Gastroesophageal adenocarcinomas (GEAs), including gastric, esophageal, and gastroesophageal junction (GEJ) adenocarcinomas, are common cancers with high morbidity and mortality. In approximately 25% of GEA cases, HER2 is overexpressed/amplified. Patients with advanced/metastatic HER2+ GEA are typically treated with trastuzumab, a HER2-targeted therapy, plus chemotherapy in the first-line setting. Preliminary data suggests that the addition of an immune checkpoint inhibitor to the treatment regimen may further improve patient outcomes. Zanidatamab is a novel, bispecific HER2-targeting monoclonal antibody (mAb) that binds to two non-overlapping extracellular domains (ECD4 and ECD2) on HER2. This bispecific binding forms HER2 clusters and induces greater internalization and downregulation of cell surface HER2 compared to trastuzumab (as observed in preclinical studies). Zanidatamab also causes growth signal reduction and triggers immune-mediated antitumor activity through antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Early studies have shown that zanidatamab has a manageable safety profile with encouraging antitumor activity in HER2+ GEA, when used both as a monotherapy and in combination with chemotherapy in later-line treatment. In the first-line setting in a phase 2 study, zanidatamab plus chemotherapy demonstrated a confirmed objective response rate (ORR) of 75%, median duration of response (DOR) of 16.4 months, and median progression-free survival (PFS) of 12.0 months. Separately, the anti-programmed cell death-1 (PD1) mAb tislelizumab has demonstrated a manageable safety profile and clinical activity in multiple cancers, including gastric and GEJ adenocarcinoma. The combination of zanidatamab with chemotherapy plus tislelizumab is being studied in an ongoing phase 1b/2 study and has recently completed accrual. HERIZON-GEA-01 (NCT05152147; EudraCT#: 2021-000296-36), is a global, randomized, open-label, active-comparator, phase 3 study that will further investigate the efficacy and safety of zanidatamab in combination with chemotherapy with or without tislelizumab as first-line treatment for patients with advanced/metastatic HER2+ GEA. Key eligibility criteria include: age ≥ 18 years, untreated, unresectable locally advanced/metastatic GEA that is HER2+ (IHC3+ or IHC2+/ISH+) per central testing, ECOG PS of 0 or 1, and adequate organ function, including LVEF > 50%. Enrolled patients will be assigned randomly (1:1:1) to either: trastuzumab (6 mg/kg IV Q3W) plus chemotherapy; zanidatamab (1800 mg IV [patient 2 IV Q3W and capecitabine 1000 mg/m2 oral BID on days 1–15) or FP (cisplatin 80 mg/m2 IV Q3W and 5-fluorouracil 800 mg/m2 continuous IV on days 1–5). The primary endpoints of the study are PFS per RECIST v1.1 assessed by blinded independent central review (BICR), and overall survival. Secondary endpoints include: BICR-assessed confirmed ORR and DOR; investigator-assessed PFS, ORR, and DOR; incidence and severity of AEs; and changes in health-related quality of life (HRQoL). 714 patients are planned to be enrolled from ∼300 sites in 30+ countries across North America (not including the US), South America, Europe, Africa, Asia, and Oceania. The study is currently recruiting patients. NCT05152147; EudraCT#: 2021-000296-36. Sponsor. This study is sponsored by Zymeworks Inc. and BeiGene, Ltd.

Metastatic HER2-expressing GEA has limited treatment options and high morbidity and mortality. Zanidatamab (zani), a HER2-targeted bispecific antibody also known as ZW25, was well-tolerated with durable responses (33% confirmed objective response rate [cORR] as monotherapy; 54% cORR with chemo) in patients (pts) with heavily pre-treated metastatic HER2-expressing GEA in a Ph 1 study. In this ongoing Ph 2 trial (NCT03929666), pts with untreated locally advanced/metastatic HER2-expressing GEA are treated with physician's choice of zani + 5FU/leucovorin/oxaliplatin (mFOLFOX6) every 2 weeks, or zani + capecitabine/oxaliplatin (CAPOX) or 5FU/cisplatin (FP) every 3 weeks. Primary objectives are to evaluate safety and antitumor activity. Response assessments are performed every 6 weeks per RECIST 1.1. As of Mar 18, 2021, 30 pts have been treated (zani + either mFOLFOX6 [14], CAPOX [14], or FP [2]; male: 87%; median age: 58 yrs). The median treatment duration was 9 cycles (range, 1-20) and 14 pts remain on treatment. All pts had a treatment-related (zani and/or chemo) adverse event (TRAE), the majority of which were Grade (Gr) 1 or 2 in severity; the most common (≥25% pts) were diarrhea, nausea, peripheral sensory neuropathy, fatigue, decreased appetite, vomiting, and hypokalemia. TRAEs with Gr 3 as the highest grade were reported in 66% of pts overall (79% zani + mFOLFOX6; 57% zani + CAPOX; 50% zani + FP); the most common (≥10% pts) were diarrhea (43%), hypokalemia (10%), and vomiting (10%). 7% experienced Gr 4 TRAEs (hypokalemia and leukopenia) and 10% discontinued treatment due to TRAEs (all in zani + mFOLFOX6). There were no treatment-related deaths. In 22 HER2+ (IHC3+ or ISH+/IHC2+) response-evaluable pts, the cORR was 68.2% and the disease control rate was 90.9%. For the 15 pts with confirmed responses, the duration ranged from 1.4 to 15.4 months, with 10 responses ongoing. Zanidatamab with standard 1L chemo shows an encouraging cORR and response durability in pts with HER2+ GEA with a manageable safety profile. Based on these results, a global Ph 3 study in 1L HER2+ GEA is planned to evaluate zani + chemo (CAPOX or FP) ± the PD-1 inhibitor tislelizumab.

We have previously demonstrated that an intracellular antibody (sFv) directed against erbB2 can achieve a specific cytotoxicity in erbB2 overexpressing cancer cells of varying histogenesis. In order to further delineate the mechanistic basis of the induced apoptosis, transient and stable cotransfections were performed. Transient cotransfection of erbB2 mutant and chimeric molecules demonstrated that the cytoplasmic domain of erbB2, or the homologous cytoplasmic domain of the epidermal growth factor receptor, is required for apoptosis induction. These results were confirmed in assays utilizing differential derivation of stable clones. To examine the effects of varying ratios of the anti-erbB2 sFv and its target erbB2 we performed additional cotransfection experiments in erbB2 negative target cells. When erbB2 levels are held constant, observed cytotoxicity is proportional to the amount of sFv added. In addition, when sFv levels are held constant, increasing levels of cotransfected erbB2 can overcome the apoptotic response. These results indicate that a minimal threshold level of the sFv and its target are required to induce cytotoxicity. To examine this phenomenon in an erbB2 positive cell line, SKOV3 ovarian carcinoma cells were utilized to derive a stable clone expressing low levels of sFv. When this cell line was compared to the parental SKOV3 cell line, it was shown that less exogenous sFv was needed to induce cytotoxicity in the clone already expressing low levels of sFv, indicating that endogenous and exogenous levels of sFv are additive. In summary, the results presented here indicate that the carboxy-terminus of the intracellular domain of the erbB2 molecule is involved in the induction of apoptosis. Furthermore, the expression levels of the sFv and its target protein need to overcome a threshold level in order to achieve a cytotoxic response.

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. Anti-HPV16 E7 intracellular single-chain antibodies (scFvs) were constructed to down-regulate HPV16 E7 oncoprotein in HPV DNA-containing cell lines. In these studies, we transfected anti-E7 scFvs into the HPV16-positive human cervical carcinoma cell lines CaSki and SiHa and tested them for their ability to inhibit cell proliferation and alter the level of HPV16 E7 oncoprotein. Our results showed that anti-HPV16 E7 scFvs inhibited cell proliferation by >85% in CaSki cells and by 95% in SiHa cells. E7 oncoprotein was down-regulated by anti-HPV16 E7 scFv, and its expression was inversely related to the amount of scFv transfected. However, there were no effects of transfecting scFvs alone in HPV-negative cell lines. These results imply that anti-HPV16 E7 scFvs only have specific anti-HPV16 E7 effects on cell proliferation and on the synthesis of virally encoded proteins in HPV-positive cell lines. Thus, transfection of HPV16 E7-positive tumors with antigen-specific scFvs may be a viable strategy for cervical cancer gene therapy.

Originally conceived and applied for the treatment of inherited monogenetic defects such as adenosine deaminase deficiency and cystic fibrosis, gene therapy was later applied to the treatment of cancer. Such a genetic strategy seemed rational given the recognition that cancer typically develops in a multistep process involving alterations of a number of different genes as demonstrated in familial polyposis and colorectal cancer through the work of Vogelstein et al. Because of the numerous alterations that may result in the eventual development of cancer, there is no obvious single choice for a therapeutic gene. Although one may view this as an obstacle, it also allows for a variety of possible therapeutic interventions. This review focuses on the known genetic defects that occur in ovarian cancer, the gene therapy strategies suggested by such defects, and the approaches under current development for the treatment of this disease. As such, this work also describes some of the approved human gene therapy protocols. Finally, an overview of the problems and directions for future growth and research is presented.

Extensive experimental evidence suggests that Bcl-2 promotes cell survival by preventing the onset of apoptosis induced by a variety of stimuli. In addition, Bcl-2 expression has been correlated with resistance and poor response to chemotherapy in a number of cell types. Therefore, this protein represents a logical target for gene therapy strategies designed to achieve selective gene product ablation, In this study, we have developed an approach based upon intracellular expression of single-chain antibodies (sFvs) to achieve modulation of Bcl-2 protein levels in target cells. Using a transient expression system, we show that this intracellular anti-Bcl-2 sFv mediates specific reduction of Bcl-2 levels. This effect significantly enhances drug-mediated cytotoxicity in Bcl-2-overexpressing tumor cells, whereas transfection of the anti-Bcl-2 sFv did not affect the growth rate of the tumor cell lines. This method thus represents a novel and efficient way to selectively abrogate the activity of Bcl-2.

We previously demonstrated that delivery of a gene encoding an anti-erbB-2 intracellular single-chain antibody (sFv) resulted in down-regulation of cell surface erbB-2 levels and induction of apoptosis in erbB-2 overexpressing ovarian cancer cells. Based upon these findings, we hypothesized that human breast carcinomas overexpressing erbB-2 would be similarly affected by this genetic intervention. We evaluated the phenotypic effects resulting from intracellular expression of the anti-erbB-2 sFv on the human breast cancer cell lines MDA-MB-361, SK-BR-3, BT-474, MCF-7 and MDA-MB-231. Recombinant adenoviruses encoding either a reporter gene (AdCMVLacZ) or the endoplasmic reticulum (ER) directed anti-erbB-2 sFv (Ad21) were delivered to various breast cancer cell lines. Cell viability was determined by a proliferation assay and fluorescent microscopy allowed visualization of apoptotic cells. An erbB-2 ELISA quantified the endogenous erbB-2 levels of each cell line. The anti-erbB-2 sFv-encoding-adenovirus, Ad21, but not the β-galactosidase encoding adenovirus, AdCMVLacZ, was cytotoxic to >95% of the tumor cells in the MDA-MB-361 and SK-BR-3 lines, and >60% of the tumor cells in the BT-474 line. In marked contrast, the MCF-7 and MDA-MB-231 cell lines showed no change in the rate of cell proliferation following this treatment. The cytotoxic effects generated in the first three lines were a consequence of the induction of apoptosis by the anti-erbB-2 sFv. An ELISA specific for erbB-2 showed that the breast cancer cell lines most susceptible to the anti-erbB-2 sFv, MDA-MB-361, SK-BR-3 and BT-474, overexpressed the erbB-2 protein while the cell lines demonstrating no response to the anti-erbB-2 sFv, MCF-7 and MDA-MB-231, expressed the lowest levels of erbB-2. These results demonstrate that targeted killing of erbB-2 overexpressing cells via intracellular knockout can be accomplished in the context of breast carcinoma. Furthermore, erbB-2 levels in breast tumor cells may be predictive of their sensitivity to sFv-mediated killing. The ability to accomplish selective cytotoxicity of breast cancer cell lines overexpressing the erbB-2 tumor marker should allow for derivation of clinical gene therapy strategies for breast cancer utilizing this approach.

We have previously established the efficacy of adenoviral gene delivery vectors for the treatment of bladder carcinoma in vivo. In the present work, we developed a gene therapy strategy for bladder cancer based on the replacement of the tumor suppressor p16, which is known to be mutated or deleted in a variety of human tumors, including those derived from the bladder. Previous reports have demonstrated that reconstitution of p16 has marked effects on the proliferative capacity of tumor cell lines both in vitro and in vivo, and that p16 expression causes resistance to some chemotherapeutic agents. In the present study, we describe the construction of the recombinant adenovirus Adp16, expressing the p16 gene, to evaluate the effects of transient p16 replacement in the context of bladder carcinoma. To identify candidate target cell lines, we screened a panel of bladder cancer cell lines for p16 and retinoblastoma (RB) protein expression. We demonstrate that Adp16 can mediate high-efficiency p16 replacement to the p16-negative cell lines EJ and UMUC-3. In addition, the reconstitution of p16 to the highly transducible p16-negative, RB-positive bladder cancer cell line EJ caused a profound inhibition of cell proliferation mediated by arrest in the G1 phase of the cell cycle. In contrast, the p16-positive, RB-negative cell line J82 was unaffected by this treatment. However, when adenovirally mediated p16 replacement was combined with the chemotherapeutics cisplatin and paclitaxel, a marked chemoresistance was observed in genetically corrected cells. This work has implications for future gene therapy strategies based on p16 replacement.

erbB-2 is known to be overexpressed in several human malignancies including lung cancer. Because of its role in neoplastic transformation as well as its association with poor prognosis, this oncogene has been targeted through various anti-cancer methodologies. In this regard, we have recently demonstrated that erbB-2-overexpressing ovarian tumor cell lines transfected with an endoplasmic reticulum form of an anti-erbB-2 single-chain antibody undergo a specific cytotoxicity through the induction of apoptosis. Since certain forms of lung cancer are also associated with overexpression of erbB-2, we evaluated the use of this novel therapeutic in this context. For these studies, several human lung adenocarcinoma cell lines were stably and transiently transfected with the anti-erbB-2 sFv gene. We demonstrate here that the anti-erbB-2 sFv can cause specific cytotoxicity in lung cancer cells. As a first step toward clinical translation of this strategy, we constructed a replication-deficient recombinant adenoviral vector expressing the anti-erbB-2 sFv construct. We further demonstrate that our anti-erbB-2 sFv-encoding adenoviral vector can accomplish high levels of cytotoxicity in lung cancer cells. Based on these results, it is proposed that this strategy of oncoprotein ablation may have use in the treatment of some forms of human lung cancer.

Methods were developed to achieve targeted eradication of the erbB-2 oncoprotein using gene constructs encoding anti-erbB-2 intracellular single-chain antibodies. This method of genetic intervention caused a marked cytocidal effect in erbB-2-overexpressing human ovarian tumor cells. Evaluation of the mechanistic basis of this phenomenon demonstrated that programmed cell death had been induced. Significantly, no cytocidal effect was observed in non-erbB-2-overexpressing tumors. The induction of apoptosis could be shown to be secondary to the intracellular antibody-mediated ectopic localization of the erbB-2 oncoprotein. Thus, the strategy of selective oncogene "knock-out" using intracellular antibodies represents a novel anticancer gene therapy strategy that offers the potential to achieve highly specific, targeted eradication of human tumor cells.