C&CB Department – Stephanie Rathmann – Ph.D. Defence Seminar
Jul 16, 2020
1:30PM to 2:30PM
Date(s) - 16/07/2020
1:30 pm - 2:30 pm
Title: CombinationTargeted Radionuclide and Immune Recruitment Therapy
Date: Thursday, July 16, 2020
Time: 1:30 p.m.
Host: Dr. John Valliant
Antibody recruiting molecules(ARMs) are a unique class of immune- stimulatory compound that contain two keyregions: the target-binding terminus (TBT), which interacts with the site ofinterest, and the antibody-binding terminus (ABT), which recruits endogenous,hapten specific antibodies. Preclinical ARM studies have demonstrated reducedgrowth rates in a variety of cancers such as melanoma, prostate and breastusing small molecule or antibody targeting vectors. There is an opportunity toenhance the immune response caused by ARMs through the addition of aradionuclide that delivers cytotoxic radiation to the site of interest.Evidence suggests that a combination therapy of immune stimulation andradiation can lead to regression of not only primary tumours, but alsosimultaneous regression and control of distant metastases.
Rather than develop aradiolabelled ARM for each biomarker, a general platform was established withthree functional regions, a tetrazine for rapid ligation to a targeting vector,a 2,4-dinitrophenyl hapten for antibody recruiting, and a DOTA chelate to bindradiometals. The tetrazine was introduced in place of the TBT, permitting abio-orthogonal inverse electron demand Diels-Alder (IEDDA) reaction with a trans-cyclooctene (TCO) labelled ligand to be employed fortargeting. The chelate DOTA was chosen due to the wide range of compatible isotopesit can bind, including alpha, beta, gamma and positron emitters. Theantibody-recruiting hapten, 2,4-dinitrophenyl (DNP), was chosen to promoteantibody-dependent cellular cytotoxicity (ADCC) by recruiting endogenousanti-DNP antibodies.
The trifunctional ligand wassynthesized and radiolabelled with beta (lutetium- 177) and alpha(actinium-225) emitting radionuclides in high yield. Biodistribution of thelutetium-ligand revealed rapid renal clearance and minimal uptake in non-targettissues with all organs and tissues containing less than 0.3 %ID/g by 24 hourspost- injection. Having determined the pharmacokinetic properties of the ligand,a biodistribution study was performed to determine the targeting potential ofthe platform. The lutetium-labelled compound was incubated with TCO-BP, a bonetargeting bisphosphonate, for 10 minutes followed by administration to mice.The biodistribution revealed activity in the bone that was significantly higherthan the ligand alone, however, there was very high off-target uptake in thelungs. In an attempt to decrease the uptake in the lungs while maintaininguptake in the bone, a pre-targeting study was performed. TCO-BP wasadministered i.v., followed an hour later by the lutetium- labelled ligandwhich allowed time for the targeting vector to accumulate at the site ofinterest while clearing non-target tissues. The study showed a significant reductionof the activity in the lungs, and only a small decrease in bone uptake. Theunlabelled ligand was shown to recruit anti-DNP antibodies both in solution andwhen linked to a surface through ELISA and flow cytometry respectively. Theligand showed promising results invitro and in vivo therefore the next step was to perform therapy studies.
ARM therapy studies require theuse of immunocompetent animals that have been immunized towards the chosenantigen. This limits the use of targeting vectors such as antibodies, as manyhumanized antibodies would cause an immune response when administered to ahealthy animal. To this end a melanoma targeted agent was synthesized andtested which resulted in low target uptake that washed out over time. Tworadiolabelled bisphosphonates were also evaluated for their uptake in a flankosteosarcoma tumour, however both agents had very low uptake. As analternative, an intratumoral (i.t.) strategy was developed through thepreparation of a TCO-bovine serum albumin (BSA) derivative. This new chemicalentity was used in both an aggregated and non-aggregated form to retain theR-ARM in the tumour after i.t. administration. Biodistribution showed highretention of the aggregated and non- aggregated BSA out to 120 hours with 167 ±94 and 81 ± 32 %ID/g respectively remaining in the tumour. An autoradiographystudy revealed the after i.t. administration the aggregated material waslocalized in specific regions within the tumour compared to the non-aggregatedmaterial which diffused throughout. The aggregated material was used in asingle and multi-dosing radiotherapy study in which the latter induced astatistically significant survival advantage compared to the control. Oneadditional multi-dosing study was performed with the non-aggregated materialwhich resulted in the largest survival advantage to date. Intratumoraladministration of TCO-BSA linked to the trifunctional tetrazine showedpromising radiotherapy results and future work on dose optimization withlutetium and actinium is required prior to the combination R- ARM therapy.
In parallel, the efficacy of theunlabelled ARM linked to TCO-BSA was interrogated in preclinical models. Thecompound was administered i.t. three times per week in a breast cancer tumourmodel, and response to therapy monitored. The immunized group showed nosurvival advantage compared to the control group comprised of nai?ve animals.Biodistribution studies were performed to determine if TCO-BSA was accessibleto the bloodstream following i.t. administration in both the aggregated andnon-aggregated forms. Saline, aggregated or non-aggregated TCO-BSA wereadministered i.t. followed by the R-ARM. The results showed very low uptake inthe tumour for all three groups, with minimal change in distribution from that ofthe native R-ARM. This suggests that after i.t. administration, the TCO-BSA wasnot available to moleculesin the bloodstream or the concentration was insufficient to promote in vivo coupling. Further work on this component of the platform isneeded before further ARM studies are performed.