Development of [⁸⁹Zr]Zr-hCD103.Fab01A and [⁶⁸Ga]Ga-hCD103.Fab01A for PET imaging to noninvasively assess cancer reactive T cell infiltration: Fab-based CD103 immunoPET

Cancer immunotherapy has revolutionized the field of immunology and oncology. At its core, immunotherapy relies on generating or augmenting adaptive immune responses against antigens preferentially or selectively expressed in cancer cells [1]. This is best exemplified by monoclonal antibody therapies that block the immune checkpoint programmed death-1 (PD-1) or its ligand (PD-L1), and their exquisite activity in patients with a high mutation burden [2]. Nevertheless, the majority of patients do not respond to immune checkpoint therapy and early response biomarkers that can guide treatment decision making are urgently needed.

One of the indicators of successful immunotherapy across tumor types is an increase in the number of tumor-infiltrating lymphocytes (TILs) [3]. TILs in tumor lesions are correlated with greater immunological responses; therefore, they represent attractive biomarkers for monitoring immunotherapy. However, not every T cell within a tumor is involved in the anti-cancer immune response [4]. In recent years, we and others have refined the definition of cancer-reactive TILs [5]. Across different phenotypes of T-lymphocytes, tissue-resident memory-like cells (T_RM-like cell) have emerged as a subset with a prognostic value across different malignancies. These noncirculating memory T cells (T_RM-like cells) are linked to an integrated immune response and a favorable outcome of immune checkpoint blockade therapy in patients [6‐8]. A primary cell surface marker of CD8+ T_RM-like cells is the αE subunit of the αEβ7 integrin complex, commonly known as CD103. This integrin mediates retention of lymphocytes in peripheral tissues by binding to E-cadherin expressed on epithelial cells [8]. Recent work has shown that in melanoma, lung and esophageal cancer patients, the number of CD103+ TILs was significantly increased during immunotherapy in responding lesions in comparison with lesions of treatment-naive patients and nonresponders [9, 10]. Thus, CD103 is an interesting biomarker for the assessment of cancer reactive T cell infiltration.

The current standard for determination of CD103+ cell infiltration is immunohistochemistry staining (IHC) on tissue biopsies. However, biopsies have several disadvantages. Among those shortcomings are tumor heterogeneity within and between lesions, poor accessibility of lesions, and sampling errors. In order to obtain accurate information about CD103+ T_RM load in all tumor lesions, noninvasive whole-body imaging techniques can be applied. The most suitable technique in the clinical setting is positron emission tomography (PET), a diagnostic tool for functional/molecular imaging. PET offers high sensitivity and fast imaging time and requires small amounts of molecular probe to be used in comparison with other imaging techniques [11]. Moreover, PET allows for repetitive and noninvasive clinical assessment. The possibility of imaging signal quantification is ideally suited for determining whole-body T_RM load using radiolabeled antibody fragments (Fabs). For monoclonal antibodies, zirconium-89 (⁸⁹Zr; t_1/2 = 78.4 h) is a suitable isotope for radiolabeling, as its physical half-life matches the time mAbs which require for achieving optimal target-to-background signals. In the case of antibody fragments, radioisotopes with a shorter half-life could also be applicable, given that Fabs are smaller in size and thus have a faster biodistribution [12]. An alternative radioisotope that was successfully used in the past for antibody-based tracer labeling is gallium-68 (⁶⁸Ga; t_1/2 = 67.7 min) [13]. Recently, a first-in-human PET imaging study with ⁸⁹Zr-labeled atezolizumab (anti-PD-L1) showed dependency of tracer uptake in response to atezolizumab treatment [14]. Noninvasive PET imaging of T cells has also been described in mouse models and clinical trials using markers such as CD3 and CD8 [15, 16]. Here, we generated [⁸⁹Zr]Zr-hCD103.Fab01A and [⁶⁸Ga]Ga-hCD103. Fab01A tracers that specifically recognize human CD103 for noninvasive immuno-PET imaging of T cell infiltration in human cancers as a potential biomarker for effective anti-cancer immune responses.

To date, PET imaging of T cells has been described in preclinical models targeting T cell surface markers CD3, CD4 and CD8, and uptake was correlated with response to immunotherapy [15, 16, 18]. The most potent effectors of the antitumor immune response are CD8+ cytotoxic T cells. Therefore, CD8+ T cell imaging is currently considered to be the most promising tool for the early identification of immune surveillance function [18]. In clinical trials, anti-CD8 imaging agents are under investigation examining patients before, during and after treatment with checkpoint inhibitors (NCT04029181, NCT03802123). However, the presence of CD8+ TILs in tumor tissue does not mean that these TILs are functional. A prominent feature of immune escape is T cell depletion. Instead of CD8, CD103⁺ resident-like tumor-infiltrating T cells have been reported as a subtype of tissue-resident memory T cells (T_RM) that have been found in the tumor microenvironment [19, 20] and have been shown to have a prognostic effect across multiple types of solid cancers, including cervical cancer [21], head & neck squamous cell carcinoma [22], lung and bladder cancer [23], cholangiocarcinoma [24], gastric cancer [25], ovarian cancer [26], esophageal squamous cell carcinoma [27], colorectal cancer [28], and melanoma [20]. Furthermore, when we compared the preclinical performance of the [⁸⁹Zr]Zr-hCD103.Fab01A tracer with that of the [⁸⁹Zr]Zr-DFO-CD8a F(ab)'2 tracer[29] in syngeneic mice, both tracers showed a comparable uptake in the xenografts at the selected scan time point ([⁸⁹Zr]Zr-hCD103.Fab01A vs [⁸⁹Zr]Zr-DFO-CD8a F(ab)’2, mean %ID/g: 2.28 vs 2.44, mean xenograft/tumor-to-blood ratio 13.33 vs 14.69). As such, CD103+ TRM cells represent an interesting biomarker for evaluating responses to immune checkpoint inhibitors and potentially discriminate responding vs. nonresponding patients, by comparing pre- and on-treatment densities of CD103+ TRM. Herein, the use of a noninvasive imaging modality such as PET offers significant advantages over classical biopsy-based approaches that are hampered by the requirement for accessible lesions and tumor heterogeneity.

The CD103+ TRM imaging strategy proposed here will have applications beyond those described for cancer. CD103+ TRM cells are implicated in a number of auto-immune diseases such as inflammatory bowel disease and multiple sclerosis. CD103+ TRM cells have also been shown to play a significant role in the rejection of transplanted organs, and strategies that deplete CD103+ cells have shown therapeutic success in preclinical models of these disease indications. Like proposed for cancer, CD103 imaging may help diagnose patients with diseases mediated by CD103+ TRM, which are frequently difficult to assess through biopsy-based methods. CD103 imaging may also help monitor therapy responses, discriminate responders from nonresponders and help guide treatment decision making in these indications. Finally, CD103 is expressed on a series of leukemic cancers, such as hairy cell leukemia [30] and may be used for diagnosis and monitoring of disease progression in response to therapy.

Recent preclinical studies have demonstrated that the use of mAbs for T cell imaging can impair T cell function, despite being administered at low doses [14, 31]. These functional effects of mAbs are likely due to their bivalent nature and interaction with specific Fc receptors. As a result of these interactions, full-length mAbs can trigger antigen crosslinking and cell- or complement-mediated effector functions. For mAbs used therapeutically in parallel to imaging, these considerations are mute. However, the requirement for parallel use of antibodies as therapeutic and imaging agents limits the potential for noninvasive imaging of clinically relevant immune subsets that do not directly represent a therapeutic target, such as CD103+ TRM cells. Here, we demonstrate that Fab derivation of a previously developed anti-CD103 imaging antibody could be used to side-step these issues while maintaining the previously described high target-to-background ratios, high target site selectivity and a high sensitivity of this mAb. While highly promising, care should be taken with regards to the generalization of our observations as the mAbs should possess sufficient single arm binding affinity to allow derivation as a Fab without compromising binding activity. Although not discussed extensively here, several of the previously developed anti-CD103 mAbs failed to exert sufficient binding affinity to allow use as Fabs as imaging agents [17].

Due to the relatively large molecular size of the full-length mAbs, their serum half-life is often more than ten days. Therefore mAbs need to be labeled by radionuclides with a longer half-life, such as ⁸⁹Zr (t_1/2 = 78.4 h) [32]. Fabs, as a fragment of the mAb, have a relatively short serum half-life of 12–20 h compared to full-length mAbs [33], which implies rapid clearance from the blood and nonspecific compartments. With its shorter serum half-life, Fabs could potentially be labeled with the shorter-lived radionuclides. However, uptake of the ⁶⁸Ga-labeled CD103 Fab tracer, was not statistically significant and a considerable amount of background was present. In further studies, other isotopes such as ⁶⁴Cu (t_1/2 = 12.7 h) or ¹⁸F (t_1/2 = 110 min) may be more suitable for Fab imaging. Labeling with ¹⁸F could be highly interesting due to usage of aluminium-[¹⁸F] fluoride with the RESCA chelator. As previously demonstrated [34], RESCA allows for the straightforward radiolabeling of proteins in mild conditions, crucial for Fabs.

In conclusion, we developed a potent high-affinity Fab tracer to image CD103+ TRM cells as a novel tool in monitoring of immunotherapy. However, optimization of the half-life of the applied radionuclide is required.

This study establishes proof-of-concept for the noninvasive assessment of tumor-resident memory T cells (TRM) through Fab-based CD103 PET imaging. In vitro, we leveraged a previously developed high-affinity anti-CD103 mAb clone to generate a Fab with high single-arm binding affinity. Next, we compared [⁶⁸Ga]Ga- and [⁸⁹Zr]Zr-labeled Fabs for anti-CD103 immuno-PET and demonstrated that this approach can be used to visualize CD103-positive cells with high target-to-background ratios, high target site selectivity and a high sensitivity in hCD103-positive xenografts.