{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T10:56:43Z","timestamp":1772535403580,"version":"3.50.1"},"reference-count":22,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T00:00:00Z","timestamp":1763424000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100013140","name":"COLORECTAL CANCER ALLIANCE, USA","doi-asserted-by":"publisher","award":["10065978"],"award-info":[{"award-number":["10065978"]}],"id":[{"id":"10.13039\/100013140","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>CCR8 chemokine receptor is a selective marker of tumor-infiltrating regulatory T cells (ti-Tregs) which interfere with the efficacy of checkpoint inhibitor immunotherapy (ICI) in many types of cancer. Eliminating CCR8+ ti-Tregs dramatically improves the results of subsequent ICI. We have recently reported using 225Actinium-labeled anti-CCR8 IgG for killing CCR8+ ti-Tregs in murine colorectal tumors which synergized with subsequent anti-CTLA4 ICI. Here, we aimed to compare the in vivo behavior of anti-CCR8 full-sized IgG and its Fab fragments to select the best antibody format for the pre-clinical development of this combination modality. Anti-CCR8 Fab fragments were generated by papain digest of the whole IgG. The whole IgG and Fab were conjugated to bifunctional chelating agent DOTA and labeled with 111Indium (111In). MC8 and CT6 murine colorectal tumor-bearing C57Bl6 and Balb\/c mice, respectively, were administered 111In-DOTA-IgG or 111In-DOTA-Fab and imaged with microSPECT\/CT at 2\u201372 h post-injection. A biodistribution was performed after the last imaging time point. Both 111In-DOTA-IgG and 111In-DOTA-Fab demonstrated high tumor uptake in both MC38 and CT26 tumors, with 111In-DOTA-IgG uptake being significantly higher from the 24 h time point and onwards. 111In-DOTA-Fab also exhibited pronounced kidney uptake which persisted even at 72 h. The kidney clearance and retention of 111In-DOTA-Fab might represent a problem during therapy employing 225Actimium or other long-lived therapeutic radionuclides by potentially causing a dose-limiting kidney toxicity. This imaging\/biodistribution evaluation not only determined that full-size anti-CCR8 IgG is the optimal antibody format for pre-clinical development but also informed on the timing of immunotherapy administration in future radioimmunotherapy and immunotherapy combination studies.<\/jats:p>","DOI":"10.3390\/molecules30224445","type":"journal-article","created":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T12:33:04Z","timestamp":1763469184000},"page":"4445","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Comparative In Vitro and In Vivo Evaluation of Anti-CCR8 Full-Sized IgG and Its Fab Fragments in Murine Colorectal Cancer Models"],"prefix":"10.3390","volume":"30","author":[{"given":"Tongshuo","family":"Hu","sequence":"first","affiliation":[{"name":"College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rubin","family":"Jiao","sequence":"additional","affiliation":[{"name":"College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin J. H.","family":"Allen","sequence":"additional","affiliation":[{"name":"College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2910-9857","authenticated-orcid":false,"given":"Connor","family":"Frank","sequence":"additional","affiliation":[{"name":"College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9118-0806","authenticated-orcid":false,"given":"Mackenzie E.","family":"Malo","sequence":"additional","affiliation":[{"name":"College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ekaterina","family":"Dadachova","sequence":"additional","affiliation":[{"name":"College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1016\/j.cell.2008.05.009","article-title":"Regulatory T cells and immune tolerance","volume":"133","author":"Sakaguchi","year":"2008","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"942","DOI":"10.1038\/nm1093","article-title":"Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival","volume":"10","author":"Curiel","year":"2004","journal-title":"Nat. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2080","DOI":"10.1111\/cas.14069","article-title":"Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target?","volume":"110","author":"Ohue","year":"2019","journal-title":"Cancer Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"E10672","DOI":"10.1073\/pnas.1810580115","article-title":"Identification and validation of a tumor-infiltrating Treg transcriptional signature conserved across species and tumor types","volume":"115","author":"Magnuson","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1889","DOI":"10.1084\/jem.20130240","article-title":"Identification of human CCR8 as a CCL18 receptor","volume":"210","author":"Islam","year":"2013","journal-title":"J. Exp. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e001749","DOI":"10.1136\/jitc-2020-001749","article-title":"Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy","volume":"9","author":"Dombrecht","year":"2021","journal-title":"J. Immunother. Cancer"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1662216","DOI":"10.3389\/fimmu.2025.1662216","article-title":"225Actinium-armed antibody targeting CCR8+ regulatory T cells synergizes with immunotherapy to promote tumor rejection in syngeneic colorectal cancer models","volume":"16","author":"Frank","year":"2025","journal-title":"Front. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/nrc3925","article-title":"Radioimmunotherapy of human tumours","volume":"15","author":"Larson","year":"2015","journal-title":"Nat. Rev. Cancer"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.2967\/jnumed.110.075101","article-title":"Renal toxicity of radiolabeled peptides and antibody fragments: Mechanisms, impact on radionuclide therapy, and strategies for prevention","volume":"51","author":"Vegt","year":"2010","journal-title":"J. Nucl. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e220157","DOI":"10.1148\/rycan.220157","article-title":"A review of theranostics: Perspectives on emerging approaches and clinical advancements","volume":"5","author":"Burkett","year":"2023","journal-title":"Radiol. Imaging Cancer"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1021\/acs.molpharmaceut.4c01071","article-title":"Preclinical Comparison of [111In]In- and [225Ac]Ac-DOTA-Trastuzumab IgG, F(ab\u2019)2 and Fab for Theranostic SPECT\/CT Imaging and \u03b1-Particle Radioimmunotherapy of HER2-Positive Human Breast Cancer","volume":"22","author":"Kondo","year":"2025","journal-title":"Mol. Pharm."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2895","DOI":"10.4049\/jimmunol.131.6.2895","article-title":"On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB\/c mice","volume":"131","author":"Parham","year":"1983","journal-title":"J. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1021\/acs.bioconjchem.8b00912","article-title":"Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity","volume":"30","author":"Kinman","year":"2019","journal-title":"Bioconjug. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/S0022-1759(00)00135-6","article-title":"Papain digestion of different mouse IgG subclasses as studied by electrospray mass spectrometry","volume":"237","author":"Adamczyk","year":"2000","journal-title":"J. Immunol. Methods"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1080\/09553002.2025.2467691","article-title":"Comparison of radiobiological effects induced by radiolabeled antibodies in human cancer cells and fungal cells","volume":"101","author":"Allen","year":"2025","journal-title":"Int. J. Radiat. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1647","DOI":"10.4049\/jimmunol.2300751","article-title":"Antibody-Mediated Depletion of Autoreactive T Lymphocytes through PD-1 Improves Disease Outcomes and Visualizes T Cell Activation in Experimental Autoimmune Encephalomyelitis","volume":"212","author":"Frank","year":"2024","journal-title":"J. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1002\/cncr.22402","article-title":"Antibody constructs in cancer therapy: Protein engineering strategies to improve exposure in solid tumors","volume":"109","author":"Beckman","year":"2007","journal-title":"Cancer"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1007\/s002590050216","article-title":"Reducing the renal uptake of radiolabeled antibody fragments and peptides for diagnosis and therapy: Present status, future prospects and limitations","volume":"25","author":"Behr","year":"1998","journal-title":"Eur. J. Nucl. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"8600","DOI":"10.1021\/acs.jmedchem.3c00258","article-title":"Reduction of the Renal Radioactivity of 111In-DOTA-Labeled Antibody Fragments with a Linkage Cleaved by the Renal Brush Border Membrane Enzymes","volume":"66","author":"Suzuki","year":"2023","journal-title":"J. Med. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"806","DOI":"10.2967\/jnumed.124.268411","article-title":"Reduced Renal Uptake of Various Radiopharmaceuticals with Sodium Paraaminohippurate Coadministration in a Rat Model","volume":"66","author":"Meckel","year":"2025","journal-title":"J. Nucl. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"755","DOI":"10.2147\/ITT.S485643","article-title":"Combination of Radioligand Therapy and Immunotherapy: How to Make It Work in Clinic?","volume":"14","author":"Jiao","year":"2025","journal-title":"Immunotargets Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"50000","DOI":"10.1021\/acsomega.4c09450","article-title":"In Vitro and In Vivo Comparison of Random versus Site-Specific Conjugation of Bifunctional Chelating Agents to the CD33-Binding Antibody for Use in Alpha- and Beta-Radioimmunotherapy","volume":"9","author":"Allen","year":"2024","journal-title":"ACS Omega"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/22\/4445\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T12:38:34Z","timestamp":1763469514000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/22\/4445"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,18]]},"references-count":22,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["molecules30224445"],"URL":"https:\/\/doi.org\/10.3390\/molecules30224445","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,18]]}}}