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Targeted α-therapy (TAT) delivers high-linear-transfer-energy α-particles to tumors with the potential to generate tumor immune responses that may be augmented by antigen-targeted immunotherapy. Methods: This concept was evaluated in immunocompetent carcinoembryonic antigen (CEA) transgenic mice bearing CEA-positive mammary or colon tumors. Tumors were targeted with humanized anti-CEA antibody M5A labeled with 225 Ac for its 10-d half-life and emission of 4 α-particles, as well as being targeted with the immunocytokine M5A-interleukin 2. Results: A dose response (3.7, 7.4, and 11.1 kBq) to TAT only, for orthotopic CEA-positive mammary tumors, was observed, with a tumor growth delay of 30 d and an increase in median survival from 20 to 36 d at the highest dose. Immunocytokine (4 times daily) monotherapy gave a tumor growth delay of 20 d that was not improved by addition of 7.4 kBq of TAT 5 d after the start of immunocytokine. However, TAT (7.4 kBq) followed by immunocytokine 10 d later led to a tumor growth delay of 38 d, with an increase in median survival to 45 d. Similar results were seen for TAT followed by immunocytokine at 5 versus 10 d. When a similar study was performed with subcutaneously implanted CEA-positive MC38 colon tumors, TAT (7.4 kBq) monotherapy gave an increase in median survival from 29 to 42 d. The addition of immunocytokine 10 d after 7.4 kBq of TAT increased median survival to 57 d. Immunophenotyping showed increased tumor-infiltrating interferon-γ-positive, CD8-positive T cells and an increased ratio of these cells to Foxp3-positive, CD4-positive regulatory T cells with sequential therapy. Immunohistochemistry confirmed there was an increase in tumor-infiltrating CD8-positive T cells in the sequential therapy group, strongly suggesting that immunocytokine augmented TAT can lead to an immune response that improves tumor therapy. Conclusion: Low-dose (7.4 kBq) TAT followed by a 4-dose immunocytokine regimen 5 or 10 d later gave superior tumor reductions and survival curves compared with either monotherapy in breast and colon cancer tumor models. Reversing the order of therapy to immunocytokine followed by TAT 5 d later was equivalent to either monotherapy in the breast cancer model., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Lipid nanodiscs (LNDs), comprising a phospholipid bilayer encircled by two molecules of a recombinant membrane scaffold protein, can be targeted to tumors with covalently attached antibodies (Abs) or their fragments. Antibody attachment to click chemistry based PEGylated lipids on LNDs including DOTA allowed PET imaging with the positron emitter 64 Cu. Carcinoembryonic antigen (CEA) positive tumors in CEA transgenic mice were chosen as a tumor target. Fab' fragments, that otherwise are rapidly cleared by the kidney due to their small size, were retained in circulation when conjugated to LNDs. Untargeted PET imaging of 64 Cu-DOTA-LNDs revealed low tumor uptake (4-5% ID/g) in the range expected for the enhanced permeability retention (EPR) effect with high liver uptake (17-21% ID/g) indicating gut clearance. Fab' targeted LNDs showed little improvement over untargeted LNDs, but intact IgG targeted LNDs gave high tumor uptake (40% ID/g) with low liver (8% ID/g), demonstrating that tumor targeting with antibody conjugated LNDs is feasible.

Real-time intraoperative image-guided cancer surgery promises to improve oncologic outcomes. Tumor-specific antibodies conjugated with near-infrared (NIR) fluorophores have demonstrated the potential to enhance visualization of solid tumor margins and metastatic disease; however, multiple challenges remain, including improvement in probe development for clinical utility. We have developed an NIR-IR800 dye on a PEGylated linker (sidewinder) conjugated to the humanized anti-carcinoembryonic antigen (CEA) antibody (M5A) with extended in vivo serum and tumor persistence. The anti-CEA M5A-sidewinder has a high dye-to-antibody ratio (average of 7 per antibody) that allows, in an orthotopic implanted human pancreatic cancer mouse model increased tumor fluorescence, higher tumor-to-background ratio and extends the surgical scheduling window compared to current antibody dye conjugates. These preclinical results demonstrate the potential of this probe for fluorescence-guided surgery of CEA-positive gastrointestinal cancers.

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.