Implication of 99mTc-sum IL-2 SPECT/CT in immunotherapy by imaging of tumor-infiltrating T cells

Abstract

Background: Although immune checkpoint blockade (ICB) and adoptive T cell transfer (ACT) therapy have achieved impressive clinical outcomes, majority of patients do not respond to immunotherapy. Tumor-infiltrating T cells, a critical factor to immunotherapy, is dynamically changing. Therefore, a reliable real-time in vivo imaging system for tumor-infiltrating T cells, but not immunohistochemical analyses, will be more valuable to predict response and guide immunotherapy. In this study, we developed a new SPECT/CT imaging probe ^99mTc-sum IL-2 targeting the IL-2Rβ/IL-2Rγ (CD122/CD132) receptor on tumor-infiltrating T cells, and evaluated its application in predicting the immune response to anti-PD-L1 (αPD-L1) therapy as well as tracking infused T cells in ACT therapy.

Methods: The binding affinity of the super mutated IL-2 (sum IL-2) in various T cell subtypes was measured. Sum IL-2 was subsequently labeled with ^99mTc through Sortase-A mediated site-specific transpeptidation. SPECT/CT imaging and biodistribution studies of ^99mTc-sum IL-2 were performed in a MC38 mouse model. Wild type IL-2 (IL-2) was used as control in the above studies. Finally, we evaluated ^99mTc-sum IL-2 SPECT/CT for the detection of tumor-infiltrating T cells in the context of αPD-L1 immunotherapy and ACT therapy.

Results: Sum IL-2 preferentially bound to CD8⁺ T cells, especially activated CD8⁺ T cells, while IL-2 showed biased binding to Treg cells. As a result, ^99mTc-sum IL-2 could detect tumor-infiltrating T cells. In the MC38 tumor model, SPECT/CT imaging showed the increased tumor uptake of ^99mTc-sum IL-2 after αPD-L1 treatment, suggesting that the treatment significantly increased tumor-infiltrating T cells, resulting in a correspondingly significant curative effect. In addition, ^99mTc-sum IL-2 SPECT/CT could also track the infiltration of antigen-specific cytotoxic CD8⁺ T cells during ACT therapy.

Conclusion: ^99mTc-sum IL-2 has great clinical potential for non-invasive and specific SPECT/CT imaging of tumor-infiltrating T cells as well as for timely prediction and evaluation of the therapeutic efficacy of ICB and ACT therapy.

Keywords: SPECT; immunotherapy; lymphocytes, tumor-infiltrating.

Figure 1

In vitro characterization of sum IL-2 and IL-2. (A) SDS-PAGE of IL-2-LPETG-10*His and sum IL-2-LPETG-10*His. (B) ELISA was used to detect the binding of IL-2 and sum IL-2 to the human IL-2 receptor α subunit or β subunit. (C) flow cytometric detection of the binding of IL-2 and sum IL-2 to CD3⁺CD8⁺CD44^high activated T cells and CD3⁺CD4⁺Foxp3⁺ Treg cells in the mouse spleen. **p<0.01. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; MFI, mean fluorescence intensity.

Figure 2

Radiochemistry of ^99mTc-sum IL-2. (A) Radiolabeling procedure for ^99mTc-sum IL-2. (B) ITLC chromatograms of the ^99mTc-sum IL-2 before and after purification. ITLC, instant thin layer chromatography.

Figure 3

^99mTc-sum IL-2 specifically detects T cells in vivo. (A) Representative small animal SPECT/CT images obtained after injection of ^99mTc-sum IL-2 in MC38 subcutaneous tumor-bearing mice without or with blocking doses of sum IL-2 protein. (White dashed circle indicates the tumor) (B) Biodistribution of ^99mTc-sum IL-2 in MC38 tumor-bearing mice, as well as a blocking study performed by co-injecting ^99mTc-sum IL-2 and cold sum IL-2 protein (n=4/group). **p<0.01, ****p<0.001. (C) Representative flow cytometry analysis of the MC38 whole tumor, CD8⁺ and CD4⁺ T cell fractions. (D) ^99mTc-sum IL-2 avidity in designated cell fractions from MC38 tumors (n=4/group). All the experiments were carried out twice. SPECT, single photon emission computed tomography; FSC-H, Forward Scatter Hight; TdLN, tumor-draining lymph node.

Figure 4

Comparison ^99mTc-sum IL-2 and ^99mTc-IL-2. (A) Representative small animal SPECT/CT images obtained at 0.5 hour after injection of ^99mTc-sum IL-2 or ^99mTc-IL-2 in MC38 subcutaneous tumor-bearing mice (white dashed circles indicate tumors). (B) Biodistribution of ^99mTc-sum IL-2 or ^99mTc-IL-2 in MC38 tumor-bearing mice at 0.5 h p.i. TdLN (n=4/group). All the experiments were carried out twice. *p<0.05, **p<0.01. SPECT, single photon emission computed tomography; TdLN, tumor-draining lymph node.

Figure 5

^99mTc-sum IL-2 SPECT/CT imaging and biodistribution of MC38 tumor-bearing mice receiving αPD-L1 therapy. (A) C57BL/6 mice bearing s.c. MC38-tumors were treated with αPD-L1 therapy three times (day 9, 13 and 17) and ^99mTc-sum IL-2 SPECT/CT imaging and biodistribution were acquired before treatment (day 8) and after second αPD-L1 treatment (day14), flow cytometry performed at day 14. (B) SPECT/CT imaging in MC38 models before and after αPD-L1 treatment. (White dashed circles indicate tumors) (C) Biodistribution of ^99mTc-sum IL-2 in αPD-L1-treated or untreated mice at 0.5 h p.i. (n=4/group). **p<0.01. (D) Flow cytometry analysis of tumors was used to determine the number of CD45⁺CD3⁺ T cells in the MC38 tumors in αPD-L1-treated and untreated mice (n=4/group). *p<0.05. (E) Tumor growth curves (mean±SD) (n=6-8/group) and (F) Kaplan-Meier survival curves (n=6-8/group). All the experiments were carried out twice. SPECT, single photon emission computed tomography; Ctrl, control; p.i, post-injection; s.c., subcutaneously; TdLN, tumor-draining lymph node.

Figure 6

^99mTc-sum IL-2 SPECT/CT imaging and biodistribution in the OT-I adoptive T cell therapy MC38 tumor model. (A) C57BL/6 mice bearing s.c. MC38-OVA/MC38 tumors were treated with OT-I ACT and ^99mTc-sum IL-2 SPECT/CT imaging and biodistribution were acquired before and after ACT. MC38 and MC38-OVA tumor cells were subcutaneously injected in the right and left flank of BABL/c nu/nu mice. One day after adoptive OT-I T cell transfer, OVA protein was intraperitoneally injected in BABL/c nu/nu mice. (B) SPECT/CT images at 0.5 hour after injection of ^99mTc-sum IL-2 in BABL/c nu/nu mice bearing MC38-OVA and MC38 tumors before transfer and 2 days after adoptive OT-I T cell transfer. (Brown dashed circles and arrows indicate MC38-OVA tumors; white dashed circles and arrows indicate MC38 tumors) (C) Biodistribution of ^99mTc-sum IL-2 in control and T cell transferred mice bearing MC38-OVA and MC38 tumors at 0.5 h p.i. (n=4/group). (D) Comparative analysis of ^99mTc-sum IL-2 uptake in tumor and TdLN before and after ACT. *p<0.05, **p<0.01, ***p<0.001. SPECT, single photon emission computed tomography; ACT, adoptive T cell transfer; OVA, ovalbumin; s.c., subcutaneously; p.i., post-injection; TdLN, tumor-draining lymph node.