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Over the last decade, immunotherapy has rapidly changed the therapeutic landscape and prognosis for many hematologic malignancies and adult solid tumors. Despite this success, immunotherapy for pediatric solid tumors remains in the early stages of development, and significant clinical benefit has yet to be realized, with anti-GD2 for neuroblastoma being the exception. The limited neoepitope expression and paucity of T-cell infiltration into the immunosuppressive tumor microenvironment have hampered current established immunotherapies. Emerging approaches to recruit T cells, to convert phenotypically “cold” into “inflamed” tumors, and to vastly improve therapeutic indices hold exceptional promise. Here, we review these approaches, highlighting the role of the tumor microenvironment and novel antibody platforms to maximize the full clinical potential of immunotherapy in pediatric oncology.

Supplemental Tables

Supplemental Table S1: Affinities relative to hu3F8-IgG1. Supplemental Table S2: Affinities to both human and mouse FcR(n). Supplemental Figure S1: Kinetic analysis of in vitro binding of antibodies to CD16A(158V) by SPR. Supplemental Figure S2: Blood clearance of the antibodies in nude mice.

Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >105-fold selectivity over normal tissues, releasing Th1 cytokines (TNFα, IFNγ, and IL2) when GD2(+) tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells. Cancer Immunol Res; 3(3); 266–77. ©2014 AACR.

Supplemental Figure S1: Tumor free survival in humanized DKO mice. Supplemental Figure S2: T cell survival in the sc tumors. Supplemental Figure S3: Efficacy of hu3F8-BsAb vs. 5HLDS(15)BA(Y) in humanized DKO mice. Supplemental Figure S4: Cytokine release from different BsAb formats.

Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26–27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic “cure”) at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42–52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

Figure S4 shows the result of CD45RO and CD25 staining using cells from TDCC assay

Supplementary Data

This table provides the MFI values of different cell lines using huA33-BsAb

This file contains supplementary material and methods for some supplementary data

Despite progress in the treatment of colorectal cancer, curing metastatic colorectal cancer remains a major unmet medical need worldwide. Here, we describe a T-cell–engaging bispecific antibody (T-BsAb) to redirect polyclonal cytotoxic T cells to eradicate colorectal cancer. A33, a murine antibody specific for GPA33, was humanized to huA33 and reformatted to huA33-BsAb, based on a novel IgG(L)–scFv platform by linking the anti-CD3 huOKT3 scFv to the carboxyl end of the light chain. This T-BsAb was stably expressed in CHO cells and purified as a stable monomer by HPLC, retaining immunoreactivity by FACS through 30 days of incubation at 37°C. In vitro, it induced activation and expansion of unstimulated T cells and elicited potent T-cell–dependent cell-mediated cytotoxicity against colon and gastric cancer cells in an antigen-specific manner. In vivo, huA33-BsAb inhibited the colon and gastric cancer xenografts, in both subcutaneous and intraperitoneal tumor models. More importantly, both microsatellite instable and microsatellite stable colorectal cancer were effectively eliminated by huA33-BsAb. These preclinical results provide further support for the use of IgG(L)–scFv platform to build BsAb, and especially one targeting GPA33 for colorectal cancer. These preclinical results also support further development of huA33-BsAb as a potential immunotherapeutic. Mol Cancer Ther; 17(10); 2164–75. ©2018 AACR.

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Supplementary Data from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

Purpose: The norepinephrine transporter (NET) is a critical regulator of catecholamine uptake in normal physiology and is expressed in neuroendocrine tumors like neuroblastoma. Although the norepinephrine analog, meta-iodobenzylguanidine (MIBG), is an established substrate for NET, 123I/131I-MIBG has several clinical limitations for diagnostic imaging. In the current studies, we evaluated meta-[18F]-fluorobenzylguanidine ([18F]-MFBG) and compared it with 123I-MIBG for imaging NET-expressing neuroblastomas.Experimental Design: NET expression levels in neuroblastoma cell lines were determined by Western blot and 123I-MIBG uptake assays. Five neuroblastoma cell lines and two xenografts (SK-N-BE(2)C and LAN1) expressing different levels of NET were used for comparative in vitro and in vivo uptake studies.Results: The uptake of [18F]-MFBG in cells was specific and proportional to the expression level of NET. Although [18F]-MFBG had a 3-fold lower affinity for NET and an approximately 2-fold lower cell uptake in vitro compared with that of 123I-MIBG, the in vivo imaging and tissue radioactivity concentration measurements demonstrated higher [18F]-MFBG xenograft uptake and tumor-to-normal organ ratios at 1 and 4 hours after injection. A comparison of 4 hours [18F]-MFBG PET (positron emission tomography) imaging with 24 hours 123I-MIBG SPECT (single-photon emission computed tomography) imaging showed an approximately 3-fold higher tumor uptake of [18F]-MFBG, but slightly lower tumor-to-background ratios in mice.Conclusions: [18F]-MFBG is a promising radiopharmaceutical for specifically imaging NET-expressing neuroblastomas, with fast pharmacokinetics and whole-body clearance. [18F]-MFBG PET imaging shows higher sensitivity, better detection of small lesions with low NET expression, allows same day scintigraphy with a shorter image acquisition time, and has the potential for lower patient radiation exposure compared with 131I/123I-MIBG. Clin Cancer Res; 20(8); 2182–91. ©2014 AACR.

MATERIALS AND METHODS Table S1. Biodistribution of [18F]-MFBG in neuroblastoma xenografts. Table S2. Biodistribution of 123I-MIBG in neuroblastoma xenografts.

Purpose: Neuroblastoma is an embryonal tumor with contrasting clinical courses. Despite elaborate stratification strategies, precise clinical risk assessment still remains a challenge. The purpose of this study was to develop a PCR-based predictor model to improve clinical risk assessment of patients with neuroblastoma.Experimental Design: The model was developed using real-time PCR gene expression data from 96 samples and tested on separate expression data sets obtained from real-time PCR and microarray studies comprising 362 patients.Results: On the basis of our prior study of differentially expressed genes in favorable and unfavorable neuroblastoma subgroups, we identified three genes, CHD5, PAFAH1B1, and NME1, strongly associated with patient outcome. The expression pattern of these genes was used to develop a PCR-based single-score predictor model. The model discriminated patients into two groups with significantly different clinical outcome [set 1: 5-year overall survival (OS): 0.93 ± 0.03 vs. 0.53 ± 0.06, 5-year event-free survival (EFS): 0.85 ± 0.04 vs. 0.042 ± 0.06, both P < 0.001; set 2 OS: 0.97 ± 0.02 vs. 0.61 ± 0.1, P = 0.005, EFS: 0.91 ± 0.8 vs. 0.56 ± 0.1, P = 0.005; and set 3 OS: 0.99 ± 0.01 vs. 0.56 ± 0.06, EFS: 0.96 ± 0.02 vs. 0.43 ± 0.05, both P < 0.001]. Multivariate analysis showed that the model was an independent marker for survival (P < 0.001, for all). In comparison with accepted risk stratification systems, the model robustly classified patients in the total cohort and in different clinically relevant risk subgroups.Conclusion: We propose for the first time in neuroblastoma, a technically simple PCR-based predictor model that could help refine current risk stratification systems. Clin Cancer Res; 18(7); 2012–23. ©2012 AACR.

Supplementary Figure from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

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PDF file - 85KB, Table S1. Biodistribution of 18F-MFBG in neuroblastoma xenografts. Table S2. Biodistribution of 123I-MIBG in neuroblastoma xenografts.

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Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26–27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic “cure”) at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42–52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

Over the last decade, immunotherapy has rapidly changed the therapeutic landscape and prognosis for many hematologic malignancies and adult solid tumors. Despite this success, immunotherapy for pediatric solid tumors remains in the early stages of development, and significant clinical benefit has yet to be realized, with anti-GD2 for neuroblastoma being the exception. The limited neoepitope expression and paucity of T-cell infiltration into the immunosuppressive tumor microenvironment have hampered current established immunotherapies. Emerging approaches to recruit T cells, to convert phenotypically “cold” into “inflamed” tumors, and to vastly improve therapeutic indices hold exceptional promise. Here, we review these approaches, highlighting the role of the tumor microenvironment and novel antibody platforms to maximize the full clinical potential of immunotherapy in pediatric oncology.

Bispecific antibodies (BsAb) that engage T cells bind to tumor cells via a tumor-associated antigen and to T cells through surface CD3. BsAbs have promising antitumor properties in vivo. Here, we describe the effects of Fc silencing on BsAb-driven T-cell trafficking to solid tumors. We used BsAbs specific for disialoganglioside GD2 or oncoprotein ErbB2 (HER2) and built on the IgG(L)-scFv platform with or without Fc silencing. We studied the kinetics of T-cell infiltration from blood into solid tumor masses when driven by these BsAbs. We also investigated the therapeutic efficacy of these BsAbs in two mouse models: immunodeficient mice xenografted with patient-derived GD2+ neuroblastoma or HER2+ breast cancer, and human CD3ϵ transgenic mice implanted with a GD2+ murine tumor. BsAbs built with intact Fc domain were unable to drive T cells to tumor, thereby failing to achieve an antitumor effect in mice. T cells became sequestered in lungs by myeloid cells or depleted in circulation. In contrast, when Fc function was silenced by N297A ± K322A mutations, T cells were able to infiltrate into subcutaneous solid tumors, a prerequisite for successful therapy outcome.

Introduction: GD2-SADA is a pre-targeted radioimmunotherapy drug-candidate designed to target radioactive lutetium-177 (in the form of 177Lu-DOTA) to GD2-expressing tumor cells (GD2-SADA:177Lu-DOTA Drug Complex). GD2-SADA is comprised of an anti-GD2 single-chain variable fragment (scFv), an anti-DOTA scFv that binds specifically to DOTA metal chelates, such as 177Lu-DOTA, and a SADA domain based on the human P53 tetramerization sequence, which can self-assemble from a monomeric 60-kDa polypeptide into 240-kDa tetrameric protein. We have previously demonstrated (1) that GD2-SADA has a unique clearance profile that allowed for the delivery of 177Lu-DOTA to tumors with minimal exposure to normal tissues like the bone marrow or kidneys and shrank established neuroblastoma in preclinical mouse models. We now provide additional mechanistic data demonstrating the importance of the SADA domain in providing this potent anti-tumor function. Methods: To study the role of the SADA domain, we designed a modified version of GD2-SADA that could not self-assemble into a tetrameric state by removing the entire SADA domain. The resulting P53(-/-)GD2-SADA protein retained binding to GD2 and Lu-DOTA but remained in a 60 kDa monomeric state in assays. P53(-/-)GD2-SADA and GD2-SADA were compared using SPR to measure antigen binding affinities, flow cytometry to evaluate cell binding, and in preclinical mouse models to evaluate tumor uptake and anti-tumor efficacy. Results: GD2-SADA demonstrated stronger binding affinity to GD2 antigen compared to P53(-/-)GD2-SADA, but comparable binding affinity to Lu-DOTA. In SPECT/CT imaging studies using tumor bearing mice, GD2-SADA demonstrated substantially higher uptake and persistence in GD2-expressing tumors compared with P53(-/-)GD2-SADA. Finally, treatment of tumor bearing mice with 3 cycles of GD2-SADA or P53(-/-)GD2-SADA and 177Lu-DOTA (once per week each for 3 weeks) resulted in potent anti-tumor responses from GD2-SADA, with only modest anti-tumor efficacy from P53(-/-)GD2-SADA at two different dose levels corresponding to equal molar doses (1 GD2-SADA to 1 P53(-/-)GD2-SADA) or equal mass doses (1 GD2-SADA to 4 P53(-/-)GD2-SADA i.e. same number of binding sites). Conclusions: The tetramerizing function of the SADA domain is critically important to the binding activity and anti-tumor efficacy of GD2 SADA. These data confirm that the SADA domain increases tumor antigen binding, uptake and persistence in tumor tissue, and markedly improves anti-tumor responses in preclinical models. References: Santich BH, Cheal SM, Ahmed M, McDevitt MR, Ouerfelli O, Yang G, et al. A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy. Clin Cancer Res Off J Am Assoc Cancer Res. 2021 Jan 15;27(2):532-41. Citation Format: Brian H. Santich, Stine Louise Kjellev, Sarah Cheal, Linlin Wang, Mette Gillberg, Mallika Vadlamudi, Amadou A. Ouattara, Nico Liebenberg, Lone Frost Larsen, Darren R. Veach, Shin Seo, Nai-Kong V. Cheung, Steven M. Larson, Steen Lisby. Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3309.