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Clinical/translational cancer immunotherapy
Original research
Toripalimab in combination with HBM4003, an anti-CTLA-4 heavy chain-only antibody, in advanced melanoma and other solid tumors: an open-label phase I trial
  1. Bixia Tang1,
  2. Yu Chen2,
  3. Yu Jiang3,
  4. Meiyu Fang4,
  5. Quanli Gao5,
  6. Xiubao Ren6,
  7. Li Yao7,
  8. Gang Huang8,
  9. Jing Chen9,
  10. Xiaoshi Zhang10,
  11. Rongqing Li11,
  12. Shuai Zhao12,
  13. Meijuan Gao12,
  14. Ruixuan Luo12,
  15. Meng Qi12,
  16. Feng Li12,
  17. Fei Zheng12,
  18. Michael Lee12,
  19. Xiaolu Tao12,
  20. Rong Duan1,
  21. Jun Guo1,
  22. Zhihong Chi1 and
  23. Chuanliang Cui1
  1. 1Peking University Cancer Hospital & Institute, Beijing, China
  2. 2Department of Medical Oncology, Fujian Medical University Cancer Hospital, Fuzhou, Fujian, China
  3. 3Department of Head and Neck Oncology, West China Hospital of Sichuan University, Chengdu, Sichuan Province, China
  4. 4Zhejiang Cancer Hospital, Hangzhou, Zhejiang, China
  5. 5Immunotherapy Department, Henan Cancer Hospital, Zhengzhou, Henan, China
  6. 6Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
  7. 7The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China
  8. 8Central South University (Hunan Cancer Hospital), Changsha, Hunan, China
  9. 9Union Hospital Tongji Medical College Huazhong University of Science and Technology, Wuhan, Hubei, China
  10. 10Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China
  11. 11Tumor Radiotherapy Department, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
  12. 12Harbour BioMed, Shanghai, China
  1. Correspondence to Dr Chuanliang Cui; 1008ccl{at}163.com

Abstract

Background HBM4003 is a novel anti-CTLA-4 heavy chain-only antibody, designed to enhance Treg ablation and antibody-dependent cell-mediated cytotoxicity while ensuring a manageable safety profile. This phase I trial investigated the safety, pharmacokinetics, immunogenicity and preliminary efficacy of HBM4003 plus with anti-PD-1 antibody toripalimab in patients with advanced solid tumors, especially focusing on melanoma.

Methods The multicenter, open-label phase I trial was divided into two parts: dose-escalation phase (part 1) and dose-expansion phase (part 2). In part 1, HBM4003 was administered at doses of 0.03, 0.1, 0.3 mg/kg in combination with toripalimab with fixed dosage of 240 mg every 3 weeks. The recommended phase II dose (RP2D) was used in the expansion phase. Primary endpoints were safety and RP2D in part 1 and objective response rate (ORR) in part 2. Biomarkers based on cytokines and multiplex immunofluorescence staining were explored.

Results A total of 40 patients received study treatment, including 36 patients treated with RP2D of HBM4003 0.3 mg/kg plus toripalimab 240 mg every 3 week. 36 participants (90.0%) experienced at least one treatment-related adverse event (TRAE), of which 10 (25.0%) patients experienced grade ≥3 TRAEs and 5 (12.5%) experienced immune-mediated adverse events (irAEs) with maximum severity of grade 3. No grade 4 or 5 irAEs occurred. Efficacy analysis set included 32 melanoma patients treated with RP2D and with available post-baseline imaging data. The ORRs of anti-PD-1/PD-L1 treatment-naïve subgroup and anti-PD-1/PD-L1 treatment-failed subgroup were 33.3% and 5.9%, respectively. In mucosal melanoma, the ORR of the two subgroups were 40.0% and 10.0%, respectively. Baseline high Treg/CD4+ratio in the tumor serves as an independent predictive factor for the efficacy of immunotherapy.

Conclusions HBM4003 0.3 mg/kg plus toripalimab 240 mg every 3 week demonstrated manageable safety in solid tumors and no new safety signal. Limited data demonstrated promising antitumor activity, especially in PD-1 treatment-naïve mucosal melanoma.

Trial registration number NCT04727164.

  • Pharmacokinetics - PK
  • Immunotherapy
  • Solid tumor

Data availability statement

All data relevant to the study are included in the article or uploaded as online supplemental information.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2024-009662

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Dual immunotherapy with anti-CTLA-4 and anti-PD-1 antibodies has been approved as a front-line treatment for several solid tumors, including melanoma, renal cell carcinoma, and non-small cell lung cancer. However, the high toxicity of anti-CTLA-4 antibody has limited its broader use. HBM4003 is a novel anti-CTLA-4 heavy chain-only antibody whose safety and antitumor efficacy in combination with anti-PD-1 antibodies require further exploration.

    WHAT THIS STUDY ADDS

    • HBM4003 at a dose of 0.3 mg/kg combined with toripalimab at 240 mg every 3 weeks has shown promising antitumor activity and manageable safety in patients with advanced melanoma, including the difficult-to-treat mucosal subtypes.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • Given the safety and initial efficacy of HBM4003 0.3 mg/kg combined with toripalimab 240 mg administered every 3 weeks in patients with advanced melanoma, further studies are required to confirm these findings in a broader melanoma patient population.

    Introduction

    The advent of immunotherapy has revolutionized the treatment strategy for solid tumors.1–3 Dual immunotherapy of anti-CTLA-4 antibody plus anti-PD-1 antibody has been approved in some types of solid tumors including melanoma, renal cell carcinoma and non-small cell lung cancer as front line,4–6 but the high toxicity of anti-CTLA-4 antibody limited the extensive application of the regimen. Although anti-CTLA-4 antibody Ipilimumab was restricted to four dosages, the treatment-related adverse events (TRAEs) of grade 3 or 4 occurred in 19%–28% of those with ipilimumab monotherapy, which was up to 56%–71% of those with dual immunotherapy.7–9

    HBM4003 is a new generation of anti-CTLA-4 recombinant fully human monoclonal heavy chain antibody. Each molecule of this antibody consists of one heavy chain variable region and two heavy chain constant regions (CH2 and CH3), with a lack of the CH1 domain. This unique structure allows HBM4003 to bind human CTLA-4 with high affinity, thereby improving the effect of Treg ablation with enhanced antibody-dependent cell-mediated cytotoxicity.10 Additionally, HBM4003 has a shorter serum half-life and less systemic drug exposure, which potentially provides an improved therapeutic window.10 In the human CTLA-4 chimeric knock-in (hCTLA-4 KI) mouse MC38 tumor-bearing model, HBM4003 shows optimal safety profiles and better preclinical in vivo efficacy than ipilimumab analogs,10 highlighting the potential benefits of using HBM4003 in combination with ICI regimens. Toripalimab is an anti-PD-1 monoclonal antibody, which has been approved in China and USA for melanoma and other tumors.11 12

    Here, we conducted this open-label, multicenter, phase I trial to assess the safety, pharmacokinetics (PK), immunogenicity, and preliminary efficacy of HBM4003 plus toripalimab in patients with advanced melanoma and other solid tumors. We also explored potential biomarkers for efficacy based on cytokines and multiplex immunofluorescence (mIF) staining.

    Methods

    Study design and participants

    This multicenter, open-label, phase I clinical trial consisted of two parts—dose-escalation phase (part 1) and dose-expansion phase (part 2). Patients were enrolled from nine centers in China.

    Eligible participants were 18 years or older with histopathologically confirmed advanced or recurrent solid tumors per American Joint Committee on Cancer eighth edition (melanoma of uveal origin was not included). In part 1, except for melanoma, patients with other solid tumors who failed at least first-line systemic standard treatment, or disease progression occurred within 6 months after adjuvant/neoadjuvant treatment were eligible. In part 2, melanoma patients were enrolled, of whom at least half had not received anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy in the advanced or metastatic stages (those who had received adjuvant/neoadjuvant treatment with anti-PD-1, anti-PD-L1 or anti-PD-L2 drugs and whose last treatment was more than 12 months prior to the first dose in this study were eligible). All participants were expected to survive for at least 3 months and had an Eastern Cooperative Oncology Group performance status score of ≤1. The detailed inclusion and exclusion criteria are presented in online supplemental material.

    Supplemental material

    Procedures

    A 21-day cycle treatment regimen was applied, and HBM4003 and toripalimab were administered on the first day of each treatment cycle.

    In part 1, toripalimab was administrated with a fixed dose of 240 mg while HBM4003 was administered at a starting dose of 0.03 mg/kg, which underwent accelerated titration. The subsequent participants underwent dose escalation or reduction in accordance with the i3+3 dose decision rule.13 The planned escalated doses of HBM4003 were 0.1, 0.3 and 0.6 mg/kg. The dose-limiting toxicity (DLT) was assessed for all treated participants during the first treatment cycle (21 consecutive days). In part 2, 30 melanoma participants were planned to be enrolled and treated with the recommended phase II dose (RP2D).

    All participants were allowed to receive study treatment for up to 2 years, until confirmed disease progression, loss of benefit from study treatment, intolerance to toxicity, or voluntary discontinuation, whichever occurred first. Follow-up visits would be performed within 28 days (±3 days) after the last dose to collect the PK, anti-drug antibody (ADA), safety, and biomarker data.

    Endpoints and assessments

    The primary endpoints of part 1 included the maximum tolerated dose (MTD) and RP2D of HBM4003 plus toripalimab. Secondary endpoints included the overall response rate (ORR), disease control rate (DCR), duration of response (DOR), duration of disease control (DDC), PK parameters of HMB4003 and immunogenicity of HBM4003 and toripalimab. The primary endpoint of part 2 was the ORR. The secondary endpoints included safety, DCR, DOR, DDC, overall survival (OS), progress-free survival (PFS), PK parameters of HMB4003 and immunogenicity of HBM4003 and toripalimab.

    During the treatment period, tumor assessments were performed by CT scan/MR every 6 weeks (±1 week) for the initial 24 weeks after the first dose of study treatment, after that for every 12 weeks (±2 weeks) until 1 year after the last dosing, or the start of a new antitumor treatment, or disease progression, whichever occurred first. Tumor assessment was performed by the investigators according to the RECIST v1.1 criteria. The ORR was estimated based on the proportion of participants who achieved complete response (CR) or partial response (PR). The DCR was defined as the proportion of participants who achieved CR, PR, or stable disease (SD) in the relevant analysis set. DOR was the time from initial response (CR or PR) to disease progression or death for any cause. DDC was the time from initial CR, PR or SD to disease progression or death for any cause. OS was the time from the first dose of study treatment to death for any cause. PFS was the time from the first dose of study treatment to disease progression or death for any cause.

    The safety evaluation included monitoring and recording of all DLTs in part 1, adverse events (AEs), serious AEs (SAEs); and changes in physical findings, vital signs, ECG, echocardiogram, laboratory test results and concomitant medications of all participants. AEs were graded according to the Common Terminology Criteria for Adverse Events version 5.0.

    The PK parameters of HBM4003 after single and multiple administrations were evaluated using non-compartmental analysis, which included the maximum concentration (Cmax), time to reach maximum concentration (Tmax), the area under the curve (AUC0-last, AUC0-tau, or AUC0-inf), half-life (t1/2), volume of distribution (Vz), and clearance (CL).

    Analysis of tumor tissue samples and blood-based biomarkers

    As an exploratory trial endpoint, pharmacodynamic effects and potential underlying immune mechanisms were investigated by profiling serial patient blood samples and tumor biopsies obtained before and during treatment. Formalin-fixed, paraffin-embedded tumor tissue samples underwent mIF profiling (Leica Bond RX). Stained slides were digitally scanned by Aperio Versa 8 scanner (Leica). Image analysis software was HALO. PD-L1 expression was evaluated by immunohistochemistry staining with the JS311 antibody which was generated by immunization of rabbits with the cytoplasmic domain of PD-L1 (Ventana Benchmark Ultra platform). Blood samples were analyzed using flow cytometry (BD FACS CantoTM II) and serum cytokine quantification (MSD V-PLEX proinflammatory panel 1(human) kit).

    Statistical analysis

    Safety analysis set included all participants who have received at least one dose of the study treatment. MTD Set included all participants from part 1 of the trial who could be fully evaluated at the time of determining the MTD during the first treatment cycle. Participants who were replaced during the MTD assessment period in part 1 of the trial are excluded from the MTD Set. RP2D melanoma set included all participants with melanoma in part 1 and part 2 who have been treated with the RP2D. Efficacy analysis set included melanoma participants treated with RP2D and with available post-baseline imaging data. PK set (PKS) includes participants who have at least one valid drug serum concentration data available. Immunogenicity set (IMGS) included participants who have at least one valid immunogenicity readout available.

    The target DLT probability at MTD was 25%, the equivalent interval of DLT probability was 20%–30%, and the maximum sample size for Part 1 was 31. After the completion of part 1, an isotonic regression was applied to select the MTD, and the RP2D was determined by consultation with the Scientific Review Committee (SRC). In part 2, the sample size was determined by estimating the likelihood of observing nine or more objective responses, corresponding to an ORR of 30% or greater. Given a sample size of 30, if the true underlying ORR is 33%, there is a 70.1% probability of achieving this outcome; if the true underlying ORR is 36%, the probability will increase to 80.8%. Conversely, if the true underlying ORR is only 20%, the probability of observing nine or more responses drops to less than 15%. Consequently, we planned to recruit 30 participants for part 2 of the study.

    Categorical variables were presented as count (n) and percents (%). Continuous variables were presented as the number of participants included in the summary, and means±SDs. ORR and DCR were estimated, and the corresponding Clopper-Pearson 95% CI was reported. Time-to-event data, such as DCR, DOR, PFS, and OS, were estimated using Kaplan-Meier curves.

    The PK parameters were calculated using Phoenix WinNonlin V.8.2 (Certara, Princeton, New Jersey, USA). All statistical analyses were performed by using SAS V.9.4 (SAS Institute).

    Patient and public involvement

    No patient was involved in the development of the research questions and outcome measures, study design or recruitment, and in the conduct of this study.

    Results

    Participants

    From March 9, 2021 to March 17, 2022, 56 patients were screened, of whom 40 (14 in part 1 and 26 in part 2) received at least one dose of study treatment, and were included in safety analysis set (figure 1). One participant (dose level: HBM4003 0.3 mg/kg plus toripalimab 240 mg every 3 weeks) in part 1 withdrew informed consent during the DLT evaluation period, thus MTD set included 13 participants. RP2D melanoma set included 34 participants and postbaseline imaging data were not available for two of them. Therefore, the efficacy analysis set included 32 participants (9 in part 1 and 23 in part 2). PKS and IMGS each included 40 participants.

    Figure 1
    Figure 1

    Study flow chart. IMGS, immunogenicity set; MTD, maximum tolerated dose; PKS, pharmacokinetic set; RP2D, recommended phase II dose.

    The baseline characteristics of all participants are shown in table 1. The mean age was 53.6±13.0 years, and 20 (50.0%) participants were female. One (2.5%), 2 (5.0%), and 37 (92.5%) participants had renal cell carcinoma, urothelial carcinoma and melanoma, respectively. The clinical stage was stage IV (n=37, 92.5%) or III (n=3, 7.5%). 15 (37.5%) participants had baseline PD-L1 expression ≥1%. 16 (40.0%) participants had not previously received systemic treatment, and 21 (52.5%) had received anti-PD-1/PD-L1 treatment.

    View this table:
    Table 1

    Patient demographics and baseline characteristics (n=40)

    RP2D of HBM4003 plus toripalimab

    In part 1, 1 participant received HBM4003 0.03 mg/kg plus toripalimab and another three received HBM4003 0.1 mg/kg plus toripalimab, and no DLT event occurred and no response had been observed in these four participants. Then, three participants were enrolled to receive HBM4003 0.3 mg/kg plus toripalimab, and one developed DLTs (grade 3 diarrhea and grade 2 immune-mediated enterocolitis). After judgment by the SRC, three additional participants were enrolled in this dose cohort to continue observing safety, among whom one patient withdrew informed consent during the DLT observation period and was replaced. Subsequently, the SRC decided to enroll three more participants to explore the safety of HBM4003 0.3 mg/kg combined with toripalimab dose, and this dose cohort included nine participants in the MTD set, all of whom completed the DLT assessment. Till SRC meeting, six patients underwent tumor assessment and one was evaluated as PR (tumor shrinkage by 37.2%), two as SD and three as progressive disease (PD). Therefore, RP2D was determined as HBM4003 0.3 mg/kg plus toripalimab 240 mg every 3 weeks.

    Safety

    The average treatment duration of HBM4003 and toripalimab was 120.2±161.3 days and 129.9±160.8 days, respectively; 14 (35.0%) and 17 (42.5%) participants received HBM4003 and toripalimab, respectively, for >4 treatment cycles, including 5 (12.5%) participants received >16 treatment cycles.

    A total of 36 (90.0%) participants experienced TRAEs, of which 10 (25.0%) participants experienced ≥grade 3 TRAEs. The commonly reported (incidence ≥10%) TRAEs were rash (30.0%), abnormal liver function (30.0%), leukopenia (25.0%), and fever (20.0%) (table 2). Immune-mediated AEs (irAEs) were reported in 22 (55.0%) participants, and 5 (12.5%) participants experienced irAEs with maximum severity of grade 3. The grade 3 irAEs were lymphocyte count decreased, rash, diarrhea, immune-mediated enterocolitis and neurotoxicity, and each was reported in one participant. No grade 4 or 5 irAEs were observed. HBM4003-related or toripalimab-related SAEs occurred in nine (22.5%) participants. irAEs occurred in ≥2 participants were rash (17.5%), diarrhea (15.0%), abnormal liver function test (12.5%), gastroenteritis (10.0%), fever (7.5%), thyroid hypofunction disorders (7.5%), vomiting (5.0%), lymphopenia (5.0%), thyroid function test abnormal (5.0%), decreased appetite (5.0%) and musculoskeletal pain (5.0%).

    View this table:
    Table 2

    Summarized safety profile for treated patients (n=40)

    Efficacy

    A total of 13 participants were included in part 1, with 1, 3 and 9 participants receiving HBM4003 0.03 mg/kg plus toripalimab 240 mg (group A), HBM4003 0.1 mg/kg plus toripalimab 240 mg (group B) and HBM4003 0.3 mg/kg plus toripalimab 240 mg (group C), respectively. In part 1, no participants in group A and group B achieved CR or PR (one case of PD in group A, two cases of PD and one case of SD in group B), and the nine participants in group C were included in the efficacy analysis set.

    In the efficacy analysis set, there were 32 participants with melanoma (9 in part 1 and 23 in part 2) who received RP2D treatment, including cohort A (N=15, anti-PD-1/PD-L1 treatment-naïve subgroup) and cohort B (N=17, anti-PD-1/PD-L1 treatment-failed subgroup). As of June 14, 2023, the median follow-up time was 12.4 months (range 3.4–22.3 months). The best changes in tumor volume from baseline are presented in figure 2A and the tumor responses of each patient are presented in figure 2B.

    Figure 2
    Figure 2

    Antitumor activity of HBM4003 combined with toripalimab. (A) Best percentage change from baseline in the sum of the longest diameters of target lesions in patients who had at least one assessable postbaseline tumor assessment (efficacy analysis set). (B) Swimmer plots of tumor responses in efficacy analysis set. PD, progressive disease; PR, partial response; SD, stable disease.

    In cohort A, the ORR was 33.3% (95% CI 26.6% to 78.7%; n=5); DCR was 73.3% (95% CI 44.9% to 92.2%; n=11); the median DOR was not reached (95% CI 4.1 to NR), and the median PFS and median OS were 4.0 (95% CI 1.4 to NR) months and NR (95% CI 10.0 to NR) months, respectively. In cohort B, the ORR was 5.9% (95% CI 0.1% to 28.7%; n=1); DCR was 29.4% (95% CI 10.3% to 56.0%; n=5); the median DOR was not reached (95% CI NR to NR) and the median PFS and median OS were 1.6 (95% CI 1.4 to 2.5) months and 13.6 (95% CI 7.5 to NR) months, respectively (table 3).

    View this table:
    Table 3

    Best response evaluation in efficacy analysis set

    The ORR in participants with cutaneous melanoma, acral melanoma, mucosal melanoma and unknown type of melanoma were 20% (95% CI 0.5% to 71.6%), 14.3% (95% CI 0.4% to 57.9%), 20.0% (95% CI 4.3% to 48.1%) and 20% (95% CI 0.5% to 71.6%), respectively. In the mucosal melanoma subgroup (n=15), 2 out of 5 anti-PD-1/PD-L1 treatment-naïve participants (40%, 95% CI 5.3% to 85.3%) achieved confirmed PR as best response. The DCR was 80.0% (95% CI 28.4% to 99.5%). The median DOR was not reached (95% CI NR to NR); the median PFS was 4.1 (95% CI 1.4 to NR) months; and the median OS was not reached (95% CI 10.0 months to NR). Of the 10 anti-PD-1/PD-L1 treatment-failed participants, 1 achieved PR (10%, 95% CI 0.3% to 44.5%) as the best response. The DCR was 30.0% (95% CI 6.7% to 65.2%); the median PFS and median OS were 1.5 (95% CI 1.2 to 2.5) months and 13.6 (95% CI 7.5 to NR) months, respectively (online supplemental table 1).

    Pharmacokinetics

    In the PK analysis of 40 participants, following a single intravenous infusion at 0.03, 0.1 and 0.3 mg/kg of HBM4003 plus toripalimab, the Cmax and AUC of HMB4003 increased generally in a dose-proportional manner (online supplemental table 2). The PK parameters of HBM4003 after multiple doses closely resemble those after the first dose. Based on the available data, no significant accumulation of HBM4003 was observed in serum after consecutive every 3 weeks intravenous infusions of HBM4003 in combination with toripalimab, and no significant impact of HB4003 on the exposure levels of toripalimab was observed.

    Immunogenicity

    Among 40 participants who had serum samples collected for immunogenicity analysis, two (5.0%) and six (15.0%) participants developed treatment-induced ADA against HBM4003 and toripalimab, respectively, with a negative ADA result at baseline but subsequent ADA development after treatment. Two participants (5.0%) developed treatment-enhanced ADA against toripalimab, defined as a baseline positive ADA result with a significant increase in detection signal (≥4 fold increase in titer compared with baseline). ADA-positive participants showed no significant differences in exposure levels of either HBM4003 or toripalimab compared with the other participants in the same group, suggesting no observed impacts of immunogenicity on the PK profiles of HBM4003 or toripalimab.

    Pharmacodynamic effects

    The longitudinal profiling of peripheral blood mononuclear cells revealed a relatively stable level of Treg (CD4+Foxp3+) cells in the circulation throughout HBM4003/toripalimab treatment (figure 3A). Significant increases in proliferating Ki-67+CD8+ and Ki-67+CD4+ T cells on-treatment were observed (figure 3B,C), indicating that HBM4003 plus toripalimab could effectively activate the participants’ T cells and induce T cells to proliferate in large numbers.

    Figure 3
    Figure 3

    Effect of HBM4003/toripalimab on circulating T-cells. Quantification of peripheral Treg cells (A), Ki67+CD4+ T cells (B), and Ki67+CD8+ (C) by flow cytometry. (D) Representative multiplex immunofluorescence of tumor immune microenvironment before and 6 weeks after HBM4003/toripalimab treatment.

    To evaluate circulating proteins known to associate with immune and inflammatory activities, an array of serum proteins was analyzed, the serum interferon (IFN)-γ level and TNF-α increased after administration in a dose and time-dependent manner (online supplemental figure 1), which was in line with the expectation of the immunomodulatory effect of HBM4003 combined with toripalimab. To evaluate the effect of HBM4003/toripalimab on tumor immune microenvironment, we assessed tumor samples collected at screening and 6 weeks after drug treatment using mIF. The spatial analysis revealed a remarkable increase in T-cells within the tumor, particularly a significant elevation in CD8+killer T-cells (figure 3D).

    Prognostic and predictive effects of baseline Treg cell infiltration

    In the efficacy analysis set, 25 participants with baseline archived/fresh tumor tissue samples were successfully profiled by mIF. The mean density of baseline tumor infiltrating Treg cells, total CD4+helper T cells and total CD8+cytotoxic T cells were 68.1, 668.9 and 193.1 cells/mm2, respectively. There was no significant difference in Treg, total CD4+or total CD8+T cell infiltration between participants with ORR and non-responders (figure 4A). Interestingly, the ratio of intratumoral Tregs to total CD4+T cell was significantly higher in participants who achieved PR (figure 4B). Survival analysis demonstrated that the participants with higher (above upper quartile) ratio of Treg infiltration/CD4+ T cells had substantially longer PFS than those with lower (below upper quartile) infiltration (median PFS, NR vs 1.64 months; HR, 0.17; 95% CI 0.07 to 0.43; p=0.004; figure 4C).

    Figure 4
    Figure 4

    T-cell infiltration in association with response to HBM4003/toripalimab. (A) Baseline tumor-infiltrating T-cell density in patients with ORR and non-responders. (B) Baseline intratumoral Treg/CD4+ratio in patients with ORR and non-responders. (C) Kaplan-Meier plots of PFS stratified by quartile Treg/CD4+ratio. ORR, overall response rate; PFS, progress-free survival.

    As our data suggested an association between the Treg/CD4+ratio and survival, we further evaluated its potential predictive and prognostic value in a multivariate analysis together with key clinical variables that have previously been implicated as prognostic. As illustrated in online supplemental table 3, participants with higher ratio of Treg infiltration/CD4+ T cells displayed a better PFS (HR 0.14; 95% CI 0.029 to 0.62; p=0.01) at univariate analyses. In multivariate analyses, high Treg/CD4+ratio remained as statistically independent factor for PFS (HR 0.13; 95% CI 0.025 to 0.71; p=0.018). Consistently, the multivariate analysis revealed that baseline Treg/CD4+ratio in the tumor serves as an independent predictive factor for the anti-CTLA-4 immunotherapeutic response.

    Discussion

    To our knowledge, this is the first phase I trial of anti-CTLA-4 heavy chain-only antibody plus anti-PD-1 antibody in advanced melanoma and other solid tumors. The results showed promising antitumor activity and manageable safety in patients with advanced melanoma at dose level of HBM4003 0.3 mg/kg plus toripalimab 240 mg every 3 weeks, which was decided as RP2D for further exploration. Potential was also observed in the mucosal melanoma subtypes.

    One of the 13 MTD evaluable patients in the dose-escalation phase developed a DLT event, which was immune-mediated enterocolitis manifesting as diarrhea, and no DLT was observed in other dose levels. The overall incidence of grade ≥3 TRAEs in the safety analysis set (n=40) was relatively low (25.0%), and 3 (7.5%) participants discontinued HBM4003 treatment due to TRAEs. The results of Checkmate 067 showed that the incidence of grade 3–4 TRAEs in patients who received ipilimumab combined with nivolumab (ipilimumab 3 mg/kg plus nivolumab 1 mg/kg every 3 weeks, followed by nivolumab 3 mg/kg every 2 weeks for cycle 3 and beyond) was 55.0%, and 36.4% discontinued treatment due to TRAEs.4 The most frequent immune-related TRAEs in our study were rash, and increased liver function tests, which were comparable with those observed in Checkmate 067.14 No new safety signals were identified compared with the available data obtained previously.4 15 16 Therefore, the combination of HBM4003 and toripalimab appears relatively safe compared with ipilimumab and nivolumab.

    This study showed that in melanoma patients who received the RP2D, the confirmed ORRs in the anti-PD-(L)1 treatment-naïve subgroup and prior anti-PD-(L)1 treatment-failed subgroup were 33.3% and 5.9%, respectively, indicating higher efficacy in anti-PD-1-naïve patients. The data were consistent with data from studies of ipilimumab combined with nivolumab in melanoma.8 17 18 In patients with untreated advanced melanoma, the ORR of ipilimumab combined with nivolumab was 58.9%,8 while the ORR of ipilimumab combined with PD-1 monoclonal antibody was about 28%–29% in melanoma patients who failed anti-PD-1/PD-L1 treatment.17 18 For other ICIs combination regimen, such as relatlimab (LAG-3-blocking antibody) plus nivolumab, the ORRs were 43.1% and 12.0% in patients with untreated melanoma and prior anti-PD-(L)1 treated melanoma, respectively.19 The results above highlight the need to further investigate the efficacy of HBM4003 plus toripalimab in patients with previously untreated melanoma.

    Response to immunotherapy varies substantially by the subtypes of melanoma. In a pooled analysis of 889 patients with nivolumab monotherapy treatment, median PFS was 3.0 months vs 6.2 months for mucosal and cutaneous melanoma, with ORR of 23.3% vs 40.9%.20 Similarly, 128 pretreated Chinese advanced melanoma patients received toripalimab, median PFS was 1.9 months vs 5.5 months for mucosal and cutaneous melanoma, with ORR of 0% vs 31%.21 In the efficacy analysis of this study, 46.7% (15/32) of the patients had mucosal melanoma, including five anti-PD-(L)1 treatment-naïve mucosal melanoma, with unconfirmed ORR of 60% (3/5) and confirmed ORR of 40% (2/5). The multicenter JMAC retrospective study conducted in Japan reported an ORR of 28.8% in 66 Japanese patients with previously untreated mucosal melanoma treated with nivolumab plus ipilimumab.22 Considering that mucosal melanomas have much poorer prognosis than other melanoma subtypes,23 the ORR of 40% in participants with untreated mucosal melanoma suggest the promising efficacy of HBM4003 combined with toripalimab in treating patients with previously untreated mucosal melanoma, which needs to be confirmed in studies of larger scale.

    Currently, studies identifying populations with potential benefits from dual immunotherapy targeting PD-L1 and CTLA-4 are limited. Our study showed an association between the Treg/CD4+ratio and survival. Treg cells with an activated phenotype are often enriched within tumors, and high levels of Tregs are typically associated with poorer prognosis in various cancer types, including melanoma.24–26 There are several promising predictive biomarkers identified for effectively identifying and stratifying melanoma patients who may benefit from anti-PD-(L)1 blockade, but there remains a gap in predictive biomarkers for anti-CTLA-4 therapy.27 28 Remarkably, we discovered that the baseline Treg/CD4+ratio within the tumor served as an independent predictive factor for the response to anti-CTLA-4 immunotherapy. This predictive capacity may stem from the higher proportion of Treg cells, which could prime the Treg-depleting effects of the HBM4003. Indeed, Treg cells exert an immunosuppressive effect through the activation of CTLA-4,29 and decreased Treg cells would reduce their immunosuppressive effects on effector T cells. Future studies should focus on validating this biomarker in larger, independent cohorts to establish its clinical utility.

    This study had some limitations. Although the number of participants was sufficient for a phase I trial according to the power analysis, the sample size was too small to obtain a definite affirmative safety and efficacy conclusion, which should be proved in large-scale and randomized trials. A relatively short follow-up period might miss potentially late-onset adverse effects, and several time-based efficacy and survival parameters were immature. Our study identified the baseline Treg/CD4+ratio within the tumor as a potential independent predictive factor for response to anti-CTLA-4 therapy. Although our data suggest a significant association between the Treg/CD4+ratio and therapeutic outcomes, it is important to note that these findings require validation in an independent cohort to confirm its predictive value.

    In conclusion, HBM4003 0.3 mg/kg plus toripalimab 240 mg every 3 weeks demonstrated promising antitumor activity and manageable safety in patients with advanced melanoma, including the difficult-to-treat mucosal subtypes. Additional studies are necessary to confirm the results of this combination therapy in patients with melanoma, especially mucosal melanoma.

    Supplemental material

    Data availability statement

    All data relevant to the study are included in the article or uploaded as online supplemental information.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and this phase 1 oncology study was approved by the Ethics Committee of Peking University Cancer Hospital and Institute (approval number 2020YW123). Participants gave informed consent to participate in the study before taking part.

    Acknowledgments

    We thank all the patients for their participation. Medical writing support was provided by Yebo He (Ph.D, Harbour BioMed).

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    Supplementary materials

    • Supplementary Data

      This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

    Footnotes

    • Contributors JG, CC, BT and XT had full access to all of the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. BT, SZ, MG, RLuo, MQ, FZ, ML, XT, RD, JG, ZC and CC were involved in the study concepts and design as well as drafting of the manuscript. All authors were involved in the acquisition, analysis and interpretation of data. Supervision: JG and XT. Statistical analysis: ML. All authors read, critically revised and approved the manuscript. The guarantor of the study was CC.

    • Funding This study was funded by Nona Biosciences (Suzhou) Co.

    • Disclaimer The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

    • Competing interests JG is a member of the advisory board/consultant of MSD, Roche, Pfizer, Bayer, Novartis, Simcere, Shanghai Junshi Bioscience, Oriengene.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.