TFX06 exhibits potent ERα antagonist and degradation activity in vitro.
In HEK293/GAL4/ERα reporter cell line, TFX06 (WM = 602.63; Fig. 1a) demonstrated highly potent ERα antagonist activity with an IC50 of 0.101 nM, which was superior to the reference agents, including GDC9545 (0.218 nM), AZD9833 (0.425 nM), fulvestrant (1.421 nM), and elacestrant (2.766 nM) (Fig. 1b). In addition, treatment of cultured ER+ MCF-7 BC cells with TFX06 reduced the expression levels of ERα protein with an IC50 of 0.24 nM more potently than AZD9833 (0.47 nM) and fulvestrant (0.24 nM) (Fig. 1c). These results suggest that TFX06 possesses more potent ERα antagonism activity in comparison with GDC9545, AZD9833, fulvestrant and elacestrant, as well as more potent ERα degradation activity in comparison with AZD9833.
The following studies were frequently conducted side-by-side with AZD9833 because the latter is the most clinically advanced oral SERD demonstrating improved efficacy and safety compared to elacestrant 13,14.
TFX06 inhibits the proliferation of ER+ MCF-7 BC cells in vitro.
TFX06 inhibited cell proliferation with a lower IC50s than AZD9833 in ER+ MCF-7 parental cells harboring ESR1WT (IC50 = 0.18 and 0.35 nM, respectively), MCF-7 cells harboring ESR1Y537S (11.17 and 31.35 nM) or ESR1D538G (2.90 and 9.32 nM) mutation (Fig. 1d-f). The results suggest that TFX06 has more potent antiproliferative activity than AZD9833 in BC cells carrying either wild-type or mutant ESR1 genes.
TFX06 demonstrates substantial single-agent antitumor activity and synergizes with palbociclib in ER+ MCF-7 BC CDX models
We then investigated whether TFX06 inhibited the tumor growth in mouse xenograft tumor models in vivo. In mice bearing ER+ MCF-7 BC CDX tumors, oral administration of TFX06 at a dose of 1, 3, 10, or 30 mg/kg exerted antitumor activity in a dose-dependent manner with TGI rates of 85.3% (3 mSD plus 5 mPD: P = 0.003), 100.9% (8 mSD; P = 0.001), 104.6% (2 mPR plus 6 mSD; P = 0.001) and 106.5% (2 mPR plus 6 mSD; P = 0.001), respectively, at the end of treatment (day 21) (Fig. 2a). The results of mRECIST analysis in eight animals of each group and statistical analyses in comparison with the vehicle control were shown in the brackets. No significant body weight changes was observed in TFX06-treated animals in comparison with the vehicle- treated animals (Fig. 2b).
In the same experiment, TGI rates of fulvestrant (5 mg/mouse), AZD9833 (3 mg/kg), and palbociclib (25 mg/kg) were 60.9% (8 mPD; P = 0.019), 57.4% (8 mPD; P = 0.029), and 42.8% (1 mSD plus 7 mPD; P = 0.219), respectively (Fig. 2c). According to the TGI assessment, fulvestrant and AZD9833 exerted antitumor effects as single agent, whereas palbociclib monotherapy demonstrated limited activity with only one of eight animals exhibiting stable disease. Compared with these three reference agents dosed at the specified regiments, TFX06 at 3 mg/kg exhibited a greater antitumor activity with a disease control rate (DCR) of 100%. In the combination arm, TFX06 (3 mg/kg) plus palbociclib led to a TGI of 110.5% (7 mPR plus 1 mSD; P = 0.001). The objective response rate (ORR) reached 87.5% (Fig. 2d). The synergy score of the combination treatment was 1.44, indicating that the combination displayed synergistic activity. No substantial body weight loss was observed in the treatment arms of the efficacy studies shown in Figure 2c and 2d (data not shown).
Overall, in comparison with AZD9833, TFX06 exerts more potent antitumor activity in the ESR1WT BC CDX model. In addition, TFX06 in combination with palbociclib demonstrates synergistic antitumor activity in the CDX model.
TFX06 exerts single-agent antitumor activity and synergizes with palbociclib in ER+ BC CDX and PDX models carrying ESR1 mutations.
We next investigated the antitumor activity of TFX06 in mice bearing ESR1-mutant xenograft tumors. First, single-agent activity of TFX06 was evaluated in ER+ MCF-7 BC CDX model carrying a ESR1D538G-mutant gene. Oral administration of TFX06 at a dose of 5, 15, or 30mg/kg led to dose-dependent antitumor activity with TGI rates of 98.9% (4 mSD plus 4 mPD; P = 0.0001), 104.5% (1 mPR plus 7 mSD; P = 0.0001), 120.5% (3 mCR plus 5 mPR; P = 0.0001), respectively (Fig. 3a). AZD9833 at 15 mg/kg showed antitumor effects with a TGI rate of 91.9% (1 mPR plus 4 mSD plus 3 mPD; P = 0.0001). The DCRs of TFX06 and AZD9833 at the same dose level were 100% and 62.5%, respectively, suggesting that TFX06 is more potent in inhibiting growth of the mutant tumors than AZD9833.
In the same experiment using the ESR1D538G-mutant CDX model, the combination treatment of TFX06 (5 mg/kg) plus palbociclib was simultaneously evaluated (Fig. 3b). Palbociclib at 50 mg/kg demonstrated single-agent antitumor activity with a TGI rate of 70.9% (4 mSD plus 4 mPD; P = 0.0001). As described above, TFX06 at 5 mg/kg caused a TGI rate of 98.9% (4 mSD plus 4 mPD; P = 0.0001). The combination treatment resulted in a remarkable TGI of 122.83% (1 mCR plus 7 mPR; P = 0.0001). Clearly, the ORR reached 100% in the combination arm in comparison with 0% in both palbociclib and TFX06 single agent arms. A synergy score of 2.21 was obtained, which indicated significant synergistic activity by using the combination treatment.
ESR1Y537S is another frequently identified mutation in patients who developed resistance to ET10. Thereby, ESR1Y537S-mutant xenograft models were next employed to investigate antitumor activity of TFX06 as single agent. In ER+ MCF-7 BC CDX model harboring ESR1Y537S mutation, TFX06 at 10 mg/kg exerted substantial antitumor activity with a TGI rate of 127.8 % (6 mPR; P = 0.0001), while AZD9833 at 10 mg/kg showed antitumor effects with a TGI rate of 120.4 % (4 mPR plus 2 mSD; P=0.0001) (Fig. 3c). The ORR of 100% and 67% were achieved by TFX06 and AZD9833, respectively, suggesting that TFX06 is more potent against ESR1Y537S-mutant tumors than AZD9833.
Furthermore, in an ER+ BC PDX model HSC-013 harboring ESR1Y537S mutation, TFX06 at 3 mg/kg exerted substantial antitumor activity with a TGI rate of 99.4% (8 mSD; P = 0.001) and DCR of 100% (Fig. 4d).
No substantial mouse body weight loss was observed in the treatment arms of efficacy studies shown in Figure 3 (data not shown).
The above results indicate that TFX06 is highly effective in overcoming drug resistance conferred by ESR1 mutations in both CDX and PDX models. In comparison with AZD9833, treatment with TFX06 results in deeper responses in the mutant tumor models.
TFX06 demonstrates an excellent pharmacokinetic profile and pharmacokinetic-pharmacodynamic correlation in subcutaneous ER+ MCF-7 CDX models in mice.
In the efficacy experiment conducted in an ER+ MCF-7 CDX model shown in Figure 2a, pharmacokinetic characteristics of TFX06 were investigated by analyzing the plasma samples collected from the tumor-bearing animals (see Methods for the details). The results showed that plasma Cmax and AUC0-last of TFX06 increased approximately in a dose proportional manner, indicating a linear pharmacokinetic profile for TFX06 (Fig. 4a and Table 1).
Table 1. Pharmacokinetic characteristics of TFX06 in mouse plasma.
Parameters
|
TFX06
|
1 mg/kg
|
3 mg/kg
|
10 mg/kg
|
30 mg/kg
|
Cmax (nM)
|
37.6
|
97.8
|
374
|
1130
|
Ctumor (nmol/kg)
|
357
|
828
|
3540
|
10500
|
Tmax (h)
|
2
|
1
|
2
|
2
|
T1/2 (h)
|
7.06
|
4.75
|
4.92
|
4.55
|
AUC0-last (nM·h)
|
436
|
950
|
2990
|
10300
|
AUC0-inf (nM·h)
|
484
|
983
|
3100
|
10600
|
MRT0-last (h)
|
8.27
|
7.09
|
6.5
|
6.93
|
MRT0-inf (h)
|
10.8
|
7.89
|
7.38
|
7.59
|
The results were presented as mean (n = 4; i.g., 8 mice were divided into 2 groups to cover various timepoints)
|
Xenograft tumor tissues collected from the efficacy experiment (Figure 2a) were subject to pharmacodynamic analysis by using Western blotting to assess target modulation by TFX06. The results showed that the treatment with TFX06 at a dose of 1, 3, 10, or 30 mg/kg induced a decrease of 63% – 37% in the expression levels of ERα protein in tumor tissues relevant to the vehicle control. The maximal pharmacodynamic activity of TFX06 was achieved at 3 mg/kg (Fig. 4b and 4c). Therefore, the target engagement of TFX06 was confirmed.
Taken together, the above pharmacokinetic-pharmacodynamic results, in combination with the antitumor activity, elucidate the correlation of pharmacokinetic-pharmacodynamic-efficacy of TFX06 in a xenograft tumor model.
To further explore the drug exposures in the tumor tissues and brain penetrating property, xenograft tissues and cerebrospinal fluid (CSF) samples collected from animals in the MCF-7 CDX efficacy study shown in Figure 2a were subjected to LC/MS/MS analysis. The results showed that TFX06 concentrations in the tumor tissues generally exceeded those in the plasma with a tumor-to-plasma ratio of approximately 9 after the tumor-bearing mice were treated with TFX06 at various doses (e.g., 1, 3, 10, 30 mg/kg) (Fig. 4d). Interestingly, TFX06 was also detected at a significant level in the cerebrospinal fluid (CSF) at the dose levels above 10 mg/kg. The results suggest that TFX06 exhibits a favorable target tissue distribution and the potential to penetrate the brain blood barrier (BBB), which may benefit the patients with brain metastases.