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Anlotinib combined with TQB2450 in patients with platinum-resistant or -refractory ovarian cancer: A multi-center, single-arm, phase 1b trial

  • Author Footnotes
    9 These authors contributed equally
    Chun-Yan Lan
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    9 These authors contributed equally
    Affiliations
    Department of Gynecologic Oncology, Sun Yat-sen University Cancer Centre, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou 510060, China
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  • Author Footnotes
    9 These authors contributed equally
    Jing Zhao
    Footnotes
    9 These authors contributed equally
    Affiliations
    Department of Radiology, Sun Yat-sen University Cancer Centre, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou 510060, China
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  • Author Footnotes
    9 These authors contributed equally
    Fan Yang
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    9 These authors contributed equally
    Affiliations
    Department of Gynecologic Oncology, Sun Yat-sen University Cancer Centre, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou 510060, China
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    9 These authors contributed equally
    Ying Xiong
    Footnotes
    9 These authors contributed equally
    Affiliations
    Department of Gynecologic Oncology, Sun Yat-sen University Cancer Centre, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou 510060, China
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    9 These authors contributed equally
    Rong Li
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    9 These authors contributed equally
    Affiliations
    Gynecological Oncology Center, Chongqing University Cancer Hospital, Chongqing 40000, China
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  • Yu Huang
    Affiliations
    Gynecological Oncology Center, Chongqing University Cancer Hospital, Chongqing 40000, China
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  • Jing Wang
    Affiliations
    Gynecological Oncology Center, Chongqing University Cancer Hospital, Chongqing 40000, China
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  • Chang Liu
    Affiliations
    Gynecology Department, The First Hospital of Lanzhou University, Lanzhou 730030, China
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  • Xue-Han Bi
    Affiliations
    Gynecology Department, The First Hospital of Lanzhou University, Lanzhou 730030, China
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  • Hai-Hong Jin
    Affiliations
    Gynecology Department, First Hospital of Qinhuangdao, Qinhuangdao 066000, China
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  • Jin Meng
    Affiliations
    Gynecology Department, First Hospital of Qinhuangdao, Qinhuangdao 066000, China
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  • Wei-Hong Zhao
    Affiliations
    Medical Oncology, Chinese PLA General Hospital, Beijing 100000, China
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  • Li Zhang
    Affiliations
    Medical Oncology, Chinese PLA General Hospital, Beijing 100000, China
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  • Ya-Fei Wang
    Affiliations
    Data Management Department, Chia Tai Tianqing Pharmaceutical Group, Nanjing 210000, China
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  • Author Footnotes
    10 These authors contributed equally
    Min Zheng
    Footnotes
    10 These authors contributed equally
    Affiliations
    Department of Gynecologic Oncology, Sun Yat-sen University Cancer Centre, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou 510060, China
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    10 These authors contributed equally
    ,
    Author Footnotes
    11 Lead contact
    Xin Huang
    Correspondence
    Corresponding author
    Footnotes
    10 These authors contributed equally
    11 Lead contact
    Affiliations
    Department of Gynecologic Oncology, Sun Yat-sen University Cancer Centre, Guangzhou 510060, China

    State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine, Guangzhou 510060, China
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  • Author Footnotes
    9 These authors contributed equally
    10 These authors contributed equally
    11 Lead contact

      Highlights

      • Anlotinib plus TQB2450 improves the response rate in platinum-resistant ovarian cancer
      • Anlotinib plus TQB2450 shows durable response in platinum-resistant ovarian cancer
      • A phase 3 randomized controlled trial to further validate our findings is ongoing

      Summary

      This is a phase Ib study of anlotinib plus a programmed death-ligand 1 (PD-L1) inhibitor TQB2450 for platinum-resistant or -refractory ovarian cancer. Thirty-four patients are enrolled and receive treatment. The objective response rate (ORR) is 47.1%, and the disease control rate is 97.1%. The median duration of response (DOR) has not been reached, and 61.3% of patients have a DOR of at least 8 months. The median progression-free survival (PFS) is 7.8 months, and the median overall survival (OS) has not been reached. The PD-L1-positive group has an ORR of 25.0%, whereas the PD-L1-negative group has an ORR of 92.9%. Treatment-related grade 3 or 4 adverse events (AEs) occur in 70.6% of patients, with the most common being hypertension (29.4%) and palmar-plantar erythrodysesthesia syndrome (29.4%). Anlotinib plus TQB2450 show promising antitumor activity and manageable toxicities in patients with platinum-resistant or -refractory ovarian cancer. A phase 3 randomized controlled trial to further validate our findings is ongoing.

      Graphical abstract

      Keywords

      Introduction

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      Results

      Between February 27, 2020, and February 26, 2021, we screened 44 patients, of whom 34 eligible patients were enrolled and received the study treatment (full analysis set [FAS] and safety analysis set). One patient (2.9%) who discontinued the treatment before the first post-baseline tumor assessment due to adverse events (AEs) was excluded from the per-protocol set (PPS) (Figure 1). As of the data cutoff (May 31, 2021), the median follow up was 8.6 (range: 2.3–15.3) months. Thirteen (38.2%) of the 34 patients were still on treatment. Twenty-one (61.8%) patients discontinued treatment due to disease progression (n = 16, 47.1%), AEs (n = 2, 5.9%), and patient refusal (n = 3, 8.8%). Baseline characteristics of the population are summarized in Table 1.
      Table 1Baseline patient characteristics
      Characteristic, n (%)All patients (n = 34)
      Age, years
      Median (range)55 (26–71)
      FIGO stage at diagnosis
      IA1 (2.9)
      IC2 (5.9)
      IIB2 (5.9)
      IIIA14 (11.8)
      IIIB5 (14.7)
      IIIC16 (47.1)
      IVB4 (11.8)
      Histologic subtype
      High-grade serous carcinoma27 (79.4)
      Low-grade serous carcinoma2 (5.9)
      Endometrioid1 (2.9)
      Clear cell4 (11.8)
      ECOG performance status
      03 (8.8)
      131 (91.2)
      Number of prior lines of systemic therapies
      1–212 (35.3)
      3–622 (64.7)
      Median (range)3 (1–6)
      Target lesion size, mm
      Median (range)48 (17–182)
      Treatment-free interval (TFI)
      Progression during the last therapy/<1 month21 (61.8)
      ≥1 and <2 months9 (26.5)
      ≥2 and <6 months4 (11.8)
      Platinum-free interval (PFI)
      <3 months11 (32.4)
      ≥3 and <6 months9 (26.5)
      ≥6 months
      These 14 patients received at least one line of single-agent therapy with a non-platinum compound after being considered platinum-resistant (defined as disease progressing within 6 months after the last platinum therapy), which made the PFI ≥6 months. Of these 14 patients, 13 (92.9%) had TFI ≤1 month and one had TFI of 4.2 months.
      14 (41.2)
      Prior bevacizumab
      Yes9 (26.5)
      No25 (73.5)
      BRCA1/2 mutation status
      gBRCA1/2 mutation4 (11.7)
      BRCA1/2 wild type16 (47.1)
      BRCA1/2 unknown14 (41.2)
      PD-L1 expression status
      Positive8 (23.5)
      Negative14 (41.2)
      Unknown12 (35.3)
      Microsatellite status
      Assessed by fluorescent multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE).
      High microsatellite instability0 (0.0)
      Microsatellite stable16 (47.1)
      Unknown18 (52.9)
      FIGO, The International Federation of Gynecology and Obstetrics; ECOG, Eastern Cooperative Oncology Group.
      a These 14 patients received at least one line of single-agent therapy with a non-platinum compound after being considered platinum-resistant (defined as disease progressing within 6 months after the last platinum therapy), which made the PFI ≥6 months. Of these 14 patients, 13 (92.9%) had TFI ≤1 month and one had TFI of 4.2 months.
      b Assessed by fluorescent multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE).

      Antitumor activity

      In the first nine patients enrolled, confirmed responses were noted in five patients. The ORR threshold for the first stage of Simon’s two-stage design was reached, and the trial continued to full accrual. In the FAS, 16 of 34 patients achieved partial response (PR), thus the ORR was 47.1% (95% confidence interval [CI], 29.8–64.9) (Table 2). An additional three patients had unconfirmed PR. Similar responses were recorded in PPS (Table 2). Seventeen (50.0%) of 34 patients achieved stable disease (SD), hence the DCR was 97.1% (95% CI, 84.7–99.9; Table 2). As shown in Figure 2A , 26 (78.8%) of the 33 patients who had an evaluable tumor assessment had some degree of tumor shrinkage.
      Table 2Antitumor activity assessed by RECIST 1.1
      Antitumor activityThe full analysis set (n = 34)The per-protocol set (n = 33)
      ORR16 (47.1)16 (48.5)
      95% CI29.8–64.930.8–66.5
      DCR33 (97.1)33 (100)
      95% CI84.7–99.989.4–100.0
      Best overall response
      CR0 (0.0)0 (0.0)
      PR16 (47.1)16 (48.5)
      SD17 (50.0)17 (51.5)
      Progressive disease0 (0.0)0 (0.0)
      No assessment
      One patient discontinued study treatment before the first scheduled post-baseline scan.
      1 (2.9)
      Data are presented as n (%) unless otherwise specified. Responses were assessed in accordance with the RECIST 1.1. Only confirmed responses were included. RECIST, Response Evaluation Criteria in Solid Tumors; ORR, objective response rate; DCR, disease control rate; CR, complete response; PR, partial response; SD, stable disease.
      a One patient discontinued study treatment before the first scheduled post-baseline scan.
      Figure thumbnail gr2
      Figure 2Tumor response assessment with waterfall plot and treatment duration with swimmer plot
      (A) Waterfall plot for best percentage change in target lesion size. The patients who had at least one post-baseline tumor assessment were included (n = 33). The dashed line at −30% change represents the RECIST 1.1 cutoff to define partial response or complete response.
      (B) Swimmer plot. The length of each bar represents the duration treatment for each patient.
      Among the 16 patients with a confirmed objective response, the median time to achieve response was 2.7 (range: 1.3–7.0) months, and the median DOR was not reached (95% CI, 5.3 to not reached; Figure 3A ). A DOR of at least 8 months was observed in 61.3% (95% CI, 25.1–84.0). Figure 2B presents a swimmer plot of the time to receive treatment across all patients.
      Figure thumbnail gr3
      Figure 3Kaplan-Meier curves of duration of response, progression-free survival, and overall survival
      Kaplan-Meier curves of (A) duration of response, (B) progression-free survival, and (C) overall survival. NE, not estimable.
      As of the data cutoff, 17 (50.0%) of the 34 patients had disease progression. The median progression-free survival (PFS) was 7.8 months (95% CI, 4.9 to not reached; Figure 3B). The proportion of patients estimated to be progression free at 6 months was 66.7% (95% CI, 46.7–80.7) and at 9 months was 38.1% (95% CI, 19.1–57.0). Nine (26.5%) patients had died. The median overall survival (OS) was not reached (95% CI, 11.3 to not reached; Figure 3C).

      Safety

      Treatment-related AEs of any grade occurred in all 34 patients (100%) (Table 3): hypertension (n = 31, 91.2%), palmar-plantar erythrodysesthesia syndrome (n = 25, 73.5%), hypertriglyceridemia (n = 20, 58.8%), and hypothyroidism (n = 17, 50.0%; Table 3). Grade 3–4 treatment-related AEs occurred in 24 (70.6%) patients; the most common were hypertension (n = 10, 29.4%), palmar-plantar erythrodysesthesia syndrome (n = 10, 29.4%), hypertriglyceridemia (n = 5, 14.7%), fatigue (n = 3, 8.8%), and weight loss (n = 3, 8.8%; Table 3). No treatment-related deaths occurred. Serious treatment-related AEs were observed in 5 (14.7%) of 34 patients, including one (2.9%) each with hypertension, fatigue, pneumonitis, and arthritis and one (2.9%) with increased aspartate aminotransferase and alanine aminotransferase (Table S1).
      Table 3Treatment-related adverse events in the total treated patients (n = 34)
      Any treatment-related adverse events, n (%)Grade 1Grade 2Grade 3Grade 4
      Hypertension2 (5.9)19 (55.9)10 (29.4)0
      Palmar-plantar erythrodysesthesia syndrome2 (5.9)13 (38.2)10 (29.4)0
      Hypothyroidism12 (35.3)5 (14.7)00
      Hypertriglyceridemia11 (32.4)4 (11.8)3 (8.8)2 (5.9)
      Proteinuria9 (26.5)5 (14.7)2 (5.9)0
      Stomach ache13 (38.2)1 (2.9)00
      Increased aspartate aminotransferase12 (35.3)1 (2.9)01 (2.9)
      Increased alanine aminotransferase12 (35.3)1 (2.9)01 (2.9)
      Diarrhea9 (26.5)3 (8.8)1 (2.9)0
      Fatigue8 (23.5)2 (5.9)3 (8.8)0
      Hoarseness8 (23.5)2 (5.9)00
      Stomatitis6 (17.6)4 (11.8)1 (2.9)0
      Headache8 (23.5)1 (2.9)00
      Hypoalbuminemia6 (17.6)2 (5.9)00
      Sore throat7 (20.6)1 (2.9)00
      Weight loss4 (11.8)3 (8.8)3 (8.8)0
      Myalgia6 (17.6)000
      Thrombocytopenia5 (14.7)01 (2.9)0
      Anorexia5 (14.7)000
      Neutropenia3 (8.8)2 (5.9)1 (2.9)0
      Rash4 (11.8)1 (2.9)00
      Increased amylase4 (11.8)01 (2.9)0
      Arthritis2 (5.9)2 (5.9)1 (2.9)0
      Dry mouth4 (11.8)000
      Pruritus3 (8.8)1 (2.9)00
      Hyperthyroidism2 (5.9)2 (5.9)00
      Cough3 (8.8)000
      Abdominal pain3 (8.8)000
      Nausea3 (8.8)000
      Gingival pain3 (8.8)000
      Gamma-glutamyltransferase increased2 (5.9)1 (2.9)2 (5.9)0
      Increased bilirubin2 (5.9)02 (5.9)0
      Insomnia2 (5.9)000
      Vomiting1 (2.9)1 (2.9)00
      Increased lipase1 (2.9)1 (2.9)00
      Pneumonitis02 (5.9)00
      Tinnitus02 (5.9)00
      Anaemia1 (2.9)000
      Myositis01 (2.9)00
      No grade 5 treatment-related adverse events were reported. See also Tables S1 and S2.
      All 34 patients received at least one complete cycle of anlotinib. Anlotinib dose interruption due to AEs occurred in 22 patients (64.7%). Sixteen (47.1%) patients required anlotinib dose reduction, of whom 12 (81.3%) required only one dose reduction and three (18.8%) required two dose reductions. The most common AE leading to anlotinib dose reduction was palmar-plantar erythrodysesthesia syndrome (10/16, 62.5%). AEs leading to anlotinib dose reduction are shown in Table S2. TQB2450 dose interruption occurred in 8 (23.5%) of the 34 patients. Eight (23.5%) patients required corticosteroids: two with pneumonitis, two with arthritis, one each with a rash, myositis, and hyperthyroidism, and one with increased aspartate aminotransferase and alanine aminotransferase. In the patients who received corticosteroids, one patient had a TQB2450 dose interruption, and one patient permanently discontinued TQB2450. Two (5.9%) patients withdrew from the study due to toxicities: one with fatigue and one with grade 4 increased aspartate aminotransferase and alanine aminotransferase.

      PD-L1 expression

      PD-L1 expression was detected in 22 patients. Eight patients were positive for PD-L1, and 14 were negative for PD-L1 (Table S3). ORR was 25% (95% CI, 3.1%–65.1%) in patients with PD-L1-positive tumors, whereas it was 92.9% (95% CI, 66.1%–99.8%) in patients with PD-L1-negative tumors (p = 0.002; Table S3). Median PFS was 8.5 months (95% CI, 2.6 to not reached) in PD-L1-positive patients and was not reached (95% CI, 6.8 to not reached) in PD-L1-negative patients (Figure 4). The difference in PFS was not statistically significant between PD-L1-positive or -negative patients (p = 0.28; Figure 4).
      Figure thumbnail gr4
      Figure 4Kaplan-Meier curves of progression-free survival of PD-L1-positive and -negative tumors. NE, not estimable; HR, hazard ratio.

      Discussion

      In this study, anlotinib plus TQB2450 demonstrated promising antitumor activity with favorable response rate, durable response, and tolerable toxicity profile in patients with platinum-resistant or -refractory ovarian cancer. To our knowledge, this is the largest cohort of patients in this setting treated with a multi-targeted TKI plus a PD-L1 inhibitor.
      Despite the immunogenic profile in ovarian cancer revealed by preclinical data, PD-1/PD-L1 inhibitor monotherapy showed only low to moderate activity in patients with recurrent ovarian cancer.
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      However, this treatment combination seemed to have less activity in the platinum-resistant setting (response rate 16.7%). Limited data have reported the clinical effect of PD-1/PD-L1 inhibitor combined with VEGFR TKI in advanced ovarian cancer. Remarkably, the efficacy observed in our study is encouraging, with 16 (47.1%) confirmed responses and three unconfirmed responses and 78.8% of the patients showing some degree of tumor shrinkage. Our data suggest that anlotinib plus TQB2450 has enhanced antitumor activity in platinum-resistant ovarian cancer, given the efficacy previously reported in anlotinib monotherapy and PD-1/PD-L1 inhibitors monotherapy in this setting.
      • Hamanishi J.
      • Mandai M.
      • Ikeda T.
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      • Kawaguchi A.
      • Murayama T.
      • Kanai M.
      • Mori Y.
      • Matsumoto S.
      • Chikuma S.
      • et al.
      Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer.
      • Matulonis U.A.
      • Shapira-Frommer R.
      • Santin A.D.
      • Lisyanskaya A.S.
      • Pignata S.
      • Vergote I.
      • Raspagliesi F.
      • Sonke G.S.
      • Birrer M.
      • Provencher D.M.
      • et al.
      Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: results from the phase II KEYNOTE-100 study.
      • Disis M.L.
      • Taylor M.H.
      • Kelly K.
      • Beck J.T.
      • Gordon M.
      • Moore K.M.
      • Patel M.R.
      • Chaves J.
      • Park H.
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      Efficacy and safety of avelumab for patients with recurrent or refractory ovarian cancer: phase 1b results from the JAVELIN solid tumor trial.
      ,
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      Anlotinib in patients with recurrent platinum-resistant or refractory ovarian carcinoma: a prospective, single-arm, single-center, phase II clinical study..
      Yang et al. identified that PD-L1 directly interacted with VEGFR2 and that it promoted the angiogenesis and metastasis of ovarian cancer by participating in the c-JUN/VEGFR2 signaling axis.
      • Yang Y.
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      Programmed death ligand-1 regulates angiogenesis and metastasis by participating in the c-JUN/VEGFR2 signaling axis in ovarian cancer.
      Furthermore, they demonstrated the PD-L1 inhibitor durvalumab combined with the VEGFR TKI apatinib enhanced the effect of anti-angiogenesis and the inhibition of cell migration and invasion. These findings may be one of the plausible explanations for the encouraging results observed in our study, where a multi-targeted TKI anlotinib was used as a partner combined with the PD-L1 inhibitor TQB2450.
      In addition, 41.2% of the patients in our study had multiple lines of single-agent therapy with a non-platinum compound after being considered resistant to the last platinum therapy, which made the platinum-free interval (PFI) ≥6 months. There is a concern that the prolonged PFI may, to some extent, better our efficacy. However, there is no evidence that the predictive and prognostic roles of PFI apply for more than two relapses, and PFI may not be a valuable criteria for molecular targeted therapy.
      • Pujade-Lauraine E.
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      Management of platinum-resistant, relapsed epithelial ovarian cancer and new drug perspectives.
      ,
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      OCEANS: a randomized, double-blind, placebo-controlled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer.
      In our study, 88.3% of patients had a treatment-free interval (TFI) <2 months, which represented a patient population with poor prognosis.
      • Pujade-Lauraine E.
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      • Pignata S.
      Management of platinum-resistant, relapsed epithelial ovarian cancer and new drug perspectives.
      Of note, the findings of our study and other trials of combination PD-1/PD-L1 inhibitor therapy demonstrated a durable clinical benefit in patients with platinum-resistant ovarian cancer. In a phase 1b study of 20 patients with platinum-resistant ovarian cancer who were treated with atezolizumab and bevacizumab, despite having a modest ORR of 15%, all three patients with PR had durable responses (11.3–18.9 months) and are still ongoing at the data cutoff, with four of eight patients with SD for >8 months.
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      Safety and clinical activity of atezolizumab plus bevacizumab in patients with ovarian cancer: a phase Ib study.
      In a phase 2 study combining nivolumab and bevacizumab in patients with recurrent ovarian cancer, only 3 of 18 patients (16.7%) with platinum-resistant disease achieved PR. However, two of them had a prolonged response of 16–18 months, with 16.7% of patients with SD lasting at least 24 weeks.
      • Liu J.F.
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      • Curtis J.
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      • et al.
      Assessment of combined nivolumab and bevacizumab in relapsed ovarian cancer: a phase 2 clinical trial.
      Similarly, in our study, the median PFS was 7.8 months, and the median DOR was not reached, with 61.3% of the patients showing a DOR of at least 8 months.
      The safety profile of anlotinib plus TQB2450 was consistent with that reported in previous studies of other selective VEGFR TKIs combined with PD-1/PD-L1 inhibitors,
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      • et al.
      Axitinib in combination with pembrolizumab in patients with advanced renal cell cancer: a non-randomised, open-label, dose-finding, and dose-expansion phase 1b trial.
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      • et al.
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      with no unexpected AEs. Although the frequency of the overall grade 3–4 AEs was as high as 70.6% in our study, the prevalence of specific grade 3–4 AEs was not so high, with hypertension and palmar-plantar erythrodysesthesia syndrome being the highest ranked with 29.4% each. These toxicities were associated with anlotinib and were manageable. Despite the grade 3–4 hypertriglyceridemia and grade 3 increased amylase observed in 14.7% and 2.9% of the patients in our study, respectively, all these patients did not have any symptoms. In the eight patients who required corticosteroids, most of them did not need TQB2450 dose interruption. In addition, the discontinuation rate due to treatment-related AEs was only 5.9% in our study. Our findings suggested that anlotinib plus TQB2450 had a manageable safety profile.
      PD-L1 is the most widely investigated biomarker for predicting response to PD-1/PD-L1 inhibitor therapy; however, the predictive value of PD-L1 in ovarian cancer remains highly controversial.
      • Hamanishi J.
      • Mandai M.
      • Ikeda T.
      • Minami M.
      • Kawaguchi A.
      • Murayama T.
      • Kanai M.
      • Mori Y.
      • Matsumoto S.
      • Chikuma S.
      • et al.
      Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer.
      ,
      • Matulonis U.A.
      • Shapira-Frommer R.
      • Santin A.D.
      • Lisyanskaya A.S.
      • Pignata S.
      • Vergote I.
      • Raspagliesi F.
      • Sonke G.S.
      • Birrer M.
      • Provencher D.M.
      • et al.
      Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: results from the phase II KEYNOTE-100 study.
      ,
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      • Gray K.P.
      • Penson R.T.
      • Horowitz N.
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      • Hill S.J.
      • Curtis J.
      • Luo W.
      • et al.
      Assessment of combined nivolumab and bevacizumab in relapsed ovarian cancer: a phase 2 clinical trial.
      In our study, patients with PD-L1-negative tumors had higher response rates than those with PD-L1-positive tumors (92.9% versus 25.0%). Several factors may explain the inconsistency between the efficacy and PD-L1 expression in our study, such as the spatial and temporal heterogeneity in PD-L1 expression, the variability between primary and metastatic tumor sites, and the influence by prior chemotherapy.
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      • et al.
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      • Fujimoto D.
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      • Kosaka Y.
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      • et al.
      Alteration of PD-L1 expression and its prognostic impact after concurrent chemoradiation therapy in non-small cell lung cancer patients.
      • Ben Dori S.
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      Spatial heterogeneity of PD-L1 expression and the risk for misclassification of PD-L1 immunohistochemistry in non-small cell lung cancer.
      It should be noted that only 22 (64.7%) patients in our study had archival tumor samples for PD-L1 staining, and the small number of patients precluded definitive conclusions. Efforts are ongoing to identify more reliable biomarkers for PD-1/PD-L1 inhibitor therapy.
      Anlotinib plus TQB2450 has promising antitumor activity and manageable toxicities in patients with platinum-resistant or -refractory ovarian cancer. Accordingly, a phase 3 multi-center randomized trial (ClinicalTrials.gov: NCT05145218) to investigate anlotinib plus TQB2450 as treatment for patients with platinum-resistant or -refractory ovarian cancer is ongoing.

      Limitations of study

      This study has limitations. This is a single-arm study lacking a comparator treatment arm; thus, selection bias could not be ruled out. Further, the small sample size reduced the certainty of effectiveness observed.

      STAR★Methods

      Key resources table

      Tabled 1
      REAGENT or RESOURCESOURCEIDENTIFIER
      Biological samples
      Formalin-fixed paraffin-embedded (FFPE) archival tumor specimensThis manuscriptN/A
      Chemicals, peptides, and recombinant proteins
      TQB2450Chia Tai Tianqing Pharmaceutical Group Co., Ltd.See Table S4
      AnlotinibChia Tai Tianqing Pharmaceutical Group Co., Ltd.See Table S5
      Critical commercial assays
      PD-L1 IHC 22C3Dako, an Agilent Technologies, Inc. company,

      Santa Clara, CA, USA
      Cat# 11174544
      Deposited data
      The data of patientsThis manuscripthttps://www.researchdata.org.cn/ (ID: RDDA2021002123)
      Software and algorithms
      SAS software version 9.4SAS Institute, Cary, NC, USAwww.sas.com

      Resource availability

      Lead contact

      Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xin Huang ( huangxin@sysucc.org.cn ).

      Materials availability

      This study did not generate new unique reagents.

      Experimental model and subject details

      Ethics statement

      The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines after approval by the China National Medical Products Administration (ID: [NMPA] 2017L04914) and local institutional review board at each participating site.

      Human subjects

      Chinese women with histologically confirmed epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer who had platinum-resistant or platinum-refractory disease, were enrolled in the study. Demographic information was provided in Table 1. All patients provided written informed consent prior to enrollment.

      Patient eligibility

      The key inclusion criteria were: (1) aged 18–70 years with histologically confirmed epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer who had platinum-resistant or platinum-refractory disease. Platinum-resistant was defined as disease progressing within 6 months after the last platinum therapy. Platinum-refractory was defined as disease progressing during the initial platinum-based therapy or within 28 days after the last dose of platinum. We also enrolled patients whose disease progressed within 6 months after the last platinum therapy and who received subsequent non-platinum treatment. (2) with radiological progression during the last treatment before enrolling in this study; (3) with measurable disease according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1); (4) an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; (5) adequate organ function. Major exclusion criteria were: (1) poorly controlled hypertension; (2) previous treatment with VEGFR-TKIs, anti-PD-1, anti-PD-L1, or anti-cytotoxic T-lymphocyte-associated antigen-4 antibodies; (3) active or documented history of autoimmune disease; (4) active brain metastases; (5) active hepatitis B or hepatitis C viral infection. All patients provided written informed consent prior to enrollment.

      Subject allocation

      The current phase Ib clinical trial was a single-arm study, with no control group, and all the patients were enrolled in one group.

      Method details

      Study design

      This study (ACTION study) is part of an open-label, multi-cohort, multicenter phase Ib trial (clinicalTrials.gov identifier: NCT04236362) evaluating the efficacy and safety of anlotinib plus TQB2450 in patients with advanced gynecologic cancer at five academic medical centers in China. Here we report the results of ovarian cancer cohort. The primary endpoint was ORR, defined as the proportion of patients with complete response (CR) or PR according to RECIST 1.1 assessed by investigators. Objective responses had to be confirmed with a repeat scan at least 4 weeks later. Secondary endpoints were PFS, OS, DOR, DCR, safety, and tolerability.

      Sample size estimation

      A Simon optimal two-stage design is used to estimate sample size. The ORR of single-agent anlotinib in platinum-resistant ovarian cancer was approximately 15%. The combination of TQB2450 and anlotinib is expected to result in an improved ORR of 35%. The null hypothesis that the true ORR was 15% would be tested against a one-sided alternative of 35%. In the first stage, nine patients were accrued. If no response or one response was observed, the study was terminated and declared negative. Otherwise, 25 additional patients would be accrued to the second stage. The study was considered positive if > 8 responders were observed among the 34 patients. This design yielded a type I error rate of 5% and power of 80% when the true response rate was 35%.

      Drug administration

      Patients received anlotinib 12 mg orally once daily on days 1–14 followed by a 7-day rest and TQB2450 1,200 mg intravenously on day 1 of a 21-day cycle. This dose was determined based on the phase Ib study of anlotinib plus TQB2450 in advanced solid tumors.
      • Cheng Y.
      • Cui H.
      • Wu C.
      • Wang Y.
      • Zhang T.
      • Xin Y.
      • Xu J.
      • Chen Y.
      • Li Z.
      • Wang Y.
      • et al.
      A phase Ib study of TQ-B2450 plus anlotinib in patients with advanced solid tumor..
      Treatment continued until disease progression, unacceptable toxicity, or withdrawal of consent. Dose interruptions and dose reductions of anlotinib were permitted for toxicities that were not relieved by supportive care. A maximum of two dose reductions were allowed in anlotinib therapy. The first dose reduction was to 10 mg once daily, and the additional reduction was to 8 mg once daily. Dose reductions in TQB2450 were not allowed.

      Assessments

      Tumor responses were assessed by investigators according to RECIST 1.1 using computed tomography (CT) or magnetic resonance imaging (MRI) at baseline, every 6 weeks for the initial 54 weeks and every 9 weeks thereafter. The primary endpoint was ORR, defined as the proportion of patients with CR or PR according to RECIST 1.1 assessed by investigators. Objective responses had to be confirmed with a repeat scan at least 4 weeks later. Secondary endpoints were PFS, OS, DOR, DCR, safety, and tolerability. PFS was defined as the time from treatment initiation to the date of first documented disease progression or death from any cause, whichever occurred first. OS was defined as the time from treatment initiation to the date of death from any cause. DOR was defined as the time from the first evidence of response to disease progression in patients who achieved PR or better. DCR was defined as the proportion of patients who achieved confirmed CR or PR or SD. AEs were monitored throughout the treatment period and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE; version 5.0). The exploratory endpoint reported here included associations between antitumor activity and PD-L1 expression.

      PD-L1 expression analysis

      PD-L1 expression in formalin-fixed paraffin-embedded (FFPE) archival tumor specimens was assessed using the PD-L1 immunohistochemistry (IHC) 22C3 assay (Dako Inc., Agilent Technologies, Santa Clara, CA, USA) and measured using a combined positive score (CPS), defined as the number of PD-L1-stained cells divided by the total number of viable tumor cells, multiplied by 100. PD-L1-positivity was defined as a CPS of ≥1. The expression of PD-L1 and its correlation with treatment efficacy were provided in Figure 4 and Table S3.

      Quantification and statistical analysis

      All statistical tests were performed using the SAS software version 9.4 (SAS Institute, Cary, NC, USA). The FAS and safety analysis set were defined as all enrolled patients who received at least one dose of study treatment. The PPS consisted all FAS patients who had at least one post-baseline tumor assessment and no major protocol deviations and completed at least one treatment cycle. The primary endpoint was analyzed in both FAS and PPS. Secondary endpoints were analyzed in the FAS. A safety analysis was performed in the safety analysis set. We summarized the proportion of patients who achieved an objective response and calculated 95% CI using the Clopper–Pearson method. DOR, PFS, and OS were analyzed using the Kaplan-Meier method. Median DOR, PFS, and OS were estimated, and 95% CIs were calculated based on Greenwood’s formula and log-log transformation. The AEs by the proportion of the total number of patients treated are summarized. In post hoc analyses, the association between ORR and PD-L1 status were assessed using the Chi-square test or Fisher’s exact test. PFS according to PD-L1 expression status was estimated using Kaplan-Meier method and compared by log-rank tests.

      Additional resources

      This study has been registered on https://clinicaltrials.gov/, ID: NCT04236362.

      Data and code availability

      • The data of patients in this study have been recorded at Research Data Deposit: http://www.researchdata.org.cn with number RDDA2021002123. The data are available from Research Data Deposit but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Research Data Deposit public platform.
      • This paper does not report the original code.
      • Any additional information required to reanalyze the data reported in this work paper is available from the Lead Contact upon request.

      Acknowledgments

      We thank all of the patients and their families who participated in this study. We thank Xu Li, Ying-Ying Li, and Ming-Xun Cao for collection of the data and provision of study materials and Ding Yu for supervision of research. This study was funded by the Chia Tai Tianqing Pharmaceutical Group Co., Ltd. The funder provided the study drug and designed the studies in collaboration with the investigators, participating in data collection. The funder sources had no role in data analysis and interpretation, preparation, review, or approval of the manuscript and decision to submit the manuscript for publication. This study was also supported, in part, by the Guangdong Basic and Applied Basic Research Fund Provincial Enterprise Joint Fund ( 2021A1515220166 ).

      Author contributions

      Conception and design, C.-Y.L. and X.H.; provision of study materials or patients, C.-Y.L., F.Y., Y.X., R.L., Y.H., J.W., C.L., X.-H.B., H.-H.J., J.M., W.-H.Z., L.Z., M.Z., and X.H.; collection and assembly of data, C.-Y.L., J.Z., F.Y., Y.X., R.L., J.W., C.L., H.-H.J., J.M., W.-H.Z., L.Z., M.Z., and X.H.; data analysis and interpretation, C.-Y.L., F.Y., Y.X., R.L., Y.H., J.W., C.L., J.M., W.-H.Z., Y.-F.W., M.Z., and X.H.; manuscript writing, C.-Y.L.; final approval of manuscript, all authors.

      Declaration of interests

      Y.-F.W. is an employee of Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

      Supplemental information

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