Trough levels of ipilimumab in serum as a potential biomarker of clinical outcomes for patients with advanced melanoma after treatment with ipilimumab

Yoshinobu Koguchi, Noriko Iwamoto, Takashi Shimada, Shu-Ching Chang, John Cha, Brendan D Curti, Walter J Urba, Brian D Piening, William L Redmond, Yoshinobu Koguchi, Noriko Iwamoto, Takashi Shimada, Shu-Ching Chang, John Cha, Brendan D Curti, Walter J Urba, Brian D Piening, William L Redmond

Abstract

Background: Immune checkpoint blockade (ICB) using anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of advanced cancer. However, ICB is effective for only a small fraction of patients, and biomarkers such as expression of PD-L1 in tumor or serum levels of CXCL11 have suboptimal sensitivity and specificity. Exposure-response (E-R) relationships have been observed with other therapeutic monoclonal antibodies. There are many factors influencing E-R relationships, yet several studies have shown that trough levels of anti-PD-1/PD-L1 correlated with clinical outcomes. However, the potential utility of anti-CTLA-4 levels as a biomarker remains unknown.

Methods: Serum was obtained at trough levels at weeks 7 and 12 (after doses 2 and 4) from patients with advanced melanoma who received ipilimumab alone (3 mg/kg every 3 weeks for four treatments) via an expanded access program (NCT00495066). We have successfully established a proteomics assay to measure the concentration of ipilimumab in serum using an liquid chromatography with tandem mass spectrometry-based nanosurface and molecular-orientation limited proteolysis (nSMOL) approach. Serum samples from 38 patients were assessed for trough levels of ipilimumab by the nSMOL assay.

Results: We found that trough levels of ipilimumab were higher in patients who developed immune-related adverse events but did not differ based on the presence or absence of disease progression. We found that patients with higher trough levels of ipilimumab had better overall survival when grouped based on ipilimumab trough levels. Trough levels of ipilimumab were inversely associated with pretreatment serum levels of CXCL11, a predictive biomarker we previously identified, and soluble CD25 (sCD25), a prognostic biomarker for advanced melanoma, as well as C reactive protein (CRP) and interleukin (IL)-6 levels at week 7.

Conclusions: Our results suggest that trough levels of ipilimumab may be a useful biomarker for the long-term survival of patients with advanced melanoma treated with ipilimumab. The association of ipilimumab trough levels with pretreatment serum levels of CXCL11 and sCD25 is suggestive of a baseline-driven E-R relationship, and the association of ipilimumab trough levels with on-treatment levels of CRP and IL-6 is suggestive of response-driven E-R relationship. Our findings highlight the potential utility of trough levels of ipilimumab as a biomarker.

Trial registration number: NCT00495066.

Keywords: CTLA-4 antigen; biomarkers; immunotherapy; melanoma; translational medical research; tumor.

Conflict of interest statement

Competing interests: YK: research support from Bristol Myers Squibb (BMS), GlaxoSmithKline, and Shimadzu. NI and TS: employees of Shimadzu Scientific Instruments. BDC: research support from AstraZeneca, Clinigen, and Galectin Therapeutics; patents/royalties: none; advisory boards: Clinigen, Nektar, and Merck. WJU: research support from BMS; royalties: data safety monitoring board for AstraZeneca. BDP: research support from Heat Biologics and Shimadzu; advisory boards: Bayer and UnitedHealthcare. WLR: research support from Galectin Therapeutics, BMS, GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr; patents/licensing fees: Galectin Therapeutics; advisory boards: Nektar Therapeutics and Vesselon.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1
Figure 1
Establishment of nSMOL assay for ipilimumab detection. (A) ClustalW sequence alignment of ipilimumab, nivolumab, avelumab, and ramucirumab. The black area indicates the common sequence for all antibodies. The red line describes the selected signature peptide for ipilimumab quantification. The peptides marked with blue and green lines did not meet the validation criteria. (B) Calibration curve of ipilimumab-spiked serum analyzed by nSMOL method. (C) MRM chromatograms of the signature ipilimumab peptide TGWLGPFDYWGQGTLVTVSSASTK. The peptide peak was observed at a retention time at 6.03 min (red arrow). Representative chromatograms of 853.5→780.4 transition from non-spiked (left) and ipilimumab-spiked (right) serum are shown. MRM, multiple reaction monitoring; nSMOL, nanosurface and molecular-orientation limited proteolysis.
Figure 2
Figure 2
Trough levels of ipilimumab were associated with irAE status but not with clinical benefit. (A) The trough levels at weeks 7 and 12 of ipilimumab in individual patients were compared. (B) The trough levels of ipilimumab at week 7 were compared between patients with or without PD (n=27 and n=10, respectively). (C) The trough levels of ipilimumab at week 12 were compared between patients with or without PD (n=22 and n=10, respectively). (D) The trough levels of ipilimumab at week 7 were compared between patients who did not develop irAE (no irAE) and those who developed irAE G1–4 (n=10 and n=27, respectively). (E) The trough levels of ipilimumab at week 12 were compared between patients who did not develop irAE (no irAE) and those who developed irAE G1–4 (n=8 and n=24, respectively). CR, complete response; ipi, ipilimumab; irAE, immune-related adverse event; irAE G1–4, grades 1–4 irAE; PD, disease progression; PR, partial response; SD, stable disease.
Figure 3
Figure 3
Patients with lower serum trough levels of ipilimumab had worse OS. Curves for OS obtained by applying a median value of ipilimumab trough levels as cut points for week 7 (A) and week 12 (B). The survival curves are significantly different by the log-rank test (A, p=0.0021; B, p=0.0177). ipi, ipilimumab; OS, overall survival.
Figure 4
Figure 4
Relationship between trough levels of ipilimumab and biomarkers of clinical outcomes and inflammation. The relationship between pre-treatment levels of CXCL11 (A), sCD25 (B), or LDH (C) and the trough levels of ipilimumab at week seven was shown. The relationship between levels of CRP (D) or IL-6 (E) at week seven and the trough levels of ipilimumab at week seven are shown. CRP, C reactive protein; ipi, ipilimumab; IL, interleukin; pre-tx, pre-treatment; sCD25, soluble CD25.

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