Anneliese Gonzalez

A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy

IntroductionRecent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.MethodsTrastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.ResultsscIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.ConclusionTrastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.

A novel therapeutic strategy to rescue the immune effector function of proteolytically-inactivated cancer therapeutic antibodies

Primary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for therapeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derangement of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading proteinase (IdeS). Similar to cancer-related matrix metalloproteinases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost antibody-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-suppressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzumab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity. Mol Cancer Ther; 14(3); 681–91. ©2014 AACR.

Distant Metastatic Disease Manifestations in Infiltrating Lobular Carcinoma of the Breast

OBJECTIVE This article reviews unusual distant metastatic patterns of infiltrating lobular carcinoma (ILC) of the breast. CONCLUSION ILC of the breast tends to spread to the gastrointestinal tract, genitourinary tract, peritoneum, retroperitoneum, and leptomeninges in addition to common visceral sites such as the liver, bone, and lung. Knowledge of these unusual metastatic manifestations and disease patterns may aid in differentiating distant metastatic disease from secondary primary cancers and help plan appropriate therapy.

Dysfunctional Antibodies in the Tumor Microenvironment Associate with Impaired Anticancer Immunity

Purpose: Studies have demonstrated that cancer-associated matrix metalloproteinases (MMP) can generate single peptide bond cleavages in the hinge region of immunoglobulin G1 (IgG1). This study investigated the cleavage of endogenous IgGs by MMPs in the tumor microenvironment and the consequences of the IgG hinge cleavage for humoral immunity. Experimental Design: We investigated the occurrence of single peptide bond cleaved IgGs (scIgG) in tumor tissues and plasma samples collected from a cohort of breast cancer patients (n = 60). Samples from healthy people (n = 20) were used as the control. Antibody hinge cleavage was detected by multiple assays, including IHC, ELISA, and flow cytometry. A correlation analysis was conducted between scIgG levels and patient clinical parameters. Results: Levels of scIgGs in tumors were significantly higher than in normal tissues. In addition, scIgG levels in tumors were enriched compared with that in the plasma of the same patients. The appearance of scIgGs in tumor tissues was associated with altered host IgG content and decreased IgG1. Increased tumor scIgGs were found to be positively correlated with adverse clinical factors, such as elevated tumor-associated macrophages, increased expression of MMP9 and other MMPs, and local metastasis to axillary lymph nodes. Conclusions: The study contributes to mounting evidence for the presence of hinge-cleaved antibodies with reduced Fc immune effector function in the tumor microenvironment. The results highlight a link between tumor scIgGs and poor patient outcomes, and reveal a component of compromised humoral immunity within tumors that could point to new immunotherapeutic strategies to rescue host immunity. Clin Cancer Res; 21(23); 5380–90. ©2015 AACR.

Evaluation Fucoidan Extracts From Undaria pinnatifida and Fucus vesiculosus in Combination With Anticancer Drugs in Human Cancer Orthotopic Mouse Models

Objective: To determine the activity of fucoidan from Undaria pinnatifida (UPF) and Fucus vesiculosus (FVF) when given in combination of chemotherapy drugs using selected human breast or ovarian cancer orthotopic mouse models. Methods: Mice were inoculated with 1 × 106 cells of TOV-112d, MCF-7, or ZR-75 subcutaneously or SKOV3-GFP-Luc intraperitoneally on day 0. MCF-7 and ZR-75 mice were administered with estradiol valerate 2 mg/kg in 0.2 mL castor oil subcutaneously two days prior to cell inoculation. Mice were randomized to one of six arms (N = 10/arm) paclitaxel, UPF/paclitaxel, FVF/paclitaxel, tamoxifen, UPF/tamoxifen, or FVF/tamoxifen. Tumors were measured three times per week for 28 days. Results: Improved activity was observed with UPF or FVF in combination with tamoxifen in both the MCF-7 and ZR-75D breast cancer mouse models. Decreased activity of paclitaxel was observed when given in combination with UPF or FVF in both breast cancer mouse models. The combination of FVF/tamoxifen in the TOV-112d ovarian cancer mouse model had improved activity but no there was difference observed with the UPF/tamoxifen in either ovarian cancer mouse model. No difference was observed with combination of UPF or FVF with paclitaxel in human ovarian cancer SKOV3 or TOV-112d orthotopic mouse models. Conclusion: This study did confirm that UPF/FVF in combination with tamoxifen did not decrease tamoxifen activity in both breast and ovarian cancer, with some potential to improve activity compared to tamoxifen alone in breast cancers. Previous in vitro studies had suggested UPF and FVF had overall synergistic activity with paclitaxel; however, in the current in vivo human cancer mouse model studies there was no change in paclitaxel activity when given in combination with UPF or FVF in either of the two human ovarian cancer models. Furthermore, this study demonstrated that UPF or FVF given in combination with paclitaxel had a potential antagonistic effect in breast cancer models. Additional studies are warranted to delineate mechanisms contributing to variation in the in vivo activity when given in combination with paclitaxel. As a first step, a clinical pharmacokinetic study evaluating impact of FVF/UPF given in combination with chemotherapy in patients with solid tumors is underway.

The use of modern imaging technologies in radiation therapy of cervical cancer

Over the past century, definitive management of unresectable cervical cancer has evolved and currently employs high-dose radiation treatment with teletherapy and intracavitary brachytherapy (ICBT) components, combined with concurrent chemotherapy. Reflecting high disease prevalence among developing nations, the International Federation of Gynecology and Obstetrics (FIGO) staging of cervical cancer relies on clinical assessment, with limited radiographic studies. However, multiple clinicopathologic analyses describe suboptimal correlation between clinical examination findings and pathologic stage. Over the past two decades, systematic evaluation of volumetric and functional imaging modalities including CT, MRI, and PET-CT has refined our ability to define disease extent and provide posttreatment surveillance. Similarly, traditional ICBT techniques relied on two-dimensional (2D) data for evaluation of target dose coverage and offered limited assessment of exposure to critical structures including the bladder, rectum, and sigmoid colon. During the last several years, investigators at leading European centers have enhanced the capabilities of existing ICBT techniques through dose optimization [high-dose rate (HDR) and pulsed dose rate (PDR)] and by incorporating volumetric imaging methods. Early results are encouraging, from both toxicity and tumor control perspectives. These techniques are currently being adopted in multiple centers. Pertinent aspects are summarized in the body of this report.

Preclinical Evaluation of Safety of Fucoidan Extracts From Undaria pinnatifida and Fucus vesiculosus for Use in Cancer Treatment

Objectives: To evaluate potential hepatic metabolism-mediated drug interactions with fucoidan from Undaria pinnatifida (UPF) or Fucus vesiculosus (FVF) and potential growth inhibition activity with either fucoidan alone or with chemotherapy. In vivo studies were done to confirm safety and investigate fucoidan-mediated immune modulation. Methods: Cytochrome P450 (CYP450) 3A4, 2C8, 2C9, and 2D6 inhibition experiments were conducted in vitro followed by an ex vivo human hepatocytes model to evaluate the CYP450 induction potential of each fucoidan at highest theoretical concentrations. Four hepatic metabolism phase II pathways—glutathione S transferase (GST), quinone oxidoreductase (QOR), catechol-O-methyltransferases (COMT), and uridine di-phosphate (UDP)-glucuronosyltransferase (UGT)—were evaluated with validated immunoassays. Growth inhibition assays were performed with each fucoidan alone and in combination with chemotherapy agents in a panel of human cancer cell lines. In vivo studies evaluated safety and immune modualtion. Results: CYP450 inhibition was observed with FVF. The GST, QOR, and UGT pathways had no changes. UPF and FVF both interacted with COMT. No growth inhibitory activity in cancer cell lines was observed. UPF and FVF had synergistic activity with paclitaxel or tamoxifen and additive activity with topotecan. In vivo, FVF decreased HeLa human cervical tumor growth and both FVF and UPF decreased TOV-112D human ovarian tumor growth. Otherwise, no significant change in tumor growth was observed. FVF immune modulation of IgG and IL-6 was observed (p<0.03). Conclusion: At higher doses, UPF and FVF may have limited potential for drug-supplement interactions, with either CYP450 or COMT hepatic metabolism pathways. Additional studies are warranted to evaluate to confirm findings of fucoidans in combination with chemotherapy.

Evaluation of Active Hexose Correlated Compound (AHCC) in Combination With Anticancer Hormones in Orthotopic Breast Cancer Models

Objective. To determine the impact on antitumor activity when active hexose correlated compound (AHCC) in combination with anticancer hormonal agents in orthotopic mouse models of human estrogen receptor positive breast cancer and evaluate impact of AHCC on aromatase activity. Methods. The study consisted of 7 treatment arms (n=10) conducted in 2 breast cancer mouse models: MCF-7 and ZR-75. Treatment groups included untreated, vehicle, AHCC 50 mg/kg, AHCC 50 mg/kg + tamoxifen 10 mg/kg, tamoxifen 10 mg/kg, AHCC 50 mg/kg + letrozole 10 µg/mouse, or letrozole 10 µg/mouse. All treatments were administered daily by oral gavage for 12 weeks. Tumors were measured 3 times a week. In vitro estrone and 17β-estradiol enzyme immunoassay was used to evaluate aromatase activity. Results. There was no difference in the activity with the combination of AHCC + tamoxifen compared with tamoxifen (P = 0.29). In the ZR-75 model (catechol-O-methyltransferase [COMT] wild-type), there was no difference in activity with the letrozole + AHCC compared with letrozole. However, in the MCF-7 model (COMT variant), AHCC + letrozole resulted in a decrease in activity compared with letrozole (P < 0.01). Immunoassay data suggested that AHCC is a potential inducer of aromatase activity. In both tumor models, there was cytotoxicity observed with AHCC compared with untreated (P < 0.02). Conclusion. AHCC did not change the activity of tamoxifen. AHCC may have some interaction with letrozole in patients with COMT variant genotype. AHCC had cytotoxicity that warrents additional studies to evaluate its potential role for consolidation/prevention of breast cancer.

Abstract P5-10-09: Correlation between germline and tumor CYP450 2D6 gene polymorphisms

Tamoxifen is used for the treatment and prevention of ER (estrogen receptor)-positive BrCa (Breast Cancer). Several studies have shown that genetic variations in the CYP2D6 gene and/or drug-dependent inhibition of CYP2D6 enzyme activity (drug-drug interactions or DDIs) are associated with significant reductions in the circulating levels of endoxifen the active tamoxifen metabolite. These studies demonstrated that changes in CYP2D6 metabolism, as predicted from CYP2D6 genotyping, affect efficacy. Recent negative results with regards to CYP2D6 genotyping from two large studies suggest that testing for CYP2D6 status has no practical clinical value. Tumor DNA was used for genotyping in a large fraction of the BrCa patients in these studies. Genetic bias may result when CYP2D6 genotyping is carried out on the tumor (somatic) genome rather than the host genome (germline DNA) since it is the host genome that determines CYP450 enzyme activity within the liver and GI tract. Also expanded CYP2D6 polymorphism coverage, using a more comprehensive genotyping panel, may improve risk stratification when using CYP2D6 genotyping as a prognosticator in BrCa patients treated with tamoxifen. We hypothesize that BrCa tissues will harbor numerous mutations, due to a mutator phenotype inherent in most cancers, including within the CYP450 family of genes and, specifically, within the CYP2D6 gene. We expect that comparisons between germline DNA isolated from peripheral blood, and mutated DNA isolated from BrCa (somatic DNA) specimens within the same patient will reveal extensive differences in CYP2D6 genotypes. The aim of this study, is to extensively genotype 70 BrCa patients using a retrospective cohort with matched blood and tumor tissue from an existing biobank at UTHealth. We plan to perform genotype to phenotype conversions on each patient, for both germline and somatic DNA. We will look for discrepancies in genotypes and phenotypes and determine the magnitude of genetic bias possible in such cohorts. Method: DNA samples were extracted from matched archived tumor cells, dissected by laser microdissection microscopy and blood. Genotyping was performed by clinically validated Taqman® discrimination assays on the most common alleles for CYP2D6 . We also studied the genotype status of these paired samples for CYP2C9 , VKORC1 , Factor II , Factor V , MTHFR , CYP3A4 and CYP3A5 genes. CYP2D6 gene copy number and gene rearrangement with CYP2D7 pseudogene were also assessed by Taqman copy number assays at 3 three different sites within gene. Genotype-phenotype conversion was performed using an in-house developed, clinically validated genotype-phenotype translator package. Genotype agreement was assessed between the two DNA sources. Results : Noticeable non-concordant results between DNA from breast tumors and blood were observed in the genotyping of polymorphisms in the CYP2D6 gene. However, strong agreement between DNA from breast tumors and blood was detected in the genotyping of polymorphisms in all other studied genes. These results suggest that previous publications refuting the association between CYP2D6 genetic polymorphisms and tamoxifen efficacy could have reached an inaccurate conclusion due to genetic bias and study design. Further research in this biomarker area is needed. Citation Format: Mehdi Dehghani, Anneliese O Gonzalez, Neda Hashemi-Sadraei, Songlin Zhang, Kevin P Rosenblatt. Correlation between germline and tumor CYP450 2D6 gene polymorphisms [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-09.

Abstract A107: Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues

Growing evidence indicates that the tumor microenvironment has suppressive immune system and immune evasion is emerging as one of the cancer hallmarks. Immunoglobulin G (IgG) is an important component of human adaptive immunity in removing pathological cells including tumor cells. Recently, we reported that the cancer therapeutic antibody trastuzumab, a humanized IgG1, can be cleaved at the hinge region by matrix metalloproteinases (MMPs) in vitro, and the hinge cleaved trastuzumab (scIgG-T) showed significant loss of immune effector functions such as antibody dependent cellular cytotoxicity (ADCC) and anti-tumor efficacy in vivo (Fan et al., Breast Cancer Research 2012). The objectives of this study are 1) to investigate if the Fc hinge cleavage of IgG occurs in tumor tissues of breast cancer patients; 2) to understand the relationship among IgG Fc hinge cleavage, MMP expression, and anticancer immunity. Methods: Surgically removed fresh tumor tissues (n=23, 11 with upfront surgery and 12 with neoadjuvant therapies) from breast cancer patients with informed consent (protocol # HSC-MS-10-0580) were snap frozen in liquid nitrogen and stored in a -80oC freezer until analysis. Normal breast tissues (n=20) were obtained from an outside vendor (Cureline Inc., CA) as snap frozen tissues. Peripheral blood mononuclear cells (PBMCs) were freshly prepared from patient blood samples collected before surgery and were preserved in liquid nitrogen storage tank until analysis. Hinge cleaved IgGs were quantified by ELISA and immuno-histochemistry (IHC) detection with a panel of anti-hinge specific antibodies developed at Johnson & Johnson. MMP expression was profiled using a reverse phase protein array method (Ray Biotech Inc., CA) and populations of immune effector cells in patient PBMCs were determined by multi-color flow cytometry. Results: Levels of single hinge cleaved IgGs (scIgGs) were significantly higher in breast cancer tumor tissue than in normal breast tissue. The elevated Fc hinge cleavage of IgGs in breast tumor tissues was positively correlated with high levels of MMPs and expression of MMP-9 showed linear correlation (R=0.78) with the level of scIgGs in the tumor tissues. Notably, Fc hinge cleavage of IgGs in residual tumor tissues from patients treated with neoadjuvant therapies was similar to the levels in the normal breast tissues, while tumor tissues from patients with up-front surgery had significantly higher Fc hinge cleavage than both normal and neoadjuvant treated tumor tissues. In comparison with PBMCs from patients with the up-front surgery, PBMCs from patients with neoadjuvant therapies had an increased percentage of NK cells (CD56+) and immune cells with Fc gamma receptor IIa (CD32a) expression. In addition, percentages of immune effector cells with CD56+/CD16+ (Fc gamma receptor RIII), CD56+/CD64+ (Fc gamma receptor RI), and CD14+/CD16+ were also higher in PBMCs from patients treated with neoadjuvant therapies than those from patients with up-front surgery. Taken together, our results suggest that the reduced scIgG levels in neoadjuvant treated tumor tissue correlated with the better profiles of immune effector cells, while the increased Fc hinge cleavage in breast tumor tissue before therapeutic intervention is linked with immune suppression in the tumor microenvironment. Citation Format: Ningyan zhang, Anneliese Gonzalez, Hui Deng, Xuejun Fan, Luis Baez Vallecillo, songlin zhang, Randall Brezski, Emily Roberson, Robert Jorden, William Strohl, Zhiqiang An. Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A107.