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Supplementary Figure S1 PDF file 1270K, Structural model of Fc�R - IgG Fc - domain B triple complex

Supplementary Information PDF file - 77K, Additional information on the HER2 Domain 4 sequence, methods and figure legends for supplemental figures

Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185her2/neu-targeting antibodies not only downregulate the p185her2/neu kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here, we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a “Grababody” that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the use of this platform recognizes p185her2/neu and includes an IgG binding domain. The recombinant scFv Grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185her2/neu. The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved antitumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications. Cancer Res; 73(8); 2619–27. ©2013 AACR.

Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1’s Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease.

Forkhead box P3 (FOXP3) is a “master regulator” of regulatory T cells (Tregs), which are a subset of T cells that can suppress the antigen-specific immune reaction and that play important roles in host tolerance and immune homeostasis. It is well known that FOXP3 forms complexes with several proteins and can be regulated by various post-translational modifications (PTMs) such as acetylation, phosphorylation, ubiquitination, and methylation. As a consequence, the PTMs change the stability of FOXP3 and its capability to regulate gene expression, and eventually affect Treg activity. Although FOXP3 per se is not an ideal drug target, deacetylases, acetyltransferases, kinases, and other enzymes that regulate the PTMs of FOXP3 are potential targets to modulate FOXP3 and Treg activity. However, FOXP3 is not the only substrate for most of these enzymes; thus, unwanted “on target/off FOXP3” side effects must be considered when inhibitors to these enzymes are used. In this review, we summarize recent progress in the study of FOXP3 cofactors and PTM proteins, and potential clinical applications in autoimmunity and cancer immunity. Keywords: Treg, Forkhead box P3 (FOXP3), Post-translational modification, Autoimmune, Cancer

The lack of progress in developing targeted therapeutics directed at protein–protein complexes has been due to the absence of well-defined ligand-binding pockets and the extensive intermolecular contacts at the protein–protein interface. Our laboratory has developed approaches to dissect protein–protein complexes focusing on the superfamilies of erbB and tumor necrosis factor (TNF) receptors by the combined use of structural biology and computational biology to facilitate small molecule development. We present a perspective on the development and application of peptide inhibitors as well as immunoadhesins to cell surface receptors performed in our laboratory.

Despite substantial clinical progress with targeted therapies, current antibody-based approaches have limited efficacy at controlling HER2/neu-positive breast cancers, especially in the absence of chemotherapies. Previously, we showed that the combination of IFNγ and anti-HER2/neu antibody synergistically reduces tumor growth in an in vivo implanted mammary tumor model. Here, we report a recombinant approach to produce an anti-HER2/neu scFv and IFNγ fusion protein using an engineered effector domain (EED) scaffold. The new molecule induces in vitro apoptosis in an IFNγ receptor-dependent manner. At a very low dose in the in vivo xenografted tumor models, the new EED-IFNγ fusion protein demonstrates superior activity over the anti-HER2/neu antibody and is even active on tumors that are resistant to anti-HER2/neu antibody therapy. Examination of tumor infiltrated macrophages and lymphocytes reveals that the fusion protein can induce changes in tumor microenvironment to support immune reactivity against tumors. Our studies have defined a targeted immunotherapy approach for the treatment of cancers.

Background We have developed a new approach to reduce the serum interference for ELISA. The purpose of this study is to investigate if we can use the optimized ELISA (MBB-ELISA) to detect serum soluble HER2/neu (sHER2) in early stage primary breast cancer and monitor its change during treatments. Findings We collected sera preoperatively from 118 primary breast cancer patients. Serum samples were also collected sequentially from a subset of patients during and after adjuvant treatment. sHER2 in these samples was measured by the MBB-ELISA. Only 16.7 % of tissue HER2 (tHER2) positive patients had significantly elevated sHER2 levels in serum. Interestingly, sera of some patients with tHER2 negative tumors, including those that were 2+ by IHC but negative by FISH, demonstrated slightly elevated sHER2 levels. Multivariate analysis demonstrated that patients with elevated sHER2 (> = 7 ng/ml) had significantly worse disease free survival. During treatments, sHER2 levels consistently fell in response to adjuvant therapies. Nevertheless, in all 4 patients who developed metastases, a steady rise in sHER2 levels was noted before metastatic disease became clinically evident. Conclusions For early stage breast cancers, sHER2 is a poor biomarker to predict tHER2 status, but may have value to supplement tissue tests to identify patients with HER2 tumors. Our results also suggest that sHER2 is worth further study as a biomarker to monitor breast cancer patients during treatments.

The integration of III-V or III-N materials onto selected regions of a silicon or silicon-on-insulator (SOI) with the goal of creating electronic-photonic circuits is examined. Epitaxial transfer and bonding methods are discussed, as well as issues of CMOS compatibility and the thermal environment of the III-V devices. Possible devices for future electronic-photonic integration are also discussed.

This is an initial report of a new method of axillary dissection via a periareolar incision and an 8 mm incision in the axilla with the da Vinci Surgical System. The 10x magnification and three-dimensional image, together with the versatility and precision of the robotic telemanipulators, has enabled us to perform nerve-sparing axillary dissection in four patients with invasive ductal carcinoma of the breast undergoing segmental (conservative) excision and level II axillary dissection. The time for the robotic axillary dissection ranged from 30 to 105 minutes (average 70.5 minutes). The average number of lymph nodes retrieved was 13 (11, 11, 13, and 17, respectively). Postoperatively all four patients recovered well and were discharged the next day. The robotic system can enhance the surgeon's ability by providing a high-definition, magnified, three-dimensional view of the operative field, intuitively controlled articulating instruments, and elimination of tremors; and it has potential benefits for the patient.

The light-emitting transistor (LET) shows promise as a fundamental circuit element for electronic-photonic integration. This paper reviews key characteristics of the transistor laser, discusses preliminary work on integration, and explores methods for heterogeneous integration of LET-based electronic-photonic circuit blocks with silicon CMOS in a wafer-scale process.

Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185her2/neu-targeting antibodies not only downregulate the p185her2/neu kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here, we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a “Grababody” that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the use of this platform recognizes p185her2/neu and includes an IgG binding domain. The recombinant scFv Grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185her2/neu. The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved antitumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications. Cancer Res; 73(8); 2619–27. ©2013 AACR.

Over-expression of the HER2/neu receptor occurs in 20 to 30 percent of breast tumors and is linked to poorer prognosis. The HER2/neu expression status determines whether or not patient will receive trastuzumab-based treatment. In clinical practice, over-expression of HER2/neu is routinely identified using Immunohistochemistry (IHC) or Fluorescence in Situ Hybridization (FISH), both of which are invasive approaches requiring tissue samples. Serum assays for the Extra Cellular Domain of HER2/neu receptor (HER2 ECD) have been reported but the use is very limited due to serum interference factors (e.g. human anti-animal immunoglobulin antibodies) that lead to false test results and inconsistency with tissue Her2 status. We have developed an ELISA based approach using an MBB buffer to eliminate false results and to obtain more accurate assessment of HER2 ECD levels. Using this refined assay we retroactively measured HER2/neu levels from breast cancer patients and controls. Abnormal HER2 ECD levels were detected in about 32% of invasive breast cancer patients but not in controls or patients with benign diseases. In addition, we also showed that patients with elevated serum HER2 levels appeared to have worse survival regardless of treatments. In a small group of 12 Ductal Carcinoma in situ (DCIS) patients who received HER2/neu peptide vaccination and surgery, only one patient showed constantly rising HER2 levels after treatment and this patient had recurrence of HER2 positive tumor within 5 years. Our studies indicate that once the serum interference issue is resolved, serum HER2 ECD can have potential clinical utility to supplement the tissue based tests.

Approximately 15-30% of breast cancers over-express the HER2/neu receptor. Historically, over-expression of HER2/neu has been identified using IHC or FISH, both of which are invasive approaches requiring tissue samples. Recent evidence has shown that some tumors identified as "negative" using these methods can respond to HER2/neu targeted therapy. Shedding of the extracellular domain (ECD) of the receptor into the circulation has led to the development of a serum test of HER2 ECD as an additional approach to probe HER2/neu overexpression. The serum test will be able to monitor the dynamic changes of HER2 status over the course of disease progression. Some studies further suggest that the serum HER2 ECD level and its change may serve as a biomarker to reflect patients' response to therapy. Yet more than 10years after the first serum HER2 ECD test was approved by the FDA, serum HER2 testing has yet to be widely used in clinical practice. In this article we will review the progress of the serum HER2 ECD test and discuss some obstacles impeding its incorporation into broad clinical practice. We will also discuss recent improvements in the sensitivity and specificity of the assay that offer some hope for the future of serum HER2 test.