Cell-dependent Cellular Cytotoxicity Research Articles

Preclinical Characterization of AMG 424, a Novel Humanized T Cell-Recruiting Bispecific Anti-CD3/CD38 Antibody

Introduction: Multiple myeloma (MM) is a highly prevalent hematological malignancy, with a worldwide incidence of over 62,000 patients in 2012. Despite significant progress in the development of MM treatments in the last decade, there is a great unmet medical need for MM patients who relapse on existing therapies. Given the greater time needed for relapses to manifest when minimal residual disease (MRD) is reduced, new therapies should be aimed at eliminating MRD. Bispecific T cell engager (BiTE®) molecules recruit T cells to cancer cells and trigger T cell-dependent cellular cytotoxicity (TDCC). BiTEs are clinically validated for the treatment of hematological malignancies and have the potential to greatly reduce MRD. CD38 is a membrane protein normally expressed by plasma cells, lymphocytes, and other immune cells and is a clinically validated tumor-associated antigen expressed at high levels on MM cells. Herein, we describe for the first time AMG 424, a novel, humanized T cell-recruiting bispecific anti-CD3/CD38 antibody containing an XmAb® Fc domain that cross-reacts with nonhuman primate (NHP) CD3 and CD38. AMG 424 was selected for its affinity for CD3 and CD38 in an effort to overcome the challenges associated with CD38 and CD3 binding.Methods: A panel of eight novel bispecific molecules that bind with various affinities to CD3 and CD38 were tested in a series of in vitro assays, including TDCC against cancer cell lines, autologous B cell depletion, cytokine release, and soluble CD38 interference assays. AMG 424 was selected from this panel and evaluated in vivo for its ability to induce tumor growth inhibition and prolong survival in an orthotopic MOLM-13-luc xenograft model in NOD/SCID gamma mice co-implanted with human T cells. AMG 424 was also tested in a repeat dose study in NHPs in which depletion of peripheral CD38-expressing target cells was monitored as a pharmacodynamic endpoint. Finally, the potential of AMG 424 to be combined with relevant standard-of-care therapies was evaluated in vitro.Results: In vitro, AMG 424 demonstrated complete redirected lysis activity against cancer cells with CD38 surface density as low as ~2,000 copies per cell. Compared with molecules with greater CD3 affinity, AMG 424 triggered the release of less IFN-γ and TNF-α in the presence of target cells. Moreover, in the presence of soluble CD38 (200 ng/mL), the in vitro potency of AMG 424 was decreased less than two-fold, whereas molecules with greater affinity for CD38 were decreased nearly ten-fold. Thus, the reduced affinity of AMG 424 for CD3 and CD38 versus other molecules may improve tolerability and pharmacokinetic variability. In tumor-bearing mice with pre-activated human T cells, AMG 424 induced 98% tumor growth inhibition and prolonged survival by more than 70%. In NHPs, AMG 424 depleted peripheral CD38-expressing target cells. In vitro, AMG 424 activity was minimally affected when combined with existing standard-of-care therapies.Conclusions: AMG 424 is a novel, humanized, bispecific T cell-recruiting anti-CD3/CD38 antibody that targets MM cancer cells through T-cell dependent redirected lysis, an approach with the potential to eliminate MRD. AMG 424 completely cleared target cells in TDCC assays and triggered depletion of target cells in vivo. These results support further investigation of AMG 424 as a single agent or combined with existing standard-of-care therapies. Disclosuresde Zafra:Amgen Inc.: Employment. Balazs:Amgen Inc.: Employment, Equity Ownership. Fajardo:Amgen Inc.: Employment, Equity Ownership. Liang:Amgen Inc.: Employment, Equity Ownership. Zhong:Amgen Inc.: Employment, Equity Ownership. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment. Winters:Amgen Inc.: Employment, Equity Ownership. Frank:Amgen Inc.: Employment, Equity Ownership. Cook:Amgen Inc.: Employment, Equity Ownership. Pearson:Amgen Inc.: Employment, Equity Ownership. Melhem:Amgen Inc.: Employment. Petrovic:Amgen Inc.: Employment, Equity Ownership. Pelham:Amgen Inc.: Employment, Equity Ownership. Friedrich:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Tan:Amgen Inc.: Employment, Equity Ownership. Moody:MedImmune, Inc.: Employment; Amgen Inc.: Equity Ownership. Stevens:Amgen Inc.: Employment, Patents & Royalties. Desjarlais:Xencor, Inc.: Employment. Coxon:Amgen Inc.: Employment, Equity Ownership. Nolan-Stevaux:Amgen Inc.: Employment, Equity Ownership.

Abstract 5654: Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer cells

Abstract Mesothelin (MSLN) is a tumor-associated antigen highly expressed in ovarian tumors. Current treatment options for ovarian cancer have only modest efficacy and there remains a large unmet need for new targeted therapies. We generated targeted T cell engager bispecific antibodies that bind MSLN on tumor cells and CD3 on T cells. The humanized and affinity-optimized MSLN x CD3 bispecific antibodies are in an XmAb® 2+1 Fab2-scFv-Fc format that bind bivalently to MSLN and monovalently to CD3. The affinity of the MSLN-targeting arms was reduced to promote avid binding to MSLN expressing cancer cell lines and to minimize reactivity on normal surrogate cell lines. We correlated the expression of MSLN on cancer cell lines to 191 biopsy cores of serous and mucinous ovarian cancer tissues by IHC to identify surrogate cell lines to test for T cell-dependent cellular cytotoxicity (TDCC) activity. MSLN-transfected A549 cells were found to stain as intensely as ovarian cancer tumors expressing high amounts of MSLN. OVCAR-8 cancer cells were found to correlate with a medium MSLN level density on ovarian cancer tumors while ASPC-1 cells represented low MSLN-expressing tumors. Finally, we correlated expression of MSLN to non-cancer adjacent tissues and identified SKOV3, HT29, MCF7, and A549 (parental) cancer cells as appropriate surrogates of normal tissues. Reduction of MSLN affinity of MSLN x CD3 XmAb® 2+1 bispecific antibodies led to preferential killing of MSLN-high target cells, with up to 45-fold more potent TDCC activity on OVCAR8 and ASPC-1 cancer cells compared to the selected normal surrogate cell lines. Engineering of selective MSLN x CD3 bispecific antibodies allows for preferential killing of cancer cells, potentially minimizing on-target/off-tumor effects that may contribute to dose-limiting toxicities. Citation Format: Veronica G. Zeng, Matthew S. Faber, Matthew J. Bernett, Kendra N. Avery, John L. Zeytounian, Rumana Rashid, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais, Michael Hedvat. Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5654.

Abstract 5663: Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models

Abstract Bispecific antibodies enable therapeutic modalities inaccessible to traditional mAbs, such as T cell engagers that bind both CD3 on T cells and a target antigen on tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Prostate cancer (PC) is one of the most prevalent cancers in men, and end stage (castration-resistant prostate cancer) has no curative treatment option. Prostate specific membrane antigen (PSMA), a type II transmembrane protein with a large extracellular domain, has long generated interest as a therapeutic target. It is highly overexpressed in PC compared to normal tissue, and its expression has been shown to correlate with malignancy. Previous attempts to target PSMA include antibody-based radiotherapy and antibody drug conjugates, which have shown some success but can be hampered by the inherent toxicity of the modality. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for PSMA and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong activity on high PSMA expressing cell lines while minimizing reactivity on low expressing cell lines, matching PSMA's differential expression pattern. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding PSMA antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Matthew Dragovich, Erik W. Pong, Veronica Zeng, Michael Hedvat, Christine Bonzon, Kendra Avery, Rumana Rashid, Connie Ardila, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5663.

Abstract 2286: XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies

Abstract Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. One underexplored TAA is ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) for renal cell carcinoma (RCC). An activation marker for basophils, ENPP3 is a type II transmembrane protein that can promiscuously hydrolyze a large variety of nucleotide-containing molecules. Although its specific function in the tumor microenvironment is unknown, bulk RNAseq data from the TCGA describes broad overexpression of ENPP3 in clear cell and papillary RCC and less frequently in hepatocellular carcinoma (HCC). This pattern of expression has been confirmed by IHC and makes ENPP3 a potential target for a CD3 bispecific approach. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for ENPP3 and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong potency on high ENPP3 expressing cell lines while minimizing reactivity on low expressing cell lines, matching ENPP3's differential expression pattern. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. In vivo potency was evaluated by xenograft tumor models in human PBMC-engrafted NSG mice. In a KU812 tumor model, the lead candidate, XmAb30819, exhibited complete responses both as a single agent and in combination with checkpoint blockade. When tested against RXF393, an RCC cell line that overexpresses ENPP3 upon tumor engraftment, XmAb30819 demonstrated complete responses at all tested doses. Tolerability in non-human primates was evaluated in an escalating dose model and then confirmed in an expansion cohort. Dose-dependent T cell margination was observed, and doses that were active in the mouse tumor studies were well-tolerated. Preclinical studies have demonstrated the safety and efficacy of XmAb30819, an XmAb 2+1 ENPP3 x CD3 bispecific antibody for RCC. Citation Format: Alex Nisthal, Sung-Hyung Lee, Yoon Kyung Kim, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Liz Bogaert, Connie Ardila, Irene W. Leung, Nicole Rodriguez, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2286.

AMG 701, a half-life extended anti-BCMA BiTE®, potently induces T cell-redirected lysis of human multiple myeloma cells and can be combined with IMiDs to overcome the immunosuppressive bone marrow microenvironment

AMG 701 is an anti-BCMA BiTE® with characteristics suitable for once-weekly dosing in multiple myeloma (MM) patients. We here evaluated AMG 701-mediated (i) T cell-dependent cellular cytotoxicity (TDCC) of MM cells with or without MM-supporting cells from the bone marrow (BM) microenvironment, (ii) changes in T cells and (iii) TDCC in combination with immunomodulatory drugs (IMiDs). AMG 701 induced TDCC of (i) BCMA+ MM cells resistant or sensitive to current anti-MM agents including bortezomib (PIs) and IMiDs, (ii) lenalidomide (len)- and pomalidomide (pom)-resistant MM cells in the presence of osteoclasts (OCs), BM stromal cells (BMSCs), or a proliferation-inducing ligand (100 ng/ml).

Abstract 2378: Differential exhaustion on cytokine release (DECREASE) by ERY974, a novel T-cell-redirecting antibody targeting glypican-3: A new type of T-cell exhaustion

Abstract Background: T cell-redirecting antibody (TRAB), which bispecifically binds to CD3 and tumor antigen, is a key player in next-generation cancer immunotherapy (CIT). Because TRAB can redirect T cells regardless of their TCR specificity, it is expected to be efficacious in immune checkpoint inhibitor-resistant tumors. The CD19-targeting bispecific T cell engager, blinatumomab, has been used for the treatment of blood cancers, and CEA-TCB and IMCgp100 have shown promising clinical efficacy in solid tumors as well. ERY974 is another promising TRAB targeting glypican-3 (GPC3) and a phase 1 study is in progress (Ishiguro et al., Sci Transl Med 2017). On the other hand, cytokine release syndrome (CRS) has been recognized as a common side effect of TRAB. Thus, mitigation of CRS is an urgent issue. Intra-patient step-up dosing regimens have been incorporated in clinical trials to reduce cytokine release, but the mechanism behind this phenomenon remains elusive. In this study, we pre-clinically explored the phenomenon and its mechanism by using ERY974. Method & Results: GPC3-expressing human cancer cells were incubated with human PBMCs and ERY974. The incubated PBMCs were harvested and mixed with newly prepared GPC3-expressing cancer cells and higher doses of ERY974. The pre-treated PBMCs showed reduced cytokine production compared to that without pre-treatment, while maintaining the same level of T cell-dependent cellular cytotoxicity (TDCC). Comprehensive gene expression analysis of the pre-treated PBMCs was also conducted. A murine cancer cell line expressing human GPC3 was established and implanted into immune competent mice. A mouse surrogate version of ERY974 (mERY974) was first administered at a low dose expected to elicit weak anti-tumor activity, and mice were then treated with a higher dose of mERY974. Anti-tumor activity was similar regardless of the pre-treatment, but plasma cytokine levels were shown to be reduced in the pre-treated mice upon administration of the higher dose of mERY974. Conclusion: Cytokine production following the high-dose ERY974 treatment was mitigated by the low-dose pre-treatment in vitro and in vivo. Cytotoxic activity did not decrease in this setting, thus demonstrating that the pre-treatment selectively suppressed only cytokine production. It is well known that repeated stimulation of TCR leads to T-cell exhaustion. The phenomenon we observed here can also be characterized as a sort of exhaustion, but is quite unconventional. Thus, we propose a new type of exhaustion and suggest calling it ‘Differential exhaustion on cytokine release (DECREASE)’. Management of CRS is critical for the clinical use of TRAB. A detailed analysis of the mechanism of DECREASE currently in progress, is expected to contribute to the development of desired dosing regimens for this new type of CIT agent. Citation Format: Mayumi Hoshino, Yuji Sano, Yasuko Kinoshita, Yumiko Azuma, Toshiaki Tsunenari, Yoko Kayukawa, Mizuho Noguchi, Takahiro Ishiguro, Shohei Kishishita, Noriaki Sawada, Mika Endo, Junichi Nezu. Differential exhaustion on cytokine release (DECREASE) by ERY974, a novel T-cell-redirecting antibody targeting glypican-3: A new type of T-cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2378.

Preclinical and Nonclinical Characterization of HPN217: A Tri-Specific T Cell Activating Construct (TriTAC) Targeting B Cell Maturation Antigen (BCMA) for the Treatment of Multiple Myeloma

About 31,000 new cases of multiple myeloma (MM) will be diagnosed in the US in 2018. In addition to chemotherapeutic agents, several targeted therapies utilizing distinct mechanisms of action, e.g., proteasome inhibitors (bortezomib, carfilzomib, ixazomib), HDAC inhibitors (panobinostat), Cullin-RING E3 ubiquitin ligase activators (thalidomide, lenalidomide, pomalidomide), and antibodies (daratumumab, elotuzumab) have become available for treating MM. However, MM remains an incurable disease. Patients who relapse after or are refractory to standard of care treatments generally have poor prognosis. In 2018, close to 13,000 patients will die of the disease in the US. Targeting the B cell maturation antigen (BCMA), a BAFF/BLyS and APRIL receptor, for treating MM patients can provide a new treatment approach complementary to existing therapies. CAR-T therapies and an antibody-drug conjugate targeting BCMA have demonstrated early clinical success in the treatment of relapsed refractory MM (RRMM).HPN217 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: an N-terminal single domain antibody (sdAb) that binds to human BCMA, a middle sdAb that binds to human serum albumin (HSA), and a C-terminal single chain Fv (scFv) that binds to CD3ε of the T cell receptor (TCR) complex. HPN217 is a highly stable single polypeptide of ~ 53 kDa expressed by CHO cells. Simultaneous engagement of BCMA on a target MM cell and CD3 on a T cell results in T cell activation, functional differentiation and the eventual lysis of the target MM cell. Engineering of an HSA binding domain into HPN217 represents a unique strategy in extending serum half-life, giving the TriTAC molecule a small molecular size and flexibility. This approach is different from Fc-engineering applied in other CD3-based bispecific T cell engaging molecules.The KD of HPN217 binding to recombinant human BCMA, HSA, and recombinant human CD3ε was determined to be 5.5 nM, 6 nM, and 17 nM, respectively, as measured by biolayer interferometry. Flow cytometric analysis on a panel of T cells from normal donors and BCMA positive and BCMA negative tumor cell lines confirmed binding of HPN217 to its native targets expressed on cell surface. The in vitro pharmacological activity of HPN217 was evaluated by T cell-dependent cellular cytotoxicity (TDCC) assays. In co-cultures of T cells from normal human or cynomolgus monkey donors, target tumor cells, and HSA, HPN217 mediated dose-dependent and BCMA-dependent cytotoxicity with EC50 values ranging from 0.05 to 0.7 nM. Killing was dependent on expression of BCMA on target tumor cells. Concomitant with target tumor cell killing, HPN217 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when BCMA positive tumor cells were presence. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, e.g., TNFα and IFNγ, were detected. Similar T cell activation could be observed using human or cynomolgus monkey whole blood as a source of T cells.Nonclinical in vivo properties of HPN217 were evaluated in xenograft models and a single dose pharmacokinetic (PK) study in cynomolgus monkeys. HPN217 mediated dose-dependent growth suppression against the RPMI-8226 MM model and Jeko-1 mantle cell lymphoma model expressing relatively low levels of 5,600 and 2,200 copies of BCMA per cell, respectively. In the PK study, a single dose of HPN217 at 0.01, 0.1, or 1 mg/kg was given to cynomolgus monkeys. HPN217 exhibited linear PK behavior over this dose range. Serum half-life was in the range of 64 to 85 hours. Serum half-life, volume of distribution, and clearance appeared to be independent of dose. HPN217 was demonstrated to be stable and remained intact up to 3 weeks in vivo as demonstrated by a functional ligand binding assay using recombinant CD3ε and BCMA, respectively, to capture and detect HPN217. Importantly, serum samples collected one week after dosing were as potent as stock HPN217 in MM tumor cell killing in TDCC assays.Collectively, preclinical and nonclinical characterization suggests that HPN217 is an efficacious novel therapeutic candidate that can provide a convenient dosing schedule for patients. A first-in-human phase 1 clinical trial is planned to evaluate HPN217 in RRMM. DisclosuresLaw:Harpoon Therapeutics: Employment. Aaron:Harpoon Therapeutics: Employment. Austin:Harpoon Therapeutics: Employment. Barath:Harpoon Therapeutics: Employment. Callihan:Harpoon Therapeutics: Employment. Evans:Harpoon Therapeutics: Employment. Gamez Guerrero:Harpoon Therapeutics: Employment. Hemmati:Harpoon Therapeutics: Employment. Jones:Harpoon Therapeutics: Employment. Kwant:Harpoon Therapeutics: Employment. Lao:Harpoon Therapeutics: Employment. Lemon:Harpoon Therapeutics: Employment. Patnaik:Harpoon Therapeutics: Employment. Sexton:Harpoon Therapeutics: Employment. Wesche:Harpoon Therapeutics: Employment. Xiao:Harpoon Therapeutics: Employment. Yu:Harpoon Therapeutics: Employment. Yu:Harpoon Therapeutics: Employment.

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell–dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell–mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

Abstract 585: Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy

Abstract BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model. Citation Format: Olivier Nolan-Stevaux, Flordeliza Fajardo, Lily Liu, Suzanne Coberly, Patricia McElroy, Aaron Nazarian, Franziska Bott, Elisabeth Nahrwold, Tobias Raum, Roman Kischel, Ralf Lutterbuese, Patrick Hoffmann, Peter Kufer. Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 585.

Abstract DDT01-05: First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen

Abstract Immune checkpoint inhibitors such as anti-PD1 antibodies have shown promising clinical responses in several solid tumors, however there remain patients who do not show an adequate response. Recent biomarker studies have revealed that the presence of neoantigens in the tumor can determine the level of response, and thus the next challenge will be how to target tumors with a neoantigen level that is too low to be recognized by endogenous cytotoxic T cells. Hope in this area is offered by a T cell-redirecting antibody (TRAB), which bispecifically engages CD3 and a tumor antigen, even at very low expression levels, to activate the inherent cytolytic potential of T cells against target tumor cells. A TRAB is highly potent because T cells are activated only in the presence of the targeted antigens and are not restricted by the specificity of the T cell receptor. Given this very potent cytotoxicity, the key to successfully achieving strong antitumor efficacy while avoiding on-target off-tumor toxicity is to select a highly tumor-selective antigen. Our fully humanized IgG TRAB recognizes CD3 and a highly tumor-selective antigen, glypican-3 (GPC3), which is a fetal protein expressed in a wide variety of tissues during development but suppressed in most adult tissues. On the other hand, an inct101e in GPC3 expression has been reported in hepatocellular carcinoma, gastric cancer, lung squamous cell carcinoma, and other cancers. In nonclinical in vitro pharmacology studies, the anti-GPC3 TRAB elicited activation and proliferation of T cells and T cell-dependent cellular cytotoxicity against a wide variety of GPC3-expressing tumor cells, and showed long-lasting in vivo efficacy against tumor expressing very low levels of GPC3 at a few thousand molecules per cell. Furthermore, in an immunocompetent mouse model using human CD3 transgenic mice, anti-GPC3 TRAB showed strong antitumor efficacy against poorly immunogenic tumors, whereas both the immune checkpoint inhibitors and a conventional ADCC-inducing antibody recognizing GPC3 did not show significant efficacy. Pharmacokinetics and toxicology studies in nonhuman primates showed a plasma half-life comparable to a standard IgG drug, allowing a QW or Q2W regimen in humans, with toxicity which was manageable and reversible; the main observations of transient cytokine elevation and associated clinical symptoms were markedly reduced by steroid premedication. Our anti-GPC3 TRAB, which is supported by proprietary antibody engineering technology (ART-Ig) that enables large-scale GMP manufacturing, has promise as a new approach in cancer immunotherapy. Citation Format: Takahiro Ishiguro, Yasuko Kinoshita, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Mika Kamata-Sakurai, Hirotake Shiraiwa, Akihisa Kaneko, Werner Frings, Shunichiro Komatsu, Junichi Nezu, Mika Endo. First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr DDT01-05.

An EpCAM/CD3 bispecific antibody efficiently eliminates hepatocellular carcinoma cells with limited galectin-1 expression.

There have been several studies suggesting that cancer stem cells (CSCs) contribute to the high rates of recurrence and resistance to therapies observed in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) has been demonstrated to be a biomarker of CSCs and a potential therapeutic target in HCC. Here, we prepared two anti-EpCAM monoclonal antibodies (1H8 and 2F2) and an anti-EpCAM bispecific T cell engager (BiTE) 1H8/CD3, which was derived from 1H8, and used them to treat HCC in vitro and in vivo. The results demonstrated that all of the developed anti-EpCAM antibodies specifically bound to EpCAM. Neither anti-EpCAM monoclonal antibody had obvious anti-HCC activities in vitro or in vivo. However, anti-EpCAM BiTE 1H8/CD3 induced strong peripheral blood mononuclear cell-dependent cellular cytotoxicity in Huh-7 and Hep3B cells but not EpCAM-negative SK-Hep-1 cells. Notably, 1H8/CD3 completely inhibited the growth of Huh-7 and Hep3B xenografts in vivo. Treatment of the Huh-7 HCC xenografts with 1H8/CD3 significantly suppressed tumor proliferation and reduced the expression of most CSC biomarkers. Intriguingly, galectin-1 (Gal-1) overexpression inhibited 1H8/CD3-induced lymphocytotoxicity in HCCs while knockdown of Gal-1 increased the lymphocytotoxicity. Collectively, these results indicate that anti-EpCAM BiTE 1H8/CD3 is a promising therapeutic agent for HCC treatment. Gal-1 may contribute to the resistance of HCC cells to 1H8/CD3-induced lysis.