Expanding the horizons of cancer therapy with next-generation 4-1BB agonists: a review of molecular and clinical strategies to maximize efficacy and ensure safety

Immune cell costimulation via 4-1BB agonism has shown anti-tumor activity in the clinic and is an important element of next-generation chimeric-antigen-receptor (CAR) adoptive T-cell therapy approaches. However, the clinical development of first-generation, 4-1BB agonistic antibodies has been hampered by significant hepatic toxicity. Activity of such first-generation, 4-1BB agonistic antibodies typically depends on their hyperclustering via Fc-gamma-receptor (FcgR)-binding. Here we describe a next generation, tumor-targeted 4-1BB agonist whose activity is independent of FcgR-binding. The molecule consists of an IgG fusion protein composed of a trimeric, human 4-1BB ligand (4-1BBL), a targeting Fab moiety recognizing fibroblast activation protein (FAP), and a heterodimeric Fc region engineered to be devoid of interactions with FcgRs and C1q. The molecule mediates potent costimulation of CD8 T-, CD4 T- and NK-cells, but only in the presence of FAP-expressing cells, such as cancer associated fibroblasts, which are highly prevalent in many solid tumors. This FAP-targeted 4-1BB agonist is significantly more potent and efficacious than first generation, standard 4-1BB agonistic antibodies when compared side-by-side in preclinical models. We show its activity in a variety of preclinical models including reporter cell assays, assays with primary T- and NK-cells, ex-vivo assays with patient tumor-derived material including cancer cells, stroma cells and tumor-infiltrating lymphocytes, fully immunocompetent murine tumor models (employing a surrogate, murinized molecule targeting murine FAP and carrying murine 4-1BBL), and in human hematopoietic stem cell-humanized mice with human tumor xenografts. We also demonstrate its activity in combination with checkpoint inhibitors and with T-cell redirecting approaches, such as a CEA-CD3 T-cell bispecific antibody. We show that hepatic toxicity of first generation, standard 4-1BB antibodies is dependent on FgR interactions and the next generation, FcgR-independent and FAP-targeted molecule described here is safe and does not induce any hepatotoxicity in preclinical models including non-human primates where it was tested at doses of up to 50 mg/kg and where it showed a long circulatory half-life. Its combination with T-cell bispecific antibodies induces a massive T cell accumulation in the tumor, accompanied with an elevated CD8/Treg ratio, as compared to the respective monotherapies. Therefore, we conclude that the tumor-targeted cross-linking of 4-1BB provides a safe and effective way for the co-stimulation of T cells for cancer immunotherapy and its combination with T-cell bispecific antibodies may provide an alternative, but more convenient, off-the-shelf approach to CAR T-cell therapies. The molecule is scheduled to enter clinical trials soon. Citation Format: Christina Claus, Claudia Ferrara, Sabine Lang, Rosmarie Albrecht, Sylvia Herter, Maria Amann, Sandra Richards-Grau, Johannes Sam, Sara Colombetti, Marina Bacac, Christian Klein, Pablo Umana. A novel tumor-targeted 4-1BB agonist and its combination with T-cell bispecific antibodies: an off-the-shelf cancer immunotherapy alternative to CAR T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3634. doi:10.1158/1538-7445.AM2017-3634

4-1BB, a member of the tumor necrosis factor receptor superfamily, is an important co-stimulatory molecule regulating the activity of immune cells across a range of physiological and pathological processes, which culminates in a potent immune response (Figure 1A and B) [1, 2]. Numerous clinical trials have been conducted utilizing 4–1BB agonists (Supplemental Table S1); however, previous and ongoing 4-1BB agonist trials are being conducted without biomarker selection, which potentially explains their modest efficacy. Interestingly, several studies suggest the potential value of utilizing transcriptomics in addition to genomics to identify the unique immunologic signature of individual tumors [3-7]. Herein, we explore the landscape of 4–1BB transcriptomic profiles in 514 patients, including 489 with advanced/metastatic cancers and clinical annotation, and we discuss the potential therapeutic implications of the observed patterns and heterogeneity. The study methods are provided in the Supplementary File. There were 514 tumors reflecting 31 different cancer types evaluated (Supplemental Table S2). Their median age was 61 (range, 24-93) years; 310 (60.3%) were women. The most frequent tumor types assessed were colorectal cancer (n = 140 samples). Of the 514 patients, 489 had confirmed metastatic or locally advanced disease, but the dates of metastatic disease for 25 patients were not documented. Overall, 489 patients with advanced/metastatic disease had fully evaluable clinical correlative data (Figure 1C). In total, 217 patients received ICIs; 199 received anti-programmed cell death 1 (PD-1)/ programmed death-ligand 1 (PD-L1) monotherapy, 2 received anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) monotherapy, and 16 received a combination of anti-PD-1/PD-L1 and anti-CTLA-4. The remaining 272 patients never received immunotherapy. Figure 1D demonstrates the variations in 4-1BB RNA expression across different cancer types: 4-1BB ribonucleic acid (RNA) expression was classified as "high" (75-100th percentile), "moderate" (25-74th percentile), and "low" (0-24th percentile). Among all cancers (n = 514), 77 (15.0%) had high, 268 (52.1%) had moderate, and 169 (32.9%) had low 4-1BB expression. Small intestine and ovarian cancers most frequently had high 4-1BB RNA expression (25.0% and 20.9% of tumors, respectively). To identify the patient population who might theoretically benefit the most from 4-1BB agonism, the proportion of patients having high 4-1BB expression along with low/moderate 4-1BB ligand (4-1BBL) expression was assessed in malignancies that had more than 20 representative samples (from a biologic point of view, high receptor with low/moderate ligand might be most amenable to treatment with 4-1BB agonists in the clinic). Malignancies with the highest proportion of patients having both high 4-1BB expression and low/moderate 4-1BBL expression were ovarian and pancreatic, with 18.6% (8/43) and 14.5% (8/55), respectively, representing the only two malignancies with >15% of patients having this expression profile (Figure 1E). Otherwise, the proportions of patients having high 4-1BB expression along with low/moderate 4-1BBL expression were 12.5% (3/24) in sarcoma, 12.5% (3/24) in uterine, 10.0% (2/20) in lung, 8.0% (2/25) in stomach, 7.9% (11/140) in colorectal, and 0% (0/49) in breast. There was variability of expression patterns both within and between tumor types. It is, therefore, conceivable that patient selection for 4-1BB agonist trials based in part on transcript levels may merit investigation. We examined 12 genes that produce immunomodulatory molecules related to the TNF receptor superfamily or interacting co-stimulatory molecules, as well as markers related to ICIs in the clinic (Supplemental Table S3). The results of the univariable and the subsequent multivariable logistic regression are shown in Figure 1F. High RNA expression of 4-1BB was not associated with any specific tumor types. Among immune markers, high 4-1BB RNA expression was significantly associated with that of PD-L2 (odds ratio [OR] 3.1, 95% confidence interval [CI] 1.56–6.47, P = 0.018), ICOS (OR 4.5, 95% CI 2.03-10.36, P = 0.003), and CD27 (OR 2.2, 95% CI 1.12-4.81, P = 0.046) (all multivariable). When analyzing the pan-cancer whole genome cohort from the ICGC and TCGA (n = 2,658), ICOS RNA expression was the most significantly correlated with 4-1BB expression among 33,855 genes (spearman correlation coefficient (r): 0.77, P < 0.001; Figure 1G). Although high RNA expression of 4-1BB was not associated with high PD-1/PD-L1 expression in our cohort (Figure 1F), 4-1BB RNA expression was significantly correlated with PD-1 expression when examined as a linear variable (spearman correlation coefficient: 0.70, P < 0.001; Supplementary Figure S1). Additionally, PD-L2 and CD27 expression were also significantly correlated with 4-1BB expression (spearman correlation coefficient: 0.69, P < 0.001; and 0.53, P < 0.001, respectively, Figure 1G). Therefore, patients with cancer that co-expresses high PD-L2 may benefit from concomitant blocking of the immune inhibitory activity of PD-L2 if a 4-1BB agonist is given; this could be achieved by co-administering an anti-PD-1 agent since PD-L2 serves as a ligand for PD-1. Survival outcomes were evaluable among 489 patients; among them, 272 patients never received immunotherapy, and 217 patients were treated with an immunotherapy-based regimen (Figure 1C). Among the 489 patients, the median overall survival (OS) was 51.7 months (95% CI, 43.3-61.3) for the high-4-1BB group and 35.7 months (95% CI, 30.3-44.0) for the low/moderate 4-1BB group, respectively, from the time of metastatic/advanced disease (hazard ratio [HR] = 0.74, 95%CI: 0.52-1.06, P = 0.103; Figure 1H). Regarding patients who never received immunotherapy, the median OS from advanced/metastatic disease was 51.7 months (95% CI, 24.4–not available [NA]) for the high-4-1BB group and 42.2 months (95% CI, 30.9-46.3) for the low/moderate 4-1BB group, respectively, from time of advanced/metastatic disease (HR = 0.83, 95%CI: 0.49-1.41, P = 0.497; Figure 1I). In patients treated with immunotherapy (n = 217), the high 4-1BB group (n = 39) showed a trend towards longer progression-free survival (PFS) (HR = 0.70, 95%CI: 0.47-1.04, P = 0.074; Figure 1J) and significantly longer OS (HR = 0.55, 95%CI: 0.33-0.89, P = 0.016; Figure 1K) compared to the low/moderate 4-1BB group (n = 178) (calculated from start of immunotherapy). However, the significance of 4-1BB did not hold as an independent factor for survival in multivariable analysis (HR 0.83, 95% CI 0.47–1.46, P = 0.515; Supplemental Table S4). In contrast, in patients treated with immunotherapy, high 4-1BBL expression was not associated with longer PFS or OS (Supplementary Figure S2). There are several limitations in this study due to its retrospective nature and limited sample size. First, although multiple types of cancer were represented, the number of patients for each type was relatively small. Secondly, our bulk analysis was not able to identify the cell types expressing 4-1BB and its ligand. In our bulk analysis, both high 4-1BB expression and high CD4/CD8 expression were observed in 10.7% of samples (Supplementary Table S5), suggesting that 4-1BB was expressed not only in T cells but also in tumor cells within the tumor microenvironment. In summary, a myriad of reasons likely account for the suboptimal outcomes observed in current trials that focus on 4-1BB targeting. This study illuminates additional factors worth investigating in subsequent clinical trials. Based on our findings, 4-1BB and its ligand show variability of expression both between and within tumor types, indicating that cancers need to be sampled and analyzed in order to establish their individual immunomic portfolios. The concomitant high expression of 4-1BB and PD-L2 suggests that a combination approach that includes anti-PD-1 agents for malignancies with high PD-L2 expression warrants exploration. Conception and design: Yuji Uehara, Shumei Kato, Razelle Kurzrock. Development of methodology: Yuji Uehara, Shumei Kato, Daisuke Nishizaki, Razelle Kurzrock. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Yuji Uehara, Shumei Kato, Daisuke Nishizaki, Razelle Kurzrock. Writing, review, and/or revision of the manuscript: Yuji Uehara, Shumei Kato, Daisuke Nishizaki, Hirotaka Miyashita, Suzanna Lee, Mary K. Nesline, Sarabjot Pabla, Jeffrey M. Conroy, Paul DePietro, Heidi Ko, Jason K. Sicklick, Razelle Kurzrock. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Suzanna Lee, Shumei Kato, Daisuke Nishizaki, Razelle Kurzrock. Study supervision: Shumei Kato, Razelle Kurzrock. Not applicable. Dr. Kato serves as a consultant for Medpace, Foundation Medicine, NeoGenomics, and CureMatch. He receives speaker's fees from Roche and Bayer, and the advisory board for Pfizer. He has research funding from ACT Genomics, Sysmex, Konica Minolta, OmniSeq, and Personalis. Dr. Sicklick receives research funding from Novartis Pharmaceuticals, Amgen Pharmaceuticals, and Foundation Medicine; consultant fees from Grand Rounds, Loxo, and Deciphera; and speaker's fees from Roche and Deciphera. He also owns stocks in Personalis. Dr. Kurzrock has received research funding from Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance and from the NCI; as well as consultant and/or speaker fees and/or advisory board/consultant for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Inc., Biological Dynamics, Caris, Datar Cancer Genetics, Daiichi, EISAI, EOM Pharmaceuticals, Iylon, LabCorp, Merck, NeoGenomics, Neomed, Pfizer, Prosperdtx, Regeneron, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc. and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch. No potential conflicts of interest were disclosed by the other authors. Dr. Kurzrock is funded in part by the National Institutes of Health (grant numbers: 5U01CA180888-08 and 5UG1CA233198-05.) All investigations were conducted in accordance with the guidelines set by the Institutional Review Board of the University of California San Diego for data collection (Study of Personalized Cancer Therapy to Determine A Response and Toxicity, UCSD_PREDICT, NCT02478931) and all relevant investigational interventions for which the patients had provided consent. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

4-1BB (CD137) is an inducible co-stimulatory receptor expressed on activated T-cells and natural killer (NK) cells. Activation of 4-1BB induces proliferation, cytokine production, cytotoxic function and survival of T and NK cells. Most importantly, it has been shown to result in tumor eradication and long-term tumor immunity in numerous experimental tumor models. Thus, 4-1BB agonists may have great potential in cancer immunotherapy. However, the 4-1BB antibodies in clinical development are associated with toxic side effects and/or poor efficacy, potentially limiting their clinical use. ATOR-1017 is an anti-4-1BB IgG4 antibody designed to overcome the limitations observed with other 4-1BB antibodies. It binds to a distinct 4-1BB epitope located in domain 2 and blocks 4-1BB ligand binding. Furthermore, its agonistic effect is dependent on crosslinking by Fcγ Receptor (FcγR) I and IIb. A potentially advantageous safety profile was supported by the lack of observed immunotoxicity in a cytokine release assay based on human PBMC using a panel of proinflammatory cytokines as readout. Furthermore, expression profiling of human tumor and normal tissue demonstrates that 4-1BB and FcγRs are highly expressed in the tumor environment. In contrast, co-expression of 4-1BB and FcγRs in non-tumor tissues, such as the liver, is low. Therefore, ATOR-1017 is expected to result in tumor-localized immune activation, thereby avoiding systemic adverse events, including hepatotoxicity, which has been observed by another, FcγR-independent 4-1BB antibody. Finally, to further investigate the safety profile of ATOR-1017, a pilot toxicology study was conducted. ATOR-1017 binds with similar affinity of approximately 0.2 nM to human and cynomolgus monkey 4-1BB, whereas no detectable binding to mouse 4-1BB is observed. Thus, cynomolgus monkeys were administered four weekly doses of 5, 15 or 50 mg/kg ATOR-1017. No clinical signs were observed at any dose, indicating a favorable safety profile. In conclusion, ATOR-1017 was designed to be highly active in the tumor environment, and to have minimal systemic effects, thereby enabling a superior safety/efficacy profile. This is supported by a preclinical data package, demonstrating dependency on FcγR-mediated crosslinking, a clean profile in cytokine release assay, 4-1BB and FcγRs co-expression in the tumor and no clinical adverse events observed in a repeat dosing pilot toxicology study. ATOR-1017 is in preclinical development with initiation of phase I anticipated in 2019. Citation Format: Eva Dahlén, Anna Rosén, Karin Barchan, Anna Dahlman, Peter Ellmark, Tina Furebring, Karin Enell Smith. ATOR-1017: A 4-1BB antibody designed for superior safety/efficacy profile in cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A183.

T cells play a central role in immune responses against cancer. Within the tumor, however, T cells are exposed to a plethora of inactivating factors causing various degrees of dysfunction, changes in metabolism and a generally reduced cellular fitness, eventually leading to tumor progression. To prevent or delay the onset of exhaustion and instead augment effector functions and persistence of functional T cells, costimulatory factors and cytokines are needed. Targeting 4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, has been shown to represent a promising strategy for inducing an activating signal in CD8+ T cells, resulting in increased pro-inflammatory cytokine secretion, cytotoxic function, and survival. Engineered arenavirus vectors based on lymphocytic choriomeningitis virus (LCMV) or Pichinde virus (PICV) have been shown previously to induce massive infiltration of tumor antigen specific CD8+ T cells into the tumor in several preclinical cancer models. To investigate whether enhanced co-stimulation via 4-1BB further improves T cell responses and/or tumor control, combination therapies with replicating LCMV based vectors (artLCMV) and 4-1BB agonists were explored. A single intravenous treatment of artLCMV encoding the tumor associated antigens (TAA) gp70 or Trp2 in B16.F10 tumor bearing mice induced TAA specific CD8+ T cells in both the periphery and the tumor, resulting in tumor growth delay and some complete responses. Combining artLCMV with agonistic anti-4-1BB significantly improved tumor control and increased the number of complete responders. Analysis of tumor infiltrating lymphocytes revealed higher absolute numbers of TAA specific CD8+ T cells in the combination group compared to the artLCMV alone group. Analyses of the TAA specific cells revealed that more cells expressed granzyme B, Ki67 and Bcl-XL when co-stimulated with anti-4-1BB compared to the group treated with artLCMV alone. Importantly, encoding 4-1BBL in addition to a TAA in artLCMV revealed similar outcomes as just summarized for the combination with agonistic antibodies. Overall, these experiments confirmed the strong antigenicity and T cell inducing capacity of the engineered arenavirus platform, leading to efficient tumor control in a stringent mouse model. Combination with 4-1BB agonists, either in form of antibodies or encoded within the vector genome, was shown to further augment TAA-specific T cell responses within the tumor, leading to better tumor growth control and a higher rate of complete responders. Citation Format: Judith Strauss, Marilies Scheinost, Theresa Kleissner, Diana Reckendorfer, Kimberly Pojar, Mohamed Habbeddine, Sarah Ahmadi-Erber, Daniela Deutschmann, Sarah Schmidt, Josipa Raguz, Igor Matushansky, Christoph Lampert, Klaus K. Orlinger, Henning Lauterbach. Evaluation of a cancer immunotherapy with engineered arenavirus vectors and 4-1BB agonists in a preclinical tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4198.

Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library

Combinatorial Therapy for Liver Metastatic Colon Cancer: Dendritic Cell Vaccine and Low-Dose Agonistic Anti-4-1BB Antibody Co-Stimulatory Signal

Toll-like Receptors (TLRs) are a class of Pattern Recognition Receptors (PRRs) capable of recognizing pathogen-associated molecular patterns (PAMPs) (1) and damage-associated molecular patterns (DAMPs) (2) to initiate immune responses. TLRs are crucial for the detection of infections, and activating downstream signaling pathways that lead to the production of pro-inflammatory cytokines and interferons, thereby initiating host defense mechanisms against a wide range of pathogens (3). Moreover, TLRs are involved in the crosstalk between the innate and adaptive immune systems, as their activation can influence the development of antigenspecific adaptive immune responses (4). TLR ligands differentially regulate the function of dendritic cells that play a central role during priming and activation of naive T cells (5). Understanding the complex interplay of TLRs in immune regulation not only contributes to our comprehension of host defense mechanisms but also has implications for the development of vaccines, immunotherapies, and treatments for various autoimmune and inflammatory disorders such as cancer.Recent developments in the field of TLRs and cancer immunity underscore the dual role of TLRs in cancer development: promoting or inhibiting tumor growth. TLR agonists are being explored as adjuvants in cancer vaccines and immunotherapies, thereby enhancing the activation of dendritic cells and adaptive immune responses against cancer. However, the intricate interplay between TLRs and the tumor microenvironment has been increasingly recognized, with some TLRs implicated in supporting an immunosuppressive milieu that benefits tumor growth. Velasco et al. demonstrated that TLR signaling blockade significantly decrease COPDlike inflammation dependent tumor onset in the lung using k-ras driven lung adeno-carcinoma model (6).Researchers are now delving into the specific TLR subtypes, their differential effects on immune cells, and the development of TLR-targeted therapies that could either enhance the immune response against cancer or attenuate immunosuppressive signals, all of which hold significant promise in advancing cancer immunotherapy strategies (7). Interestingly, Liu et al. showed that LPS signaling enhanced tumor apoptotic activity of IAP targeting therapy, suggesting TLR signaling may use "synthetic lethal" approach for reducing its toxicity (8).In recent years much scientific interest has focused on improving the efficacy of cancer immunotherapy. In the realm of cancer immunotherapy, TLRs are garnering increased attention as potential therapeutic targets. TLR agonists are being explored for their capacity to stimulate innate immune responses, such as the activation of dendritic cells and natural killer cells, which can bolster antitumor immunity. Combining TLR agonists with other immunotherapies like checkpoint inhibitors or adoptive cell therapies holds promise in creating synergistic effects to enhance cancer treatment outcomes (7). However, there is ongoing research to better understand the nuances of TLR signaling in the context of different cancer types and patient populations to optimize their use in personalized cancer immunotherapies. Furthermore, efforts to mitigate potential side effects and enhance the specificity of TLR-targeted treatments are also underway, indicating a dynamic and evolving field within cancer immunotherapy. Ota et al. tried to achieve efficacy-safety margin by utilizing small molecule TLR7 specific agonist which shows rapid clearance from the body (9).The therapeutic prospects for TLRs are exceptionally promising, as they represent a critical avenue in both the understanding and application of immunotherapy. In this topic there are 3 review articles discussing the strategy of clinical application by targeting TLR signaling, especially in the oncology field. Hoden et al. focuses on TLR agonists for use in lung cancer (7). More broadly, Yang et al. focuses more on the mechanisms and cutting-edge technologies of various tumor types (10) and Mukheerjee et al. explores cancer type specific strategies (11). Ongoing research is likely to uncover novel TLR subtypes and ligands, shedding light on their nuanced roles in diverse disease contexts, including cancer, infectious diseases, and autoimmune disorders. Additionally, the development of more refined TLR-targeted therapies, such as selective agonists or antagonists, offers the potential for precise modulation of immune responses, minimizing unwanted side effects. Harnessing TLRs as adjuvants in combination with emerging immunotherapies and personalized medicine approaches may further revolutionize the treatment of various conditions, ultimately paving the way for more effective and tailored therapeutic interventions. This evolving field holds great promise for the continued advancement of immunotherapy and precision medicine in the years to come.

Cancer immunotherapy, encompassing both experimental and standard-of-care therapies, has emerged as a promising approach to harnessing the immune system for tumor suppression. Experimental strategies, including novel immunotherapies and preclinical models, are actively being explored, while established treatments, such as immune checkpoint inhibitors (ICIs), are widely implemented in clinical settings. This comprehensive review examines the historical evolution, underlying mechanisms, and diverse strategies of cancer immunotherapy, highlighting both its clinical applications and ongoing preclinical advancements. The review delves into the essential components of anticancer immunity, including dendritic cell activation, T cell priming, and immune surveillance, while addressing the challenges posed by immune evasion mechanisms. Key immunotherapeutic strategies, such as cancer vaccines, oncolytic viruses, adoptive cell transfer, and ICIs, are discussed in detail. Additionally, the role of nanotechnology, cytokines, chemokines, and adjuvants in enhancing the precision and efficacy of immunotherapies were explored. Combination therapies, particularly those integrating immunotherapy with radiotherapy or chemotherapy, exhibit synergistic potential but necessitate careful management to reduce side effects. Emerging factors influencing immunotherapy outcomes, including tumor heterogeneity, gut microbiota composition, and genomic and epigenetic modifications, are also examined. Furthermore, the molecular mechanisms underlying immune evasion and therapeutic resistance are analyzed, with a focus on the contributions of noncoding RNAs and epigenetic alterations, along with innovative intervention strategies. This review emphasizes recent preclinical and clinical advancements, with particular attention to biomarker-driven approaches aimed at optimizing patient prognosis. Challenges such as immunotherapy-related toxicity, limited efficacy in solid tumors, and production constraints are highlighted as critical areas for future research. Advancements in personalized therapies and novel delivery systems are proposed as avenues to enhance treatment effectiveness and accessibility. By incorporating insights from multiple disciplines, this review aims to deepen the understanding and application of cancer immunotherapy, ultimately fostering more effective and widely accessible therapeutic solutions.

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity

Cancer immunotherapy with 4-1BB agonists has limited further clinical development because of dose-limiting toxicity. Here, we developed a bispecific antibody (bsAb; B7-H3×4-1BB), targeting human B7-H3 (hB7-H3) and mouse or human 4-1BB, to restrict the 4-1BB stimulation in tumors. B7-H3×m4-1BB elicited a 4-1BB-dependent antitumor response in hB7-H3-overexpressing tumor models without systemic toxicity. BsAb primarily targets CD8 T cells in the tumor and increases their proliferation and cytokine production. Among the CD8 T cell population in the tumor, 4-1BB is solely expressed on PD-1+Tim-3+ "terminally differentiated" subset, and bsAb potentiates these cells for eliminating the tumor. Furthermore, the combination of bsAb and PD-1 blockade synergistically inhibits tumor growth accompanied by further increasing terminally differentiated CD8 T cells. B7-H3×h4-1BB also shows antitumor activity in h4-1BB-expressing mice. Our data suggest that B7-H3×4-1BB is an effective and safe therapeutic agent against B7-H3-positive cancers as monotherapy and combination therapy with PD-1 blockade.

BackgroundResistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy.MethodsHK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys.ResultsHK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies.ConclusionWe generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.

This talk will describe a new drug candidate for cancer immunotherapy, FAP-4-1BBL, a next generation, tissue-targeted agonist of the 4-1BB costimulatory receptor. Agonizing 4-1BB to enhance the activity of antitumor T cells has been successfully exploited by 2nd and 3rd generation CAR T-cell therapies for hematological malignancies. However, the clinical development of first-generation, 4-1BB agonistic antibodies for systemic administration has been hampered by significant hepatic toxicity. Here we describe a next generation, fibroblast activated protein (FAP)-targeted 4-1BB agonist whose activity is independent of Fc-gamma-receptor (FcγR)-binding, is safe in preclinical models (including non-human primates using doses of up to 50 mg/kg) and is significantly more potent and efficacious than first generation, standard 4-1BB agonistic antibodies when compared side-by-side in preclinical models. In preclinical models, combination of FAP-4-1BBL with T-cell bispecific antibodies leads to significant increases in T cell infiltration into the tumor, CD8/Treg ratio and anti-tumoral efficacy. We conclude that the tumor-targeted cross-linking of 4-1BB provides a safe and effective way for co-stimulation of T cells for cancer immunotherapy and its combination with T-cell bispecific antibodies may provide a convenient “off-the-shelf,” systemic cancer immunotherapy approach for many tumor types. Citation Format: Pablo Umana. FAP-4-1BBL: A next generation, targeted costimulatory agonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr DDT02-01.

TPS3106Background: Programmed death-1 receptor ligand (PD-L1) is a key therapeutic target in the reactivation of the immune response against multiple cancers. Additionally, 4-1BB agonists have been...

Following the clinical success of checkpoint inhibitors, cancer immunotherapy is rapidly expanding into combination treatments to enhance response rates and duration. Engagement of co-stimulatory molecules from the tumor necrosis factor receptor (TNFR) superfamily, including 4-1BB, may be a promising approach to enhance the benefits of cancer immunotherapy. Agonistic antibodies against the costimulatory receptor 4-1BB (CD137) have been shown to effectively enhance the anti-tumor activity of checkpoint inhibitors and other agents in preclinical animal models. However, the clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. We generated a DARPin® therapeutic candidate, MP0310 (AMG 506), which comprises domains binding to 4-1BB and fibroblast activation protein (FAP). MP0310 triggers 4-1BB activation only if bound and clustered via FAP which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. In vitro functional assays indicate that MP0310 is a potent T cell co-stimulator in the presence of FAP-expressing cells. In vivo activity of tumor-targeted 4-1BB agonism was assessed in the HT-29 colon carcinoma xenograft model in PBMC humanized mice in combination with a T cell engager. Consistent with tumor targeting, MP0310 enhanced intra-tumoral CD8 T cell expansion while showing only limited systemic activity. We used a translational pharmacokinetic-pharmacodynamic (PK-PD) modeling approach to integrate in vitro and in vivo data to support the estimation of a minimal anticipated biological effect level (MABEL) and the relevant dose range for first in human (FIH) studies. In addition to a PK model describing the MP0310 concentrations over time in mouse and monkey, direct and indirect response models were used to describe receptor occupancy (RO), intra-tumoral CD8 T cell infiltration and peripheral CD8 count kinetics. Predictions from the combined PK and PD models using average intra-tumoral and peripheral drug concentrations (Cav) provide a MABEL dose with minimal expected systemic PD effects at 20% RO as well as the anticipated therapeutic dose range in humans. In conclusion, our PK/PD-modeling approach provides support for the selection of a safe starting dose for MP0310 (AMG506) in patients. It provides a rationale for selecting the maximum tested dose, and may help reduce the number of cancer patients receiving sub-therapeutic doses. MP0310 (AMG 506) is currently being evaluated in a Ph1 clinical study. Citation Format: Alexander Link, Laurent Juglair, Heïdi Poulet, Guy Lemaillet, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Robinson, Niina Veitonmäki, Jörg Herbst, Keith Dawson, Christof Zitt, Camila de Almeida, Rik de Greef, Victor Levitsky, Michael T. Stumpp, Hong Ji, Elmar Vom Baur. Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 (AMG 506) using a pharmacokinetic/pharmacodynamics modeling approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2273.

Preclinical studies in mice and recent clinical trials have highlighted the promise of immune costimulation in cancer immunotherapy. Not unexpectedly, murine studies have also shown that systemic administration of immune stimulatory antibodies can be associated with adverse effects, mostly on-target autoimmune pathologies resulting from the activation of autoreactive T cells. This was underscored in a recent phase I clinical trials of an agonistic 4-1BB antibody that was associated with high frequencies of objective responses but also accompanied by adverse effects that became significant at the highest dose tested causing liver toxicity resulting in two fatalities, by-and-large precluding the dose escalation necessary to fully exploit the therapeutic potential of this otherwise promising drug. Arguably, targeting costimulatory ligands to the disseminated tumor lesions of the patient would reduce drug associated toxicities. With the goal of developing a clinically feasible and broadly applicable approach to reduce the aforementioned dose limiting toxicities we are developing an oligonucleotide aptamer platform consisting of bi-specific aptamer conjugates whereby costimulatory aptamer ligands (the therapeutic agents) are conjugated to aptamers that bind to tumor-specific aptamers (the targeting ligands). The chemically synthesized oligonucleotide aptamers offer significant advantages over antibodies in terms of synthesis, cost, conjugation chemistry, and reduced risk of neutralizing immunogenicity. We have previously shown that systemic delivery of a bi-specific aptamer composed of an agonistic 4-1BB binding aptamer conjugated to a an aptamer that bound to a product expressed on the surface of a tumor cell, led to inhibition of tumor growth, and exhibited a superior therapeutic index compared to nontargeted costimulation with 4-1BB antibodies or 4-1BB aptamers. Nonetheless, the main limitation of tumor targeted costimulation in its current form stems from the fact that the costimulatory (aptamer) ligand, has to be displayed on the surface of the targeted tumor cells. Consequently one is limited to target receptors that do not internalize upon ligand binding. While such receptor-ligand interactions exist, most receptor-ligand complexes are internalized, and consequently the targeting choices are severely limited. To address this limitation, and broaden the scope of tumor-targeted costimulation, the 4-1BB aptamers were targeted to products secreted in the tumor stroma by conjugation to aptamers that bind to either VEGF or osteopontin (OPN). This approach was predicated on the premise that by targeting the costimulatory ligands to products secreted into the tumor stroma the T cells will be costimulated prior to their engagement of the MHC/peptide complex on the tumor cell, thereby obviating the need to target the costimulatory ligands to non-internalizing cell surface products expressed on the tumor cells. We have shown that the stroma targeted 4-1BB aptamers exhibited a superior therapeutic index compared to an agonistic 4-1BB antibody, and engendered potent antitumor immunity against multiple unrelated tumors in subcutaneous, post surgical metastasis, and carcinogen-induced tumor models. We are currently developing increasingly potent stroma targeted platforms using multi-valent dendrimers and are exploring the use of peptides as alternative targeting ligands. In addition we are developing stroma targeted strategies with stimulatory CD27 and OX40 aptamers and blocking PD-1 aptamers. Citation Format: Brett Schrand, Alexey Berezhnoy, Randall Brenneman, Anthony Williams, Agata Levay, Ling-Yuan Kong, Ganesh Rao, Shouhao Zhou, Amy Heimberger, Eli Gilboa. Targeting 4-1BB costimulation to the tumor stroma with bispecific aptamer conjugates enhances the therapeutic index of tumor immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A88.