The antibody–drug conjugate targeting ROR1,NBE-002,is active in high-grade serous ovarian cancer preclinical models

ROR1 ADCs construction

NBE-002 (huXBR1-402-G2-EDA-PNU) was generated as described. ³⁹ NBE-002 and its isotype control ADC (Qb3-LC-G2-EDA-PNU) were diluted in phosphate-buffered saline (PBS). Stock solution was stored at −80°C and the working dilutions were prepared freshly. ADCs were prepared in a 3.5-fold serial dilution starting from 8 µg/mL. PA-1 was plated in 96-well plates and incubated with ADCs for 6 days. CellTiter-Glo cell viability assay (#G9243; Promega, Madison, Wisconsin, USA) was applied to assess the dose–response following the treatment of the ADCs.

Cell culture

The ovarian teratocarcinoma cell line PA-1 was cultured in MEM complete medium (supplemented with 10% foetal bovine serum, 1× GlutaMax and 1% Pen/Strep). HGSOC cell lines OVCAR4, Kuramochi, PEO1, PEO4 and OAW28 were cultured in RPMI complete medium. All cell lines were incubated at 37°C and 5% carbon dioxide, and were tested routinely for mycoplasma contamination.

PA-1 cell line xenograft model

Female NMRI-Foxn1nu (BomTac:NMRI-Foxn1nu) mice were purchased from Taconic, Denmark and allowed to acclimate for at least 5 days before the start of the experiments. All animals were housed in Macrolon^® type III cages in groups of 8–10 animals under standardised conditions (21.5 ± 1.5°C temperature, 55% ± 10% humidity, 12 h light–dark cycles), and received a standardised diet (PROVIMI KLIBA) and autoclaved tap water ad libitum. Each animal was identified using subcutaneous microchips implanted under isoflurane anaesthesia. On the day of injection, PA-1 cells were harvested by centrifugation, washed and resuspended in 5% FBS (in PBS 1×) at a concentration of 1 × 10⁸ cells/mL. The cell suspension was mixed at a 1:1 ratio with Matrigel (#356231; Corning, New York, USA) to obtain a final concentration of 5 × 10⁷ cells/mL and 100 µL of the cell suspension was injected subcutaneously into the right flank of ~7-week-old mice. Tumour volumes (in mm³) were determined three times a week via calliper measurements and the following formula: Tumour volume = length * width2 * π/6. Once tumours reached volumes of 117.18–226.82 mm³, mice were randomly distributed across treatment and control groups by tumour size using the data storage system Sepia. Group sizes were determined by sample size calculation and animals were housed by treatment groups. All compounds and vehicle controls were administered i.v. at a volume of 5 mL/kg, by experimenters that were not blinded to the treatment. To monitor treatment side effects, mice were inspected daily and body weight was measured thrice a week. Animals were euthanised by cervical dislocation when their tumours exceeded a volume of 1500 mm³, in which case the tumour volume was carried forward in the analysis. In the experiment depicted in Figure 1(b)–(d), three animals reached humane endpoint prior to the end of the experiment (day 18): two animals in the isotype ctrl ADC, 0.11 mg/kg group (one emaciated mouse on day 14 and one mouse with a tumour volume >1500 mm³ on day 16) and one animal in the isotype ctrl ADC, 0.33 mg/kg group (tumour volume >1500 mm³ on day 16) had to be sacrificed. The lowest dose of NBE-002 was adjusted from 0.11 mg/kg (first experiment; Figure 1(b)–(d)) to 0.165 mg/kg (second experiment, Figure 1(e)–(i)) to directly compare the efficacy of the 0.33 mg/kg weekly dose administered according to two dose schedules: once and twice split-dose regimen per week. A 0.11 mg/kg dose given 3 times/week to maintain the same total weekly dose was deemed impractical due to potential stress on the animals. In Figure 1(e)–(i), tumour volume comparisons were performed on day 16, as the vehicle and isotype control groups had reached a humane endpoint by this time. Additionally, two animals had to be euthanised before the experiment endpoint in the NBE-002, 0.165 mg/kg, q7d group (one on day 25 due to body weight loss and one on day 39 due to tumour volume >1500 mm³), two in the NBE-002, 0.33 mg/kg, q7d group (one on day 42 and one on day 65 due to tumour volume >1500 mm³), two in the NBE-002, 0.33 mg/kg, single dose group (both on day 23 due to tumour volume >1500 mm³) and two in the NBE-002, 0.165 mg/kg, q3/4d group (one on day 23 and one on day 65 due to tumour volume >1500 mm³).

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Figure 1.

ROR1 ADC showed anti-tumour effects in ROR1-high PA-1 models. (a) Dose–response curves of PA-1 to a range of ROR1 ADCs at day 6. (b) Tumour growth (left panel; mean ± SEM) and average body weight change (right panel) of mice subcutaneously injected with the PA-1 ovarian teratocarcinoma cell line (xenograft) and treated with either NBE-002 or its isotype control ADC at the indicated doses and schedules. (c) Individual tumour volume in mm³ on day 18 (last day of the experiment) and (d) Change from baseline (tumour volume on day 18 normalised to tumour volume at the start of treatment) from the experiment depicted in (c). Each dot or bar represents one animal; the horizontal lines in (d) represent the mean tumour volume. (e) Tumour growth (left panel; mean ± SEM) and average body weight change (right panel) of mice subcutaneously injected with the PA-1 ovarian teratocarcinoma cell line (xenograft) and treated with either NBE-002 or its isotype control ADC at the indicated doses and schedules. The last treatment was administered on day 22, after which tumour regrowth was monitored. (f) Individual tumour volume in mm³ on day 16 (last day of the vehicle control group) and (g) change from baseline (tumour volume on day 16 normalised to tumour volume at the start of treatment) from the experiment depicted in (e). Each dot or bar represents one animal. (h) Individual tumour volume in mm³ on day 44 (when the first NBE-002-treated group was stopped) and (i) change from baseline (tumour volume on day 44 normalised to tumour volume at the start of treatment) from the experiment depicted in (e). Each dot or bar represents one animal. The horizontal lines in (f) and (h) represent the mean tumour volume. One-sided non-parametric Mann–Whitney–Wilcoxon U tests with Bonferroni–Holm adjustments; for (c) and (f), top row: comparison to PBS control, bottom row: comparison to the respective isotype control groups. Change from baseline values is capped at 100%.

Ascites-derived primary cell lines

Four HGSOC patient ascites-derived cell lines (OC012, OC017, OC018 and OC023) were established following the procedure outlined in Shepherd et al. ⁴⁰ In summary, the ascites fluid was transferred into the culture flasks with the addition of an equal volume of the complete RPMI medium. The medium was replenished every 3–4 days once most of the cells were established. Cells were trypsinised and passaged when confluent. Only early passages of the cells (less than four passages) were applied for cell assays to minimise the risk of morphological or other changes that may occur with prolonged passaging.

Western blot

Protein was extracted from the cell lines using cell lysis buffer (Cell Signalling Technology, Danvers, Massachusetts, USA) and supplemented with protease and phosphatase inhibitors (Cell Signalling Technology, Danvers, Massachusetts, USA). The protein concentration was determined using the Pierce bicinchoninic acid assay kit (ThermoFisher Scientific, Waltham, Massachusetts, USA). Subsequently, Western Blot analysis was performed as previously outlined.41 Primary antibodies, specifically monoclonal rabbit anti-ROR1 (#AF2000; R&D Systems, Minneapolis, Minnesota, USA) and monoclonal mouse anti-α-Tubulin (#3873; Cell Signalling Technology, Danvers, Massachusetts, USA), were employed in this analysis.

ROR1 surface flow cytometry

Cell surface ROR1 levels on the HGSOC cells were assessed using flow cytometry. In brief, the cells were initially blocked with Human TruStain FcX (#422302; BioLegend, San Diego, California, USA) for 20 min at 4°C. Subsequently, they were incubated with either a PE anti-human ROR1 antibody (#357804; BioLegend, San Diego, California, USA) or an isotype control antibody (#400112; BioLegend, San Diego, California, USA) for 60 min at 4°C in the dark. Cell fluorescence of ROR1 (PE channel) was measured using the flow cytometer (BD FACSCanto II, BD Biosciences, San Jose, California, USA). BD Quantibrite beads (#340495; BD Biosciences, San Jose, California, USA) were run in parallel as internal standards to quantify the PE levels as per the manufacturer’s protocol.

Dose–response assays

Cells were plated in 96-well plates within their specific media and incubated overnight. On the following day, cells were treated with NBE-002 using 1:2 serially diluted doses ranging from 0.1 to 20 µg/mL, or vehicle control (0.1% DMSO) in triplicates. Annexin V Red Dye at a dilution of 1:400 was also applied to stain apoptotic cells. Cell confluency and red fluorescent cells were monitored for a total period of 120 h in the IncuCyte S3 Live Imaging system (Sartorius, Goettingen, Germany). Both fluorescent and phase images were captured every 3 h at a 10× magnification. For each cell line, three independent tests, each with triplicates, were conducted. Cell confluence of each time point was normalised against the baseline. The relative cell apoptosis was calculated by dividing the red fluorescence area over the phase confluence area. A non-linear regression model was fit to estimate the half maximal inhibitory concentration (IC50) dose at 120 h.

Interactive analysis of combined treatments

To evaluate the combined effect of NBE-002 in conjunction with commonly used therapies, we subjected HGSOC cell lines (OVCAR4, Kuramochi, PEO1 and PEO4), along with a chemoresistant primary cell line (OC023), to various treatment conditions. Specifically, we treated these cells with different concentrations of NBE-002 (0.625, 1.25, 2.5, 5, 10 µg/mL) either alone or in combination with carboplatin (0, 3.125, 6.25, 12.5, 25, 50 µM), paclitaxel (0, 1.25, 2.5, 5, 10, 20 nM) or olaparib (0, 3.125, 6.25, 12.5, 25, 50 µM). SynergyFinder 2.0 ⁴² was applied to visualise and estimate the combination effects, employing the Bliss independence model to calculate combination factors.

ROR1 immunohistochemistry

Formalin-fixed paraffin-embedded PDX tumour samples were sectioned and automated immunohistochemistry was performed on the BOND RX (Leica Biosystems, Nussloch, Germany) with anti-ROR1 antibody. ¹⁷ IHC slides were scanned digitally at 20× magnification using the Pannoramic Scan II scanner (3DHISTECH Ltd., Budapest, Hungary) and high-definition images were uploaded into CaseCenter (3DHISTECH Ltd., Budapest, Hungary). Membranous ROR1 staining was scored by a gynaecological pathologist and an H-score was determined; H-score = (%strong positive × 3) + (%moderate positive × 2) + (%weak positive × 1).

PDX generation and in vivo treatment

All animal research was performed according to the Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition, 2013 (updated 2021) and was approved by the WEHI Animal Ethics Committee (2019.024 and 2022.030).

Female NSG (NOD-scid IL2Rg^null) mice 6–10 weeks of age were obtained from the WEHI-specific pathogen-free breeding facility and allowed to acclimate for at least 3 days before the start of the experiments. Animals were housed in Tecniplast Emerald IVC enclosures under standard conditions (21 ± 3°C temperature, 55% ± 15% humidity, 14 h/10 h light–dark cycles). Each animal was identified using subcutaneous microchips implanted under isoflurane anaesthesia.

The following HGSOC PDX models have been described previously: #56, #62, #201, ⁴³ #32, #111, #931 ⁴⁴ and #56 pp. ⁴⁵ PDX models #22, #419 and #448 were generated from fresh biopsy or surgical specimens, which were cut into small fragments and transplanted subcutaneously on the flank of NSG mice. ⁴³ PDX models were validated by histological review, conducted by a gynaecological pathologist, and by BROCA panel sequencing (mutation match with patient sample). Relevant clinical data and key molecular features are presented in Supplemental Table 2.

Reagents for in vivo treatments were human immunoglobulin (also known as IVIg; Privigen; CSL Behring, Parkville, Victoria, Australia), NBE-002, Qb3-LC-G2-EDA-PNU (referred to as isotype control; anti-Qb3 antibody recognises a bacteriophage Qβ protein), carboplatin (Pfizer, New York City, New York, USA) and olaparib (MedChemExpress, Monmouth Junction, New Jersey, USA). Olaparib was prepared in 10% DMSO and 10% hydroxypropyl-β-cyclodextrin. The vehicle for all other treatments was Dulbecco’s phosphate-buffered saline (DPBS). NSG mice bearing passage 5–9 tumours (180–300 mm³ in size) were randomly assigned to the following treatment regimens, weekly cycles for 4 weeks. Rolling enrolment was used, with mice being assigned equally across the treatment arms as tumours reached the treatment volume. Tumour measurement, randomisation and treatments were each performed by separate researchers or technicians, with technicians blinded to the study hypothesis, design and data analysis. Studylog software (Studylog Systems Inc., San Diego, California, USA) was used to record and track tumour volume and Animal Management System software (WEHI, Melbourne, Victoria, Australia) to manage animal procedures and ethics.

Digital calliper tumour measurements were taken twice weekly until tumour volume reached >700 mm³ (humane endpoint for tumour volume, 1000 mm³) or mice reached the experimental endpoint, 120 days post-treatment. Additionally, humane endpoints were reached for 10 mice that were assessed by a veterinarian. Mice were euthanised and post-mortems were conducted: three mice had thymic lymphomas (days 35, 88 and 108 from the start of treatment), which are known to occur occasionally in NSG mice; one mouse had an ulcerated tumour (day 43); three mice had weight loss that reached humane endpoint (−20%; days 9, 63, 84) and three mice were hunched and appeared unwell but post-mortem did not identify a cause (days 31, 108 and 134). Five mice with weight loss or that were hunched were on a carboplatin treatment regimen, one mouse single agent carboplatin, three mice isotype control + carboplatin and one mouse NBE-002 + carboplatin. One mouse with weight loss received NBE-002 + olaparib treatment. These events are censored on the Kaplan–Meier survival curves as the tumour volume endpoint was not reached.

Statistical analysis

One-tailed non-parametric Mann–Whitney–Wilcoxon U test was performed to compare the effect of the treatments on the vehicle control group, as well as to compare the effect of each dose and schedule of NBE-002 to its respective isotype control ADC treatment group in the PA-1 xenograft models. Within each subtopic, the p values of the efficacy parameters were adjusted for multiple comparisons according to Bonferroni–Holm. Group analysis was stopped when <70% of the animals within the group remained and curves were truncated on the graphs past those timepoints (in Figure 2 on day 60 for the NBE-002, 0.165 mg/kg, q7d group, on day 44 for the NBE-002, 0.33 mg/kg, single dose group and on day 70 for the 0.165 mg/kg, q3/4d group). Two-way ANOVA (time and treatment as factors) was performed to compare cell confluence or tumour growth between various treatments. Post hoc Tukey tests were applied to correct for multiple comparisons. Kaplan–Meier survival analysis (with tumour volume at the endpoint) was performed using the log-rank test. Statistical analysis and figure generation were carried out using GraphPad Prism (v10.2.0; GraphPad Software, San Diego, California, USA). Statistical significance representations: *p < 0.05, **p < 0.01, ***p < 0.001. Data were presented as mean ± standard deviation.

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Figure 2.

NBE-002 treatment inhibited the growth of ROR1-expressing HGSOC cell lines in vitro. (a) Western blot of ROR1 (Polyclonal Goat IgG, R&D systems, AF2,000) in HGSOC cell lines, as well as high and low ROR1 expressed control cell lines PA-1 and OAW28. (b) Cell surface ROR1 expression level of the cell lines was analysed with PE anti-ROR1 antibody (BioLegend, clone 2A2) using BD Quantibrite Beads. (c)–(i) Cell confluency of the HGSOC cell lines following ROR1 ADC treatment by IncuCyte S3 imaging for a total period of 120 h. Two-way ANOVA (time and treatment as two factors) was performed to assess the mean differences of cell confluency across treatments, with Tukey’s multiple comparison test used to adjust the p value. (j) Dose–response curves and IC50 of NBE-002 at 120 h. For panels (c)–(j), n = 3, the error bar represents the standard deviation.

Single-agent NBE-002 was active even at a low dose in vitro and in vivo in ROR-high expressing preclinical models

We constructed a range of ROR1 ADCs, with different ROR1-targeting antibodies and payloads, (DAR shown in Figure 1(a)). The effects of the ADCs were evaluated using the high ROR1-expressing ovarian teratocarcinoma cell line, PA-1. ROR1 ADCs (adc1923, adc1155) exhibited a higher level of sensitivity than did their corresponding isotype control mAbs targeting CD30 (Figure 1(a)). Notably, adc1155, with the LC-G2-EDA-PNU payload, exhibited greater activity than adc1923, which had a vc-PAB-MMAE payload. Consequently, adc1155, referred to as NBE-002 henceforth, was selected for subsequent downstream analysis. In the PA-1 xenograft model, NBE-002 administered at 0.11 or 0.33 mg/kg every 7 days (q7d) significantly suppressed the tumour growth at day 18 relative to the respective isotype control ADC groups (Figure 1(b)–(d), p = 0.02 and 0.0003, respectively). Additionally, no average body weight loss was observed in any of the treatment groups (Figure 1(b), right panel). In the same PA-1 xenograft model, the efficacy of NBE-002 with 0.33 mg/kg weekly dose was assessed according to two dose schedules. Mice received 0.33 mg/kg either as a single dose or repeated once a week (q7d x3) or 0.165 mg/kg, either as a single dose or repeated twice a week (q3/4d x3). All tested dosing regimens resulted in a significant inhibition of tumour growth when compared to the vehicle control or the corresponding isotype control ADC (at day 16; Figure 1(e)–(g)). Following treatment discontinuation, the most prolonged response across all tested doses and schedules was observed in the animal group which received 0.33 mg/kg NBE-002 on a once weekly schedule. The responses were sustained overtime (at day 44; Figure 1(h) and (i)).

Single-agent NBE-002 treatment inhibited HGSOC cell growth in vitro across a range of HGSOC cell lines

ROR1 expression was profiled in the HGSOC cell lines, OVCAR4, Kuramochi, PEO1, PEO4. Derived from the same BRCA2-mutated HGSOC, from samples collected at different times in the patient trajectory, PEO1 is chemosensitive and HRD, whilst PEO4 is chemoresistant and HRP, due to treatment pressure resulting in a secondary BRCA2 reversion mutation. In addition, three HGSOC cell lines, OC012, OC017 and OC018, were established from patient ascites fluid and also used for subsequent experiments. Patient clinicopathological characteristics are summarised in Supplemental Table 3. The ovarian teratocarcinoma cell line PA-1 and the HGSOC cell line OAW28 were included as ROR1 high and low controls, respectively. Most of the HGSOC cell lines exhibited a moderate to low level of ROR1 expression (Figure 2(a) and (b)). Notably, the more recently derived OC cell lines demonstrated higher levels of ROR1 expression in terms of surface and total protein, compared with long-established commercial cell lines. Subsequently, we monitored the real-time growth of four HGSOC cell lines (OVCAR4, Kuramochi, PEO1, PEO4) as well as three cell lines recently derived from ascites (OC012, OC017 and OC018), following treatment with NBE-002 by IncuCyte S3 platform imaging for a total period of 120 h (Figure 2(c)–(i)). Treatment with NBE-002 at doses of 1 and 10 µg/mL significantly inhibited cell growth in all cell lines, except for OC017. Notably, for Kuramochi and ascites cell lines OC012, a dose of NBE-002 as low as 0.1 µg/ml was sufficient to significantly suppress cell growth. Commercial cell lines exhibited a lower IC50 compared with more recently derived ascites cell lines (1.89 ± 0.99 vs 4.1 ± 1.53 µg/mL, Figure 2(j), relevant p values included).

Single-agent NBE-002 treatment induced apoptosis in HGSOC cell lines

To investigate apoptosis following NBE-002 treatment, cells were incubated with Annexin V red dye (Sartorius, Goettingen, Germany) during the IncuCyte run. A significantly higher number of red-stained cells, indicative of apoptosis, was observed in HGSOC cell lines when exposed to NBE-002 at doses ranging from 0.1 to 10 µg/mL (Figure 3(a)–(n), relevant p values included). At the 10 µg/mL dose, a significant portion of cells had detached and displayed red staining in all cell lines tested; these cells are not included in the IncuCyte analysis. Kuramochi, the cell line exhibiting the highest sensitivity based on the IC50 (Figure 2(j)), demonstrated a substantial induction of apoptosis from the start of treatment (1 h prior to the beginning of the IncuCyte run), with cells detaching from the plate at the 0.1 µg/mL NBE-002 dose (Figure 3(d)).

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Figure 3.

NBE-002 induced apoptosis of HGSOC cell lines in vitro. Figures (a), (c), (e), (g), (i), (k) and (m) displayed a red Annexin V positive proportion of HGSOC cell lines over time when incubated with NBE-002 or vehicle control (n = 3 independent experiments), as detected by the IncuCyte S3 platform. Figures (b), (d), (f), (h), (j), (l) and (n) include representative images captured by the IncuCyte at 120 h, scale bar 200 µm, magnification 10×. The images were selected from one of the three independent experiments. Two-way ANOVA (time and treatment as two factors) was performed to assess the mean differences of cell apoptosis% across treatments, with Tukey’s multiple comparison test used to adjust the p value.

NBE-002 was synergistic in combination with SoC therapies in HGSOC cell lines

To assess the combination effect of NBE-002 with SoC therapies used in the clinic, we applied the Bliss model within the SynergyFinder 2.0 tool to estimate combinatorial effects (Figure 4). Since most of the ascites-derived cell lines were nearly depleted in earlier experiments, we substituted a fourth cell line, derived from a chemo-resistant HGSOC patient with ROR1 expression, in the combination analysis. A synergistic effect was observed when NBE-002 was combined with carboplatin in two out of five cell lines (PEO4 and OC023, chemo-resistant), and when NBE-002 was combined with the PARPi, olaparib, in one out of five cell lines (PEO1, BRCA2 mutated, HR deficient). No synergy was observed for NBE-002 in combination with paclitaxel in any cell line. For the majority of cell lines, combining NBE-002 with SoC therapies showed an additive effect: three out of five cell lines for NBE-002 and carboplatin combination, four out of five for NBE-002 and paclitaxel combination and four out of five for NBE-002 and olaparib combination.

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Figure 4.

Analysis of interactions between NBE-002 and standard-of-care therapies in high-grade serous ovarian cancer cell lines at 120 h. Dose–response matrices with Bliss synergy scores (values located in the bottom right corner of each panel) estimated via SynergyFinder indicated synergistic (>10, highlighted in red), subadditive (<−10, highlighted in green) or additive (−10–10) effect of the drug combination.

The activity of NBE-002 in HGSOC PDX models was dependent on ROR1 expression level and HR status

ROR1 expression was assessed in 26 PDX models of HGSOC (Supplemental Table 2). These PDX models cover the major contexts of the HGSOC patient journey, from chemo-naïve, to post-chemotherapy (following a range of numbers of lines (1–7 lines)) and post-PARPi; as well as covering the major molecular categories relevant for the clinic: HRP, HR deficient and HR reversion, having originally been HRD. ROR1 expression was scored by a pathologist, from absent to high expression (0–3) and the percentage of the corresponding tumour area was estimated. H-scores were derived, with scores ranging from 0 to 285 being observed; with more than half of the 26 distinct PDX models (62%) having low expression with an H-score <100 (median H-score 86.5). Chemo-naïve PDX models had a numerically greater representation in the ROR1 medium-to-high expression group: H-score >100, 5 out of 10 chemo-naïve (50%) versus H-score <100, 5 out of 16 chemo-naïve (31%). Five out of seven (71%) functionally HRD models (i.e. those models that were responsive to PARPi treatment, as well as being molecularly consistent with HRD), had a ROR1 H-score >100; conversely, only one of eight (12.5%) post-PARPi HRD reversion to HRP PARPi-resistant models had a ROR1 H-score >100 (Chi-square test p = 0.02). Therefore, it appeared that ROR1 expression likely decreased with disease progression. This was supported by a matched PDX pair #56 (H-score 190) and #56pp (H-score 83), generated from chemo-naïve and post-PARPi samples, respectively, collected 7 years and five lines of treatment apart, from the same patient.

Ten PDX models with a range of ROR1 IHC intensity and known HRP or HRD status were selected for in vivo investigation (Figure S1). The number of PDX models and the body weights of each experimental group are shown in Supplemental Tables 4, 5 and Figure S2, respectively. As NOD-scid IL2Rg^null (NSG) mice are highly immunodeficient and lack production of immunoglobulin, 24 h prior to treatment with NBE-002, immunoglobulin (Ig) was administered to block Fc receptors. This step reduced the Fc-mediated binding of NBE-002, or any ADC, to myeloid cells in nontarget organs. ⁴⁶ NBE-002 significantly suppressed tumour growth and improved OS in the ROR1 high HRD model PDX#62 (ROR1 H-score 178), compared with the mAb isotype control treatment (Figure 5(a) and (k); Supplemental Tables 4 and 5). Single-agent NBE-002 treatment suppressed tumour growth of PDX#56 (ROR1 H-score 190, HRD) and PDX#22 (ROR1 H-score 90, HRD reversion) without improving survival compared to the isotype control, which had activity in these models (Figure 5(b), (f) and (k); Supplemental Tables 4 and 5). The combination of NBE-002 with carboplatin resulted in a greater reduction in tumour volumes for PDX#62, 931, 22 and 201 compared with treatment with either isotype control plus carboplatin or NBE-002 (Figure 5(l)). In comparison to either single-agent carboplatin or NBE-002, PDX#56 and 56pp also showed combinatorial activity for NBE-002 delivered together with carboplatin (Figure 5(b) and (e); Supplemental Table 4).

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Figure 5.

NBE-002 inhibited tumour growth as a single agent in ROR1-high HRD and post-HRD reversion PDX models and combination with carboplatin. (a–j) Tumour volume (left, mean ± SEM) and Kaplan–Meier survival curves (right) for in vivo PDX treatments ((a–b) for HRD models, (c–f) for HRD reversion models, (g–j) for HRP models). The ROR1 H-score is shown in each panel adjacent to the PDX ID. Mice were treated for 4 weeks with weekly cycles of: IVIg (30 mg/kg) day 1 + DPBS day 2, IVIg (30 mg/kg) day 1 + Isotype control (0.33 mg/kg) day 2, IVIg (30 mg/kg) day 1 + NBE-002 (0.33 mg/kg) day 2, IVIg (30 mg/kg) day 1 + Isotype control (0.33 mg/kg) day 2 + carboplatin (50 mg/kg) day 2, IVIg (30 mg/kg) day 1 + NBE-002 (0.33 mg/kg) day 2 + carboplatin (50 mg/kg) day 2, IVIg (30 mg/kg) day 1 + Isotype control (0.33 mg/kg) day 2 or carboplatin (50 mg/kg) q7d. The PDX models #56 and #56pp (pp: post-PARPi) were a matched PDX pair from the same individual: #56 derived from a chemo-naïve sample and #56pp derived from a sample collected after multiple lines of treatment, including PARP inhibitor. The red bar below the x-axis indicates the treatment interval. For n per treatment arm and Kaplan–Meier Log-Rank analysis p values, see Supplemental Table 5. (k, l) The mean difference in tumour growth between treatments. Two-way ANOVA (time and treatment as two factors) was performed to assess the mean differences in tumour volume across treatments, with Tukey’s multiple comparison test used to adjust the p value (Supplemental Table 4). (m) Percentage change in tumour volume from baseline for NBE-002 and isotype control-treated tumours at day 28, end of treatment (or earlier for rapidly growing PDX models).

Apart from two ROR1-high PDX models that were resistant to all treatments, PDX #111 and #448, the response to single-agent NBE-002 correlated with ROR1 expression #56 > #62 > #22. For models with lower ROR1 expression, such as #201 (H-score 60) and #56pp (H-score 83), no single-agent NBE-002 activity was observed. However, synergy in combination with carboplatin suggested that these models were not completely resistant to PNU, perhaps due to a lower dose/delivery of NBE-002, indicating the expression of ROR1 was not high enough for single-agent activity. As PDX #62 and #56 were sensitive to both NBE-002 and carboplatin as single agents, lower doses of each agent would need to be explored to uncover the full potential of this combination. In both PDX, however, the combination of NBE-002 and carboplatin was the most active treatment (Supplemental Tables 4 and 5).

Response to the isotype control ADC (the treatment arm of IVIg plus isotype control) was seen in 2 out of 10 PDX: in PDX #56 (ROR1 H score 190) but not #56pp (ROR1 H score 83) and in PDX #22 (ROR1 H score 90) and was unexpected. Perhaps as PDX #56 is very sensitive to all therapies, in keeping with the very long time for the patient, from first diagnosis to ultimate PD on PARPi (3 years), following which she retained platinum sensitivity (another 2 years), lowering the dose of NBE-002 and of the isotype control might have revealed the specific ROR1-targeted response. Similarly, the isotype control + carboplatin arm responded better than did the carboplatin-alone arm in 4 out of 10 PDX, spanning the full range of carboplatin response-sensitive, mid-responsive and refractory. Despite this apparent increased sensitivity to carboplatin in the setting of isotype control, it was clear that the combination of NBE-002 and carboplatin was the best or equal to the best therapeutic arm in all 10 PDX.

Additionally, we explored the combination effect of NBE-002 with olaparib in two PDX models #62 and #931 representing HRD and HRD reversion. Combining NBE-002 with olaparib led to a decreased tumour size in the HRD model PDX #62 compared with treatment with either the isotype control plus olaparib or with NBE-002 (p < 0.001), albeit without a significant enhancement in survival compared with either monotherapy, as all mice survived until the experimental endpoint (120 days; Figure 6 and Supplemental Table 5).

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Figure 6.

A combination effect was observed in an HRD model in vivo upon treatment with NBE-002 and olaparib. (a, b) Tumour volume (left, mean ± SEM) and Kaplan–Meier survival curves (right) for HRD PDX #62 (a) and HRD reversion PDX #931 (b). ROR1 H-score is shown in each panel next to the PDX ID. (c) The mean difference in the tumour growth between treatments. Two-way ANOVA (time and treatment as two factors) was performed to assess the mean differences in tumour volume across treatments, with Tukey’s multiple comparison test used to adjust the p value (Supplemental Table 4). For n and Kaplan–Meier log-rank analysis p values, see Supplemental Table 5.