197gHg and 197mHg are attractive radionuclides for radioimmunotherapy of cancer due to their abundant Auger electron emissions (23.2 and 19.4 electrons/decay, respectively). Our aim was to study the cytotoxicity and DNA-damaging properties of panitumumab labeled with 197gHg/197mHg on a panel of human breast cancer cells expressing different levels of epidermal growth factor receptor (EGFR) (104 to 106 EGFR/cell) and evaluate the tumour and normal tissue uptake of these radioimmunoconjugates in mice with EGFR-overexpressing MDA-MB-468 human breast cancer xenografts (106 EGFR/cell). Panitumumab was conjugated to 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetamide (TCMC) or a sulphur-rich (NS4) bifunctional chelator to complex 197gHg/197mHg or directly labeled with 197gHg/197mHg through endogenous mercury binding sites. All radioimmunoconjugates bound specifically (87–91

PORPHYSOMES (PS) are multifunctional porphyrin-lipid nanoparticles for fluorescence-guided photochemical tumour ablation and immune stimulation. PS can be radiolabelled with Copper-64 (64Cu-PS) to permit tracing of their whole-body distribution and uptake in tumours with nuclear imaging. Herein we characterised the physicochemical and radiochemical properties of PS and 64Cu-PS and evaluated their pharmacology and toxicology in rats and dogs. 64Cu-PS labelling procedure was optimised and developed into a “one pot” kit format. The plasma pharmacokinetics (PK), tissue distribution, and single and repeat-dose toxicity of dose escalating PS was evaluated after intravenous injection in healthy Fisher 344 rats and Beagle dogs. Radiation dosimetry from single 64Cu-PS dose was estimated in humans. Kit prepared 64Cu-PS had high labelling yields and mean specific activities of 18.5 MBq/mg (11.4 GBq/mg max). The plasma PK of PS was linear between doses 0.5–15 mg/kg in both rats and dogs. Tissue distribution was primarily to the liver. The estimated effective dose from 64Cu-PS in humans was 38–47 µSv/MBq. Single doses of 32.6 mg/kg PS did not have detectable toxicities in rats. In dogs, mild transient infusion reactions were observed following single and repeat-doses of PS starting at ≥ 10 mg/kg PS. Doses up to 15 mg/kg PS were tolerated in dogs. The pharmacology and safety profiles of PS and 64Cu-PS in rats and dogs did not raise concerns regarding their use in humans. Radiolabelled 64Cu-PS will permit accurate imaging-based assessments of nanoparticle PK and tumour uptake in imaging studies in cancer patients.

The use of chemically matched theranostic radiometals in nuclear medicine presents a paradigm shift in personalized medicine with immense potential to treat advanced cancers. The nuclear isomers, mercury-197g (197gHg, half-life 64.14 h) and mercury-197m (197mHg, half-life 23.8 h) possess optimal physical decay properties to be applied in theranostic radiopharmaceuticals; however, their use has been limited due to the lack of suitable bifunctional chelators (BFCs) capable of attaching the radionuclides to disease targeting biomolecules. Herein we report the development and evaluation of two novel 197m/gHg BFCs derived from a 15-membered thiacrown ether macrocycle (NS4) bearing isothiocyanate (-NCS) or tetrazine (-Tz) bifunctional handles to allow conjugation to biomolecules. Both chelators were synthesized and radiolabeled with 197m/gHg, assessed for complex stability, and bioconjugation to trastuzumab (TmAb), a monoclonal antibody targeting HER2 receptors. NS4-Tz efficiently and stably complexes [197m/gHg]Hg2+ and exhibited excellent in vitro stability in both glutathione and human serum. In contrast, NS4-NCS showed lower radiometal incorporation yields and reduced complex stability, likely attributed to non-specific interactions of the isothiocyanate group with Hg2+. NS4-Tz was successfully conjugated to transcyclooctene-modified TmAb with favourable chelator-to-antibody ratios and subsequently radiolabeled. Due to non-specific Hg2+ binding to TmAb observed during direct labeling, a two-step labeling strategy was employed to improve selectivity. The resulting [197m/gHg]Hg-NS4-Tz-TmAb construct demonstrated specific binding to HER2-positive SK-BR-3 cells in vitro and, in the first in vivo study of a [197m/gHg]Hg-labeled immunoconjugate, confirmed tumour-specific uptake in a SKOV-3 xenograft mouse model. Biodistribution and SPECT/CT studies of the BFC complex alone, [197m/gHg]Hg-NS4-Tz, revealed high hepatic and splenic accumulation, with some renal uptake possibly due to transchelation or tracer pharmacokinetics. While long-term in vivo stability of the radioimmunoconjugate remains a challenge, NS4-Tz shows significant promise for applications with faster-clearing vectors such as peptides or small molecules. Future work will focus on improving hydrophilicity and further optimizing chelator design for mercury-based theranostics.

Head and neck squamous cell carcinoma (HNSCC) expresses epidermal growth factor receptors (EGFR) in 90% of cases. Here we studied PET/CT imaging of three clinically relevant HNSCC patient derived xenografts (PDX) with low (#61531), moderate (#73191) or high (#88955) EGFR expression in NRG mice using anti-EGFR [64Cu]Cu-DOTA-panitumumab F(ab')2. We further assessed the effectiveness of β-particle-emitting [177Lu]Lu-DOTA-panitumumab F(ab')2 for radioimmunotherapy (RIT) of these PDX. All PDX were visualized by PET/CT at 24 h postinjection (p.i.) of [64Cu]Cu-DOTA-panitumumab F(ab')2. In addition, in mice with PDX #88955, an axillary lymph node metastasis was imaged by PET and lung metastases were imaged by SPECT/CT at 3 to 12 d p.i. of [177Lu]Lu-DOTA-panitumumab F(ab')2. Tumor uptake of [64Cu]Cu-DOTA-panitumumab F(ab')2 at 24 h p.i. was directly correlated with EGFR expression (6.7 ± 3.5%, 13.5 ± 2.5% and 16.7 ± 1.0% ID/g for PDX #61531, #73191 and #88955, respectively). Intravenous administration of 5.0 MBq (50 μg) of [177Lu]Lu-DOTA-panitumumab F(ab')2 to healthy NRG mice caused no hematologic, liver or kidney toxicity or decrease in body weight. RIT with 4.0-5.0 MBq (50 μg) of [177Lu]Lu-DOTA-panitumumab F(ab')2 decreased the tumor growth rate vs 0.9% NaCl by 2, 9 and 3.2-fold, respectively in mice with PDX #61531, #73191 and #88955 (P = 0.014, P < 0.001, P = 0.004). Treatment of mice with unlabeled DOTA-panitumumab F(ab')2 did not decrease the tumor growth rate of PDX#61531 (P = 0.457) but modestly decreased the tumor growth rate of PDX #73191 by 1.3-fold (P = 0.024) and PDX #88955 by 1.4-fold (P = 0.027). RIT was EGFR-specific as irrelevant anti-HER2 [177Lu]Lu-DOTA-trastuzumab F(ab')2 was not effective for treatment of PDX #73191 vs 0.9% NaCl (P = 0.282). RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2 was 3-fold more effective for treating moderately EGFR-expressing PDX #73191 than PDX #88955 with high EGFR expression. This may be explained by the human papilloma virus (HPV) positivity of PDX #73191 since HPV-positive HNSCC is more responsive to external radiation beam treatment. We conclude that [64Cu]Cu-DOTA-panitumumab F(ab')2 and [177Lu]Lu-DOTA-panitumumab F(ab')2 are a promising theranostic pair for PET/CT imaging and RIT of HNSCC.

Abstract Introduction: Our aim was to optimize the therapeutic index of TART of HER2-positive BC in NRG mice by studying the effectiveness and normal tissue toxicity of trastuzumab IgG, F(ab’)2 or Fab modified with DOTA for complexing the α-particle emitter, 225Ac. Methods: The toxicity of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 and Fab were assessed in NRG mice (n=5) injected i.v. with 2 and 4 kBq (total 80 μg) separated by 8 d. Body weight was monitored and complete blood cell (CBC) counts and alanine aminotransferase (ALT) and creatinine (CRE) were measured at 14 d post-injection (p.i.). TART was performed in NRG mice (n=7) with s.c. HER2-positive 164/8-1B/H2N.luc+ xenografts injected i.v. with 2 and 4 kBq (total 80 μg) separated by 8 d of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 or Fab. Control mice received irrelevant [225Ac]Ac-DOTA-IgG, trastuzumab or saline. The tumor growth index (TGI=tumor volume/initial tumor volume) was measured and Kaplan-Meier median survival estimated. Tumor and normal tissue uptake (%ID/g) of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 and Fab (4 kBq) in tumor-bearing NRG mice were measured up to 14 d p.i.. Results: [225Ac]Ac-DOTA-trastuzumab F(ab’)2 and Fab caused no decrease in CBC, while [225Ac]Ac-DOTA-trastuzumab IgG decreased white blood cells by 4.5-fold, platelets by 7.5-fold, red blood cells by 1.2-fold and hematocrit by 1.2-fold compared to saline-treated mice. [225Ac]Ac-trastuzumab F(ab’)2 or Fab caused no increase in ALT or CRE. Body weight was not decreased in all groups of mice. [225Ac]Ac-DOTA-trastuzumab IgG, F(ab’)2 or Fab inhibited tumor growth (TGI at 15 d = 2.5, 1.8, and 1.9, respectively) vs. saline or trastuzumab (TGI= 6.3 and 5.2; P=0.0047 and 0.0028). Median survival was increased to 46 d for mice treated with [225Ac]Ac-DOTA-trastuzumab F(ab’)2 vs. 29 d for Fab (P=0.008), 22 d for IgG (P=0.0005) and 15 d for saline (P=0.0005). Median survival for mice treated with [225Ac]Ac-DOTA-IgG was 20 d and for trastuzumab was 22 d. Tumor uptake of [225Ac]Ac-DOTA-trastuzumab IgG and F(ab’)2 at 48 h p.i. were 10.6 ± 0.6 and 8.7 ± 0.8, respectively, while uptake of [225Ac]Ac-DOTA-trastuzumab Fab at 18 h p.i. was 3.1 ± 0.5 %ID/g. Elimination from the blood was slowest for [225Ac]Ac-DOTA-trastuzumab IgG followed by F(ab’)2 then Fab. Spleen and liver uptake were greatest for [225Ac]Ac-DOTA-trastuzumab IgG but much lower for F(ab’)2 and Fab. Kidney uptake was highest for [225Ac]Ac-DOTA-trastuzumab Fab. Conclusion: [225Ac]Ac-DOTA-trastuzumab F(ab’)2 provided the highest therapeutic index, inhibiting tumor growth and improving survival while minimizing toxicity. TART with [225Ac]Ac-DOTA-trastuzumab F(ab’)2 is a promising new treatment for HER2-positive BC that could be more effective than trastuzumab. Citation Format: Misaki Kondo, Zhongli Cai, Conrad Chan, Raymond M. Reilly. Optimizing the therapeutic index of targeted α-particle radioimmuotherapy (TART) of HER2-positive breast cancer tumors in NRG mice with 225Ac-labeled trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6030.

The objective of this research was the development and evaluation of 203Pb-labelled panitumumab (203Pb-PSC-panitumumab) as an immuno-SPECT radioligand for the detection of EGFR + head and neck squamous cell carcinoma (HNSCC) in a patient-derived xenograft (PDX) mouse model. The 51.9 h physical half-life and favourable γ-emission (279 keV; 81

Radioimmunotherapy (RIT) with α-particle-emitting, 225Ac complexed to trastuzumab may offer an alternative treatment for patients who progress on HER2-targeted therapies. Moreover, RIT with [225Ac]Ac-DOTA-trastuzumab could be combined with SPECT/CT imaging with [111In]In-DOTA-trastuzumab in a theranostic approach. In this study, we compared DOTA-conjugated trastuzumab IgG, F(ab')2 or Fab complexed to 111In or 225Ac for SPECT/CT imaging and α-particle RIT of subcutaneous (s.c.) HER2-positive 164/8-1B/H2N.luc+ human BC tumors in NRG mice. SPECT/CT imaging and tumor and normal tissue uptake were compared in NRG or NOD-SCID mice coinjected i.v. with [111In]In-DOTA-trastuzumab IgG, F(ab')2 or Fab and [225Ac]Ac-DOTA-trastuzumab IgG, F(ab')2 or Fab. Radiation absorbed doses in the tumor and normal organs for [225Ac]Ac-DOTA-trastuzumab IgG, F(ab')2 or Fab were estimated based on the biodistribution of the [111In]In-DOTA-trastuzumab IgG, F(ab')2 or Fab. Normal tissue toxicity was assessed by hematology and blood biochemistry analyses and monitoring body weight in NRG mice injected i.v. with 2 and 4 kBq of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab')2 or Fab separated by 8 d. RIT studies were performed in NRG mice with s.c. 164/8-1B/H2N.luc+ tumors injected i.v. with 2 kBq and 4 kBq of [225Ac]Ac-DOTA-trastuzumab IgG, F(ab')2 or Fab separated by 8 d or irrelevant [225Ac]Ac-DOTA-IgG1, two doses of unlabeled trastuzumab IgG or 0.9% NaCl. A tumor growth index (TGI) was plotted vs time (d) and Kaplan-Meier median survival estimated. [111In]In-DOTA-trastuzumab IgG or F(ab')2 exhibited 4.1-fold and 3.3-fold significantly greater tumor uptake at 2 d postinjection (p.i.) than Fab at 24 h p.i. However, spleen uptake at 2 d p.i. for [111In]In-DOTA-trastuzumab IgG was 3.3-fold significantly higher than F(ab')2 and 13.2-fold higher than Fab at 24 h p.i. [111In]In-DOTA-trastuzumab F(ab')2 and Fab exhibited higher kidney uptake than IgG. Tumors were imaged by SPECT/CT with [111In]In-DOTA-trastuzumab IgG and F(ab')2 but were not well-visualized with [111In]In-DOTA-trastuzumab Fab. The absorbed dose in the tumor was 2.2-fold greater for [225Ac]Ac-DOTA-trastuzumab F(ab')2 than IgG and 3.4-fold greater than Fab. Hematological toxicity was observed for [225Ac]Ac-DOTA-trastuzumab IgG but not for [225Ac]Ac-DOTA-trastuzumab F(ab')2 or Fab. No kidney or liver toxicity or decreased body weight was observed for any RIT agent. Tumor growth was significantly inhibited by [225Ac]Ac-DOTA-trastuzumab IgG, F(ab')2 or Fab but [225Ac]Ac-DOTA-trastuzumab F(ab')2 was most effective for increasing median survival (46 d vs 22 d for IgG and 29 d for Fab). We conclude that [111In]In- and [225Ac]Ac-DOTA-trastuzumab F(ab')2 exhibited superior properties for theranostic imaging and α-particle RIT of HER2-positive human BC xenografts in NRG mice.

Background Trastuzumab (Herceptin) has improved the outcome for patients with HER2-positive breast cancer (BC) but brain metastases (BM) remain a challenge due to poor uptake of trastuzumab into the brain. Radioimmunotherapy (RIT) with trastuzumab labeled with α-particle emitting, 225 Ac may overcome this challenge by increasing the cytotoxic potency on HER2-positive BC cells. Our first aim was to synthesize and characterize [ 111 In]In-DOTA-trastuzumab and [ 225 Ac]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC, respectively. A second aim was to estimate the cellular dosimetry of [ 225 Ac]Ac-DOTA-trastuzumab and determine its cytotoxicity in vitro on HER2-positive BC cells. A third aim was to study the tumour and normal tissue uptake of [ 225 Ac]Ac-DOTA-trastuzumab using [ 111 In]In-DOTA-trastuzumab as a radiotracer in vivo in NRG mice with s.c. 164/8-1B/H2N.luc + human BC tumours that metastasize to the brain. Results Trastuzumab was conjugated to 12.7 ± 1.2 DOTA chelators and labeled with 111 In or 225 Ac. [ 111 In]In-DOTA-trastuzumab exhibited high affinity specific binding to HER2-positive SK-BR-3 human BC cells (K D = 1.2 ± 0.3 × 10 –8 mol/L). Treatment with [ 225 Ac]Ac-DOTA-trastuzumab decreased the surviving fraction (SF) of SK-BR-3 cells dependent on the specific activity (SA) with SF < 0.001 at SA = 0.74 kBq/µg. No surviving colonies were noted at SA = 1.10 kBq/µg or 1.665 kBq/µg. Multiple DNA double-strand breaks (DSBs) were detected in SK-BR-3 cells exposed to [ 225 Ac]Ac-DOTA-trastuzumab by γ-H2AX immunofluorescence microscopy. The time-integrated activity of [ 111 In]In-DOTA-trastuzumab in SK-BR-3 cells was measured and used to estimate the absorbed doses from [ 225 Ac]Ac-DOTA-trastuzumab by Monte Carlo N-Particle simulation for correlation with the SF. The dose required to decrease the SF of SK-BR-3 cells to 0.10 (D 10 ) was 1.10 Gy. Based on the D 10 reported for γ-irradiation of SK-BR-3 cells, we estimate that the relative biological effectiveness of the α-particles emitted by 225 Ac is 4.4. Biodistribution studies in NRG mice with s.c. 164/8-1B/H2N.luc + human BC tumours at 48 h post-coinjection of [ 111 In]In-DOTA-trastuzumab and [ 225 Ac]Ac-DOTA-trastuzumab revealed HER2-specific tumour uptake (10.6 ± 0.6% ID/g) but spleen uptake was high (28.9 ± 7.4% ID/g). Tumours were well-visualized by SPECT/CT imaging using [ 111 In]In-DOTA-trastuzumab. Conclusion We conclude that [ 225 Ac]Ac-DOTA-trastuzumab exhibited potent and HER2-specific cytotoxicity on SK-BR-3 cells in vitro and HER2-specific uptake in s.c. 164/8-1B/H2N.luc + human BC tumours in NRG mice, and these tumours were imaged by SPECT/CT with [ 111 In]In-DOTA-trastuzumab. These results are promising for combining [ 111 In]In-DOTA-trastuzumab and [ 225 Ac]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC.

The effectiveness and normal tissue toxicity of a novel nanoparticle depot (NPD) brachytherapy seed incorporating gold nanoparticles (AuNPs) labeled with β-particle emitting, 90Y (termed a "radiation nanomedicine"), were studied for the treatment of 4T1 triple-negative murine mammary carcinoma tumors in Balb/c mice and for inducing an abscopal effect on a distant non-irradiated tumor alone or combined with anti-PD-L1 immune checkpoint antibodies. Balb/c mice with two subcutaneous 4T1 tumors─a primary tumor and a distant secondary tumor were implanted intratumorally (i.t.) in the primary tumor with NPD incorporating 3.5 MBq of 90Y-AuNPs (1 × 1014 AuNPs) or unlabeled AuNPs, alone or combined with systemically administered anti-PD-L1 antibodies (200 μg i.p. three times/week for 2 weeks) or received anti-PD-L1 antibodies alone or no treatment. The primary tumor was strongly growth-inhibited over 14 d by NPD incorporating 90Y-AuNPs but only very modestly inhibited by NPD incorporating unlabeled AuNPs. Anti-PD-L1 antibodies alone were ineffective, and combining anti-PD-L1 antibodies with NPD incorporating 90Y-AuNPs did not further inhibit the growth of the primary tumor. Secondary tumor growth was inhibited by treatment of the primary tumor with NPD incorporating 90Y-AuNPs, and growth inhibition was enhanced by anti-PD-L1 antibodies. Treatment of the primary tumor with NPD incorporating unlabeled AuNPs or anti-PD-L1 antibodies alone had no effect on secondary tumor growth. Biodistribution studies showed high uptake of 90Y in the primary tumor [516-810% implanted dose/g (%ID/g)] but very low uptake in the secondary tumor (0.033-0.16% ID/g) and in normal tissues (<0.5% ID/g) except for kidneys (5-8% ID/g). Very high radiation absorbed doses were estimated for the primary tumor (472 Gy) but very low doses in the secondary tumor (0.13 Gy). There was highdose-heterogeneity in the primary tumor with doses as high as 9964 Gy in close proximity to the NPD, decreasing rapidly with distance from the NPD. Normal organ doses were low (<1 Gy) except for kidneys (4 Gy). No normal tissue toxicity was observed, but white blood cell counts (WBC) decreased in tumor-bearing mice treated with NPD incorporating 90Y-AuNPs. Decreased WBC counts were interpreted as tumor response and not toxicity since these were higher than that in healthy non-tumor-bearing mice, and there was a direct association between WBC counts and 4T1 tumor burden. We conclude that implantation of NPD incorporating 90Y-AuNPs into a primary 4T1 tumor in Balb/c mice strongly inhibited tumor growth and combined with anti-PD-L1 antibodies induced an abscopal effect on a distant secondary tumor. This radiation nanomedicine is promising for the local treatment of triple-negative breast cancer tumors in patients, and these therapeutic effects may extend to non-irradiated lesions, especially when combined with checkpoint immunotherapy.

Background 111 In[In]-BnDTPA-trastuzumab-NLS is a radiopharmaceutical with theranostic applications for imaging and Meitner-Auger electron (MAE) radioimmunotherapy (RIT) of HER2-positive breast cancer (BC). Nuclear localization sequence (NLS) peptides route the radiopharmaceutical to the nucleus of HER2-positive BC cells following receptor-mediated internalization for RIT with subcellular range MAEs. The γ-photons emitted by 111 In permit tumour imaging by SPECT. Our aim was to formulate a kit under Good Manufacturing Practices conditions to prepare 111 In[In]-BnDTPA-trastuzumab-NLS injection for a first-in-human clinical trial. Results Trastuzumab was derivatized with p-SCN-BnDTPA to introduce Bn-DTPA for complexing 111 In, then modified with maleimide groups for conjugation to the thiol on cysteine in NLS peptides [CGYGPKKKRKVGG]. BnDTPA-trastuzumab-NLS (5 mg in 1.0 mL of 0.05 M ammonium acetate buffer, pH 5.5) was dispensed into unit dose sterile glass vials to produce kits for labeling with 100–165 MBq of 111 In[In]Cl 3 . The kits met specifications for protein concentration (4.5–5.5 mg/mL), volume (0.95–1.05 mL), pH (5.5–6.0), appearance (clear, pale-yellow, particulate-free), BnDTPA substitution level (2.0–7.0 BnDTPA/trastuzumab), purity and homogeneity (SDS-PAGE and SE-HPLC), 111 In labeling efficiency (> 90%), binding to HER2-positive SK-BR-3 human breast cancer cells (K a = 1–8 × 10 8 L/mmol; B max = 0.5–2 × 10 6 sites/cell), NLS peptide conjugation (upward band shift on SDS-PAGE), sterility (USP Sterility Test) and endotoxins (USP Bacterial Endotoxins Test). 111 In-BnDTPA-trastuzumab-NLS injection met specifications for pH (5.5–6.5), radiochemical purity (≥ 90%), radionuclide purity (≥ 99%), appearance (clear, colourless, particle-free) and sterility (retrospective USP Sterility Test). Kits were stable stored at 2–8 °C for up to 661 days (d) meeting all key specifications. Protein concentration remained within or just slightly greater than the specification for up to 139 d. 111 In[In]-BnDTPA-trastuzumab-NLS injection was stable for up to 24 h. An expiry of 180 d was assigned for the kits and 8 h for the final radiopharmaceutical. Conclusion A kit was formulated under GMP conditions for preparing 111 In[In]-BnDTPA-trastuzumab-NLS injection. This radiopharmaceutical was safely administered to 4 patients with HER2-positive BC to trace the uptake of trastuzumab into brain metastases before and after MRI-guided focused ultrasound (MRIg-FUS) by SPECT imaging.

We report the pharmaceutical stability of trastuzumab stored for a short time (12 h) at room temperature (RT; 20-25 degrees C) compared to trastuzumab stored at 2-8 degrees C. The physicochemical properties were evaluated by UV-visible and FTIR spectroscopy, SDS-PAGE and size-exclusion HPLC (SE-HPLC). Trastuzumab was reacted with benzylisothiocyanate diethylenetriaminepentaacetic acid (BzDTPA) to complex In-111. The HER2-binding affinity of In-111-BzDTPA-trastuzumab synthesised from trastuzumab stored at RT or at 2-8 degrees C was measured using HER2-positive SK-Br-3 human breast cancer (BC) cells. The tumour and normal tissue uptake of (111)InBzDTPA-trastuzumab was studied by microSPECT/CT imaging and biodistribution studies in CD1 athymic mice with s.c. HER2-positive SK-Ov-3 human ovarian cancer xenografts. There were no differences in lambda(max) or molar absorptivity (epsilon) values in the UV-visible spectra of trastuzumab stored at RT or at 2-8 degrees C. FTIR spectroscopy suggested no differences in secondary structure. SDS-PAGE revealed protein bands corresponding to the expected molecular weights. SE-HPLC showed identical properties for trastuzumab stored at RT or at 2-8 degrees C. The dissociation constant (K-d) for binding of In-111-BzDTPA-trastuzumab to HER2 on SK-Br-3 cells (2.2-4.4 nM) was not significantly different when the radioimmunoconjugates were synthesised from trastuzumab stored at RT or at 2-8 degrees C. MicroSPECT/CT demonstrated high uptake in SK-Ov-3 tumours in mice that was not significantly different using trastuzumab stored at RT or at 2-8 degrees C (33.7 +/- 8.8% vs. 22.2 +/- 8.1% i.d./g, respectively; P = 0.36). There were no significant differences in normal tissue uptake or in tumour/normal tissue (T/NT) ratios. We conclude that short-term storage of trastuzumab at RT for 12 h did not affect the physicochemical or biological properties of the drug.

Introduction: Our objective was to determine the feasibility of extending our previously reported PET imaging study of pancreatic cancer (PnCa) with [Cu-64]Cu-NOTA-panitumumab F(ab')(2) to radioimmunotherapy (RIT) by exploiting the beta-particle and Auger electron emissions of Cu-64 (PET theranostic concept). To enhance the effectiveness of [Cu-64]Cu-NOTA-panitumumab F(ab')(2), we further combined RIT with radiosensitizing gemcitabine (GEM) and the poly(ADP)ribose polymerase inhibitor (PARPi), rucaparib. Methods: Normal tissue toxicity was assessed in non-tumor-bearing NOD-sad mice injected i.v. with [Cu-64]Cu-NOTA-panitumumab F(ab')(2) (1.85-9.25 MBq; 10 mu g) or [Cu-64]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')(2) (12.95 MBq). Body weight was monitored, and hematopoietic (CBC), liver (ALT) and kidney [creatinine (SCr)] toxicity were assessed. RIT studies were performed in NOD-sdd mice with s.c. OCIP23 human PnCa patient-derived xenografts (PDX) administered [Cu-64]Cu-NOTA-panitumumab F(ab')(2) (3.7 MBq; 10 mu g), unlabeled panitumumab F(ab')(2) (10 mu g) or normal saline every two weeks. Subsequent studies evaluated RIT with [Cu-64] Cu-NOTA-panitumumab F(ab')(2) (12.95 MBq; 10 mu g) administered alone or combined with GEM and the PARPi, rucaparib administered on a 14-day treatment cycle for up to 6 cycles in NOD-scid mice with s.c. PANC-1 human PnCa xenografts. The radiation absorbed dose in PANC-1 tumors and normal organs in mice after a single i.v. injection of [Cu-64]Cu-NOTA-panitumumab F(ab')(2) (12.95 MBq; 10 mu g) was estimated based on previously reported biodistribution studies of [Cu-64]Cu-NOTA-panitumumab F(ab')(2). Results: No normal tissue toxicity was observed in non-tumor-bearing NOD-scid mice administered up to 3.7 MBq (10 mu g) of [Cu-64]Cu-NOTA-panitumumab F(ab')(2) but slightly increased ALT was noted at 9.25 MBq. Administration of [Cu-64]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')(2) (12.95 MBq; 10 mu g) caused some hematopoietic toxicity but no increase in ALT or SCr or decreased body weight. A slight tumor growth delay and increased survival was noted in NOD-scid mice with s.c. OCIP23 PDX treated with [Cu-64]Cu-NOTA-panitumumab F(ab')(2) (3.7 MBq; 10 mu g) or unlabeled panitumumab F(ab')(2) (10 mu g) compared to normal saline treated mice. RIT with [Cu-64]Cu-NOTA-panitumumab F(ab')(2) (12.95 MBq; 10 mu g) combined with GEM + PARPi for up to 6 cycles was most effective for the treatment of PANC-1 tumors. Tumor doubling time increased to 13.3 +/- 0.9 days vs. 7.8 +/- 3.7 days for RIT alone and 9.3 +/- 22 days for normal saline treatment. Median survival was significantly longer (P< 0.05) than in mice treated with normal saline (35 days) for RIT + GEM PARPi (71 days), GEM + PARPi (44 days) and RIT + GEM (43 days) but not for RIT alone (25 days). RIT - GEM + PARPi provided a longer median survival than RIT (P < 0.01), GEM 4- PARPi (P = 0.01) but not RIT + GEM (P = 023). Nonetheless, PANC-1 tumors grew exponentially in all treatment groups. The absorbed dose in PANC-1 tumors after a single i.v. injection of [Cu-64]Cu-NOTA-panitumumab F(ab')(2) (12.85 MBq; 10 mu g) was 0.8 Gy, while the dose in normal organs ranged from 0.6-12 Gy. Conclusions: We conclude that RIP with [Cu-64]Cu-NOTA-panitumumab F(ab')(2) did not cause significant normal tissue toxicity but was not effective when administered alone for treatment of PnCa xenografts in NOD-scid mice. Combining RIT with GEM and the PARPi, rucaparib enhanced its effectiveness but tumors continued to grow exponentially. Our results suggest that Cu-64 is not feasible for RIT of PnCa due to low tumor absorbed doses. Lu-177 which has a higher abundance of moderate energy beta-particle emissions may be more effective than Cu-64. The hematopoietic toxicity of [Cu-64]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')(2) may be mediated by binding to mouse EGFR expressed on some hematopoietic stem cells. Advances in knowledge and implications for patient care: Direct extension of PET with Cu-64(Cu)-NOTA-panitumumab F(ab')(2) to RFT exploiting the beta-particie and Auger electron emissions of Cu-64 is not feasible. Theranostic approaches that combine PET with Lift employing Lu-177 may be more promising and should be explored. (C) 2020 Elsevier Inc. All rights reserved.

T cells are essential in adaptive immunity and play an indispensable role to eliminate abnormal or virus infected cells. By merging antibody technology and cell engineering, T cells equipped with a chimeric antigen receptor (CAR) can be redirected to the target in a non-MHC restricted fashion. Within the past few years, clinical trials using CAR T cells engineered to recognize B cell cancers have shown high rates of response (70%-90%) and durability of response that are unprecedented in acute and chronic leukaemia. However, severe toxicity has been observed due to massive, and to some extent nonspecific, T cell activation. Although CARs have been improved and investigated heavily, the extent to which a CAR is similar to its parental TCR extra- or intracellularly, and whether critical elements involved in normal TCR signalling are kept or rewired in CAR signalling, are unclear. A better understanding of these questions would greatly facilitate improvements in CAR technology and its usefulness in clinical practice. A CAR targeting a peptide-MHC complex (using an Fv from an antibody that recognizes MHC-peptide: in other words, a TCR-like Ab) mimics the process of TCR recognition and takes advantage of CAR technologies developed so far. It is thus an excellent study object to compare TCR and CAR signalling. Well-built research methods on TCR can be transferred immediately to TCR-like CAR, while the knowledge and findings generated through the studies of TCR-like CAR can in turn renew our perceptions on TCR. TCR-like antibodies targeting the EBV epitopes LMP1125-133, LMP2A426-434 or EBNA1562-570, were engineered as CARs. We find that conformation change, if any, upon ligand binding is not enough to activate downstream TCR signalling, but oligomerization is a crucial factor. Although CAR with a TCR-like specificity can recruit CD8 co-receptor to the immunological synapse, it is dispensable for activation of the T cell. Furthermore, the activation kinetics of CAR and TCR are distinctly different. TCR shows a pulse-like activation, whereas CAR shows an activation that gradually plateaus and remains steady. These unique properties of CAR identified in our study demonstrate that CAR signalling properties may be amenable to modifications leading to better specificity and activity in vivo. Our long-term goal is that the TCR-like CAR can be used to combat EBV induced Nasopharyngeal Carcinoma. Citation Format: Ling Wu, Joanna Brzostek, Shvetha Sankaran, Triscilla Tan, Conrad Chan, Jiawei Yap, Junyun Lai, Paul MacAry, Nicholas Gascoigne. Chimeric antigen receptors based on T cell receptor-like antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1425.

We aimed to investigate the feasibility of conjugating synthetic hexahistidine peptides (His(6)) peptides to panitumumab Fab (PmFab) to enable labeling with [Tc-99m(H2O3(CO)(3)](+) complex and study these radio-immunoconjugates for imaging EGFR-overexpressing tumor xenografts in mice by microSPECT/CT. Fab were reacted with a 10-fold excess of sulfo-SMCC to introduce maleimide functional groups for reaction with the terminal thiol on peptides [CGYGGHHHHHH] that harbored the His(6) motif. Modification of Fab with His(6) peptides was assessed by SDS-PAGE/Western blot, and the number of His(6) peptides introduced was quantified by a radiometric assay incorporating I-123-labeled peptides into the conjugation reaction. Radiolabeling was achieved by incubation of PmFab-His(6) in PBS, pH 7.0, with [Tc-99m(H2O3(CO)(3)](+) in a 1.4 MBq/mu g ratio. The complex was prepared by adding [(TcO4)-Tc-99m](-) to an Isolink kit (Paul Scherrer Institute). Immunoreactivity was assessed in a direct (saturation) binding assay using MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Tumor and normal tissue uptake and imaging properties of Tc-99m-PmFab-His(6) (70 mu g; 35-40 MBq) injected i.v. (tail vein) were compared to irrelevant Tc-99m-Fab 3913 in NOD/SCID mice engrafted subcutaneously (s.c.) with EGFR-overexpressing MDA-MB-468 or PANC-1 human pancreatic ductal carcinoma (PDCa) cell-line derived xenografts (CLX) at 4 and 24 h post injection (p.i.). In addition, tumor imaging studies were performed with Tc-99m-PmFab-His(6) in mice with patient-derived tumor xenografts (PDX) of TNBC, PDCa, and head and neck squamous cell carcinoma (HNSCC). Biodistribution studies in nontumor bearing Balb/c mice were performed to project the radiation absorbed doses for imaging studies in humans with Tc-99m-PmFab-His(6). PmFab was derivatized with 0.80 +/- 0.03 His(6) peptides. Western blot and SDS-PAGE confirmed the presence of His(6) peptides. Tc-99m-PmFab-His(6) was labeled to high radiochemical purity (>= 95%), and the K-d for binding to EGFR on MDA-MB-468 cells was 5.5 +/- 0.4 x 10(-8) mol/L. Tumor uptake of (99m)TcPmFab-His(6) at 24 h p.i. was significantly (P < 0.05) higher than irrelevant Tc-99m-Fab 3913 in mice with MDA-MB-468 tumors (14.9 +/- 3.1%ID/g vs 3.0 +/- 0.9%ID/g) and in mice with PANC-1 tumors (5.6 +/- 0.6 vs 0.5 +/- 0.1%ID/g). In mice implanted orthotopically in the pancreas with the same PDCa PDX, tumor uptake at 24 h p.i. was 4.2 +/- 0.2%ID/g. Locoregional metastases of these PDCa tumors in the peritoneum exhibited slightly and significantly lower uptake than the primary tumors (3.1 +/- 0.3 vs 4.2 +/- 0.3%ID/g; P = 0.02). In mice implanted with different TNBC or HNSCC PDX, tumor uptake at 24 h p.i. was variable and ranged from 3.7 to 11.4%ID/g and 3.8-14.5%ID/g, respectively. MicroSPECT/CT visualized all CLX and PDX tumor xenografts at 4 and 24 h p.i. Dosimetry estimates revealed that in humans, the whole body dose from administration of 740-1110 MBq of Tc-99m-PmFab-His(6) would be 2-3 mSv, which is less than for a Tc-99m-medronate bone scan (4 mSv).

INTRODUCTION:Our objective was to evaluate the effectiveness and normal tissue toxicity of nimotuzumab labeled with the Auger electron (AE)-emitter, 111In ([111In]In-Bn-DTPA-nimotuzumab) for radioimmunotherapy (RIT) of human triple-negative breast cancer (TNBC) or trastuzumab-resistant HER2-positive BC tumors overexpressing epidermal growth factor receptors (EGFR) in athymic mice. METHODS:Normal tissue toxicity was studied in non-tumor-bearing Balb/c mice i.v. administered 9.0 or 28.6 MBq (3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab, unlabeled nimotuzumab (3 mg/kg) or normal saline. A complete blood cell count (CBC) and serum alanine aminotransferase (ALT) and creatinine (Cr) were measured at 14 days. Body weight was monitored. RIT studies were performed in CD-1 athymic mice engrafted s.c. with MDA-MB-468 human TNBC tumors or TrR1 HER2-positive but trastuzumab-resistant BC tumors. Mice were i.v. administered two amounts (15.5 MBq; 3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab separated by 14 days. Control mice received unlabeled Bn-DTPA-nimotuzumab (3 mg/kg) or anti-HER2 [111In]In-Bn-DTPA-trastuzumab or normal saline. Tumor growth and body weight were measured for 6 weeks. A tumor growth index (TGI) and body weight index (BWI) were calculated to compare the tumor size and body weight post-treatment with the pre-treatment values. A tumor doubling ratio (TDR) was calculated for each treatment group compared to control mice receiving normal saline. RESULTS:There was no loss of body weight or decreased red blood cells (RBC) or platelets (PLT) or increased serum ALT or Cr in Balb/c mice administered 9.0 or 28.6 MBq (3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab compared to mice treated with unlabeled Bn-DTPA-nimotuzumab (3 mg/kg) or normal saline. There was a significant decrease in white blood cell (WBC) counts in Balb/c mice receiving 28.6 MBq but not 9.0 MBq of [111In]In-Bn-DTPA-nimotuzumab. Based on these results, an administered amount of 15.5 MBq (3 mg/kg) was selected for RIT studies. Administration of two amounts (15.5 MBq; 3 mg/kg) separated by 14 days to CD-1 athymic mice with s.c. MDA-MB-468 xenografts strongly inhibited tumor growth. The TDR for mice treated with [111In]In-Bn-DTPA-nimotuzumab was 2.15 compared to control mice receiving normal saline. In contrast, treatment with unlabeled Bn-DTPA-nimotuzumab or [111In]In-Bn-DTPA-trastuzumab had no significant effect on tumor growth (TDR = 0.96 and 1.08, respectively). RIT with [111In]In-Bn-DTPA-nimotuzumab also strongly inhibited the growth of TrR1 tumors in athymic mice (TDR = 2.13) compared to unlabeled Bn-DTPA-nimotuzumab (TDR = 0.91). There were no losses in body weight over 6 weeks in tumor bearing mice receiving [111In]In-Bn-DTPA-nimotuzumab, unlabeled Bn-DTPA-nimotuzumab, [111In]In-Bn-DTPA-trastuzumab or normal saline. CONCLUSIONS:[111In]In-Bn-DTPA-nimotuzumab was effective for treatment of TNBC or trastuzumab-resistant HER2-positive human BC tumors in mice that overexpress EGFR at administered amounts that caused no decrease in body weight or normal tissue toxicity in non-tumor-bearing Balb/c mice. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE:Our results suggest that Auger electron RIT with [111In]In-Bn-DTPA-nimotuzumab may provide a novel therapeutic option for patients with TNBC or trastuzumab-resistant HER2-positive BC that overexpresses EGFR. The low normal tissue toxicity of this approach may allow combination with other targeted therapies such as antibody-drug conjugates (ADCs).

Our aim was to synthesize Zr-89-labeled trastuzumab-emtansine (Zr-89-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of Zr-89-DFO-T-DM1 with Zr-89-DFO-trastuzumab. T-DM1 was modified with 3.0 +/- 0.2 desferrioxamine (DFO) chelators for complexing Zr-89 by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl3. SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with Zr-89 at a specific activity of 0.55 MBq/mu g in a 2 M Na2CO3/0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. Zr-89-DFO-T-DM1 exhibited high affinity HER2 binding (K-d = 3.7 +/- 0.4 nM) that was not significantly different than Zr-89-DFO-trastuzumab (4.4 +/- 0.5 nM; P = 0.06). The optimal time for tumor imaging with Zr-89-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with Zr-89-DFO-T-DM1 (10 mu g, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake of Zr-89-DFO-T-DM1 was 4.1-fold significantly higher than Zr-89-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 +/- 4.3%ID/g vs 10.6 +/- 5.4%ID/g, respectively; P < 0.001). Tumor uptake of Zr-89-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher than 89Zr-DFO-trastuzumab (10.1 +/- 3.6%ID/g vs 2.7 +/- 0.5%ID/g; P < 0.001). The higher tumor uptake of 89Zr-DFO-T-DM1 compared to 89Zr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that 89Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET with 89Zr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that 89Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.