The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R–targeted antibody–drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety.
Antibody–drug conjugates (ADC) are a class of anticancer agent that combine a human or humanized mAb with a potent cytotoxic drug in a single molecule via a chemical linker (1–3). The development of ADCs takes advantage of the specificity of the mAb to target antigen-positive tumor cells while augmenting its ability to produce a cytotoxic effect. The chemical linker between the antibody and the cytotoxic drug helps the antibody to selectively deliver the cytotoxic drug to tumor cells and accurately release the cytotoxic drug at the tumor site (4). ADCs thus enable selective delivery of a highly cytotoxic drug to tumor cells while minimizing systemic toxicity, providing a broader therapeutic window and improved clinical benefit to patients (2, 3). To date, 4 ADCs have been approved by the FDA and the European Medicines Agency for the treatment of metastatic or hematologic cancers, including acute myeloid leukemia and acute lymphoblastic leukemia (2). ADCs are a major focus for oncology research, and approximately 80 ADCs are currently being investigated in clinical trials (1–3, 5, 6), including 9 in late-stage clinical development (phase III or pivotal phase II studies; ref. 7). Of the ADCs in clinical development, approximately 70% use inhibitors of tubulin polymerization, such as maytansinoid and auristatin, or their derivatives, as the cytotoxic agent (1, 8, 9).
The insulin-like growth factor type 1 receptor (IGF-1R) is a transmembrane tyrosine kinase receptor. It consists of two extracellular alpha subunits and two transmembrane beta subunits, which have tyrosine kinase activity (10). The IGF-1R has long been recognized for its role in tumorigenesis and tumor growth (11–14), and it is overexpressed in several cancer types, including non–small cell lung cancer (15), head and neck squamous cell carcinoma (16), estrogen receptor–positive breast cancer (17), prostate cancer (18), and osteosarcoma (19). To date, therapeutic approaches targeting the IGF-1R based on mAbs and tyrosine kinase inhibitors (TKI) have shown rather disappointing results, with clinical benefit seen only in selected patient populations rather than general unselected populations (11, 20–25).
We and others have reported that IGF-1R can be internalized and degraded in lysosomes (26). This property of IGF-1R makes it an attractive target for an ADC strategy, as it is localized on the cell surface, it is overexpressed in many cancers, and it could deliver the drug directly into tumor cells. Using IGF-1R as a target in some cancer types could thus enable the delivery of a cytotoxic agent into the tumor and efficiently kill cancer cells.
W0101 is a first-in-class ADC designed for the treatment of patients with tumors overexpressing the IGF-1R. W0101 consists of three components. Firstly, a humanized mAb (hz208F2-4) that acts as a vector and binds specifically to the IGF-1R, triggering a rapid and strong internalization of the receptor and the cytotoxic drug within cells that overexpress this receptor. Secondly, a cytotoxic derivative of auristatin, an antimitotic agent that inhibits cell division by blocking the polymerization of tubulin. This compound was selected for its very potent antimitotic properties. Thirdly, a noncleavable maleimidocaproyl (mc) linker, which is used in many ADCs currently investigated in clinical trials and known to be catabolized to Cysteine-mc-Drug in the intracellular lysosomal compartment (1). On average, 4 drug-linker molecules were coupled per mAb molecule, giving a drug-to-antibody ratio (DAR) of 4 (27). W0101 is being developed as a novel immunochemotherapeutic agent for the treatment of advanced or metastatic solid cancers. Here, we report the results of in vitro and in vivo preclinical studies to characterize and assess the specificity and efficacy of W0101 in mice bearing human tumors, supporting the ongoing clinical trial in patients.
Materials and Methods
MCF-7, NCI-H2122, NCI-H292, NCI-H460, NCI-H358, BxPC3, CaoV3, and Hs746t cells were obtained from the ATTC. SBC5 cell line was obtained from JCRB. Normal human urothelial cells (HUC) and epithelial prostatic cells (HPEpiC) were purchased from ScienCell, renal epithelial cells (HREpC), aortic endothelial cells (HAoEC), and bronchial smooth muscle cell (HBSMC) were purchased from PromoCell GmbH. Cells were maintained in an incubator at 5% CO2, 90% humidity, and 37°C in standard cell culture medium as recommended by the supplier. The cell lines were authenticated using short tandem repeat DNA profiling (LGC Standard) and routinely tested for absence of mycoplasma. Passages were limited to 20 for experimental procedures with tumor cell lines (<3 months) and were limited to 5 for experimental procedures with normal cells.
The mAbs specific for IGF-1R, hz208F2-4, and m816C12 were generated using hybridoma technology. Balb/c mice were immunized with recombinant human IGF-1R (rhIGF-1R) protein (R&D Systems) in the presence of Freund adjuvant. Spleens were fused with the SP2/0 myeloma fusion partner. After cloning by limit dilution, MCF-7 binding and internalization ability were confirmed, and the isotype determined. Binding specificity was checked by ELISA using rhIGF-1R, recombinant human insulin receptor (rhIR), and murine IGF-1R (R&D Systems). The m208F2 antibody was then humanized by complementarity determining region (CDR) grafting leading to human IgG1 antibody and expressed in Chinese hamster ovary cells for full pharmacology characterization. In contrast, m816C12 antibody was kept as a murine IgG 1k antibody and produced using Celline flasks (IBS Integra Biosciences) for IHC.
ADC production and characterization
Mild reduction of mAb hz208F2-4 and coupling of the linker-payload were performed as previously described (28, 29). Briefly, to target a DAR of 4, hz208F2-4 was reduced with 2.5 molar equivalents of tris(2-carboxyethyl)phosphine. The reduced antibody was then treated with 7 molar equivalents of linker-payload. At the end of the conjugation, the ADC was concentrated 10-fold by tangential flow filtration using a KrossFlo system (Spectrumlabs) equipped with a 30 kDa cutoff filter cassette (Pellicon, Sartorius) and then diafiltered against 15 volumes of a 25 mmol/L histidine pH 6.5 buffer containing 150 mmol/L NaCl and 6% sucrose. Tween 80 (Histidine buffer) was then added to obtain a final concentration of 0.005% (v/v), and the ADC concentration was adjusted to 5 mg/mL before sterile filtration (0.2 μm Stericup filter, Millipore). The ADC was stored at 4°C until use.
Then, ADC W0101 was characterized as described previously (29). It was analyzed by SDS-PAGE under reducing and nonreducing conditions to confirm drug conjugation and by size exclusion chromatography (SEC) to determine the content of monomers and aggregated forms. SEC assays were performed with a Superdex 200 Increase 10/300 GL column (GE Healthcare). Briefly, 50 μg of samples were injected and eluted at a flow rate of 0.4 mL/min with 25 mmol/L Histidine pH 6.5 buffer containing 150 mmol/L NaCl and 6% sucrose as mobile phase. The protein concentration was determined by using the bicinchoninic acid assay. The average DAR was calculated by quantifying the various loaded forms based on the 280 nm UV peaks area obtained after analysis by hydrophobic interaction chromatography (HIC) on TSKgel butyl-NPR (4.6 x 35 mm column, Tosoh Bioscience). The mobile phase consisted of 1.5 mol/L ammonium sulfate in 25 mmol/L potassium phosphate, pH 7.0 (buffer A), and 25 mmol/L potassium phosphate, pH 7, 25% isopropanol (buffer B). Separation was obtained with a linear gradient of 10% to 100% buffer B over 12 minutes at flow rate of 0.8 mL/min. Sample amount of 20 μg was injected after 2-fold dilution in mobile phase A.
The specificity of m816C12 was validated using various formalin-fixed, paraffin-embedded xenografted tissues, positive and negative for IGF-1R. The staining procedure was performed using the Ventana Discovery Ultra platform (Roche Diagnostics). Briefly, after deparaffinization and rehydration, tissue sections were treated for antigen retrieval with tris-EDTA pH 6 (Agilent) over 45 minutes at 98°C and incubated with the primary antibody m816C12 or the isotype control mouse antibody for 30 minutes. Expression was detected using the OmniMap anti-mouse HRP system (Roche Diagnostics).
For staining analysis, slides were scanned using the iScan HT scanner (Roche Diagnostics), and IGF-1R staining was quantified using Virtuoso software (Roche Diagnostics). For tissue analysis, 1 to 4 fields of view per tumor with more than 50 cells were scored to increase the statistical accuracy of the algorithm. Tissue microarrays and tumor xenograft models were scored following the American Society of Clinical Oncology/College of American Pathologists testing guidelines.
IGF-1R expression was determined as described elsewhere (26). Briefly, cells were incubated with a murine anti–IGF-1R antibody at 4°C and washed before incubation with a secondary labeled antibody. Cell surface receptor expression was determined by flow cytometry using Qifikit as recommended by the manufacturer (Agilent).
To assess antibody internalization, cells were incubated with anti–IGF-1R antibody at 4°C or 37°C for 120 minutes. At the selected time point, cells were washed and incubated with an Alexa Fluor 488–labeled secondary anti–IGF-1R antibody for 20 minutes at 4°C to measure the remaining cell surface antibody. The cells were then washed and analyzed by flow cytometry (30). Total antibody, both cell surface and internalized, was determined after cell permeabilization using saponin as described previously (26).
Propidium iodide (PI) staining was used to assess the cell-cycle distribution through flow cytometry as previously described (31). Briefly, cells were incubated with naked antibodies (isotype control and hz208F2
-4), ADCs (isotype control ADC and W0101), and monomethyl auristatin E (MMAE) in complete medium for 24, 48, or 72 hours. Cells were harvested and washed with complete medium once and then with PBS. The cell pellet was resuspended with 250 μL of PBS, and 2 mL of cold ethanol was added. The mixture was stored for at least 2 hours at −20°C. Cells were centrifuged and washed with PBS. RNAse 200 μL was added to the cell pellet, which was incubated for 30 minutes at 37°C. PI 200 μL (Interchim, FluoProbes) + 0.1% Triton X-100 was then added and the mixture incubated for 1 hour at 4°C before cell-cycle analysis. Data were analyzed using the ModFit DNA analysis program.
Cells were incubated in complete medium in the presence of 10 μg/mL of antibody or MMAE (equivalent to 10 μg/mL ADC) for 3 days. Supernatants were collected, and attached cells were trypsinized and washed. Cells and their corresponding supernatants were mixed in Annexin V buffer (140 mmol/L NaCl, 5 mmol/L CaCl2, and 10 mmol/L Hepes; pH7.4) and incubated with Annexin V–FITC (Invitrogen #A13199) for 20 minutes. Cells were washed before addition of PI (Interchim, FluoProbes). Cells were then analyzed by flow cytometry. Viable cells were Annexin V–/PI–, apoptotic cells were Annexin V+/PI–, necrotic cells were Annexin V+/PI+, and late necrosis was characterized by Annexin V–/PI+ cells (32).
Evaluation of W0101 internalization and colocalization
MCF-7 cells were labeled with W0101 10 μg/mL for 20 minutes at 4°C, washed, and incubated for 30 minutes at 37°C. The localization of W0101 was determined by immunofluorescence staining and visualized by confocal microscopy. W0101 was revealed using an Alexa Fluor 488–labeled secondary antibody (green), lysosomes were stained with anti-LAMP1 antibody (red; Cell Signaling Technology # 9091), and nuclei were counterstained with DRAQ5 (blue; Cell Signaling Technology # 4084). Slides were examined with an LSM510 laser scanning microscope (Carl Zeiss, Jena) equipped with 63 objectives, 1.4 numerical aperture, using LSM software.
Tumor and normal cells were plated in 96-well flat bottomed microplates (100 μL/well) in cell culture medium and incubated overnight at 37°C in 5% CO2. The next day, increasing concentrations of W0101 or isotype control ADC (0–10 μg/mL) were added into 3 replicate wells containing cells (10 μL/well). Plates were incubated for 6 days at 37°C in 5% CO2. Cell viability was determined by measuring ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was read using a Multimode Microplate Reader (Mithras LB940, Berthold Technologies). The percentage cell viability was calculated for each concentration considering 0 μg/mL ADC as 100% viability. The IC50 was calculated using Prism software. Three independent experiments were performed.
Determination of in vivo activity
All experimental protocols were approved by the Pierre Fabre Institute Animal Care and Use Committee. For the breast cancer model, 7-week-old female Swiss nude mice (Charles River Laboratories) were engrafted subcutaneously with 5 × 106 MCF-7 cells 1 day after subcutaneous implantation of 0.72 mg 17β-estradiol 60-day releasing pellets (Innovative Research of America). For the ovarian cancer model, 7-week-old female SCID mice (Charles River Laboratories) were engrafted subcutaneously with 10 × 106 CaoV3 cells. For the lung cancer models, 7-week-old female athymic nude mice (Envigo) were engrafted subcutaneously with 7 × 106 SBC5 cells, and 7-week-old female athymic nude mice (Envigo) were engrafted subcutaneously with 7 × 106 NCI-H2122 cells. For the gastric cancer model, 7-week-old male SCID mice (Charles River Laboratories) were engrafted subcutaneously with 7 × 106 Hs746t cells.
After randomization, treatment with intravenous administration of W0101, isotype control ADC, or ADC vehicle (histidine buffer, pH 6.5) was initiated when tumors reached a size of approximately 150 mm3 (n = 5 or n = 6 mice per group). Tumor volume (length × width × height × 0.52) was measured using an electronic caliper at least twice weekly, and the treatment response was defined using RECIST (33).
The percentage of regression values was calculated using the formula
Regression % = 100 ×ΔT/Tinitial
where T = the tumor volume in the treated group, ΔT = the tumor volume in the treated group on the study day of interest minus the tumor volume in the treated group on the initial day of dosing, and Tinitial = the tumor volume in the treated group on the initial day of dosing. Progressive disease (PD) was defined as an increase in tumor size > 20%. Partial regression (PR) was defined as a decrease in tumor size > 30%. No tumor growth, or a slight decrease (<30%), or a small increase (<20%) in tumor size was defined as stable disease (SD), and an absence of any palpable tumor mass was defined as complete regression (CR).
Determination of in vivo activity in an MCF-7 docetaxel-resistant model
Mice bearing MCF-7 tumors were injected i.v. with docetaxel 9 mg/kg every 2 weeks for a total of 5 injections. When tumors became resistant to docetaxel and relapsed, mice were divided in 2 groups of 5 animals: group 1 received docetaxel 9 mg/kg once every 2 weeks and group 2 received a single injection of W0101 3 mg/kg.
The statistical significance of the differences in tumor growth between the treatment groups was determined using a Mann–Whitney U test performed with SigmaStat 3.5 (Systat Software Inc.).
IGF-1R expression on tumor and normal tissues
Expression of IGF-1R was analyzed by IHC staining on a panel of 10 cases of tumor and normal breast tissues microarrays, 56 cases of tumor and normal lung tissues microarrays, and 33 cases of larynx tumor tissue microarrays (Supplementary Table S1). IGF-1R was highly expressed on breast cancer, with almost 60% of the core scored 3+, on squamous cell lung carcinoma, with 47% of the core scored 3+ (Fig. 1A), and on larynx squamous cancer, with 30% of the core scored 3+. The expression in vital organs of normal individuals was limited to cytoplasmic expression on kidney, adrenal gland, heart, colon, pancreas, and jejunum (Fig. 1B). No normal tissue was stained 3+; urothelium, prostate epithelium, placenta, proliferative, and secretory endometrium were scored 2+ (Fig. 1C).
IGF-1R expression on the cell surface of tumor and normal cells
To identify IGF-1R expression on the cell surface of cancer cells, 9 cell lines expressing various levels of the receptor were tested. Expression was evaluated by flow cytometry. High expression of IGF-1R was detected only in the MCF-7 cell line. Four cell lines, NCI-H2122, NCI-H358, NCI-H292, and CaoV3, showed a medium level of expression, three cell lines were identified with a low level of expression (NCI-H460, BxPC3, and SBC5), and only one, Hs746t, had no expression of IGF-1R on its surface. Normal primary cells of lung, aortic, renal, prostatic, and urothelial origin were also evaluated for their IGF-1R expression level. No normal cell line was identified with a high level of IGF-1R, HUC showed a medium expression level, corresponding to that observed with normal urothelium, and 4 cell lines expressed a low level or no expression of IGF-1R (Supplementary Table S2).
Expression on T cells, B cells, monocytes, and neutrophils was also evaluated (Supplementary Table S2), indicating a very low level of expression of IGF-1R on those cell types compared with tumor cells.
Antibody and ADC characterization
The mAb m208F2 was selected from 578 hybridomas, based on its binding properties and its internalization ability in MCF-7 cells (Supplementary Figs. S1 and S2). As expected from the screening process, m208F2 showed no detectable binding to hIR. The m208F2 antibody partially inhibited IGF-1 binding on IGF-1R (Supplementary Fig. S3A) and inhibited IGF-1 signaling (Supplementary Fig. S3B). The selected antibody was humanized by classical CDR grafting and conjugated to an auristatin derivative, via a noncleavable maleimidocaproyl linker (Fig. 2A), resulting in W0101 with an average DAR of 4 (Fig. 2B; Supplementary Table S2). W0101 had a monomer content > 97%, as determined by SEC (Fig. 2C). Both unconjugated hz208F2 antibody and W0101 exhibited high affinity binding (IC50 = 3.10–10 mol/L) and equivalent internalization properties in the MCF-7 cell line (Supplementary Fig. S4).
Binding specificity to rhIGF-1R but not to rhIR was assessed by ELISA. Binding specificity to IGF-1R was also documented by flow cytometry on MCF-7, with no binding on negative Hs746t cells.
In vitro characterization
To assess the internalization kinetics of the ADC, MCF-7 cells were exposed to the W0101 ADC and incubated at 37°C to allow internalization. Cells were washed, and the remaining ADC on the cell surface was determined. After 10 minutes, 49% of W0101 was already internalized, after 30 minutes internalization was 72%, and after 1 hour it reached approximately 80% (Fig. 3A). Although W0101 on the cell surface rapidly decreased, total ADC remained stable after incubation for 1 hour at 37°C (Fig. 3B). Therefore, W0101 is rapidly internalized into the cells. This was confirmed by confocal microscopy (Fig. 3C). Before incubation, W0101 was located on the cell surface of MCF-7 cells and after 15 minutes of incubation a large proportion of the ADC was in intracellular vesicles. After 30 minutes of incubation, W0101 colocalized in lysosomes and little remained on the cell surface. After 2 hours, the W0101 level decreased, mainly due to degradation in lysosomes. This lysosomal degradation was demonstrated using a specific inhibitor of lysosome that inhibits the vacuolar proton pump V-type ATPase, which prevents acidification of the endosome compartments required for lysosomal protease maturation (Fig. 3D). Indeed, total W0101 decreased after 2 hours of incubation with MCF-7 cells, but this level remained stable in the presence of the lysosome inhibitor bafilomycin A1, confirming that W0101 internalization occurred via lysosomes.
We further investigated the potential of W0101 to kill tumor cells. In MCF-7 and NCI-H2122 cell lines expressing high levels of IGF-1R, W0101 displayed very potent cytotoxic activity, leading to near 100% cell death, with an IC50 of 5.1 × 10–11 and 6.25 × 10–10 mol/L, respectively (Fig. 4A). In cells expressing lower levels of IGF-1R, W0101 was unable to induce 100% mortality (Fig. 4A). As expected, no cell killing was measured in cell lines that do not express IGF-1R, such as Hs746t (Fig. 4A). The urothelial cells HUC and the epithelial prostatic cells HPEpiC, which expressed the highest level of IGF-1R within the normal cell panel, were sensitive to W0101, with 30% of cytotoxicity obtained. In normal cells expressing very low level of IGF-1R, no significant cytotoxicity was observed (Fig. 4B). To further characterize the in vitro effect of W0101, its impact on the cell cycle was evaluated by flow cytometry analysis of MCF-7 cells. After incubation for 72 hours with W0101, cells were blocked massively in the M phase, whereas no effect on the cell cycle was noted using an isotype control ADC (Supplementary Fig. S5A). This result was consistent with the known activity of MMAE and its derivatives to induce G2–M-phase cell-cycle arrest via its uptake in cells (Supplementary Fig. S5A; Supplementary Table S3; ref. 34), confirmed in MCF-7 cells. To assess the ADC mechanism of MCF-7 killing, apoptosis and cell death were evaluated by flow cytometry. After incubation for 72 hours with W0101, a significant increase of apoptotic and late necrotic cells was observed, compared with the naked antibody or the isotype control ADC (Supplementary Fig. S5B).
In vivo characterization
The expression level of IGF-1R was evaluated in several xenograft models by IHC using the m816C12 antibody. Five models were selected based on IGF-1R expression from the highest to the lowest expression level of IGF-1R: MCF-7 (3+), NCI-H2122 (2+), CaoV3 (2+), SBC5 (1+), and Hs746t (negative model; Fig. 5A).
To analyze the in vivo potency of W0101, we first evaluated its efficacy in the IGF-1R 3+ breast cancer model MCF-7 (Fig. 5A), in a once every 4 days for 4 cycles (Q4d4) administration schedule. In contrast to the isotype control ADC, W0101 3 mg/kg induced CR in 4 mice of 6 and PR in the 2 remaining mice (Fig. 5B). The antitumor activity of W0101 was then evaluated at doses of 3, 1, and 0.5 mg/kg with different administration schedules (Fig. 5C). Whatever the tested dose, administration of W0101 once every 4 days for 6 cycles (Q4d6) or once every 7 days for 7 cycles (Q7d7) resulted in PR or CR, except at a dose of 0.5 mg/kg Q7d7. A dose schedule of once every 14 days for 3 cycles (Q14d3) still induced CR when W0101 was dosed at 3 mg/kg. When dosed at 1 mg/kg, W0101 induced CR in 4 mice of 6, and 2 mice had PD. At a dose of 0.5 mg/kg, no CR was observed, tumors were in PR (2 mice of 6), SD (3 mice of 6), or PD (1 mouse). Finally, when administered once every 21 days for 3 cycles (Q21d3), a CR was obtained in 5 mice of 6 at a dose of 3 mg/kg, whereas SD, PR, or PD were obtained at a dose of 1 and 0.5 mg/kg. In the 0.5 mg/kg dose group, 1 mouse was missing because the animal was euthanized for ethical reasons. The lowest dose sufficient to induce a CR was determined to be 0.5 mg/kg with a Q4d6 administration schedule.
The naked antibody hz208F2-4 was also evaluated at 7 mg/kg in a Q7d2 schedule, and no significant tumor growth inhibition was observed (Supplementary Fig. S6A).
During all the course of the experiment, animals were monitored daily for general aspect and twice a week for body weight loss. No significant body weight loss nor degradation in the general aspect were noticed.
To assess the duration of the tumor growth inhibition induced by a single injection of W0101, mice were injected with a single dose of 3 mg/kg of the ADC and monitored for 3 months. The regrowth of tumors was not observed before 90 days (5 mice of 6), indicating strong and long-lasting antitumor activity (Fig. 5D).
The potency of W0101 was also evaluated in models expressing different levels of IGF-1R from 0 to 2+. In the 2+ models NCI-H2122 (Fig. 5E) and CaoV3 (Fig. 5F), W0101 3 mg/kg with a Q4d4 administration schedule resulted in a significant tumor growth inhibition (P < 0.05), with 3 mice of 6 having SD in the NCI-H2122 model and all 6 mice having PR in the CaoV3 model. Similar to the experiment performed in the MCF-7 model, neither the naked antibody in the NCI-H2122 model (Supplementary Fig. S6B) nor the isotype control ADC in both models (Fig. 5E and F) induced tumor growth inhibition. In the 1+ SBC5 model (Fig. 5G) and the negative Hs746t model (Fig. 5H), W0101 3 mg/kg did not induce any tumor growth inhibition. These results, combined with the absence of activity of the nontargeting ADC, indicate that the activity of W0101 is selective and specific for IGF-1R.
To further evaluate the therapeutic potential of W0101 in breast cancer, W0101 was investigated in the MCF-7 docetaxel-resistant model. After 70 days, tumors became resistant to docetaxel and relapsed (Fig. 6A). Administration of a single dose of W0101 3 mg/kg in mice with docetaxel-resistant tumors induced strong and significant tumor growth inhibition (P < 0.05; Fig. 6B), with CR in 1 mouse and PR in 3 mice of 5.
The key role of IGF-1R in cancer development has been known for decades. The IGF-1 axis is implicated in the development of resistance to several agents used as standard of care in oncology (11, 20–25, 35). Several approaches to inhibit IGF-1R signaling have been explored, including mAbs against IGF-1R, IGF-1R ligand, and IGF-1R TKI. However, none of the strategies developed for inhibiting the IGF-1R pathway have demonstrated a clear clinical benefit for patients (25). In this study, we confirmed that IGF-1R is overexpressed in many types of cancer but has restricted expression in normal tissues, and thus constitutes an attractive target for the development of an ADC. Indeed, one of the key parameters for an ideal target antigen is a highly and homogenously expressed target in tumors with a low expression in normal tissues (36). Thus, as IGF-1R expression corresponds to this requirement, W0101 was designed in order to overcome the lack of a clear benefit seen with naked anti–IGF-1R antibodies in clinical trials.
In this study, a novel anti–IGF-1R antibody was generated following a precise selection process, which led to the selection of the murine anti–IGF-1R mAb, m208F2. This novel mAb m208F2 had a high affinity, rapid internalization rate, and preferred intracellular trafficking into lysosomes upon binding to IGF-1R. It did not recognize IR nor activate IGF-1R. This specific and careful selection process should limit potential side effects related to insulin pathway.
W0101 has shown to be rapidly internalized from the cell surface to the cytosol and colocalized within the lysosome. The internalization of W0101 led to its delivery to the lysosomal compartment, where cleavage and release of the cytotoxic occurred, leading to cell-cycle arrest and cell death through the induction of apoptosis, as reported for auristatin derivatives (34, 37, 38). No effect was measured in the IGF-1R–negative cell line Hs746t, indicating target selectivity. Altogether, our results clearly show that the cytotoxicity of W0101 is mainly driven by IGF-1R and effective lysosomal degradation. Related to its structural profile, the drug-linker compound did not penetrate cells efficiently and did not induce cytotoxicity in vitro. Moreover, the major compound resulting from the cleavage of W0101 was identified and tested for its ability to induce cytotoxicity in vitro in MCF7 and NCI-H2122 cells, and no major cell mortality was observed. This property of the selected payload suggested that limited bystander effect is expected, consecutive to drug release after killing of highly expressive IGF-1R cells. The limiting bystander effect of W0101 was also demonstrated in a coculture experiment (Supplementary Fig. S7). Furthermore, no major side effects were noted neither in mice bearing IGF-1R–negative tumors treated with either W0101 or with the isotype control ADC, nor during the toxicity study in NHP. However, the absence of bystander effect could have an impact of the efficacy of W0101 on tumor with a nonhomogeneous expression of IGF-1R. Finally, W0101 did not show any ADCC properties.
It is known that the most frequently adverse events reported with anti–IGF-1R antibodies at high antibody concentrations (10 or 20 mg/kg) were as hyperglycemia, anemia, nausea, neutropenia, and thrombocytopenia (39). We cannot exclude similar adverse events with W0101; however, ADC are usually used at lower doses than naked antibody due to the toxicity of the payload. Altogether, the limited bystander effect and the absence of any major toxicity in NHP suggested that low doses of W0101 should be relatively well tolerated in patients.
W0101 resulted in dose-dependent tumor regression in an MCF-7 breast cancer model with an IGF-1R expression level of 3+, leading to almost complete tumor regression. All regimens were well tolerated with no morbidity and no body weight loss over the course of the study, even with repeat-dosing regimens at a dose of 3 mg/kg. Complete tumor regression was observed even at a dose of 0.5 mg/kg administered Q4d6. Higher doses could be used with fewer cycles. Indeed, CR was obtained with 1 mg/kg Q7d7 and with 3 mg/kg Q21d3. In addition, a single injection of W0101 3 mg/kg was sufficient to produce a long-lasting antitumor effect (>90 days). These results show that fractionated dosing could be an alternative to limit potential toxicity even if no toxicity was observed in treated mice. In two mouse models with an IGF-1R-expression level of 2+ (CaOV3 and NCI-H2122), tumor growth was inhibited for 20 days, whereas in a model with an IGF-1R expression level of 1+ (SBC5), no tumor regression was seen at any of the tested W0101 doses. Because the potency of ADCs depends on several parameters, including target biology, internalization kinetics, intracellular processing, and payload efficacy (36), a linear relationship between the level of target expression and the efficacy of ADCs was not anticipated. However, the sensitivity of tumors to W0101 observed in this study showed that the expression level of IGF-1R was a key parameter for inhibiting tumor growth. The therapeutic potential of W0101 was highlighted by the sustained tumor regression it induced in the mouse models. This effect was specific to W0101, as neither the naked antibody hz208F2-4 nor the control ADC induced tumor regression.
W0101 was shown to be cytotoxic in tumor cell lines highly expressing the IGF-1R, and a slight cytotoxicity was observed in normal cells. A linear correlation between cell cytotoxicity and IGF-1R expression level was also noted in vitro. At least 50,000 receptor sites were mandatory on the tumor cell surface to observe dramatic cytotoxicity in vitro. Interestingly, W0101 demonstrated better in vivo activity compared with in vitro. Indeed, no or slight cytotoxicity was measured with W0101 on CaoV3 in our in vitro assay, whereas tumor growth of xenograft CaOV3 tumors was significantly reduced after W0101 treatment. Potential explanations could be a differential expression level of IGF-1R in in vivo models versus in vitro, a differential conformation of the epitope in the acidic tumor microenvironment (40), or IGF-1R recycling (41). However, even if HUC cells expressed similar level of IGF-1R as CaoV3 cells, no toxicity on urothelial cells is expected. Indeed, the mechanism of action of auristatin derivative is well described acting on dividing cells, and the proliferative index of such kind of cells in situ is lower than 5%.
Taxanes are one of the most potent and effective classes of chemotherapeutic agents and are widely used for the treatment of a variety of cancers including lung, head and neck, prostate, and breast. However, resistance to these agents is common (42). In a docetaxel-resistant MCF-7 tumor model, W0101 induce inhibition of tumor growth. W0101 could therefore be a potential treatment option for patients with taxane-resistant tumors.
In conclusion, these preclinical studies demonstrate the specificity of the ADC W0101 for the IGF-1R and the absence of binding to the IR. W0101 is a promising new potential therapeutic option for patients with tumors overexpressing the IGF-1R. A companion diagnostic test is under development to improve patient selection, and the first in-human clinical trial of W0101 (ClinicalTrials.gov Identifier: NCT03316638) is currently ongoing in all type of solid tumors to find the MTD and to investigate its clinical safety.
Disclosure of Potential Conflicts of Interest
E. Chetaille is Head of Oncology Innovation Unit at Pierre Fabre. N. Corvaia is Head of Research at CIPF. No potential conflicts of interest were disclosed by the other authors.
Conception and design: M. Broussas, N. Loukili, A. Beck, M. Perez, N. Corvaia
Development of methodology: M. Broussas, N. Loukili, C. Beau-Larvor, N. Boute, C. Dreyfus
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Broussas, N. Loukili, A. Robert, C. Beau-Larvor, M. Malissard, N. Boute, T. Champion, J.-F. Haeuw, A. Beck
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Akla, M. Broussas, N. Loukili, A. Robert, C. Beau-Larvor, N. Boute, T. Champion, A. Beck, C. Dreyfus, E. Chetaille
Writing, review, and/or revision of the manuscript: M. Broussas, N. Loukili, J.-F. Haeuw, A. Beck, C. Dreyfus, M. Pavlyuk, E. Chetaille, N. Corvaia
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Broussas, N. Loukili, A. Beck
Study supervision: M. Broussas, M. Pavlyuk, E. Chetaille, N. Corvaia
The authors would like to thank all past and present members of the Pierre Fabre team that have contributed to this work.
These studies were sponsored by Pierre Fabre.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.