Introduction:

The tumor-selective cell surface overexpression of Delta-like ligand 3 (DLL3) makes this an attractive feature for a variety of therapeutic strategies. [1] This protein is aberrantly overexpressed in neuroendocrine cancers like small-cell lung cancer (SCLC), which have a reputation for being highly aggressive and diagnosed at an advanced stage. [2] Though there are treatment options available for SCLC patients, most patients do not achieve long-term disease control due to the inevitable development of resistance and relapse following first-line chemotherapy and/or immunotherapy. [1] The anti-DLL3 construct [¹⁷⁷Lu]Lu-DTPA-CHX-A”-N12 has previously been developed in our lab, showing promising therapeutic efficacy. To further increase the therapeutic impact, the current focus includes utilizing other therapeutic radionuclides such as alpha emitters.

Targeted alpha therapy (TAT) is an increasingly popular strategy for therapeutics in nuclear medicine. Alpha-emitting radionuclides possess notable features such as high linear energy transfer and minuscule penetration depth, which translates to potent therapeutic efficacy and potentially minimizes damage to surrounding healthy tissues. [3,4] Of particular interest is the alpha-emitter, Actinium-225 (²²⁵Ac), which has a half-life of 9.92 days and a decay chain yielding four net alpha and two beta particles. These characteristics make ²²⁵Ac adequate for TAT, especially for targeting vectors with matched biological half-lives such as monoclonal antibodies (mAb). [3]

The purpose of this study was to utilize the anti-DLL3 N12 mAb to generate two ²²⁵Ac-labelled constructs. Here, macropa analogs, a macrocyclic chelator suited for ²²⁵Ac, were employed, allowing for short reaction times and mild radiolabeling conditions. [5,6] The two macropa-based chelators were either (1) directly conjugated utilizing Macropa-PEG₄-TFP or via (2) click reaction (inverse electron-demand Diels–Alder) with [²²⁵Ac]Ac-Macropa-PEG₈-Tz following previously reported methods. [7] Both constructs were evaluated for their ex vivo properties and in vivo biodistribution. 

Methods and Results:

For the direct approach, N12 was conjugated to Macropa-PEG₄-TFP and subsequently labeled with ²²⁵Ac (Fig. 1A). For the click method, N12 was first conjugated to transcyclooctene (TCO). Macropa-PEG₈-Tz was then radiolabeled with ²²⁵Ac and the TCO-modified N12 was added as the final step of the reaction (Fig. 1B). For both approaches, radiochemical purities of >98 % were achieved.

Biodistribution studies were performed for both radioimmunoconjugates in female nude mice at 24-, 72-, and 168 hrs. Tumoral uptake increased over time for [²²⁵Ac]Ac-Macropa-PEG₄-TFP-N12 with the final uptake of 64.3 ± 13.4 % ID/g at 168 hr post-injection. In contrast, the blood circulation time of this construct steadily decreased over time; from 18.3 ± 7.6 % ID/g at 24 hr post-injection to 6.2 ± 1.1 % ID/g at 168 hr post-injection. The ratio of tumor-to-liver uptake was estimated to be 3:1 at 72 hr post-injection of [²²⁵Ac]Ac-Macropa-PEG₄-TFP-N12 (Fig. 1C). The click-assembled N12, [²²⁵Ac]Ac-Macropa-PEG₈-Tz-TCO-N12, also demonstrated increasing tumoral uptake over time, however, it was significantly lower compared to the direct approach, which reached 10.6 ± 2.3 % ID/g at 168 hr post-injection. Liver uptake had the highest uptake of the non-tumor bearing organs for the [²²⁵Ac]Ac-Macropa-PEG₈-Tz-TCO-N12 with the value of 6.0 ± 1.9 % ID/g at 168 hr post-injection. (Fig. 1D).

Conclusions:

Macropa is a suitable chelator for ²²⁵Ac radiolabeling of N12 utilized for the treatment of DLL3-expressing SCLC. Of the two constructs explored, [²²⁵Ac]Ac-Macropa-PEG₄-TFP-N12 exhibited excellent biodistribution properties and will be further validated in therapeutic studies.

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Image/Figure Caption:

Figure 1. Chemical structures of (A) [²²⁵Ac]Ac-Macropa-PEG₄-TFP-N12 and (B) [²²⁵Ac]Ac-Macropa-PEG₈-Tz-TCO-N12. Biodistribution studies of (C) [²²⁵Ac]Ac-Macropa-PEG₄-TFP-N12 (0.04 Mbq, 52-60 μg protein) and (D) [²²⁵Ac]Ac-Macropa-PEG₈-Tz-TCO-N12 (0.02 Mbq, 52-60 μg protein) in female nude mice bearing subcutaneous H82 tumors. %ID/g, percentage of injected dose per gram.

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