5T4 Antigen Research Articles

Abstract 2623: Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index

Abstract TUB-030 is a novel Antibody-Drug Conjugate (ADC) targeting the oncofetal 5T4 antigen, a glycoprotein, that is functionally involved in regulation of the Wnt signalling pathway and promoting cellular motility and adhesion. Immunohistochemical analysis identified 5T4 overexpressed in many types of cancers, including bladder, lung, breast, stomach, esophagus, head and neck, colon, and ovarian. Analysis in fresh frozen healthy human tissue with the therapeutic mAb candidate of TUB-030 revealed limited cross reactivity. Ethynylphosphonamidate conjugation chemistry was used to build TUB-030 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting 5T4 using a cleavable linker system. Overall, TUB-030 contrasts previous ADC designs targeting 5T4, implementing several layers of differentiation in terms of antibody, payload and conjugation technology. In vitro, TUB-030 is characterized by sub-nanomolar affinity to 5T4, efficient target-mediated internalization and strong cytotoxicity towards cancer cells from different tumor indications with a broad range of 5T4 expression levels. Differently to previous 5T4-targeting ADCs, TUB-030 has efficient bystander activity against co-cultured target-negative cells, which facilitates targeting of tumors with heterogenous 5T4 expression. Pharmacokinetic analysis showed that TUB-030 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation and antibody-like clearance profile contrasting maleimide-conjugated ADCs and enabling most efficient and continued delivery of Exatecan to the tumor site. Single-dose treatment with TUB-030 results in long-lasting tumor regression with high complete remission rates across a variety of both cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) cancer models of multiple tumor indications in vivo, including models with low target expression. Preliminary repeat-dose toxicological assessment of TUB-030 in a pharmacologically relevant non-human primate species demonstrated that TUB-030 is well tolerated. The preclinical results underline that TUB-030 with novel ADC technology has the potential to enable 5T4 as a therapeutic target and provide treatment options in numerous solid cancer indications with high unmet medical need supporting further development towards the clinic. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index [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 2623.

Abstract 2620: BCG016, a first-in-class bispecific antibody-drug conjugate targeting 5T4 and MUC1, demonstrates potent preclinical anti-tumor activity

Abstract 5T4 is a highly expressed tumor-associated antigen associated with adverse clinical outcomes in solid tumors, and is an attractive therapeutic target due to low expression on normal adult tissues. While several therapeutic agents targeting 5T4 antigen are currently being evaluated in human clinical trials, none have yet entered the market. Another target, MUC1, is currently under clinical investigation, with most drugs in clinical trials showing limited efficacy due to shedding of the target antigen MUC1-N. 5T4 and MUC1 are commonly co-expressed in various solid tumors, including lung, breast, colorectal and pancreatic cancers, suggesting that targeting both antigens with a single drug could be a promising therapeutic strategy. We previously identified and evaluated antibodies targeting human 5T4 and MUC1-C (cleaved MUC1) using RenLite® fully human common light chain mice. Here, selected antibodies were constructed into 5T4 × MUC1 bispecific antibodies (bsAb). The 5T4-MUC1 bsAb demonstrated high avidity in multiple human tumor cell lines by flow cytometry. Notably, the parental 5T4 antibody also binds strongly to many tumor cell lines. Internalization assays demonstrate that the internalization of 5T4-MUC1 bsAb in breast cancer cells was enhanced compared to the parental anti-5T4 and anti-MUC1 monoclonal antibodies, as measured by Incucyte® live cell imaging. Subsequently, the 5T4-MUC1 bsAb was then conjugated with monomethyl auristatin E (MMAE) to generate 5T4 x MUC1 bsADC (BCG016). Cytotoxicity assays indicate that BCG016 improved tumor cell killing in vitro compared to benchmark ADCs, and in vivo efficacy studies showed superior anti-tumor efficacy of BCG016 in patient-derived NSCLC xenografts compared to benchmark ADCs and parental ADC combination therapy. In summary, we have generated a novel bispecific ADC targeting 5T4 and the cleaved MUC1-C protein, which remains membrane-bound on tumor cells. The 5T4-MUC1 bsADC showed superior anti-tumor efficacy in PDX models and has the potential to be a novel therapeutic for 5T4 and MUC1 co-expressing tumors. Citation Format: Zhenyan Han, Chengzhang Shang, Yifu Zhang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG016, a first-in-class bispecific antibody-drug conjugate targeting 5T4 and MUC1, demonstrates potent preclinical anti-tumor activity [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 2620.

Abstract LB213: Identification of DM004, a first-in-class anti-5T4/MET bispecific antibody-drug conjugate

Abstract Aberrant MET signaling is frequently found in various types of solid tumors, and is correlated with oncogenic transformation, treatment resistance and poor prognosis. While MET remains an attractive therapeutic target, it is widely expressed on the surface of epithelial and endothelial cells, including normal tissues and tumors. To date, MET-targeting agents are associated with adverse clinical effects, including hypoalbuminemia, peripheral edema and pneumonitis, indicating that alternate treatments and/or modalities are needed. Intriguingly, MET antigen is commonly co-expressed with the oncofetal antigen 5T4 in various cancer types, including head and neck, lung and pancreatic cancer. While 5T4 is highly expressed on primary and metastatic cancers and is associated with adverse clinical outcomes in solid tumors, expression on normal adult tissues is very limited. Although several therapeutic agents targeting 5T4 antigen are currently being evaluated in human clinical studies, none have yet entered the market. To address these challenges, we hypothesized that targeting both MET and 5T4 with a bispecific antibody-drug conjugate (BsADC) could provide a more targeted therapeutic strategy to effectively eliminate tumor cells and reduce systemic toxicity. Here, we report that we have successfully generated two bispecific antibody candidates targeting both 5T4 and MET. The candidates were conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker to generate DM004 BsADCs, i.e., Top1-MMAE and Top2-MMAE. In vitro, DM004 BsAbs demonstrated enhanced internalization in the NCI-H226 cell line compared to its parental monoclonal and monovalent anti-5T4 and anti-MET antibodies. In vivo, DM004 BsADCs exhibited robust anti-tumor activity in cell line-derived and patient-derived xenografts of gastric cancer and lung cancer, respectively. In particular, DM004 Top2-MMAE outperformed benchmark ADCs in lung BP0508 PDX models. In summary, we have identified a novel BsADC which may be a promising future treatment for cancers co-expressing 5T4 and MET. Citation Format: Zhenyan Han, Chengzhang Shang, Wenjuan Dai, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. Identification of DM004, a first-in-class anti-5T4/MET bispecific antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB213.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Abstract 2552: Preclinical development of T-cell receptor therapy targeting the 5T4 tumor antigen on renal cell carcinoma

Abstract Purpose: The cancer-testis tumor antigen 5T4 is expressed on ~ 95% of primary RCC tumors as well as a variety of other tumor types but not on healthy tissue. Consequently, 5T4 has been targeted by antibody and vaccine therapies in cancer patients. Our group has previously isolated high-avidity human CD8+ T-cell clones specific for 5T417-25 and HLA-A2 that are capable of recognizing RCC tumor in vitro and as a murine xenograft. These clones provide the source for T-cell receptor alpha (TRA) and beta (TRB) genes to develop engineered T cells with re-directed cytotoxic specificity for tumors expressing the 5T4 antigen. Experimental Procedures: Targeted single-cell RNA-seq was performed on 5T417-25 specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of TRA and TRB genes. Full length 5T417-25 specific TRA and TRB sequences were then re-constructed and modified by codon optimization and introduction of transmembrane domain cys-cys covalent crosslinking to augment correct alpha and beta chain pairing. TRA and TRB genes were cloned into third generation lentiviral vectors and transduced into CD8+ T cells from healthy donors. Re-directed effector function for target cells expressing 5T4 was measured by chromium release assay and IFN-gamma ELISA. CRISPR/Cas9 gene editing techniques to augment effector activity of engineered T cells by suppressing native TCR expression and disrupting inhibitory co-receptor genes for PD-1, LAG-3, or TIM-3, are also under development. Results: Seven unique TRA-TRB pairs were identified from a panel of 38 5T417-25 specific CD8+ T cell clones. Lentiviral mediated expression of all seven 5T417-25 specific TCR transgene constructs was detectable by 5T417-25/HLA-A2 tetramer immunostaining with transduction efficiency from 30% to 80%. 5T4-TCR transduced CD8+ T cells demonstrated re-directed cytotoxic activity to 5T4+/HLA-A2+ RCC, breast, and colorectal tumor cells, but not to 5T4-/HLA-A2+ normal cells. CRISPR/Cas9 targeting of a conserved sequence in the native TRA constant region was sufficient to suppress native TCR expression on polyclonal CD8+ T cells derived from healthy donors. Characterization of the proliferation kinetics, differentiation phenotypes and effector functions of 5T4-TCR transduced T cells modified by gene editing inhibition of native TRA or inhibitory co-receptors (PD-1, LAG-3 or TIM-3) is ongoing. Conclusions: Transgenic T cell products can be re-directed to recognize a broadly shared epitope of the 5T4 tumor antigen and to kill 5T4+ tumor lines in vitro. Further augmentation of effector activity and specificity by CRISPR/Cas9 gene editing techniques is a developmental priority. Transgenic 5T4-reactive T cells are of interest for future testing as a therapeutic cellular immunotherapy product in patients with 5T4+ tumors. Citation Format: Yuexin Xu, Alicia J. Morales, Michael J. Cargill, Andrea Towlerton, Edus H. Warren, Scott S. Tykodi. Preclinical development of T-cell receptor therapy targeting the 5T4 tumor antigen on renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2552.

Trophoblast Glycoprotein is Associated With a Favorable Outcome for Mesothelioma and a Target for Antibody Drug Conjugates.

The prognosis for patients with mesothelioma is poor, which prompts the need for the development of better treatment options. Antibody drug conjugates (ADCs) are gaining interest as a therapeutic strategy in mesothelioma. Trophoblast glycoprotein (5T4) is an oncofetal protein overexpressed in mesothelioma with low expression in normal tissue and therefore a good candidate for ADC treatment. Here,we evaluated and manipulated 5T4 as a suitable antigen for ADC targeted therapy in patients with mesothelioma. Expression of the 5T4 antigen is evaluated in (primary) mesothelioma cell lines and biopsy specimens, and correlated with clinical outcome. Internalization was assessed in 5T4 expressing cells. The cytotoxicity of three different 5T4-targeting ADCs was tested on (primary) mesotheliomacells. 5T4 was expressed in 10 of 12 (primary) cell lines. Most biopsy specimens stained positive for the 5T4 antigen, with marked differences in staining intensity and percentage of positive cells. High expression correlated with long progression-free survival. Both free antibody and ADCs targeting 5T4 were internalized and entered lysosomal compartments. Cytotoxicity experiments showed that cell lines with a high expression for 5T4 were sensitive to two of three ADCs. Lack of efficacy for the third ADC could be restored by neutralizing lysosomal compartments with chloroquine. The 5T4 antigen is expressed in mesothelioma and 5T4-based ADCs are internalized in lysosomes. Two of three ADCs were capable of killing the mesothelioma cells; the third ADC required additional lysosomal neutralization for its effect. 5T4-based ADCs would be a selective strategy for the treatment of mesothelioma.

Tumor uptake of pegylated diabodies: Balancing systemic clearance and vascular transport

The accumulation, dissemination and clearance of monoclonal antibody-based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)-modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG-modified diabodies improved the PK of the parental diabody from a half-life of 40 min to over 40 h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody-PEG5K showing peak accumulation early on, but with the larger diabody-PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody-PEG20K-B with a hydrodynamic radius (Rh) of 6 nm had superior tumor uptake than the larger diabody-PEG40K-B with Rh of 12 nm, indicating that beyond 6 nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ~6 nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.

Abstract B109: ASN004, a novel 5T4-targeted Dolaflexin ADC, causes complete and durable tumor regression in a variety of tumor xenograft models

Abstract The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors, while showing very limited expression in normal adult tissues. This selective pattern of expression establishes 5T4 as an attractive therapeutic target for the Antibody-Drug Conjugate (ADC) strategy. ASN004 is an anti-5T4 ADC that incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and cytotoxic dolastatin (auristatin) analog warheads, with a drug/antibody ratio of ~15:1. ASN004 shows high affinity for the 5T4 antigen (Kd < 30 pM), with selective binding and internalization to 5T4-expressing tumor cells. Potent, selective cytotoxicity has been determined for a diverse panel of tumor cell lines. ASN004 provides complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a range of 5T4 expression levels. Tumor-free survivors could be achieved in tumor models with a single dose of ASN004, as low as 1 mg/kg iv. The broad, superior activity of ASN004 was demonstrated in a head-to-head study against trastuzumab-DM1, in a low-5T4/high-HER2 expressing tumor model. Finally, efficacy of ASN004 in patient-derived tumor xenograft (PDX) models will be presented. In summary, ASN004 has been established as a promising ADC therapeutic for multiple tumor types, encompassing a wide range of 5T4-expression levels. IND-enabling studies are ongoing in preparation for advancement to clinical development. Citation Format: Roger A. Smith, David J. Zammit, Sanjeeva P. Reddy. ASN004, a novel 5T4-targeted Dolaflexin ADC, causes complete and durable tumor regression in a variety of tumor xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B109.

Abstract 43: ASN004, a novel 5T4-targeted Dolaflexin ADC, achieves complete regressions and tumor-free survivors in a broad variety of solid tumor models

Abstract ASN004 is an Antibody Drug Conjugate (ADC) that targets the 5T4 oncofetal antigen (trophoblast glycoprotein). High expression of 5T4 is observed in a wide range of malignant tumors, while very limited expression is found in normal tissues. ASN004 incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and several cytotoxic dolastatin (auristatin) analog warheads per ADC molecule with a drug/antibody ratio of ~15:1. ASN004 shows high affinity for the 5T4 antigen (Kd < 30 pM) and 5T4-expressing tumor cells; rapid cellular internalization; and potent, selective cytotoxicity. ASN004 provides complete tumor regressions and tumor-free survivors in multiple tumor xenograft models, derived from human tumor cell lines having both high and low 5T4 expression levels. Tumor-free survivors were achieved at doses that are well-tolerated, based on xenograft body weight measures and exploratory toxicology studies in pharmacologically-relevant non-human primates. As well, tumor-free survivors were achieved in xenograft tumor models following administration of a single dose of ASN004, as low as 1 mg/kg iv. The broad activity of ASN004 has been demonstrated by its superior efficacy in a head-to-head study against trastuzumab-DM1, in a low-5T4, high-HER2 expressing tumor xenograft model. Finally, tumor xenografts that initially responded to ASN004 and later showed tumor regrowth, had not developed resistance and responded well to subsequent treatment with ASN004. Overall, ASN004 is demonstrated to be an ADC with high therapeutic potential in multiple tumor types, encompassing a wide range of 5T4-expression levels. IND-enabling toxicology studies are ongoing, in preparation for advancement to clinical studies in patients with solid tumors. Citation Format: Roger A. Smith, David J. Zammit, Sanjeeva P. Reddy. ASN004, a novel 5T4-targeted Dolaflexin ADC, achieves complete regressions and tumor-free survivors in a broad variety of solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2017-43

Abstract 2064: Tumor cells treated with a PBD-based antibody-drug conjugate targeting 5T4 develop acquired resistance due to 5T4 down-regulation and response to alternate therapeutics

Abstract Resistance to antibody-drug conjugates (ADC) has been observed in both preclinical models and in the clinic. It has been previously reported that an ADC targeting CD33 with a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer was active in models of multidrug-resistant (MDR) AML, suggesting that PBDs, delivered as part of a molecular-targeted cancer therapy may be effective in MDR settings. However it could still be possible to develop resistance to PBD-conjugated ADCs though other mechanisms that remain unknown. Studies were conducted to determine if tumor cells have innate resistance to PBDs or an anti-5T4 ADC delivering PBD warhead. It was found that both the ADC and its PBD warhead, SG3199, completely eradicated MDA-MB-361 breast cancer cells and NCI-N87 gastric cancer cells in culture, suggesting a lack of intrinsic resistance in these cell lines. The potential emergence of acquired resistance was investigated by culturing MDA-MB-361 cells in gradually-increasing concentrations of either the ADC or SG3199 for over a year and the variants resistant to either the ADC (361-ADCr) or the PBD warhead (361-PBDr) were established as determined by decreased sensitivity in cytotoxicity assays. Transcriptional profiling characterization of these two cell models demonstrated that neither of them had significant upregulation of P-glycoprotein (P-gp) or other drug efflux transporter genes. Significant 5T4 antigen down-regulation was observed in the 361-ADCr cells but not the 361-PBDr cells and these cells also remained sensitive to alternate treatments including ADCs targeting a different surface antigen with the same PBD warhead, suggesting that down-regulation of target was likely the main driver of resistance in 361-ADCr cells. Further transcriptional profiling analyses conducted on 361-PBDr cells suggest a multi-factorial process may be needed to develop resistance to PBD warheads as differential expression of genes associated with DNA damage and repair, drug metabolism and cell cycle was observed. Similar gene expression profiles were observed in both cultured cells and in tumors derived from these cells. Studies to validate these differentially expressed genes, including proteomic and phosphoproteomic profiling are currently being conducted. Collectively these data could identify how resistance to ADCs conjugated with PBD payloads may develop in the clinic and more importantly, provide insight into developing strategies to overcome such resistance. Citation Format: Shenlan Mao, Andrew Garcia, Tracy Chen, Raghothama Chaerkady, Allison Marreno, Helen Zhong, Dorin Toader, Nazzareno Dimasi, Maureen Kennedy, Philip Howard, Changshou Gao, Herren Wu, Ronald Herbst, David Tice, Jay Harper. Tumor cells treated with a PBD-based antibody-drug conjugate targeting 5T4 develop acquired resistance due to 5T4 down-regulation and response to alternate therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2064. doi:10.1158/1538-7445.AM2017-2064

Abstract 1693: ASN004, a novel 5T4-targeted Dolaflexin™ antibody drug conjugate, causes complete regression in multiple solid tumor models

Abstract ASN004 is an Antibody Drug Conjugate (ADC) that targets the 5T4 oncofetal antigen (trophoblast glycoprotein), which is highly expressed in a wide range of malignant tumors, while having very limited expression in normal tissues. ASN004 incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and several cytotoxic dolastatin (auristatin) analog warheads per ADC molecule (drug/antibody ratio ∼15). ASN004 shows high affinity for the 5T4 antigen and for 5T4-expressing tumor cells. As well, ASN004 shows potent cytotoxicity that is selective for 5T4-expressing tumor cells. ASN004 provides strong tumor regression and tumor-free survivors in multiple tumor xenograft models, at well-tolerated doses as low as 0.3 mg/kg iv. Furthermore, ASN004 causes tumor regression when administered to xenografts bearing more advanced (500 mm3) tumors. Robust, potent efficacy for ASN004 has also been demonstrated in head-to-head comparison studies with relevant reference ADCs. A favorable pharmacokinetics profile was observed in rodents and primates. Dose-range finding exploratory toxicology studies have been completed in both pharmacological and non-pharmacological non-clinical species. Efforts toward IND-enabling safety studies with this promising new agent are in progress. Citation Format: Roger A. Smith, Nitin K. Damle, Sanjeeva P. Reddy, Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Dmitry Gumerov, Elena Ter-Ovanesyan, Liu Qin, Peter U. Park, Timothy B. Lowinger, Sandeep Gupta. ASN004, a novel 5T4-targeted Dolaflexin™ antibody drug conjugate, causes complete regression in multiple solid tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2015-1693

The Tumor Targeted Superantigen ABR-217620 Selectively Engages TRBV7-9 and Exploits TCR-pMHC Affinity Mimicry in Mediating T Cell Cytotoxicity

The T lymphocytes are the most important effector cells in immunotherapy of cancer. The conceptual objective for developing the tumor targeted superantigen (TTS) ABR-217620 (naptumomab estafenatox, 5T4Fab-SEA/E-120), now in phase 3 studies for advanced renal cell cancer, was to selectively coat tumor cells with cytotoxic T lymphocytes (CTL) target structures functionally similar to natural CTL pMHC target molecules. Here we present data showing that the molecular basis for the anti-tumor activity by ABR-217620 resides in the distinct interaction between the T cell receptor β variable (TRBV) 7-9 and the engineered superantigen (Sag) SEA/E-120 in the fusion protein bound to the 5T4 antigen on tumor cells. Multimeric but not monomeric ABR-217620 selectively stains TRBV7-9 expressing T lymphocytes from human peripheral blood similar to antigen specific staining of T cells with pMHC tetramers. SEA/E-120 selectively activates TRBV7-9 expressing T lymphocytes resulting in expansion of the subset. ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells. Furthermore, ABR-217620 activates TRBV7-9 expressing T cell line cells in the presence of cell- and bead-bound 5T4 tumor antigen. Surface plasmon resonance analysis revealed that ABR-217620 binds to 5T4 with high affinity, to TRBV7-9 with low affinity and to MHC class II with very low affinity. The T lymphocyte engagement by ABR-217620 is constituted by displaying high affinity binding to the tumor cells (KD approximately 1 nM) and with the mimicry of natural productive immune TCR-pMHC contact using affinities of around 1 µM. This difference in kinetics between the two components of the ABR-217620 fusion protein will bias the binding towards the 5T4 target antigen, efficiently activating T-cells via SEA/E-120 only when presented by the tumor cells.

Identification of Pre- and Post-Treatment Markers, Clinical, and Laboratory Parameters Associated with Outcome in Renal Cancer Patients Treated with MVA-5T4

The recent approvals of immunotherapeutic agents (Sipuleucel-T and Ipilimumab) for the treatment of different solid tumors gave a boost to the growing cancer immunotherapy field, even though few immunotherapy studies have demonstrated convincingly that there is a direct link between the predicted mode of action of an immunological compound and therapeutic benefit. MVA-5T4 (TroVax®) is a novel vaccine combining the tumor-associated antigen 5T4 to an engineered vector-modified vaccinia Ankara (MVA). MVA helps to express the oncofetal 5T4 antigen and subsequently trigger a tumor-directed immune reaction. The safety and clinical benefit reported in multiple phase I and II clinical trials using MVA-5T4 were encouraging; immune responses were induced in almost all treated patients, and associations between 5T4-specific cellular or humoral responses and clinical benefit were reported in most of the nine phase II trials. In particular, clinical studies conducted in renal cell carcinoma (RCC) patients have demonstrated an association between 5T4-specific (but not MVA) antibody responses and enhanced survival. This review describes the clinical studies using MVA-5T4 conducted in RCC that convincingly demonstrated that an antigen-specific immune response induced by vaccination is associated with enhanced patient survival and is not simply a function of the general “health” of patients. We will also provide our expert opinions on possible future better-designed clinical trials based on relevant biomarkers. In addition, various combinations of MVA-5T4 and different and newer immunomodulator agents with promising clinical benefit will be discussed.

CD8+ T-cell Clones Specific for the 5T4 Antigen Target Renal Cell Carcinoma Tumor-initiating Cells in a Murine Xenograft Model

The tumor antigen 5T4 is frequently expressed at high levels on renal cell carcinoma (RCC) and other epithelial carcinomas. Surveys of normal tissues demonstrate abundant 5T4 expression on placental trophoblast cells with limited expression elsewhere. 5T4 is the target for a therapeutic cancer vaccine (MVA-5T4) that elicits 5T4-specific serological, proliferative, and cytotoxic T lymphocyte (CTL) responses. However, the antitumor activity of 5T4-specific CTL has not been extensively characterized. CD8 T cells from HLA-A2 healthy donors (n=4) or RCC patients (n=2) were stimulated in vitro with the HLA-A2-binding nonamer peptides 5T417-25 or 5T497-105 and screened by flow cytometry with specific tetramers (TET). CD8/TET T-cell clones specific for 5T417-25 or 5T497-105 peptide were isolated from 4/6 and 1/4 donors, respectively. A subset of clones specific for 5T417-25 was cytolytic for MVA-5T4-infected HLA-A2 EBV-transformed lymphoblastoid cell line target cells and for constitutively HLA-A2-expressing and 5T4-expressing RCC tumor cell lines (including A498 RCC). In a xenoengraftment assay, the coinoculation of a representative 5T417-25-specific CTL clone with A498 RCC tumors cells into immune-deficient mice completely prevented growth of A498 tumors. Taken together, these data demonstrate high-avidity CD8 CTL able to recognize the naturally processed 5T417-25 epitope on RCC tumor cells including putative tumor-initiating cells are present in peripheral blood of both healthy donors and RCC patients. CD8T-cell immunity targeting 5T417-25 is therefore of substantial interest both as a potential target for further development of vaccination or adoptive cellular immunotherapy and for immune monitoring studies in association with nonspecific immunotherapies.

Abstract 2530: Long-term tumor regression induced by a novel antibody drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells

Abstract Antibody-drug conjugates (ADCs) represent a promising therapeutic modality for the clinical management of cancer. We have developed a novel ADC that targets 5T4 (also known as TPBG), a tumor-associated antigen that rapidly internalizes. In preclinical models of NSCLC, 5T4 was identified as a marker of undifferentiated tumorigenic cells that express properties of tumor-initiating cells (TICs) and was associated with a highly proliferating (Ki67 positive) cell phenotype. Based on the biological properties of 5T4, we designed the ADC by conjugating an internalizing humanized anti-5T4 antibody (Ab), A1, to the tubulin inhibitor monomethylauristatin F via a maleimidocaproyl (mc) linker (A1mcMMAF) with a drug/antibody ratio of ∼ 4. The A1 Ab has a Kd of 0.9 nM against the 5T4 antigen, binds to a broad range of 5T4 expressing cell lines, and rapidly internalizes (66% internalized within 4h). As an ADC, A1mcMMAF retains similar properties to the unconjugated Ab. In vitro, A1mcMMAF exhibited potent and target-specific cytotoxic activity against a panel of 5T4+ cell lines with an IC50 of 5 ng Ab/ml against high 5T4 expressing cells. In vivo, A1mcMMAF exhibited potent anti-tumor activity in all five tumor models tested (with a broad range of 5T4 expression) and caused long-term regressions monitored up to 100 days after the last dose. Strikingly, animals were cured of disease in every model with doses as low as 3 mg Ab/kg Q4d x 4. In a lung cancer patient-derived xenograft in which 5T4 is not uniformly expressed, but instead is preferentially expressed on the less differentiated sub-population of tumor cells, A1mcMMAF treatment resulted in sustained tumor regression. This result highlights the clinical potential of ADCs that target aggressive cell subpopulations such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered at 10 mg Ab/kg/cycle x 2 to cynomolgus monkeys and had a half-life of ∼5 days. The encouraging efficacy and safety data of the conjugate resulting in high therapeutic index in preclinical models warrants further clinical testing of this ADC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2530. doi:1538-7445.AM2012-2530

Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2+, CD8+ T-cell line, developed against the 5T417–25 peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8+ T-cells were able to kill four out of six HLA-A2+ MPM cell lines but not an HLA-A2− cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients.

Clinical Efficacy of TroVax in the Treatment of Progressive Castration-resistant Prostate Cancer

With approximately 240,890 new cases expected in 2011, prostate cancer remains the leading cause of non-melanoma cancer deaths in men. Immunotherapies using viral vector-based delivery systems targeting tumor-specific antigens are being studied. Viral vector-based delivery systems present tumor-targeted antigens (TAAs) to the immune system while breaking self-tolerance. Modified vaccinia ankara has been combined with the oncofetal antigen 5T4 to create TroVax for the treatment of castration-resistant prostate cancer (CRPC). The 5T4 antigen is highly expressed in a large number of carcinomas, including prostate cancer, but is rarely expressed in healthy tissue. TroVax has been demonstrated to be safe and highly immunogenic, both as monotherapy and in combination with other standard of care therapies in colorectal, renal cell, and prostate cancer. With minimal side effects and the ability to produce a strong immunogenic response, TroVax (MVA-5T4) is a viable addition to the treatment of prostate cancer.

Waif1/5T4 Inhibits Wnt/β-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.

Abstract A215: Antitumor activity of an antibody-drug conjugate targeting 5T4, a putative tumor-initiating cell antigen.

Abstract 5T4 (also known as TPBG) is a transmembrane, tumor-associated protein that rapidly internalizes. In preclinical models of NSCLC, 5T4 was identified as a marker of undifferentiated tumorigenic cells that express properties of tumor-initiating cells (TICs) and was associated with a highly proliferating (Ki67 positive) cell phenotype. In addition, 5T4 has been associated with a worse clinical outcome in NSCLC (Damelin et al, Cancer Res, 2011). We constructed an ADC with a humanized anti-5T4 Ab (A1) linked to the tubulin inhibitor monomethylauristatin F through the noncleavable maleimidocaproyl (mc) linker attached to cysteine residues (A1mcMMAF) with a DAR of approximately 4 in order to target 5T4 expressing tumor cells. Anti-5T4 A1 Ab has a Kd of 0.9 nM against the 5T4 antigen, binds to a broad range of 5T4 expressing cell lines, and rapidly internalizes (66% internalized within 4h). As an ADC, A1mcMMAF retains similar properties to the unconjugated Ab. In vitro, A1mcMMAF exhibited receptor-density dependent cytotoxic activity against a panel of 5T4 + cell lines with an IC50 of 5 ng Ab/ml against high 5T4 expressing cells. A1mcMMAF exerts no inhibitory effect on 5T4 negative cells demonstrating it does not exert off-target activity in vitro. In vivo, A1mcMMAF causes long-term (100 days) tumor regressions at 10 mg Ab/kg in MDAMB435/5T4 tumor xenografts (Q4d×2, high 5T4 expression by IHC) and in the NSCLC patient-derived xenograft 37622A model (Q4d×4, high 5T4 expression by IHC though only a portion of the cells express 5T4). Significant anti-tumor activity was demonstrated at 1 mg/kg. Against the low 5T4 expressing H1975 tumor xenograft, A1mcMMAF causes potent tumor regression in spite of its lack of in vitro inhibitory activity against this cell line. Bioimaging of the H1975 tumor demonstrated good penetration of Alexa 750 labeled A1 Ab into the xenograft. A1mcMMAF exhibited no overt toxicities when administered at 10 mg Ab/kg/cycle × 2 to cynomolgus macques and possessed a half-life of 4 to 7 days. The observation that A1mcMMAF regresses H1975 tumor xenografts while demonstrating poor activity against H1975 in vitro demonstrates the attractiveness of targeting 5T4 in the tumor environment and may be explained by 5T4's principal association with proliferating cells in the tumor. It is also interesting that A1mcMMAF was able to completely regress 37622A xenografts since this patient-derived xenograft presents heterogeneous 5T4 expression, i.e. not all cells demonstrate detectable levels of 5T4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A215.

Highlights of This Issue

Kwon and colleagues explored the role of the nuclear protein HMGB2 in hepatocellular carcinoma (HCC). The mRNA and protein expression levels of HMGB2 were significantly elevated in HCC and increased expression of HMGB2 was associated with poor prognosis. Further, silencing of HMGB2 by siRNA decreased HCC cell survival and overexpression of HMGB2 suppressed cisplatin- and etoposide-induced apoptotic cell death in vitro. These findings suggest that HMGB2 expression has biological and clinical implications as an important prognostic factor associated with antiapoptotic pathways in HCC.The EGFR T790M mutation and MET amplification are the two main molecular mechanisms responsible for acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in patients with non–small cell lung carcinoma (NSCLC). To explore their relationship, Suda and colleagues analyzed lung cancer cell lines and gefitinib-refractory tumors from autopsy patients. They found that these two resistance mechanisms exhibited a reciprocal and complementary relationship, with a MET amplification of four-fold or greater and EGFR T790M being almost mutually exclusive. These findings have implications in both the prevention and therapy of secondary resistance to EGFR TKIs in NSCLC.The 5T4 antigen is expressed on the surface of most solid tumors. Amato and colleagues conducted a phase III study in which modified vaccinia Ankara (MVA) was used to deliver 5T4 (MVA-5T4) to patients with renal cancer. MVA-5T4 was well tolerated, but did not result in significantly enhanced survival relative to controls. Exploratory analyses showed that a 5T4 antibody response was associated with enhanced survival and that several pretreatment hematologic factors may identify patients who receive significant benefit from MVA- 5T4. These observations could lead to the development of an early biomarker of efficacy and help to select patients most likely to benefit from treatment.There has been limited study of dendritic cell (DC) vaccines in the adjuvant setting after resection of metastatic tumors, in general, and colorectal cancer, in particular. Barth and colleagues conducted a clinical trial in which patients were treated with a DC vaccine in the adjuvant setting after resection of metastatic disease. They found that an autologous tumor lysate–pulsed DC vaccine could induce immune responses against autologous tumor in over 60% of patients. Patients who developed an autologous tumor– specific immune response had improved recurrence-free survival.