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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.

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

Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g., of streptavidin–antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive [GD2(+)] tumors. For this purpose, an IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with β-particle–emitting radiometals such as 177Lu and 90Y. A three-step regimen, including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days), was optimized in immunocompromised mice carrying subcutaneous human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were approximately 85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indices (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5/group; tumor volume, 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3,400 cGy), revealed complete tumor response in 5 of 5 animals, with no recurrence up to 28 days after treatment. Tumor ablation was confirmed histologically in 4 of 5 mice, and normal organs showed minimal overall toxicities. All nontreated mice required sacrifice within 12 days (>1.0-cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate subcutaneous GD2(+)-NB in mice while sparing kidney and bone marrow. Mol Cancer Ther; 13(7); 1803–12. ©2014 AACR.

This file contains supplementary material and methods for some supplementary data

Supplementary Figure S1. Modeling comparison of two different therapy regimens. Supplementary Figure S2. Positron-emission tomography imaging data for I-124 labeled bispecific antibody in tumor-bearing mice. Supplementary Figure S3. Biodistribution data I-124 labeled bispecific antibody in tumor-bearing mice. Supplementary Tables 1 and 2 are biodistribution data following various pretargeting experiments with the bispecific antibody. Supplementary Table 3 contains hematological and clinical chemistry data from treatment study.

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.

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.

summary of toxicity data from 177Lu SADA-PRIT experiment against a neuroblastoma cell line

2-step SADA-PRIT imaging by PET/CT

2-step IgG-PRIT imaging by PET/CT (no CA)

Supplementary Data (Tables and Figure legends)

Purpose:Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index.Experimental Design:The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA–BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses.Results:SADA–BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA–BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver.Conclusions:The SADA–BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA–BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377

3-step IgG-PRIT imaging by PET/CT

Immuno-positron emission tomography (immuno-PET) is a powerful tool to noninvasively characterize the in vivo biodistribution of engineered antibodies (Abs). Methods L1 cell adhesion molecule (L1CAM)-targeting HuE71 immunoglobulin G1 (IgG1) and immunoglobulin G4 (IgG4) Abs bearing identical variable heavy and light chain sequences but different fragment crystallizable (Fc) portions were radiolabeled with Zirconium-89 (89Zr) and the in vivo biodistribution was studied in SKOV3 ovarian cancer xenografted nude mice. Results In addition to showing uptake in L1CAM-expressing SKOV3 tumors like its parental counterpart HuE71 IgG1, the afucosylated variant having enhanced Fc-receptor (FcR) affinity showed high nonspecific uptake in lymph nodes. On the other hand, aglycosylated HuE71 IgG1 with abrogated FcR binding did not show lymphoid uptake. The use of IgG4 subclass showed high nonspecific uptake in the kidneys, which was prevented by mutating serine at position 228 to proline (S228P) in the hinge region of the IgG4 Ab to mitigate in vivo fragment antigen-binding (Fab) arm exchange. Conclusion Our findings highlight the influence of Fc-modifications and the choice of IgG subclass on the in vivo biodistribution of Abs and the potential outcomes thereof.

Radioimmunotherapy (RIT) delivered through the cerebrospinal fluid (CSF) has been shown to be a safe and promising treatment for leptomeningeal metastases. Pharmacokinetic models for intraOmmaya antiGD2 monoclonal antibody 131I-3F8 have been proposed to improve therapeutic effect while minimizing radiation toxicity. In this study, we now apply pharmacokinetic modeling to intraOmmaya 131I-omburtamab (8H9), an antiB7-H3 antibody which has shown promise in RIT of leptomeningeal metastases. Serial CSF samples were collected and radioassayed from 61 patients undergoing a total of 177 intraOmmaya administrations of 131I-omburtamab for leptomeningeal malignancy. A two-compartment pharmacokinetic model with 12 differential equations was constructed and fitted to the radioactivity measurements of CSF samples collected from patients. The model was used to improve anti-tumor dose while reducing off-target toxicity. Mathematical endpoints were (a) the area under the concentration curve (AUC) of the tumor-bound antibody, AUC [CIAR(t)], (b) the AUC of the unbound “harmful” antibody, AUC [CIA(t)], and (c) the therapeutic index, AUC [CIAR(t)] ÷ AUC [CIA(t)]. The model fit CSF radioactivity data well (mean R = 96.4%). The median immunoreactivity of 131I-omburtamab matched literature values at 69.1%. Off-target toxicity (AUC [CIA(t)]) was predicted to increase more quickly than AUC [CIAR(t)] as a function of 131I-omburtamab dose, but the balance of therapeutic index and AUC [CIAR(t)] remained favorable over a broad range of administered doses (0.48–1.40 mg or 881–2592 MBq). While antitumor dose and therapeutic index increased with antigen density, the optimal administered dose did not. Dose fractionization into two separate injections increased therapeutic index by 38%, and splitting into 5 injections by 82%. Increasing antibody immunoreactivity to 100% only increased therapeutic index by 17.5%. The 2-compartmental pharmacokinetic model when applied to intraOmmaya 131I-omburtamab yielded both intuitive and nonintuitive therapeutic predictions. The potential advantage of further dose fractionization warrants clinical validation. ClinicalTrials.gov , NCT00089245.

BackgroundSuccess of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of T cell engaging bispecific antibody (BsAb) treatment relies on the successful trafficking and cytolytic activity of T cells in solid tumors. Normalization of tumor vasculature using vascular endothelial growth factor (VEGF) blockades could improve efficacy of BsAb-based T cell immunotherapy.MethodsAnti-human VEGF (bevacizumab, BVZ) or anti-mouse VEGFR2 antibody (DC101) was used as VEGF blockade, and ex vivo armed T cells (EATs) carrying anti-GD2, anti-HER2, or anti-glypican3 (GPC3) IgG-(L)-scFv platformed BsAb were used. BsAb-driven intratumoral T cell infiltration and in vivo antitumor response were evaluated using cancer cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) carried out in BALB-Rag2-/-IL-2R-γc-KO (BRG) mice. VEGF expression on human cancer cell lines was analyzed by flow cytometry, and VEGF levels in mouse serum were measured using VEGF Quantikine ELISA Kit. Tumor infiltrating lymphocytes (TILs) were evaluated using flow cytometry and by bioluminescence; both TILs and tumor vasculature were studied using immunohistochemistry.ResultsVEGF expression on cancer cell lines increased with seeding density in vitro. BVZ significantly reduced serum VEGF levels in mice. BVZ or DC101 increased high endothelial venules (HEVs) in the TME and substantially enhanced (2.1–8.1 fold) BsAb-driven T cell infiltration into neuroblastoma and osteosarcoma xenografts, which was preferential for CD8(+) TILs versus CD4(+) TILs, leading to superior antitumor effects in multiple CDX and PDX tumor models without added toxicities.ConclusionsVEGF blockade using specific antibodies against VEGF or VEGFR2 increased HEVs in the TME and cytotoxic CD8(+) TILs, significantly improving the therapeutic efficacy of EAT strategies in preclinical models, supporting the clinical investigation of VEGF blockades to further enhance BsAb-based T cell immunotherapies.

BackgroundThe prognosis for metastatic Ewing sarcoma family of tumors (EFT) is still poor despite high-dose chemotherapy and radiation treatment. Immunotherapies hold promise, but cancer antigen-targeting immunotherapies have largely failed to induce effective T cell receptor-mediated antitumor response. However, T cell-engaging bispecific antibodies (T-BsAbs) have yet to be adequately explored.MethodsRehumanized STEAP1-IgG was used to build T-BsAb (named BC261) using the 2+2 IgG-[L]-scFv platform carrying the anti-CD3 huOKT3 scFv as the second specificity. Its binding epitope mapping, species cross-reactivity, tumor cell line staining, and in vitro cytotoxicity were investigated thoroughly. Its potency in driving tumor-infiltrating lymphocytes (TILs) was quantified using bioluminescence, correlated with in vivo antitumor response against cell line-derived or patient-derived xenografts (CDXs or PDXs) and compared with anti-STEAP1 T-BsAbs built on representative antibody platforms.ResultsBC261 binding epitope was mapped to its second extracellular domain of STEAP1 shared among canine and primate orthologs. BC261 induced potent cytotoxicity against panels of EFT, prostate cancer, and canine osteosarcoma cell lines despite their low antigen density. BC261 drove significantly more TILs into tumors (30-fold) and exerted superior antitumor effects compared with the other standard BsAb platforms. The antitumor efficacy of BC261 was consistent against EFT and prostate cancer CDXs and PDXs.ConclusionsBC261 was highly efficient in driving T cell infiltration and tumor ablation. Either as stand-alone therapeutics or for ex vivo armed T cells, this novel anti-STEAP1 T-BsAb BC261 has therapeutic potential.

Clearing agents (CAs) can rapidly remove non-localized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multi-step pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a non-radioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying sixteen terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y(3+); molecular weight: 9059 Da). Pretargeting [(177)Lu]LuDOTA-Bn with CCA α-16-DOTA-Y(3+) to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [(177)Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.

Background Humanized 3F8-bispecific antibody (hu3F8-BsAb) using the IgG(L)-scFv format (where scFv is single-chain variable fragment), where the anti-CD3 huOKT3 scFv is fused with the carboxyl end of the hu3F8 light chain, has potent antitumor cytotoxicity against GD2(+) tumors. To overcome the insufficient number and function of T cells in cancer patients, they can be rejuvenated and expanded ex vivo before arming with hu3F8-BsAb for adoptive transfer, potentially reducing toxic side effects from direct BsAb administration. Procedure T cells from normal volunteers were expanded and activated ex vivo using CD3/CD28 beads for 8 days. Activated T cells (ATCs) were harvested and co-incubated with a Good Manufacturing Practice grade hu3F8-BsAb at room temperature for 20 min. These armed ATCs were tested for cytotoxicity in vitro and in vivo against human GD2(+) cell lines and patient-derived xenografts in BALB-Rag2-/- IL-2R-γc-KO mice. Results Hu3F8-BsAb armed ATCs showed robust antigen-specific tumor cytotoxicity against GD2(+) tumors in vitro. In vivo, T cells armed with hu3F8-BsAb were highly cytotoxic against GD2(+) melanoma and neuroblastoma xenografts in mice, accompanied by T-cell infiltration without significant side effects. Only zeptomole (10-21 ) quantities of BsAb per T cell was required for maximal antitumor effects. Tumor response was a function of T-cell dose. Conclusion BsAb armed T cells may have clinical utility as the next generation of cytotherapy combined with recombinant BsAb against human tumors for both adult and pediatrics, if autologous T cells can be activated and expanded ex vivo.

The role of T cells in controlling human cancers is well known. Their success requires continued persistence in vivo and efficient trafficking to tumor sites, requirements shared by other effectors such as Natural Killer (NK) cells. To date, cytokine IL2 remains the only clinically approved cytokine therapy available to expand, maintain, and activate these effector lymphoid cells, but toxicities can be severe. Cytokine IL15 offers similar T cell proliferation and activation properties, but without the unwanted side-effects seen with IL2. Several IL15-cytokine fusion proteins have been developed to improve their in vivo function, typically exploiting the IL15Rα to complex with IL15, to extend serum half-life and increase affinity for IL15β receptor on immune cells. Here we describe a novel IL15 complex incorporating the full-length IL15Rα to complex with wild type IL15 to form spontaneous trimers of dimers (6 IL15 + 6 IL15Rα) during co-expression, resulting in a substantial increase in serum half-life and enhancement of in vivo cytokine effect on IgG or T cell engaging antibody-dependent cell-mediated cytotoxicities, when compared to alternative strategies.

Purpose: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. Experimental Design: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA–BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. Results: SADA–BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA–BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. Conclusions: The SADA–BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA–BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose. See related commentary by Capala and Kunos, p. 377

T cell bispecific antibodies (BsAb) couple cytotoxic T lymphocytes to tumor cells, inducing their destruction. Although there are over 60 classes of BsAb in development, the relative importance of parameters such as interdomain spacing or spatial configuration are largely unknown. Here, we dissected a symmetric dual bivalent BsAb platform (IgG-[L]-scFv: anti-tumor IgG with anti-CD3 scFv fused to the light chains) to explore the importance of valency and spatial configuration for BsAb-induced T cell cytotoxicity. Our results revealed that placing tumor and T cell binding domains on the same side of a BsAb (cis configuration) elicited substantially stronger anti-tumor activity, in vitro and in vivo, compared to positioning them on opposite sides (trans configuration). Moreover, using two cis-modules in the same BsAb further improved cytotoxicity (up to 2,000-fold). Additionally, separating antigen-binding components with a single Ig domain (C(L)) dramatically enhanced cytokine release and in vivo tumor responses compared to smaller (G(4)S(1)) or larger (C(H1)-C(H2)-C(H3)) spacers. These findings provide guidelines for improving BsAb function and highlight the importance of spatial configuration and dual bivalency as development parameters.

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 sufficient TIs and absorbed tumor doses to achieve a high probability of cures of established human breast cancer xenografts while sparing critical organs of significant radiotoxicity.

Objectives Epithelial ovarian carcinoma (EOC) is a common and lethal gynecologic malignancy that frequently presents as advanced staged disease, such as peritoneal carcinomatosis (PC). We previously reported cures in BT-474 murine xenografts with Pretargeted Radioimmunotherapy (PRIT) using 177Lu radiohaptens targeting Her2 via a bispecific antibody (BsAb). Here-in we report the use of PRIT with alpha emitter 225Ac labelled PrDOTA to treat a PC model of SKOV3, a Her2+ cell line of EOC. Methods 6 wk old female athymic nude mice inoculated IP with 1E5 luciferase/GFP-transfected SKOV3 cells were separated into 5 groups (n=10). Treatment mice received 1 or 2 cycles of Anti-Her2-C825 BsAb + [225Ac]PrDOTA (Her2-Targeted), at 14 and 21 days after inoculation, respectively. Control groups received Anti-Her2 BsAb only, Anti-GPA33 BsAb + [225Ac]PrDOTA (Off-Targeted) or no treatment. On cycle day 1, the mice were injected IP with 0.25mg (1.19nmol) BsAb. On cycle day 2, 25µg (2.76nmol) CCA16-DOTAY clearing agent (CA) was given IV 22h from BsAb. Mice in therapy groups were injected IP with 1µCi (0.74-0.79nmol) [225Ac]PrDOTA-Bn 4h after CA. Weekly weights and BLIs with IP cavity ROIs were obtained and normalized to the respective values for each mouse at week 0 pre-treatment. End points: weight loss >20% baseline, moribund, or severe abdominal distension. At 154 days, 15 surviving treatment and 1 untreated control mice were submitted for hematology and histopathology. Results Histologic cures and prolonged survival were demonstrated in treatment mice (17/20 at 133 days) as compared to control mice (12/27 at 133 d, Logrank p0.05; all weeks). BLI of mice treated with 1 and 2 cycle of Her2 PRIT decreased 47% when compared to baseline within 1 week (T test p=0.04), suggesting treatment effects as early as 1 week. There were no differences in weights when compared to baseline (2-way ANOVA p>0.05). While the untreated mouse had high peritoneal adenocarcinoma tumor burden, there was no histologic evidence of viable neoplasia in 15/15 submitted treatment mice. Treatment mice had moderate renal tubular degenerative lesions on histology, but this did not affect renal function based on serum BUN or Cr. All hematologic parameters were within normal limits for treated mice. Conclusions 1 and 2 cycles of [225Ac]PrDOTA-PRIT against Her2 resulted in histologic cures and prolonged survival in IP SKOV3 xenografts with minimal toxicity. The anti-Her2 PrDOTA-PRIT system is a promising theranostic approach for otherwise incurable PC. Citation Format: Sebastian K. Chung, Christopher S. Chandler, Daniela Burnes Vargas, Shin H. Seo, Michael R. McDevitt, Darren Veach, Blesida Punzalan, Xu Hong, Hong-fen Guo, Garrett M. Nash, Andrea Cercek, Nai-Kong V. Cheung, Steven M. Larson, Sarah M. Cheal. Pretargeted radioimmunotherapy using 225Ac for intraperitoneal Her2-expressing epithelial ovarian carcinoma xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P168.

Therapies for acute myeloid leukemia (AML) has barely changed over 30 years, while treatment for other blood cancers have made remarkable leaps forward. Recent advances in genomics have allowed molecular targeted therapies (i.e. FLT3-ITD, IDH, c-KIT inhibitors) extending survival, but most patients still succumb (Burd et al. Nat Med 2020). Therefore, developing more efficient, less toxic, and immune-based therapies for AML is an urgent unmet need. Previous studies showed that stromal cell-derived IL-33 stimulates myeloproliferative neoplasms (Mager et al. J Clin Invest 2015). Stimulation-2 (ST2), IL-33 receptor, contributes to leukemia stem cells (LSCs) survival in Cbfb-MYH11 mice (Wang et al . Sci Rep 2019). We showed that ST2 blockade enhanced graft versus leukemia activity against MLL-AF9 egfp AML after hematopoietic cell transplantation (Zhang et al . Sci Transl Med 2015). We and others, also, found that ST2 is expressed on normal murine and human hematopoietic stem cells (HSCs), respectively (Capitano et al. Blood Cells Mol Dis 2020; Alt et al. Biol Blood Marrow Transplant 2019). These data suggest a leukemia-promoting role of ST2/IL-33 signaling. To determine clinical relevance of ST2 in AML, we generated Kaplan-Meier curves using TCGA (n=173) and TARGET AML (n=187) databases. Decreased survival was observed in patients with high IL1RL1 (ST2 gene) which was validated in an independent database (AMLCG 1999 trial, n=417) (Fig. 1A). Since ST2 is expressed on HSCs, we interrogated if ST2 is expressed on LSCs defined as CD34 +CD38 - in the Princess Margaret Leukemia biobank (n=192), and found ST2 is higher on LSCs vs CD34 -CD38 +/- cells (Fig. 1B). We then sought to analyze ST2 on bone marrow samples comparing complete responders vs refractory patients to note that ST2 expression was increased in refractory patients' LSCs (Fig. 1C). To scrutinize the role of ST2 in initiating leukemogenesis, we performed limiting dilution transplantation using 500, 200, and 50 Lin -Sca-1 +c-KIT +-sorted LSCs from WT vs ST2 -/- MLL-AF9 egfp transduced cells. Frequency of LSCs in ST2 -/- cells was decreased by ~15-fold as opposed to WT cells [1:2141 (1:546-1:8405) vs 1:145 (1:75-1:283), p=3.37e-05] (Fig. 2A). We also tested leukemia maintenance, secondary transplantations from the primary recipients resulted in leukemia growth delay in ST2 -/- vs WT cells which was confirmed in tertiary transplantations (Fig. 2B-E). Self-renewal ability of LSCs is correlated to reactive oxygen species (ROS) (Testa et al. Exp Hematol 2016), and we found that ROS levels in ST2 -/- leukemic cells are markedly diminished in contrast to WT leukemic cells (Fig. 2F). ST2 deficiency in leukemic cells arrests G2/S/M cell cycle progression in LSCs (Fig. 2G-J). These data indicated that ST2 is indispensable for initiating and maintaining LSCs in MLL-AF9 AML. We next developed murine and human Fc-silenced-bispecific antibodies engaging mouse or human ST2 and CD3 (BsAb) built on the IgG[L]-scFv platform with proven ability to drive T cells into human tumors for effective tumor ablation (Santich et al. Sci Transl Med 2020; Park et al. J Immunother Cancer 2021) (Fig. 3A, 3E). Both BsAbs showed >90% purity by HPLC, stability under heat stress and low endotoxin. Animals did not exhibit any in vivo toxicity at BsAb doses of 0.4, 2, 5, 10 μg ip q 3 days x 6 doses (not shown). In the immunocompetent MLL-AF9 mice, murine anti-ST2 BsAb (BC281) treatment (10 μg i.p, 4 days post-AML challenge and given every three days for a total of 6 injections) resulted in extended survival compared to isotype control mice (Fig. 3B). Leukemic cells and LSCs were accordingly decreased in treated vs control group (Fig. 3C-D). We modeled humanized leukemic mice with MOLM-14 egfp cells and weekly injection of human CD8 + T cells in NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice (Fig. 3F). Animals treated with human anti-ST2 BsAb (BC282), using a similar regimen as for the immunocompetent model, led to better survival when compared to animals treated with mutated non-functional anti-ST2 BsAb (BC283) (Fig. 3G). Frequency of MOLM-14 egfp cells was lower in the BC282 vs BC283 group (Fig. 3H). These results suggested that anti-ST2 BsAbs can inhibit AML growth to improve survival. We concluded that ST2 is a potential therapeutic target, and ST2-specific T cell engaging BsAbs represent promising immunotherapeutics for AML. Figure 1 Figure 1. Disclosures Cheung: Medical University of South Carolina: Patents & Royalties: inventor on the ST2 bispecific antibody patent application; Y-mabs Therapeutics and Abpro-Labs Inc: Patents & Royalties: inventor on multiple patents filed by MSK, including those licensed to Ymabs Therapeutics, Biotec Pharmacon, and Abpro-labs; Eureka Therapeutics: Membership on an entity's Board of Directors or advisory committees. Paczesny: Medical University of South Carolina: Patents & Royalties: inventor on the ST2 bispecific antibody patent application.

Objectives: There is increasing interest in the potential role of radioimmunotherapy in the treatment of peritoneal carcinomatosis (PC), locoregional metastasis throughout the peritoneal cavity. We recently described a SADA platform when applied to GD2-specific PRIT using beta and alpha emitting radioisotopes achieved exceptional therapeutic indices (TI) achieving tumor cures without myelo-, renal-, neuro-, or hepatic toxicities (1). We applied SADA technology to a tumor associated antigen GPA33 expressed by colorectal cancer in a PC model, and compared that to a 3 step PRIT which requires a clearing agent (CA), previously reported to produce durable responses including histologic cures of GPA33-expressing SW1222 subcutaneous (2) and intraperitoneal (ip) colorectal xenograft models (3). Methods: SADA BsAb built from a humanized anti-GPA33 single chain Fv (scFv) and a humanized anti-DOTA scFv were expressed using the Expi293 Expression System and purified by affinity chromatography (1). Biochemical purity of >90% was confirmed via size exclusion HPLC and antigen binding confirmed by surface plasmon resonance and by flow cytometry on GPA33(+) cell lines. 8 wk old female athymic nude mice were inoculated ip with 5E6 cells of stably luciferase/GFP-transfected SW1222 in 200 µL media. PC tumor progression was monitored by in vivo BLI. GPA33-SADA and control GD2-SADA were injected intravenously (iv) on day 26, followed 48 hr later with 1 mCi (37 MBq,200 pmol) [177Lu]Lu-DOTA-Bn ip. In the 3 step PRIT group, mice were treated with 250µg (1.19nmol) GPA33-BsAb (n=4) followed 24 hr later by 20 µg (2.21 nmol) of CA iv, before the injection of 1 mCi (37 MBq,200 pmol) [177Lu]Lu-DOTA-Bn ip 4 hr later. In an additional control group, no SADA was given before 1 mCi (37 MBq,200 pmol) [177Lu]Lu-DOTA-Bn ip. Biodistribution was performed 24 hr after injection of ip 177Lu radiohapten. Results: Radioactivity absorbed in harvested ip SW1222 xenograft tumors treated with SADA-GPA33 (4.92 + 1.59 [mean + SD] percentage injected dose per gram [%ID/g]) was greater than that of harvested tumors treated with the traditional 3 step PRIT to GPA33 (2.68 + 0.179 %ID/g, p=0.043) or with negative control SADA-GD2 (0.513 + 0.293 %ID/g, p Conclusions: SADA-PRIT targeting GPA33 has favorable TI for colorectal PC. When compared to 3 step PRIT, SADA showed more selectivity and TI. {{References}}: 1. Santich BH, Cheal SM, Ahmed M, et al. Clin Cancer Res. 2020; 2. Cheal SM, Xu H, Guo HF, et al. Eur J Nucl Med Mol Imaging. 2016;43(5):925-937.3. Chandler C, Cheal SM, Nash G, et al. Journal of Nuclear Medicine. 2020;61. Citation Format: Sebastian Chung, Mao Wang, Darren Veach, Sarah Cheal, Daniela Burnes Vargas, Shin Hyuk Seo, Blesida Punzalan, Brian Santich, Hong-fen Guo, Hong Xu, Garrett Nash, Andrea Cercek, Christopher Chandler, Steven Larson, Nai-Kong Cheung. Self-assembling and disassembling bispecific antibody platform for pretargeted radioimmunotherapy against GPA33 in a xenograft model of colorectal peritoneal carcinomatosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1391.

Radioimmunotherapy of solid tumors using antibody-targeted radionuclides has been limited by low therapeutic indices (TIs). We recently reported a novel 3-step pretargeted radioimmunotherapy (PRIT) strategy based on a glycoprotein A33 (GPA33)–targeting bispecific antibody and a small-molecule radioactive hapten, a complex of 177Lu and S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (177Lu-DOTA-Bn), that leads to high TIs for radiosensitive tissues such as blood (TI = 73) and kidney (TI = 12). We tested our hypothesis that a fractionated anti-GPA33 DOTA-PRIT regimen calibrated to deliver a radiation absorbed dose to tumor of more than 100 Gy would lead to a high probability of tumor cure while being well tolerated by nude mice bearing subcutaneous GPA33-positive SW1222 xenografts. Methods: We treated groups of nude mice bearing 7-d-old SW1222 xenografts with a fractionated 3-cycle anti-GPA33 DOTA-PRIT regimen (total administered 177Lu-DOTA-Bn activity, 167 MBq/mouse; estimated radiation absorbed dose to tumor, 110 Gy). In randomly selected mice undergoing treatment, serial SPECT/CT imaging was used to monitor treatment response and calculate radiation absorbed doses to tumor. Necropsy was done on surviving animals 100–200 d after treatment to determine frequency of cure and assess select normal tissues for treatment-related histopathologies. Results: Rapid exponential tumor progression was observed in control treatment groups (i.e., no treatment or 177Lu-DOTA-Bn only), leading to euthanasia due to excessive tumor burden, whereas 10 of 10 complete responses were observed for the DOTA-PRIT–treated animals within 30 d. Treatment was well tolerated, and 100% histologic cure was achieved in 9 of 9 assessable animals without detectable radiation damage to critical organs, including bone marrow and kidney. Radiation absorbed doses to tumor derived from SPECT/CT (102 Gy) and from biodistribution (110 Gy) agreed to within 6.9%. Of the total dose of approximately 100 Gy, the first dose contributes 30%, the second dose 60%, and the third dose 10%. Conclusion: In a GPA33-positive human colorectal cancer xenograft mouse model, we validated a SPECT/CT-based theranostic PRIT regimen that led to 100% complete responses and 100% cures without any treatment-related toxicities, based on high TIs for radiosensitive tissues. These studies support the view that anti-GPA33 DOTA-PRIT will be a potent radioimmunotherapy regimen for GPA33-positive colorectal cancer tumors in humans.

BackgroundAcute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain.MethodsIn this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo.ResultsIn vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models.ConclusionsThese data support further clinical development of this BsAb for first-in-human phase I clinical trial.

The affinity of therapeutic antibodies for Fcγ receptors (FcγRs) strongly influences their antitumor potency. To generate antibodies with optimal binding and immunologic efficacy, we compared the affinities of different versions of an IgG1 Fc region that had an altered peptide backbone, altered glycans, or both. To produce IgG1 with glycans that lacked α1,6-fucose, we used CHO cells that were deficient in the enzyme UDP-N-acetylglucosamine: α-3-d-mannoside-β-1,2-N-acetylglucosaminyltransferase I (GnT1), encoded by the MGAT1 gene. Mature N-linked glycans require this enzyme, and without it, CHO cells synthesize antibodies carrying only Man5-GlcNAc2, which were more effective in antibody-dependent cell-mediated cytotoxicity (ADCC). Our engineered IgG1, hu3F8-IgG1, is specific for GD2, a neuroendocrine tumor ganglioside. Its peptide mutant is IgG1-DEL (S239D/I332E/A330L), both produced in wild-type CHO cells. When produced in GnT1-deficient CHO cells, we refer to them as IgG1n and IgG1n-DEL, respectively. Affinities for human FcγRs were measured using Biacore T-100 (on CD16 and CD32 polymorphic alleles), their immunologic properties compared for ADCC and complement-mediated cytotoxicity (CMC) in vitro, and pharmacokinetics and antitumor effects were compared in vivo in humanized mice. IgG1n and IgG1n-DEL contained only mannose and acetylglucosamine and had preferential affinity for activating CD16s, over inhibitory CD32B, receptors. In vivo, the antitumor effects of IgG1, IgG1-DEL, and IgG1n-DEL were similar but modest, whereas IgG1n was significantly more effective (P < 0.05). Thus, IgG1n antibodies produced in GnT1-deficient CHO cells may have potential as improved anticancer therapeutics. Cancer Immunol Res; 4(7); 631–8. ©2016 AACR.

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.

GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn–(radiolanthanide metal) complex. PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens 177Lu-or 86Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq 177Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 – 700 mm3) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 – 3.0 based on time to 500-mm3 tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten 86Y-DOTA-Bn. We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts.

Neuroblastoma, a malignancy of multipotent embryonic neural crest cells, is the most common extracranial solid cancer in childhood and most common cancer in infancy. Cellular phenotype has been shown to be an important determinant of the malignant potential in human neuroblastoma cells and tumors. Whereas neuroblastic (N-type) are moderately malignant and nonneuronal (S-type) cells are nonmalignant, I-type stem cells are highly tumorigenic, irrespective of N-myc amplification status. In the present study, we sought to determine which genes were overexpressed in the I-type cells which might characterize and maintain the stem cell state and/or malignancy of human neuroblastoma cancer stem cells. We used a microarray platform to compare the steady-state expression levels of mRNAs from 13 human neuroblastoma cell lines representing the three cellular phenotypes. Using qRT-PCR and Western blot analyses, we identified seven genes whose expression is consistently elevated exclusively in neuroblastoma cancer stem cells: CD133, KIT, NOTCH1, GPRC5C, PIGF2, TRKB, and LNGFR. Moreover, we show that the genes are phenotype specific, as differentiation of I-type BE(2)-C cells to either an N- or S-type morphology results in significantly reduced mRNA expression. Finally, we show that NOTCH1 plays an important role in maintaining the stem cell phenotype. The identification and characterization of these genes, elevated in highly malignant neuroblastoma stem cells, could provide the basis for developing novel therapies for treatment of this lethal childhood cancer.

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development.

Acute myeloid leukemia (AML), the most common acute leukemia in adults and the second most common cancer in children, is still a lethal disease in the majority of patients, but immunologic approaches have improved outcome. Bispecific antibodies (BsAbs) are novel immunotherapeutics that can redirect immune cells against AML. We now report a tetravalent (2+2) humanized BsAb in the immunoglobulin G light chain single chain fragment variable [IgG(L)-scFv] format to engage polyclonal T cells to kill CD33+ AML targets. In vitro, this BsAb demonstrated strong antigen-specific T-cell-dependent cell-mediated cytotoxicity (TDCC) with an 50% effective concentration (EC50) in the femtomolar range that translated into treatment of established human AML IV xenografts in vivo. Importantly, it could redirect intraperitoneally injected T cells to ablate established and rapidly growing extramedullary subcutaneous AML xenografts in vivo. Furthermore, internalization of CD33 upon BsAb binding was identical to that of a bivalent (1+1) heterodimer, both being substantially less than anti-CD33 IgG. In contrast to the heterodimer, the tetravalent IgG-scFv BsAb was >10-fold more efficient in TDCC of AML cells in vitro and in vivo. This BsAb did not react with and did not kill CD38-CD34+ hematopoietic stem cells from cord blood. We conclude that the novel anti-CD33 IgG(L)-scFv BsAb construct reported here is a potential candidate for clinical development.