Theranostic pretargeted radioimmunotherapy of internalizing solid tumor antigens in human tumor xenografts in mice: Curative treatment of HER2-positive breast carcinoma

Abstract

In recent reports, we have shown that optimized pretargeted radioimmunotherapy (PRIT) based on molecularly engineered antibody conjugates and 177Lu-DOTA chelate (DOTA-PRIT) can be used to cure mice bearing human solid tumor xenografts using antitumor antibodies to minimally internalizing membrane antigens, GPA33 (colon) and GD2 (neuroblastoma). However, many solid tumor membrane antigens are internalized after antibody binding and it is generally believed that internalizing tumor membrane antigens are not suitable targets for PRIT. In this study, we tested the hypothesis that DOTA-PRIT can be performed successfully to target HER2, an internalizing membrane antigen widely expressed in breast, ovarian, and gastroesophageal junction cancers.Methods: DOTA-PRIT was carried out in athymic nude mice bearing BT-474 xenografts, a HER2-expressing human breast cancer, using a three-step dosing regimen consisting of sequential intravenous administrations of: 1) a bispecific IgG-scFv (210 kD) format (BsAb) carrying the IgG sequence of the anti-HER2 antibody trastuzumab and the scFv “C825” with high-affinity, hapten-binding antibody for Bn-DOTA (metal) (BsAb: anti-HER2-C825), 2) a 500 kD dextran-based clearing agent, followed by 3) 177Lu-DOTA-Bn. At the time of treatment, athymic nude mice bearing established subcutaneous BT-474 tumors (medium- and smaller-sized tumors with tumor volumes of 209 ± 101 mm3 and ranging from palpable to 30 mm3, respectively), were studied along with controls. We studied single- and multi-dose regimens. For groups receiving fractionated treatment, we verified quantitative tumor targeting during each treatment cycle using non-invasive imaging with single-photon emission computed tomography/computed tomography (SPECT/CT).Results: We achieved high therapeutic indices (TI, the ratio of radiation-absorbed dose in tumor to radiation-absorbed dose to critical organs, such as bone marrow) for targeting in blood (TI = 28) and kidney (TI = 7), while delivering average radiation-absorbed doses of 39.9 cGy/MBq to tumor. Based on dosimetry estimates, we implemented a curative fractionated therapeutic regimen for medium-sized tumors that would deliver approximately 70 Gy to tumors, which required treatment with a total of 167 MBq 177Lu-DOTA-Bn/mouse (estimated absorbed tumor dose: 66 Gy). This regimen was well tolerated and achieved 100% complete responses (CRs; defined herein as tumor volume equal to or smaller than 4.2 mm3), including 62.5% histologic cure (5/8) and 37.5% microscopic residual disease (3/8) at 85 days (d). Treatment controls showed tumor progression to 207 ± 201% of pre-treatment volume at 85 d and no CRs. Finally, we show that treatment with this curative 177Lu regimen leads to a very low incidence of histopathologic abnormalities in critical organs such as bone marrow and kidney among survivors compared with non-treated controls.Conclusion: Contrary to popular belief, we demonstrate that DOTA-PRIT can be successfully adapted to an internalizing antigen-antibody system such as HER2, with suffi...

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses

“…Female athymic nude mice (strain: Hsd:Athymic Nude-Foxn 1nu , Envigo, aged 6-8 weeks, average weight for six-week-old and eight-week-old animals: 18.6 and 21.0 g, respectively) were used for all experiments. The subcutaneous BT-474 tumor model 20 , SW1222 tumor model 18 , 19 , and IMR32 or luciferase gene reporter transfected IMR32 (IMR-32/luc) tumor model 17 (~50-900 mm 3 by caliper measurement, assuming ellipsoid geometry for calculation of tumor volume) was used for targeting of HER2, GPA33, or GD2 antigens, respectively, with minor modifications (described in SI). All animal experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center following National Institutes of Health guidelines for animal welfare.…”

“…For all DOTA-PRIT studies, mice were given three separate intravenous (i.v.) injections: (1) 250 µg (1.19 nmol) of anti-tumor/C825 BsAb (anti-GPA33: huA33-C825 18 , 19 ; anti-HER2: anti-HER2-C825 20 ; anti-GD2: hu3F8-C825 17 ) 28 h prior to radiohapten administration; and (2) CA (dextran-CA: 62.5 µg, 0.125 nmol dextran, 7.625 nmol (Y)DOTA (unless otherwise noted) or, alternatively, dendrimer-CA: 25 µg, 2.76 nmol (Y)DOTA 30 ) 4 h prior to administration of (3) radiohapten (as described). Note: the dendrimer-CA was recently developed by our group as an alternative to the dextran-CA in anticipation of clinical translation, and was found to have a similar impact on in vivo pretargeting of [ 177 Lu]LuDOTA-Bn 30 .…”

“…For initial pretargeting of [ 225 Ac]DOTA-Bn, the GD2 system was used as a model. The GD2 model was used for preliminary testing of DOTA-PRIT with [ 225 Ac]DOTA-Bn since we determined for [ 177 Lu]LuDOTA-Bn, the TI for tumor-to-blood to be highest in that model (TI = 142) 17 in comparison to HER2 (TI = 28) 20 and GPA33 (TI = 73) 19 . Ten IMR-32 tumor-bearing mice were injected with BsAb, dextran-CA (100 µg, 0.2 nmol dextran, 29 nmol of (Y)DOTA), and then an equimolar amount of either [ 225 Ac]DOTA-Bn 31 or, for comparison, [ 177 Lu]LuDOTA-Bn 17 (1.85 MBq and 3.7 MBq of 177 Lu and 225 Ac, respectively, 8-10 pmol; n = 5/radiohapten).…”

“…We have successfully applied DOTA-PRIT to a variety of solid tumor-associated antigens for high-TI targeting of β-emitting isotopes, including GD2 17 , GPA33 18 , 19 , and HER2 20 (β-DOTA-PRIT). In our hands, β-DOTA-PRIT now reliably leads to cures in animal tumors with minimal radiotoxicity.…”

Combination therapy with B7H3-redirected bispecific antibody and Sorafenib elicits enhanced synergistic antitumor efficacy

“…Recently, immunotherapy has emerged as a beneficial tool for cancer therapy by activating the immune system to fight against tumor cells, including antibodies, vaccines and engineered cells 6 - 8 . Among these treatments, bispecific T-cell-engaging antibody (BiTE), which promotes T cell infiltrating in tumors and facilitating the efficacy of tumor-killing, constitutes the most promising approach 9 - 11 .…”