A phase 1b study of the allosteric extracellular FGFR2 inhibitor alofanib in patients with pretreated advanced gastric cancer

Alofanib is a small-molecule allosteric extracellular FGFR2 inhibitor. We report safety and preliminary efficacy from the first-in-human phase 1b study of alofanib in heavily pretreated patients with advanced gastric cancer. The standard dose-escalation design 3+3 aimed to establish the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Alofanib was administered daily intravenously 5 days on, 2 days off. There were five dose levels (50-350 mg/m2). All patients received alofanib until disease progression or unacceptable toxicity. 21 patients were enrolled. Patients were predominantly male (71%), 67% had 2 and more metastatic sites, including liver metastases (43%), 19% had ECOG PS 2, and were heavily pretreated (86% had previous 2 and more treatment lines). During dose escalation, no dose-limiting toxicities were observed, and MTD was not defined. 15 (71.4%) patients had at least one adverse event associated with the treatment (TRAE). Grade 3 or higher TRAEs were observed in 6 patients (28.6%). The most common TRAEs included reactions immediately after administration, diarrhea, thrombocytopenia, arthralgia, and headache. The median progression-free survival and overall survival was 3.63 (95% CI 1.58–5.68) and 7.0 (95% CI 3.82–10.18) months, respectively. The 6- and 12-month overall survival rates were 57.1% and 33.3%. Disease control rate was 68% with one durable partial response. The MTD has not been reached and dose of 350 mg/m2, 5 days on, 2 days off has been declared as RP2D. Alofanib showed acceptable tolerability and preliminary signs of clinical activity in the late-line treatment of metastatic gastric cancer. (ClinicalTrials.gov identifier: NCT04071184).


Introduction
Despite some progress, gastric cancer remains one of the most common malignant tumors of the digestive tract and continues to be a major public health problem in terms of Presented in part as a Best Poster in Track at the 2022 ASCO Gastrointestinal Cancers Symposium in San Francisco, CA, USA. frequency, aggressiveness, and poor overall survival at an advanced stage. The instability of the fibroblast growth factor receptor 2 (FGFR2) signaling pathway due to the accumulation of genetic and molecular modifications is associated with the development and progression of gastric cancer [1]. Recent clinical data suggest that FGFR2-positive gastric adenocarcinomas have poorly differentiated histology and exhibit a higher rate of lymph node and distant metastases [2,3]. Compared with patients with FGFR2-negative tumors, patients with gastric cancer harboring FGFR2 amplification or expression showed significantly worse survival [4,5].
The FGFR2 gene undergoes alternative splicing in the third immunoglobulin domain, leading to different isoforms of the FGFR2 receptor, FGFR2b and FGFR2c, with different FGF ligand binding [6]. Altered FGFR isoform switching is involved in epithelial-mesenchymal transition and cancer progression [7,8]. Moreover, FGFR isoform switching can affect the activity of FGFR2-neutralizing antibodies [9,10]. On other hand, mechanisms of FGFR2 tyrosine kinase inhibitors resistance include gatekeeper mutations [11]. Therefore, the development of novel compounds with activity regardless of FGFR2 isoforms and mutations seems reasonable.
Alofanib (RPT835) is a small-molecule allosteric inhibitor that binds to the non-active site of FGFR2 extracellular domain. In the preclinical models, alofanib inhibited phosphorylation of FRS2α with the half maximal inhibitory concentration (IC50) values of 7 and 9 nmol/l in cancer cells expressing different FGFR2 isoforms [12,13]. Preclinical results are consistent with alofanib being a predominantly FGFR2-selective small molecular inhibitor with potent activity in gastric cancer, breast cancer, lung cancer, and ovarian cancer models. Inhibition of FGF signaling by alofanib also suppressed tumor angiogenesis [13].
Here, we report safety and preliminary efficacy data from the first-in-human phase 1b study of alofanib as monotherapy in heavily pretreated patients with advanced gastric cancer.

Patients
All patients were ≥18 years old with a histologically confirmed gastric adenocarcinoma that was refractory to currently available therapies, for which no standard therapy was possible or the patient declined standard therapy. All patients had measurable disease per Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1, an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 2 with adequate bone marrow, renal, cardiac and hepatic function at screening. Patients were excluded if they had symptomatic brain metastases, a history of secondary malignancy and any other clinically significant disease or were about to conceive. All patients provided written informed consent.

Study design, endpoints and assessments
This was an open-label, multicenter Phase 1b study (RPT-835GC1B; ClinicalTrials.gov identifier: NCT04071184) at 7 centers in the Russian Federation between October 1, 2019 and December 20, 2021. The standard dose-escalation design "3+3" aimed to establish the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) as a primary endpoint. Dose escalation was defined by the study protocol and included 5 dose levels. The last fifth dose level involved the administration of the alofanib at a dose of 350 mg/m2. This dose was significantly higher than the therapeutic dose in terms of animals, therefore, further escalation of the targeted drug was not required. The dose of the last level in the absence of dose-limiting toxicities (DLTs) was defined as the RP2D.
The starting dose of alofanib was 50 mg/m2 with planned escalation to 350 mg/m2 intravenously for 5 days followed by a 2-day interval (rest) till disease progression or unacceptable toxicity. An additional 6 patients were included in the last dose level cohort.
Dosing decisions were based on the rate of dose-limiting toxicities (DLTs) observed during the first 28-day treatment cycle. The prespecified DLTs were grade ≥3 non-hematologic toxicity with the exception of alopecia and nausea/vomiting, which were controlled by an optimal antiemetic therapy, any grade non-hematologic treatment-related toxicity that required interruption of alofanib therapy for more than 14 days, grade ≥3 febrile neutropenia or grade ≥4 neutropenia, grade 4 thrombocytopenia requiring platelet transfusion or leading to a life-threatening bleeding event.
Safety parameters were assessed as secondary endpoints and included adverse events (AE), serious AEs (SAE), laboratory evaluations, and vital signs. Pharmacokinetic (PK) parameters for alofanib were also assessed as secondary endpoints and included area under the plasma concentration-time curve (AUC), maximum observed plasma concentration (Cmax), time to reach Cmax (Tmax), half-life time (t1/2), clearance (CL), and volume of distribution (Vd). Blood samples for PK analysis of alofanib were collected pre-dose and at 5 min, 30 min, 1, 2, 4, 8, 24, 48, and 72 hours after administration of alofanib on day 1 and day 8.
Secondary efficacy endpoints were objective response rate (ORR), disease control rate (DCR), duration of response (DOR) and stable disease (DORS), progression-free survival (PFS), and overall survival (OS) as determined by the investigator. Tumor response was assessed by computed tomography per RECIST v.1.1 at screening and repeated every 8 weeks.
Exploratory endpoints included rates of FGFR2 amplification and overexpression. Archived tumor tissue was collected from all patients. GmbH, Germany) and FGFR2 expression was accessed by immunohistochemistry with antibody 1G3 (#ab5820, Abcam, USA) and antibody EPR24075-418 (#ab289968, Abcam, USA). Antitumor activity related to FGFR2 molecular changes was also evaluated.
The study was approved by institutional review boards and independent ethics committees at each center. The study was conducted according to all applicable local legal and regulatory requirements, as well as the general principles set forth in the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Guidelines for Good Clinical Practice, and the Declaration of Helsinki.

Statistical analyses
Overall, 21 patients were planned for enrollment. The analysis included all patients who received ≥1 dose of alofanib. All safety analyses were based on the safety data set and summarized descriptively. All AEs and laboratory abnormalities were graded using Common Terminology Criteria for Adverse Events (CTCAE) version 4.03. All efficacy analyses were based on the full examination set. ORR was based on confirmed responses and 2-sided 95% confidence intervals (CI) calculated using the Pearson exact binomial method. DOR, DORS, PFS, and OS were estimated using the Kaplan-Meier method and 2-sided 95% CIs. All PK analyses were based on the PK analysis set. Non-compartmental PK parameters were estimated for each patient and were summarized descriptively. No formal tests were performed on the biomarker analyses.
Statistical analyses were conducted using SPSS Statistics 27 (IBM, USA). The data cutoff date for all analyses was November 23, 2022.

Patients
Between October 21, 2019, and August 8, 2021, 21 patients were enrolled. All patients received the therapy at the recommended dose and were included in the analysis. The study involved 15 (71.4%) men and 6 (28.6%) women. Although patients of any race were allowed to be included, 100% of patients were Caucasian. The median age was 56 years (range 38-75). Eight (38%) patients were over 60 years old and 2 (9.5%) were over 70 years old. Five (23.8%), 12 (57.1%), and 4 (19%) patients had an ECOG PS of 0, 1, and 2, respectively. Nineteen (90.5%) patients had symptoms of the disease of varying severity. Fourteen (66.7%) patients had metastases in two and more organs. Liver metastases were identified in 9 (42.9%) patients. Bone metastases were observed in 3 (14.3%) patients. The median time from the diagnosis of gastric cancer to enrollment in the study was 27.3 months. (range 3.7 -85.5).
Surgery was performed in 15 (71.4%) patients with a history of local or locally advanced gastric cancer. All patients had gastric adenocarcinoma (N=21, 100%). There were no patients with gastroesophageal junction cancer or esophageal adenocarcinoma. The number of patients who received 2 or more previous lines of therapy for metastatic disease was 18 (85.7%). Four (19%) patients had HER2-positive gastric cancer treated with trastuzumab in previous lines. Ramucirumab with paclitaxel or immunotherapy with nivolumab were used in the second-and subsequent lines in 5 (23.8%) and 2 (9.5%) patients, respectively. Other treatments included various chemotherapy options with platinum-and fluoropyrimidinebased regimens (N=21, 100%). Patient and treatment characteristics are presented in Table 1.

Safety
During dose escalation, no DLTs were observed, and MTD was not defined. Per protocol, the maximum administrated dose was 350 mg/m2, 5 days on, 2 days off till disease progression or unacceptable toxicity. This dose will be RP2D. Any grade adverse events, regardless of treatment with alofanib, were observed in 20 (95.2%) patients. Adverse events (both associated and not associated with alofanib) of grade ≥3 were found in 10 (47.6%) patients.
Fifteen (71.4%) patients had at least one adverse event associated with the treatment with alofanib (TRAE, Table 2). Grade 3 or higher TRAEs were observed in 6 patients (28.6%) and were not related to dose level (Table 3). Grade 3-4 TRAEs included elevated levels of ALT/AST at a dose of 50 mg/m2, neutropenia at a dose of 50 mg/m2, anemia at a dose of 50 mg/m2, diarrhea at a dose of 165 mg/m2, headache at a dose of 165 mg/m2, elevated serum amylase levels at 350 mg/m2, and general reactions immediately after intravenous administration of alofanib (facial flushing, dizziness, weakness, sweating, and sinus tachycardia) at 350 mg/ m2. The most common TRAEs of all grades included reactions immediately after intravenous administration, diarrhea, thrombocytopenia, arthralgia, headache, and hyperkalemia. ECOG PS and body weight changes did not differ significantly at different points of observation. Hyperphosphatemia (grade 2), an on-target off-tumor adverse event of FGFR inhibition, was found in only 1 (4.8%) patient.
At the discretion of the investigators, 2 (9.5%) patients discontinued treatment due to TRAEs while receiving alofanib for more than 30 days. Withdrawal was due to uncontrolled grade 3 diarrhea in one patient and grade 4 reaction immediately after intravenous administration of alofanib in the second patient. Treatment was interrupted due to TRAEs (elevated levels of ALT/AST and neutropenia) in 2 (9.5%) patients. These TRAEs occurred more than one month after the start of alofanib therapy and did not represent DLTs. None of the patients withdrew consent on their own due to tolerability or the regimen of alofanib administration.
SAEs were recorded in 6 patients, which accounted for 28.6%. In one patient (4.8%) SAE (general reaction immediately after intravenous administration) was associated with the use of alofanib, the association was not established in 5 (23.8%). There were no deaths due to toxicity of trial treatment.

Antitumor activity
As of the last on-study imaging performed on November 2022, the objective response rate was 5.26% (1 of 19 assessed patients; 95% CI 0-15.8%). Patient had a partial response. The time to response was 56 weeks. The patient continued to receive alofanib till development of acute anastomositis as a previous surgical complication and emergency hospitalization. Withdrawal of alofanib therapy resulted in a relapse at 79th week (one month after discontinuation of alofanib), which was confirmed by biopsy and subsequent histological examination. The DOR was 18.53 months. This patient had no any FGFR2 molecular changes.
The rate of radiographically confirmed stable disease was 63.2% (   control was found in the study (partial response was found at a dose of 50 mg/m2, disease control rates in cohorts 1-5 were 100%, 66.7%, 66.7%, 66.7%, 57.1%, respectively). Patients with 2 and more organs with metastases had numerically lower DCR comparing to patients with single organ affected by metastases (57.1% vs. 71.4%; P=0.5). There was no difference in DCR in patients with or without liver metastases (66.7% and 70%; P=0.88). As an example, one patient with multiple large liver metastases had a tumor mass reduction of 24% and 7%. This was a 53-year-old male with heavily pretreated FGFR2-IHC-positive/FGFR2-FISH-negative gastric cancer. He received first-line therapy with mDCF (11 cycles), second-line with FOLFIRI (18 cycles), third-line with paclitaxel and ramucirumab (3 cycles), fourth-line with capecitabine (4 cycles), fifth-line with nivolumab (5 doses), and sixth-line with leucovorin + etoposide + 5FU (2 cycles). At the time of inclusion in the study, the patient had primary tumor of the stomach, multiple metastases in lymph nodes and liver. Further disease progression during therapy with alofanib was due to the appearance of new metastases in the lymph nodes. At a median follow-up of 19.5 months, 1 (4.76%) patient was alive and remained on therapy without signs of disease progression. The median OS was 7.0 (95% CI 3.82-10.18) months. The median OS in patients achieved disease control was 10.08 (95% CI 4.00-16.15) months. The 6-and 12-month OS rates were 57.1% and 33.3%. The median PFS was 3.63 (95% CI 1.58-5.68). Table 4 summarizes efficacy data.

Pharmacokinetics and biomarkers
The geometric mean values of Cmax, AUC0-t, T1/2, Vd increased and CL, Kel decreased approximately doseproportionally after single dosing (Table 5), similar to previous preclinical studies [14]. The decrease in the mean value of the Vd for a dose of 350 mg/m2 may be associated with a significant increase in AUC0-t. No correlations between PK values and objective response rate, PFS, and OS as well as in patients with liver metastases were found (all P >0.1).
After evaluating IHC expression in the first patients, further study was carried out only using the Abcam EPR24075-418 assay due to pronounced nuclear staining with Abcam 1G3 assay. Three of 17 (17.6%) patients had FGFR2 2+/3+ expression by IHC EPR24075-418 test. Two of 17 (11.8%) patients had FGFR2 amplification by FISH. Compared with the positive FISH results, only one test was positive in same patient by IHC. There was intratumoral heterogeneity in primary tumor samples. Figure 1 shows examples of FGFR2 FISH/IHC positive and negative samples. One IHC/FISH-positive patient and one IHC-positive/FISH-negative patient had stable disease with PFS of 7.0 and 4.7 months, respectively.

Discussion
This phase 1b clinical trial was designed to evaluate the safety and preliminary efficacy of alofanib, a first-in-class allosteric extracellular FGFR2 inhibitor. Patients with metastatic gastric cancer who had previously progressed on all standard treatments received alofanib till unacceptable toxicity or disease progression. Over the past 5 years, the development of FGFR inhibitors has attracted attention, and patients with FGFR-aberrant gastrointestinal tumors have been included in several clinical trials [15]. In contrast to alofanib, these small molecule inhibitors blocked the tyrosine kinase domain of receptor and all types of FGFRs, which could lead to a variety of adverse events. For example, 64% of patients treated with pemigatinib and infigratinib, pan-FGFR tyrosine kinase inhibitors, had grade ≥3 adverse events [16,17]. Moreover, hyperphosphataemia, which is mainly associated with FGFR1 inhibition, was the most common all-grade adverse event irrespective of cause (60%-77%) [16,17]. In five dose cohorts, alofanib was well tolerated with no DLT. Only a third of the patients had treatmentrelated toxicity of grade 3 or higher and only one patient had grade 2 hyperphosphatemia. Commonly reported TRAEs included diarrhea, thrombocytopenia, arthralgia, headache, and hyperkalemia. Intravenous administration of alofanib caused a hypersensitivity reaction, including facial flushing, dizziness, weakness, sweating, and sinus tachycardia. Similar reactions to alofanib have been observed in preclinical studies [18]. However, these adverse events resulted in the discontinuation of treatment in a single patient. In other cases, the hypersensitivity reaction passed by itself or was resolved with diphenhydramine. The study did not record any treatmentrelated deaths. Low toxicity of alofanib makes it an attractive candidate for combination with chemotherapy.
Heavily pretreated patients might have less benefit and worse tolerability from systemic therapy. In the current study alofanib provides clinically meaningful disease control with good tolerability in 13 out of 19 patients across all doses, with one durable partial response. Discontinuation of therapy due to adverse events unrelated to the alofanib resulted in disease progression. This fact suggests that alofanib therapy should be continued even in the event of a prolonged response or stable disease. In our study, the median PFS was 3.63 months. Patients treated in the third or later lines had a median OS of 7.0 months and those with disease control had a median OS of 10.1 months. Seven out of 21 (33%) heavily pretreated patients were alive after 1 year. The standard of care for patients with advanced gastric cancer after platinum-and ramucirumabbased therapy is trifluridine/tipiracil [19]. The TAGS study, in which patients with metastatic gastric adenocarcinoma treated with at least two prior lines of chemotherapy were recruited, reported a median OS of 5.7 months (95% CI 4.8-6.2) and 1-year OS rate of 21.2% [20,21]. Median PFS was 3.1 and 1.9 months in the third-line and fourth-or later-line treatment groups, respectively [22].  Previous analysis suggests that chemotherapy has little efficacy in patients with FGFR2 expression or rearrangements [15,23]. In the randomized phase 2 trial (FIGHT), first-line FGFR2b monoclonal antibody bemarituzumab in combination with mFOLFOX6 significantly improved PFS compared with chemotherapy alone (HR=0.68) in patients with FGFR2/isoform IIb-selected metastatic gastric adenocarcinoma [23]. When our study was initiated in 2019, predictors of extracellular FGFR2 inhibitor sensitivity were limited. In addition, there were no biomarkers for compounds that bind outside the active site of the receptor. Therefore, patients were included in the study, regardless of the FGFR2 expression or amplification as determined by IHC and FISH tests. The frequency of FGFR2 molecular changes was as described previously. However, a small number of FGFR2-positive patients does not allow to determine the predictive role of molecular changes and limits an accurate conclusion. The single-arm, open-label design might be another study limitation, but a randomized phase 2 trial design is currently being developed in which alofanib will be studied in combination with first-line chemotherapy.
Taken together, subsequent therapy with alofanib in patients with advanced gastric cancer was tolerable and with manageable toxicity. The RP2D was determined to be 350 mg/m2, 5 days on, 2 days off schedule. Alofanib has demonstrated preliminary antitumor activity, strongly supporting further biological and clinical studies.