Globo H (GH), a hexasaccharide, is expressed at low levels in normal tissues but is highly expressed in multiple cancer types, rendering it a promising target for cancer immunotherapy. OBI-999, a novel antibody–drug conjugate, is derived from a conjugation of a GH-specific mAb with a monomethyl auristatin E (MMAE) payload through a site-specific ThioBridge and a cleavable linker. OBI-999 high homogeneity with a drug-to-antibody ratio of 4 (>95%) was achieved using ThioBridge. OBI-999 displayed GH-dependent cellular internalization and trafficked to endosome and lysosome within 1 and 5 hours, respectively. Furthermore, OBI-999 showed low nanomolar cytotoxicity in the assay with high GH expression on tumor cells and exhibited a bystander killing effect on tumor cells with minimal GH expression. Tissue distribution indicated that OBI-999 and free MMAE gradually accumulated in the tumor, reaching maximum level at 168 hours after treatment, whereas OBI-999 and free MMAE decreased quickly at 4 hours after treatment in normal organs. Maximum MMAE level in the tumor was 16-fold higher than in serum, suggesting that OBI-999 is stable during circulation and MMAE is selectively released in the tumor. Excellent tumor growth inhibition of OBI-999 was demonstrated in breast, gastric, and pancreatic cancer xenograft or lung patient–derived xenograft models in a dose-dependent manner. The highest nonseverely toxic dose in cynomolgus monkeys is 10 mg/kg determined by a 3-week repeated-dose toxicology study demonstrating an acceptable safety margin. Taken together, these results support further clinical development of OBI-999, which is currently in a phase I/II clinical study in multiple solid tumors (NCT04084366). OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibition in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers, warranting further investigation in the treatment of solid tumors.

The carbohydrate antigen expression profile on tumor cells is different from that on normal tissues; these antigens are known as tumor-associated carbohydrate antigens (TACAs). Globo H (GH) has become one of the well-known TACAs following its discovery in 1983 (1–3). Many antibodies targeting TACAs have been developed or are currently under development as potential cancer therapeutics, such as Lewis-Y antigen (4, 5), Sialyl-Thomsen-nouveau (6, 7), Tn, and the ganglioside GD2 (8, 9).

GH, a hexasaccharide (Fucα1–2Galβ1–3GalNAcβ1–3Galα1–4Galβ1–4Glc) originally isolated as a ceramide-linked glycolipid from the human breast cancer cell line MCF-7, is one of the most prevalent TACAs (1, 10). GH is highly overexpressed in several cancers, including breast, ovarian, gastric, lung, prostate, pancreatic, endometrial, and colon (2, 11, 12). However, in normal tissues, GH expression and prevalence are very low and limited to the secretory borders of apical epithelial cells (13). GH is therefore a promising target for developing anticancer therapeutics such as vaccines, mAbs, and antibody–drug conjugates (ADCs).

Several mechanisms for the role of GH in promoting tumor growth and survival have been proposed. Chuang and colleagues indicated that the Globo series glycosphingolipids recruit the FAK/CAV1/AKT/RIP complex (the FAK complex) on the cell surface to trigger the downstream β-catenin signaling pathway and promote cancer cell survival. The association of GH with the FAK complex also prevents caspase-3 activation and the subsequent apoptosis, thereby enhancing tumor survival and metastasis in breast carcinoma cells (14). In another study, addition of GH ceramides to cancer cells triggered endothelial cell migration, tube formation, and intracellular calcium ion mobilization to promote angiogenesis (15). Tsai and colleagues demonstrated that GH ceramides may act as an immune checkpoint that modulates immune activity against tumors. Furthermore, immune cell proliferation and cytokine secretion of human peripheral blood mononuclear cells and murine splenocytes upon CD3/CD28 stimulation were inhibited in the presence of GH ceramides (16). GH ceramides shed from tumor cells into the tumor microenvironment (15) compromise tumor-infiltrated immune cell function. Consequently, GH may contribute to tumor growth through triggering survival signaling of tumor cells as well as modulating the tumor microenvironment. Taken together, inhibition of GH ceramide functions may suppress tumor development and survival.

Several GH-targeting cancer vaccines, from monovalent to multivalent, have entered clinical trials as therapeutics for patients with advanced prostate, breast, ovarian, and many other types of cancers (17–21). Among them, adagloxad simolenin is the most advanced GH-targeting cancer vaccine and is currently in a global phase III clinical trial. In addition, a phase I/II clinical trial evaluating the GH-targeting therapeutic mAb (OBI-888) was initiated in 2018. Both GH-targeting therapeutics were well tolerated at the doses used in these trials.

In this study, we report the results from multiple preclinical studies evaluating a novel ADC, OBI-999, which is composed of the GH-targeting antibody (OBI-888) plus a novel linker (ThioBridge) and a known cytotoxic payload, the tubulin polymerization inhibitor (monomethyl auristatin E, MMAE). The ThioBridge is designed to form site-specific disulfide bonds through cross-linking to the reduced cysteines in the Fab and hinge regions of the antibody rendering a more homogeneous drug-to-antibody ratio (DAR). OBI-999 was evaluated for its antigen specificity, internalization process, bystander effect, pharmacokinetics, biodistribution, antitumor efficacy, and safety profile. Results from these preclinical studies suggest that OBI-999 has the desired characteristics of an antitumor agent that warrants further clinical development in multiple cancers.

Preparation of ADC

OBI-999 was generated based on methods previously described, including reagents, linker–payload synthesis, and conjugation procedures (22, 23). A relevant control ADC (Ctrl-ADC) of anti-CD30 antibody conjugated with MMAE was produced with exact same method as OBI-999. Briefly, OBI-888 or anti-CD30 antibody interchain disulfide bonds were fully reduced by excessive (7 molar equivalent per mAb) tris(2-carboxyethyl)phosphine hydrochloride. The ThioBridge linker–payload was added to the reduced OBI-888 with a 6 molar equivalent linker–payload/mAb ratio and incubated for 16 hours (23). The resulting mixture was purified by preparative hydrophobic interaction chromatography (HIC) to give OBI-999 with a DAR of 4. The analytical results are listed in Supplementary Table S1. HIC analysis along with peak determination is illustrated in Supplementary Figs. S1 and S2 and Supplementary Table S2. The OBI-999 structure of heavy-chain and light-chain sequence was as listed in patent WO2018094414A1 Seq. ID No. 17 and No. 18 respectively.

Cell lines

Human cancer cell lines MDA-MB-231, HCC-1428, SK-BR-3, ES-2, HPAC, and NCI-N87 were purchased from the ATCC. MCF-7 cells were obtained from Academia Sinica. ES-2/GFP cells were purchased from GeneTarget Inc. The culturing conditions of each cell line were based on the provider's instructions. Each cell line was utilized in experiments within a month after thawing. All media were supplemented with 10% FBS and antibiotics under 5% carbon dioxide. Mycoplasma detection was performed routinely using a PCR detection kit (Applied Biological Materials, Inc.), and negative results were obtained for all the cell lines tested.

Flow cytometry

To evaluate the level of GH expression, cells were suspended and incubated with the anti-GH antibody (clone VK9; Thermo Fisher) or mouse IgG isotype control antibody (BioLegend) at 4°C for 1 hour. After being washed once with PBS, cells were then incubated with goat anti-mouse IgG-PE (BioLegend) followed by analysis with flow cytometry. Because anti-CD30 ADC was used as a control ADC (Ctrl-ADC) in this study, the level of CD30 expression was also evaluated using an anti–CD30-FITC antibody (BioLegend). Each cell line used in the study showed negative CD30 expression.

Binding affinity and specificity of OBI-999

The binding affinities of OBI-888 and OBI-999 were determined by a customized GH Human IgG ELISA Kit (Biocheck) following the manufacturer's instructions. A series of concentrations of OBI-888 and OBI-999 were prepared in a 2-fold serial dilution from 3,125 to 0.1 ng/mL and introduced to a GH ceramide–coated plate. The plates were read by SpectraMax M2 (Molecular Devices) at optical density (OD) 450. The EC50 calculated from triplicate wells of each concentration were averaged and analyzed by the Student t test. The result is representative of two independent experiments.

For evaluation of binding specificity against GH, OBI-999 or the Ctrl-ADC was incubated with HCC-1428, MDA-MB-231, or SK-BR-3 cells, which exhibited high, low, and negligible expression of GH, respectively. Cells at a density of 2 × 105 cells were suspended in FACS buffer that contained 1% BSA in PBS and kept in an ice bath for 1 hour followed by the addition of a series of concentrations of ADC (0 to 3,125 nmol/L). After being washed once with FACS buffer, the cells were incubated with anti-human IgG-FITC (Sigma-Aldrich) as a secondary antibody. The resulting cells were washed once with FACS buffer and analyzed by BD FACSCanto II (BD Biosciences).

Internalization of OBI-999

To monitor the internalization process of OBI-999, HCC-1428 cells were seeded in a 24-well culture plate at a density of 5 × 104 per well. After overnight incubation at 37°C, 20 μg/mL of OBI-999 or the Ctrl-ADC was added to the cells at 4°C and incubated for 1 hour to ensure synchronization of the internalization process. Cells were then transferred and incubated at 37°C. The cells were collected at 0, 1, 2.5, and 5 hours for OBI-999 and EEA1 costaining, and at 0, 5, 8, and 24 hours for OBI-999 and LAMP-1 costaining. Mouse anti–EEA-1 IgG (Cell Signaling Technology) and rabbit anti-LAMP1 IgG (Becton Dickinson) were used as endosome and lysosome primary antibody, respectively. AF568-goat anti-mouse IgG and AF568-goat anti-rabbit IgG (Thermo Fisher Scientific) were used as the secondary antibodies. The images were acquired by Leica SP8 confocal microscopy and processed with LAS X software. A cell-based internalization assay for flow cytometry detection was modified from procedures described previously (24). Detailed procedures are described in the Supplementary Information.

Bystander effect of OBI-999

MCF-7 and ES-2/GFP cell lines with high (97%) and negligible (<1%) GH expression, respectively, were used to study the bystander effect of OBI-999. MCF-7 and ES-2/GFP cells were cocultured at a 0:1 or 8:1 ratio with a constant ES-2/GFP cell number of 1 × 103. Cells were treated with 46 or 94 nmol/L OBI-999 or the Ctrl-ADC for 13 days followed by measurement of ES-2/GFP cell viability. The ES-2/GFP cell viability was evaluated based on the GFP intensity detected by SpectraMax M2 (Molecular Devices). The Student t test was used to assess statistical significance between study groups. The detailed procedures of another approach using culture medium transfer method was listed in the Supplementary Information.

Serum stability of OBI-999

To assess the serum stability in vitro, OBI-999 and Adcetris (brentuximab vedotin; 20 μg/mL) were incubated in mouse and human serum at 37°C for 0, 1, 3, 5, and 7 days. Pooled CD-1 male mouse serum (BioLASCO; Lot. BLT122817003) and pooled male human AB plasma (Sigma-Aldrich; Lot. SLBR90007V) were used for this study. The MMAE concentrations in the serum samples were quantified by LC/MS/MS. The maximum percentage of released MMAE was calculated based on an average DAR of 4. LC/MS/MS analysis of MMAE in the serum samples was accomplished by initial separation on a high-performance liquid chromatography (HPLC) column followed by detection with the mass spectrometer. The LC/MS/MS system was an Agilent 1200 HPLC equipped with binary pump, autosampler module, and column heater. Mass spectrometric analysis was performed using the SCIEX 4000 QTRAP with an electrospray ionization ion source in a positive mode.

Pharmacokinetic and tissue distribution studies of OBI-999 in tumor-bearing mice

The pharmacokinetics and distribution of OBI-999 were evaluated in NCI-N87 gastric tumor–bearing mice (female BALB/c nu/nu). After a single i.v. injection of OBI-999 at 5 mg/kg, the mouse serum, organs (including liver, lung, spleen, kidney, and heart), and tumor mass were harvested at 1, 4, 8, 24, 72, 168, and 336 hours after injection for analysis of total GH antibody, OBI-999, and free MMAE. The total antibody and OBI-999 concentrations were determined using an ELISA-based assay. The free MMAE concentration was determined by LC/MS/MS, and detailed procedures are as described in Supplementary Materials and Methods along with Supplementary Figs. S3 and S4. Biodistribution study of Indium111-labeled OBI-999 was performed on NCI-N87 tumor–bearing mice, and detailed procedure is listed in Supplementary Information along with Supplementary Fig. S5.

In vivo efficacy studies of OBI-999

The antitumor efficacy of OBI-999 was evaluated in breast (MCF-7), gastric (NCI-N87), and pancreatic (HPAC) cancers in xenograft models, and a lung cancer (LU-01-0266) patient-derived xenograft (PDX) model. GH expression was evaluated by anti-GH antibody (Thermo Fisher) staining using flow cytometry before inoculation. The animal studies were conducted at several facilities based on the availability of the animal models. Standard protocol review and regular institutional animal care and use committee approval were followed at the corresponding animal facilities. In each animal study, the antitumor efficacy was evaluated with doses of 1, 3, or 10 mg/kg of OBI-999 via i.v. injection. In NCI-N87 xenograft model, treatment groups of MMAE 0.191 mg/kg and Ctrl-ADC 3 mg/kg were also included. Each study utilized 6 to 8 mice per group. The tumor volume was measured regularly (Cn and Tn represent tumor volume measured in the control and treatment groups at day n, respectively). Percent tumor growth inhibition (TGI) was calculated based on the equation: TGI = (1 − [(Tn – T1)/(Cn – C1)]) × 100%. Two-way ANOVA and Bonferroni test were used to assess the statistical significance of groups compared with the respective vehicle control.

Safety study of OBI-999

The safety of OBI-999 was evaluated in cynomolgus monkeys (Macaca fascicularis). Groups of six animals (three males and three females) were administered OBI-999 via intravenous infusion at 0, 2, 5, or 10 mg/kg on days 1 and 22. Animals were sacrificed on day 29 for toxicity evaluation. For the vehicle control group (0 mg/kg) and the high-dose group (10 mg/kg), an additional two males and two females were included and sacrificed on day 43 to assess the reversibility of the toxic effects and potential delayed onset of toxicities. Blood samples were collected on days 1 and 22 for toxicokinetic analysis and immunogenicity testing. On day 1, samples were collected at predose and 1, 4, 8, 24, 72, 168, 336, and 504 hours after dose. On day 22, samples were collected at 1, 4, 24, 72, and 168 hours after dose. Data for each sex were analyzed separately. The significance of differences between groups was analyzed using ANOVA and pairwise comparison. The Levene test was used to test the equality of variances between groups.

The structure and binding specificity of OBI-999

OBI-999 is derived from conjugation of the GH-targeting mAb OBI-888 with MMAE through a ThioBridge and a protease cleavable peptide-based linker, as shown in Fig. 1A. The linker contains a valine-citrulline-p-aminobenzyl (PAB) system to ensure stability in the bloodstream and specific cleavage by cathepsin B after internalization. The ThioBridge, which is a bis-sulfone functionalized drug linker, was site-specifically attached to OBI-888 by cross-linking to the reduced cysteines in the Fab and hinge regions of the antibody (Fig. 1A). Upon further purification with HIC, OBI-999 exhibited as a homogeneous ADC with a DAR of 4 (>95%; Supplementary Fig. S1A) as opposed to other ADCs such as Adcetris with a DAR in the range of 1 to 8 (Supplementary Fig. S1B and S1C; refs. 22, 23). To reduce the hydrophobicity of OBI-999, PEG24 is linked to the glutamic acid in the linker. Compared with currently approved ADCs, OBI-999 is more homogenous and more stable than ADCs that utilize a maleimide linker (25).

Figure 1.

Structure and binding specificity of OBI-999. A, Chemical structure of OBI-999. Linker and payload MMAE were conjugated to cysteine residues as illustrated on the parental antibody OBI-888. B, Binding affinity of OBI-999 or OBI-888 to GH ceramide (0.2 μg/well) based on ELISA assay. C, Binding specificity of OBI-999 or Ctrl-ADC (anti-CD30 ADC) to cancer cells with different level of GH expression based on flow cytometry. Breast cancer cell lines HCC-1428, MDA-MB-231, and SK-BR-3 represented high (98.8%), low (10.6%), and negligible level (0.1%) GH-expressing cells, respectively. GH expression level was determined by anti-GH antibody staining using flow cytometry. MFI, mean fluorescence intensity.

Figure 1.

Structure and binding specificity of OBI-999. A, Chemical structure of OBI-999. Linker and payload MMAE were conjugated to cysteine residues as illustrated on the parental antibody OBI-888. B, Binding affinity of OBI-999 or OBI-888 to GH ceramide (0.2 μg/well) based on ELISA assay. C, Binding specificity of OBI-999 or Ctrl-ADC (anti-CD30 ADC) to cancer cells with different level of GH expression based on flow cytometry. Breast cancer cell lines HCC-1428, MDA-MB-231, and SK-BR-3 represented high (98.8%), low (10.6%), and negligible level (0.1%) GH-expressing cells, respectively. GH expression level was determined by anti-GH antibody staining using flow cytometry. MFI, mean fluorescence intensity.

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The binding affinity of OBI-999 to the antigen GH ceramide was evaluated by ELISA assay, which showed comparable binding affinity with its parental antibody OBI-888 (Fig. 1B) with EC50 at 0.46 and 0.29 nmol/L, respectively. To investigate the binding specificity of OBI-999 to GH on the surface of tumor cells, three breast cancer cell lines, HCC-1428, MDA-MB-231, and SK-BR-3, expressing high (98.8%), low (10.6%), and negligible levels of GH (0.1%), respectively, were used in this study. The FACS staining results indicate that OBI-999 has the highest signals with HCC-1428 cells, and much lower signals with MDA-MB-231 and SK-BR-3 cells even at doses of up to 3,125 nmol/L (500 μg/mL) as shown in Fig. 1C. The antigen specificity was further confirmed with confocal microscopy where OBI-999 showed high level of fluorescence with high GH expressing cancer cells, and no fluorescence was detected for human IgG and Ctrl-ADC (anti–CD30-MMAE; Supplementary Fig. S6). These results indicate that OBI-999 is highly specific to GH-expressing tumor cells.

Internalization of OBI-999

The internalization process of OBI-999 was monitored by a double staining of OBI-999 with endosome marker EEA-1 or lysosome marker LAMP-1 on HCC-1428 cells using confocal microscopy. OBI-999 was only detectable on the cell surface at the beginning of incubation and was internalized within 1 hours of incubation (Fig. 2A). After 2.5 hours of incubation, no signal was detected on the cell surface, and a punctate signal was observed intracellularly. As shown in Fig. 2A, OBI-999 was internalized and trafficked to EEA-1+ endosome as early as 1 hour after treatment. After 5 hours of incubation, OBI-999 was colocalized with lysosome marker (LAMP-1) and was retained in the lysosome for at least 24 hours (Fig. 2B). In contrast, when immunofluorescence staining was conducted on GH low-expressing MDA-MB-231 cells, or the Ctrl-ADC (anti–CD30-MMAE), no surface binding or internalization was observed (Supplementary Fig. S7). These results suggest that OBI-999 is internalized and trafficked through endosome and lysosome compartments efficiently, rendering the proteolytic cleavage of the linker and the release of MMAE intracellularly. More importantly, the internalization of OBI-999 is GH-dependent.

Figure 2.

Internalization of OBI-999 to GH-expressing tumor cells. HCC-1428 breast cancer cells were incubated with 20 μg/mL OBI-999 or anti-CD30 ADC (Ctrl-ADC) at 4°C for 1 hour and then transferred to 37°C and incubated for various time periods. The cells at a specific time point were removed and stained for (A) endosome marker EEA-1 and OBI-999 or Ctrl-ADC, and (B) lysosome marker LAMP-1 and OBI-999 or Ctrl-ADC. OBI-999 and Ctrl-ADC, EEA-1, and LAMP-1 were detected using anti-human IgG-AF488 (green), anti-EEA-1 IgG-AF568 (red), and anti–LAMP-1 IgG-AF568 (red) antibodies, respectively. Cell nuclei were stained with DAPI (blue). The images were acquired by Leica SP8 and processed with LAS X software. The scale indicates 10 μm. C, Kinetics of internalization were assessed using FACS staining on HCC-1428 cells under the same incubation procedures with 2 μg of Alexa-488–conjugated OBI-999 or isotype control antibody. The internalized signal was determined by incubation with 50 μg/mL anti–Alexa-488-quenching antibody at 4°C for 30 minutes followed by measuring the fluorescence intensity. The percentage of internalization was calculated based on MFI of the surface quenched and unquenched cells measured by FACSCanto II.

Figure 2.

Internalization of OBI-999 to GH-expressing tumor cells. HCC-1428 breast cancer cells were incubated with 20 μg/mL OBI-999 or anti-CD30 ADC (Ctrl-ADC) at 4°C for 1 hour and then transferred to 37°C and incubated for various time periods. The cells at a specific time point were removed and stained for (A) endosome marker EEA-1 and OBI-999 or Ctrl-ADC, and (B) lysosome marker LAMP-1 and OBI-999 or Ctrl-ADC. OBI-999 and Ctrl-ADC, EEA-1, and LAMP-1 were detected using anti-human IgG-AF488 (green), anti-EEA-1 IgG-AF568 (red), and anti–LAMP-1 IgG-AF568 (red) antibodies, respectively. Cell nuclei were stained with DAPI (blue). The images were acquired by Leica SP8 and processed with LAS X software. The scale indicates 10 μm. C, Kinetics of internalization were assessed using FACS staining on HCC-1428 cells under the same incubation procedures with 2 μg of Alexa-488–conjugated OBI-999 or isotype control antibody. The internalized signal was determined by incubation with 50 μg/mL anti–Alexa-488-quenching antibody at 4°C for 30 minutes followed by measuring the fluorescence intensity. The percentage of internalization was calculated based on MFI of the surface quenched and unquenched cells measured by FACSCanto II.

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The kinetics of internalization were evaluated by FACS staining with HCC-1428 cells. No intracellular signal was detected upon the initial treatment of OBI-999. After being incubated for 1 hour, 19% of internalization was observed followed by a time-dependent increase of internalization to a maximum of 67% at 23 hours, whereas no internalization signal was detected in the control antibody group (Fig. 2C). Both confocal and flow cytometry results showed that the internalization of OBI-999 occurred within 1 hour of incubation with the target cells.

Bystander effect of OBI-999

MCF-7 cells with high GH expression (97%) and ES-2/GFP cells with negligible GH expression (<1%) were used to investigate a potential bystander effect of OBI-999. The EC50 of OBI-999 against the MCF-7 and ES-2/GFP cells were 45 and 255 nmol/L, respectively (Fig. 3A). When cell cultures containing both MCF-7 and ES-2/GFP cells at a MCF-7/(ES-2/GFP) ratio of 0:1 or 8:1 were treated with 46 nmol/L OBI-999 for 13 days, the viability of ES-2/GFP cells in the coculture was 27.7%, which was much lower than that of ES-2/GFP cells alone treated with OBI-999, 88.7%. In addition, when treated with OBI-999 at 94 nmol/L, the viability of ES-2/GFP cells was 13.1% in the coculture, which was much lower than that of ES-2/GFP cells alone treated with OBI-999, 57.8%, as shown in Fig. 3B. These results suggest that the free MMAE released from OBI-999–treated high GH expression cells can trigger a bystander effect to kill ES-2/GFP cancer cells with negligible GH expression in the coculture experiments. The ES-2/GFP cell viability was minimally affected upon treatment with 94 nmol/L of Ctrl-ADC under the same coculture conditions (Fig. 3B).

Figure 3.

In vitro bystander effect of OBI-999. A, GH expression level and cytotoxicity of MMAE or OBI-999 on MCF-7 and ES-2/GFP cells. The GH expression was determined using FACS surface staining, and cell viability was evaluated using CellTiter-Glo viability assay kit with luminescence detection after 6 days of OBI-999 or MMAE treatment at various doses (0.1–6,000 nmol/L). B, Cell viability of ES-2/GFP cells treated with either OBI-999 (blue) or Ctrl-ADC (gray), or cocultured with MCF-7 cells and treated with either OBI-999 (green) or Ctrl-ADC (dark gray) for 13 days. ES-2/GFP cell viability was calculated from fluorescence intensity of treated cells relative to corresponding untreated cells (0 nmol/L). Statistically significant differences were indicated (***, P > 0.05). ns, not significant.

Figure 3.

In vitro bystander effect of OBI-999. A, GH expression level and cytotoxicity of MMAE or OBI-999 on MCF-7 and ES-2/GFP cells. The GH expression was determined using FACS surface staining, and cell viability was evaluated using CellTiter-Glo viability assay kit with luminescence detection after 6 days of OBI-999 or MMAE treatment at various doses (0.1–6,000 nmol/L). B, Cell viability of ES-2/GFP cells treated with either OBI-999 (blue) or Ctrl-ADC (gray), or cocultured with MCF-7 cells and treated with either OBI-999 (green) or Ctrl-ADC (dark gray) for 13 days. ES-2/GFP cell viability was calculated from fluorescence intensity of treated cells relative to corresponding untreated cells (0 nmol/L). Statistically significant differences were indicated (***, P > 0.05). ns, not significant.

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Another approach was also used to evaluate the bystander effect of OBI-999 by transferring the conditioned medium from high GH expression MCF7 cells treated with OBI-999 to low GH expression (8.4%) MDA-MB-231 cells. Significantly higher cytotoxicity was observed for cells incubated with the conditioned medium compared with cells treated with OBI-999 directly (Supplementary Fig. S8). These results indicate that OBI-999 is capable of triggering a bystander effect upon releasing free MMAE from high GH expression cells to kill surrounding cells with a low level of GH.

In vitro serum stability of OBI-999

OBI-999 and Adcetris at concentrations of 20 μg/mL were incubated in mouse or human serum at 37°C for 0, 1, 3, 5, and 7 days. The stability of the ADC was evaluated based on the level of MMAE released from the ADC. The level of MMAE released from Adcetris was much higher than that from OBI-999 in mouse serum, indicating that OBI-999 may be more stable than Adcetris in mouse serum. The levels of MMAE released from both OBI-999 and Adcetris in human serum were below 0.5% based on a DAR of 4, indicating that the levels of free MMAE were limited and that both OBI-999 and Adcetris were stable in human serum (Fig. 4A).

Figure 4.

Pharmacokinetic profile of OBI-999. A,In vitro serum stability of OBI-999 in mouse or human serum was conducted at 37°C for 1, 3, 5, and 7 days. MMAE release was determined by LC/MS/MS. B,In vivo pharmacokinetic profiles of OBI-999 (5 mpk) with NCI-N87 gastric tumor–bearing mice (n = 5). C and D, Organs and tumor tissues from tumor-bearing mice were harvested and homogenized to evaluate OBI-999 and free MMAE concentration at various time points from 0 to 336 hours after injection. OBI-999 total antibody and ADC concentrations were determined by ELISA, and MMAE was evaluated by LC/MS/MS.

Figure 4.

Pharmacokinetic profile of OBI-999. A,In vitro serum stability of OBI-999 in mouse or human serum was conducted at 37°C for 1, 3, 5, and 7 days. MMAE release was determined by LC/MS/MS. B,In vivo pharmacokinetic profiles of OBI-999 (5 mpk) with NCI-N87 gastric tumor–bearing mice (n = 5). C and D, Organs and tumor tissues from tumor-bearing mice were harvested and homogenized to evaluate OBI-999 and free MMAE concentration at various time points from 0 to 336 hours after injection. OBI-999 total antibody and ADC concentrations were determined by ELISA, and MMAE was evaluated by LC/MS/MS.

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Pharmacokinetics and tissue distribution of OBI-999 in tumor-bearing mice

OBI-999 was administered intravenously to NCI-N87 gastric cancer tumor (97% GH)–bearing BALB/c nude mice with a single injection at a dose of 5 mg/kg to assess the pharmacokinetics and tissue distribution of the ADC. The mean concentration of total GH antibody (tAb), OBI-999, and MMAE in serum and tumor is illustrated in Fig. 4B. The concentration of tAb and OBI-999 in serum decreased gradually, whereas the free MMAE in serum was minimal (<0.0018 μg/mL) throughout the study, indicating OBI-999 was highly stable in mouse serum.

In contrast, both OBI-999 and free MMAE in the tumor cells reached a maximum level of 3.3 and 0.029 μg/g at 72 and 168 hours, respectively, and was maintained at approximately these levels for up to 335 hours. Peak concentration (29 ng/g) of free MMAE in tumor homogenates was approximately 16 times higher than that of free MMAE in serum, indicating an efficient binding of OBI-999 on the GH-expressing tumor followed by internalization and subsequent release of MMAE at the tumor site.

Distribution of OBI-999 and free MMAE in tumor and organs is illustrated in Fig. 4C and D. OBI-999 was mainly distributed to the highly perfused organs at the first hour (i.e., liver, kidney, and spleen) and declined quickly over time. On the other hand, OBI-999 accumulated gradually in tumor cells, reaching a peak level at 72 hours, and maintained at a similar level for up to 168 hours (Fig. 4C). Similar distribution profiles of free MMAE in organs and tumor cells were also observed, where free MMAE quickly disappeared in organs upon initial distribution and gradually accumulated in tumor cells for up to 168 hours (Fig. 4D). The accumulation of OBI-999 and free MMAE in tumor cells clearly demonstrated the specificity of OBI-999 toward GH-positive tumor cells, resulting in growth inhibition of the tumor as described subsequently. Detailed pharmacokinetic parameters are summarized in Supplementary Table S3.

In addition, indium111-labeled OBI-999 was also administered to normal and tumor-bearing mice to investigate its biodistribution. Similar to the pattern described above, the majority of indium111-labeled OBI-999 was detected in the tumor (Supplementary Fig. S9). Taken together, these results demonstrate that OBI-999 is highly specific to GH-expressing tumor cells.

Antitumor efficacy of OBI-999 in multiple cell line–derived xenograft and PDX models

The antitumor efficacy of OBI-999 was evaluated in animal models using GH-expressing cancer cells. In an MCF-7 breast cancer xenograft model, the expression of GH was more than 90% based on FACS staining on the day before inoculation. Mice were treated with OBI-999 once weekly at doses of 1 and 3 mg/kg (mpk) for a total of six injections, and 10 mpk for a total of two injections. TGI reached 77% at 1 mpk and >100% at both doses of 3 and 10 mpk (Fig. 5A). In gastric cancer NCI-N87 xenograft model under a similar treatment protocol (once weekly for four injections at 1, 3, and 10 mpk), an 83% TGI was achieved at a dose of 1 mpk and 97% at both doses of 3 and 10 mpk. Ctrl-ADC (anti-CD30 MMAE) and free MMAE treatment groups were included in this study as negative controls. Ctrl-ADC (3 mpk) or free MMAE (0.19 mpk: mole equivalent to 10 mpk ADC) exhibited low level of TGI, whereas OBI-999 with target specificity achieved complete tumor suppression at the same dose level, suggesting that tumor suppression effect is antigen dependent (Fig. 5B).

Figure 5.

Antitumor efficacy of OBI-999 on multiple xenograft models. TGI activity was evaluated in MCF-7, NCI-N87, HPAC xenograft models, and LU-01-0266 lung cancer PDX model. A, MCF-7 xenograft mice were intravenously treated with 1 or 3 mg/kg OBI-999 once a week for six injections, or 10 mg/kg OBI-999 once a week for two injections. B, NCI-N87 xenograft mice were intravenously treated with 1, 3, or 10 mg/kg OBI-999, or MMAE 0.191 mg/kg, or Ctrl-ADC 3 mg/kg weekly for four injections. C and D, HPAC and LU-01-0266 xenograft mice were intravenously treated with 1, 3, or 10 mg/kg OBI-999 weekly for four injections. E, MCF7 xenograft mice were intravenously treated with 3 mg/kg OBI-999 weekly for four injections (QWx4) or once every 3 weeks for two injections (Q3Wx2). In MCF-7, NCI-N87, and LU-01-0266 studies, the treatment was initiated on the day after cell implantation with average tumor volume of 100 to 175 mm3. In the HPAC study, the treatment was initiated 7 days after cell implantation, as indicated by arrows. The GH expression levels of MCF-7, NCI-N87, HPAC, and LU-01-0266 were 97%, 96%, 97%, and 26% respectively, which were detected by FACS on the inoculation day.

Figure 5.

Antitumor efficacy of OBI-999 on multiple xenograft models. TGI activity was evaluated in MCF-7, NCI-N87, HPAC xenograft models, and LU-01-0266 lung cancer PDX model. A, MCF-7 xenograft mice were intravenously treated with 1 or 3 mg/kg OBI-999 once a week for six injections, or 10 mg/kg OBI-999 once a week for two injections. B, NCI-N87 xenograft mice were intravenously treated with 1, 3, or 10 mg/kg OBI-999, or MMAE 0.191 mg/kg, or Ctrl-ADC 3 mg/kg weekly for four injections. C and D, HPAC and LU-01-0266 xenograft mice were intravenously treated with 1, 3, or 10 mg/kg OBI-999 weekly for four injections. E, MCF7 xenograft mice were intravenously treated with 3 mg/kg OBI-999 weekly for four injections (QWx4) or once every 3 weeks for two injections (Q3Wx2). In MCF-7, NCI-N87, and LU-01-0266 studies, the treatment was initiated on the day after cell implantation with average tumor volume of 100 to 175 mm3. In the HPAC study, the treatment was initiated 7 days after cell implantation, as indicated by arrows. The GH expression levels of MCF-7, NCI-N87, HPAC, and LU-01-0266 were 97%, 96%, 97%, and 26% respectively, which were detected by FACS on the inoculation day.

Close modal

In pancreatic cancer HPAC xenograft model, the antitumor efficacy was evaluated at 1, 3, and 10 mpk doses with once-weekly dosing for 4 weeks, which showed a TGI of 4%, 36%, and >100%, respectively (Fig. 5C). In a PDX LU-01-0266 lung cancer model, OBI-999 showed TGI in a dose-dependent manner with 66%, 86%, and >100%, respectively, under the same dosing conditions. Although LU-01-0266 cancer cells exhibit low prevalence in GH expression (26%), the tissue IHC staining showed high intensity on cell membrane (Supplementary Fig. S10), which may contribute to higher binding affinity of OBI-999 to the cells. The excellent efficacy of OBI-999 in this model may be attributed to the efficient internalization and bystander effects of the ADC, suggesting that the therapeutic effect of OBI-999 may not be limited to high GH expression tumors (Fig. 5D). It is noteworthy that tumor regression lasted up to 2.5 months after dosing ceased, and no tumor recurrence was observed (Fig. 5A–D).

The dosing frequency of OBI-999 in animal models was also investigated. The antitumor efficacy was compared between once-weekly injection for a total of four injections (QWx4) and once-every-3-week injection for a total of two injections (Q3Wx2) in a MCF-7 xenograft model. The efficacy was comparable between the two dosing regimens, suggesting a 3-week treatment interval of OBI-999 can still exert a long-lasting TGI effect (Fig. 5E).

Safety of OBI-999 in cynomolgus monkeys

The safety of OBI-999 was evaluated in male and female cynomolgus monkeys. The cynomolgus monkey was considered a relevant species because the monkey tissues also express GH, which can be recognized by parental anti-GH mAb OBI-888 using IHC. Monkeys were administered at dose of 0, 2, 5, or 10 mg/kg of OBI-999 via intravenous infusion once every 3 weeks for a total of two injections. The toxicological findings are summarized in Table 1. No lethal toxicity was found.

Table 1.

Summary of toxicologic and toxicokinetic studies in a repeat-dose toxicity findings of OBI-999 in cynomolgus monkeys.

Toxicities of OBI-999
Parameter/organ systemEffects
Body weight Decreased changes in body weight at 10 mg/kg/dose 
Food consumption Decreases in food consumption at 10 mg/kg/dose 
Hematology A cycle of bone marrow suppression 5 to 7 days after dose with a regenerative hematopoietic effort 2 to 3 weeks after dose 
White blood cell populations Minimal to mild transient decreases at ≥5 mg/kg dose 
Red blood cell populations Minimal to mild changes followed by a reticulocyte rebound at ≥5 mg/kg dose 
Bone marrow Minimal to mild decreased M:E ratio at 10 mg/kg dose 
Liver Minimal increases in AST levels and mild decreases in albumin levels at 10 mg/kg dose 
Toxicokinetic parameters at 10 mg/mL on day 22 (mean ± SD) 
 Total antibody OBI-999 ADC MMAE (pg/mL)a 
 Male Female Male Female Male Female 
Cmax (μg/mL) 185 ± 16.0 183 ± 46.4 187 ± 11.9 201 ± 30.6 456 ± 94.3 666 ± 458 
AUC (h*μg/mL) 7,890 ± 816 6,610 ± 2,660 8,820 ± 876 8,530 ± 1,960 55,300 ± 8,680 56,600 ± 13,100 
T1/2 (h) 44.4 ± 4.6 40.3 ± 11.1 46.8 ± 2.5 48.0 ± 3.1 88.8 ± 12.3 72.8 ± 24.6 
Toxicities of OBI-999
Parameter/organ systemEffects
Body weight Decreased changes in body weight at 10 mg/kg/dose 
Food consumption Decreases in food consumption at 10 mg/kg/dose 
Hematology A cycle of bone marrow suppression 5 to 7 days after dose with a regenerative hematopoietic effort 2 to 3 weeks after dose 
White blood cell populations Minimal to mild transient decreases at ≥5 mg/kg dose 
Red blood cell populations Minimal to mild changes followed by a reticulocyte rebound at ≥5 mg/kg dose 
Bone marrow Minimal to mild decreased M:E ratio at 10 mg/kg dose 
Liver Minimal increases in AST levels and mild decreases in albumin levels at 10 mg/kg dose 
Toxicokinetic parameters at 10 mg/mL on day 22 (mean ± SD) 
 Total antibody OBI-999 ADC MMAE (pg/mL)a 
 Male Female Male Female Male Female 
Cmax (μg/mL) 185 ± 16.0 183 ± 46.4 187 ± 11.9 201 ± 30.6 456 ± 94.3 666 ± 458 
AUC (h*μg/mL) 7,890 ± 816 6,610 ± 2,660 8,820 ± 876 8,530 ± 1,960 55,300 ± 8,680 56,600 ± 13,100 
T1/2 (h) 44.4 ± 4.6 40.3 ± 11.1 46.8 ± 2.5 48.0 ± 3.1 88.8 ± 12.3 72.8 ± 24.6 

Abbreviations: AST, aspartate aminotransferase; AUC, area under the concentration–time curve; Cmax, maximum concentration; M:E, myeloid to erythroid; T1/2, half-life.

aMMAE concentrations were measured as pg/mL (Cmax = pg/mL; AUC = h*pg/mL).

The major adverse effects of OBI-999 were hematologic changes, including decreased counts of neutrophils, monocytes, lymphocytes, and red cell mass that occurred predominantly at the 10 mg/kg dose. These effects were reversible; an increased reticulocyte count occurred approximately 2 to 3 weeks after the dose. As a result, the highest nonseverely toxic dose (HNSTD) was determined to be 10 mg/kg in cynomolgus monkeys. The corresponding exposures of OBI-999, total antibody, and free MMAE at 10 mg/kg on day 22 are listed in Table 1.

In this study, we demonstrated that OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibitory effects in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers. The impressive preclinical efficacy and safety can be attributed to the high specificity of OBI-999 to GH-expressing tumor cells both in vitro and in vivo; the efficient internalization and trafficking of OBI-999 through endosome and lysosome compartments to release free MMAE, which is capable of exerting a bystander effect to kill surrounding low GH-expressing tumor cells; and the preferential accumulation and retention of OBI-999 and the released MMAE in the tumor region. In addition to these desirable properties, OBI-999 demonstrated an acceptable safety profile in monkeys.

As a result, a phase I/II clinical trial (NCT04084366) has been initiated to evaluate the MTD and efficacy profile of OBI-999 in patients with advanced solid tumors, which includes IHC prescreening for GH expression.

OBI-999 used a unique ThioBridge linker system to conjugate with the payload MMAE. The conjugation is designed to be site-specific to cysteine residues at the Fab and hinge regions of the antibody through interchain disulfide bond reduction to make OBI-999 a homogenous ADC with a DAR of 4 (22). There are several advantages of using this conjugation system. First, the site-specific conjugation potentially improves the therapeutic efficacy of the ADC. A head-to-head comparison study conducted by Bai and colleagues in 2019 demonstrated the superiority in efficacy of site-specific cysteine-conjugated ADC compared with a lysine-conjugated ADC (26). Second, bridging the disulfides produces a homogenous ADC with minimal impact on the antibody structure, which increases the overall stability of ADC. Reports have shown that the homogeneity of DARs contributes to the efficacy of the ADC (22, 26). This result is consistent with our observation in OBI-999 pharmacokinetic study, indicating that only a limited amount of free MMAE was present in the serum fraction (Fig. 4D).

Another feature of OBI-999 is the conjugation of multiple PEGs on the branch of the linker. In 2015, Lyon and colleagues showed that the PEG conjugation to an ADC with an auristatin drug could decrease the hydrophobicity of the ADC. In addition, there was an inverse correlation between hydrophobicity and the antitumor activity of the ADC in animal studies (27), which may be ascribed to reducing the aggregation propensity of the ADC. As a result, the linker systems used in OBI-999 may enhance its stability in the circulation, which, in turn, improves its efficacy.

MMAE is an antineoplastic agent that has been used in many ADCs approved by the FDA, including Adcetris, Polivy (polatuzumab vedotin-piiq), and Padcev (enfortumab vedotin-ejfv; ref. 28). In addition to the cytotoxicity, the membrane-permeable property of MMAE gives it an additional advantage in triggering a bystander effect, which is important for killing tumors that express heterogeneous levels of target antigen (29). Okeley and colleagues demonstrated that the release of free MMAE from CD30-positive cells could mediate CD30-negative cell death upon treatment with Adcetris, which provided strong evidence of a bystander effect (30). Consistent with their observation, we showed that the bystander effect was also observed in the study where high and negligible GH-expressing cells were cocultured and treated with OBI-999. The viability of GH-negative ES-2/GFP cells in the coculture experiment was much lower than the viability of ES-2/GFP cells alone (i.e., without coculture) when treated with OBI-999, suggesting that free MMAE can be released from high GH-expressing tumor cells and subsequently kill surrounding low GH-expressing tumor cells. This effect may strengthen the overall antitumor efficacy of OBI-999.

We also conducted experiments to determine if the activity of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) could potentially contribute to the efficacy of OBI-999. However, the binding activity of OBI-999 toward various Fcγ receptors was not as strong as its parental antibody OBI-888 (Supplementary Fig. S11); hence, no significant ADCC or CDC activity was observed (Supplementary Fig. S12). Our observation is consistent with previous publications, suggesting that PEG conjugation to an ADC might contribute to the loss of affinity to Fcγ receptors (31, 32).

When comparing the serum stability of OBI-999 with that of Adcetris, the high percentage (>10% at day 7) of free MMAE detected in mouse serum may have resulted from the presence of carboxylesterase 1C in mouse serum. The valine–citrulline peptide linker used in OBI-999 and Adcetris is known to be susceptible to extracellular carboxylesterase (33). The linker of OBI-999 contains a glutamic acid to avoid premature cleavage by increasing steric hindrance (34), which may contribute to the better serum stability of OBI-999 than that of Adcetris in mouse serum.

In the in vivo pharmacokinetic studies, ADCs usually display a shorter half-life and faster clearance compared with the total antibody (35, 36). However, pharmacokinetic profiles of the total antibody and OBI-999 in mice were almost superimposable. The fact that the clearance and half-life of OBI-999 were comparable with those of the total antibody indicates that OBI-999 is stable in vivo. Levels of MMAE released from both OBI-999 and Adcetris were below 0.5% of the calculated maximal release (%) in human serum, suggesting that OBI-999 is stable in human serum with minimal release of MMAE.

In summary, OBI-999 exhibited many desirable properties as a novel GH-targeting ADC: a homogeneous DAR, efficient internalization rate, tumor-specific ADC accumulation, and payload release against GH-expressing cancer cells, a bystander effect, excellent efficacy in animal models, and an acceptable safety margin in monkeys. These results warrant the further investigation of this novel ADC in patients with solid tumors, and a phase I/II clinical trial (NCT04084366) of OBI-999 in these patients with cancer is currently ongoing.

W.-F. Li reports a patent for WO2018094414A1 issued. I.-J. Chen reports a patent for WO2015/157629 issued, a patent for WO2017/062792 issued, and a patent for WO2018094414A1 issued. J.-S. Lai reports a patent for WO2018094414A1 pending. M.-T. Lai reports a patent for WO2018094414A1 issued. No disclosures were reported by the other authors.

M.-C. Yang: Conceptualization, supervision, writing–original draft, writing–review and editing. C.-S. Shia: Data curation, formal analysis, writing–original draft, writing–review and editing. W.-F. Li: Data curation, supervision, methodology, writing–original draft, project administration, writing–review and editing. C.-C. Wang: Data curation, supervision, investigation, methodology, writing–review and editing. I.-J. Chen: Data curation, supervision, investigation, methodology, writing–review and editing. T.-Y. Huang: Conceptualization, data curation, supervision, methodology, writing–original draft, writing–review and editing. Y.-J. Chen: Data curation, methodology, writing–original draft, writing–review and editing. H.W. Chang: Data curation, methodology, writing–original draft, writing–review and editing. C.-H. Lu: Data curation, methodology, writing–original draft, writing–review and editing. Y.-C. Wu: Data curation, methodology, writing–original draft, writing–review and editing. N.-H. Wang: Conceptualization, data curation, supervision, methodology, writing–original draft, writing–review and editing. J.-S. Lai: Supervision, methodology. C.-D. Yu: Conceptualization, data curation, supervision, methodology, writing–original draft, writing–review and editing. M.-T. Lai: Conceptualization, data curation, supervision, methodology, writing–original draft, writing–review and editing.

This work was supported by OBI Pharma Inc. The authors would like to thank Wei-Han Lee and Pin-Tzu Chiu for their contributions on study coordination and technical assistance.

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.

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