Probes for HER2

Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

Gene-protein assay (GPA), a combination of immunohistochemistry and dual in situ hybridization, allows simultaneous visualization of HER2 protein and gene on a single slide. We aimed to clarify the clinical significance of HER2 intratumoral heterogeneity (ITH) using GPA. We investigated the relationships between various HER2 ITH indicators and clinical course in 102 patients with HER2-positive breast cancer, treated with neoadjuvant trastuzumab and chemotherapy. Five representative microscopic images were captured from each GPA slide of pre-therapeutic biopsy materials. All evaluable cancer cells in the images were individually assessed for HER2 gene copy number and protein expression. Mean and coefficient of variation (CV) of both gene copy number and protein category were calculated, and each was divided into negative, equivocal, and positive. Based on their combined status, cancer cells were classified into nine types. Pathological complete response (pCR) to neoadjuvant treatments showed positive relationships to mean gene copy number (P < 0.001), mean protein category (P < 0.001), and proportion of gene- and protein-positive tumor cells (P < 0.001) and showed negative relationships to the CV of protein category (P < 0.001) and the proportion of gene-amplified but protein-negative tumor cells (P = 0.002). Two diagnostic models, created by combining clinicopathological factors and ITH indicators, showed excellent potential diagnostic ability for pCR (mean gene copy number and protein category CV; AUC = 0.837, proportion of gene- and protein-positive tumor cells; AUC = 0.831). HER2 ITH quantified by GPA is a potential predictive indicator for efficacy of HER2-targeted treatment.

Background: Adoptive T cell therapies have led to remarkable advances in hematologic cancers but with less effect in ST. Actively recruited tumor associated macrophages (TAM) are abundant in the ST microenvironment (TME) and typically display immunosuppressive behavior. Macrophages engineered to be proinflammatory may be an ideal vector for adoptive ST cellular therapy. Engineered CAR-M selectively recognize and phagocytose antigen overexpressing cancer cells, reprogram TME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis in many cancers (Table 1). CT-0508 is a cell product comprised of autologous monocyte-derived proinflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies show that CT-0508 induces targeted cancer cell phagocytosis while sparing normal cells, decreases tumor burden and prolongs survival, and was safe and effective in a semi-immunocompetent mouse model of human HER2-overexpressing ovarian cancer. Methods: This FIH Phase 1 study is evaluating safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary efficacy in 18 subjects with locally advanced/unresectable or metastatic ST overexpressing HER2, with progression on available therapies, including anti-HER2 therapies. Filgrastim is used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis prior to CT-0508 CAR macrophage infusion. Group 1 subjects receive CT-0508 on D1, 3, & 5. Group 2 subjects will receive full dose on D1. A Safety Review Committee will review dose limiting toxicities. Pre/post-treatment biopsies and blood samples will be collected for correlative analysis of immunogenicity, trafficking (PCR, RNA scope), CT-0508 persistence in blood and tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Table 1. Her2 Overexpression Across Tumor Types Tumor HER2 Overexpression (%) Bladder 8-70 Salivary duct 30-40 Gastric 7-34 Ovarian 26 Breast 11-25 Salivary mucoepidermoid 17.6 Esophageal 12-14 Gallbladder 9.8-12.8 Cholangiocarcinoma 6.3-9 Colorectal 1.6-5 Cervical 2.8-3.9 Uterine 3 Testicular 2.4 Citation Format: Kim A. Reiss, Yuan Yuan, Naoto T. Ueno, Yara Abdou, Debora Barton, Ramona F. Swaby, Amy Ronczka, Daniel J. Cushing, Sascha Abramson, Thomas Condamine, Michael Klichinsky, E. Claire Dees. A phase 1, first in human (FIH) study of autologous anti-HER2 chimeric antigen receptor macrophages (CAR-M) in HER2-overexpressing solid tumors (ST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT524.

Although RNA plays a vital role in the process of gene expression, it is less used as an in situ biomarker for clinical diagnostics compared to DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules themselves. To tackle this issue, methods that are sensitive and specific are needed. Here we present an RNA single molecule chromogenic in situ hybridization assay based on DNA probe proximity ligation and rolling circle amplification. When the DNA probes hybridize into close proximity on the RNA molecules, they form V shape structure and mediate the circularization of circle probes. Thus, our method was termed vsmCISH. We not only successfully applied our method to assess HER2 RNA mRNA expression status in invasive breast cancer tissue, but also to investigate the utility of albumin mRNA ISH for differentiating primary from metastatic liver cancer. The promising results on clinical samples indicates the great potential of our method to be applied in the diagnosis of disease using RNA biomarkers.

TPS668 Background: Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and are abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically display immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR on the macrophages allow them to selectively recognize and phagocytose antigen overexpressing cancer cells. CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis in many cancers (Table). CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden and prolonged survival in relevant models. CT-0508 cells were safe and effective in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. Methods: This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking and preliminary evidence of efficacy of investigational product CT-0508 in 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2, who have failed available therapies, including anti-HER2 therapies when indicated. Filgrastim is being used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product is manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. Group 1 subjects receive CT-0508 infusion split over D1, 3 and 5. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and in the tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Clinical trial information: NCT04660929. [Table: see text]