AMG 509 (Xaluritamig), an Anti-STEAP1 XmAb 2+1 T-cell Redirecting Immune Therapy with Avidity-Dependent Activity against Prostate Cancer.

The tumor-associated antigen STEAP1 is a potential therapeutic target that is expressed in most prostate tumors and at increased levels in metastatic castration-resistant prostate cancer (mCRPC). We developed a STEAP1-targeted XmAb 2+1 T-cell engager (TCE) molecule, AMG 509 (also designated xaluritamig), that is designed to redirect T cells to kill prostate cancer cells that express STEAP1. AMG 509 mediates potent T cell-dependent cytotoxicity of prostate cancer cell lines in vitro and promotes tumor regression in xenograft and syngeneic mouse models of prostate cancer in vivo. The avidity-driven activity of AMG 509 enables selectivity for tumor cells with high STEAP1 expression compared with normal cells. AMG 509 is the first STEAP1 TCE to advance to clinical testing, and we report a case study of a patient with mCRPC who achieved an objective response on AMG 509 treatment. SIGNIFICANCE: Immunotherapy in prostate cancer has met with limited success due to the immunosuppressive microenvironment and lack of tumor-specific targets. AMG 509 provides a targeted immunotherapy approach to engage a patient's T cells to kill STEAP1-expressing tumor cells and represents a new treatment option for mCRPC and potentially more broadly for prostate cancer. See related commentary by Hage Chehade et al., p. 20. See related article by Kelly et al., p. 76. This article is featured in Selected Articles from This Issue, p. 5.

Characterization of an Anti-CD70 Half-Life Extended Bispecific T-Cell Engager (HLE-BiTE) and Associated On-Target Toxicity in Cynomolgus Monkeys.

Bispecific T-cell engager (BiTE) molecules have great potential to treat cancer. Nevertheless, dependent on the targeted tumor antigen, the mechanism of action that drives efficacy may also contribute to on-target/off-tumor toxicities. In this study, we characterize an anti-CD70 half-life extended BiTE molecule (termed N6P) which targets CD70, a TNF family protein detected in several cancers. First, the therapeutic potential of N6P was demonstrated using in vitro cytotoxicity assays and an orthotopic xenograft mouse study resulting in potent killing of CD70+ cancer cells. Next, in vitro characterization demonstrated specificity for CD70 and equipotent activity against human and cynomolgus monkey CD70+ cells. To understand the potential for on-target toxicity, a tissue expression analysis was performed and indicated CD70 is primarily restricted to lymphocytes in normal healthy tissues and cells. Therefore, no on-target toxicity was expected to be associated with N6P. However, in a repeat-dose toxicology study using cynomolgus monkeys, adverse N6P-mediated inflammation was identified in multiple tissues frequently involving the mesothelium and epithelium. Follow-up immunohistochemistry analysis revealed CD70 expression in mesothelial and epithelial cells in some tissues with N6P-mediated injury, but not in control tissues or those without injury. Collectively, the data indicate that for some target antigens such as CD70, BiTE molecules may exhibit activity in tissues with very low antigen expression or the antigen may be upregulated under stress enabling molecule activity. This work illustrates how a thorough understanding of expression and upregulation is needed to fully address putative liabilities associated with on-target/off-tumor activity of CD3 bispecific molecules.

Cancer cell-intrinsic resistance to BiTE therapy is mediated by loss of CD58 costimulation and modulation of the extrinsic apoptotic pathway.

BACKGROUND: Bispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated, efficacy of T-cell engagers (TCEs) in solid tumors settings, molecular determinants of response, and underlying mechanisms of resistance to BiTE therapy require more investigation. METHODS: To uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity, we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies, we validated the roles of two common pathways identified in our screen, T-cell costimulation pathway and apoptosis pathway, as key modifiers of BiTE activity. RESULTS: Our genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy, T-cell costimulation, the apoptosis pathway, chromatin remodeling, and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably, loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor), a gene frequently altered in cancer, led to decreased TCE-mediated cytotoxicity, T-cell activation and antitumor efficacy in vitro and in vivo. Moreover, the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor), whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing, indicating non-redundant costimulatory mechanisms between the two pathways. Additionally, loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator), BCL2L1, and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs, indicating that key regulators of apoptosis, which are frequently altered in cancer, impact tumor responsiveness to BiTE therapy. CONCLUSIONS: This study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo, findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy.

Immunotherapy combinations overcome resistance to bispecific T cell engager treatment in T cell-cold solid tumors.

Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3ε chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8+ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4+ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell-infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8+ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule-mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.

The PSMA-targeting Half-life Extended BiTE Therapy AMG 160 has Potent Antitumor Activity in Preclinical Models of Metastatic Castration-resistant Prostate Cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. EXPERIMENTAL DESIGN: We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. RESULTS: AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6-42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68-labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. CONCLUSIONS: The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675.

Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38/CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release.

PURPOSE: Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell-redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell-recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma. EXPERIMENTAL DESIGN: Anti-CD38/CD3 XmAb T-cell-recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties. RESULTS: AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow-invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented. CONCLUSIONS: These findings support the clinical development of AMG 424, an affinity-optimized T-cell-recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment.

Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.

Endoglin requirement for BMP9 signaling in endothelial cells reveals new mechanism of action for selective anti-endoglin antibodies.

Endoglin (ENG), a co-receptor for several TGFß-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFß and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFß1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFß only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFß1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.

Imaging guided trials of the angiogenesis inhibitor sunitinib in mouse models predict efficacy in pancreatic neuroendocrine but not ductal carcinoma.

Preclinical trials in mice represent a critical step in the evaluation of experimental therapeutics. Genetically engineered mouse models (GEMMs) represent a promising platform for the evaluation of drugs, particularly those targeting the tumor microenvironment. We evaluated sunitinib, an angiogenesis inhibitor that targets VEGF and PDGF receptor signaling, in two GEMMs of pancreatic cancer. Sunitinib did not reduce tumor burden in pancreatic ductal adenocarcinoma (PDAC), whereas tumor burden was reduced in the pancreatic neuroendocrine tumor (PNET) model, the latter results confirming and extending previous studies. To explore the basis for the lack of pathologic response in PDAC, we used noninvasive microbubble contrast-enhanced ultrasound imaging, which revealed that sunitinib reduced blood flow both in PDAC and in PNET, concomitant with a reduction in vessel density; nevertheless, PDAC tumors continued to grow, whereas PNET were growth impaired. These results parallel the response in humans, where sunitinib recently garnered FDA and European approval in PNET, whereas two antiangiogenic drugs failed to demonstrate efficacy in PDAC clinical trials. The demonstration of on-target activity but with discordant benefit in the PDAC and PNET GEMMs illustrates the potential value of linked preclinical and clinical trials.

Differential contribution to neuroendocrine tumorigenesis of parallel egfr signaling in cancer cells and pericytes.

Factors associated with tumor sensitivity to epidermal growth factor receptor (EGFR) inhibitors in the context of wild-type EGFR remain elusive. This study investigates the mechanistic basis of responsiveness to EGFR inhibitors in the RIP1-Tag2 (RT2) mouse model of pancreatic neuroendocrine tumorigenesis (PNET). Upon treatment of RT2 mice with EGFR inhibitors, PNET tumors harboring wild-type, nonamplified alleles of Egfr grow at a markedly reduced rate and display a significant increase in tumor cell apoptosis, as well as reduced neovascularization. The authors identify Tgf-α and Hb-egf as key limiting mediators of separable pathological functions of Egfr in neuroendocrine tumor progression: Tgf-α mutant tumors present with an elevated apoptotic index, whereas Hb-egf mutant lesions exhibit decreased angiogenic switching and neovascularization. This study not only associates Tgf-α and Hb-egf expression with wild-type Egfr oncogenicity but also ascribes the proangiogenic activity of Egfr in this tumor model to a novel mesenchymal Hb-egf/Egfr signaling axis, whereby endothelial and pericyte-derived Hb-egf activates Egfr specifically in tumor-associated perivascular cells, leading to increased pericyte coverage of the tumor endothelium and enhanced angiogenesis.

GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh-Ptch-Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh-Ptch-Smo signaling and is regulated by TGF-beta and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells.