Research ArticleFEATURED BASIC SCIENCE ARTICLE

Detection of IL12/23p40 via PET Visualizes Inflammatory Bowel Disease

Farzaneh Rezazadeh, Nicholas Ramos, Allen-Dexter Saliganan, Najeeb Al-Hallak, Kang Chen, Bashar Mohamad, Wendy N. Wiesend and Nerissa T. Viola
Journal of Nuclear Medicine November 2023, 64 (11) 1806-1814; DOI: https://doi.org/10.2967/jnumed.123.265649

Abstract

Inflammatory bowel disease (IBD), which includes both Crohn disease and ulcerative colitis, is a relapsing inflammatory disease of the gastrointestinal tract. Long-term chronic inflammatory conditions elevate the patient’s risk of colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires a bowel-preparatory regimen, adding to patient burden. Interleukin 12 (IL12) and interleukin 23 (IL23) play key roles in inflammation, especially in the pathogenesis of IBD, and are established therapeutic targets. We propose that imaging of IL12/23 and its p40 subunit in IBD via immuno-PET potentially provides a new noninvasive diagnostic approach. Methods: Our aim was to investigate the potential of immuno-PET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate by targeting IL12/23p40 with a 89Zr-radiolabeled anti-IL12/23p40 antibody. Results: High uptake of the IL12/23p40 immuno-PET agent was exhibited by dextran sodium sulfate–administered mice, and this uptake correlated with increased IL12/23p40 present in the sera. Competitive binding studies confirmed the specificity of the radiotracer for IL12/23p40 in the gastrointestinal tract. Conclusion: These promising results demonstrate the utility of this radiotracer as an imaging biomarker of IBD. Moreover, IL12/23p40 immuno-PET can potentially guide treatment decisions for IBD management.

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract. It is believed to be caused by dysregulation of immune response to pathogens. IBD comprises 2 major forms: ulcerative colitis and Crohn disease (1). The global prevalence of IBD increased from 3.7 million in 1990 to more than 6.8 million in 2017 (2). The detailed etiology of IBD is still unclear, but genetic predisposition, intestinal dysbiosis, various proinflammatory cytokines, T-helper cells, and interleukins are implicated in its pathogenesis (3,4). In early-stage IBD, a loss of intestinal epithelial barrier function is observed, increasing bacterial translocation and thus activating the mucosal immune system and intestinal inflammation (5). If left unmanaged, patients with ulcerative colitis have a 2% increased incidence of colorectal cancer (CRC) at 10 y after diagnosis, 8% at 20 y, and about 18% at 30 y, compared with those without IBD. So, careful monitoring of ulcerative colitis patients is recommended (6). IBD-associated CRC carries a particularly poor prognosis (mortality > 50%) (7) compared with sporadic CRC, for which the prognosis is improving. Unlike sporadic CRC, which progresses through an adenoma–carcinoma pathway, IBD CRC evolves from an inflammation–dysplasia–carcinoma sequence (8). Accordingly, there are currently no tools specifically designed to help clinicians track the initiation and progression of IBD CRC pathology.

Detecting and tracking chronic inflammation in the gastrointestinal tract is critical to improving outcomes among patients with IBD. Current diagnostic and surveillance methods for IBD involve clinical manifestations (e.g., bloody diarrhea) in conjunction with physical examination, endoscopy, and pathologic findings (9). Molecular assays that detect elevated biomarkers of inflammation in blood and stool samples reinforce its evaluation and diagnosis, albeit with no acquisition of locoregional information on inflamed areas (10). Currently, endoscopy with biopsy is the gold standard for the diagnosis of IBD and for staging its activity (11). However, endoscopy is invasive and can result in toxic megacolon if performed during exacerbation. Another drawback of endoscopy is that it is limited to imaging segmental areas of the intestine and the superficial mucosal surface. Furthermore, it cannot provide detailed molecular information on the disease (12). Less invasive imaging modalities, including CT, ultrasound, and MRI, are also used clinically for IBD diagnosis (13,14). However, these modalities provide information only on the anatomic integrity of gastrointestinal tissues. None of these available standard-of-care diagnostic tools, whether used alone or in combination, completely meets the need for safe, accessible, reliable, quantitative visualization of gastrointestinal inflammation with high spatial and molecular specificity. A less invasive and quantitative tool that can generate both molecular and morphologic information on specific pathologic processes would be useful in determining the extent of IBD activity (15).

Numerous studies have explored the utility of [18F]-FDG PET in the assessment of IBD. High uptake of [18F]-FDG in intestinal tissues due to increased immune metabolic activity allows the delineation of inflammatory sites in IBD patients (16,17). However, although [18F]-FDG has been shown to be sensitive in the detection of IBD, its specificity leaves much to be desired because of physiologic uptake in the normal bowel (18). To date, there are no specific PET tracers for imaging IBD that are Food and Drug Administration–approved. Other than [18F]-FDG, novel PET agents developed for IBD diagnosis are limited to preclinical studies (12,19–21), with only 3 new agents in patient trials (NCT03546868, NCT04507932, and NCT03414788). Thus, there is a clear need to develop a molecular imaging agent that can detect and visualize active inflammation on-site.

The cytokines interleukin 12 (IL12) and interleukin 23 (IL23) are associated with mediating inflammation. Both cytokines are members of the IL12 family and share the p40 subunit (22). IL12/23p40 has become a clinically relevant target in IBD treatment because of its central role in inducing and sustaining inflammation (23). For example, ustekinumab (Stelara; Janssen), an IL12/23p40 antagonist, has been Food and Drug Administration–approved for treatment, with clinical data showing that patients experienced symptom relief and achieved clinical remission (24,25). Thus, IL12/23p40 is an appealing target not only for treatment but also for diagnosis and surveillance of IBD.

Here, we present the development of an immuno-PET imaging agent by radiolabeling an antibody targeting IL12/23p40 with the radioisotope 89Zr (half-life, ∼3.27 d). We characterized the radiotracer’s in vitro specificity and stability and evaluated its potential to visualize the cytokine in a dextran sodium sulfate (DSS)–induced acute model of gastrointestinal inflammation in immune-competent mice. We performed in vivo competitive binding studies to demonstrate the tracer’s specificity and determined its whole-body tissue distribution. We also validated the uptake of the radiotracer from the genetic expression of IL12/23p40 from excised tissues. Finally, we explored the correlation between tracer uptake in gastrointestinal tissues and IL12/23p40 serum levels.

MATERIALS AND METHODS

General

All chemicals and supplies were purchased from commercial suppliers and used without further manipulation except when otherwise stated. [89Zr]Zr-oxalate was obtained from 3D Imaging, LLC. Antimouse IL12/23p40 monoclonal antibody (mAb) (rat IgG2a) was purchased from Bio X Cell (catalog number BE0051).

Chemical Induction of Colitis via DSS

All animal experiments were conducted in compliance with the Institutional Animal Care and Use Committee at Wayne State University. Male and female BALB/c mice aged 10–14 wk (weight range, 20–30 g) were purchased from Charles River Laboratories. Colitis was induced by replacing normal drinking water with 3% (w/v) DSS (molecular mass, 40 kDa; Alfa Aesar) for 7 d alongside access to a chow diet (PicoLab Laboratory Rodent Diet 5L0D) ad libitum. The mice were weighed daily and assessed for clinical manifestations of colitis. The severity of the colitis was determined by a disease activity index according to Freise et al. (19): weight loss (0, none; 1, 1%–4%; 2, 5%–10%; 3, 11%–20%; 4, 0.20%), fecal blood (0, none; 2, blood present in stool; 4, gross bleeding from anus), and stool consistency (0, normal; 1, moist/sticky; 2, soft; 3, diarrhea; 4, bleeding).

Antibody Conjugation and 89Zr Radiolabeling

p-Benzyl-isothiocyanate-deferoxamine (DFO-Bz-SCN, Macrocylics) was conjugated to anti-IL12/23p40 by a similar protocol to that previously reported (26). Briefly, DFO-Bz-SCN in DMSO was added to the mAb at a ratio of 1:5 (mAb:DFO) in 0.9% saline, pH approximately 9, at 37°C for 1 h. Subsequent purification using a centrifugation column filter (molecular weight cutoff, 30 kDa) and 0.9% saline as the mobile phase eliminated unconjugated DFO-Bz-SCN. 89Zr-radioabeling of DFO-anti-IL12/23p40 antibody proceeded in a neutral pH environment in saline. Approximately 37 MBq (1 mCi) of [89Zr]Zr-oxalate previously neutralized to pH 7.0–7.2 were added to a solution of DFO-anti-IL12/23p40 (0.2 mg, 1.3 nmol) and incubated for 1 h at room temperature. The radiolabeling reaction and efficiency were monitored via radio–instant thin-layer chromatography (Mini-Scan/FC; Eckert and Ziegler) using instant thin-layer chromatography glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, catalog number SGI0001) as the stationary phase and 50 mM ethylenediaminetetraacetic acid as the mobile phase. The reaction was quenched with 5 μL of 50 mM ethylenediaminetetraacetic acid and purified through spin column centrifugation (molecular weight cutoff, 30 kDa) with sterile saline as the eluent. Stability studies of [89Zr]Zr-DFO-anti-IL12/23p40 were conducted in saline at 37°C and monitored over 96 h.

In Vitro Binding and Blocking Studies

Saturation binding studies were conducted to determine the dissociation constant of the [89Zr]Zr-DFO-anti-IL12/23p40. Mouse IL12/23p40 protein (Bio-Techne) (5 μg/mL in bicarbonate buffer) was coated onto a 96-well strip plate and incubated at 4°C overnight. The wells were then washed 3 times with 0.05% polysorbate-20 in ×1 phosphate-buffered saline and incubated for 2 h at room temperature with 200 μL of blocking buffer (1% bovine serum albumin in wash buffer). The IL12/23p40-coated wells were incubated with varying concentrations of radiolabeled antibody (0.5–5,000 nM) or subjected to blocking by coincubation of the radiotracer with a 100-fold excess of unmodified IL12/23p40 antibody in triplicate. After 1 h at 37°C, the unbound radiotracer was removed and the bound activity for each well was measured with a γ-counter. The dissociation constant was calculated by nonlinear regression using GraphPad Prism, version 9.2. Specific binding was measured by subtracting nonspecific binding from total bound activity expressed as counts per minute. To evaluate specificity, a separate group of IL12/23p40-coated wells was treated with an excess of cold anti-IL12/23p40 mAb (500 nM) 30 min before the addition of the radiotracer (5 nM, 0.012 MBq [0.34 μCi] per well) and incubation for 1 h at 37°C. Unbound radiotracer was removed, and the wells were washed twice with ×1 phosphate-buffered saline. All experiments were performed in triplicate. The bound activity for each well was measured by a γ-counter and expressed as counts per minute.

PET/CT Imaging

[89Zr]Zr-DFO-anti-IL12/23p40 (6.66–9.25 MBq, 36–50 μg, 0.24–0.33 nmol) in sterile saline was administered intravenously to mice (5 per sex) in the lateral tail vein 5 d after DSS treatment. A separate cohort of DSS-treated mice was injected with an 89Zr-labeled nonspecific isotype-matched IgG antibody to control for specificity. Images were then acquired using a Bruker Albira Si PET/CT scanner at 24, 48, and 96 h after injection while the mice were anesthetized with 2% isoflurane. Subsequent imaging scans were acquired at 48 h after injection, the time point that was identified to achieve optimum contrast. Corresponding CT scans were acquired at 45 kV and 400 μA after each PET scan. Images were reconstructed through maximum-likelihood expectation maximization with 12 iterations and a 0.75-mm voxel resolution, registered on CT, and analyzed by PMOD software, version 4.3. Volumes of interest were acquired via isocontouring and are expressed as the mean percentage injected dose per cm3 of tissue (%ID/cm3). After imaging, the mice were euthanized, and the colons were excised and laid out for ex vivo PET imaging.

Biodistribution and In Vivo Blocking Studies

The tissue distribution of [89Zr]Zr-DFO-anti-IL12/23p40 was assessed in healthy and DSS-treated mice by intravenous injection of 0.37–0.92 MBq (10–25 μCi, 2–5 μg) in the lateral tail vein of the mice. To assess the specificity of target binding, we performed an in vivo blocking study on a separate group of mice in which a ×100 excess of unlabeled anti-IL12/23p40 mAb was coinjected with the radiotracer. Euthanasia was performed via CO2 asphyxiation 48 h after injection. Blood was immediately collected via cardiac puncture. Select organs were collected and weighed. Bound radioactivity was measured using a γ-counter (2480 Wizard2; Perkin Elmer) and expressed as %ID/g.

IL12/23p40 Enzyme-Linked Immunosorbent Assay

Blood was collected via the cheek vein before (baseline or day 0) and during (day 5) DSS treatment. Sera were obtained and stored at −80°C to allow for decay before analysis. Levels of IL12/23p40 were evaluated using a commercial enzyme-linked immunosorbent assay kit (catalog number ELM-IL12p40p70; RayBiotech). Serum levels were then correlated with the radiotracer uptake data from the biodistribution studies.

In Situ Hybridization

To investigate the expression of IL12/23p40 in DSS-treated mice, RNA in situ hybridization for IL12/23p40 messenger RNA (RNAscope probe Mm-IL12b; ACD Bio) was performed using an RNAscope 2.5 HD detection assay (ACD Bio). The RNA spatial expression of IL12p35 (IL12a; Mm IL12a; ACD Bio) and IFN-γ (RNAscope Probe-Mm-Ifng-C2; ACD Bio) were also examined. In brief, intestinal tissues were harvested, fixed, and stored in 10% formalin until decayed. Once decayed, the formalin was removed and replaced with 70% ethanol before paraffin embedding and sectioning. Tissues were sliced into 5-μm sections and mounted on glass slides. Paraffin-embedded tissue sections were deparaffinized and subjected to antigen retrieval using an RNAscope antigen retrieval kit (ACD Bio) followed by hybridization, signal amplification, and chromogenic detection procedures, which were performed according to the manufacturer’s instructions (RNAscope 2.5 HD duplex detection; ACD Bio). The messenger RNA for IL12/23p40 and IFN-γ was detected using red chromogen and that of IL12p35 was detected using green chromogen. Images (×40 objective) were captured under an optical microscope (Carl Zeiss), and signal quantification was analyzed by HALO software, version 4.1.1 (Indica Labs). At least 5 fields per section were analyzed.

Statistical Analysis

GraphPad Prism, version 9.02, was used to perform statistical analyses unless otherwise stated. Data are presented as the mean ± SD. Mann–Whitney t tests were performed for the tumor-blocking competitive studies. Two-way ANOVA multiple-comparison analyses were performed for the tissue distribution studies. The significance of correlations was determined by Pearson analysis. A P value of less than 0.05 was considered statistically significant.

RESULTS

Successful Labeling and Target Binding of [89Zr]Zr-DFO-Anti-IL12/23p40

Radiolabeling of the DFO immunoconjugate was straightforward, with radiolabeling yields of more than 95%. A specific activity of 180 ± 10 MBq/mg was established. The radiotracer remained moderately intact (>90%) in saline over a 96-h incubation period (Supplemental Fig. 1). The binding of [89Zr]Zr-DFO-anti-IL12/23p40 to mouse IL12/12/23p40 protein was evaluated by saturation binding assays, showing total and nonspecific binding (Supplemental Fig. 2), which confirmed the high affinity (dissociation constant, ∼9.8 ± 1.9 nM) of the radiolabeled mAb for its target (Fig. 1A). The in vitro blocking study, in which [89Zr]Zr-DFO-anti-IL12/23p40 was incubated with a 100-fold mass excess of the unmodified nonradiolabeled antibody, demonstrated significantly decreased binding of [89Zr]Zr-DFO-anti-IL12/23p40 to IL12/23p40 protein (P = 0.0001) (Fig. 1B), confirming the in vitro specificity of the radiotracer.

FIGURE 1.
FIGURE 1.

In vitro characterization of [89Zr]Zr-DFO-anti-IL12/23p40. (A) Nonlinear regression analysis determination of binding affinity (dissociation constant [Kd]) of tracer for IL12/23p40. (B) Competitive inhibition with ×100 excess of unmodified antibody coadministered with radiotracer in IL12/23p40-coated wells. CPM = counts per minute. ***P < 0.001.

Induction and Confirmation of Colitis

Colitis was induced in BALB/c mice by providing DSS ad libitum for 7 d, followed by recovery with regular drinking water on day 8 (Fig. 2A). DSS-treated mice showed weight loss on day 5 (Fig. 2B). Disease activity index scores for stool consistency and fecal bleeding were higher in the DSS-treated mice than in the healthy mice (Supplemental Table 1). On day 8, scores of more than 2 were recorded for DSS-treated mice according to the presence of diarrhea and blood in their stools.

FIGURE 2.
FIGURE 2.

(A) Scheme showing days of DSS treatment, injection of [89Zr]Zr-DFO-anti-IL12/23p40, and PET imaging. (B) Changes in body weight after 5 d of DSS treatment (5 per sex). Vertical dashed line indicates beginning of body weight loss. ****P = 0.0001.

In Vivo Delineation of Intestinal Inflammation by IL12/23p40 Immuno-PET

[89Zr]Zr-DFO-anti-IL12/23p40 delineated the intestinal tract of a mouse with severe gastrointestinal inflammation, whereas no clear delineation was observed in healthy control mice (Fig. 3A). The planar sections of the PET images for these mice and those with mild IBD are shown in Supplemental Figures 3A and 3B. Uptake of the radiotracer in the colons of both healthy and DSS-treated mice was recorded and classified according to sex (Fig. 3B). For both male mice (DSS, 5.99 ± 0.65 %ID/cm3, vs. healthy, 3.98 ± 0.58 %ID/cm3; P = 0.0002) and female mice (DSS, 8.53 ± 1.47 %ID/cm3, vs. healthy, 6.56 ± 0.85 %ID/cm3; P = 0.014), DSS treatment led to higher uptake in the colon than that for the healthy group at 24 h after injection or day 6. A comparative analysis between the sexes in healthy and DSS mice revealed significant differences in radiotracer uptake between male and female (Fig. 3C). The time–activity curve of [89Zr]Zr-DFO-anti-IL12/23p40 taken from volumes of interest of the heart, liver, and muscle uptake from 24 to 96 h after injection (days 6–9) showed decreasing nonspecific binding of the radiotracer over time (Fig. 3D).

FIGURE 3.
FIGURE 3.

IL12/23p40 immuno-PET/CT imaging of healthy and DSS-treated mice. (A) Representative maximum-intensity projection images of healthy (left) and DSS-treated (right) mice acquired at 24 h (day 6) and 48 h (day 7) after injection of [89Zr]Zr-DFO-anti-IL12/23p40. (B) Plotted volumes of interest of animals administered [89Zr]Zr-DFO-anti-IL12/23p40 exhibiting higher uptake in colons of male and female mice treated with DSS than in healthy mice. (C) Sex differences in pooled analysis of uptake of IL12/23-40 radiotracer (n = 5 per sex) in both healthy and DSS-treated groups. (D) Time–activity curves of [89Zr]Zr-DFO-anti-IL12/23p40 binding in heart, liver, and muscle in healthy and DSS-treated groups. (E) Planar section of DSS-treated mice imaged with [89Zr]Zr-DFO-IgG (3 per sex) vs. [89Zr]Zr-DFO-anti-IL12/23p40 demonstrating minimal binding of nonspecific radiotracer in colon at day 7 (arrows). (F) Higher colon uptake of [89Zr]Zr-DFO-anti-IL12/23p40 (anti-IL12/23p40) in male (n = 3) and female groups (n = 3) than in those imaged with [89Zr]Zr-DFO-IgG (IgG) at 48 h after injection. All cohorts were 5 per sex unless otherwise stated. MIP = maximum-intensity projection. *P < 0.05. **P < 0.01. ***P < 0.001.

The specificity of the cytokine radiotracer was assessed by imaging a separate cohort of DSS-treated mice with a 89Zr-radiolabeled isotype control antibody. Unlike colitis mice imaged with [89Zr]Zr-DFO-anti-IL12/23p40, colitis mice imaged with [89Zr]Zr-DFO-IgG did not show specific accumulation in the colon (Fig. 3E). Colitis mice injected with [89Zr]Zr-DFO-IgG showed lower uptake in the colon at 48 h after injection (day 7) of the radiotracer than did DSS mice injected with [89Zr]Zr-DFO-anti-IL12/23p40 (Fig. 3F). Selected healthy organs (e.g., heart, liver, and muscle) displayed lower binding of [89Zr]Zr-DFO-IgG than of the IL12/23p40-specific radiotracer (Supplemental Figs. 3C–3D).

Ex Vivo Imaging of Excised Colons

We next performed ex vivo imaging to assess the specific focal uptake of the tracer in the colon. Tracer uptake was increased in the cecum, proximal colon, and mid colon of DSS-treated mice injected with [89Zr]Zr-DFO-anti-IL12/23p40, whereas a lower accumulation of [89Zr]Zr-DFO-IgG was observed in similar regions (Fig. 4A). The lengths of the small (Fig. 4B) and large (Fig. 4C) intestines were measured after imaging. The small intestines of DSS-treated mice appeared longer than those of healthy mice, whereas the large intestines were collectively shorter, which agrees well with previous literature (19). Interestingly, no significant difference in intestinal lengths (healthy vs. DSS) was displayed after the DSS-treated mice were switched to regular drinking water, suggesting quick recovery.

FIGURE 4.
FIGURE 4.

Ex vivo immuno-PET and tissue distribution studies. (A) Representative PET images of excised intestinal tissues from DSS-administered mice demonstrating increased accumulation of IL12/23p40 tracer (middle) in cecum, proximal colon, and mid colon vs. radiolabeled nonspecific isotype antibody (right). Schematic representation of mouse colon depicts colon sections (left) (created via Biorender). (B and C) Lengths of small (B) and large (C) intestines from separate cohorts were measured on last day of DSS treatment (day 7) and 2 d after switching to regular drinking water (day 9) (5 per sex). Small intestines in DSS-treated group were longer than those in healthy controls. Colons from DSS-treated mice were significantly shorter than those from healthy group on day 7, but there was no significant difference in length after switching to normal drinking water on day 9. (D) Tissue distribution of IL12/23p40 radiotracer in healthy and DSS-treated mice at 48 h after injection. Separate DSS-treated cohort was coinjected with excess of unmodified anti-IL23p40 for competitive binding with radiotracer (5 female animals per cohort). (E) Uptake of tracer in small intestines, colon, and cecum. *P < 0.05. ***P < 0.001. ****P < 0.0001.

Tissue Biodistribution and Competitive Binding

Tissue biodistribution of [89Zr]Zr-DFO-anti-IL12/23p40 was analyzed in both healthy and DSS-treated mice at 48 h after injection (Fig. 4D). Uptake in the large intestine was higher in the DSS group than in the healthy mice (3.0 ± 0.4 %ID/g vs. 1.7 ± 0.35 %ID/g, P = 0.0008), whereas no significant differences in the cecum and small intestines were observed between the 2 cohorts.

To confirm specificity, blocking experiments were performed in which at least a 100-fold excess of the unmodified, nonradioactive mAb was coinjected with the radiotracer into DSS-treated mice. Blocking significantly decreased uptake in the small intestines (1.3 ± 0.2 %ID/g vs. 0.4 ± 0.1 %ID/g, P < 0.0001), colon (3.0 ± 0.4 %ID/g vs. 0.8 ± 0.2 %ID/g, P < 0.0001), and cecum (1.6 ± 0.5 %ID/g vs. 1.0 ± 0.3 %ID/g, P = 0.0132) (Fig. 4E). We also observed a decrease in accumulation of the radiotracer in the nontarget tissues of the blocked versus unblocked DSS cohorts (Fig. 4D), suggesting that the excess mAb attenuated the binding of the radiotracer in endogenously expressed and systemically circulating IL12/23p40. Furthermore, another rationale for the observed decreased binding is the fact that the mAb is inherently therapeutic, suggesting that the mass injected for blocking (∼200 μg) may have depleted the cytokine at 48 h after injection, resulting in lower tracer binding.

Correlation Between Tracer Uptake and Serum Expression of IL12/23p40

To confirm expression of IL12/23p40 in colitis mice, sera were sequentially collected on day 0 before radiotracer injection (the baseline before DSS treatment) and on day 5. Enzyme-linked immunosorbent assay results showed elevated total IL12/23p40 on day 5 compared with day 0 for both male and female groups (Fig. 5A) (male, 1.85 ± 0.28 vs. 1.22 ± 0.33 ng/mL [P = 0.0125]; female, 3 ± 0.58 vs. 2.04 ± 0.05 ng/mL [P = 0.0473]). A correlation between IL12/23p40 levels in sera on day 5 and [89Zr]Zr-DFO-anti-IL12/23p40 uptake in the small intestine and cecum (measured from the ex vivo distribution study) indicated a direct relationship between IL12/23p40 concentration and radiotracer uptake in the male group (Fig. 5B). A positive correlation, albeit not significant, was observed between IL12/23p40 in the serum and radiotracer uptake in the colon (Supplemental Fig. 4A; supplemental materials are available at http://jnm.snmjournals.org). In the female group, correlations between the serum level of IL12/23p40 and radiotracer uptake in the colon, cecum, and small intestine were not significant (Supplemental Fig. 4B).

FIGURE 5.
FIGURE 5.

Correlation of serum IL12/23p40 with [89Zr]Zr-DFO-anti-IL12/23p40 tissue uptake. (A) IL12/23p40 in serum showing increased expression 5 d after DSS administration in male (n = 5) and female (n = 3) mice. (B) Strong positive correlation between IL12/23p40 serum concentration and radiotracer uptake in male group. *P < 0.05.

High Transcript Expression of IL12/23p40 in the Intestinal Tissues Validating PET Radiotracer Uptake

To further detect IL12/23p40 cytokine in healthy and DSS mice, in situ hybridization was performed to visualize RNA expression per cell (Fig. 6A). A high percentage of cells showed increased expression of IL12b, the gene for IL12/23p40 (percentage of cells positive for IL12b, 27% ± 7.5%), in the cecum of DSS-treated mice compared with healthy tissue (Fig. 6B). To show that the p40 subunit is the main driver of inflammation, we examined for IL12p35 (IL12a), the other subunit specific to IL12 alone. IL12a was not as markedly expressed in inflamed tissues (percentage of cells positive for IL12a, 2.75% ± 0.22%; P = 0.0012) (Supplemental Fig. 5), suggesting that the inflammation in this mouse model is mediated by the p40 subunit. Interestingly, IFN-γ, a downstream molecule produced by both IL12 and IL23 that is considered to stimulate massive leukocyte infiltration and mucosal damage in IBD (5), showed elevated transcript expression in the DSS-treated intestines but was not as elevated as IL12/23p40.

FIGURE 6.
FIGURE 6.

In situ hybridization to detect transcripts of IL12/23p40 (IL12b) in intestinal tissues of healthy and DSS mice. (A) Higher IL12b expression (red staining) in DSS-treated mice (top left) than in healthy mice (top right). Bottom figures represent individual cells analyzed by HALO software of healthy (left) and DSS-treated (right) colons. Purple outlines represent positive cells expressing IL12b, and cell nuclei are marked in blue. (B) Quantitative analysis of messenger RNA levels of IL12/23p40 (≥5 fields of view from 1 tissue section). Cytokine expression is higher in DSS-treated than healthy mice. **P < 0.01.

DISCUSSION

The global increase in IBD incidence is anticipated to impose increasing health and economic burdens (2). Patients from industrialized western countries are those primarily affected by IBD. However, the prevalence and incidence are increasing worldwide, specifically in newly industrialized countries (2). On ultrasound alone, approximately 3.1 million adults were diagnosed with this autoimmune condition in 2015, with an increased prevalence observed for people over 45 y old, Hispanic and non-Hispanic white people, and those with low levels of education (27). Thus, addressing this autoimmune condition before it becomes a global issue is critical. In addition, management and surveillance of IBD are crucial because it predisposes patients to a higher risk of CRC (28). Colitis is further observed as an immune-related adverse event associated with immune checkpoint inhibitors (29) and can be life-threatening at higher grades (30).

The current diagnostic standard of care is reliant on endoscopy or colonoscopy to assess mucosal inflammation. However, endoscopy or colonoscopy can image only superficial tissue architectures. These approaches reveal very little about the molecular characteristics of a patient’s disease, especially when inflammation is occurring deep within the mucosal layer before anatomic changes are observed (31). Moreover, this approach carries the risk of bowel perforation, which can lead to bleeding. Noninvasive molecular imaging techniques can potentially overcome these limitations and improve IBD staging and diagnosis.

Here, we have presented the development of a noninvasive and quantitative imaging agent via immuno-PET that is directed against IL12/23p40, successfully achieving detection of acute inflammation in a DSS-induced mouse model of colitis. We have shown that [89Zr]Zr-DFO-anti-IL12/23p40 was able to detect IL12/23p40 in the colons of DSS-treated mice. We have demonstrated the radiotracer’s specificity in both in vitro and in vivo competitive binding studies as well as when compared with a radiolabeled nonspecific antibody, which failed to delineate intestinal inflammation. The results of the biodistribution study clearly demonstrated increased uptake in the gastrointestinal tissues of the colitis mice, reflecting the PET imaging results.

In our hands, intestinal inflammation developed in the mice 5 d after administration of DSS, as marked by significant weight loss and high disease activity index scores. Different small- and large-intestine lengths in healthy versus DSS mice confirmed the intestinal inflammatory status of the latter, with measurements that agree well with prior reports (19). The radiotracer [89Zr]Zr-DFO-anti-IL12/23p40 was injected on day 5. We have determined that the optimum imaging time point at which a high signal-to-noise ratio can be achieved is between 24 h (day 6) and 48 h (day 7) after injection. The ex vivo imaging of excised large intestines displayed sporadic accumulation in the cecum and in different sections of the colon. We first endeavored to remove the feces. However, on removal, the PET signal decreased to almost background. We posit that the tracer is not bound to the gastrointestinal wall because it binds to soluble IL12/23p40, released in the extracellular milieu. Moreover, the cytokine binds to its receptor, which is present on trafficking immune cells. Thus, the PET signal from the images taken on different days is not localized (Fig. 3A).

One of the challenges we encountered in this study lies in the fact that inflammation is a dynamic immune event. The immune microenvironment during a state of inflammation is transient, with immune cells trafficking between the gut and peripheral lymphoid tissues to initiate and sustain inflammatory activity (32). Moreover, the severity of inflammation can vary in location and per animal, despite ensuring similar age and environmental conditions. This was evidenced through ex vivo images of the large intestines (Supplemental Fig. 3D) and through the disease activity index, where clinical manifestations (e.g., fecal blood, stool consistency) varied per mouse (Supplemental Table 1). As a result, discrepancies in radiotracer uptake between separate groups of animals used for imaging and those used for biodistribution can be misappropriated as discordant.

A growing body of evidence indicates that circulating IL23 may be a biomarker of disease severity in patients with IBD (33). Lucaciu et al. have shown that for differentiating mild from severe inflammation, IL23 (from serum) is a diagnostic marker superior to current standard-of-care inflammation markers such as C-reactive protein and fecal calprotectin (34). Another study has shown that the enhanced serum levels of IL12p40 significantly correlate with clinical and endoscopic disease activity in patients with IBD (35). In our study, we showed that elevated IL12/23p40 was detected by the radiotracer within the intestinal site and displayed a positive correlation between the ceca and small intestines with serum IL12/23p40 in male mice. We did not find a correlation between serum levels of IL12/23p40 and uptake of [89Zr]Zr-DFO-anti-IL12/23p40 in the colons of DSS-treated male mice, although a positive but nonsignificant trend was observed (Supplemental Fig. 3A). This is likely due to heterogeneity in immune activity and varying severities of inflammatory response, which can differ among mice despite their similar genetic backgrounds and housing conditions.

A sex disparity in inflammatory response to mucosal injury was evident in our study, and it was clearly delineated by the radiotracer. DSS-induced colitis in male mice resulted in significantly higher inflammation than that in female rodents, as demonstrated by disease activity index scores, a finding that parallels findings by others (36). Sex differences have long been recognized in IBD susceptibility and disease severity, but they remain poorly understood (37). The estrus cycle has been shown to influence IBD pathogenesis by controlling intestinal permeability, which is lower at the proestrus stage when estrogen peaks (38). Ogawa et al. reported that the gut of female rodents withstands the damaging effects of hypoxia and acidosis better than the gut of male rodents, but this disparity was eliminated on administration of estradiol to male rats, demonstrating the protective role of estrogen (33). Moreover, estrogen has been shown to exert an antiinflammatory function in the gut by reducing immune infiltration and cytokine secretion during colitis, which leads to significantly lower inflammation, as demonstrated by Houdeau et al. and Harnish et al. (39,40).

Other molecular agents to image IBD have been investigated. To date, clinical SPECT and PET imaging of IBD is restricted to using 111In- or 99mTc-radiolabeled leukocytes and [18F]-FDG, none of which provides clinical information (17,41). Leukocyte SPECT imaging suffers from poor-quality images, and the radiolabeling is a time-consuming process (42). [18F]-FDG PET can identify inflammation, but its highly variable tissue uptake limits its utility as a diagnostic tool (43). [18F]-FDG marks cellular metabolism, providing information about the energy consumption of mucosal-layer–infiltrating immune cells within the inflamed tissue. This information can be useful for imaging glycolytic activity of immune cells but not for assessing mediators of inflammation (16). A significant drawback of using [18F]-FDG in IBD is that many individuals have progressive physiologic uptake in the gut, particularly in the large bowel, which may make it difficult to diagnose IBD in colonic segments (44). Additionally, patients with diabetes who use antidiabetic medications (such metformin) may experience significant absorption in the large bowel, affecting the inflammatory readout from the tracer (45). New immuno-PET agents were developed and have shown excellent target selectivity; however, to date, their application to IBD detection is limited to preclinical studies. Freise et al. showed delineation of CD4+ T cells that are present in inflamed colons of DSS mice using a murine CD4 diabody (GK1.5) radiolabeled with 89Zr (19). Imaging CD4 precludes assessment of other proinflammatory immune cell phenotypes that contribute to this autoimmune condition (46,47). The innate immune markers CD11b and IL-1β were detected by 89Zr-conjugated antibodies targeting these molecules. Both immuno-PET agents detected colonic inflammation, but [89Zr]Zr-anti-IL-1β was more specifically accumulated in the gastrointestinal tract than was [89Zr]Zr-anti-CD11b, which was dispersed to other tissues (12). However, the role of IL-1β in IBD is far from clear, stemming from the lack of positive response in patients given the targeted blockade (12). Recently, an immuno-PET agent was developed for targeting tumor necrosis factor-α in DSS-induced colitis mice using 89Zr-labeled mAb infliximab. Elevated levels of [89Zr]Zr-DFO-infliximab were observed in the colon of colitis mice compared with the healthy control and blocked groups (21). However, 30%–50% of patients eventually relapse from tumor necrosis factor-α blockade due to IL23-mediated resistance, which makes this new radiotracer more relevant as a predictive biomarker of response to therapy (48).

Our previous work developed and assessed an immuno-PET imaging agent specific for IL12p70 that detected the cytokine globally in an induced inflammation model (49). Compelling studies have shown that IL23 rather than IL12 plays a major proinflammatory role in IBD, which eliminates the targeting of IL12p70 as a method for imaging IBD. In this regard, the p40 subunit shared between IL12 and IL23 was demonstrated to be one of the mediators of inflammation and may be an appealing target for diagnosis and treatment of IBD. In fact, ustekinumab is an antihuman IL12/23p40 mAb currently deployed as the standard of care for Crohn disease patients and those with moderate to severe ulcerative colitis. Thus, the development of a companion diagnostic is an apparent rational next step (23).

Mouse IL12/23p40 is only 70% homologous to the human version (50). Consequently, we rationalize that proof-of-concept studies—as for all immunologic studies that use antibodies—will be better achieved if we use antibodies that target the host’s protein (e.g., mouse IL12/23p40) and will eliminate variables that can confound the study. Indeed, using an antimouse IL12/23p40 antibody to target mouse IL12/23p40 precludes a straightforward application of the tracer in humans and limits the application in mice. However, our data establish proof that the tracer can delineate gastrointestinal inflammation, laying the groundwork for developing a tracer for human application.

A clear limitation of this radiotracer is its bispecificity for IL12 and IL23 because both share the p40 subunit, making it difficult to provide an absolute measure of IL23. IL12 (through IL12a) expression is 10-fold lower than that of IL23, suggesting that it is indeed IL23 that mediates the inflammation in this mouse model. Another limitation lies in the potential of the antibody to neutralize IL12/23p40, consequently decreasing IL12 and IL23 expression, which we have observed during in vivo competitive binding studies and during imaging studies. To address this, work is under way to develop nonneutralizing second-generation mAb fragments that are specific to IL23.

Our findings demonstrate that our IL12/23p40 tracer constitutes a novel approach to delineate IBD selectively and robustly in this model of acute inflammation. The promising findings from this study have major implications in IBD treatment as a noninvasive, quantitative, and molecular technique for its diagnosis, potentially improving the standard of care. Although there is a risk associated with radiation produced by radiopharmaceuticals agents, the advantages of PET imaging—in this case, informing inflammatory activity by imaging IL12/23p40—far outweigh exposure of patients to radiation, which can easily be mitigated by administering safe doses. Furthermore, IL12/23p40 immuno-PET can provide useful insight for patient treatment, including those who will potentially respond to its blockade.

CONCLUSION

To the best of our knowledge, this is the first report illustrating the preliminary development of an IBD immuno-PET imaging tool specific for IL12/23p40. This new imaging technology can potentially facilitate detection and accurate staging of IBD in patients via generation of a global in vivo inflammation map of the entire gastrointestinal tract.

DISCLOSURE

The study was supported by NIH/NCI R37 CA220482 (Nerissa Viola). The Microscopy, Imaging, and Cytometry Resources Core (MICR) and the Animal Modeling and Therapeutics Core, which provided technical assistance, are supported, in part, by NIH Cancer Center grant P30 CA022453 to the Karmanos Cancer Institute at Wayne State University. No other potential conflict of interest relevant to this article was reported.

KEY POINTS

QUESTION: What is the potential of IL12/23p40 immuno-PET to visualize IBD?

PERTINENT FINDINGS: IL12/23p40 immuno-PET selectively and robustly delineated acute inflammation in an induced-colitis mouse model via monitoring IL12/23p40, making it a novel imaging technology that can potentially facilitate early detection of IBD.

IMPLICATIONS FOR PATIENT CARE: The findings of this study are significant because it potentially represents a new category of diagnostic tool for surveillance and diagnosis of IBD. Imaging IL12/23p40 is an innovative and minimally invasive approach for identifying the dominant signaling pathway to guide clinical decisions on appropriate treatment.

ACKNOWLEDGMENTS

We thank Dr. Lisa Polin and Michael Bradley for suggestions and advice on animal studies. We are further grateful to Dr. Freddy Escorcia for helpful discussions and review of the manuscript. We also thank Dr. Todd Sasser for assistance in the PET/CT image analysis.

Footnotes

  • Published online Jul. 20, 2023.

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  • Received for publication December 22, 2022.
  • Revision received June 20, 2023.
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