CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

Otano, Itziar; Glez-Vaz, Javier; Medina-Echeverz, José; Cortés-Domínguez, Iván; Ortiz‐de‐Solórzano, Carlos; Ellmark, Peter; Fritzell, Sara; Hernández-Hoyos, Gabriela; Nelson, Michelle H.; Ochoa, María C.; Bolaños, Elixabet; Cuculescu, Doina; Jáuregui, Patricia; Sánchez-Gregorio, Sandra; Etxeberría, Iñaki; Rodríguez-Ruiz, María E.; Sanmamed, Miguel F.; Teijeira, Álvaro; Berraondo, Pedro; Melero, Ignacio

doi:10.1038/s41467-021-27613-w

Cited by 42 publications

(23 citation statements)

References 47 publications

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“…According to this hypothesis, the IFNγ released by PRS-344–/S095012-stimulated T cells can locally increase PD-L1 expression on tumor cells in trans and create a positive feedback loop, ultimately enhancing the activity of PRS-344/S095012 ( 46 ). At the same time, bridging of effector with target cells could stabilize the immunologic synapse, potentially enhancing activation and facilitating the killing of target cells ( 47 ). The latter hypothesis is supported by the data we generated in our T-cell killing experiments.…”

Section: Discussionmentioning

confidence: 99%

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

Peper-Gabriel

Pavlidou

Pattarini

et al. 2022

Clinical Cancer Research

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

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Section: Discussionmentioning

confidence: 99%

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

Peper-Gabriel

Pavlidou

Pattarini

et al. 2022

Clinical Cancer Research

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“…To quantitatively assess these phenomena, we covalently coated microbeads with a fixed amount of anti-CD3ε and graded amounts of anti-CD137 mAb that were fluorescently labeled in a manner that Flow Cytometry permitted to estimate the gradual coating of the beads ( figure 1E ). 37 Using such microbeads as stimulators for human CD8 T cells, we could observe a dose ( figure 1F ) and time dependent ( figure 1G ) induction of soluble and membrane bound CD137 in addition to other activation markers such as CD25, CD69 Ki67 and granzyme B. These results are further indicative of the value of CD137 to quantitatively estimate the intensity of CD137 costimulation.…”

Section: Resultsmentioning

confidence: 75%

“…Non-fluorescent irrelevant MOPC-1 mAb was added to adjust for the total amount of mAb in the coupling reaction. 37 For fluorescence-dye labeling of the antibodies, Alexa Fluor 488/647 Antibody Labeling Kits (ThermoFisher) were used according to manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Glez-Vaz

Olivera

Cirella

et al. 2022

J Immunother Cancer

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BackgroundOn the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic.MethodsWe used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored on treatment with agonist anti-CD137 monoclonal antibodies (mAbs). Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 mAb (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISA and Luminex. Flow cytometry was used to monitor CD137 surface expression.ResultsCD137 costimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 costimulation inside subcutaneously implanted Matrigel plugs. The CD137 signaling domain-containing CAR T cells readily released sCD137 and acquired CD137 surface expression on antigen recognition. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137.ConclusionsCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.

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“…In keeping with these advantageous properties of pCAR T-cells, administration of an anti-4-1BB antibody boosts anti-tumor activity of CD28-containing 2G CAR T-cells ( 26 ). Moreover, provision of either CD28 or 4-1BB co-stimulation to an activated T-cell in cis is markedly more potent than when either is provided in trans ( 27 ).…”

Section: Discussionmentioning

confidence: 99%

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

Halim

Das²,

Larcombe-Young

et al. 2022

Front. Immunol.

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Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.

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CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

Cited by 42 publications

References 47 publications

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

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

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

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