MTX-13, a Novel PTK7-Directed Antibody–Drug Conjugate with Widened Therapeutic Index Shows Sustained Tumor Regressions for a Broader Spectrum of PTK7-Positive Tumors

This article is featured in Selected Articles from This Issue, p. 1113

Dysregulation of Wnt signaling, including both canonical and noncanonical pathways, has been linked to cancer development (1). The mediators of noncanonical Wnt signaling include receptor tyrosine kinase family members ROR1, ROR2, RYK, and PTK7. All of these are pseudokinases that lack phosphotransferase activity (2). Among them, PTK7 is overexpressed in many tumors including advanced non–small cell lung cancer (NSCLC), ovarian cancer, triple-negative breast cancer (TNBC), colon cancer, and gastric and esophageal cancers (3–7). PTK7 overexpression has been associated with a poor prognosis, tumor metastasis, and worse overall survival in patients (8–13), due to its enrichment on tumor-initiating cells (TIC) or cancer stem cells (CSC; refs. 14, 15). Thus, PTK7 is an attractive target that may facilitate elimination of cancer cells and the TICs responsible for tumor recurrence and progression. PTK7 is not amenable for small-molecule inhibitor targeting due to a lack of catalytic activity. However, its extracellular domain (ECD) can be targeted by antibody-based modalities such as bispecific antibody or antibody–drug conjugate (ADC).

ADCs provide tumor cell–targeted delivery of cytotoxic drugs (“payloads”) on antibody carriers and the antitumor activity of ADCs mainly depends on cell-surface target expression, representing a viable strategy for targeting cell-surface targets regardless of their biological function (16, 17). PF-06647020/h6M24-vc0101 (cofetuzumab pelidotin) is a PTK7-targeting ADC comprising a humanized anti-PTK7 mAb (hu6M024, IgG1) joined to an auristatin microtubule inhibitor payload, auristatin-0101 (Aur0101), by a cleavable valine-citrulline (vc)-based linker (18, 19). In addition to PF-06647020, HER2-targeting ADC trastuzumab-pelidotin (PF-06804103) and TROP2-targeting ADC PF-06664178 (RN927C) have also used Aur0101 as payload (20, 21). However, Aur0101-based ADCs are hindered by their systemic toxicity and limited clinical efficacy. PF-06647020 showed modest therapeutic activity in moderate or high PTK7 expression tumors with an overall objective response rate (ORR) of 10%–30% in patients with ovarian cancer, NSCLC, and TNBC at a dose range of 2.1–3.2 mg/kg in a phase I clinical trial (22). PF-06804103 showed a preliminary ORR of about 50% in the HER2⁺ patients and observed dose-limiting toxicities (DLT), including arthralgia, neuropathy, myalgia, and osteomuscular pain (23). PF-06664178 was terminated due to excess toxicity and a lack of meaningful response in patients despite potent activity in a preclinical study (21, 24).

On the other hand, HER2-targeting ADC DS-8201a (T-DXd, DAR 8) using the less potent payload topoisomerase I (Top I) inhibitor DXd showed an ORR of 80% in HER2⁺ patients and a 50% ORR even in HER2-low expression breast cancer patients (25, 26). Similarly, TROP2-targeting ADCs using DXd (datopotamab deruxtecan, DAR4, phase I–II) or another Top I inhibitor SN-38 (IMMU-132/Trodelvy, DAR 8, regulatory approval) showed impressive efficacy with better tolerability when compared with RN927C (27, 28). Top I inhibitors are 10–100 times less potent than microtubule inhibitors and induce tumor cell apoptosis by binding to and inhibiting Top I-DNA complexes, leading to the inhibition of DNA replication, cell-cycle arrest, and cell apoptosis (29). By using payloads with a strong bystander killing effect, DS-8201a/IMMU-132/datopotamab deruxtecan are administered at a higher clinical maximum tolerated dose of 5–10 mg/kg in order to achieve improved clinical efficacy in patients. Improving a PTK7 targeting ADC by adopting similar design principles (lower potency payload, higher DAR, high stability linker, stronger bystander killing effect), might produce a higher patient response in broader tumor types.

We have recently introduced a class of ADCs, T moiety–exatecan conjugates, with improved antitumor performance and therapeutic index (30). Exatecan, the precursor of DXd, has a superior payload profile characterized by higher potency, reduced sensitivity to multidrug resistance (MDR), and higher cell permeability for bystander killing effect than DXd (31, 32). Exatecan was conjugated to an antibody through a novel, hydrophilic self-immolative moiety T1000 to overcome the high hydrophobicity of exatecan (32). T moiety–exatecan conjugates are characterized by improved stability, potency, bystander killing capability, and intratumoral pharmacodynamic response (30). Here we present a T moiety–exatecan–based PTK7-targeting ADC, MTX-13, with an improved therapeutic index compared with PF-06647020, ability to overcome tumor resistance, and potent antitumor efficacy in PTK7-expressing solid tumors. MTX-13 (Ab13-Val-Ala-T1000-exatecan) has eight exatecan molecules (DAR 8) and is homogeneous, hydrophilic, and stable both in vitro and in vivo. MTX-13 shows potent therapeutic activity in mouse xenograft tumor models of ovarian cancer and TNBC, small cell lung cancer, and squamous cell carcinoma (SCC) in the lungs, head and neck, and esophagus, tumors that are often resistant to benchmark ADC h6M24-vc0101. In addition, MTX-13 significantly inhibited tumor growth and metastasis in an orthotopic colon cancer xenograft model. Pharmacokinetics and toxicological studies in monkeys demonstrated that MTX-13 possesses a favorable terminal half-life and stability and is well tolerated. MTX-13 is a PTK7-targeting ADC with an expanded therapeutic index, and our data build a case in favor of clinical investigation into MTX-13 as an agent against PTK7-expressing malignancies, particularly in tumors that currently lack effective ADCs.

Six- to 8-week-old female BALB/c mice were used for the immunization process in which antigenic peptides were injected repeatedly for 6 times over 6 weeks. One day after the final immunization, total splenocytes were isolated and fused with the myeloma cell line SP2/0 (TCM18, stem cell bank, Chinese Academy of Sciences) by using PEG for cell fusion and HAT medium for hybridomas selection. After 7 to 10 days, single hybridoma clones were isolated and antibody-producing hybridomas were selected by screening supernatants for antigen binding using ELISA. All positive clones were injected into mice for ascites generation. Ascites fluid was purified in protein A/G (MabSelect, GE Healthcare Lifesciences) gravity columns and screened again using flow cytometry. One clone, mAb13, was selected for subsequent sequencing, humanization, and further development into the final antibody Ab13.

Anti-PTK7 antibody h6M24, anti-B7H3 antibody hM30, anti-DLL3 antibody SC16 were generated in CHO expression system and purified by MabSelect Sure Resin (GE Healthcare Lifesciences) using a gravity column. The sequences of h6M24 (33), SC16 (34), and hM30 (35) were obtained from the corresponding patents and were summarized in Supplementary Table S1. Isotype IgG was purchased from Beijing Solarbio Science and Technology Co., Ltd. ADCs were synthesized as described below, and their DAR values were determined by reverse-phase chromatography and LC-MS. The drug distribution was analyzed by hydrophobic interaction chromatography (HIC). DS7300a was made in house using linker-payload directly purchased from MedChemExpress: MC-VC-PABC-Aur0101(HY-128955, MedChemExpress) and MC-GGFG-DXd (HY-114233, MedChemExpress). Etoposide (HY-13629) and cisplatin (HY-17394) were all purchased from MedChemExpress.

A solution of naked mAb (10 mg/mL in PBS (7.0) + 2 mmol/L EDTA) was treated with 7 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 2 hours at 37°C. Then 14 molar equivalents of linker-drug (from a 10 mmol/L DMA stock solution) were added to the fully reduced antibody, while keeping residual DMA concentration below 10% (v/v). The mixture was incubated at room temperature for an hour followed by purification over a Zeba spin desalting column (Thermo Fisher Scientific) to remove excess reagents. In this step, the resulting ADC was also buffer exchanged into formulation buffer (20 mmol/L histidine, 150 mmol/L NaCl, pH5.5). Moreover, the excess linker-drug could be removed thoroughly by treating the product with activated charcoal (30 mg of charcoal to 1 mL ADC solution) followed by vortexing for 2 hours at room temperature. The charcoal was then removed via sterile filtration (0.2 μm PES filters) and the final ADC was stored at −80°C.

The concentration of mAb and conjugated drug in the antibody–drug conjugate was calculated by measuring UV absorbance of an aqueous solution of the antibody–drug conjugate at two wavelengths of 280 nm and 370 nm, as the total absorbance at any given wavelength is equal to the sum of the absorbance of all light-absorbing chemical species that are present in a system (additivity of absorbance). Equations were as follows.

A280 = AD,280 + AA,280 = εD,280CD +εA,280CA (1) and

A370 = AD,370 + AA,370 = εD,370CD +εA,370CA (2).

The values of εA,280, εA,370, εD,280, and εD,370 were estimated based on calculating the amino acid sequence of the antibody or obtained by UV measurement of the compound according to Lambert-Beer's law. Solving the simultaneous equations (1) and (2) by substitution of the above values and then CA and CD were determined. Dividing CD by CA, then the average number of conjugated drug molecules per antibody was determined.

Aggregation was assessed by size exclusion chromatography which was performed on an Agilent 1260 Infinity II HPLC system with UV detection at 280 nm. Column was a TSKgel G3000SWXL column (Tosoh Bioscience). Samples were injected at 50 μg load. Mobile phase was 200 mmol/L sodium phosphate and 150 mmol/L sodium chloride (pH 7.0). Also, 15% (v/v) isopropanol was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and furthermore prevent bacterial growth. The flow rate was 0.75 mL/minutes and column temperature was set at room temperature.

Antibody–drug conjugates with a different number of drugs per antibody were separated using a butyl HIC column (TSK-gel Butyl-NPR 4.6 × 35 mm 2.5 μm, Tosoh Bioscience). HIC was also performed on an Agilent 1260 Infinity II HPLC system with UV detection at 280 nm. Mobile phase A was 1.5 M (NH₄)₂SO₄, 50 mmol/L K₂HPO₄, pH7.0 and mobile phase B was 21.3 mmol/L KH₂PO₄, 28.6 mmol/L K₂HPO₄, 25% (v/v) isopropanol, pH7.0. Gradient program was as follows: B %: 0%–25% (0–1 minute, 0.8 mL/minute), 25% (1–3 minutes, 0.6 mL/minute), 25%–80% (3–13 minutes, 0.6 mL/minute), 80% (13–17 minutes, 0.6 mL/minute), 80%–0% (17–17.10 minutes, 0.5 mL/minute), 0% (17.10–25 minutes, 0.7 mL/minute).

The human cell lines were purchased from the ATCC or stem cell bank, Chinese Academy of Sciences (Shanghai, China). Cell lines were identified by short tandem repeat analysis and mycoplasma was detected.

According to ATCC culture standards, MDA-MB-468 and FaDu cells were kept in DMEM, supplemented with 10% FBS. SNU-16, PC-9, NCI-H2170, NCI-H226, NCI-H520, OVCAR3, KYSE-150, PC9, BXPC-3, and NCI-H1975 were cultured in RPMI-1640 medium with 10%FBS. SK-OV-3 and HCT116-luc cells are cultured in McCoy's 5A medium with 10% FBS. A549 is cultured in Ham's F-12K medium with 10% FBS. All cell lines were maintained in a humidified incubator at 37°C, 5% CO₂.

SNU-16 (RRID: CVCL_0076) cell lines were purchased from the ATCC in 2020. SK-OV-3 (RRID: CVCL_0532), PC-9 (RRID: CVCL_B260), NCI-H226 (RRID: CVCL_1544), OVCAR3 (RRID: CVCL_0465), KYSE-150 (RRID: CVCL_1348), MDA-MB-468 (RRID: CVCL_0419), NCI-H1975 (RRID: CVCL_1511), and A549 (RRID: CVCL_0023) cell lines were purchased from a stem cell bank, Chinese Academy of Sciences (Shanghai, China) in 2018. NCI-H2170 (RRID: CVCL_1535) and NCI-H520 (RRID: CVCL_1566) cell lines were purchased from Nanjing Cobioer Biosciences Co., Ltd. HCT-116 luc (RRID: CVCL_J254) is purchased from WuXi AppTec (Suzhou) Co., Ltd. All patient-derived xenograft (PDX) models were obtained from Lidebiotech Co., Ltd. in 2021. Cells were maintained in RPMI-1640 medium or DMEM or F-12K medium or McCoy's 5A medium supplemented with 10% heat-inactivated FBS (as introduced in Cell Culture) and cultured at 37°C and 5% CO₂ atmosphere. When cells were grown to ∼80% confluence, they were dissociated from culture plates by treatment with 1× PBS and 1 mmol/L EDTA for 10 to 20 minutes. All cell lines were authenticated by short tandem repeat analysis according to suppliers’ information. All cells were tested to ensure that they were negative for mycoplasma by using Mycoplasma PCR Detection Kit (MP004, GeneCopoeia) prior to conducting cell-based assays. And all cells and PDX models were used for the experiments within 5 to 15 passages and 2 to 5 passages, respectively, after collection.

For cell-surface proteins, IF and flow cytometry assays were performed under nonpermeable conditions without detergent in the buffers. Antibody binding signal was detected using Alexa Fluor 488 and 594 goat anti-mouse IgG secondary antibodies (Lot. 115-545-003 and Lot. 115-585-003, Jackson ImmunoResearch) or Fluor 488 goat anti-human IgG secondary antibody (Lot. 109-545-008, Jackson ImmunoResearch). Briefly, cells attached on coverslips (IF assays) or suspended in 1× PBS (Flow cytometry assays) were first fixed in 4% paraformaldehyde (PFA) for 10 minutes. PFA was then removed, and cells were rinsed three times with 1× PBS. Cells were blocked overnight at 4°C in blocking buffer (1× PBS containing 10% normal goat serum). After removing the blocking buffer, cells were incubated with primary antibody (dilution in the blocking buffer at 1:100 to 1,000 in accordance with each instruction) for 2 hours at room temperature. Cells were rinsed three times in 1× PBS before being incubated with fluorescence-labeled secondary antibody (diluted in blocking buffer at 1:500 dilution ratio with 1:10,000 Hoechst 33258; Sigma) for 1 hour. Last, cells were rinsed three times with 1× PBS. IF images were recorded with OLYMBUS CKX53 fluorescence microscope. The three-dimensional reconstruction of the IF results was performed in ImageJ [National Center for Biotechnology Information, NIH (NCBI) free software]. The FACS data were collected using a BD Accuri C6 Plus system. A control sample without primary antibody and another sample with isotype control antibody were used. The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude dead cells. Relative mean fluorescence intensity (rMFI) was calculated by the ratio of MFI of anti-target Ab-FITC/MFI of isotype control-FITC.

For the internalization of antibody or ADC in vitro, cancer cells were first grown on 10-cm tissue culture plates until confluent. Cells were detached by 1 mmol/L EDTA and collected by centrifugation at 1,500 rpm. Cells were resuspended in blocking buffer (1× PBS+10% goat serum) and incubated at 37°C for 30 minutes. After washing with 1× PBS for 3 times, 1 μg/mL of MabJ253 antibody was added to the cells and incubated at 4°C for 30 minutes. Cells were then washed and moved to a 37°C incubator, and at each time point (0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, and 8 hours) a fraction of cells was removed, washed, and fixed in 4% paraformaldehyde for 10 minutes. The cells were then washed and resuspended in PBS and kept on ice. Once samples of all the time points were collected, the cells were incubated with FITC-conjugated goat anti-mouse secondary antibody in a blocking buffer before FACS analysis.

Cells were seeded to a 96-well plate at an appropriate density depending on the cell line (typically 1,000–3,000 cells per well in a 100 μL of appropriate culture media). After overnight incubation, a concentration series of small molecule or naked antibodies or ADC drugs and controls were added. Cell viability was evaluated after 5 days. CCK-8 (10%, v/v, HY-K0301, MedChemExpress) or CellTiter-Glo (CTG) buffer (G7570, Promega) was added into the wells, and incubation was continued for 1 to 2 hours at 37°C or 10 minutes, respectively. Absorbance values were measured on a Thermo Scientific Multiskan EX microplate reader using a wavelength of 450 nm for CCK-8 assay and luminescence detection for CTG assay. The IC₅₀ values compared with untreated cells or treated with other controls were determined using inhibition dose-response curve fitting (GraphPad Prism 9). Bystander killing efficacy of MTX-13 or isotype control IgG-T1000–exatecan ADC was measured using an in vitro cell culture system with single or cocultures of PTK7⁻ (A549, copy number < 1,000/cell) and PTK7⁺ (NCI-H520, copy number > 70,000/cell) cell lines. After 5 days of incubation with corresponding treatments in 6-well plates, the adherent cells were collected, and cell number and ratio of PTK7‐positive and PTK7‐negative cells were determined by a cell counter (Countess3, Invitrogen) and a flow cytometer (BD Accuri C6 Plus), respectively.

All tumor models were established in female BALB/c nude mice which were purchased from Shanghai Family Planning Research Institute. All in vivo studies were performed in accordance with the local guidelines of the Institutional Animal Care and Use Committee of Multitude Therapeutics and with the approval of the committee. Briefly, 4-to-6-week-old mice were housed together in sterilized cages and maintained under required pathogen-free conditions. Each cell suspension or tumor fragment was inoculated subcutaneously into female nude mice. When the tumor had grown to an appropriate volume, the tumor-bearing mice were randomized into treatment and control groups based on the tumor volumes, and dosing was started. Antibodies and ADCs were administered intravenously to the mice. Small-molecule drugs were administered orally.

Tumor volume defined as 1/2 × length × width² was measured twice (3–4 days) a week. TGI (%) was calculated as follows: TGI (%) = [1 – (mean of treatment group tumor volume on evaluation day)/(mean of control group tumor volume on evaluation day)] × 100. The mice were euthanized with CO₂ gas when they reached endpoints (tumor volume exceeding 3,000 mm³, >10% reduction of body weight, or clinical signs indicating that mice should be euthanized for ethical reasons). To establish the orthotopic HCT116-luc cell-derived xenograft model, HCT116-luc cells were surgically implanted into the cecal wall of 4-week-old male BALB/c nude mice. Thirteen days after the implantation, mice showed comparable bioluminescence intensity of the tumors and were divided into five groups (5 mice/group) with tumor growth being monitored by in vivo bioluminescence imaging twice a week. The surgically inoculated mice were weighed and intraperitoneally administered luciferin at a dose of 150 mg/kg. After the animals were completely anesthetized with the mixture gas of oxygen and isoflurane, the mice were moved into the imaging chamber for bioluminescence measurements with an IVIS (Lumina III) imaging system. The bioluminescence of the whole animal body, including primary and metastatic tumors, was measured and recorded once per week according to protocol. Tumor growth curve is plotted with bioluminescence intensity (photons/second). At the end of the experiment (day 39 after first dosing), major organs (lungs, liver, spleen, kidneys, pancreas, diaphragm, and mesentery) from all experimental groups were excised for anti-metastasis evaluation with bioluminescence imaging. Organ metastasis rate refers to the ratio of the number of animals with metastases in a certain organ in the group to the number of animals in the group.

Concentrations of ADC and the total antibody in plasma were determined with a validated ligand-binding assay; the lower limit of quantitation was 0.02 μg/mL. Briefly, immune plates were coated with 1 μg/mL human PTK7 protein, His Tag (ER3-H5223, Acrobiosystems Inc.) in coating buffer and kept overnight at 4°C. After washing, the plates were blocked and each serially diluted sample was added to the wells. After incubation for 1 to 2 hours at 37°C, the plates were washed and incubated with HRP-conjugated anti-human IgG Fc secondary antibody (ab99759, Abcam Inc.) for total antibody measurement or a biotin-labeled anti-exatecan/DXd antibody (ADC-Ab0001, Abmart) for ADC measurement. After reaction at 37°C for 1 to 2 hours, TMB solution (Lot. 50-85-05, KPL Inc.) was added directly or after incubated with streptavidin protein, HRP (21124, Thermo Fisher Scientific Inc.) for 40–60 minutes at 37°C. A450 in each well was measured with a microplate reader. MTX-13 was intravenously administered at 10.0 mg/kg to NCI-H1975 cell-derived xenograft tumor-bearing BALB/c-nu nude mouse. Plasma concentrations of ADC and total antibody were measured up to 21 days after dose.

For a binding assay, immune plates were coated with 1 μg/mL Human His tag PTK7 protein, Cynomolgus His tag PTK7 protein, and Mouse His tag PTK7 protein that were all purchased from Acrobiosystems in coating buffer and kept overnight at 4°C. After washing, the plates were blocked and each serially diluted substance was added to the wells. After incubation for 1 to 2 hours at 37°C, the plates were washed and incubated with HRP-conjugated anti-human IgG secondary antibody for 1 hour at 37°C. After washing, TMB solution was added, and A₄₅₀ in each well was measured with a microplate reader.

Cells were treated with each substance and lysed with RIPA lysis buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (78428, Thermo Fisher Scientific Inc.). The samples were loaded and separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were blocked and probed overnight with anti-cleaved Caspase-3 (Asp175) antibody (#9661, Cell Signaling Technology), anti-H2AX antibody (T55269M, Abmart), and anti-γH2AX antibody (T56572M, Abmart), anti-KAP1 antibody (T55173M, Abmart), and anti-pKAP1 antibody (T59666M, Abmart), at 40°C, anti–β-tubulin antibody (R20005, Abmart) at 4°C. Then, the membranes were washed and incubated with HRP-labeled secondary antibodies for 1 hour and visualized using the luminescent analyzer Tanon 4600 (Tanon, Inc).

The formalin-fixed, paraffin-embedded (FFPE) sections were generated from various cell lines, xenografted tissues. After deparaffinization and rehydration, sections were treated with antigen retrieval buffer (DAKO) and incubated with corresponding primary antibodies for 1 hour. Then the sections were washed and incubated with HRP-labeled secondary antibody (DAKO) for 1 hour. Target expression was then detected by using DAB staining buffer and hematoxylin (Harris). A histochemical scoring system (H-score) was used for target expression intensity evaluation. The expression grade was classified as 0 (H-score 0–14), 1+ (H-score 15–99), 2+ (H-score 100–199), and 3+ (H-score 200–300).

MTX-13 was intravenously administered at 3-week intervals over a 6-week period on days 1, 22, and 43 (total 3 doses) to cynomolgus monkeys (Table 1). Animals were monitored daily for responsiveness, food consumption, bodyweight, temperature, pulse, and others. Urine and blood samples were collected according to the schedule. A necropsy was conducted a week after the last administration. The reversibility of the toxic changes was assessed in a subsequent one-week recovery period in cynomolgus monkeys.

All statistical analysis except for toxicity studies was performed using SAS System Release 9.1.3 and 9.2 (SAS Institute Inc.). Statistical analysis in toxicity studies was performed using MUSCOT (YukmsCo., Ltd.). All IC₅₀ and ED₅₀ values were determined by a Sigmoid Emax model, and dose dependency was evaluated by a Spearman rank correlation coefficient hypothesis test.

Tests to determine significance are detailed in the relevant figure legends. Briefly, t tests or one-way ANOVAs were used to determine the statistical significance of treatment conditions and two-way ANOVA to discriminate between the contributions of both dose and transformation.

Correspondence and requests for data underlying the findings or materials should be addressed to Tao Meng (PhD): megatrump@foxmail.com.

A pool of anti-PTK7 mouse monoclonal antibodies (mAb) was generated using a recombinant protein fragment derived from the extracellular domain (ECD) of the protein as an antigen for a series of mouse immunizations (Supplementary Fig. S1A and S1B). One of the mAbs, mAb13, was selected based on screening using target overexpression immunofluorescence, PTK7-positive and -negative cell line flow cytometry, antibody sequence analysis, and expression-level assessment. mAb13 specifically bound to overexpressed PTK7 in 293T cells and the target-positive cell line OVCAR-3, but not the target-negative cell line Ramos (Fig. 1A; Supplementary Fig. S1C and S1D). The humanized variants of mAb13 were derived from its mouse antibody precursor by CDR grafting and back mutation and were fused to human C-kappa and gamma 1 constant regions. One of the variants, Ab13 (13H3L3) was selected for ADC development based on affinity (EC₅₀ by flow cytometry) and transient expression-level evaluation (Supplementary Fig.S1E). Ab13 showed around three times higher affinity binding (EC₅₀ = 21 pmol/L) on OVCAR-3 with flow cytometry than the benchmark antibody h6M24 (EC₅₀ = 60 pmol/L; Fig. 1B; ref. 19). Through immunofluorescence, Ab13 showed specific binding to overexpressed cynomolgus monkey PTK7 (Supplementary Fig. S1F). Ab13 was cross-reactive to mouse PTK7, although the binding affinity was 25 times lower, whereas h6M24 was not cross-reactive to mouse PTK7, which was consistent with previous results (Supplementary Fig. S1G; ref. 19).

For ADC programs for preclinical and clinical studies, a high-quality IHC antibody is often necessary. We screened the mAb pool generated for PTK7 and identified that mAb13 produces low but specific membranous staining in multiple tissues—including esophagus, cervix, stomach, and colon—while remaining negative in most normal tissues, a pattern that is largely consistent with previous observations (Fig. 1C; Supplementary Fig.S2A and S2B; ref. 19). Expression profiling of PTK7 expression was expanded to include more normal and tumor tissue types from previous surveys (9, 11–13, 19). PTK7 expression in esophagus and cervical tissues was mainly observed in the basal layer cells (Fig. 1C; Supplementary Fig.S2A). PTK7 was expressed in stroma cells in certain tissues, including the tongue, bladder, and thymus (Supplementary Fig.S2B). Overall PTK7 was expressed in more than 40% of over 30 normal tissues surveyed, although the expression was generally low (1+; Supplementary Fig. S2C and S2D).

PTK7 was overexpressed at a much higher level (typically 3+ or 2+) than normal tissues in all tumor types surveyed (Supplementary Fig. S3A). PTK7-positive tumors were expanded to include small cell lung cancer (SCLC) and liver (hepatocellular) cancer (Fig. 1D; Supplementary Fig. S3A). PTK7 was highly overexpressed in tumors of squamous cells including lung, head and neck, cervix, and esophagus (Fig. 1E). PTK7 was significantly overexpressed in metastatic colorectal (74%) and rectal cancer (90%) compared with primary tumors (55% and 56% for colorectal and rectal cancer, respectively), which is consistent with previous observation of PTK7 association with tumor metastasis (refs. 4, 11; Fig. 1F; Supplementary Fig. S3B). Metastatic pancreatic cancer also showed a higher positivity (60%) versus primary tumors (40%; Supplementary Fig. S3C). PTK7 expression was found in stroma cells of liver, pancreatic, gastric, and kidney cancers in both tumor cell line xenograft (CDX) and PDX samples (Supplementary Fig. S3D and S3E), consistent with previous results (Supplementary Fig. S3F; ref. 18). Some PTK7-positive stromal cells also expressed FAP, a marker for cancer-associated fibroblasts or CAFs (Supplementary Fig. S3G). The correlation of PTK7 overexpression with tumor metastasis and the concurrent expression of PTK7 in stroma cells imply the potential application of PTK7-targeted ADC in metastasis and stroma-rich tumor types.

The number of PTK7 molecules expressed on the surface of individual cells was measured by a QIFIKIT using mAb13 in a panel of cancer cell lines representative of diverse solid tumors (Fig. 1G; Supplementary Fig. S3H). The average copy number/cell was 35,000 with a range of a few hundred to >100,000. Compared with HER2 with an average of 120,000 copies/cell, PTK7 was a low-to-medium abundance cell-surface target protein (Fig. 1G, left). Noteworthily, PTK7 expression in ovarian cancer, lung cancer, and colon cancer was relatively higher (Fig. 1G, right), indicating potentially better ADC therapeutic outcomes in those tumor types.

Ab13 was attached to exatecan using dipeptide Val-Ala (VA) linker and a novel, modified self-immolative PABC spacer called T1000 (Fig. 2A). T1000 is a self-immolative PABC modified with a polysarcosine sidechain to increase hydrophilicity masking effect in order to provide a stability shield for the highly hydrophobic and sterically challenging exatecan (30, 32). T1000 dramatically reduced hydrophobicity and aggregation (about 2%) to produce homogeneous and hydrophilic Ab13–exatecan conjugate, MTX-13 of DAR 8 (Fig. 2B and C; Supplementary Fig. S4A). The conjugation yield was >90%. The benchmark antibody h6M24 was readily conjugated to T1000–exatecan (MTX-624 or h6M24–T1000–exatecan) and was slightly more hydrophobic than MTX-13 (Fig. 2B and C). Conversely, Ab13 was conjugated to vc0101 similarly as h6M24 with a DAR of 3–4 (Supplementary Fig. S4B). The attachment of exatecan to Ab13 did not affect PTK7 antibody binding to the PTK7-positive cell line PC9, with an EC₅₀ of 180 pmol/L versus 290 pmol/L, whereas h6M24 conjugation with either vc0101 or T1000–exatecan also yielded molecules with similar binding affinity of about 1 nmol/L to PTK7-PC9 (Fig. 2D). Ab13/MTX-13 binding affinity to PTK7 was 3–5 times higher than h6M24/h6M24-vc0101/MTX-624, similar to the affinity difference observed on OVCAR-3 (Fig. 1B), although the absolute binding affinity were lower in PC9 than in OVCAR-3.

The binding and internalization of Ab13 and MTX-13 were evaluated in a panel of cancer cell lines with different levels of PTK7 expression (Fig. 2E; Supplementary Fig. S4C). Both Ab13 and MTX-13 internalized efficiently with a T_1/2 of 0.5–4 hours in different cell lines, and there was a proportional relationship between PTK7 expression level and internalization rate. H6M24 and its ADC h6M24-vc0101 also internalized similarly to Ab13/MTX-13 (Fig. 2E).

The topoisomerase I inhibition effect of MTX-13 was assessed on the basis of DNA damage (assessment of CHK1-dependent phosphorylation of Kap1 and Histone H2A.X) in PC9 cells in the presence of Ab13, MTX-13, and exatecan (Supplementary Fig. S4D). MTX-13 (10 μg/mL) induced the phosphorylations of Kap1 and Histone H2A.X, similar to exatecan (at 72 hours), whereas Ab13 did not (Supplementary Fig. S4D). This confirms that there is topoisomerase I inhibition activity in the exatecan released from MTX-13.

Bystander killing efficacy of MTX-13 was measured using an in vitro cell culture system with single or cocultures of PTK7⁻ (A549, copy number <1,000/cell) and PTK7⁺ (H520, copy number >70,000/cell) cell lines (Fig. 2F; Supplementary Fig. S4E–S4G). After 5 days of incubation, cells were sorted for PTK7 expression by flow cytometry (Supplementary Fig. S4F and S4G). MTX-13 killed PTK7-A549 cells efficiently (Fig. 2F; Supplementary Fig. S4F). Negative control ADCs did not induce cell killing compared with untreated wells, confirming the bystander-killing effect of MTX-13.

MTX-13′s in vivo pharmacokinetics was evaluated in H1975 tumor-bearing mice model. There was no clear difference in plasma concentration of MTX-13 and total Ab (Fig. 2G), MTX-13 is stable with a similar half-life (T_1/2 7.99 ± 1.57 days) as naked Ab13 (T_1/2 7.46 ± 1.04 days; Supplementary Fig. S4H).

In vitro, h6M24-vc0101 showed high potency and PTK7-specific cytotoxicity in a panel of cancer cell lines, often outperforming MTX-13 (Supplementary Fig. S5A). These findings were consistent with the generally higher cytotoxic potency of microtube inhibitors than Top I inhibitors. The functional equivalence of PF-06647020 from the original publication and h6M24-vc0101 prepared in this study was confirmed in a cell line–derived xenograft (CDX) model of OVCAR-3 (19). h6M24-vc0101 showed a similar tumor inhibition rate as PF-06647020 from the publication under the same drug dosing scheme (Supplementary Fig. S5B) (19). Four ADCs were prepared using an orthogonal combination of antibodies and linker-payload pairs (Supplementary Fig. S5C). In the OVCAR-3 model, T1000–exatecan ADC MTX-13 or MTX-624 showed significantly better tumor inhibition than h6M24-vc0101, and Ab13 in conjugation with vc0101 also produced a more efficacious ADC than h6M24-vc0101, demonstrating the combination of Ab13 and T1000–exatecan may have the potential to build more efficacious PTK7 targeting ADC (Fig. 3A; Supplementary Fig. S5D). Because PTK7 was found to be expressed in tumor stromal cells, we tested whether a T1000–exatecan-based ADC was more effective at targeting tumor stromal cells than its vc0101 counterpart. In gastric cancer cell line SNU-16, PTK7 was strongly expressed in stromal cells but not in the cancer cells (Supplementary Fig. S5E). In vivo, MTX-13 produced modest but slightly better antitumor efficacy than Ab13-vc0101 in the SNU-16 xenograft model (Supplementary Fig. S5F), suggesting the potential capability of T1000–exatecan ADC for targeting stromal cells. Similarly, MTX-13 outperformed h6M24-vc0101 in another ovarian cancer SK-OV-3 xenograft mouse model (Fig. 3A; Supplementary Fig. S5G).

A dose of 5 mg/kg of h6M24-vc0101 administered 1–4 times was selected for efficacy study, corresponding to a 1-to-1 ratio of potency dose to reported maximally tolerated dose (5 mg/kg) of in h6M24-vc0101 in monkeys (19). On the other hand, dosing at 10 mg/kg (1–2 times) for MTX-13 was about one third of its maximally tolerated dose of 30 mg/kg in a pilot monkey toxicity study (see below). In a series of TNBC CDX and PDX models, 5 mg/kg or 10 mg/kg of MTX-13 produced comparable or significantly better antitumor efficacy than h6M24-vc0101, and statistically different compared with isotype IgG–T1000–exatecan control (Fig. 3B–E). MTX-13 was effective in TNBC tumors with low to medium (IHC 1+ or 2+) PTK7 expression level that was more resistant to h6M24-vc0101 (Fig. 3D and E). Together, the above data demonstrated MTX-13’s improved antitumor activity in vivo against ovarian cancer and TNBC (Fig. 3A and F). Because h6M24-vc0101 showed modest clinical efficacy (overall response rate of 20%–30% in medium and high PTK7 expression tumors) in ovarian cancer and TNBC presumably due to the narrow therapeutic index indicated by DLT and potential tumor resistance, the improved antitumor activity of MTX-13 may improve patient responses in those indications (Supplementary Fig. S5H).

An initial survey in PDX samples suggested that PTK7 was highly expressed in SCLC with an overall positivity >80% (Fig. 1D). A recent study classified four SCLC subtypes defined by differential expressions of transcription factors ASCL1, NEUROD1, and POU2F3, or low expression of all three transcription factor signatures accompanied by an inflamed gene signature (SCLC-A, N, P, and I, respectively; ref. 36). In a panel of human patient SCLC samples, we observed similar percentages of each subtype and ASCL1, NEUROD1, and POU2F3 expression patterns (Fig. 4A; Supplementary Fig. S6A). PTK7 showed similar overall positivity and H-score distribution as the PDX samples (Fig. 4B). PTK7 was uniformly overexpressed in all subtypes (Fig. 4B). PTK7 showed a higher percentage of 2+ and 3+ expression (70%–80%) in SCLC than previously reported targets DLL3 and B7H3 (generally in 30%–50%; refs. 37, 38).

The antitumor efficacy of MTX-13 was tested in an SCLC-A subtype PDX model with a high PTK and DLL3 but a relatively low B7H3 expression (Supplementary Fig. S6B). In this model, one dose of MTX-13 (10 mg/kg) caused tumor regression for almost 2 months before the tumor started to regrow (Fig. 4C). In comparison, the DLL3-targeting ADC (SC16–T1000–exatecan) that includes the same antibody as Rova-T (SC16) and T1000–exatecan, as well as the B7H3-targeting ADC DS-7300a (hM30-GGFG-DXd) both resulted in tumor regression, but were persistent for a shorter duration of approximately 40 days (Fig. 4C). h6M24-vc0101 was ineffective in this model suggesting that SCLC may be more sensitive to TOP I inhibitors than microtubule inhibitors. The standard-of-care drug combination of etoposide plus cisplatin was ineffective in this PDX (Fig. 4C). MTX-13 showed similarly potent tumor inhibition in two other SCLC-A PDX models (Fig. 4D and E; Supplementary Fig. S6C and S6D). Overall, MTX-13 showed the most consistent tumor regression out of the ADCs, including DS-7300a and SC16–T1000–exatecan (Fig. 4F). Our data suggested that PTK7 was an actionable target in SCLC and MTX-13 may be a superior ADC for SCLC due to higher PTK7 expression and sensitivity to exatecan in SCLC.

PTK7 showed particularly high positivity (over 70%) and strong overexpression in SCCs of the lungs, cervix, esophagus, and head and neck (Fig. 1E). However, the clinical activity of h6M24-vc0101 in those tumors has not been reported (Supplementary Fig. S7A). Representative PDX models of SCCs were treated with MTX-13 and h6M24-vc0101. MTX-13 showed significantly better tumor inhibition in three lung SCCs (Fig. 5A and B; Supplementary Fig. S7B and S7C). Similarly, MTX-13 outperformed h6M24-vc0101 in SCCs of the head and neck as well as of the esophagus (Fig. 5C–H; Supplementary Fig. S7D). MTX-13 frequently caused tumor regression, whereas tumors in h6M24-vc0101–treated groups continued to grow (Fig. 5D, F, and G; Supplementary Fig.S7D). The consistently better potency of MTX-13 in causing tumor regression in SCCs of diverse origin boded well for its clinical efficacy potential in this important group of tumors that are more challenging for current PTK7-targeting ADC.

PTK7 is highly expressed in colorectal cancer and is implicated in tumor metastasis (4, 11). PTK7 was found to have higher positivity and expression intensity in metastatic than primary colorectal tumors (Fig. 1F; Supplementary Fig. S3B). No clinical activity of h6M24-vc0101 has been reported in colon cancer (Supplementary Fig. S8A). To explore the possibility of using MTX-13 in inhibiting colon cancer metastasis, we investigated the in vivo antitumor activity of MTX-13 in an orthotopic colon cancer model of HCT116-luc. The orthotopic HCT116-luc xenograft model establishment, treatment regimen, and bioluminescence imaging schedule were illustratively shown in Fig. 6A. MTX-13 showed a significantly better inhibition effect on tumor growth and metastasis than h6M24-vc0101 as indicated by the bioluminescence intensity and whole-body bioluminescence imaging on day 39 (Fig. 6B and C). Tumor metastasis was observed in all the organs evaluated in the control group (Fig. 6E; Supplementary Fig. S8B), indicating the successful development of this orthotopic metastasis colon model. Tumors in the h6M24-vc0101 treatment groups showed obvious metastasis to the liver, pancreas, lung, diaphragm, and mesentery, whereas MTX-13 treatment groups showed no metastasis to those organs and undetectable bioluminescence signal at the primary tumor in the colon (Fig. 6D and E; Supplementary Fig. S8B).

Finally, we tested whether MTX-13 was useful for inhibiting large-size and metastatic tumors at advanced stage to mimic the advanced colon cancer patient situation. On day 39 of the above experiment, two groups (n = 4) of remaining naïve orthotopic HCT116-luc mice with large tumor burden and obvious metastasis were given two doses of MTX-13 or h6M24-vc0101 (see Day39, also T0 of Supplementary Fig. S8C and S8D). MTX-13 showed significantly better inhibition activity of tumor growth and metastasis than h6M24-vc0101 (one animal died after D14 of first dosing due to heavy tumor burden from general body metastasis) during the observation period, resulting in superior survival/living status (Supplementary Fig. S8C and S8D).

MTX-13 toxicity and pharmacokinetics in cynomolgus monkeys (cross-reactive species) were conducted by a repeated intravenous dosing (3 doses every 3 weeks at 15 mg/kg and 30 mg/kg) study. MTX-13 was found to be well tolerated in cynomolgus monkeys. Abnormal changes were not observed grossly for daily activity, body weight, body temperature, food consumption, vision, breath, and urine samples. Target organs of toxicity are summarized in Table 1. Major toxicity findings were found in the bone marrow, thymus, liver, gallbladder, and stomach in monkeys. All these findings were minimal or mild. Almost all treatment-related changes showed recovery or a trend of reversibility after 1-week withdrawal period. Reticulocyte counts showed increase and decrease beyond the normal range, whereas changes of other hematologic indicators were largely within the normal range (Supplementary Fig. S9A–S9I). Serum chemistry also remained normal as shown in minimal changes of ALT (alanine aminotransferase) and AST (aspartate aminotransferase; Supplementary Fig. S9H and S9I). On the basis of the toxicology findings for MTX-13, the highest non-severely toxic dose (HNSTD) for monkeys was considered to be at least 30 mg/kg. MTX-13 showed much fewer toxicities than h6M24-vc0101 with an HNSTD of 3–5 mg/kg Supplementary Fig. S9J; ref. 19).

The toxicokinetic profile following all three dosings were simultaneously studied. Serum concentrations of ADC, total antibody and exatecan versus time after treatment with 15 mg/kg and 30 mg/kg of MTX-13 are shown in Supplementary Fig. S9K. The pharmacokinetic parameters of MTX-13 and total antibody for each dose over cycle 1 are summarized in Supplementary Fig. S9K, and those of ADC, total antibody, and free exatecan in all cycles are summarized in Supplementary Table S2. Exposure of ADC and total antibody showed good dose-proportionality and no drug accumulation was observed after multiple administration. PK was nonlinear, the clearance (CL) decreased, and the T_1/2 was prolonged with increasing doses. Low concentrations of free exatecan were observed, and no apparent differences were observed in PK parameters between ADC and the total antibody regardless of the dose. MTX-13 was a highly stable ADC with a T_1/2 of 6–8 days (Supplementary Fig. S9K). In comparison, h6M24-vc0101 was less stable with a much shorter T_1/2 of less than 3 days (19).

MTX-13 is a PTK7-targeting ADC demonstrating superior antitumor activity, favorable pharmacokinetics, and high tolerability in non-human primates. MTX-13 was based on a novel linker-payload platform (T1000–exatecan) we recently established (30). T1000–exatecan enabled the generation of hydrophilic and homogeneous ADC by conjugating 4–8 molecules of TOP I inhibitor exatecan to an antibody using a modified self-immolative pABC structure called T1000. MTX-13 consisted of a high-affinity PTK7-specific antibody, dipeptide VA linker, and T1000–exatecan with a DAR 8. MTX-13 translated exatecan's superior payload features into improved antitumor performance. In vitro cytotoxic and in vivo antitumor activity of MTX-13 was mediated by released exatecan through DNA damage and apoptosis induction, which had a strong bystander killing effect and expanded therapeutic index. MTX-13 exhibited deep and durable antitumor response in both low-target expressing xenograft and heterogeneous PDX models, including ones resistant to a current PTK7-targeting ADC h6M24-vc0101, indicating its ability to overcome tumor resistance from microtubule inhibitors. Despite its higher potency, MTX-13 was well tolerated in the non-human primate toxicity study, with mostly reversible digestive system toxicity (diarrhea) typical of the classic Topo I inhibitor class. MTX-13 showed an HNSTD of at least 30 mg/kg and was significantly better tolerated than h6M24-vc0101 with an HNSTD of 3–5 mg/kg (19). MTX-13’s more potent antitumor efficacy combined with higher tolerability resulted in a higher therapeutic index than h6M24-vc0101, boding well for its potential to elicit responses in broad solid tumor types that are challenging for current ADCs. To our knowledge, MTX-13 may be the first PTK7-targeting ADC using a payload of the TOP I inhibitor class that has already found clinical successes in multiple ADCs including DS-8201a and Trodelvy (25, 28).

We expanded the coverage of tumor types for PTK7. SCLC represents a challenging tumor, and only modest improvement in progression-free survival and overall survival was shown with the addition of immunotherapy to platinum-based first-line chemotherapy (39). A DLL-3 targeting ADC Rova-T, using highly potent DNA-damaging pyrrolobenzodiazepine (PBD) dimer, failed in phase III of the clinical trial due to the narrow therapeutic window for PBD-based ADCs (40, 41). However, a TOP I inhibitor-based ADC DS-7300a targeting B7H3 became the first ADC with promising clinical efficacy and limited toxicities (33). Compared with DLL-3 and B7H3, PTK7 showed higher overall expression positivity (>90%) and intensity (70%–80% of IHC 2+, 3+) in SCLC. PTK7 was uniformly overexpressed in all subtypes of SCLC. In multiple SCLC PDX models resistant to SCLC standard-of-care treatment of etoposide plus cisplatin, MTX-13 caused tumor regression and outperformed DS-7300a and an ADC consisting of the antibody from Rova-T (SC16) and linker-payload T1000-exatecan. H6M24-vc0101 was not effective in SCLC, reflecting the insensitivity of highly proliferating SCLC tumor cells toward microtubule inhibitors. Together, our data suggested that PTK7 is an actionable target in SCLC and MTX-13 may be a therapeutic option for a majority of SCLC patients.

PTK7 was shown to be overexpressed in a majority of patients with SCCs of diverse origins. SCCs arising from stratified epithelial cells are the leading causes of cancer and are known to occur in the skin, head and neck, esophagus, lung, cervix, and other organs (42). Although extensive genomic and proteomic analysis of SCCs has highlighted both common and unique molecular alterations of potential therapeutic interest, therapeutic targeting of SCCs has largely been unsuccessful. Lung squamous cell cancer, accounting for approximately 15% to 20% of non–SCLC cases, is challenging to treat, and targeted treatments are rarely approved for mutations/alterations (43). About 90% of all head and neck cancers are SCCs (HNSCC) that are heterogeneous, aggressive, and a genetically complex collection of malignancies with a dismal 5-year patient survival rate (44). Traditionally, the treatment for SCCs commonly follows anatomic divisions; for example, head and neck SCCs (HNSCC) are treated separately from SCCs affecting the reproductive regions (45). The universal and uniform overexpression of PTK7 in SCCs of different anatomic divisions suggested a common tumor target for SCCs. In a large panel of xenograft and PDX models representative of SCCs of the lung, esophagus, head and neck, and cervix, PTK7 showed potent antitumor activity leading to tumor regression, consistently outperforming h6M24-vc0101. PTK7 represents a promising target for heterogeneous SCCs and MTX-13 warrants further clinical investigation as a targeted therapeutic without consideration of anatomic features in SCCs.

PTK7 was implicated in tumor metastasis including lymph node metastasis and distant metastasis (11). As a matter of fact, PTK7 was found to have a higher overexpression in metastatic tumors than in primary tumors. Anti-metastatic effect of MTX-13 treatment was demonstrated in an orthotopic colon cancer xenograft model at both the early stage and advanced stage of metastasis. MTX-13 also showed efficacy in liver, gastric, and EGT cancer that warrants further investigation. Finally, MTX-13 showed better tumor inhibition in ovarian cancer and TNBC for which h6M24-vc0101 has shown moderate clinical efficacy (22). MTX-13 may improve the overall response rate of ovarian cancer and TNBC patients due to its higher efficacy and expanded therapeutic index, including tumors with a lower PTK7 expression.

In conclusion, we have developed a PTK7-targeting ADC with an improved therapeutic index, ability to overcome tumor resistance, and potential to inhibit tumor metastasis against PTK7-positive tumors. PTK7 is an attractive therapeutic target across a broad spectrum of tumors due to its high overexpression positivity and its role in tumor development and metastasis. Clinical efficacy demonstrated by h6M24-vc0101 highlights the potential of PTK7 as a valid therapeutic target. MTX-13 has the potential to outperform h6M24-vc0101 in clinical trials and expand the therapeutic opportunities of PTK7-positive tumors.

No disclosures were reported.

C. Kong: Resources, investigation, visualization, methodology. J. Pu: Resources, investigation, visualization, methodology. Q. Zhao: Resources, investigation, visualization, methodology. W. Weng: Investigation, visualization. L. Ma: Investigation, visualization. Y. Qian: Investigation, visualization. W. Hu: Resources, investigation, visualization. X. Meng: Conceptualization, resources, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. T. Meng: Conceptualization, resources, formal analysis, validation, investigation, visualization, writing–original draft, writing–review and editing.

This study was funded by Mabcare, Multitude Therapeutics, and Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery (2019B030301005). We thank Y.F. Tang of Multitude for supporting clinical development for this project. We thank technical assistance from Y. Sheng, B. Zhou, W. Chen, and L.J. Wang at Multitude. We thank X.X. Meng for reading and editing the manuscript.