Toxicology, pharmacokinetics, and immunogenicity studies of CCR4-IL2 bispecific immunotoxin in rats and minipigs

Abstract

We have developed a diphtheria toxin-based recombinant human CCR4-IL2 bispecific immunotoxin (CCR4-IL2-IT) for targeted therapy of cutaneous T-cell lymphoma (CTCL). CCR4-IL2-IT demonstrated superior efficacy in an immunodeficient mouse CTCL model. Recently, we have compared the in vivo efficacy of CCR4-IL2-IT versus Brentuximab (FDA approved leading drug in CTCL market) in the same immunodeficient mouse CTCL model. The comparison demonstrated that CCR4-IL2-IT was significantly more effective than Brentuximab. In this study, we have performed non-GLP (Good Laboratory Practice) toxicology, pharmacokinetics, immunogenicity studies of CCR4-IL2-IT in both rats and minipigs. CCR4-IL2-IT demonstrated excellent safety profiles in both rats and minipigs. The maximum tolerated dose of CCR4-IL2-IT was determined as 0.4 mg/kg in both rats and minipigs. Complete blood count and chemistry analysis did not show significant difference for all measured parameters between the blood samples of pre-injection versus post-injection from the five-day toxicology studies of CCT4-IL2-IT in both rats and minipigs. Histology analysis did not show difference between the PBS treatment group versus CCR4-IL2-IT treatment group at 50 μg/kg in both rats and minipigs. The half-life of CCR4-IL2-IT was determined as about 45 min in rats and 30 min in minipigs. The antibodies against CCR4-IL2-IT were detected in about two weeks after CCR4-IL2-IT treatment. CCR4-IL2-IT did not induce cytokine release syndrome in a peripheral blood mononuclear cell derived humanized mouse model. The depletion of CCR4⁺ cell and CD25⁺ cell (two target cell populations of CCR4-IL2-IT) was observed in minipigs. The excellent safety profile promoted us to further develop CCR4-IL2-IT towards clinical trials.

1. Introduction

CCR4 and CD25 are highly expressed on the surface of cutaneous T-cell lymphoma (CTCL). The two receptors can be used for targeted therapy of CTCL. We have developed a truncated diphtheria toxin-based recombinant human CCR4-IL2 bispecific immunotoxin (CCR4-IL2-IT) for targeted therapy of CTCL (Wang et al., 2020). CCR4-IL2-IT demonstrated superior efficacy to prolong the survival of the tumor-bearing mice in an immunodeficient mouse CTCL model (Wang et al., 2020). Recently, we have compared the in vivo efficacy of CCR4-IL2-IT versus Brentuximab (an antibody–drug conjugate targeting CD30⁺ cancers, FDA approved leading drug in CTCL market) in the same immunodeficient mouse CTCL model. The comparison demonstrated that CCR4-IL2-IT was significantly more effective than Brentuximab and the combination treatment of CCR4-IL2-IT and Brentuximab was more effective than either CCR4-IL2-IT or Brentuximab alone (Wang et al., 2023). Since CCR4 and CD25 are also highly expressed on the cell surface of tumor-infiltrating effector Tregs, CCR4-IL2-IT also has potential to deplete tumor-infiltrating effector Tregs for broad-spectrum cancer immunotherapy. Our current goal is to prepare an investigational new drug data package including good manufacturing practice production and good laboratory practice (GLP) toxicology studies. However, outsourced GLP-toxicology studies in contract research organization facilities could be quite costly. To de-risk the GLP-toxicology studies, we first performed the non-GLP toxicology studies including maximal tolerant dose, pharmacokinetics, and immunogenicity in both rats (as rodent model) and minipigs (as non-rodent model) at University of Colorado Anschutz Medical Campus Animal Facility.

2. Materials and Methods

2.1. Immunotoxins and antibodies

CCR4-IL2-IT, Ontak^®-like human IL2 fusion toxin (IL2-IT), fold-back diabody anti-human CCR4 immunotoxin (CCR4-IT) were all produced in our laboratory using a unique diphtheria toxin-resistant yeast Pichia pastoris expression system as previously reported (Wang et al., 2020), (Peraino et al., 2014), (Wang et al., 2015), (Liu et al., 2003). Antibodies used in this study were listed in Table 1.

Table 1.

Antibodies used in this study

	Antibody	Cat#	Clone#
ELISA Antibodies	Goat anti Diphtheria Toxin	Meridian #B65701G	Polyclonal
	Mouse anti-Human IL2	BD 555051	5344.111
	HRP Goat Anti-Mouse IgG	Southern biotech 1030–05	Polyclonal
	HRP Goat Anti-Rat IgG	Southern biotech 3051–05	Polyclonal
	HRP Goat Anti-Porcine IgG	Southern biotech 6050–05	Polyclonal
Immuno-Phenotyping & Binding Antibodies	FITC Mouse Anti-Porcine CD3ε	Homemade	898H2–6–15
	PerCp-Cy5.5 mouse Anti Porcine CD4a	BD 561474	74–12–4
	PE Mouse Anti-Pig CD8a	BD 559584	76–2–11
	PE Mouse Anti-Human CD21	BD 557327	B-ly4
	FITC Mouse anti Pig CD1	Bio-Rad MCA6098F	76–7–4
	FITC Mouse anti-Rat CD3	BioLegend 201403	1F4
	PE-Cy7 Mouse Anti-Rat CD45	BD 561588	OX-1
	APC Mouse anti-Rat CD45RA	BioLegend 202314	OX-33
	PE-Cy7 Mouse Anti-Human CCR4	BioLegend 359410	L291H4
	AlexaFlour 647 Mouse Anti-Pig CD25	Bio-Rad MCA1736A647	K231.3B2
	PE Mouse Anti-Human CCR4	Bio-Techne FAB1567P	205410
	Biotin Mouse Anti-Rat CD25	BioLegend 202111	OX-39
CRS Antibodies	FITC Rat anti-Mouse CD45	BioLegend 103108	30-F11
	PE Mouse anti-Human CD45	BioLegend 304058	HI30
	APC Mouse anti-Human CD3	BioLegend 300458	UCHT1

2.2. Rats and minipigs

Sprague Dawley Rats (250–350 g) were purchased from Charles River Laboratories (Wilmington, MA, USA). Yucatan miniature pigs (15–20 kg) were purchased from Sinclair Bio-Resource (Auxvasse, MO, USA). Rats and minipigs were housed in the Animal Research Facility of University of Colorado Anschutz Medical Campus. All animal experiments were approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee.

2.3. Immunotoxin injection and blood sample collection in both rats and minipigs

After one week of acclimation, the Sprague Dawley rats were used for the non-GLP toxicology studies. The main procedures included intravenous immunotoxin injection and blood sampling. The rat was under general anesthesia by inducing with 5% isoflurane and maintaining with 2% isoflurane, and then placed in a restrainer and the tail was immersed in warm water for 5 minutes, cleaned with 70% ethanol prep. The lateral tail vein was used for intravenous immunotoxin injection and blood collection using a 25G blood collection needle (BD, Franklin Lakes, NJ, USA). The blood samples were collected into a K₂ EDTA and serum separation vacutainer blood collection tube (BD, Franklin Lakes, NJ, USA).

Two intravenous catheters were placed separately for immunotoxin administration and blood collection in minipigs. After one-week of acclimation, the minipig was fasted for 12 hours prior to surgery. The single lumen central intravenous catheter (CR Bard, New Providence, NJ, USA) was placed into the external jugular vein and then fixed by two 4–0 silk ligatures around the vein. After skin closure, two layers of tubular elastic net retainer dressing (McKesson, Richmond, VA, USA) were applied on the minipig to secure the catheter. Each intravenous catheter was maintained by daily flushing with 3 mL of 100 units/mL heparin saline (Excelsior Medical, NEPTUNE, NJ, USA) until the study endpoint. K₂ EDTA and serum separation vacutainer blood collection tubes were used to collect the blood samples.

2.4. Single-dose escalation toxicology study of CCR4-IL2-IT in both rats and minipigs

Single-dose escalation toxicology studies of CCR4-IL2-IT in rats were conducted with six Sprague Dawley rats including three males and three females. Four escalating doses (0.1, 0.2, 0.4, and 0.8 mg/kg) were intravenously injected on day 1, 4, 7, and 10. Body weight and clinical observation were closely monitored. The body weight was monitored on day 1, 4, 7, 8, 10, 11, and 12. The escalation doses were designed based on the preclinical toxicology study data of Ontak^® (diphtheria toxin based human IL2 fusion toxin).

Single-dose escalation toxicology study of CCR4-IL2-IT in minipigs were conducted using four minipigs including two males and two females. Four escalating doses (0.1, 0.2, 0.4, and 0.8 mg/kg) were intravenously injected on day 1, 4, 7, and 10. The escalation doses were also based on the preclinical toxicology study data of Ontak^®. Pharmacokinetic (PK) following each dose escalation were measured at six time points (pre-injection, 15, 30, 60, 90, 120 min post injection). Blood samples were also collected before each dose, after the last dose, and then once every 3-days for complete blood count (CBC) and chemistry analysis, until the study endpoint.

2.5. Repeat dose for five-day toxicology studies of CCR4-IL2-IT in both rats and minipigs

Four groups of rats including a vehicle control group (n=6) and three dose groups of CCR4-IL2-IT with 2.5 μg/kg as low dose (n=6), 25 μg/kg as middle dose (n=6), and 50 μg/kg as high dose (n=6) were designed in this study. Selection of the three CCR4-IL2-IT doses was based on the dose ranges used for other diphtheria toxin-based immunotoxins including Ontak^®, Elzonris^® (human IL3 fusion toxin) and Resimmune^® (anti-human CD3 immunotoxin). CCR4-IL2-IT or phosphate buffered saline (PBS, as vehicle) was injected intravenously for five consecutive days. Blood samples were collected from lateral tail vein at seven time points (pre-injection, 5, 15, 30, 60, 90, 120 min post injection) after dose 1 and dose 5 for PK measurements (CCR4-IL2-IT concentration in the collected serum samples). Blood samples were also collected from pre-study, and day 14 for CBC and chemistry analysis. Rats were euthanized on day 14. Liver, kidney, spleen, thymus, and lung were collected for histology analysis. For rat immunogenicity analysis, rat blood samples were collected from pre-injection, day 7, 14, 21, 28, 35, 42, 49, 56, and 63 (study endpoint) for measurements of antibodies against CCR4-IL2-IT in the collected serum samples. The rats used for immunogenicity study were euthanized on day 63.

Four groups of minipigs were used for this study including a vehicle control group (n=3) and three dose groups of CCR4-IL2-IT with 2.5 μg/kg as low dose (n=4), 25 μg/kg as middle dose (n=4), and 50 μg/kg as high dose (n=4). Selection of the three CCR4-IL2-IT doses was informed by the dose ranges used for other diphtheria toxin-based immunotoxins including Ontak^®, Elzonris^® and Resimmune^®. CCR4-IL2-IT or PBS was injected intravenously through the central catheter for five consecutive days. Blood samples were collected at six time points (pre-injection, 15, 30, 60, 90, 120 min post injection) from central catheter after dose 1 and dose 5 injections for PK measurements. Blood samples were also collected from pre-study, week one (day 7/8), week two (day 14/15), and day 22 for CBC, chemistry, and immunogenicity analysis. Minipigs were euthanized on day 22. Liver, kidney, spleen, and lung were collected for histology analysis.

2.6. Immunophenotyping by flow cytometry

Rat blood samples were collected from pre-injection and day 14 for immunophenotyping by flow cytometry analysis. 100 μL of anticoagulated rat blood was placed in a 75×12 mm tube and incubated with 3.5 mL of Red Blood Cell Lysing Buffer Hybri-Max^™ (Millipre-Sigma, Burlington, MA, USA) for 15 min at room temperature with rocking. The tube was centrifuged at 400 ×g for 5 min at room temperature, and then washed twice with 2 mL of the FACS buffer (1×Hanks Balanced Salt Solution with Ca⁺ and Mg⁺, 0.1% Bovine serum albumin and 0.1% sodium azide). After centrifugation, the supernatant was gently discarded. 10 μL of the Fc blocker and conjugated antibodies (anti-rat CD3, anti-rat CD45, anti-rat CD45RA) were added to the tube and incubated at 4°C for 30 min. The cells were washed twice with 2 mL of the FACS buffer and centrifuged at 400 ×g for 5 min at room temperature. The supernatant was discarded, 300 μL of the FACS buffer was added into the tube and stored at 4°C in the dark until running on the CytoFLEX Flow cytometer (Beckman Coulter, Brea, CA, USA).

Minipig blood samples were collected from pre-injection, day 5 and 22 for immunophenotyping by flow cytometry analysis. 100 μL of collected anticoagulation minipig blood was placed in 75×12 mm tube and washed with 2 mL of the FACS buffer followed by centrifugation at 400 ×g for 5 min at room temperature. The supernatant was gently discarded. 10 μL of the Fc blocker and conjugated antibodies were added to the tube and incubated at 4°C for 30 min. The cells were washed once with 2 mL of the FACS buffer at 400 ×g for 5 min at room temperature. The supernatant was discarded, and cells were suspended in 2 mL of lysing buffer (cat# 349202, BD BioSciences, San Jose, CA, USA) and incubated for 15 min at room temperature. After centrifugation, the supernatant was discarded gently, and the cells were washed twice with 2 mL of FACS buffer and centrifuged at 400 ×g for 5 min in room temperature. The supernatant was discarded and 300 μL of the FACS buffer was added into the tube and stored at 4°C in the dark until running on the CytoFLEX Flow cytometer. All flow data was analyzed using FlowJo software (Flowjo, LLC, Ashland, OR, USA).

2.7. Complete blood count and chemistry analysis

For CBC analysis, rat or minipig blood samples were collected into K₂ EDTA tube and analyzed by HemaTrue Veterinary Hematology Analyzer (HESKA, Loveland, CO, USA). The CBC profile includes white blood cell count, lymphocyte count, monocyte count, granulocyte count, red blood cell count, hematocrit, red blood cell distribution width, mean corpuscular volume, hemoglobin, mean corpuscular hemoglobin, platelets, and mean platelet volume.

For chemistry analysis, serum separation was required. Rat or minipig blood samples were collected into serum separator tube and standing up straight for 30 min at room temperature. The tubes were then centrifuged at 3000 ×g for 10 min at 4°C. The supernatant serum was harvested for analysis. Chemistry was analyzed by Catalyst One Chemistry Analyzer (IDEXX, Westbrook, ME, USA).

The chemistry panel includes kidney relevant parameters: creatinine, blood urea nitrogen, glucose; liver relevant parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALKP), total bilirubin (TBIL); electrolytes: calcium, sodium, potassium, chloride; pancreas relevant parameters: amylase (AMYL), lipase (LIPA); fat metabolism: cholesterol (CHOL); inorganic phosphate; protein relevant parameters: albumin, globulin, total protein (TP).

2.8. Pharmacokinetics analysis

A sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure CCR4-IL2-IT concentration in the collected serum samples from both rats and minipigs. Polyclonal goat anti-DT antibody (Meridian Bioscience Memphis, TN, USA) was used as capturing antibody and diluted at 5 μg/mL in the coating buffer (cat# C304, Sigma-Aldrich, St. Louis, MO, USA). 100 μL per well of the diluted antibody solution was coated on microplates (Cat#3590, Corning, Vineland, NJ, USA) for 16 hours at 4°C. The wells were washed for three times with a wash buffer [Tris buffered saline (TBS) with 0.05% Tween-20] and blocked with 150 μL of the blocking buffer (1% BSA in TBS) for one-hour at room temperature. The wells were then washed and loaded with 100 μL of standards or unknown samples diluted at 1:25 with diluent buffer (TBS with 1% BSA and 0.05% Tween-20). CCR4-IL2-IT was serially diluted with diluent buffer at 1000, 333, 111, 37, 12, 4, 1.4, and 0 ng/mL as standards. After 16-hours incubation at 4°C, wells were rewashed, and loaded with mouse anti-human IL2 antibody at 1 μg/mL for one-hour incubation at room temperature. The wells were rewashed and incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:4000, SouthernBiotech, Birmingham, AL, USA) for one hour. The wells were rewashed and developed with 100 μL of TMB substrate (Thermo Scientific, Waltham, MA, USA) for 15 min. Color change was stopped with 50 μL of 1N H2SO4. Absorbance was measured at 450 nm using BioTek Synergy LX Multi-Mode Reader (BioTek, Winooski, VT, USA). Each test was performed in duplicate, and the average of the data points were plotted using Prism 9.4.1. Plotted standards were fitted to a sigmoidal (four parameters) curve to calculate CCR4-IL2-IT concentration of the unknown serum samples. In vivo half-life was calculated using one phase decay equation of Prism. Y= IF (X<X₀, Y₀, Plateau + (Y0 - Plateau)*exp(−K*(X-X0))). X₀ is the time at which decay begins. In this model we constrain X₀ = 0 min. Y₀ is the immunotoxin concentration at time X₀. Plateau is the Y value at infinite time. In this model we constrain Plateau = 0. K is the rate constant, expressed in inverse minutes. Half-life is in the time units of the X axis. It is computed as ln (2)/K.

2.9. Immunogenicity analysis

Indirect ELISA was used to measure the anti-CCR4-IL2-IT antibody concentration in the collected serum samples from both experimental rats and minipigs. The microplate was coated with 100 μL/well CCR4-IL2-IT (5 μg/mL) in the coating buffer and incubated for 16-hours at 4°C and then blocked as described above. The unknown serum samples were diluted at 1:100, 1:1000, 1:10,000 and 1:100,000. Murine anti-DT antibody was serially diluted with diluent buffer at 1000, 500, 250, 125, 62.5, 31, 15, 0 ng/mL and used as standard. 100 μL/well of the diluted unknown sample or standard was incubated for two-hours at room temperature. The wells of the unknown serum samples were then incubated with HRP-conjugated goat anti-rat IgG secondary antibody (1:4000, SouthernBiotech, Birmingham, AL, USA) or HRP-conjugated goat anti-porcine IgG secondary antibody (1:4000 SouthernBiotech, Birmingham, AL, USA) for one hour at room temperature. Standards were then incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:4000) for one hour at room temperature. All wells were developed, stopped and the absorbances were read as described above. Each ELISA analysis was repeated, and the average data points were plotted using Prism 9.4.1. Plotted standards were fitted to a sigmoidal (four parameters) curve to calculate anti-CCR4-IL2-IT antibody concentration from unknown serum samples.

2.10. Histology analysis

The experimental animals were euthanized on day 14 for rats and day 22 for minipigs. Necropsy was performed to collect liver, kidney, lung, spleen, and thymus (thymus only from rats) for limited histopathology evaluation. The collected organ samples were preserved in formalin and stained with hematoxylin and eosin. Among the collected organ samples, two groups (50 μg/kg group versus PBS group) were evaluated by a professional pathologist.

2.11. In vivo cytokine release syndrome study in a PBMC-humanized mouse model

NSG mice (6–8-week-old) from Jackson Laboratory (Cat#005557, Bar Harbor, ME, USA) were used for this study. NSG mice were irradiated on day 0 at 200 cGy. Four hours after the irradiation, 20 million human peripheral blood mononuclear cells (PBMCs) from a healthy donor (Vitalant, Scottsdale, AZ, USA) were injected intravenously. On day 5, 50 μL of the blood sample was collected from the orbital sinus in heparin tube and analyzed by flow cytometry to confirm human PBMC chimerism. On day 6, PBMC-humanized NSG mice were injected intravenously with CCR4-IL2-IT (8.43×10⁻¹⁰ mol/kg, n=4), anti-human CD3 mAb (OKT3, 0.5 mg/kg, BioLegend, San Diego, CA, USA, n=4) as positive control, IL2-IT (Ontak^®-like human IL2 fusion toxin, 8.43×10⁻¹⁰ mol/kg, n=4) and CCR4-IT (8.43×10⁻¹⁰ mol/kg, n=4) as mono-specific immunotoxin controls, PBS (n=4) as negative control. Six hours after the immunotoxin injection, the mice were euthanized. Blood samples were collected from cardiac punch for serum preparation. Spleens were collected to confirm the human PBMC chimerism (murine CD45, human CD45, and human CD3). Serum samples were analyzed for human cytokine release (human IL-2, human IL-6, human IL-10, human TNF and human IFN-γ) using a BD Cytometric Bead Array Human Th1/Th2 Cytokine kit II (BD-Biosciences, San Jose, CA). The cytokine capture bead complexes (capture bead + analyte + detection reagent) were analyzed using flow cytometry, and the acquired flow cytometry data was analyzed using FlowJo software. OKT3 stimulated serum was diluted as 1:2 for cytokine release analysis of human IL-2, human IL-6, human IL-10, and human TNF. 1:10 dilution of the OKT3-stimulated serum was used for human IFN-γ analysis. No dilution was performed for the serum samples of IL2-IT, CCR4-IT, and CCR4-IL2-IT.

2.12. Flow cytometry analysis for CRS study

Human immune cell populations were monitored in PBMC-engrafted mice using mAbs specific for the following human and murine antigens: human CD45-PE (clone HI30, BioLegend), murine CD45-FITC (clone 30-F11, BioLegend), and human CD3-APC (clone UCHT1, BioLegend). Whole blood was collected from orbital sinus in heparin-containing tube. The PBMC were isolated using Histopaque-1083 (Sigma-Aldrich, St. Louis, MO). 50 μL of the whole blood was diluted with 150 μL of HBSS and overlaid on 600 μL of Histopaque-1083 and centrifuged for 4 min at 6000 ×g. Buffy layer was harvested into a new tube and washed twice with PBS. The spleen was harvested, smashed by syringe back and incubated in 6 ml of ACK buffer (Lonza, Walkersville, NC, USA) for 5 min to lyse the red blood cells. The splenocytes were washed twice with PBS. The immune cells isolated either from the whole blood or spleen were finally re-suspended in FACS buffer (HBSS supplemented with 1% BSA and 0.01% sodium azide), and pre-incubated with rat anti-mouse CD16/32 mAb (clone 93, BioLegend) as FcR blocker. Specific mAbs were then added to the samples and incubated for 30 minutes at 4°C. The stained samples were washed twice with FACS buffer and analyzed using flow cytometry (Beckman Coulter, Brea, CA, USA). The acquired flow cytometry data were analyzed using FlowJo software.

2.13. Statistical analysis

For CRS studies, statistical analyses were performed using GraphPad Prism. Data significance (P values) was calculated using t-test between the compared two groups. The significance (P values) of rat chemistry and immunophenotyping data was calculated using Two-Way ANOVA between pre-injection and day 14 for each treatment group. The significance (P values) of rat CBC, minipig CBC chemistry and immunophenotyping data were calculated using Two-Way ANOVA Dunnett’s multiple comparisons of different post-treatment time points with pre-treatment in each treatment group. Significance is defined as P < 0.05.

3. Results

3.1. In vitro binding analysis of CCR4-IL2-IT to rat, minipig and human PBMC

Rat was used as rodent animal model and minipig as non-rodent large animal model for the toxicology studies of CCR4-IL2-IT. We performed in vitro binding analysis of CCR4-IL2-IT to rat, porcine and human PBMCs. As shown in Fig. S1, we have activated the PBMC (human, porcine and rat) to upregulate the expression of CD25 and CCR4 using species-specific interleukin-2 at 800 units/mL for 5 days (Zhang et al., 2006) to perform the in vitro binding analysis of CCR4-IL2-IT to human, porcine and rat CD25 and CCR4. CCR4-IL2-IT demonstrated cross-species binding to both porcine CCR4⁺ and CD25⁺ populations. The binding affinity of CCR4-IL2-IT to porcine PBMCs is comparable to that observed with human PBMCs. While in rats, CCR4-IL2-IT bound to the CD25⁺ population but did not show binding to the CCR4⁺ population. The binding affinity to rat PBMCs was relatively lower in comparison.

3.2. Single-dose escalation toxicology study of CCR4-IL2-IT in rats

The goal of this single-dose escalation toxicology study was to determine the maximum tolerated dose (MTD) of CCR4-IL2-IT in rats. As shown in Fig.1, since more than 15% body weight loss was observed following injection of 0.8 mg/kg, the MTD of CCR4-IL2-IT in rats was determined as 0.4 mg/kg.

Fig.1.

Single-dose escalation toxicology studies of CCR4-IL2-IT in rats (n=6). 0.1, 0.2, 0.4 and 0.8 mg/kg of CCR4-IL2-IT was intravenously injected into 6 rats (3 males and 3 females) with 3-day separation between the escalations on day 1, 4, 7 and 10. Each body weight was normalized with pre-injection level. Error bars represent the SD.

3.3. Repeat-dose for five-day toxicology study of CCR4-IL2-IT in rats

PK study was performed to measure the CCR4-IL2-IT concentration in the collected serum samples of the experimental rats at 7 time points on day 1 and 5 after the intravenous injection of CCR4-IL2-IT for the 5-day treatment. CCR4-IL2-IT was detected by ELISA dose-dependently on day 1 and 5 after intravenous injection of CCR4-IL2-IT (Fig. 2). CCR4-IL2-IT was not detected in 2.5 μg/kg group probably due to the sensitivity of the in vitro ELISA assay. The half-life of CCR4-IL2-IT in rats was determined as approximately 45 min.

Fig. 2.

PK analysis of the collected serum samples from the five-day treatment of CCR4-IL2-IT in rats (n=6). CCR4-IL2-IT concentration in rat serum samples were analyzed by ELISA. Error bars represent the SD.

CBC analysis was performed for the blood samples collected pre-injection, and on day 7 and 14 post-injection of CCR4-IL2-IT for the 5-day treatment in rats (PBS, 2.5 μg/kg, 25 μg/kg, and 50 μg/kg). As shown in Fig. 3A–B, the values of each parameter in the CBC panel were statistically analyzed using Two-Way ANOVA. No significant difference was observed with any parameter of the blood samples collected pre-injection versus the blood samples collected on day 7 and 14 post-injection. We observed that the base values of white blood cell count, lymphocyte count and granulocyte count in the female rats were much lower than that in the male rats. Therefore, we presented the CBC data of male and female rats separately.

Fig. 3.

CBC and chemistry analysis of the collected blood or serum samples from the five-day treatment of CCR4-IL2-IT in rats (n=6). The blood or serum samples were collected at pre-injection and on day 7 and 14 post-injection of CCR4-IL2-IT. The shadow areas represent the reference range. A) CBC analysis of the collected blood samples from the male rats (n=3). B) CBC analysis of the collected blood samples from the female rats (n=3). C) Chemistry analysis of the collected serum samples from all experimental rats. Significance is defined as P < 0.05. P values of CBC data were calculated using Two-Way ANOVA Dunnett’s multiple comparisons of different post treatment time points with pre-treatment in each treatment group. P values of chemistry data were calculated using Two-Way ANOVA between pre-injection and day 14 in each treatment group. Error bars represent the SD.

Chemistry analysis was performed for the collected serum samples from pre-injection and on day 14 post-injection for the 5-day treatment of CCR4-IL2-IT in rats (PBS, 2.5 μg/kg, 25 μg/kg, 50 μg/kg). As shown in Fig. 3C, the functions of the kidney, liver, pancreas, and metabolism were monitored by the chemistry panel. The values of each parameter of the chemistry panel were statistically analyzed using Two-Way ANOVA. No significant difference was observed with any parameter between the collected serum samples from pre-injection versus the collected serum samples on day 14 post-injection of CCR4-IL2-IT.

Histology analysis was performed for the collected organ samples on day 14 for the 5-day treatment of CCR4-IL2-IT in rats. As shown in Fig. 4A, the organ samples from two groups (50 μg/kg group versus PBS group) were evaluated by a professional pathologist and there was no difference between the two groups. As shown in Fig. 4B, immunogenicity analysis was performed for the collected rat serum samples at pre-study and weekly post 5-day treatment (PBS, 2.5 μg/kg, 25 μg/kg, 50 μg/kg). Rat anti-CCR4-IL2-IT antibody was detected in about two weeks in 25 μg/kg and 50 μg/kg groups. Anti-CCR4-IL2-IT antibody was not detected in 2.5 μg/kg group probably due to the sensitivity of the ELISA assay. The anti-drug antibody level was significantly lower in the high-dose (50 μg/kg) group than that in the mid-dose (25 μg/kg) group. We speculate that the drug levels might have interfered with the assay.

Fig. 4.

Histology and immunogenicity analysis for the 5-day treatment of CCR4-IL2-IT in rats. A) Histology analysis of the harvested organs on day 14 from the 5-day treatment of CCR4-IL2-IT in rats. Upper panel: 50 μg/kg of CCR4-IL2-IT, once daily for 5 consecutive days. Down panel: PBS vehicle treatment, once daily for 5 consecutive days. B) Immunogenicity analysis of the collected serum samples from the 5-day treatment of CCR4-IL2-IT in rats. The collected serum samples were analyzed by indirect ELISA to measure the antibodies against CCR4-IL2-IT. Error bars represent the SD.

Immunophenotyping, including T cell, B cell, and NK cell counts and frequency, was performed for the collected blood samples from pre-injection and day 14 post injection of CCR4-IL2-IT. As shown in Fig. 5, no significant difference was observed between pre-injection and day 14 post-injection of CCR4-IL2-IT in both male and female rats. In the rat study, we only performed CBC analysis for the blood samples collected on day 7 due to the limited blood volume.

Fig. 5.

Immunophenotyping of the collected blood samples at pre-injection and day 14 post injection of CCR4-IL2-IT in rats for the 5-day treatment. Cell count and frequency of T cell, B cell and NK cell were analyzed by flow cytometry. Significance is defined as P < 0.05. P values were calculated using Two-Way ANOVA between pre-injection and day 14 in each treatment group.

3.4. Single-dose escalation study to assess tolerability of CCR4-IL2-IT in minipigs

The goal of this single-dose escalation study was to determine the MTD of CCR4-IL2-IT in minipigs. CBC and chemistry analysis were used to determine the MTD. The blood samples were collected from pre-injection and post each escalating dose of CCR4-IL2-IT in minipigs (0.1, 0.2, 0.4, 0.8 mg/kg) for the CBC and chemistry analysis. As shown in Fig. 6A, the CBC profile demonstrated that the white blood count, monocyte count and granulocyte count were transiently increased out of the normal standard range after injection of CCR4-IL2-IT at 0.8 mg/kg. As shown in Fig. 6B, the chemistry profile demonstrated that after injection of CCR4-IL2-IT at 0.8 mg/kg, the glucose, AST, TBIL and lipase were transiently increased out of the normal standard range and significantly higher than that of pre-injection. Therefore, the MTD of CCR4-IL2-IT in minipigs was determined as 0.4 mg/kg.

Fig. 6.

Single-dose escalation studies of CCR4-IL2-IT in four minipigs (two males: #1198 and #1199; two females: #3913 and #3921). A) CBC and B) chemistry analyses were used to determine the MTD. The shadow areas represent the reference range. Note: Pig#3921 received a partial 0.8 mg/kg dose and Pig#3913 did not receive 0.8 mg/kg dose due to possible type I hypersensitivity reaction.

PK study was performed for the collected serum samples from the single-dose escalating studies of CCR4-IL2-IT in minipigs. As shown in Fig. 7A, the serum samples were collected post each escalating intravenous injection dose of CCR4-IL2-IT in minipigs (0.1, 0.2, 0.4 or 0.8 mg/kg). CCR4-IL2-IT was detected by ELISA, and the half-life of CCR4-IL2-IT in minipigs was determined to be approximately 30 min (Fig. 7A).

Fig. 7.

PK analysis of the collected serum samples from the treatment of CCR4-IL2-IT in minipigs. A) PK analysis of the collected serum samples following each single-dose escalation study in minipigs. Note: Pig#3921 received a partial 0.8 mg/kg dose and Pig#3913 did not receive 0.8 mg/kg dose due to possible type I hypersensitivity reaction. Error bars represent the SD. B) PK analysis of the collected serum samples from the five-day treatment of CCR4-IL2-IT in minipigs (n=4 for 50, 25 and 2.5 μg/kg groups, n=3 for PBS group). CCR4-IL2-IT concentration in the porcine serum samples were analyzed by ELISA.

3.5. Five-day (one cycle) tolerability study of CCR4-IL2-IT in minipig

As shown in Fig. 7B, PK study was performed for the collected serum samples on day 1 and 5 at 6 time points after intravenous injection of CCR4-IL2-IT for the 5-day treatment in minipigs. CCR4-IL2-IT was detected dose-dependently by ELISA on day 1 and 5 after intravenous injection of CCR4-IL2-IT. CCR4-IL2-IT was not detected in the collected serum sample from 2.5 μg/kg group due to the sensitivity of the in vitro ELISA assay. The half-life of CCR4-IL2-IT in minipigs was determined to be about 30 min (Fig. 7B).

CBC analysis was performed for the collected blood samples from the 5-day treatment including vehicle, 2.5 μg/kg, 25 μg/kg, and 50 μg/kg in minipigs. As shown in Fig. 8A, the values of each parameter in the CBC panel were statistically analyzed using Two-Way ANOVA. No significant difference was observed with any parameter of the blood samples collected from pre-injection versus the blood samples collected post treatments on day 5, 7/8, 14/15 and 22. A transient lymphopenia was observed post-dose 5 in the 50 μg/kg treatment group, but it rebound to normal level in 3~4 days. Some animals showed increased white blood cell count and granulocyte count, which might indicate infections.

Fig. 8.

CBC and chemistry analysis of the collected blood or serum samples from the five-day treatment of CCR4-IL2-IT in minipigs (n=4 for 50, 25 and 2.5 μg/kg groups, n=3 for PBS group). The blood or serum samples were collected at pre-dose 1, post-dose 5, day 7/8, 14/15, 22. The shadow areas represent the reference range. A) CBC analysis of the collected blood samples from the minipigs. B) Chemistry analysis of the collected serum samples from the minipigs. Significance is defined as P < 0.05. P values of CBC and Chemistry data were calculated using Two-Way ANOVA Dunnett’s multiple comparisons of different post-treatment time points with pre-treatment in each treatment group. Error bars represent the SD.

Chemistry analysis was performed for the collected serum samples from the 5-day treatment including vehicle, 2.5 μg/kg, 25 μg/kg, and 50 μg/kg in minipigs. The serum samples were collected on the following 5 time points including pre-dose 1 treatment (day 1), post-dose 5 treatment (day 5), week 1 (day 7/8), week 2 (day 14/15) and study endpoint (day 22). As shown in Fig. 8B, the values of each parameter of the chemistry panel were statistically analyzed using Two-Way ANOVA. No significant difference was observed with any parameter between pre-treatment versus post-treatment on day 5, 7/8, 14/15, 22.

Histology analysis was performed for the collected organ samples on day 22 for the 5-day treatment of CCR4-IL2-IT in minipigs. As shown in Fig. 9A, the organ samples from two groups (50 μg/kg group versus PBS group) were evaluated by a professional pathologist and no difference was observed between the two groups. Immunogenicity analysis was performed for the collected serum samples on the following four time points including pre-first dose treatment (day 1), week 1 (day 7/8), week 2 (day 14/15) and study endpoint (day 22). Anti-CCR4-IL2-IT antibody was detected about 10 days after the CCR4-IL2-IT treatment in the minipigs (Fig. 9B). Immunophenotyping, including T cell, CD4⁺ T cell, CD8⁺ T cell, B cell, and NK cell counts and frequency, was conducted on collected blood samples at pre-injection, day 5 and 22 post injection of CCR4-IL2-IT in minipigs. As shown in Fig. 10A–B, no significant difference was observed with T cells, CD4⁺ T cells, CD8⁺ T cells, B cells and NK cells in frequency on day 5 and 22. However, the absolute number of T and B cells decreased significantly on day 5. This could be due to transient lymphopenia. All cell populations rebounded back to normal level at the study endpoint compared to the pre-treatment level. We have also monitored the depletion of CCR4-IL2-IT to the target cell populations (CCR4⁺ and CD25⁺ cells). As shown in Fig. 10C, significant depletion was observed with CCR4⁺ population in both frequency and absolute count on day 5 in both 50 and 25 μg/kg treatment groups. Significant depletion was also observed with CD25⁺ population in absolute count in 50 μg/kg treatment group. However, no significant decrease was observed with CD25⁺ population in frequency in 50 μg/kg treatment group.

Fig. 9.

Histology and immunogenicity analysis for the 5-day treatment of CCR4-IL2-IT in minipigs. A) Histology analysis of the harvested organs on day 22 from the 5-day treatment of CCR4-IL2-IT in minipigs. Upper panel: 50 μg/kg of CCR4-IL2-IT, once daily for 5 consecutive days. Down panel: PBS vehicle treatment, once daily for 5 consecutive days. B) Immunogenicity analysis of the collected serum samples from the 5-day treatment of CCR4-IL2-IT in minipigs. The collected serum samples were analyzed by indirect ELISA to measure the antibodies against CCR4-IL2-IT. Error bars represent the SD.

Fig. 10.

Immunophenotyping of the collected blood samples at pre-injection, day 5 and 22 (endpoint) post injection of CCR4-IL2-IT in minipigs for the 5-day treatment. Cell count and frequency of each cell population were analyzed by flow cytometry. A) Immunophenotyping of T cell, B cell and NK cell. B) CD4⁺ T cell and CD8⁺ T cell. C) Depletion analysis of CCR4⁺ and CD25⁺ cells (the target cell populations of CCR4-IL2-IT). Significance is defined as P < 0.05. P values were calculated using Two-Way ANOVA Dunnett’s multiple comparisons of different post-treatment time points with pre-treatment in each treatment group. Error bars represent the SD.

3.6. CCR4-IL2-IT did not induce cytokine release syndrome in a PBMC-humanized mouse model

To further de-risk the clinical development of CCR4-IL2-IT, we have employed a PBMC-humanized mouse model to rapidly evaluate the human cytokine release response of CCR4-IL2-IT as reported by Ye et al. 2020 (Ye et al., 2020). Human PBMC chimerism was confirmed using the PBMC-humanized murine blood sample on day 5 or using the PBMC-humanized murine spleen on day 6. Only the mice which shows >70% of human CD45 cell population frequency would be considered as humanized. OKT3 served as positive control. CCR4-IT and IL2-IT (Ontak^®-like human IL2 fusion toxin) were included as monospecific immunotoxin controls (Peraino et al., 2014; Wang et al., 2016b; Wang et al., 2015; Wang et al., 2017). As shown in Fig. 11, no cytokine release of human IL-2, human IL-6, human IL-10 and human TNF-γ was observed for CCR4-IL2-IT and its two-monospecific immunotoxin controls (IL2-IT and CCR4-IT). CCR4-IL2-IT induced minimal human IFN-γ release. Of note, negative control PBS, CCR4-IT and IL2-IT also induced minimal IFN-γ release. It is not significant between the IFN-γ induced by CCR4-IL2-IT versus the background IFN-γ by negative control PBS (p=0.2021), CCR4-IT (p=0.2198) or IL2-IT (p=0.3099). In contrast, positive control OKT3 induced significantly strong human cytokine release for all monitored human cytokines indicating that the in vivo CRS mouse model was functional. We have repeated the in vivo CRS study two more times and comparable results were observed (data not shown). Taken together, the data demonstrated that CCR4-IL2-IT and its two monospecific immunotoxin controls (CCR4-IT and IL2-IT) did not induce CRS in the PBMC-humanized mouse model.

Fig. 11.

Cytokine release response of CCR4-IL2-IT in PBMC-humanized mouse model. N=4 for each treatment group. OKT3 was included as positive control and PBS as negative control. CCR4-IT and IL2-IT were included as monospecific immunotoxin controls. OKT3, anti-human CD3 mAb; N/D, not detectable; IFN-γ, human interferon gamma; IL6, human interleukin 6; IL10, human interleukin 10; IL2, human interleukin 2; TNF, human tumor necrosis factor. The results are representative of three independent assays. Error bars represent the SD. Significance was defined as P < 0.05. P values were calculated using t-test between two groups.

4. Discussion

The non-GLP toxicology studies in both rats and minipigs were in alignment with International Conference on Harmonization and FDA guidance, with regards to expectations on the type, design, and timing of studies. The design of the CCR4-IL2-IT toxicology studies was informed by precedent molecules including Ontak^®, Elzonris^®, Lumoxiti^® (Pseudomonas exotoxin A based CD22 immunotoxin), and Poteligeo^® (Mogamulizumab, CCR4 monoclonal antibody). The development program for these drugs provided valuable information on specific toxicity concerns as well as provided useful guidance on the types of studies to conduct. We anticipated similar findings might occur for CCR4-IL2-IT. However, we did not expect the findings to be completely replicated for CCR4-IL2-IT. The dose ranges employed in toxicology studies for these drugs also provided useful guidance for what CCR4-IL2-IT studies should bracket during the initial pilot dose ranging work. In the toxicology studies of CCR4-IL2-IT in both rats and minipigs, special attention was directed towards the toxicities identified in the precedent drugs. Ontak^® key toxicities in non-human primates were liver, kidney, spleen, lungs and concerning adverse effects in humans included hepatotoxicity, renal toxicity, vascular leakage, thrombotic events, and hypersensitivity reactions. Additionally, the Ontak^® label described warning/precautions for infusion reactions, capillary leakage syndrome and loss of visual acuity and color visions. Elzonris^® adverse effects highlighted in the labels warning and precautions were capillary leakage syndrome, hepatotoxicity, and hypersensitivity. Non-human primate studies identified deficits in food intake, body weight, and reduced red cell mass with compromised regeneration, inflammatory response, liver, kidney, and choroid plexus brain-degeneration. Poteligeo^® adverse findings highlighted in the labels warning and precaution section included dermatotoxicity of moderate to severe magnitude (not reproduced in animal models), infusion reactions, infections, autoimmune complications, and graft versus host concerns. We did not observe the toxicities in either rat or minipig with CCR4-IL2-IT. Ontak^® and Elzonris^® were produced using the E. coli expression system. Ontak^® was discontinued due to purification problems related to this expression system. We speculate that some of the toxicities of Ontak^® and Elzonris^® might contribute to the protein quality related to the E. coli expression system. Of course, we could not rule out the possibility of species difference between minipigs versus non-human primates.

Sprague Dawley rat was used as rodent animal model for the non-GLP toxicology studies of CCR4-IL2-IT. The binding analysis demonstrated that CCR4-IL2-IT cross-species reacted to rat CD25 receptor and did not react to rat CCR4 receptor. Therefore, Sprague Dawley rat is a half relevant rodent animal model for the toxicology studies of CCR4-IL2-IT, which is one limitation of this study. Minipig was used as a non-rodent large animal model for the toxicology studies of CCR4-IL2-IT. We had previously demonstrated that CCR4-IT cross-species reacted to porcine CCR4 receptor (Wang et al., 2016a). The binding analysis in this study demonstrated that CCR4-IL2-IT cross-species reacted to both porcine CCR4 and porcine CD25 receptors. The depletion analysis demonstrated that CCR4-IL2-IT depleted both porcine CCR4⁺ and porcine CD25⁺ cells (two target cell populations of CCR4-IL2-IT). Therefore, minipig is a fully relevant non-rodent large animal model for the toxicology studies of CCR4-IL2-IT. In the minipig single-dose escalating toxicology study, earlier sample analyses showed CCR4-IL2-IT related changes as each daily dose increased. However, it is not clear which of the three components (DT390, IL2 or CCR4 scFv) was responsible. Anti-drug antibody (ADA) against CCR4-IL2-IT was developed in both rats and minipigs in about two weeks as previously reported by our lab for CCR4-IT in cynomolgus monkeys (Wang et al., 2018). ADA was also developed for FDA-approved Ontak^® and Elzonris^® (FDA-approved two diphtheria toxin-based fusion toxins). Ontak^® and Elzonris^® were FDA-approved for multiple courses of cancer treatment. Hopefully, CCR4-IL2-IT can also be administered for multiple courses of cancer treatment. As we predicted, the half-life of CCR4-IL2-IT was short (~45 min in rats and ~30 min in minipigs) like Ontak^®, Elzonris^®, and Resimmune^® (Woo et al., 2008). CCR4-IL2-IT did not induce CRS in a PBMC-humanized mouse model. The CRS negative data further guaranteed the safety of CCR4-IL2-IT towards clinical trials. No CRS was observed with Ontak^®-like human IL2 fusion toxin (IL2-IT) indicating that CCR4-IL2-IT and IL2-IT were killers, rather than stimulators to IL-2 receptor bearing immune cells. In the single-does escalating studies, Pig#3921 only received a partial 0.8 mg/kg dose and pig#3913 did not receive 0.8 mg/kg dose due to possible type I hypersensitivity reaction. The blood chemistry parameters including blood glucose, TBIL, AMYL and LIPA increased 30 min after the injection of CCR4-IL2-IT and came back to normal level in 48 hours, indicating that it is a type I hypersensitivity, not the immunotoxin toxicity. Later, we injected 0.8 mg/kg of CCR4-IL2-IT into a new minipig without any adverse response. Therefore, the observed possible type I hypersensitivity reaction was just an individual case. Given the possible clinical dose of CCR4-IL2-IT is very low (<30 μg/kg), the MTD as 0.4 mg/kg is more than sufficient to support the clinical development of CCR4-IL2-IT. We observed that the base values of white blood cell count, lymphocyte count, and granulocyte count in female rats were much lower than that in male rats. Therefore, we presented the CBC data of male and female rats separately. One limitation of this study is that the intra-animal single-dose escalating studies were not appropriate to determine the MTD of CCR4-IL2-IT in both rats and minipigs. Therefore, we have decided that we will repeat the actual single dose escalating studies in both rats and minipigs in the future to confirm the MTDs. In summary, the toxicology studies in both rats and minipigs demonstrated that CCR4-IL2-IT is safe for further development towards human clinical trials. We have demonstrated that minipigs (not rats) are a good toxicology model for CCR4-IL2-IT. However, we still need to consider the low cross-species reactivity when we design the clinical dose of CCR4-IL2-IT for human clinical trials.

Supplementary Material

1

Fig. S1. In vitro binding analysis of CCR4-IL2-IT to rat, minipig and human PBMC by flow cytometry. Anti-human CCR4 mAb (Bio-Techne FAB1567P) and species-specific CD25 mAb were used to characterize the binding of CCR4-IL2-IT to the two target cell populations of CCR4-IL2-IT: CCR4⁺ cell and CD25⁺ cell.

Highlights:

• CCR4-IL2-IT demonstrated excellent safety profiles in both rats and minipigs

• The maximum tolerated dose of CCR4-IL2-IT was determined as 0.4 mg/kg

• Half-life of CCR4-IL2-IT was determined as ~45 min in rats and ~30 min in minipigs

• Anti-CCR4-IL2-IT antibody was detected two weeks after CCR4-IL2-IT treatment

• CCR4-IL2-IT did not induce cytokine release syndrome