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. 2022 Mar;10(3):e003532.
doi: 10.1136/jitc-2021-003532.

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Javier Glez-Vaz #  1   2 , Arantza Azpilikueta #  1   2 , Irene Olivera  1   2 , Assunta Cirella  1   2   3 , Alvaro Teijeira  1   2   4 , Maria C Ochoa  1   2   3   4 , Maite Alvarez  1   2   3   4 , Iñaki Eguren-Santamaria  1   2 , Carlos Luri-Rey  1   2 , Maria E Rodriguez-Ruiz  1   2   3   4 , Xinxin Nie  5 , Lieping Chen  5   6 , Sonia Guedan  7 , Miguel F Sanamed  1   2   3   4 , Jose Luis Perez Gracia  1   2   3   4 , Ignacio Melero  8   2   3   4
Affiliations

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Javier Glez-Vaz et al. J Immunother Cancer. 2022 Mar.

Abstract

Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic.

Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both 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.

Results: CD137 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.

Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.

Keywords: biomarkers; costimulatory and inhibitory T-cell receptors; immunologic; receptors; tumor.

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Conflict of interest statement

Competing interests: IM acknowledges grants from Roche, Alligator, Genmab, BMS, AstraZeneca, Pharmamar and Bioncotech, as well as consultancy fees from BMS, Roche, Genmab, Numab, Gossamer, Alligator, AstraZeneca and Pharmamar. MFS receives a grant from Roche.

Figures

Figure 1
Figure 1
Soluble and transmembrane CD137 are upregulated by CD137 costimulation. (A) Quantitative RT-PCR measurements of mRNAs encoding transmembrane and sCD137 in isolated human CD8 T cells from three different donors stimulated by anti-CD3ε mAb bound to the culture plate in conjunction or not with anti-CD137 or anti-CD28 as indicated. (B) Measurement of the sCD137 concentration in the 48 hours tissue culture supernatants. (C) Surface expression levels of CD137 on CD8 T lymphocytes stimulated as in part A. (D) Surface expression levels of CD25 and CD69 as well as intracellular staining levels of Ki67 and Granzyme B in experiments as in part B. (E) Microbeads were covalently cocoated with fixed concentrations of anti-CD3ε tagged with AlexaFluor647 and serially increasing concentrations of anti-CD137 mAb tagged with AlexaFluor488 (0.5–8 µg of mAb per 50 µL of beads). Flow cytometry was used to show levels of microbead coating by relative fluorescence intensity units. F shows stimulation of CD8 T cells isolated from four healthy donors cocultured with the microbeads as indicated measuring sCD137 in culture supernatants or the intensity of surface expression of CD137, as well as other markers of T cell activation as indicated. Results were assessed at 72 hours of culture. G shows a time-course of these parameters in the cultures using the beads coated with the highest content of anti-CD137. Bars indicate mean±SEM. Experiments were performed with four independent donors. Statistical significance was assessed by Student’s paired t-test in parts A, B, C and D, and Wilcoxon test in part D (CD25 MFI). *p<0.05, **p<0.01. MFI, mean fluorescence intensity; ns, not significant.
Figure 2
Figure 2
CD137 transcription and protein expression are costimulated by CD137 on mouse CD8 T cells. (A) Mouse CD8 splenocytes were cultured with plate-bound anti-CD3ε mAb in conjunction or not with anti-CD28 or anti-CD137 mAbs and the levels of soluble and transmembrane-encoding mRNAs were measured by quantitative RT-PCR. (B) sCD137 concentrations in the tissue culture supernatants and (C) surface expression of CD137 on the corresponding T cells. (D) As a control of the cell costimulation, the immunostaining levels of CD25, CD69 as well as the % of positive cells for Ki67 and PD1 assessed by immunostaining and flow cytometry are provided. Bars indicate mean±SEM. Statistical significance was assessed by Student’s standard and ratio paired t-test in parts A, B, C, and D, as appropriate. *p<0.05. These experiments were repeated three times. mAb, monoclonal antibodiy; MFI, mean fluorescence intensity; ns, not significant.
Figure 3
Figure 3
sCD137 increases in peripheral blood of tumor-bearing mice undergoing treatment with agonist anti-CD137 mAb. (A) Experimental treatment and blood sample collection in mice bearing MC38-derived tumors. (B) Tumor size follow-up and fractions of complete rejections attained in mice (n=13 per group) treated with anti-CD137 mAb or control antibody and the corresponding overall survival (C) are shown. (D) Plasma sCD137 concentrations (mean±SD) in sequential follow-up overtime measured by ELISA in the groups of mice treated as indicated. (E) Plotted statistical correlation of plasma sCD137 on day +9 since tumor cell inoculation and the size of the MC38-derived tumors on day +20. (F) Similar experimental design in Balb/c mice bearing syngeneic CT26 tumors. (G) Individual tumor size follow-up and fractions of mice that completely rejected their tumors. (H) Overall survival of such mice. (I) Plasma sCD137 concentrations (mean±SD) in sequential follow-up overtime of the mice treated in part F. Statistical significance was assessed by log-rank (Mantel-Cox) test in figure parts C and H, and two-way ANOVA in figure parts D and I. *p<0.05, **p<0.01, ****p<0.0001. ANOVA, analysis of variance; ns, not significant.
Figure 4
Figure 4
sCD137 is produced by human T cells transiently engrafted in immunodeficient mice. (A) Experimental setting implanting subcutaneous Matrigel plugs under the skin of Rag2−/− IL2Rγ−/− mice and subsequent excision/resolubilization of the plugs as well as serial plasma collection. (B) Human PBMCs were mixed with microbeads coated with mIgG1, anti-CD3+mIgG1 or anti-CD3+anti-CD137. (C) H&E staining of sections of the Matrigel plugs showing the lymphocytes and microbeads indicated in the rectangle area that is shown under higher power magnification. (D) Concentrations of sCD137 in plasma from individual mice and in the resolubilized Matrigel plugs (E). Bars indicate mean±SEM. Statistical significance was assessed by two-way ANOVA in D and Welch’s test in part E. *p<0.05, **p<0.01. ANOVA, analysis of variance; ns, not significant.
Figure 5
Figure 5
Circulating human sCD137 is induced both in hCD137 knock-in mice and in cancer patients treated with Urelumab. (A) Scheme of treatment and plasma collections from knock-in human CD137 mice in C57BL/6 background treated intratumorally with control mAb or Urelumab. (B) Sequential follow-up of circulating concentrations of human sCD137 in these mice intraperitoneally treated with Urelumab or control antibody. (C) Scheme of patients undergoing intratumoral injections on 8 mg of Urelumab inside accessible tumor lesions, as performed in the six first patients treated in the INTRUST clinical trial (NCT03792724). (D) sCD137 was determined in samples drawn on day +14 per protocol, showing increases over baseline of sCD137 that were measured by ELISA. (E) determinations sCD137 in the plasma of patients treated with ipilimumab+nivolumab determined baseline and on day +14 following treatment administration as assessed by luminex. Bars indicate mean±SEM. Statistical significance was assessed by two-way ANOVA in part B and Student’s paired t-test in parts D and E, as appropriate. *p<0.05. mAb, monoclonal antibody; ns, not significant.
Figure 6
Figure 6
CD137 encompassing CARs induce the release of sCD137 on activation. (A) Antimesothelin second-generation CARs encoding cytoplasmic sequences of either CD137 or CD28 were lentivirally transduced to activated human CD8 T lymphocytes. Such resulting T cell cultures could be activated by microbeads decorated with chemically linked recombinant mesothelin-Fc. (B) sCD137 concentration in the culture of the corresponding CAR T cells stimulated or not with mesothelin-coated microbeads 24 and 48 hours following of exposure to the beads. (C) CD137 surface expression in the corresponding lymphocyte cultures. (D) IFNγ concentrations in the supernatants. Individual results from three or four independent donors whose individual values are linked by dotted lines are shown and the color-coded bars indicate the value of the means. CAR, chimeric antigen receptor.
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