP expressing NIH-3T3 cells (mean specific lysis: 62.8±7.3%; ±SEM; n=4). Concomitantly, the FAP-4-1BBL antibody fusion protein led to increased expression of the activation molecules CD25 (MFI ratio: 22.1±5.3 vs. 10.4±1.3; ±SEM; n=4) and 4-1BB (MFI ratio: 10.4±6.0 vs. 2.1±0.3; ±SEM; n=4) on CD3 + T cells. Furthermore, lysis was accompanied by increased frequencies of granzyme B expressing T cells (45.0±2.5% vs. 16.1±5.3%; n=3). Importantly, the FAP-4-1BBL antibody fusion protein led to improved T-cell proliferation, especially of CD8 + T cells (fold change on day 4 vs day 0: 5.7±2.2 vs. 1.0±0.3; ±SEM; n=4). Overall similar observations were made in the presence of low FAP expressing NIH-3T3 cells.Taken together, we have established an in vitro model system mimicking the immunoprotective bone marrow TME using NIH-3T3 cells resulting in impaired AML cell lysis. Providing additional T-cell co-stimulation by a tumor-stroma targeted 4-1BB agonist, however, restored WT1-TCB-mediated cytotoxicity of primary AML cells in the presence of FAP expressing cell lines. Importantly, the combination overcame the immunosuppressive effect of the NIH-3T3 cells on T cells as further demonstrated by improved T-cell activation and expansion. The tumor-stroma targeted 4-1BB agonist therefore represents a promising combinatorial approach to enhance T-cell activity at the local tumor site and warrants further investigations in an in vivo model system. DisclosuresPulko: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Claus: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Buecklein: Pfizer: Consultancy, Honoraria, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Miltenyi: Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Umana: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Klein: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; Klinikum der Universität München: Current Employment; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Avelumab in Combination Regimens for Relapsed/Refractory DLBCL: Results from the Phase Ib JAVELIN DLBCL Study

BackgroundRelapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) is associated with a poor prognosis despite the availability of multiple treatment options. Preliminary evidence suggests that DLBCL may be responsive to programmed death ligand 1 (PD-L1)/programmed death 1 inhibitors.ObjectiveThe JAVELIN DLBCL study was conducted to assess whether a combination of agents could augment and sustain the antitumor immunity of avelumab, an anti-PD-L1 antibody, in R/R DLBCL.MethodsThis was a multicenter, randomized, open-label, parallel-arm study with a phase Ib and a phase III component. Reported here are the results from the phase Ib study, wherein 29 adult patients with DLBCL were randomized 1:1:1 to receive avelumab in combination with utomilumab (an immunoglobulin G2 4-1BB agonist) and rituximab (arm A), avelumab in combination with utomilumab and azacitidine (arm B), or avelumab in combination with bendamustine and rituximab (arm C). The primary endpoints were dose-limiting toxicities and objective response as assessed by the investigator per Lugano Response Classification criteria.ResultsOf the seven patients in arm A, one (14.3%) experienced two grade 3 dose-limiting toxicities (herpes zoster and ophthalmic herpes zoster); no dose-limiting toxicities were reported in arms B or C. No new safety concerns emerged for avelumab. One partial response was reported in arm A, three complete responses in arm C, and no responses in arm B. Given the insufficient antitumor activity in arms A and B and the infeasibility of expanding arm C, the study was discontinued before initiation of the phase III component.ConclusionsThe low level of clinical activity suggests that PD-L1 inhibitor activity may be limited in R/R DLBCL.ClinicalTrials.gov IdentifierNCT02951156.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11523-021-00849-8.

Taking a BiTE out of tumors

Cancer Bispecific T cell engagers (BiTEs) and other forms of T cell–engager therapies represent a promising, off-the-shelf treatment for many types of cancer. However, it is unclear why some types of tumors are particularly resistant to BiTE treatment. Belmontes et al. evaluated pharmacokinetics, pharmacodynamics, and immune correlates of responsiveness to BiTE treatment. In multiple mouse tumor models, they found that pretreatment T cell density in tumors was associated with BiTE efficacy. In T cell–cold tumors, which lack abundant T cells, coadministration of immune checkpoint blockade antibodies or a 4-1BB agonist overcame resistance to BiTE treatment. Together, these findings identify combination therapeutic strategies for BiTES and other T cell engagers. Sci. Transl. Med. 13 , eabd1524 (2021).

Context-Dependent Immunomodulatory Effects of MEK Inhibition Are Enhanced with T-cell Agonist Therapy.

MEK inhibition (MEKi) is proposed to enhance antitumor immunity but has demonstrated mixed results as an immunomodulatory strategy in human clinical trials. MEKi exerts direct immunomodulatory effects on tumor cells and tumor-infiltrating lymphocytes (TIL), but these effects have not been independently investigated. Here we modeled tumor-specific MEKi through CRISPR/Cas-mediated genome editing of tumor cells [MEK1 knockout (KO)] and pharmacologic MEKi with cobimetinib in a RAS-driven model of colorectal cancer. This approach allowed us to distinguish tumor-mediated and tumor-independent mechanisms of MEKi immunomodulation. MEK1 KO tumors demonstrated upregulation of JAK/STAT signaling, enhanced MHCI expression, CD8+ T-cell infiltration and T-cell activation, and impaired tumor growth that is immune dependent. Pharmacologic MEKi recapitulated tumor-intrinsic effects but simultaneously impaired T-cell activation in the tumor microenvironment. We confirmed a reduction in human peripheral-lymphocyte activation from a clinical trial of anti-PD-L1 (atezolizumab) with or without cobimetinib in biliary tract cancers. Impaired activation of TILs treated with pharmacologic MEKi was reversible and was rescued with the addition of a 4-1BB agonist. Collectively, these data underscore the ability of MEKi to induce context-dependent immunomodulatory effects and suggest that T cell-agonist therapy maximizes the beneficial effects of MEKi on the antitumor immune response.

Abstract 1586: Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models

Abstract Oncolytic virotherapy is an emerging treatment modality that may hold promise as a therapeutic option for cancer patients who do not respond to checkpoint inhibitors. Oncolytic viruses preferentially replicate in tumor cells, offer the ability to express transgenes and have been shown to modify the local and distant tumor microenvironments from immunosuppressive to immunostimulatory. We generated HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12, an oncolytic virus based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce murine FMS-like tyrosine kinase 3 ligand (mFLT3L) to enhance dendritic cell expansion and T cell priming, and murine interleukin-12 (mIL12) to potentiate type I helper and cytotoxic T cell responses. We confirmed the expression and bioactivity of mFLT3L and mIL12 in vitro and evaluated the efficacy of HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 alone and in combination with anti-PD-1 or anti-4-1BB treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used and only one tumor was treated via intratumoral injection with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12. Anti-tumor responses were evaluated in the injected tumor (local response), the uninjected tumor (abscopal) and via splenic immune profiling (systemic). HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 treatment alone led to tumor growth inhibition in both treated and contralateral tumors, and a significant increase in overall survival in the A20, Neuro2a and MC38 models. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with anti-PD-1 blocking antibody further improved tumor growth inhibition in both treated and contralateral tumors in the MC38 syngeneic model, and significantly increased median overall survival compared to either agent as a monotherapy. In addition, HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with an agonistic antibody targeting 4-1BB significantly increased overall survival compared to either agent as a monotherapy in the MC38 model. To understand antigen-specific anti-tumor T cell responses, MC38 tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell responses were measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 induced and/or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that treatment with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 can cause direct tumor lysis and induce an adaptive, systemic T cell-mediated anti-tumor immune response that can be further enhanced by concurrent PD-1 blockade or 4-1BB agonism in multiple syngeneic mouse tumor models. Citation Format: Keegan Cooke, Walter H. Meisen, Petia Mitchell, Juan Estrada, Jinghui Zhan, Jessica Orf, Peng Li, Christina de Zafra, Jing Qing, Jason DeVoss. Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic 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 1586.

Abstract LB135: Simultaneous costimulatory T-cell engagement and checkpoint inhibition by PRS-344/S095012, a PD-L1/4-1BB bispecific compound for tumor localized activation of the immune system

Abstract Background. Preclinical and clinical data suggest that 4-1BB (CD137), a costimulatory immunoreceptor mainly expressed by cytotoxic cells, represents a promising therapeutic target in cancer. A combination of checkpoint blockade with further T-cell activation mediated by 4-1BB co-stimulation may increase response rates and durability of response. However, the efficacy of systemic 4-1BB stimulation is limited by on target peripheral toxicity events. PRS-344/S095012 has been designed to provide the potential of a combinatorial therapy in one molecule and favor the localized stimulation of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. Methods. Anticalin® proteins are 18 kDa protein therapeutics derived from human lipocalins. We utilized phage display technologies to generate an Anticalin protein that binds to 4-1BB with high affinity and specificity. PRS-344/S095012 was generated by recombinant fusion of two 4-1BB-specific Anticalin proteins to a PD-L1-targeting monoclonal antibody with a modified IgG4 backbone that avoids interaction with Fc gamma receptors and restricts 4-1BB agonism to PD-L1 positive tissues. The activity and potency of PRS-344/S095012 were investigated in binding assays, functional in vitro assays with human primary immune cells, as well as in a human 4-1BB knock in mouse model. Results. The bispecific fusion protein PRS-344/S095012 is capable of binding 4-1BB and PD-L1 simultaneously. We show that the bispecific compound retains its ability to block PD-1/PD-L1 receptor-ligand interaction with similar potency to the parental PD-L1 antibody. In relevant in vitro cell-based assays, PRS-344/S095012 enhances T cell effector functions only in the presence of PD-L1 positive cells, in line with the desired mechanism of action. In vitro, we show that PRS-344/S095012 activity is superior to PD-L1 antibodies, and to the combination of clinically relevant 4-1BB and PD-L1 benchmark antibodies. In a human 4-1BB knock in mouse model, subcutaneously implanted with a human PD-L1 expressing tumor, PRS-344/S095012 showed clear superiority to anti-PD-L1 alone and a robust antitumor response that leads to the complete regression of implanted tumors and extension of survival. Conclusion. We report potent costimulatory T cell engagement of the immunoreceptor 4-1BB in a PD-L1-dependent manner, utilizing the PD-L1/4-1BB bispecific compound PRS-344/S095012. This approach has the potential to provide a localized costimulation of the immune system with high efficacy and reduced peripheral toxicity. Furthermore, dual mechanism of action combining PD-L1-dependent 4-1BB agonist activity with simultaneous PD-1/PD-L1 pathway blockade provides an additional therapeutic benefit in preclinical models. Taken together our in vitro and in vivo data outlines proof of concept functionality of PRS-344/S095012 and supports further development of this promising compound. Citation Format: Aizea Morales-Kastresana, Marina Pavlidou, Janet Peper, Lucia Pattarini, Christian Barthels, Eva-Maria Hansbauer, Rachida Bel Aiba, Birgit Bossenmaier, Alix Scholer-Dahirel, Thomas Jaquin, Catherine Gallou, Véronique Blanc, Christine Rothe, Shane Olwill. Simultaneous costimulatory T-cell engagement and checkpoint inhibition by PRS-344/S095012, a PD-L1/4-1BB bispecific compound for tumor localized activation of the immune system [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 LB135.

Abstract 1846: DuoBody-CD40×4-1BB (GEN1042) induces dendritic-cell maturation and enhances T-cell activation and effector functions in vitro by conditional CD40 and 4-1BB agonist activity

Abstract The clinical success of immune checkpoint inhibitor monoclonal antibodies (mAbs) has demonstrated potential for re-activation of anti-tumor immunity in cancer treatment. DuoBody®-CD40×4-1BB (GEN1042) is a first-in-class bispecific agonistic antibody that combines targeting of costimulatory molecules CD40 and 4-1BB, resulting in priming and (re-)activation of tumor-specific immunity. We previously showed that DuoBody-CD40×4-1BB induces CD40 and 4-1BB agonist activity that is conditional, i.e., dependent on crosslinking of CD40- and 4-1BB-expressing cells. Here we further elucidate the preclinical mechanism of action of DuoBody-CD40×4-1BB in a broad range of functional immune cell assays. Fixed cell microscopy and live cell imaging of the immune synapse was performed using co-cultures of activated T cells and monocyte-derived dendritic cells (DC). DuoBody-CD40×4-1BB co-localized with LFA-1 at the interface between DC and activated T cells. By binding to both targets, DuoBody-CD40×4-1BB enhanced DC/T cell interactions and increased the contact duration between these cells compared to the combination of monovalent CD40- and 4-1BB-specific antibodies. In mixed lymphocyte reactions (MLR) comprised of naïve CD8+ T cells co-cultured with immature (iDC) or LPS-matured monocyte-derived DCs (mDC), DuoBody-CD40×4-1BB induced DC maturation and increased T cell functionality, shown by a dose-dependent increase in HLA-DR/CD86 expression and production of IFNγ, CXCL10 and granzyme B. DC maturation induced by DuoBody-CD40×4-1BB was also observed in monocultures of iDCs expressing both targets, and was associated with a concurrent dose-dependent increase in TNFα production. Furthermore, in antigen-specific T-cell assays (CLDN6-TCR+CD8+ T cells co-cultured with CLDN6+CD40+ DCs), DuoBody-CD40×4-1BB conditionally enhanced T-cell proliferation and production of IFNγ and TNFα, and induced upregulation of the degranulation marker CD107a and granzyme B. Finally, DuoBody-CD40×4-1BB enhanced clonal expansion of human TILs derived from non-small cell lung and colon cancer patients ex vivo in a dose-dependent manner. In conclusion, by enhancing the formation of the immune synapse between CD40+ DC and 4-1BB+ T cells, DuoBody-CD40×4-1BB induced conditional activation of both cell types, resulting in DC maturation, T-cell activation and effector functions in vitro. Thereby, DuoBody-CD40×4-1BB provides a unique combination of priming and enhancing existing anti-tumor immunity. DuoBody-CD40×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04083599). Citation Format: Alexander Muik, Rachelle Kosoff, Friederike Gieseke, Isil Altintas, Kristina Schödel, Saskia Burm, Eliana Stanganello, Fulvia Vascotto, Aras Toker, Homer Adams, III, Esther Breij, Mustafa Diken, Tahamtan Ahmadi, Kate Sasser, Özlem Türeci, Mark Fereshteh, Ugur Sahin, Maria Jure-Kunkel. DuoBody-CD40×4-1BB (GEN1042) induces dendritic-cell maturation and enhances T-cell activation and effector functions in vitro by conditional CD40 and 4-1BB agonist activity [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 1846.

The Factors Affecting Expansion of Reactive Tumor Infiltrating Lymphocytes (TIL) From Bladder Cancer and Potential Therapeutic Applications.

Tumor infiltrating lymphocytes (TIL) therapy was shown to provide durable objective response in patients with metastatic melanoma. As a fundamental first step to bring TIL therapy to clinical use, identification of patients whose tumors yield optimal numbers of reactive TIL is indispensable. We have previously shown that expansion of tumor reactive TIL from primary bladder tumors and lymph node metastases is feasible. Here, we performed TIL harvesting from additional surgical specimens (additional 31 primary tumors and 10 lymph nodes) to generate a heterogenous cohort of 53 patients with bladder cancer (BC) to evaluate the tumor characteristics that lead to tumor-reactive TIL expansion. Among a total of 53 patients, overall TIL growth from tumor samples were 37/53 (69.8%) and overall anti-tumor reactive TIL were 26/35 (74.3%). Mixed urothelial carcinoma is associated with higher anti-tumor reactivity of expanded TIL than pure urothelial carcinoma (89.5% vs. 56.3%, p=0.049). The anti-tumor reactivity of expanded TIL from primary tumors previously treated with BCG immunotherapy were lower (33.3% vs. 82.6%, p=0.027) although T-cell phenotype (CD3+, CD4+, CD8+, and CD56+) was similar regardless prior of BCG therapy. Addition of agonistic 4-1BB antibody in culture media with IL-2 improved the number of expanded TIL from primary tumors previously treated with BCG immunotherapy. There was no significant difference between basal and luminal subtype tumors in terms of viable and reactive TIL growth. Our study demonstrates that TIL expansion is feasible across all BC patients and BC subtypes, and we suggest that TIL therapy can be a reasonable treatment strategy for various manifestations of BC.