Therapeutic checkpoint inhibitors on tumor-infiltrating lymphocytes (TIL) are being increasingly utilized in the clinic. The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and natural killer cells. The TIGIT signaling pathway is an alternative target for checkpoint blockade to current PD-1/CTLA-4 strategies. Elevated TIGIT expression in the tumor microenvironment correlates with better therapeutic responses to anti-TIGIT therapies in preclinical models. Therefore, quantifying TIGIT expression in tumors is necessary for determining whether a patient may respond to anti-TIGIT therapy. PET imaging of TIGIT expression on TILs can therefore aid diagnosis and in monitoring therapeutic responses.
Antibody-based TIGIT imaging radiotracers were developed with the PET radionuclides copper-64 (64Cu) and zirconium-89 (89Zr). In vitro characterization of the imaging probes was followed by in vivo evaluation in both xenografts and syngeneic tumor models in mouse.
Two anti-TIGIT probes were developed and exhibited immunoreactivity of >72%, serum stability of >95%, and specificity for TIGIT with both mouse TIGIT-expressing HeLa cells and ex vivo–activated primary splenocytes. In vivo, the 89Zr-labeled probe demonstrated superior contrast than the 64Cu probe due to 89Zr's longer half-life matching the TIGIT antibody's pharmacokinetics. The 89Zr probe was used to quantify TIGIT expression on TILs in B16 melanoma in immunocompetent mice and confirmed by ex vivo flow cytometry.
This study develops and validates novel TIGIT-specific 64Cu and 89Zr PET probes for quantifying TIGIT expression on TILs for diagnosis of patient selection for anti-TIGIT therapies.
This article is featured in Highlights of This Issue, p. 1825
Checkpoint blockade therapies have expanded to new inhibitory receptors where stratification for patient selection and therapeutic monitoring strategies are needed. This work develops, validates, and applies PET imaging probes for the T-cell immunoreceptor with Ig and ITIM domains (TIGIT). TIGIT is an inhibitory receptor expressed by tumor-infiltrating lymphocytes (TIL) including T and natural killer cells, and is a therapeutic target in a range of solid tumors. Current methods to assess TIGIT expression such as biopsy are invasive and do not provide whole-body quantification of TIGIT expression. To address this need, TIGIT-specific immunoPET imaging probes are developed and validated. These probes are shown to specifically target TIGIT in both xenograft and allograft murine models. TIGIT expression on TILs in a melanoma allograft model is quantified using PET, confirmed via flow cytometry and ex vivo biodistribution studies. Our data suggest that this approach can act as a companion diagnostic for current anti-TIGIT therapies.
Immune checkpoint receptors expressed on activated T and natural killer (NK) cells play a key role in maintaining physiologic self-tolerance (1). However, these same regulating pathways are exploited by tumor cells to evade immune surveillance (2). This tumor survival strategy has been targeted with checkpoint blockade therapies such as anti-PD-1 and anti-CTLA-4 agents. While these therapies have shown remarkable responses in a subset of patients, primary and adaptive resistance often occurs (3). This is attributed to a variety of mechanisms including downregulating MHC to prevent antigen presentation and upregulation of other inhibitory receptors such as TIM-3, LAG-3, and VISTA (4).
To reduce the occurrence of tumor escape, other inhibitory targets that act through alternative pathways are being extensively explored. One such checkpoint inhibitor is the T-cell Ig and ITIM domain receptor (TIGIT). TIGIT is an inhibitory receptor expressed on CD8+ cytotoxic T cells, CD4+ Th cells, FOXP3+ regulatory T cells, and NK cells (5). Both TIGIT and PD-1 are expressed in >70% of human melanoma tumor microenvironments, with PD-1 mainly expressed on CD8+ T cells and TIGIT expressed on CD8+ T cells, regulatory T cells, and NK cells (6). The ratio of PD-1 to TIGIT expressed by tumor-infiltrating lymphocytes (TIL) is variable, ranging from 0.75 to 4.0 in colorectal cancer(5). TIGIT can inhibit immune responses of effector T and NK cells by acting through inhibitory signals (7–9) and affecting effector functions of T cells and antigen-presenting cells (10, 11). TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation on TILs (12), making TIGIT a potential key addition to current immunotherapies. For this reason, a number of clinical trials in solid tumors are combining TIGIT therapies with current checkpoint blockade therapies (13–15).
The expression of TIGIT on TILs has been reported via IHC, Western blotting, and PCR on hepatocellular carcinoma (16), follicular lymphoma (17), squamous cell cancers (5), Hodgkin lymphoma (18), renal cell carcinoma (19), small cell lung cancer (20), and melanoma (21). TIGIT has been validated as a therapeutic target and anti-TIGIT therapies are currently being evaluated in multiple clinical trials (Supplementary Table S1). An antibody specific for the TIGIT receptor (AB154) is being evaluated in a variety of solid cancers (NCT03628677). This phase I trial is evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of AB154 as either a monotherapy or in combination with an anti-PD-1 antibody (AB122). In another phase I trial, the anti-TIGIT agent MK-7684 and pembrolizumab was administered in 34 patients with advanced solid tumors for whom standard treatment options had failed (NCT02720068). The response rate was reported as 19% and the disease control rate was 47% (22). While these results are promising for a subset of patients, patient stratification for these therapies would benefit from a quantitative, whole-body measure of TIGIT expression.
Generally high but variable TIGIT expression is seen in solid tumors, making this therapy likely applicable to many cancer types. However, to determine TIGIT expression, invasive biopsy sampling followed by IHC is currently required. While this procedure is the current gold standard, it does not allow accurate quantitation in heterogeneous samples or on distant metastases. A noninvasive imaging agent could detect and quantify TIGIT within the tumor microenvironment and aid in determining what patients would benefit from TIGIT immunotherapies (23). This approach has been tested with PET agents for PD-1, PD-L1, and Ox40, all being developed as clinical diagnostics (24–26). These PET tracers allow noninvasive, whole-body, quantitative assessment of receptor expression to aid in patient stratification. Here we develop TIGIT-specific antibody PET tracers and evaluate their application in quantifying TIGIT expression in vivo (Fig. 1).
Materials and Methods
Antibodies, chelators, and radiometals
Purified anti-mouse TIGIT mAb (TIGITmAb, clone 1G9) raised in mouse was purchased from Bio X cell (catalog no. BE0274) or BioLegend (catalog no. 142102). NHS ester-DOTA (NHS-DOTA, catalog no. B-280) and p-SCN-Deferoxamine (DFO-NCS, catalog no. B705) were purchased from Macrocyclics. Copper-64 (64Cu; t1/2 = 12.7 hours, 17.76–162.8 GBq/μmol) was obtained from the University of Wisconsin (Madison, WI). Zirconium-89 (89Zr) in 1.0 mol/L oxalic acid (t1/2 = 3.3 days, purity >99%; 2–3 GBq/mL) was obtained from the University of Alabama at Birmingham (Birmingham, AL). All other general reagents were obtained from Sigma-Aldrich unless otherwise stated.
Cell lines ATCC
B16-F10 (murine melanoma) and HeLa (human cervical) cells were purchased from ATCC (CRL-6475). A HeLa cell line expressing the TIGIT receptor was generated using a plasmid from VectorBuilder consisting of the mouse TIGIT sequence driven by the EF1α promoter, eGFP driven by the cytomegalovirus promoter, and a puromycin sequence for selection (Supplementary Fig. S1). Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were sorted three times on a FACSAria II sorter with the top 1% of TIGIT staining collected. The transfected HeLa cells (TIGIT-HeLa) were kept under selection using DMEM supplemented with 10% FBS, 1% Antibiotic-Antimycotic, and 40 μmol/mL of puromycin. All cell lines were screened to exclude infection with Mycoplasma and preserved for a period of 20 passages in culture.
Qualitative and quantitative flow cytometry on transfected HeLa cells
Qualitative flow cytometry was performed on TIGIT-HeLa cells and nontransfected HeLa cells by staining with PE-mTIGIT antibody (2 μL/106 cells) and DAPI (1 μL of 10 mg/mL/106 cells). Quantitative flow cytometry was performed on an LSR II instrument to determine the average number of TIGIT receptors per cell using a QIFIKIT (Agilent).
TIGIT expression on isolated mouse lymphocytes
Flow cytometry was completed on isolated, activated C57B/l6J (Jackson Laboratory) mouse splenocytes to assess antibody affinity for mouse TIGIT. Splenocytes were activated via phorbol 12-myristate 13-acetate (PMA) and calcium ionomycin (50:500 ng/mL for 4 hours) at 37°C (27). Cell staining was performed in 96-well microtiter plates (U-bottom). A total of 100 μL of the antibody was mixed with 100 μL cell suspension containing 2 × 105 splenocytes and incubated for 1 hour at 4°C. Cells were washed three times with FACS buffer, followed by a secondary stain with 100 μL of anti-mouse IgG-APC (BioLegend, catalog no. 409305) prediluted to 1:100. The plate was incubated for 30 minutes on ice in the dark and washed three times. Propidium iodide was used as a live/dead stain. Analysis was carried out on a flow cytometer (FACS Aria III, BD Biosciences) and data were analyzed by FlowJo FACS analysis software (Tree Star).
DOTA- and DFO-TIGITmAb conjugation
DOTA was conjugated to mouse TIGITmAb at pH 7.3, 1 mg/mL Ab concentration, and a 20:1 DOTA:TIGITmAb ratio at 4°C for 14 hours. The DOTA-TIGITmAb conjugate was purified using 50 kDa spin filters (Millipore) with 0.1 mol/L ammonium acetate (pH 5.4) as the buffer. DOTA-TIGITmAb was washed three times and stored at 4°C at a concentration of 1–2 mg/mL. DFO-TIGITmAb was conjugated to mouse TIGITmAb at pH 8.5 in HEPES buffer, 6 mg/mL Ab concentration, and a 5:1 DFO:TIGITmAb ratio at 37°C for 14 hours. The DFO-TIGITmAb conjugate was purified using a SEC 2000 high-performance liquid chromatography (HPLC) with 0.1 mol/L ammonium acetate buffer (pH 7) as the mobile phase eluted at 1 mL/minute. The purified DFO-TIGITmAb was concentrated to 5 mg/mL using a Vivaspin 30 kDa cut-off centrifugal filter and stored in 100 μL aliquots in 0.1 mol/L ammonium acetate buffer (pH 7.0) at −20°C.
Radiolabeling DOTA- and DFO-TIGITmAb
64Cu was added to DOTA-TIGITmAb (1 mg/mL) in 0.1 mol/L ammonium acetate (pH 5.3) at a ratio of 0.37 MBq/μg Ab and incubated at 37°C for 30–45 minutes. A total of 10 μL of 50 mmol/L ethylenediaminetetraacetic acid (EDTA) was added for 15 minutes to scavenge free 64Cu. 64Cu-TIGITmAb was purified using 50 kDa spin filters with saline as the buffer. 89Zr in 1.0 mol/L oxalic acid was first brought to a pH of 7.0–7.4 with 1.0 mol/L sodium carbonate. 89Zr (74–75 MBq; 200 μL) was then added to DFO-TIGITmAb (400 μg; 5 mg/mL) in 1× PBS (pH 7.3) and incubated at 37°C for 1 hour. After incubation, 10 μL of 50 mmol/L EDTA was added for 15 minutes to scavenge free 89Zr. Purification of 89Zr-TIGITmAb was achieved via size exclusion (SE) HPLC in PBS buffer [0.1 mol/L NaCl, 0.05 mol/L sodium phosphate (pH 7.4)] at a flow rate of 1.0 mL/minute. The 89Zr-TIGITmAb peak corresponding to the antibody was collected and concentrated using a Vivaspin 30 kDa centrifugal filter and centrifuged at 3,000 × g for 15 minutes. The final product was filtered through a 0.2-micron filter into a sterile vial. Serum stability for 64Cu-TIGITmAb and 89Zr-TIGITmAb was conducted by addition of 3.7 MBq radiotracer to 100 μL of mouse or human serum with time points taken every 24 hours.
In vitro binding assays
The immunoreactivity of 64Cu-TIGITmAb and 89Zr-TIGITmAb was assessed via Lindmo assay (28). 64Cu-TIGITmAb and 89Zr-TIGITmAb were prepared at a concentration of 0.037 MBq in 2 mL of PBS with 1% BSA. A total of 50 μL of the radiolabeled antibody was added to a serial dilution of TIGIT receptor–positive HeLa cells ranging from 5×104 to 5×106 cells per vial (500 μL in PBS with 1% BSA added). After an incubation at room temperature for 30 minutes, the solutions were centrifuged at 300 × g, supernatant removed, and pellets counted using a gamma counter. Nontransfected HeLa cells were also tested with the radiolabeled antibodies, as described above.
89Zr-TIGITmAb binding was also assessed on isolated splenocytes. Two sets of experiments were performed (blocking and nonblocking; n = 3). Blocking samples received a 25-fold excess of cold TIGITmAb prior to the addition of 89Zr-TIGITmAb (2.3 μCi/mL; 200 μL/tube). The solutions were incubated for 1 hour at 25°C, washed, and activity bound to the cell pellets were counted on a gamma counter.
Small animal PET/CT imaging
Animal studies were performed in compliance with and approval from the Administrative Panel on Laboratory Animal Care at Stanford University (Stanford, CA). PET/CT imaging was performed on a Siemens Inveon small animal multimodality PET/CT system (Preclinical Solutions; Siemens Healthcare Molecular Imaging). CT imaging was performed at 80 kVp at 500 μA, second bed position, half scan 220 degrees of rotation, and 120 projections per bed position with a cone beam micro-X-ray source (50 μmol/L focal spot size) and a 4064 × 4064–pixel X-ray detector. The data were reconstructed using Shepp-Logan filtering and cone-beam filtered backprojection.
The small animal PET scanning (static scans with a coincidence timing window of 3.4 ns and energy window of 350–650 keV) was performed at the following time points after the tracer injection: 4, 24, and 48 hours for 10 minute, and 72 hours for 15-minute scans. The acquired images were reconstructed with two-dimensional ordered-subset expectation maximization (OSEM 2D) algorithm. PET/CT images and three-dimensional regions of interest (ROI) of organs of interest were computed using Inveon Research Workplace software. The mean pixel values within the ROI volume was converted to radioactivity concentration in counts per milliliter per minute (cpm) by using a predetermined conversion factor. The percentage-injected dose per gram of tissue (%ID/g) was determined by dividing each tissue's cpm obtained from the ROI by the injected dose. At each time point before imaging, animals were anesthetized and scanned at the same conditions as detailed above. A Hidex gamma counter was used for ex vivo radioanalysis.
64Cu- and 89Zr-TIGITmAb PET/CT imaging of TIGIT-expressing xenografts
Six- to 10-week-old nu/nu female mice (average weight = 23.0 ± 2.0 g) were injected in the flank with 7.5×105 cells in 100 μL matrigel. TIGIT-positive HeLa cells were injected into the left flank and TIGIT-negative HeLa cells were injected into the right flank. Xenografts were allowed to develop for 10 days. 64Cu-TIGITmAb (or 64Cu-mouse IgG, n = 4/probe) was injected via tail vein at 2.2–3.7 MBq (10–15 μg) in 100 μL of saline and imaged at 24 and 48 hours followed by a terminal ex vivo biodistribution study. 89Zr-TIGITmAb (or 89Zr-mouse IgG, n = 4 per probe) were injected via tail vein at 1.85–3.7 MBq (15–20 μg) in 100 μL of saline and imaged at 24, 48, and 72 hours followed by a terminal ex vivo biodistribution study. Gamma counter values were background subtracted and decay corrected to the time of injection and the %ID/g for each tissue sample was calculated by normalization to the total activity injected. Animal experiments were conducted twice.
89Zr-TIGITmAb PET/CT imaging of TIGIT-expressing TILs in melanoma allografts
Six- to 8-week-old female B6 mice were implanted with 1 × 106 B16F10 melanoma cells in the right shoulder and developed allografts over 10–12 days. Two groups of mice (blocking and nonblocking; n = 5) received 89Zr-TIGITmAb (200 μL, 1.7–2.1 MBq, 15–16 μg of DFO-TIGITmAb) via tail vein injection. Blocking was completed by injection of cold TIGIT (200 μg in 100 μL) 2 hours prior to 89Zr-TIGITmAb administration. The mice were imaged at 4, 24, 48, and 72 hours after injection and immediately followed by a terminal biodistribution study.
Ex vivo flow cytometry of mTIGIT on melanoma allografts
B6 mice (n = 5) were implanted with B16F10 melanoma allografts as described above. After 10 days of tumor growth, mice were euthanized and tumors were removed. Liberase TL (300 μg/mL) and DNase I (100μg/mL) in 5 mL Hank's Balanced Salt Solution (HBSS) were added to dissociated tumors for 1 hour at 37°C with gentle mixing every 15–20 minutes followed by washing in HBSS. Subsequently, ACK buffer was added to samples for 10 minutes to lyse red blood cells followed by two washes in cell stain buffer. Single-cell suspensions were prepared and stained for mTIGIT (2 μL Ab/106 cells) and DAPI (10 μg/mL final concentration) as a viability stain as described above. Samples were run in triplicate, and at least 2×105 live cells per sample were collected. Analysis was completed on an LSR II flow cytometer. Ex vivo flow cytometry was completed twice.
Unpaired Student t test (two-tailed, unequal variance) was used for data comparisons. P values of less than 0.05 were considered statistically significant.
Mouse TIGIT expression on engineered cell lines and activated splenocytes
Following three rounds of cell sorting under puromycin selection, >98% of HeLa cells expressed the mouse TIGIT receptor (Fig. 2A; Supplementary Fig. S2) with 95,200 ± 15,900 TIGIT receptors per cell (Fig. 2B). Splenocytes activated ex vivo were positive for TIGIT and DFO-conjugated TIGITmAb showed similar affinity as the unconjugated TIGITmAb for activated splenocytes, demonstrating that DFO conjugation did not adversely affect binding (Fig. 2C). These results validate the expression of TIGIT in transfected HeLa cells, as well as the specificity of TIGITmAb for the TIGIT receptor on primary splenocytes.
Synthesis and radiolabeling of immunoconjugates
DOTA and DFO were successfully conjugated to the TIGITmAb with 1.59 ± 0.30 DOTA molecules and 0.90 ± 0.17 DFO molecules per TIGITmAb (Supplementary Table S2), as determined by MALDI-MS (Supplementary Fig. S3). The radiochemical yield of 64Cu-TIGITmAb was 60%–75% and the specific activity of 0.222–0.370 MBq/μg, while 89Zr-TIGITmAb had a radiochemical yield of 60%–80% and a specific activity of 0.074–0.185 MBq/μg. The final purity of both radiotracers was >98% (Supplementary Fig. S4). The serum stability of 64Cu-TIGITmAb was >95% out to 48 hours (Fig. 3A) and that of 89Zr-TIGITmAb was >95% out to 96 hours (Fig. 3D). Finally, 64Cu-TIGITmAb was further evaluated using SE HPLC and showed a radiochemical purity of 98.3 ± 1.5% (mean ± SD; Supplementary Fig. S4C).
64Cu- and 89Zr-TIGITmAb binding assays demonstrate high specificity and immunoreactivity
64Cu-TIGITmAb and 89Zr-TIGITmAb showed at least 40-fold higher binding to TIGIT-HeLa cells, compared with the radiolabeled isotype control and low nonspecific binding to TIGIT-negative HeLa cells (P < 0.05; Fig. 3B and E). 64Cu-TIGITmAb and 89Zr-TIGITmAb had immunoreactivities of 72.7 ± 1.7% and 80.5 ± 2.2%, respectively (Fig. 3C and F). 89Zr-TIGITmAb demonstrated high specificity on activated splenocytes (Fig. 3G).
64Cu- and 89Zr-TIGITmAb PET Imaging of TIGIT-expressing xenografts in nu/nu mice demonstrate the specificity of probes uptake
Both PET probes showed specific uptake in HeLa-TIGIT–expressing xenografts (n = 4), compared with TIGIT-negative xenografts or isotype controls (P < 0.05). 64Cu-TIGITmAb showed specific uptake in HeLa-TIGIT at 24 hours, with increased uptake at 48 hours (Fig. 4A and B). The 48 hours ex vivo biodistribution (Fig. 4C) of 64Cu-TIGITmAb and 64Cu-mIgG showed 64Cu-TIGITmAb uptake in HeLa-TIGIT xenografts of 21.9 ± 3.5%ID/g, compared with 64Cu-mIgG uptake of 9.8 ± 2.2%ID/g (P < 0.05). However, blood activity levels at 48 hours were 13.4 ± 0.8%ID/g due to the pharmacokinetics of the antibody.
At 72 hours postinjection, 89Zr-TIGITmAb demonstrated higher tumor-to-background signal ratios than 64Cu-TIGITmAb due to higher tumor uptake and lower blood activity (Fig. 5A). This was confirmed by ex vivo biodistribution results at 72 hours (Fig. 5B), where 89Zr-TIGIT uptake in HeLa-TIGIT xenografts was 29.3 ± 4.5%ID/g, compared with 89Zr-mIgG uptake of 8.8 ± 0.8%ID/g (P < 0.05). Blood activity levels at 72 hours were 9.7 ± 1.0%ID/g, significantly lower than 64Cu-TIGITmAb at 48 hours (P < 0.05). 89Zr-TIGITmAb was chosen for further studies due to the combination of higher tumor uptake and lower blood activity levels compared with 64Cu-TIGITmAb at later time points.
89Zr-TIGITmAb PET/CT imaging of TIGIT-expressing TILs in melanoma allografts demonstrates quantification of TIGIT in the tumor microenvironment
89Zr-TIGITmAb PET signals at the tumor site indicated the presence of TIGIT on TILs. Representative axis and coronal immunoPET images of mice bearing melanoma at various time points (4, 24, and 72 hours after injection of tracer) are shown in Fig. 6A. PET ROI quantification (Fig. 6B) correlated with biodistribution results at 72 hours after injection (Fig. 6C). 89Zr-TIGITmAb uptake by TIGIT-bound TILs in nonblocking and blocking mice at 72 hours was (mean %ID/g ± SD) 7.4 ± 0.9 and 3.81 ± 0.75, respectively (P < 0.05). Tumor-to-muscle uptake ratios for 89Zr-TIGITmAb in nonblocking mice were 4.1 ± 0.2 and 7.4 ± 0.7 at 4 and 72 hours (black bar), respectively (P < 0.05; Fig. 6D).
Ex vivo flow cytometry on melanoma allografts and immunofluorescence staining
Flow cytometry on single-cell suspensions of B16 tumors from five mice showed TIGIT expression on infiltrating TILs. A total of 10.91 ± 2.22% of live cells in the tumor microenvironment were positive for TIGIT (Fig. 6E), showing TIGIT expression in this tumor microenvironment as shown previously (29). Furthermore, from a separate group (n = 4), melanoma allograft tissue was harvested and prepared several sections from intermelanoma and intramelanoma. These FFPE-tissue slides were stained to determine the expression pattern of both TILs and TIGIT by using CD45 and TIGITmAb (Supplementary Fig. S5). Low TIGIT expression correlated with low CD45 staining (Supplementary Fig. S5D) with the majority of TIGIT expression associated with the CD45+ TILs (Supplementary Fig. S5C).
This work develops and validates TIGIT-specific PET agents for quantification of TIGIT expression in the tumor microenvironment. We demonstrate that 64Cu-TIGITmAb and 89Zr-TIGITmAb are highly specific molecular imaging probes for assessing TIGIT expression in vivo. While both probes detect the TIGIT receptor in TIGIT-HeLa xenografts, 89Zr-TIGITmAb showed superior contrast due to better matching of full-length antibody pharmacokinetics, which are slow to clear from circulation. 89Zr-TIGITmAb was therefore evaluated in immunocompetent B6 mice with B16 melanoma allografts and demonstrated the ability to quantitatively image TIGIT expression from 4 to 72 hours after injection. Blocking studies with excess cold TIGITmAb decreased the PET signals in the tumor, demonstrating specificity of 89Zr-TIGITmAb. Flow cytometry and ex vivo immunofluorescence confirmed TIGIT expression on TILs in the tumor microenvironment. These results indicate 89Zr-TIGITmAb as a potential companion diagnostic for current and upcoming TIGIT therapies. The ability to quantify TIGIT expression in solid tumors could aid in treatment strategies prior to starting a particular immunotherapy regimen.
While this work has demonstrated the ability of quantitative TIGIT imaging in solid tumors, it must be noted that the murine TIGIT receptor was targeted. This was necessary to test the probe in TIGIT-expressing immune cells in immunocompetent mice; however, developing a human TIGIT imaging probe is necessary for clinical translation. A companion diagnostic for TIGIT therapy, much like 64Cu-pembrolizumab for human PD-1 could aid in therapeutic decisions prior and throughout the regimen. These results warrant the future development of a human TIGIT PET imaging agent for quantifying TIGIT expression in the tumor microenvironment for stratification of patients for anti-TIGIT therapy.
In conclusion, we developed and radiolabeled novel anti-TIGIT probes for immunoPET imaging of TIGIT expression in xenograft and melanoma allograft murine models. Furthermore, specific in vivo binding of anti-TIGIT probes were confirmed via quantitative flow cytometry and ex vivo biodistribution studies. This study demonstrates the potential for clinical translation of these radiotracers.
No disclosures were reported.
T. Shaffer: Conceptualization, formal analysis, validation, writing–original draft. A. Natarajan: Conceptualization, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. S.S. Gambhir: Conceptualization, resources, project administration, writing–review and editing.
This work was supported by a Cancer-Translational Nanotechnology Training grant (T32 CA196585, to T. Shaffer) and a Stanford Bio-X seed grant. Small animal PET/CT imaging and gamma counter measurements were performed in the SCi3 Stanford Center for Innovation In Vivo Imaging. Data were collected on an instrument in the Shared FACS Facility obtained using NIH S10 Shared Instrument Grant S10RR027431-01.
The co-authors would like to dedicate this work to S.S. Gambhir (1962–2020) for his excellent mentoring and tremendous support through the years.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.