Costimulatory Receptor Research Articles

TNFR2 Costimulation Differentially Impacts Regulatory and Conventional CD4+ T-Cell Metabolism.

CD4+ conventional T cells (Tconvs) mediate adaptive immune responses, whereas regulatory T cells (Tregs) suppress those responses to safeguard the body from autoimmunity and inflammatory diseases. The opposing activities of Tconvs and Tregs depend on the stage of the immune response and their environment, with an orchestrating role for cytokine- and costimulatory receptors. Nutrient availability also impacts T-cell functionality via metabolic and biosynthetic processes that are largely unexplored. Many data argue that costimulation by Tumor Necrosis Factor Receptor 2 (TNFR2) favors support of Treg over Tconv responses and therefore TNFR2 is a key clinical target. Here, we review the pertinent literature on this topic and highlight the newly identified role of TNFR2 as a metabolic regulator for thymus-derived (t)Tregs. We present novel transcriptomic and metabolomic data that show the differential impact of TNFR2 on Tconv and tTreg gene expression and reveal distinct metabolic impact on both cell types.

Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+ T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+ T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL’s bioactivity.

The Role of Cd28 Costimulatory Receptors in Signalling Pathways

Naive T cells require extracellular stimulus signals through the T-cell receptor cascade (TCR). This TCR then recognized the antigens present on the molecules of the major histocompatibility complex (MHC) using the CD4 and CD8 receptor. Upon recognition, TCR- induced signal transduction propagates signals through different molecules and induced secondary signalling. Consequently, intracellular signaling cascades determine the characteristics of immune responses mediated by T- cells. Consequently, rigorous regulation of T-cell activation is crucial for T-cell homeostasis and appropriate immune responses. Dysregulation of TCR signalling can result in energy or autoimmunity. In this review article, we summarize current knowledge on the signalling pathways that govern how the TCR complex transmits signals into cells and the roles of effector molecules that are involved in these pathways.

Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization.

ΔfosB is an alternatively spliced product of the FosB gene that is essential for dopamine-induced reward pathways and that acts as a master switch for addiction. However, the molecular mechanisms of its generation and regulation by dopamine signaling are unknown. Here, we report that dopamine D1 receptor signaling synergizes with the activin/ALK4/Smad3 pathway to potentiate the generation of ΔFosB mRNA in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) via activation of the RNA-binding protein PCBP1, a regulator of mRNA splicing. Concurrent activation of PCBP1 and Smad3 by D1 and ALK4 signaling induced their interaction, nuclear translocation, and binding to sequences in exon-4 and intron-4 of FosB mRNA. Ablation of either ALK4 or PCBP1 in MSNs impaired ΔFosB mRNA induction and nuclear translocation of ΔFosB protein in response to repeated co-stimulation of D1 and ALK4 receptors. Finally, ALK4 is required in NAc MSNs of adult mice for behavioral sensitization to cocaine. These findings uncover an unexpected mechanism for ΔFosB generation and drug-induced sensitization through convergent dopamine and ALK4 signaling.

Lenalidomide bypasses CD28 co-stimulation to reinstate PD-1 immunotherapy by activating Notch signaling

Programmed cell death protein 1 (PD-1) checkpoint blockade therapy requires the CD28 co-stimulatory receptor for CD8+ Tcell expansion and cytotoxicity. However, CD28 expression is frequently lost in exhausted Tcells and during immune senescence, limiting the clinical benefits of PD-1 immunotherapy in individuals with cancer. Here, using a cereblon knockin mouse model that regains invivo Tcell response to lenalidomide, an immunomodulatory imide drug, we show that lenalidomide reinstates the anti-tumor activity of CD28-deficient CD8+ Tcells after PD-1 blockade. Lenalidomide redirects the CRL4Crbn ubiquitin ligase to degrade Ikzf1 and Ikzf3 in Tcells and unleashes paracrine interleukin-2 (IL-2) and intracellular Notch signaling, which collectively bypass the CD28 requirement for activation of intratumoral CD8+ Tcells and inhibition of tumor growth by PD-1 blockade. Our results suggest that PD-1 immunotherapy can benefit from a lenalidomide combination when treating solid tumors infiltrated with abundant CD28- Tcells.

ICOS-Positive Regulatory T Cells in Hepatocellular Carcinoma: The Perspective from Digital Pathology Analysis

Introduction: Inducible co-stimulator (ICOS), an important co-stimulatory receptor on effector T cells (Teffs), may also contribute to tumor growth due to its high expression on regulatory T cells (Tregs). This study explored the clinical significance of ICOS-expressing Tregs in hepatocellular carcinoma (HCC). Methods: Tumor tissues from HCC patients who received curative hepatectomy were obtained at a referral center. Dual immunohistochemistry was performed to evaluate the expression of ICOS and Foxp3. The cell densities and proximities between stained cells in regions of interest were measured by digital pathology and the associations with clinical outcome were analyzed. Results: A total of 142 patients (male:female = 112: 30, median age of 61.0 years) were enrolled. Among them, 87 (61.3%) had chronic hepatitis B virus infection and 33 (23.2%) had chronic hepatitis C infection. Low α-fetoprotein level (<20 ng/mL) and early-stage were significantly associated with improved overall survival (OS). The density of ICOS⁺Foxp3⁺ cells and the ratio of ICOS⁺Foxp3⁺/total Foxp3⁺ cells were significantly higher (p < 0.001) in the tumor center than in the peritumor area. Patients with a high density of ICOS⁺Foxp3⁺ cells or a high ratio of ICOS⁺Foxp3⁺/total Foxp3⁺ cells in the tumor center trended to have a shorter OS. A shorter distance between ICOS⁺Foxp3⁺ cells and ICOS⁺Foxp3⁻ cells (likely Teffs) in the tumor center was significantly associated with a shorter OS (p = 0.030), suggesting active immunosuppression of ICOS⁺ Tregs on ICOS⁺ Teffs. Conclusion: An increased abundance of ICOS⁺ Tregs in the tumor center in comparison to the peritumor area indicates a strong immunosuppressive tumor microenvironment of HCC. A high proportion of ICOS⁺Foxp3⁺ cells and a shorter distance between ICOS⁺ Tregs and other ICOS⁺ cells were associated with a poor OS, suggesting that depleting ICOS⁺ Tregs might provide clinical benefit for patients with HCC.

Abstract 1875: Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy

Abstract Programmed death 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T-cell immune function. Blocking the interaction of PD-1 with its ligand PD-L1 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 blockade has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies combining PD-1 checkpoint inhibitors with a broad range of clinically active treatments including those targeting other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays as existing methods rely heavily on primary immune cells and are highly variable. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibody targeting human PD-1/PD-L1, or bispecific molecules target PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to address preclinical study and assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), PD-1 and the second IC receptor, and an artificial antigen presenting cells (aAPCs). Particularly, the combination bioassays are able to measure the synergetic effect of PD-1 blockade with a second IC inhibitor receptor blockade or a costimulatory receptor activation. These reporter-based assays reflect the mechanisms of action for the therapeutic drugs of interest and are sensitive and quantitative. They are prequalified according to ICH guideline and demonstrated the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assay for product release and stability studies during immunotherapy drug development and manufacture. Citation Format: Jamison Grailer, Julia Gilden, Jun Wang, Denise Garvin, Pete Stecha, Jim Hartnett, Steven Edenson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy [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 1875.

Abstract SY19-02: A convergence of myeloid cells and T cells controls cancer immunity

Abstract Although the advent of checkpoint inhibitors has completely transformed both oncology practice and the way we think about the immune system in cancer, their underlying mechanisms of action are only now starting to become clear. Recently, there has been increasing interest in the combination of antibodies blocking two co-inhibitory receptors on T cells, PD-1 and TIGIT, with randomized phase II results in non-small cell lung cancer having yielded significant overall survival benefit. We have investigated the mechanistic rationale for this combination and found that the inhibition of both co-inhibitory receptors is required to most effectively re-activate the two co-stimulatory receptors controlled by PD-1 (CD28) and TIGIT (CD226). Indeed, both co-stimulatory receptors serve as clients of PD-1 which recruits cytosolic phosphatases (eg SHP2) to inactivate the ITAM motifs in both CD28 and CD226. TIGIT acts to inhibit CD226 function in T cells solely by outcompeting it for ligand (CD155/PVR) binding. Unexpectedly, preclinical studies have demonstrated that ligand blocking antibodies to TIGIT are most effective when they contain a functional Fc domain. This observation suggests that at some level, Fc receptor-bearing cells play a role in mediating the function of anti-TIGIT blockade. Although this may reflect a modulation of T regulatory cell or NK cell function, both of which are TIGIT-positive, there are several observations that implicate the involvement of myeloid cells, namely macrophages and dendritic cells. One such observation is the fact that in the case of PD-1 blockade, the most relevant pool of PD-L1 turns out to be expressed by dendritic cells: in mice, genetic elimination of PD-L1 expression by tumor cells or macrophages exerts little effect on tumor growth, whilst PD-L1 elimination from dendritic cells greatly enhances endogenous anti-tumor immunity (Oh et al., 2020, Nature Cancer). This result further suggests, as is now becoming more widely accepted, that PD-L1/PD-1 blockade primarily acts by enhancing the activation of tumor-specific T cells during antigen presentation by dendritic cells, rather than by the rejuvenation of exhausted intratumoral T cells. Given the client relationship between PD-1 and CD28/CD226, it seems reasonable that TIGIT blockade similarly works at the level of antigen-presenting myeloid cells, either dendritic cells or macrophages. In the case of TIGIT, the requirement for an intact Fc domain further implies that T cell-bound anti-TIGIT may work as an opsonin that concomitantly triggers Fcγ receptor signaling to alter the functional status of the myeloid compartment. Thus, not only is there a convergence between the PD-1 and TIGIT pathways, but also a convergence between T cells and myeloid cells that may be essential for the function of these regulatory pathways and for the therapeutic effect associated with their blockade. Citation Format: Ira Mellman. A convergence of myeloid cells and T cells controls cancer immunity [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 SY19-02.

Abstract 4214: LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation

Abstract Although increased TIGIT expression on TILs is associated with poor survival in patients with cancer, monotherapy TIGIT antibody blockade has not yet demonstrated significant clinical activity. PD-1 is commonly co-expressed on TILs, and PD-1 mediated SHP-2 signaling inhibits DNAM-1 (CD226), preventing PVR co-stimulation in the setting of TIGIT blockade. The interaction between PD-1 and CD226 explains why TIGIT blockade only translates to clinical benefit in the setting of co-blockade of PD-1/L1, however does not explain why co-blockade of TIGIT and PD-1/L1 does not provide benefit in advanced and PD-1/L1 resistant tumors. In advanced and PD-1/L1 resistant tumors, progressive downregulation of CD226 has been reported. Thus, we hypothesized that the lack of CD226 may underlie the lack of clinical responses to combined TIGIT/PD-1/L1 blockade in PD-1/L1 experienced tumors and we sought to identify alternative costimulatory receptors with high-expression. Analysis of TCGA, as well as single-cell RNASeq from TILs, identified HVEM and LTβR as two costimulatory receptors with higher expression in advanced cancers as compared to CD226. HVEM and LTβR directly activate CD8+ T, natural killer (NK), and myeloid cells when bound by their TNF superfamily ligand, known as LIGHT. LIGHT potentiates effector lymphocyte function through signaling that obviates the co-stimulatory role of DNAM-1. We demonstrated that when the extracellular domain (ECD) of LIGHT is combined with the ECD of TIGIT on a TIGIT-Fc-LIGHT bi-functional fusion protein, the simultaneous blockade of PVR, PVRL2, PVRL3, and Nectin-4 and immune co-stimulation by LIGHT increased CD8+ T and NK infiltration into tumors. This translated into tumor cell killing, regression of established tumors, and improved survival in preclinical models of checkpoint primary and acquired resistance. The translation of TIGIT-Fc-LIGHT activity was evaluated in cynomolgus macaques and was well tolerated at doses up to 40 mg/kg. A series of adaptive immune and proinflammatory cytokines, including IL-2, CCL2, CCL4, IL-10, CXCL10, and CCL17, were induced within two hours of TIGIT-Fc-LIGHT infusion. Additionally, receptor engagement led to the rapid margination of HVEM+CD8+ T cells from the periphery into secondary immune tissues. This network of cytokines and post-dose immune cell margination identified in the monkey, was identical to findings in murine models that ultimately translated into significant anti-tumor responses. Lastly, the combination of TIGIT-Fc-LIGHT with anti-PD(L)1 broadened anti-tumor activity of the checkpoint antibodies in aggressive CPI-resistant tumors. These results suggest that LIGHT could be the differentiator that extends the clinical activity of conventional CPIs into PD-L1 low or CPI acquired resistance tumors. Citation Format: Kyung Jin Yoo, Louis Gonzalez, Kellsey Johannes, Jayalakshmi Miriyala, Karen Lenz, Kristen Campbell, Suresh de Silva, Taylor H. Schreiber, George J. Fromm. LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte 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 4214.

Abstract 4228: Inhibition of B7-H3 by enoblituzumab elicits antitumor immune modulation in both innate and adaptive immunity

Abstract Introduction: B7-H3, a B7 family member, is widely expressed in multiple cancers and is associated with poor clinical prognosis. Enoblituzumab (also known as MGA271 or TJ271) is an investigational, Fc-optimized monoclonal antibody that targets B7-H3 and exhibits enhanced antibody-dependent cell-mediated killing of B7-H3 expressing cancer cells. Here we identified the immuno-modulatory function of TJ271 in different cell subsets and pathways as a new mechanism for cancer treatment. Experiments: The function of B7-H3 and TJ271 in regulating immune response was investigated in human PBMCs stimulated with anti-CD3 antibody with or without plate-coated B7-H3 in the presence or absence of TJ271. Gene expression profiles and immunophenotyping of PBMCs after treatments were evaluated by Nanostring nCounter platform and CyTOF mass cytometry, respectively. IFN-γ production from cell supernatant was measured by ELISA. In vitro tumor killing activity by TJ271 and urelumab, a 4-1BB agonist, was evaluated by co-culture of B7-H3 expressing tumor cells and PBMCs stimulated with anti-CD3. The tumor cell killing activity was evaluated by 7-AAD staining on the Incucyte platform. Results: Gene expression of cell lineage markers in antigen-presenting cells, including macrophages and neutrophils was induced, while those in natural killer (NK) cells was suppressed by B7-H3 stimulation, respectively. Furthermore, expression of activation markers and co-stimulatory receptors, including IFNγ, ULBP2, GITR, and 4-1BB, was down-regulated while expression of exhaustion markers in CD8+ T cells and NK cells, including PD-L1, PD-L2, and IDO1, and of genes associated with M2-like macrophages were up-regulated by B7-H3. All this B7-H3-induced differential gene expression pattern was reversed by TJ271 treatment. As revealed by CyTOF, TJ271 treatment abolished B7-H3-mediated increase in the frequency of M2-like monocytes (B7-H3+CD68+CD206+CD16lo or B7-H3-CD68+CD163+CD16+) and led to increased frequency of 4-1BB-expressing CD4, CD8, and NK cells. TJ271 exhibited strong tumor killing activity against ES-2, a B7-H3-expressing ovarian cancer cell line in vitro. Consistent with the TJ271-associated increased 4-1BB expression in T and NK cells, activation of 4-1BB by urelumab further enhanced TJ271-mediated tumor killing activity. Conclusion: Our data confirm the immunosuppressive function of B7H3 and demonstrate the immunoregulatory function of TJ271 as evidenced by the activation of cytolytic T cell and NK cells, reinvigoration of exhausted cells, and suppression of M2-like myeloid cells. These findings provide rationale for combination therapy with blockade of B7-H3 by TJ271 with other immunotherapies to achieve increased clinical efficacy against cancers. Citation Format: Yanni Zhang, Xuejun Liu, Xinyun Zhu, Zhen Sheng, Yan Liu, Zhengyi Wang, Taylor B. Guo. Inhibition of B7-H3 by enoblituzumab elicits antitumor immune modulation in both innate and adaptive immunity [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 4228.

Abstract CT122: A phase 2, multi-center, open-label study of cinrebafusp alfa (PRS-343) in combination with ramucirumab and paclitaxel in patients with HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma and in combination with tucatinib in patients with HER2 low gastric or gastroesophageal junction (GEJ) adenocarcinoma

Abstract Background: For patients (pts) with HER2-overexpressing metastatic gastric cancer, trastuzumab + chemotherapy +/- pembrolizumab is a standard first-line option but only provides an incremental overall survival (OS) benefit vs chemotherapy. Anticalin® proteins are recombinant human proteins based on lipocalins. Cinrebafusp alfa, a first-in-class bispecific antibody-Anticalin fusion protein, targets HER2 and the co-stimulatory immune receptor 4-1BB on T cells. In a previous Phase 1 study cinrebafusp alfa monotherapy was generally well tolerated and showed deep and durable responses in patients with HER2-positive gastrointestinal malignancies at doses of 8mg/kg Q2W and 18mg/kg Q2W. Significant induction of plasma 4-1BB as well as increase of CD8+ cells was observed in on-treatment tumor biopsies at active dose levels (Piha-Paul, SITC 2020). Based on pharmacokinetics (PK), pharmacodynamics (PD) and clinical efficacy data, a phase 2 dose of 18mg/kg Q2W in C1 followed by 8mg/kg Q2W maintenance was chosen. Methods: This is a global, open-label, multicenter, two-arm phase 2 trial of cinrebafusp alfa in patients with metastatic gastric or gastroesophageal junction cancer. Arm 1 is enrolling patients with HER2 high (Immunohistochemistry (ICH) 3+ or IHC 2+ with HER2/neu gene amplification) disease. Pts who have received one prior treatment regimen for metastatic disease, including HER2-directed therapy such as trastuzumab are eligible. Pts will receive cinrebafusp alfa in combination with ramucirumab and paclitaxel. Arm 2 is enrolling patients with HER2 low (IHC 1+ or 2+ without HER2/neu gene amplification) disease. Pts who have received at least one prior treatment regimen for metastatic disease are eligible. Pts will receive cinrebafusp alfa in combination with tucatinib. After a run-in consisting of 3 pts in each arm, an additional 17 patients will be enrolled in each arm. For Arm 1, an additional 40 patients may be enrolled after a futility analysis has been conducted. Treatment will continue until disease progression, unacceptable toxicity, or consent withdrawal. Primary endpoint is confirmed overall response rate per RECIST 1.1 and key secondary endpoints are duration of response, progression free survival, overall survival, safety, PK, and immunogenicity. Recruitment is ongoing. Approximately 10 sites in 3 countries in US, Asia and Europe are expected to participate. Citation Format: Sarina A. Piha-Paul, Mukul Gupta, Do-Youn Oh, Yeul Hong Kim, Sun Young Rha, Yoon-Koo Kang, Marc Diez Garcia, Tania Fleitas Kanonnikoff, Virginia Arrazubi Arrula, Kayti Aviano, Tim Demuth, Geoffrey Ku. A phase 2, multi-center, open-label study of cinrebafusp alfa (PRS-343) in combination with ramucirumab and paclitaxel in patients with HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma and in combination with tucatinib in patients with HER2 low gastric or gastroesophageal junction (GEJ) adenocarcinoma [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 CT122.

Abstract 2076: Opal is a conditional 4-1BB agonistic fusion protein comprising trimerized 4-1BB ligand and a high affinity variant of the extracellular domain of PD-1

Abstract 4-1BB is a co-stimulatory TNF receptor family member upregulated on activated T cells, where it supports proliferation and enhanced functionality. 4-1BB agonizing monoclonal antibodies have thus far failed to progress beyond early clinical development, either due to hepatic toxicities caused by FcγR-crosslinking (urelumab) or due to low clinical activity (utomilumab). An attractive next-generation approach to 4-1BB agonistic therapeutics are multi-functional biologics that conditionally activate 4-1BB only in the tumor microenvironment, thereby localizing activity and minimizing toxicity. Furthermore, there is a strong rationale to combine 4-1BB agonism with checkpoint inhibition. Opal is a novel multi-specific construct that, in a single molecule, combines (i) a single-chain, trimeric 4-1BB ligand (sc4-1BBL); (ii) an engineered variant of the PD1 extracellular domain (ECD) with affinity-enhancing mutations (mutPD1); and (iii) a serum half-life extension domain. By fusing sc4-1BBL and mutPD1, 4-1BB agonism became conditional on the presence of PDL1 or PDL2. By using the mutPD1 ECD rather than an anti-PDL1 antibody, the specificity of Opal is expanded to PDL2-expressing cancer cells with low to undetectable levels of PDL1. The use of trimeric 4-1BBL is expected to optimally agonize the 4-1BB receptor by engaging its natural binding site. Opal omits a functional Fc gamma domain to avoid the hepatic toxicities associated with urelumab. Opal properties were characterized using biophysical and cellular assays. We showed by surface plasmon resonance spectroscopy that compared to wildtype (wt) PD1, mutPD1 had affinities for PDL1 and PDL2 that were enhanced by &amp;gt;2000-fold and &amp;gt;200-fold, respectively. In a competition ELISA assay, Opal effectively blocked wt PD1 from binding to PDL1 and PDL2. PDL1/2-conditional activation of 4-1BB was shown in two independent assays. First, using 4-1BB reporter cells, 4-1BB activity was enhanced &amp;gt;5x when Opal was pre-complexed with recombinant PDL1-Fc. Second, when activated PBMCs were co-cultured in the presence of HEK293T-PDL1 or -PDL2 cells with serial dilutions of Opal, induction of IFNγ secretion was detectable only in the presence of PDL1 or PDL2. Importantly, variants of Opal with abrogated PD1 or 4-1BBL domains failed to trigger IFNγ production. To evaluate the efficacy and toxicity of Opal in vivo, MC38 tumor cells expressing human PDL1 were inoculated in the flanks of mice expressing human 4-1BB. When tumor-bearing mice were treated IP with Opal at 1 mg/kg, 7/10 mice exhibited durable cures without significant body weight loss. Taken together, the preclinical data suggests that Opal may exhibit single-agent activity in solid tumors by conditionally activating effector cells in the tumor microenvironment via 4-1BB agonism while concurrently blocking the PD1/PDL1/PDL2 axis. Citation Format: Naveen K. Mehta, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson. Opal is a conditional 4-1BB agonistic fusion protein comprising trimerized 4-1BB ligand and a high affinity variant of the extracellular domain of PD-1 [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 2076.

Abstract 619: Functional evaluation of immuno-oncology drug candidates in customized and fine-tuned T cell bioassays to assess therapeutic potential

Abstract Re-activation of tumor-reactive T cells with so-called immune checkpoint inhibitors (ICIs) has translated into significant clinical breakthroughs. Specifically, antibodies directed against CTLA-4 and PD-L1/PD-1 have yielded long-term remission and cure in many solid as well as hematological malignancies. However, as not all cancer types respond to current ICI therapies, the quest for novel strategies to re-enable anti-tumor immunity by blocking immune checkpoints or by activating prominent co-stimulatory receptors is crucial. The field of cancer immunology has developed rapidly over the past decades, with numerous studies adding up to the complexity of immune-related interactions taking place in the tumor microenvironment. Compounds or combinations of compounds designed to target traditional or novel pathways require early evaluation of effectiveness on in vitro assays using primary immune cells or representative mouse models. On top of classical T cell bioassays, expanding on current methods and the development novel strategies to assess the modulation of cancer-related immunological networks is a continuously important process. One classical approach is the functional evaluation of compounds in a mixed lymphocyte reaction (MLR). Here, the potency of drug candidates to promote physiological T cell responses can be evaluated. Antigen-specific immune responses can be further assessed in Cytomegalovirus (CMV) recall- or in Staphylococcal enterotoxin B (SEB) activation assays. Additionally, it might also be beneficial to generate tumor-associated antigen-specific T cell pools or clones to be used in functional assays. The classical MLR assay could be expanded on by e.g., inclusion of specific tumor cell populations or T regulatory (Treg) cells, as evaluating reduced cancer cell viability, or reversing loss of immune effector cell functionality can be of interest. Furthermore, real-time evaluation of cancer cell killing by T cells upon addition of drug candidates could also provide pivotal information on the compound’s functional dynamics. With our deepening understanding of cancer-related immunological networks it becomes clear that different drug candidates may require customized and fine-tuned T cell bioassays to assess their full therapeutic potential. Moreover, quality controlled primary immune cells are an essential factor for the robustness of these assays and to yield reproducible and consistent results. Collectively, taking in mind these aspects will ease decision making and accelerate the drug discovery pipeline. Citation Format: Thibaut J. Janss, Martijn Vlaming, Simon Lefevre, Johan Arnold, Ellen Boelen, Sofie Pattijn. Functional evaluation of immuno-oncology drug candidates in customized and fine-tuned T cell bioassays to assess therapeutic potential [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 619.

Abstract 1139: A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) exhibits the optimal efficacy and superior safety profile through tumor-directed 4-1BB agonism

Abstract Although HER2-targeted therapies such as Herceptin® (trastuzumab) have dramatically improved outcomes for solid cancer patients with HER2 overexpression, it remains a challenge to cure the HER2 positive cancer patients with resistance to current HER2-targeted therapies. Therefore, new approaches need to treat them. One approach would be to combine with immunotherapy. 4-1BB (CD137) is a key costimulatory receptor expressed on activated T-cells and natural killer (NK) cells and a promising therapeutic target in cancer. In this study, we present the results from a preclinical study on the efficacy and safety of HER2/4-1BB bispecific antibody, YH32367 (ABL105). YH32367 has been designed to overcome the challenges with HER2 resistance via tumor-directed 4-1BB agonism. Its activity was determined using cell-based 4-1BB bioassay and co-culture assay with human peripheral blood mononuclear cells (hPBMC). In vivo anti-tumor efficacy of YH32367 was assessed in both HER2-expressing tumor-bearing hPBMC humanized mice and h4-1BB knock-in mice. In addition, 4-week repeated dose toxicity study of YH32367 was conducted in cynomolgus monkeys at the dose of 10, 30 and 100 mg/kg. YH32367 stimulated IFN-γ secretion and thereby eliciting tumor cell lysis in co-culture assay with hPBMC and HER2-expressing tumor cells. YH32367 also showed a long-term tumor immunity as well as superior efficacy on tumor eradication in MC38/hHER2-bearing h4-1BB knock-in mouse models, compared to the equimolar dosing of trastuzumab and benchmark 4-1BB agonistic antibody. Even a single dosing of YH32367 at the doses of 1, 3 and 10 mg/kg elicited complete tumor regression in more than 80% of mice bearing MC38/hHER2. In GLP 4-week toxicity study in monkeys, there were no YH32367-related effects on mortality, body weight, hematology, clinical chemistry, urinalysis, microscopic observations, cardiovascular system and immunotyping/cytokine analysis in any doses, indicating a favorable safety profile. In conclusion, YH32367 exhibited potent in vitro and vivo effects via tumor immunity and it was well tolerated and safe in the 4-week toxicity study. These results suggest that YH32367 could be a promising therapeutic for HER2-positive cancer patients, especially with HER2 drug resistance as an effective HER2-targeted therapy. Phase I clinical study is expected to begin in 2022. Citation Format: Eunjung Lee, Hyejin Chung, Yangsoon Lee, Eun-jung Lee, Young Bong Park, Ju Young Park, Sujin Ahn, Junhwan Kim, Kyoung Kyu Ahn, Kyeongsu Park, Wonjun Son, Donghoon Yeom, Jaeho Jung, Jonghwa Won, Se-Woong Oh. A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) exhibits the optimal efficacy and superior safety profile through tumor-directed 4-1BB agonism [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 1139.

Abstract 6268: Discovery and characterization of the CD96 antibody GSK6097608, a high-affinity, antagonistic anti-CD96 antibody for cancer immunotherapy

Abstract The overall therapeutic benefit of blocking the first generation of immune checkpoint pathways (PD-1 and CTLA-4) has been demonstrated across multiple tumor types, yielding long term protection in some patients. However, most patients do not respond to these single-agent approaches. Thus, collaborative strategies that seek to engage novel pathways and cell types may provide therapeutic options for patients wherein pre-existing host and tumor microenvironment factors do not favor current immunotherapeutic agents or in tumors where adaptive resistance has occurred. In recent years, the CD226 (DNAX Accessory Molecule-1 [DNAM-1]) axis has emerged as a relevant regulatory node in for natural killer (NK) and T cells - particularly in the context of tumor immunology. Similar to the competitive interplay between CTLA-4/CD28 and B7 (CD80/86), inhibitory receptors within the axis (e.g., CD96 [TACTILE]) effectively compete with the co-stimulatory receptor CD226 for binding to shared ligands (e.g., CD155), thereby impairing the initiation and/or promotion of ongoing antitumor immune responses. Indeed, genetic or monoclonal antibody (mAb)-based co-inhibition of CD96 with other immune checkpoints has proven efficacious in several nonclinical tumor models. CD96 has been shown to impact both T cell and NK cell function, offering a level of versatility as a target for cancer immunotherapy. For example, in the setting of anti-PD-1 neoadjuvant treatment, significantly improved survival of pancreatic ductal adenocarcinoma (PDAC) tumor-bearing mice was observed following enhancement of NK cell activity by CD96 antibody treatment. Equally, pronounced effects on primary tumor growth following anti-PD-1, -TIGIT, and -CD96 mAb treatment in a colorectal carcinoma tumor model (CT26) was found to be dependent on CD8+ T cells. GSK6097608 is a clinical-stage fully-human immunoglobulin G1 (IgG1)κ mAb that targets the inhibitory immune receptor CD96. GSK6097608 was identified, in part, for its ability prevent and disrupt CD96:CD155 interactions, thereby promoting T and NK cell function. GSK6097608 demonstrated concentration-dependent rescue of human immune cell activity following exposure to plate-bound recombinant CD155 (cognate receptor/ligand); an effect that was improved with concomitant TIGIT blockade. Notably, functional activity was found to be dependent on intact Fc-FcγR co-engagement, as potentiation of primary human T and NK cell function was lost when GSK6097608 was grafted on an Fc-attenuated backbone. Here, we describe some of the biophysical and functional characteristics that support the rationale for clinical evaluation of GSK6097608. Citation Format: Yan Degenhardt, Jun Guan, Peter Morley, David Jones, Michael Conner, Stephen Eastman, Wei Wang, Andrew Sanderson, Anand Ravindran, Julie Krueger, Iris Roth, James Smothers, Jeremy D. Waight. Discovery and characterization of the CD96 antibody GSK6097608, a high-affinity, antagonistic anti-CD96 antibody for cancer immunotherapy [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 6268.

Abstract 4245: QL301, a PD-L1 dependent 4-1BB agonist with enhanced preclinical anti-tumor efficacy and minimal liver toxicity

Abstract 4-1BB (CD137, TNFRSF9) is a potent co-stimulatory receptor found on T and NK cells. Activation of 4-1BB requires receptor clustering, which is naturally mediated by the endogenous trimeric 4-1BB ligand. Cross-linking via agnostic monoclonal antibodies can also activate 4-1BB but has generally resulted in unwanted side effects, mainly liver toxicity. To address the shortcomings of 4-1BB agonists, we have developed QL301, a PD-L1 x 4-1BB bispecific antibody that conditionally activates 4-1BB only when concurrently engaged to PD-L1, an immune checkpoint mediator elevated in the immuno-suppressive tumor microenvironment. The IgG1 backbone of QL301 was mutated to eliminate Fc gamma receptor-dependent functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), thereby preventing depletion of effector immune cells and non-specific activation of 4-1BB through Fc gamma receptor binding. In addition, QL301 blocks the suppressive interaction of PD-1 with PD-L1. Therefore, through a combination of 4-1BB agonism and PD-L1 blockade, QL301 provides both a co-stimulatory signal and the release of a suppressive signal to reinvigorate anti-tumor immune response. In vitro, QL301 induced robust cytokine release and expansion of CD8+ T cells conditional on the presence of tumor or antigen presenting cells expressing PD-L1. In xenograft and transgenic mice models, QL301 had potent anti-tumor effect that was superior to PD-L1 monoclonal antibodies. Tumor regression correlated with the expansion of CD8+ T cells. In addition, QL301 potentiated tumor cell killing mediated by an EGFR targeted CD3 bispecific antibody, providing a basis for combination therapy. In non-human primates, QL301 was well tolerated up to 30 mg/kg. There was no chronic elevation of serum AST and ALT levels, and histopathology analysis showed minimal inflammation of liver tissue. A phase 1 clinical trial to evaluate the safety, tolerability, and early efficacy of QL301 is currently ongoing. Citation Format: Shenda Gu, Irene Tang, Shirley Mihardja, Wei Wei Prior, Hieu V. Tran, Allan Chan, Anna McClain, Aaron Kurtzman, Shihao Chen, Youguang Luo, Xiaoyan Kang, Xiaoran Wu, Qingmei Zheng, Guodong Jia. QL301, a PD-L1 dependent 4-1BB agonist with enhanced preclinical anti-tumor efficacy and minimal liver toxicity [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 4245.

Abstract 2877: CB307: A novel selective 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. As such, CD137 agonists have demonstrated promising pre-clinical anti-tumor effects, however their clinical utility has been limited due to dose-limiting hepatic toxicity. PSMA is highly upregulated on tumor cells and tumor neovasculature in prostate cancer and other solid tumors, including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 is a half-life extended Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). CB307 is trispecific with VH components selected for optimal binding to PSMA, CD137 and human serum albumin (HSA). We present here the non-clinical pharmacology and toxicology of CB307. Methods: CD137 agonism and immune cell activation were evaluated in vitro using coculture assays with PSMA-expressing tumor cells as assessed by NFκB-driven luciferase and cytokine secretion. CB307 was evaluated as a single agent and in combination with enzalutamide, PD1 or PDL1 inhibitors. In vivo experiments used a syngeneic mouse system transgenic for the human CD137 immune cell target, and human PSMA expressed on mouse tumor cells. Nonclinical toxicology and pharmacokinetics of CB307 were assessed in cynomolgus macaques. Results: In reporter assays and PBMC cocultures, CB307 activated CD137 only in the presence of PSMA-expressing cells leading to enhanced immune cell activation. Enzalutamide pre-treatment of 22Rv1 (PSMA-low) cells induced a 1.5-fold increase in surface PSMA expression leading to enhanced CB307 activity. CB307 or PD1/PDL1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD1 or PDL1 inhibition. In an in vivo pharmacology study in immuno-competent mice, CB307 demonstrated statistically significant inhibition of tumor growth. CB307 was well-tolerated in a GLP-toxicology study in cynomolgus macaques, with an observed half-life supporting weekly clinical administration. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity in the presence of PSMA-positive cells. Our data demonstrates CB307 immunological pharmacology both in vitro and in vivo which has supported the initiation of the phase I ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Clare Song, Sophie Archer, Phillip D. Bartlett, Philip Bland-Ward. CB307: A novel selective CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [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 2877.

Abstract 3514: Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing

Abstract A primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex (MHC) based antigen presentation, which renders tumor cells invisible to αβ T cells, but not γδ T cells. Recently, a two-step model of γδ T cell activation has emerged, wherein one butyrophilin (BTN, i.e., BTN2A1) directly binds the γδ TCR but is only activated if certain molecular patterns (e.g., phosphoantigens) facilitate recruitment of a second BTN (i.e., BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the Vγ9Vδ2 T cells found in the peripheral blood in response to phosphoantigen build up in tumor cells. The unique mechanism of action and specificity of γδ TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific γδ T cell populations to tumor cells which lack expression of high affinity neoantigens or MHC class I molecules. In this study, we generated bispecific γδ T cell engagers (GADLEN) containing heterodimeric BTN2A1 and BTN3A1 extracellular domains (ECD) fused via inert Fc linkers to scFv domains targeting a tumor-antigen (CD19 or CD33) to test their ability to modulate Vγ9Vδ2 T cells and to promote tumor cell killing in co-culture assays. GADLEN induced proliferation, degranulation and cytokine production including IFNγ and TNFα in Vγ9Vδ2 T cells but requires co-stimulation of a natural cytotoxicity receptor (via anti-NKG2D) or T-cell co-stimulatory receptor (via anti-CD28), highlighting the parallels of signal 1 and signal 2 requirements of TCR activation in αβ- and γδ- T cells. In Vγ9Vδ2 T cells and tumor co-culture assays, the addition of GADLEN alone enhanced killing of CD19+ lymphoma cells such as Daudi and Raji, expressing known ligands for CD28 (e.g., CD80 and CD86). Similarly, GADLEN enhanced Vγ9Vδ2 T cell-mediated killing of K562 cells engineered to express CD19, which express MICA/B (ligands for NKG2D) on the cell surface. In addition, the CD33-targeted GADLEN alone was able to specifically kill acute myeloid leukemia cells expressing CD33 and costimulatory ligands (e.g., CD80 and CD86) in a mechanism similar to the CD19-targeted GADLEN construct. These results highlight the ability of GADLENs in promoting targeted killing of tumor cells by providing the “active” heterodimeric BTN2A1 and BTN3A1 that is critical to enhance cytotoxic killing by Vγ9Vδ2 T cells. Furthermore, this study provides mechanistic insights into BTN-mediated activation of Vγ9Vδ2 T cells and offers proof of concept for using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer. Citation Format: Anne Lai, Arpita Patel, Faraha Brewer, Kinsley Evans, Kellsey Johannes, Louis Gonzalez, Kyung Jin Yoo, George Fromm, Keith Wilson, Taylor H. Schreiber, Suresh de Silva. Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing [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 3514.

Abstract 4160: Promoting immunogenicity of synthetic long peptide vaccines based on in vivo IgG complex formation: Preclinical evaluation and clinical entry of the TET platform

Abstract Vaccines based on synthetic long peptides, targeting immunogenic region(s) of tumor associated proteins, are becoming increasingly used in oncology. Synthetic long peptides require endogenous processing by antigen presenting cells to be presented to T cells, thus reducing the risk of antigen presentation by non-professional antigen presenting cells lacking co-stimulation receptors. The formulation and delivery strategies require further development as peptides degrade rapidly in vivo. Herein we make use of a novel targeting and adjuvant strategy to deliver peptides to antigen presenting cells. Tumor-derived peptides are coupled to a B cell epitope element (the MTTE sequence, a tetanus toxin-derived universal B cell epitope) which induce the formation of peptide-conjugate -complex formation in vivo. This strategy, further named the TET technology, makes use of pre-existing antibodies to improve the bioavailability, stability, and immunogenicity of tumor-derived peptide therapeutics. The TET technology is here used in TENDU, a first-in-class prostate cancer vaccine candidate. TENDU incorporates selected immunogenic prostate cancer epitopes derived from PAP and PSMA. The peptides are designed to include proteasome processing elements along with a unique TAP translocation element. To assess the design, a conjugate with a known DR4 epitope embedded in a longer peptide was evaluated for T cell priming in HLA-DR4 animals and displayed T cell expansion in response to a prime/boost vaccination. To ensure that prostate cancer patients respond with antibodies to the MTTE sequence after a tetanus toxoid (TTd) vaccination, both healthy controls and patients (age and gender matched) were vaccinated using a TTd-based vaccine regime. Anti-MTTE antibody titers were evaluated. Patients and HCs responded with increased IgG1 type antibodies against MTTE after TTd vaccination. Safety with regards to hypersensitivity in response to the vaccine platform was evaluated in rabbits and a human whole blood loop system without any identified adverse events. Based on preclinical data the no-observed-adverse-effect level (NOAEL) was set to 1440µg/dose. The vaccine is currently assessed in a First-in-Human clinical study (TENDU-101, 3+3 study design) in patients with relapse after a primary radical prostatectomy. No safety concerns were noted at the lowest dose level and the 400 µg dose cohort has been initiated with the first patient dosed with no reported adverse events. The study’s primary endpoints are safety and tolerability. Secondary endpoints involve cellular immune responses, systemic PSA and PAP levels along with dose level selection. In conclusion, TENDU, a first-in-class vaccine candidate is under clinical development evaluating safety and tolerability for the TET vaccine platform technology. Citation Format: Erika Fletcher, Iliana Kerzeli, Robert Cordfunke, Aikaterini Nasi, Gunilla Törnqvist, Robert Valentijn, Frida Lindqvist, Inken Dillmann, Martin Lord, Neanke Bouwman, Jacques Neefjes, Natasja Dolezal, Kees Franken, Stephanie McArdle, Murrium Ahmad, Silvia Johansson, Ferry Ossendorp, Michael Haggman, Wolfgang Lilleby, Gustav Ullenhag, Sam Ladjevardi, Justyna Jarblad, Jan Wouter Drijfhout, Sara Mangsbo. Promoting immunogenicity of synthetic long peptide vaccines based on in vivo IgG complex formation: Preclinical evaluation and clinical entry of the TET platform [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 4160.

Optimal target saturation of ligand-blocking anti-GITR antibody IBI37G5 dictates FcγR-independent GITR agonism and antitumor activity

SummaryGlucocorticoid-induced tumor necrosis factor receptor (GITR) is a co-stimulatory receptor and an important target for cancer immunotherapy. We herein present a potent FcγR-independent GITR agonist IBI37G5 that can effectively activate effector T cells and synergize with anti-programmed death 1 (PD1) antibody to eradicate established tumors. IBI37G5 depends on both antibody bivalency and GITR homo-dimerization for efficient receptor cross-linking. Functional analyses reveal bell-shaped dose responses due to the unique 2:2 antibody-receptor stoichiometry required for GITR activation. Antibody self-competition is observed after concentration exceeded that of 100% receptor occupancy (RO), which leads to antibody monovalent binding and loss of activity. Retrospective pharmacokinetics/pharmacodynamics analysis demonstrates that the maximal efficacy is achieved at medium doses with drug exposure near saturating GITR occupancy during the dosing cycle. Finally, we propose an alternative dose-finding strategy that does not rely on the traditional maximal tolerated dose (MTD)-based paradigm but instead on utilizing the RO-function relations as biomarker to guide the clinical translation of GITR and similar co-stimulatory agonists.