Logo: to the web site of Uppsala University

Targeted radionuclide therapy is an emerging potent therapeutic strategy in oncology. The cell surface antigen CD44v6 is a potential pan-cancer target for radionuclide therapy. This study aimed to evaluate the therapeutic efficacy, biodistribution, dosimetry, and safety profile of AKIR001, an antibody targeting CD44v6 labeled with ¹⁷⁷Lu.

Methods: The biodistribution and preclinical dosimetry of [¹⁷⁷Lu]Lu-AKIR001 were calculated in the highly CD44v6-expressing A431 murine xenograft model, with subsequent extrapolation to predict human dosimetry. Therapeutic efficacy was evaluated across 3 xenograft models, 2 with high and 1 with moderate levels of CD44v6, using multiple dosing levels, fractionation regimens, and combinations with cisplatin. Preclinical toxicology was evaluated in a cross-reactive rabbit model and complemented by a PET imaging study using ⁶⁸Ga-labeled AKIR001 in a cynomolgus macaque.

Results: Biodistribution studies confirmed the high and selective tumor uptake of [¹⁷⁷Lu]Lu-AKIR001, resulting in favorable dosimetry predictions for clinical application. Therapeutic evaluations demonstrated significant dose-dependent efficacy in all tested xenograft models, with fractionated dosing (2 doses) resulting in complete tumor regression in 80% of the animals in a radioresistant xenograft model. Biodistribution in rabbits demonstrated low uptake in normal tissues, and a good-laboratory-practice study using an excessive dose of AKIR001 was well tolerated, with no signs of adverse effects. PET imaging in a cynomolgus macaque corroborated these findings.

Conclusion: Collectively, these data strongly support the therapeutic efficacy, safety, and dosimetry of [¹⁷⁷Lu]Lu-AKIR001, justifying its advancement into clinical trials. A phase 1 clinical trial of [¹⁷⁷Lu]Lu-AKIR001for CD44v6-positive solid cancers (NCT06639191) is currently recruiting patients.

Aim:

This study aimed to improve the efficacy of the CD44v6-targeting antibody UU-40 for molecular radiotherapy through affinity maturation and IgG subclass optimization.

M&M:

A panel of affinity-matured antibody candidates was generated and characterized as both human IgG4 and IgG1 with LALA mutations. Surface plasmon resonance and LigandTracer analyses identified several candidates with superior affinity and off-rates compared to the parental UU-40. Biodistribution studies in xenograft models using Lutetium-177 (177Lu)-labeled antibodies showed improved tumor retention for selected candidates, particularly UU-A-155. Species cross-reactivity assays confirmed binding to cynomolgus and rabbit v6-peptides, supporting future toxicity studies.

Results:

The IgG1 LALA format demonstrated reduced binding to Fc gamma receptors, potentially improving the safety profile. UU-A-155 emerged as the lead candidate for clinical translation, showing superior performance in both affinity and tumor retention. Our findings highlight the importance of comprehensive in vitro and in vivo assessments in antibody development, and provides valuable insights into optimizing antibody-based molecular radiotherapy.

Background

CD44v6 is a membranous antigen upregulated in solid tumors and a promising molecular radiotherapy target, especially in anaplastic thyroid carcinoma (ATC). A Phase 1 trial recently launched to evaluate the lutetium-labeled anti-CD44v6 antibody [¹⁷⁷Lu]Lu-DOTA-AKIR001 in CD44v6-positive solid tumors. Given limited data in non-ATC, we assessed CD44v6 immunoreactivity in tumors that may progress to a radioiodine-refractory state.

Materials and Methods

An exploratory cohort of 33 tumors (30 papillary thyroid carcinomas [PTCs], 3 poorly differentiated thyroid carcinomas [PDTCs]) was screened using the VFF-7 antibody, supported by detailed iodine concentration, genetic, and RNA sequencing data. A validation cohort of 40 oncocytic thyroid carcinomas (OTCs), 28 additional PDTCs, and one differentiated high-grade thyroid carcinoma was also screened using two antibody clones, VFF-7 and VFF-18.

Results

In the exploratory cohort, 10 of 33 tumors (30%) showed focal or diffuse CD44v6 expression, while the rest were negative. Among OTCs in the validation cohort, 15 of 40 (38%) were partially or diffusely positive, and in PDTCs, 14 of 28 (50%) showed focal or diffuse staining. The VFF-7 and VFF-18 clones produced similar patterns.

Conclusions

Substantial subsets of non-ATCs express CD44v6, indicating that some patients may be candidates for [¹⁷⁷Lu]Lu-DOTA-AKIR001 radiotherapy, particularly when conventional treatments are exhausted.

Multiple myeloma (MM) is a hematological disease of the plasma cell that remains clinically challenging despite the development of novel therapies. Epigenetic alterations have been demonstrated to contribute to MM pathogenesis, yet comprehensive studies into the links between different epigenetic regulatory systems in myeloma progression and drug resistance, though clinically relevant, are largely lacking. G9a and the DNA methyltransferases (DNMTs) are epigenetic modifiers that exhibit increased activity in MM, correlating with poor prognosis. To investigate the partnership between G9a and DNMTs, we used a combinatorial treatment approach involving small-molecule inhibitors. In-depth molecular analysis of the histone H3 lysine dimethylation distribution, the DNA methylome and the transcriptome of MM revealed a silencing mechanism involving G9a and DNMTs that represses key tumor suppressor genes. Moreover, dual inhibition of G9a and DNMTs reduced cell viability in primary MM cells and induced apoptosis in MM cell lines. This was accompanied by increased expression of apoptosis-related genes and decreased protein levels of the MM-associated oncoproteins IRF4, XBP1, and MYC. To assess the translational relevance of our in vitro findings, we evaluated the combination therapy in an in vivo preclinical xenograft MM model. Specifically, we demonstrate that the G9a inhibitor A366 synergizes with the DNMTs inhibitor decitabine to promote a robust tumor regression in vivo. Together, these data provide new insights into the cooperative role of G9a and the DNMTs in regulating gene silencing in MM, and support dual epigenetic inhibition as a promising therapeutic strategy.

Anaplastic thyroid cancer (ATC) is a rare but severe form of thyroid cancer responsible for approximately 50% of thyroid cancer deaths. Consequently, the identification of innovative therapies remains crucial for the effective treatment of ATC. Molecular radiotherapy is a rapidly growing field within oncology, and the cell surface antigen CD44v6, which is overexpressed in several cancers, is a plausible target for molecular radiotherapy of ATC. IHC of 39 patient samples with ATC was evaluated for CD44v6 expression. Biodistribution and dosimetry of iodine-125 (¹²⁵I)–/lutetium-177 (¹⁷⁷Lu)–labeled UU-40, a CD44v6-specific antibody, followed by in vivo efficacy in two ATC xenograft models with varying target expression levels (ACT-1 and BHT-101), accompanied by single-photon emission computed tomography (SPECT) imaging, evaluated radiolabeled UU-40 for therapeutic efficiency in ATC xenografts. The IHC revealed CD44v6 immunoreactivity in 46% of patient samples with ATC. The biodistribution favored ¹⁷⁷Lu-labeled UU-40 over the ¹²⁵I-labeled antibody and confirmed the in vivo specificity of both radioconjugates. The in vivo efficacy and accompanied SPECT imaging of a moderate CD44v6-expressing xenograft model (BHT-101) verified the tumor specificity, as well as the target-specific effect of ¹⁷⁷Lu-labeled UU-40 on tumor growth and survival. A 100% complete response rate was demonstrated as a result of therapy using a single dose of 16 MBq ¹⁷⁷Lu-labeled UU-40 in a high CD44v6-expressing xenograft model (ACT-1), and SPECT imaging revealed excellent tumor uptake of the radioconjugate at 14 days after injection. This study verifies the expression of CD44v6 in ATC and strengthens the superiority and promise of ¹⁷⁷Lu-labeled UU-40 over ¹³¹I-labeled UU-40 for antibody-based molecular radiotherapy of CD44v6-positive ATC.

Objective. The cell surface antigen CD44v6 is a promising target for several cancers, with favorable in vivo characteristics such as high affinity and suitable biodistribution. In normal tissues, expressions are restricted to basal epithelial cells in skin and mucosa. Consequently, previous clinical studies have reported varying degrees of toxicity in these tissues. To support new radioimmunotherapies (RIT), we developed small-scale internal dosimetry models for abdominal skin and esophageal mucosa. Using published biodistribution data, we compared absorbed doses to epithelial tissues and bone marrow from four clinically relevant radionuclides: rhenium-186, lutetium-177, terbium-161, and actinium-225. Approach. From the Genotype-Tissue Expression database, 288 H&E-stained sections of abdominal skin and esophageal mucosa were obtained from donors aged 21-70 and segmented to generate voxelized models. Monte Carlo simulations were conducted for the radionuclides across multiple source/target combinations. A compartment model generated serum, skin, and bone marrow biodistributions to estimate AD to the epithelium and bone marrow. Main results. Absorbed dose estimates to the basal layer of the skin were highest for 161Tb at 23.7 Gy GBq-1, followed by 177Lu and 186Re, with values of 9.5 and 5.3 Gy GBq-1, respectively, whereas the alpha emitter 225Ac delivered a dose of 4.9 Gy MBq-1. For the beta-emitters, three methods produced absorbed-dose estimates for the red marrow (RM) consistent within 5% of each other, with the highest values in the hip bone: 0.36, 0.25, and 0.39 Gy GBq-1 for 186Re, 177Lu, and 161Tb, respectively, compared to 0.66 Gy MBq-1 for 225Ac. Significance. This study presents a dosimetry framework for CD44v6-targeted RIT, demonstrating that, at clinically relevant administered activities, short-ranged emitters like 161Tb and 225Ac deliver substantially higher doses to basal epithelial layers compared to 186Re. RM doses were comparable for 161Tb and 186Re, lower for 177Lu, and highly uncertain for 225Ac due to daughter redistribution. Overall, these results support the use of 177Lu for initial clinical trials.

About 50% of all cancers carry a mutation in p53 that impairs its tumor suppressor function. The p53 missense mutation p53^R175H (p53^R172H in mice) is a hotspot mutation in various cancer types. Therefore, monoclonal antibodies selectively targeting clinically relevant mutations like p53^R175H could prove immensely value. We aimed to evaluate the in vitro and in vivo binding properties of two novel anti-p53^R175H monoclonal antibodies and to assess their performance as agents for molecular imaging. In vitro, ¹²⁵I-4H5 and ¹²⁵I-7B9 demonstrated long shelf life and antigen-specific binding. Our in vivo study design allowed head-to-head comparison of the antibodies in a double tumor model using repeated SPECT/CT imaging, followed by biodistribution and autoradiography. Both tracers performed similarly, with marginally faster blood clearance for ¹²⁵I-7B9. Repeated molecular imaging demonstrated suitable imaging characteristics for both antibodies, with the best contrast images occurring at 48 h post-injection. Significantly higher uptake was detected in the mut-p53-expressing tumors, confirmed by ex vivo autoradiography. We conclude that molecular imaging with an anti-p53^R175H tracer could be a promising approach for cancer diagnostics and could be further applied for patient stratification and treatment response monitoring of mutant p53-targeted therapeutics.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these ‘design rules’ to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

Immunotherapy has revolutionized cancer treatment but its efficacy depends on a robust immune response in the tumor. Silencing of the tumor suppressor p53 is common in tumors and can affect the recruitment and activation of different immune cells, leading to immune evasion and poor therapy response. We found that the p53 activating stapled peptide MDM2/MDMX inhibitor Sulanemadlin (ALRN-6924) inhibited p53 wild-type cancer cell growth in vitro and in vivo. In mice carrying p53 wild-type CT26.WT tumors, monotherapy with the PD-1 inhibitor DX400 or Sulanemadlin delayed tumor doubling time by 50% and 37%, respectively, while combination therapy decreased tumor doubling time by 93% leading to an increased median survival time. Sulanemadlin treatment led to increased immunogenicity and combination treatment with PD-1 inhibition resulted in an increased tumor infiltration of lymphocytes. This combination treatment strategy could potentially turn partial responders into responders of immunotherapy, expanding the patient target group for PD-1-targeting immunotherapy.

Purpose

Molecular radiotherapy is a treatment modality that is highly suitable for targeting micrometastases and [¹⁷⁷Lu]Lu-DOTATATE is currently being explored as a potential novel treatment option for high-risk neuroblastoma. p53 is a key player in the proapoptotic signalling in response to radiation-induced DNA damage and is therefore a potential target for radiosensitisation.

Methods

This study investigated the use of the p53 stabilising peptide VIP116 and [¹⁷⁷Lu]Lu-DOTATATE, either alone or in combination, for treatment of neuroblastoma tumour xenografts in mice. Initially, the uptake of [¹⁷⁷Lu]Lu-DOTATATE in the tumours was confirmed, and the efficacy of VIP116 as a monotherapy was evaluated. Subsequently, mice with neuroblastoma tumour xenografts were treated with placebo, VIP116, [¹⁷⁷Lu]Lu-DOTATATE or a combination of both agents.

Results

The results demonstrated that monotherapy with either VIP116 or [¹⁷⁷Lu]Lu-DOTATATE significantly prolonged median survival compared to the placebo group (90 and 96.5 days vs. 50.5 days, respectively). Notably, the combination treatment further improved median survival to over 120 days. Furthermore, the combination group exhibited the highest percentage of complete remission, corresponding to a twofold increase compared to the placebo group. Importantly, none of the treatments induced significant nephrotoxicity. Additionally, the therapies affected various molecular targets involved in critical processes such as apoptosis, hypoxia and angiogenesis.

Conclusion

In conclusion, the combination of VIP116 and [¹⁷⁷Lu]Lu-DOTATATE presents a promising novel treatment approach for neuroblastoma. These findings hold potential to advance research efforts towards a potential cure for this vulnerable patient population.