Introduction
The epidermal growth factor receptor (EGFR) has evolved over the years into a main molecular target for the treatment of different cancer entities. EGFR is a glycosylated transmembrane protein involved in regulating cell growth, differentiation and survival of malignant cells [
1]. This receptor is often overexpressed in various malignancies such as head and neck squamous cell carcinoma (HNSCC), gastrointestinal/abdominal carcinoma, lung and reproductive tract carcinomas, melanomas, glioblastomas and thyroid carcinoma [
2]. Despite some controversies, the overexpression of EGFR is often associated with a poor clinical prognosis and resistance to radiation therapy [
3,
4]. Therefore, therapeutic strategies involving monoclonal antibodies against EGFR, such as cetuximab, have been used pre-clinically and clinically. Cetuximab is a chimeric monoclonal antibody, which binds specifically with a high affinity to the extracellular domain of the EGFR. Cetuximab is used alone or in combination and its therapeutic indications are (i) colorectal and HNSCC and (ii) HNSCC with external radiotherapy [
5]. While the combination of cetuximab with radiotherapy showed improved survival [
5], tumor response remains heterogeneous and cetuximab failed to show benefit over chemoradiotherapy [
6,
7]. Hence, radioimmunotherapy based on cetuximab labeled with therapeutic radionuclides appears as a promising strategy allowing the delivery of radiation dose specifically to tumor cells expressing high level of EGFR while sparing normal tissues. In this regard, theranostic strategy based on monoclonal antibodies and their deriving structures could represent an exciting approach. This strategy relies on the combination of detection and treatment in one integrated approach [
8]. Indeed, it is possible to convert a purely imaging probe into a single-entity theranostic agent by simply switching the radionuclide from a γ-emitter (e.g.:
111In) to a β-emitter (e.g.:
90Y) or using a radionuclide gathering both imaging and therapeutic properties (e.g.:
177Lu). We assume that such agents should be safer, more efficient and innovative through their capabilities in sequentially or simultaneously diagnosing, monitoring, and treating disease. The other major theranostic approach relies on the use of a molecular probe to detect and follow-up a given biomarker during a pharmacotherapy, which is, for example, commonly performed in clinical routine with
18F-FDG and PET in oncology [
8]. The relevance of using cetuximab radiolabeled with
111In in diagnostic imaging has been demonstrated in several animal models mainly due to its high tumor targeting property and its adequate half-life [
9‐
12].
177Lu is a theranostic radionuclide of choice to target small tumors or metastatic deposits due to the medium energy of the emitted beta particles and their tissue penetration of 1.5 mm. In addition,
177Lu emits γ-rays which allow diagnostic imaging. The therapeutic efficacy of cetuximab radiolabeled with
177Lu has been demonstrated on various tumor types [
13‐
16].
A major concern with the use of a β-emitter such as
177Lu as radionuclide is the selection of a chelating agent that forms a sufficiently stable complex to prevent in vivo loss of the radiometal. This choice is crucial to avoid toxicity induced by the delivery of undesired amount of radiation mainly to the radio sensitive bone marrow. Several bifunctional chelating agents (BFCA) have been used for the radiolabeling of cetuximab including DTPA and DOTA derivatives. Several reports highlight the higher in vitro and in vivo stability of DOTA conjugates to sequester
111In,
177Lu but also
90Y, resulting in lower bone incorporation and marrow toxicity [
14,
17‐
21]. Our lab previously made the proof of concept that trastuzumab and cetuximab radiolabeled with
111In through our new bifunctional chelating agent DOTAGA anhydride [
22], were able to bind to their target both in vitro and in vivo [
23]. Nevertheless, full-sized antibodies (about 150 kDa) need to overcome some obstacles before penetrating into a tumor. Indeed, tumor penetration can be hampered by some physiological barriers, such as high interstitial pressure and a “binding site barrier” [
24,
25]. To circumvent these drawbacks, it is possible to generate fragments [Fab and F(ab′)
2] of the monoclonal antibody to improve its penetration within the tumor. These fragments can be prepared through enzymatic cleavages [papain for Fab and pepsin for F(ab′)
2] or by genetic engineering. Given their pharmacokinetic properties, F(ab′)
2 (110 kDa) could be valuable theranostic agents. Indeed, they are bivalent as the native monoclonal antibody but they are less immunogenic, show a shorter blood clearance, higher tumor-to-background ratios and reduced non-specific distribution [
26].
In the present work, our aims were to design F(ab′)
2-cetuximab-based theranostic agents with both diagnostic and therapeutic capabilities and to assess them in murine preclinical cancer models. First, we characterized in vitro the F(ab′)
2 fragment of cetuximab radiolabeled with
111Indium (
111In-DOTAGA-F(ab′)
2-cetuximab) in comparison with whole cetuximab used as a reference (
111In-DOTAGA-cetuximab). Then, we evaluated the stability of DTPA- and DOTAGA-radiolabeled F(ab′)
2-cetuximab. We made the proof of concept that
111In-DOTAGA-F(ab′)
2-cetuximab was suitable for monitoring the down-regulation of EGFR as a biomarker of the efficacy of a targeted therapy with a HSP90 inhibitor. In cancer cells, the stability of EGFR is promoted by the chaperon protein HSP90 [
27]. The chemical inhibition of HSP90 leads to an increase in EGFR degradation hampering oncologic signaling pathways [
27]. Finally, we designed a strategy based on radioisotopic switching to perform radioimmunoscintigraphy (
111In-DOTAGA-F(ab′)
2-cetuximab) prior to radioimmunotherapy (
177Lu-DOTAGA-F(ab′)
2-cetuximab) in colorectal tumor-bearing mice.
177Lu-DOTAGA-F(ab′)
2-cetuximab showed a good tolerance and efficacy for reducing tumor volume in our model.
Discussion
Colorectal cancer is nowadays the third most commonly diagnosed and the fourth leading cause of cancer-related deaths worldwide [
29‐
31]. Current treatment options offer a limited benefit on survival of patients with stage IV colorectal cancer with a 5 years survival of less than 10%, highlighting the urgent need for innovative therapeutic strategies [
31]. Theranostic approaches based on radiolabeled antibodies allow the selective targeting of tumor cell antigens for diagnostic, evaluation of therapy efficacy and delivery of ionizing radiation to tumor sites depending on the chosen radionuclide. The upregulation of EGFR has been reported in colorectal cancer and is associated with poor prognosis and resistance to radiation therapy. Thus, EGFR is nowadays an important target for therapy with anti-EGFR antibodies [
32]. Cetuximab is a monoclonal antibody approved for treatment of colorectal cancer which selectively binds EGFR to prevent the binding of natural EGFR-ligands and promote antibody-receptor complex internalisation [
33]. However, the pharmacokinetics of whole cetuximab is slow, due to its size, and ranges from 63 to 230 h in patients [
34]. This slow pharmacokinetics is not optimal for rapid and dynamic EGFR imaging. Moreover, the extended presence of whole cetuximab in the blood often induces misinterpretation of the amount of EGFR. In mice the biological half-life of F(ab′)
2 fragments of cetuximab is six times shorter than that of whole cetuximab (12 and 70 h, respectively [
35,
36]). Thus, we designed for the current study F(ab′)
2-cetuximab radiolabeled with
111Indium (
111In-F(ab′)
2-cetuximab) which overcomes issues raised with whole cetuximab by displaying fast blood clearance, rapid tumor accumulation and enables earlier molecular imaging [
11,
37]. Our results are in accordance with previous studies which report a peak in tumor uptake of F(ab′)
2-cetuximab at 24 h post injection [
36,
37]. Moreover, the immunoreactivity and affinity of
111In-DOTAGA-F(ab′)
2-cetuximab and
111In-DOTAGA-cetuximab found in the current study are also in accordance with the literature. Indeed, Van Dijk et al. [
37] reported an immunoreactivity of F(ab′)
2-cetuximab of 58% compared to 50% for
111In-DOTAGA-F(ab′)
2-cetuximab and
111In-DOTAGA-cetuximab in our study. These results highlight the fact that immunoreactivity is not disturbed by DOTAGA incorporation and radiolabeling. In addition, the affinity of native cetuximab for HER1 has been reported to range from 0.62 to 1.7 nM [
38,
39]. In our study,
111In-DOTAGA-F(ab′)
2-cetuximab and
111In-DOTAGA-cetuximab displayed an affinity of 0.9 and 1.7 nM, respectively, thus demonstrating that F(ab′)
2 fragments of cetuximab retain their affinity for HER1 after DOTAGA incorporation and radiolabeling. Therefore, F(ab′)
2-cetuximab represents a tool of interest for theranostic application by retaining high affinity for HER1 and better pharmacokinetic properties than whole cetuximab for imaging/quantification of EGFR.
Another crucial issue in designing a radioimmunotherapeutic tool for theranostic use is the stability of the radionuclide in vitro and in vivo. Hence, the choice of the bifunctional chelating agent (BFC) used for radiolabeling is essential to minimize toxicity of the radionuclide to normal tissues. DTPA derivatives remain the most commonly used chelators for cetuximab radiolabeling in preclinical studies [
9,
10] and in human [
40,
41]. However, macrocyclic chelators such as DOTA derivatives have been shown to form more stable antibody-conjugates in vitro and in vivo for a wide range of radionuclides including
111In [
20],
177Lu [
14] and
90Y [
18,
19]. Importantly, Camera et al. [
19] reported a lower bone uptake of
90Y-DOTA conjugates over
90Y-DTPA suggesting a better in vivo stability of DOTA conjugates limiting
90Y toxicity. Similarly, the in vitro stability of
177Lu-DOTA conjugates over
177Lu-DTPA has been clearly demonstrated. Whether DOTA is also superior in vivo for
177Lu, it remains controversial. Milenic et al. [
21] observed no significant differences between DOTA and DTPA conjugates, suggesting similar in vivo stabilities. On the contrary, Brouwers et al. [
14] showed that uptake of
177Lu-DTPA was slightly higher in most tissues, including bone, and markedly higher in liver and spleen compared with
177Lu-DOTA suggesting a difference in in vivo stability between these two conjugates. In addition, a less complicated labeling procedure and an improved labeling efficiency often leads to a preference for DTPA as the chelator for radiolabeling with
177Lu for radioimmunotherapy applications [
21]. Indeed, the use of DOTA derivatives induces slower complex formation rates which can limit radiolabeling yields and efficiency. In addition, radiolabeling conditions to perform complexation often require extensive timeframe and high temperatures which are not acceptable for protein conjugates [
42]. Nevertheless, our group developed and characterized in a previous study a DOTAGA-anhydride chelator as a powerful tool for bioconjugation [
23]. DOTAGA is a DOTA derivative that leaves four acetate pendant arms intact and can be easily synthesized in good yield. Moreover, in the current study effective conjugation of DOTAGA-anhydride to cetuximab and F(ab′)
2-cetuximab was achieved in conditions suitable for protein conjugates. In addition, DOTAGA conjugation did not disrupt cetuximab and F(ab′)
2-cetuximab immunoreactivity/affinity and showed greater stability than corresponding DTPA conjugates as previously described for trastuzumab [
23]. Moreover,
177Lu-DOTAGA complex has recently been shown to be highly stable in preclinical model of prostate cancer [
43] and in patients showing high efficacy and low toxicity [
44,
45]. BM toxicity, including long-term haematological toxicities, has been reported in 11% of patients with metastatic neuroendocrine tumours treated with
177Lu-DOTA-Tyr3-octreotate (
177Lu-DOTATATE, [
46]). In addition, 40% of patients with prostate cancer receiving
177Lu-DKFZ-617 showed hematotoxicity in a small cohort of 10 patients [
47]. Interestingly, Baum et al. [
44] reported no hematotoxicity of
177Lu-DOTAGA-PSMA complex in patients with prostate cancer with no worsening of anemia and leukocytopenia after therapy and no grade three or four hematologic toxicity in any of the patients. Thus, DOTAGA-anhydride chelator appears as a suitable tool for bioconjugation enabling a strong sequestration of radionuclides preventing their toxicity in vivo.
In the current study, we demonstrate that
111In-F(ab′)
2-cetuximab specifically target colorectal tumors expressing HER1. Our results are comparable to what was found in previous publication mainly on head and neck cancer [
11,
36,
37]. Surprisingly, the tumor targeting of
111In-F(ab′)
2-cetuximab in colorectal tumors remains not well documented. Van Dijk et al. [
11,
37] demonstrated in a murine model of head and neck squamous cell carcinoma that
111In-F(ab′)
2-cetuximab showed good tumor-to-background contrast on microSPECT imaging, allowing noninvasive assessment of EGFR expression in vivo. The same group also established that
111In-F(ab′)
2-cetuximab was able to monitor the effects of EGFR inhibition or irradiation much better than
18F-FDG PET confirming the added value of
111In-F(ab′)
2-cetuximab to follow treatment efficacy [
48]. In our study,
111In-DOTAGA-F(ab′)
2-cetuximab was used as a diagnostic tool for colorectal cancer to follow the efficacy of a HER1 targeted therapy by the HSP90 inhibitor, 17-DMAG. HSP90 is a chaperon protein that has been demonstrated to bind and stabilize HER1 and whose inhibition causes a decrease in EGFR in cancer cells [
27]. In addition to reducing membrane expression of EGFR, HSP90 inhibition significantly prevented tumor growth in head and neck squamous cell carcinoma animal models [
27]. Interestingly, Spiegelberg et al. [
49] recently showed by PET imaging with
124I-labeled cetuximab that HSP90 inhibition induced a decreased in EGFR expression in head and neck squamous cell carcinoma. Our results confirm, in a colorectal cancer model, that SPECT imaging of
111In-F(ab′)
2-cetuximab is able to monitor EGFR downregulation and the prevention of tumor growth caused by HSP90 inhibition in vivo. Indeed, HSP90 inhibition induced a decrease in tumor uptake as well as a decrease in tumor growth compared to control mice. Interestingly, we demonstrate here that tumor volume physically measured with a clipper strongly correlated with tumor volumes assessed directly by SPECT imaging of
111In-F(ab′)
2-cetuximab further validating the accuracy of imaging to follow tumor growth upon targeted therapy. Thus, tumor uptakes can be accurately expressed in regards to tumor volumes assessed directly by SPECT imaging in % ID/mm
3. However, a statistical difference between 17-DMAG and control group was found from D44 when tumor volumes were manually measured, while significance was reached only from D51 when tumor volumes were measured by SPECT imaging. Due to its specificity, imaging should be more accurate than manual measurements but in our experiments only four mice per group were imaged while ten mice per group were used for manual measurements. This difference in the number of animal can explain the discrepancy between SPECT imaging and manual measurements in the time to reach significance.
One of the advantages of the
111In-F(ab′)
2-cetuximab probe is the possibility to perform a radionuclide switch from
111In to
177Lu to obtain a therapeutic tool
177Lu-F(ab′)
2-cetuximab. β
− particles emitted by
177Lu have a medium energy and a subsequent tissue penetration of 1.5 mm supporting its therapeutic use favored in treatment of small tumors while limiting irradiation of normal tissue [
50]. Another asset of
177Lu, in comparison to other therapeutic radionuclide such as
90Y, is the low energy γ-emission that allows imaging. The combination of imaging and therapeutic properties makes
177Lu a theranostic tool of choice increasingly used in preclinical and clinical studies [
15,
44,
51,
52]. We demonstrate here that radioimmunotherapy with
177Lu-F(ab′)
2-cetuximab significantly inhibited colorectal tumor growth when 4 and 8 MBq are injected, but not significantly at 2 MBq. Our results confirm a previous study performed by Song et al. [
16] with the whole form of cetuximab in a model of esophageal cancer. As mentioned above
177Lu induces myelotoxicity. Interestingly, even if we did not evaluate hematologic toxicity, we noticed weight loss in mice receiving 4 MBq 6 days after injection, suggesting toxicity. Our results are in accordance with the findings of Fischer et al. [
51] who evaluated experimentally the maximal tolerated dose of
177Lu in nude mice bearing human ovarian cancer around 12 MBq. Nevertheless, even if 4 MBq seemed to show early signs of toxicity (with no effect on survival), other doses (2 and 8 MBq) did not induce toxicity and showed efficacy in reducing tumor growth. Thus, a dose of 2 MBq could be sufficient to achieve significant efficacy on tumor growth prevention without toxicity. The low energy and a low tissue penetration of
177Lu favored its use for treatment in metastatic cancer. This could be of great interest in the treatment of peritoneal carcinomatosis of colorectal cancer origin in which EGFR upregulation has been demonstrated to be a factor of poor prognosis [
53]. Cetuximab is already widely used for metastasized colorectal cancer but no clinical data on its specific use in peritoneal carcinomatosis is nowadays available [
53].
177Lu-F(ab′)
2-cetuximab could allow peritoneal carcinomatosis early diagnosis by localizing metastasis formation and could also represent an important therapeutic strategy by specifically delivering ionizing radiation during early metastasis invasion.