PC61, a rat IgG1k mAb purified from hybridoma PC61 5.3,20 was conjugated to tesirine (SG3249) to generate CD25-ADC, essentially as previously described for camidanlumab tesirine.4 In brief, antibody was buffer exchanged into a proprietary histidine buffer at pH 6 using tangential flow filtration, pH was adjusted to 7.5 using a TRIS/EDTA pH 8.5 buffer, and the solution was reduced with Tris (2-carboxyethyl) phosphine reductant. Dimethylacetamide and SG3249 (threefold excess relative to antibody) were added to the solution. The conjugation reaction was incubated, then quenched with threefold molar excess of N-acetyl cysteine and incubated again. The pH was then decreased to 6.0 using histidine hydrochloride solution and the generated CD25-ADC was purified by tangential flow filtration, filtered, and stored at −70°C. Final yield was estimated by ultraviolet–visible spectrophotometry based on starting antibody.

The rat isotype control ADC (C0010-5-SG3249) and the non-binding control ADC (B12-SG3249) were generated by stochastic conjugation of C0010-5 (rat isotype antibody, Crown Bioscience, California, USA) and B12 (human IgG1 non-binding antibody, produced in house), respectively, to SG3249.

Characterization of synthesized CD25-ADC was performed by size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and reduced reverse-phase (RP) liquid chromatography, using standard techniques as previously described.4 5

Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories). Optical density was measured using an Envision plate reader at 450 nm.

Analysis of CD25 expression on murine cell lines was performed by flow cytometry. Data were analyzed using ForeCyt software, and the data were reanalyzed using Flow-Jo software (V.10) to determine the half-maximal effective concentration (EC₅₀) values.

The source of cell lines used in this study along with cell growth media is shown in online supplementary table S6.

Cytotoxicity of CD25-ADC, the free warhead SG3199, and isotype control ADC C0010-5-SG3249 was determined. In brief, cell lines were incubated with serial dilutions of CD25-ADC, the isotype control ADC, or the free warhead SG3199 for 5 days at 37°C in a 5% carbon dioxide-gassed, humidified incubator. Cell viability was measured with CellTiterGlo luminescent assay (Promega Corporation) according to manufacturer’s instructions. The data were normalized to vehicle-treated cells. EC₅₀ values were determined by using GraphPad software (GraphPad). The mean and standard errors of three independent EC₅₀ values were determined.

The Institutional Animal Care and Use Program at Charles River Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, which assures compliance with accepted standards for the care and use of laboratory animals.

MC38 tumors were established in 9-week-old, female C57BL/6 mice (Charles River, Massachusetts, USA) by implanting 5×10⁵ MC38 cells subcutaneously (s.c.) into their flanks. CT26 tumors were established in 9-week-old, female BALB/c mice (Charles River) by implanting 3×10⁵ CT26 cells s.c. into their flanks. When group mean tumor volumes reached specified target ranges, animals were randomly sorted into groups and treatments were initiated. CD25-ADC, the non-binding control ADC (B12-SG3249) or isotype control ADC (C0010-5-SG3249) were administered on day 1, intraperitoneally (i.p.) as a single dose at 0.1, 0.5, or 1 mg/kg, either alone or in combination with an anti-PD-1 (clone RMP1-14) antibody (given at 5 mg/kg on days 2, 5, and 8). A vehicle-treated group served as a control. After dosing on day 1, tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 1000 mm³ or at the end of the study, whichever came first.

The coefficient of drug interaction (CDI) was assessed for subadditive, additive, or supra-additive (synergism) properties on the last day when at least 50% of the animals remained on study, as previously described.21

Tumor-free survivors (TFS) from these studies were rechallenged with a new s.c. implant (contralateral to the original cell implant) of MC38 cells or CT26 cells. Tumor formation was monitored over 30 days and 50 days in the MC38 and CT26 rechallenge studies, respectively. A group of treatment-naïve mice were implanted with MC38 cells or CT26 cells in respective studies, as a control.

To confirm the association of CD25-ADC antitumor activity with CD8+ T cells, MC38 tumor-bearing mice were sorted into groups (n=10/group) to receive treatment with CD25-ADC (0.5 mg/kg) either alone or in combination with anti-CD8 (clone 2.43) antibody (10 mg/kg on days 0, 5, 8, and 13) and/or anti-PD-1 antibody (5 mg/kg on days 2, 5, and 8). A vehicle-treated group served as control. Tumor volume was monitored over 30 days.

A partial response (PR) was defined as a tumor volume ≤50% or less than its day 1 volume for three consecutive measurements during the course of the study, and ≥13.5 mm³ for one or more of these three measurements. A complete response (CR) was defined as a tumor volume <13.5 mm³ for three consecutive measurements during the study. Any animal with a CR at the end of the study was additionally classified as a TFS. Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied.

Treatment tolerability was assessed by body weight measurements and by frequent observation for clinical signs of treatment-related side effects.

Seven-week-old, female C57BL/6 non-tumor-bearing mice were sorted into groups (n=24/group) to receive treatment with a single dose of CD25-ADC (0.5 mg/kg), isotype control ADC C0010-5-SG3249 (0.5 mg/kg), or no treatment (control). Spleen, lymph nodes, blood, and thymus were collected from six mice/timepoint and cell suspensions were processed for flow cytometry.

MC38 tumors were established as previously described in 9-week-old, female C57BL/6 mice and treatments were started at specified group mean tumor volumes. Mice were sorted into groups to receive treatment with CD25-ADC (0.5 mg/kg on day 1), and/or anti-PD-1 antibody (5 mg/kg on days 2, 5, and 8), or vehicle control (phosphate buffer saline (PBS)). For the efficacy analysis, 10 mice/group were monitored for tumor volume over 36 days. For the T-cell immunophenotype analysis, untreated mice (n=8) were collected on day 1 (pre-dose). Samples of tumor, blood and thymus were collected from all groups including vehicle (n=8 per group) at specified timepoints. Cell suspensions were processed for flow cytometry.

Blood samples were processed by centrifugation and lysis of the red blood cells. Immediately following blood collection, tumor and /or selected organs were harvested from each animal and processed for flow cytometry. Mouse tumor samples were dissociated according to the manufacturer’s instructions using the gentle MACS protocol “Tumor Dissociation Kit.” Samples were filtered through a 70 µm cell strainer and rinsed twice in PBS/2.5% fetal bovine serum buffer to remove enzymatic buffer. Spleen, lymph node and thymus samples were dissociated by gently grinding the tissue across a 70 µm cell strainer with RPMI-1640 media, followed by lysis of the red blood cells.

The final single-cell suspensions were prepared in Staining Buffer (2.5% fetal bovine serum, 0.09% sodium azide, in PBS, pH 7.4) at 2×10⁷ cells/mL. Briefly, 100 µL of single-cell suspensions were pelleted, resuspended in Live/Dead Aqua (Life Technologies) as per manufacturer’s instructions, and stained for 30 min at 4°C. After two washes with Staining Buffer, Fc receptors were blocked using Mu TruStain fcX (Biolegend) for 10 min at 4°C, washed twice with Staining Buffer and probed with antibody panels for 30 min at 4°C protected from light. Details on cell surface markers used for flow cytometry are shown in online supplementary table S7 along with representative gating strategy used to measure T_regs. All data were collected on an LSR Fortessa Cell Analyzer (BD) and analyzed with FlowJo software. Intranuclear staining of FoxP3 and Ki67 was performed using the FoxP3 Transcription Factor Staining Buffer Set (eBioscience). For intracellular staining of cytokines, cells were restimulated with phorbol 12-myristate 13-acetate (PMA, 20 ng/mL), ionomycin (500 ng/mL) and GolgiPlug in complete Roswell Park Memorial Institute (RPMI) medium for 5 hours at 37°C. For quantification of absolute number of cells, a defined number of fluorescent beads (CountBright Absolute Counting Beads, ThermoFisher) was added to each sample before acquisition and used as a counting reference.

PK analysis of CD25-ADC was performed in 7-week-old, female C57BL/6 non-tumor-bearing mice. Serum samples were collected for each timepoint after a single-dose administration of CD25-ADC (0.1, 0.5, or 1 mg/kg). Quantitation of total (unconjugated and conjugated) antibody was determined by electrochemiluminescence immunoassay using recombinant mouse CD25 as capture and a biotin-labeled polyclonal goat antirat IgG (mouse adsorbed; Bio-Rad) in combination with sulfoTAG streptavidin as detector. For each dose group, six mice were intravenously injected with CD25-ADC; serum was collected from three animals/group at 1, 6, 48, 96, 168, and 504 hours following dosing, and from the other 3 animals at 3, 24, 72, 120, and 336 hours following dosing. PK parameters were determined by non-compartmental analysis (Phoenix WinNonLin).

Statistical analyses were done using GraphPad Prism V.8.1 (Log-rank test and analysis of variance with a Tukey post-hoc test, followed by Kruskal-Wallis test with Dunn’s post-hoc test) or JMP software (Dunn method for joint ranking). Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01; ***, p≤0.001.

No patients were involved in this study.

CD25-ADC is an ADC composed of PC61, a rat IgG1k anti-CD25 antibody directed against mouse CD25, stochastically conjugated to SG3249 (tesirine; figure 1A). The resulting ADC was determined to be 96.5% monodisperse by SEC, with a DAR of 2.1 by RP liquid chromatography and 2.0 by HIC. As a control, an isotype ADC composed of a rat isotype IgG1 antibody stochastically conjugated to tesirine (C0010-5-SG3249) was prepared (98% Monomer, DAR-RP 2.01, DAR-HIC 1.87).

The PC61 antibody demonstrated strong binding affinity to both recombinant mouse CD25 (EC₅₀ 1.182 nM; figure 1Bi) and to CD25 expressed on the surface of a mouse lymphoma (Yac-1) cell line (EC₅₀ 0.9 nM); however, the isotype control antibody did not bind to CD25 (figure 1Bii).

The in vitro cytotoxicity of CD25-ADC was determined in a panel of murine tumor-derived cell lines including CD25-positive Yac-1 and CD25-negative MC38, CT26, 4T1, LL/2, and B16-F10. CD25-ADC showed potent and specific cytotoxicity in the CD25-expressing Yac-1 cell line (CD25-ADC EC₅₀ 11.5 pM (±1.56); isotype-ADC EC₅₀ 8575 pM (±1072); figure 1Bv), but it did not bind or have any specific activity in the CD25-negative cell lines (figure 1Biii,iv,vi,vii and online supplementary figure S1i–vi). In contrast, SG3199, the warhead component of CD25-ADC, showed potent cytotoxicity in all the CD25-negative cell lines tested, in line with its non-targeted mode of action (figure 1Bvi,vii and online supplementary figure S1ii,iv,vi).

CD25-ADC had strong, dose-dependent, and durable antitumor activity in established s.c. syngeneic tumor models. In the CD25-negative MC38 colon cancer model, a single dose of CD25-ADC at 0.5 and 1 mg/kg resulted in rapid antitumor responses, sustained for the 60-day study period, with 8/10 TFS and 2/10 PR at both 0.5 and 1 mg/kg (figure 2vi,vii and online supplementary table S1) and significant increased survival compared with the vehicle group (p<0.001, figure 2xi and online supplementary figure S2). Strong synergy (CDI: 0.3) was observed when a suboptimal single dose of 0.1 mg/kg of CD25-ADC was combined with an anti-PD-1 antibody (figure 2viii) resulting in significant increased survival compared with each monotherapy group (p<0.05, figure 2xi and online supplementary figure S2). No additional benefit was obtained from adding an anti-PD-1 antibody to CD25-ADC at 0.5 mg/kg and 1 mg/kg doses owing to the strong antitumor activity of CD25-ADC monotherapy at these doses (figure 2ix–x). Vehicle alone had no effect on disease activity, with no responses observed (figure 2i). Anti-PD-1 antibody administered alone at 5 mg/kg gave 0/10 PR and 3/10 TFS (figure 2ii). A non-binding control ADC (B12-SG3249; 97.8% Monomer, DAR-RP 2.08, DAR-HIC 1.79) dosed at 1 mg/kg had some activity alone (2/10 PR and 2/10 TFS) and in combination with anti-PD-1 antibody (2/10 PR and 5/10 TFS; online supplementary table S1); however, this combination was not synergistic (CDI: 2.1) and the activity was inferior to that observed with the same dose of CD25-ADC (figure 2iii,iv and online supplementary figure S2).

In the CD25-negative CT26 colorectal cancer model, a single dose of CD25-ADC (0.1, 0.5, or 1 mg/kg) showed dose-dependent antitumor activity (figure 3v–vii) and significant increased survival compared with the vehicle group (p<0.001, figure 3xi and online supplementary figure S3). Synergistic activity was observed when an anti-PD-1 antibody was combined with a low dose (0.1 mg/kg) of CD25-ADC (CDI: 0.3; figure 3viii) resulting in significant increased survival compared with each monotherapy group (p<0.001, figure 3xi and online supplementary figure S3). The antitumor activity of CD25-ADC at 0.5 mg/kg and 1 mg/kg doses was further increased on combining with anti-PD-1 antibody (7/10 TFS in the group treated with 0.5 mg/kg of CD25-ADC + anti-PD-1 antibody and 8/10 TFS in the group treated with 1 mg/kg of CD25-ADC + anti-PD-1 antibody; figure 3ix,x and online supplementary table S2). There were no responses in the group treated with vehicle (figure 3i), anti-PD-1 alone (figure 3ii), or isotype control ADC, although minor antitumor activity was observed when the isotype control ADC was combined with the anti-PD-1 antibody (1/10 TFS; figure 3iii,iv and online supplementary table S2).

In both studies, all regimens were well tolerated, with minimal body weight loss (online supplementay figure S4) and no clinical observations reported.

We first investigated whether treatment with CD25-ADC induces immunological memory. Following treatment with CD25-ADC alone or in combination with anti-PD-1 antibody, TFS mice were rechallenged on the opposite flank with the same tumor cells as originally implanted (MC38 or CT26). Tumor-naïve mice were also implanted and used as controls. None of the TFS mice developed new tumors as opposed to all treatment-naïve controls (online supplementary figure S5), indicating that CD25-ADC induced antitumor immunity. Interestingly, following treatment with the control ADCs, either monotherapy or in combination with anti-PD-1 antibody, the few TFS animals from both the MC38 and CT26 studies also did not develop new tumors on rechallenge (online supplementary figure S5).

Next, we evaluated whether CD8+ T_effs contribute to the mode of action of CD25-ADC. A CD8-depleting antibody was administered together with CD25-ADC to mice bearing established MC38 tumors. Depletion of CD8+ T cells abolished the antitumor activity of CD25-ADC both alone and together with anti-PD-1 (figure 4 and online supplementary table S3), indicating that CD25-ADC antitumor activity is mediated by CD8+ T_effs and that CD8+ T_effs are not negatively impacted by CD25-ADC.

Finally, we performed a longitudinal T-cell immunophenotype study following a single dose of CD25-ADC (0.5 mg/kg), either alone or in combination with an anti-PD-1 antibody, in mice bearing established MC38 tumors. CD25-ADC monotherapy had greater antitumor activity than vehicle or anti-PD-1 treatment (online supplementary figure S6), and synergistic activity was observed when CD25-ADC treatment was combined with anti-PD-1 (CDI: 0.133), with the combination resulting in 4/10 TFS at the end of the study (online supplementary table S4). The immunophenotype analysis was carried out at 2, 8, and 11 days post dose of CD25-ADC, which was 1 day after the first dose of anti-PD-1 and 1 and 4 days after the last dose of anti-PD-1, respectively. A single dose of CD25-ADC, either alone or in combination with anti-PD-1, induced robust and durable intratumoral T_regs depletion, which was still maintained at 11 days post dose (figure 5A, top panel). CD8+ T_effs levels were not negatively impacted by CD25-ADC, and, in fact, there was a statistically significant increase in the absolute number of tumor-infiltrating CD8+ T cells on day 11, which was further increased when CD25-ADC was combined with anti-PD-1 antibody (figure 5A, mid panel). Following CD25-ADC treatment, the CD8+ T_effs/T_regs ratio continued to increase throughout the time course analyzed, and a further increase in the ratio was observed in the combination therapy group (figure 5A, lower panel), in line with the increased antitumor activity of CD25-ADC and anti-PD-1 combination treatment. Interestingly, tumor-infiltrating CD8+ T_effs from the CD25-ADC-treated groups (either alone or in combination with anti-PD-1) had significantly higher activation (p<0.01, figure 5B, top panels) and proliferation rates compared with CD8+ T_effs from the control groups on day 11 (p<0.01, figure 5B, middle panels) and their interferon-γ production peaked on day 8, although the increase did not reach statistical significance compared with the controls (figure 5B, bottom panels). CD25-ADC treatment also reduced the number of circulating T_regs, but the effect was not durable, with values returning to control levels by day 11 post dose (figure 6A, top panel). Circulating CD8+ T_effs levels were not affected by CD25-ADC treatment, either alone or in combination with anti-PD-1 antibody, and the CD8+ T_effs/ _Tregs ratio returned to control levels by day 11 post dose (figure 6A, middle and low panels). In the thymus, CD25-ADC induced a temporary small increase in T_regs, but the CD8+ T_effs/T_regs ratio did not change during the time course analyzed (figure 6B).

The T_regs depletion activity of CD25-ADC was further assessed in non-tumor-bearing immunocompetent mice over a period of 21 days. A single dose of CD25-ADC (0.5 mg/kg) caused a substantial but transient depletion of T_regs in spleen (87% reduction; p≤0.001), lymph nodes (85% reduction; p≤0.001), and blood (96% reduction; p<0.001) compared with the vehicle and isotype control ADC at 6 days post dose, with no significant impact on T_eff levels (p>0.05; figure 7A,B and online supplementary figure S7). Conversely, CD25-ADC induced a significant increase in both T_regs (328%; p≤0.001) and T_effs (254%; p≤0.01) in the thymus at day 6 post dose (figure 7C). T_reg levels in spleen, lymph nodes, blood, and thymus were restored to similar levels as the control by day 13 post CD25-ADC dosing. No significant body weight loss (online supplementary figure S8) or clinical observations were recorded throughout the duration of the study in any treatment group.

PK analysis of CD25-ADC in non-tumor-bearing mice showed dose-dependent, target-mediated drug disposition with non-linear PK at low-dose levels and linear PK at higher dose levels (online supplementary figure S9). A dose-dependent increase in exposure (area under curve, AUC_[tau]) was observed, ranging from 14,572 ng×hour/mL to 751,771 ng×hour/mL (online supplementary table S5).