PURPOSE:To conduct a phase I and biodistribution study of the EphA3 antibody ifabotuzumab and zirconium-89-labeled ifabotuzumab (89Zr-ifabotuzumab) in patients with glioblastoma (GBM). PATIENTS AND METHODS:This multisite study was conducted in adults with recurrent GBM whose tumors were measurable according to Response Assessment in Neuro-Oncology (RANO) criteria and whose Eastern Cooperative Oncology Group performance status was 0 to 1. Patients underwent a biodistribution study with PET scans with 89Zr-ifabotuzumab, followed by three infusions of ifabotuzumab at either 3.5 or 5.25 mg/kg before undergoing a second study with 89Zr-ifabotuzumab PET scans. Resected patient diagnostic tumor samples were collected for multiplex immunofluorescence and spatial transcriptomics analyses. RESULTS:Twelve patients were recruited, of which six were treated with 3.5 mg/kg and six with 5.25 mg/kg of ifabotuzumab. 89Zr-ifabotuzumab and associated PET scanning were well tolerated, as was ifabotuzumab. There were no objective responses, but one patient had prolonged stable disease. In addition, two patients showed changes in peritumor edema that were suggestive of modulation of tumor vasculature. 89Zr-ifabotuzumab scans showed highly specific tumor uptake in all patients concordant with disease sites on MRI and PET imaging, without evidence of nonspecific binding. Spatial transcriptomics and immunofluorescence analyses of the patient's archival tissue specimens showed that EphA3 was expressed in the tumor microenvironment in all patients and tumor cells with different transcriptional states. CONCLUSIONS:Targeting EphA3 with ifabotuzumab in patients with GBM is safe and attractive, showing chronologic stable expression across both tumor compartments (particularly in cells with a mesenchymal phenotype) and nontumor compartments (particularly the vascular compartment) with evidence of target modulation.

mAb104 does not affect EGF-independent (left) EGF-dependent (right) downstream signaling in the MAPK and AKT pathways (A, B) NCI-N87 and (C, D) OE-19 cells in-vitro as monotherapy or in combination with trastuzumab or pertuzumab. Cells incubated in serum-depleted media were treated with 100µg/mL of mAb104, trastuzumab, pertuzumab alone or in combination for 24 hours prior to undergoing whole cell lysis. Equal amounts of lysates were then loaded and resolved on 4%-12% gel before transfer to nitrocellulose membrane. Membranes were immunoblotted as shown. Results are representative of two experiments.

mAb104 binding and anti-tumor effects in gastric OE-19 cancer xenograft (A) Binding of 10μg/mL of Trastuzumab, Pertuzumab and mAb104 (green) to OE-19 cells. B, Mice bearing OE-19 xenografts (n = 5) were treated with Trastuzumab, Pertuzumab and mAb104 for 3 weeks (indicated by broken orange line) as (B) monotherapy or (C) in combinations. Data shown in growth curve represents mean tumor volume ± S.E. ***p < 0.001, ****p < 0.0001

Background: The novel anti-HER2 antibody 104 (mAb104) targets a unique tumour-specific epitope, lacks normal tissue binding and can internalise into tumour cells, thus supporting its development into antibody drug conjugates (ADCs). Methods: We now describe the binding properties and preclinical activity of mAb104-ADCs developed through the conjugation of mAb104 via linkers to the anti-microtubule drug maytansoinoid ematansine (DM1-SMCC; DM1), topoisomerase I inhibitor, exatecan derivative (MC-GGFG-DX8951; DX8951) or microtubule disruptor monomethyl auristatin E (MC-vc-PAB-MMAE; MMAE). Results: Mab104-ADCs demonstrate dose-dependent cytotoxicity in vitro. The safety of single-dose mAb104-DX8951 was demonstrated in vivo at doses up to 10 mg/kg. MAb104-ADCs also demonstrated potent and prolonged anti-tumour activity in a range of tumour types with variable HER2 expression. Mab104-DX8951 showed significant responses in trastuzumab-resistant HER2-positive breast cancer, low HER2-expressing cancers, as well as HER2-overexpressing cancers. Conclusion: These findings indicate the potential for tumour-specific targeting of HER2-expressing tumours with mAb104-ADCs.

We generated a novel HER2 mAb104, which binds to an epitope in domain II of HER2 that is conformationally exposed in tumors in response to HER2 amplification or activation but is not accessible to antibody binding in normal tissues. Consistent with other studies that evaluated antibodies targeting conformationally exposed epitopes, mAb104 lacked in vitro activity but showed potent antitumor activity in vivo. The antitumor effect in vivo was similar in magnitude to trastuzumab and pertuzumab, and combination with trastuzumab was superior to trastuzumab alone. IHC screening of normal and tumor tissues with mAb104 showed that mAb104 did not bind to normal tissues, confirming the tumor specificity of mAb104. In vivo biodistribution and imaging data demonstrated specific tumor targeting of mAb104 in HER2-expressing tumors. Confocal microscopy clearly demonstrated the internalization of mAb104 into the tumor cells, consistent with mAb104:HER2 trafficking. mAb104 is tumor-specific, exhibits potent antitumor activity in HER2-positive models, and internalizes into HER2-positive tumor cells. These results demonstrate the potential of mAb104 as a novel HER2-targeting therapy, both as a naked antibody for signaling abrogation therapy and for payload delivery as an antibody-drug conjugate or for β/α particle therapy.

Abstract: Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are highly radiosensitive with immune-driven abscopal responses reported. Programmed cell death 1/programmed cell death ligand 1 (PD-L1) inhibitors are relatively ineffective in DLBCL/FL; however, evidence suggests synergy with radiotherapy (RT), but no clear biomarkers. This phase 1 study examined the safety of escalating RT dose and treated volumes with durvalumab (PD-L1 inhibitor) in 34 adults with relapsed/refractory DLBCL and relapsed/refractory FL, and the role of immune-cell subsets on outcomes. Patients received external-beam RT (2.5-30 Gray [Gy], 5 or 10 fractions up to 3 target sites) plus durvalumab from RT day 2, until progression. Novel positron emission tomography (PET) biodistribution studies of 89Zr-durvalumab and CD8 T-cell minibody-89Zr-Df-crefmirlimab were incorporated. The RT recommended phase 2 dose was 10 Gy/5 fractions and 30 Gy/10 fractions to 3 sites for FL and DLBCL, respectively. The most common grade 3 to 4 toxicities included anemia (9%), neutropenia (11%), and liver dysfunction (5%). Overall response was 60% in FL (3/5; complete response, 40% [2/5]), and 14% in DLBCL (4/27; complete response, 7% [2/27]). Distinct peripheral blood and tumor T-cell features, including CD8 PET–determined intratumoral CD8 T-cells, correlated with response (P < .05). RT-durvalumab with 30 Gy/10 fractions of RT to 3 disease sites is safe, and offers promising responses in FL. Intratumoral and peripheral blood CD8 T-cell dysregulation correlate with treatment response. This trial was registered at www.clinicaltrials.gov as #NCT03610061.

Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β “trap”) and human immunoglobulin 1 antibody which blocks programmed cell death ligand 1 (PD-L1). This trial aimed to investigate the biodistribution of 89Zr-bintrafusp alfa in patients with NSCLC with PD-L1 expressing tumors. Five lung cancer patients were recruited with PD-L1 staining more than 1

Comparative binding of mAb104, mAb105, mAb106 and mAb107 to the HER2 extracellular domain, circularized and linear peptide immunogens which the antibodies were generated against, or control irrelevant peptide, using an ELISA based assay. Specificity was confirmed by lack of binding to control-KLH conjugated peptide. Results are representative of two experiments

Novel radiation sensitizers, including inhibitors targeting DNA damage response, have been developed to enhance the efficacy of anticancer treatments that induce DNA damage in cancer cells. Peposertib, a potent, selective, and orally administered inhibitor of DNA-dependent protein kinase, impedes the nonhomologous end-joining mechanism for DNA double-strand break (DSB) repair. We investigated radioimmunotherapy alone or with peposertib in preclinical models of renal cell carcinoma (RCC) or prostate cancer. Methods: 177Lu-DOTA-girentuximab (targeting carbonic anhydrase IX) or 177Lu-DOTA-rosopatamab (targeting prostate-specific membrane antigen) was used to deliver β-radiation to tumors via a single intravenous dose (3 or 6 MBq) in mice bearing SK-RC-52 RCC or LNCaP prostate cancer xenografts, respectively. Peposertib (50 mg/kg daily for 14 d) was administered via oral gavage. Biodistribution and in vivo imaging of 177Lu-based radioimmunotherapy were performed for both preclinical models. Tumor growth and body weight were monitored until the endpoint. Assessment of DNA damage was performed by measuring DSBs through analysis of γH2AX foci formation in tumor sections. Results: Ex vivo biodistribution and in vivo SPECT/MRI revealed excellent tumor uptake of each radiopharmaceutical. Mouse body weight was stable in all treatment arms. Peposertib alone did not show a significant antitumor effect. The addition of peposertib to 177Lu-DOTA-girentuximab showed enhanced antitumor efficacy compared with 177Lu-DOTA-girentuximab alone in the SK-RC-52 animal model, with a 4 of 4 complete response rate in the 177Lu-DOTA-girentuximab (6 MBq) plus peposertib arm. Peposertib combined with low-dose 177Lu-DOTA-girentuximab (3 MBq) demonstrated antitumor activity comparable to 177Lu-DOTA-girentuximab (6 MBq) monotherapy. In the LNCaP prostate cancer model, the combination of 177Lu-DOTA-rosopatamab (6 MBq) and peposertib achieved a 3 of 4 complete response rate. Increased DSBs were observed with the addition of peposertib to 177Lu-based radioimmunotherapy. Conclusion: The combination of peposertib with 177Lu-based radioimmunotherapy was well tolerated in preclinical models of RCC and prostate cancer. Our findings suggest a synergistic effect between peposertib and 177Lu-based radioimmunotherapy, wherein peposertib enhanced the efficacy of radioimmunotherapy. This synergy indicates the potential to reduce the necessary dose of radioimmunotherapy for effective cancer treatment.

FACS analysis using 10μg/mL antibodies confirming small shift in mAb104 binding in high HER2-expressing (A) NCI-N87, (B) BT-474, (C) SK-BR-3 and (D) NCI-H2170 but not in low HER2-expressing (E) NCI-H838, (F) NCI-H522 and (G) NCI-H1650 cell lines. Another murine-anti-HER2 antibody was included at the same concentration alongside Trastuzumab in (A-C).

Immunohistochemical staining with mAb104 and anti-HER2 antibody in (x400) normal (A) brain, (B) large bowel, (C) renal tissue, (D) lung and tumor tissues (E) adenocarcinoma bowel, (F) adenomacarcinoma lung, (G) squamous carcinoma lung, (H) renal cell carcinoma, (I) hepatocellular carcinoma.

CI-8993 is a fully human IgG1κ monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Phase I safety has been established in patients with advanced cancer (NCT02671955). To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop 89Zr-labelled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial. CI-8993 and human isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabelling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [89Zr]Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabelled CI-8993, as well as isotype control [89Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsirtm1.1(VSIR)Geno, huVISTA KI) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumours; and in BALB/c nu/nu mice bearing pancreatic Capan-2 tumours. Stable constructs with an average chelator-to-antibody ratio of 1.81 were achieved. SDS-PAGE and SE-HPLC showed integrity of CI-8993 was maintained after conjugation; and ELISA indicated no impact of conjugation and radiolabelling on binding to human VISTA. PET imaging and biodistribution in MB49 tumour-bearing huVISTA KI female mice showed specific localisation of [89Zr]Zr-Df-CI-8993 to VISTA in spleen and tumour tissues expressing human VISTA. Specific tumour uptake was also demonstrated in Capan-2 xenografted BALB/c nu/nu mice. We radiolabelled and validated [89Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.

Purpose ATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89 Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo. Methods ATG-101, anti-PD-L1, and an isotype control were conjugated with p -SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89 Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [ 89 Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [ 89 Zr]Zr-labelled antibodies. The specificity of [ 89 Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies. Results The Df-conjugation and [ 89 Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [ 89 Zr]Zr-Df-ATG-101 and [ 89 Zr]Zr-Df-anti-PD-L1. Tumour uptake of [ 89 Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo. Conclusion [ 89 Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [ 89 Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [ 89 Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101.

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

INTRODUCTION:Anti-ASCT2 antibody drug conjugate (ADC) MEDI7247 has been under development as a potential anti-cancer therapy for patients with selected relapsed/refractory hematological malignancies and advanced solid tumors by MedImmune. Although promising efficacy was observed in the clinic, pharmacokinetic (PK) analyses observed low exposure of MEDI7247 in phase I hematological patients. To investigate the biodistribution properties of MEDI7247, MEDI7247 and control antibodies were radiolabeled with zirconium-89 and in vitro and in vivo properties characterized.METHODS:MEDI7247 (human anti-ASCT2 antibody conjugated with pyrrolobenzodiazepine (PBD)) and MEDI7519 (MEDI7247 without PBD drug conjugate) and an isotype control antibody drug conjugate construct were conjugated with p-isothiocyanatobenzyl-deferoxamine (Df) and radiolabeled with zirconium-89. In vitro studies included determining the radiochemical purity, protein integrity, immunoreactivity (Lindmo analysis), apparent antigen binding affinity for ASCT2-positive cells by Scatchard analysis and serum stability of the radiolabeled immunoconjugates. In vivo studies included biodistribution and PET/MRI imaging studies of the radiolabeled immunoconjugates in an ASCT2-positive tumor model, HT-29 colorectal carcinoma xenografts.RESULTS:Conditions for the Df-conjugation and radiolabeling of antibody constructs were determined to produce active radioimmunoconjugates. In vivo biodistribution and whole body PET/MRI imaging studies of [89Zr]Zr-Df-MEDI7519 and [89Zr]Zr-Df-MEDI7247 radioimmunoconjugates in HT-29 colon carcinoma xenografts in BALB/c nude mice demonstrated specific tumor localization. However, more rapid blood clearance and non-specific localization in liver was observed for [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 compared to isotype control ADC. Except for liver and bone, other normal tissues demonstrated clearance reflecting the blood clearance for all three constructs and no other abnormal tissue uptake.CONCLUSIONS AND ADVANCES IN KNOWLEDGE:Preclinical biodistribution analyses of [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 showed the biodistribution pattern of anti-ASCT2 ADC MEDI7247 was similar to parental MEDI7519, and both antibodies showed specific tumor uptake compared to an isotype control ADC. This study highlights an important role nuclear medicine imaging techniques can play in early preclinical assessment of drug specificity as part of the drug development pipeline.