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The receptor tyrosine kinase HER2 is overexpressed in approximately 20% of breast cancer, and its amplification is associated with reduced survival. Trastuzumab emtansine (Kadcyla, T-DM1), an antibody–drug conjugate that is comprised of trastuzumab covalently linked to the antimitotic agent DM1 through a stable linker, was designed to selectively deliver DM1 to HER2-overexpressing tumor cells. T-DM1 is approved for the treatment of patients with HER2-positive metastatic breast cancer following progression on trastuzumab and a taxane. Despite the improvement in clinical outcome, many patients who initially respond to T-DM1 treatment eventually develop progressive disease. The mechanisms that contribute to T-DM1 resistance are not fully understood. To this end, we developed T-DM1–resistant in vitro models to examine the mechanisms of acquired T-DM1 resistance. We demonstrate that decreased HER2 and upregulation of MDR1 contribute to T-DM1 resistance in KPL-4 T-DM1–resistant cells. In contrast, both loss of SLC46A3 and PTEN deficiency play a role in conferring resistance in BT-474M1 T-DM1–resistant cells. Our data suggest that these two cell lines acquire resistance through distinct mechanisms. Furthermore, we show that the KPL-4 T-DM1 resistance can be overcome by treatment with an inhibitor of MDR1, whereas a PI3K inhibitor can rescue PTEN loss–induced resistance in T-DM1–resistant BT-474M1 cells. Our results provide a rationale for developing therapeutic strategies to enhance T-DM1 clinical efficacy by combining T-DM1 and other inhibitors that target signaling transduction or resistance pathways. Mol Cancer Ther; 17(7); 1441–53. ©2018 AACR.

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688–97. ©2016 AACR.

Human epidermal growth factor receptor family members (EGFR, HER2, HER3, and HER4) play important roles in tumorigenesis and response to cancer therapeutics. In this study, we evaluated the capacity of the dual-target antibody MEHD7945A that simultaneously targets EGFR and HER3 to modulate radiation response in lung and head and neck cancer models. Antitumor effects of MEHD7945A in combination with radiation were evaluated in cell culture and tumor xenograft models. Mechanisms that may contribute to increased radiation killing by MEHD7945A, including DNA damage and inhibition of EGFR–HER signaling pathways, were analyzed. Immunohistochemical analysis of tumor xenografts was conducted to evaluate the effect of MEHD7945A in combination with radiation on tumor growth and microenvironment. MEHD7945A inhibited basal and radiation-induced EGFR and HER3 activation resulting in the inhibition of tumor cell growth and enhanced radiosensitivity. MEHD7945A was more effective in augmenting radiation response than treatment with individual anti-EGFR or anti-HER3 antibodies. An increase in DNA double-strand breaks associated γ-H2AX was observed in cells receiving combined treatment with MEHD7945A and radiation. Immunohistochemical staining evaluation in human tumor xenografts showed that MEHD7945A combined with radiation significantly reduced the expression of markers of tumor proliferation and tumor vasculature. These findings reveal the capacity of MEHD7945A to augment radiation response in lung and head and neck cancers. The dual EGFR/HER3–targeting action of MEHD7945A merits further investigation and clinical trial evaluation as a radiation sensitizer in cancer therapy. Mol Cancer Ther; 14(9); 2049–59. ©2015 AACR . This article is featured in Highlights of This Issue, [p. 1973][1] [1]: /lookup/volpage/14/1973?iss=9

Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC. Cancer Immunol Res; 3(2); 173–83. ©2014 AACR.

The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcγRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function–enhanced antibodies for solid tumor therapy. Cancer Res; 70(11); 4481–9. ©2010 AACR.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Breast cancers (BC) with HER2 overexpression (referred to as HER2 positive) progress more aggressively than those with normal expression. Targeted therapies against HER2 can successfully delay the progression of HER2-positive BC, but details of how this overexpression drives the disease are not fully understood. Using single-molecule biophysical approaches, we discovered a new effect of HER2 overexpression on disease-relevant cell biological changes in these BC. We found HER2 overexpression causes deformation of the cell membranes, and this in turn disrupts epithelial features by perturbing cell–substrate and cell–cell contacts. This membrane deformation does not require receptor signalling activities, but results from the high levels of HER2 on the cell surface. Our finding suggests that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signalling-independent manner., HER2 is frequently overexpressed in breast cancer in association with increased metastatic potential. Here, the authors show that HER2 overexpression causes deformation of cell membranes in a signalling-independent manner that contributes to the disease phenotype by disrupting epithelial features.

Dimerization is essential for activity of human epidermal growth factor receptors (HER1/EGFR, HER2/ErbB2, HER3/ErbB3, and ErbB4) and mediates intracellular signaling events leading to cancer cell proliferation, survival, and resistance to therapy. HER2 is the preferred dimerization partner. Activation of HER signaling pathways may be blocked by inhibition of dimer formation using a monoclonal antibody (MAb) directed against the dimerization domain of HER2. The murine MAb 2C4 that specifically binds the HER2 dimerization domain was cloned as a chimeric antibody, humanized using a computer-generated model to guide framework substitutions, and variants were tested as Fabs. Pharmacokinetics and toxicology were evaluated in rodents and cynomolgus monkeys. Cloning the variable domains of MAb 2C4 into a vector containing human kappa and CH1 domains allowed construction of a mouse-human chimeric Fab. DNA sequencing of the chimeric clone permitted identification of CDR residues. The full-length IgG1 of variant F-10 was equivalent in binding to chimeric IgG1 and was designated pertuzumab (rhuMAb 2C4; Omnitarg). Pertuzumab pharmacokinetics was best described by a two-compartment model with a distribution phase of1 day, terminal half-life of approximately 10 days, and volume of distribution of approximately 40 mL/kg that approximates serum volume. With the exception of diarrhea, pertuzumab was generally well tolerated in cynomolgus monkeys. Pertuzumab, a recombinant humanized IgG1 MAb, is the first of a new class of agents known as HER dimerization inhibitors. Inhibition of HER dimerization may be an effective anticancer strategy in tumors with either normal or elevated expression of HER2.

Purpose Pertuzumab, a recombinant humanized monoclonal antibody (2C4), binds to extracellular domain II of the HER-2 receptor and blocks its ability to dimerize with other HER receptors. Pertuzumab represents a new class of targeted therapeutics known as HER dimerization inhibitors. A clinical study was conducted to investigate safety and pharmacokinetics of pertuzumab and to perform a preliminary assessment of HER dimerization inhibition as a treatment strategy. Patients and Methods Patients with incurable, locally advanced, recurrent or metastatic solid tumors that had progressed during or after standard therapy were recruited to a dose-escalation study of pertuzumab (0.5 to 15 mg/kg) given intravenously every 3 weeks. Results Twenty-one patients received pertuzumab and 19 completed at least two cycles. Pertuzumab was well tolerated. Overall, 365 adverse events were reported and 122 considered to be possibly drug related. Of these, 116 were of grade 1 to 2 intensity. The pharmacokinetics of pertuzumab were similar to other humanized immunoglobulin G antibodies, supporting a 3-week dosing regimen. Trough plasma concentrations were in excess of target concentrations at doses greater than 5 mg/kg. Two patients, one with ovarian cancer (5.0 mg/kg) and one with pancreatic islet cell carcinoma (15.0 mg/kg), achieved a partial response. Responses were documented by Response Evaluation Criteria in Solid Tumors after 1.5 and 6 months of pertuzumab therapy, and lasted for 11 and 10 months, respectively. Stable disease lasting for more than 2.5 months (range, 2.6 to 5.5 months) was observed in six patients. Conclusion These results demonstrate that pertuzumab is well tolerated, has a pharmacokinetic profile which supports 3-week dosing, and is clinically active, suggesting that inhibition of dimerization may be an effective anticancer strategy.

Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.

Erlotinib HCl (Tarceva; Genentech, Inc, South San Francisco, CA) is an orally available, highly selective, reversible inhibitor of epidermal growth factor receptor (HER1/EGFR) tyrosine kinase. Inhibition of tyrosine kinase activity prevents HER1/EGFR phosphorylation, the associated downstream signaling events, and may block tumorigenesis mediated by inappropriate HER1/EGFR signaling. In vitro and in vivo studies show that erlotinib has activity against human colorectal, head and neck, non-small cell lung, and pancreatic tumor cells. Recent preclinical studies suggest that erlotinib may also have activity against tumors that are dependent on HER2 activation for growth and/or survival. Preclinical studies have addressed the feasibility of using erlotinib in combination with various chemotherapeutic agents, radiotherapy, and targeted agents. Combining agents that have different mechanisms of action has the potential to improve efficacy and inhibit the development of resistance. For example, in preclinical studies, combining erlotinib with cisplatin, doxorubicin, gemcitabine, or low-dose paclitaxel has an additive effect on antitumor activity with no increase in toxicity. Preclinical data provide a strong rationale for investigating erlotinib in the clinical setting. However, additional studies are required to gain further insights into the processes that regulate or influence the antitumor activity of erlotinib.

ErbB2 is a ligand-less member of the ErbB receptor family that functions as a coreceptor with EGFR, ErbB3, and ErbB4. Here, we describe an approach to target ErbB2's role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders ErbB2's recruitment into ErbB ligand complexes. Inhibition of ligand-dependent ErbB2 signaling by 2C4 occurs in both low- and high-ErbB2-expressing systems. Since the ErbB3 receptor contains an inactive tyrosine kinase domain, 2C4 is very effective in blocking heregulin-mediated ErbB3-ErbB2 signaling. We demonstrate that the in vitro and in vivo growth of several breast and prostate tumor models is inhibited by 2C4 treatment.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

ErbB2 is a unique member of the ErbB family of receptor tyrosine kinases that is distinguished by the fact that no ligand has yet been identified. Due to the absence of an ErbB2 ligand, alternative mechanisms are used for ErbB2 activation. As such, when ErbB2 is overexpressed, kinase activation occurs in the absence of ligand because of constitutive homodimerization. However, at normal expression levels ErbB2 acts as the shared coreceptor for the ErbB family, and these heterodimeric complexes are activated in response to the partner ligand. While the extracellular domain and transmembrane domains are necessary for ErbB2 transactivation, the carboxy terminus is also required. Specifically, ligand-dependent ErbB2 transactivation requires a discrete three-amino-acid segment, located at the C-terminus of ErbB family members ErbB3, ErbB4, and the epidermal growth factor receptor. Transactivation of ErbB2 via the three-amino-acid segment likely represents a conserved mechanism for regulated signaling by the ErbB family of receptors.

Background & Aims: The cyclooxygenase 2 (COX-2) and ErbB/HER pathways are important modulators of cancer cell growth. We sought to determine the effects of treatment with a specific COX-2 inhibitor and/or a monoclonal antibody against the ErbB receptor subtype HER-2/ neu on carcinoma cell growth. Methods: A cell-proliferation assay was used to determine the response of HCA-7 cells to the HER-3/HER-4 ligand heregulin β-1 (HRGβ-1). Both in vitro and in vivo assays were used to determine the effects of the selective COX-2 inhibitor, celecoxib, and/or an anti–HER-2/ neu monoclonal antibody (either Herceptin [Genetech Inc., S. San Francisco, CA] or 2C4) on cell growth. Results: HCA-7 cells express HER-2/ neu messenger RNA and protein, and exposure of these cells to HRGβ-1 results in a significant stimulation of cell growth. Celecoxib or Herceptin inhibits HCA-7 cell growth in vitro and in vivo. Combination therapy with celecoxib plus Herceptin or celecoxib plus 2C4 resulted in additive effects that resulted in almost complete inhibition of tumor growth. Conclusions: Combined treatment with COX-2 and HER-2/ neu inhibitors more effectively reduces colorectal carcinoma growth than either agent alone. Therefore, targeting of both the COX-2 and ErbB signaling pathways may represent a novel approach for the treatment and/or prevention of colorectal cancer in humans. GASTROENTEROLOGY 2001;120:1713-1719

Aims: Using a standardized immunohistochemical assay we have evaluated 575 primary neoplasms of different histogenesis to determine the incidence of HER2 overexpression in some of the most common categories of human solid neoplasms. This study addresses the variable incidence of HER2 overexpression previously published for some tumour types. Methods and results: The immunohistochemical staining was performed on paraffin sections of surgical specimens and a well-defined scoring system based upon numbers of HER2 receptors expressed on the cell surface was applied. Overexpression of HER2 as defined as a HER2 score of equal or greater than 2 was seen in breast cancer (22%), pulmonary adenocarcinoma (28%), colorectal adenocarcinomas (17%), pulmonary squamous (11%) and gastric adenocarcinomas (11%). As expected, the proportion of cases with a HER2 score of 3 was highest in breast cancer. Contrary to published results prostate and pancreas adenocarcinomas showed a very low incidence of HER2 overexpression. Conclusions: Overexpression of HER2 is detected immunohistochemically in a proportion of epithelial neoplasms of diverse histogenesis in addition to ductal breast cancer. The standardized format of the assay will allow comparative analyses of studies performed at different institutions.

The human epidermal growth factor receptor (HER) family consists of four distinct receptors: HER1 (epidermal growth factor receptor), HER2, HER3, and HER4. Their specific activating ligands are collectively known as neuregulins (NRG). We hypothesized that one member of the NRG family, NRG-1, and the HER family would play a role in fetal lung development. To test this hypothesis, we defined NRG-1 and HER gene expression in mid-trimester human fetal lung tissue. HER2 and HER3 messenger RNA and protein were detected in the fetal lung, but HER4 expression was not detected. Immunohistochemical staining of fetal lung tissue localized HER2 and HER3 protein to the developing lung epithelium. NRG-1 expression was not found in freshly isolated human fetal lung, but it was observed in fetal lung explants after 2 d of explant culture. Immunohistochemistry of cultured human fetal lung explants revealed that NRG-1 protein was also expressed in pulmonary epithelial cells. Exposing human fetal lung to recombinant NRG-1 activated the HER receptor complex as measured by approximately 4-fold increases in receptor phosphotyrosine content. In addition, NRG-1 increased explant epithelial cell volume density approximately 2-fold (P < 0. 03); increased epithelial cell proliferation approximately 2-fold, as determined by bromodeoxyuridine labeling (P = 0.002); and reduced surfactant protein-A (SP-A) levels by 53% (P < 0.05). These data are consistent with an autocrine regulatory process mediated by NRG-1 activation of HER2/HER3 heterodimers expressed on developing human fetal lung epithelial cells. Receptor activation results in increased lung epithelial cell proliferation and volume density, and decreased SP-A production, a marker of type II pneumocyte differentiation.

The heregulins are a family of ligands with ability to induce phosphorylation of the p185HER-2/neu receptor. Various investigators have reported a variety of responses of mouse and human breast and ovarian cells to this family of ligands including growth stimulation, growth inhibition, apoptosis and induction of differentiation in cells expressing the HER-2/neu receptor. Some of the disparity in the literature has been attributed to variations in the cell lines studied, ligand dose applied, methodologies utilized or model system evaluated (i.e. in vitro or in vivo). To evaluate the effects of heregulin on normal and malignant human breast and ovarian epithelial cells expressing known levels of the HER-2/neu receptor, this report presents the use of several different assays, performed both in vitro and in vivo, in vitro proliferation assays, direct cell counts, clonogenicity under anchorage-dependent and anchorage-independent conditions, as well as the in vivo effects of heregulin on human cells growing in nude mice to address heregulin activity. Using a total of five different biologic assays in nine different cell lines, across two different epithelia and over a one log heregulin dose range, we obtained results that clearly indicate a growth-stimulatory role for this ligand in human breast and ovarian epithelial cells. We find no evidence that heregulin has any growth-inhibitory effects in human epithelial cells. We also quantitated the amount of each member of the type I receptor tyrosine kinase family (RTK I, i.e. HER-1, HER-2, HER-3 and HER-4) in the cell lines employed and correlated this to their respective heregulin responses. These data demonstrate that HER-2/neu overexpression itself affects the expression of other RTK I members and that cells expressing the highest levels of HER-2/neu have the greatest response to HRG.

ErbB3 is unique among other members of the receptor tyrosine kinase family of growth factor receptors in that its kinase domain is enzymatically impaired. This renders it incapable of transducing a signal in response to ligand binding. However, in conjunction with ErbB2, ErbB3 is a potent mediator of signaling by the growth factor heregulin. Heregulin binding to ErbB3 induces formation of a heterodimeric complex with ErbB2, and this results in transactivation of the ErbB2 kinase. Although interaction between the extracellular domains of these receptors is an essential part of this process, it was not clear whether interaction between the cytoplasmic domains is also necessary for transactivation. By examining the abilities of a series of cytoplasmic domain mutants of ErbB3 to activate ErbB2, we have found a discrete sequence of three amino acid residues (LVI), located at the carboxyl-terminal end of the impaired ErbB3 kinase region, that is obligatory for transactivation. We conclude that formation of a functional ErbB2-ErbB3 signaling complex requires the presence of a specific structural feature within the ErbB3 cytoplasmic domain and suggest that ErbB2 transactivation results from a physical interaction between the cytoplasmic domains of these receptors.

We describe the identification of Neuregulin-3 (NRG3), a novel protein that is structurally related to the neuregulins (NRG1). The NRG1/neuregulins are a diverse family of proteins that arise by alternative splicing from a single gene. These proteins play an important role in controlling the growth and differentiation of glial, epithelial, and muscle cells. The biological effects of NRG1 are mediated by receptor tyrosine kinases ErbB2, ErbB3, and ErbB4. However, genetic studies have suggested that the activity of ErbB4 may also be regulated in the central nervous system by a ligand distinct from NRG1. NRG3 is predicted to contain an extracellular domain with an epidermal growth factor (EGF) motif, a transmembrane domain, and a large cytoplasmic domain. We show that the EGF-like domain of NRG3 binds to the extracellular domain of ErbB4 in vitro . Moreover, NRG3 binds to ErbB4 expressed on cells and stimulates tyrosine phosphorylation of this receptor. The expression of NRG3 is highly restricted to the developing and adult nervous system. These data suggest that NRG3 is a novel, neural-enriched ligand for ErbB4.

The activation of autocrine loops involving proto-oncogene related receptor tyrosine kinases has led to the analysis of a large number of growth factor systems in human glioma specimens and cell lines. The ErbB-2 system, also called HER-2 or neu, is analogous to the epidermal growth factor receptor system (EGF-R, ErbB-1). Neuregulins consist of a large family of proteins arising from alternative mRNA splicing of a single gene located at 8p22-p11. Activation of ErbB-2 by neuregulins occurs in heterodimeric complexes with ErbB-3 and ErbB-4. A panel of human glioma cell lines, which had previously been analyzed for ErbB-2 expression, was examined for ErbB-3 and ErbB-4 expression. Coordinate expression of ErbB-2, -3 or -4 was not observed in these cell lines. Despite the presence of a complete system capable of signaling in about half the cell lines, no constitutive activation of ErbB-2, -3 or -4 was observed, and autophosphorylation of ErbB-2 in response to heregulin was observed only in one cell line from the panel, NCE-G84. Moreover, the addition of recombinant heregulin or antibodies capable of disrupting ErbB-2/ErbB-3 complexes had no effect on cell proliferation. We conclude that the role of neuregulins and its receptors in the control of glioma cell proliferation may be limited or may be context dependent on in situ conditions which are lost in vitro. Alternatively, neuregulins may be involved in cell differentiation or survival in the central nervous system. Data supporting these conclusions are described in more detail herein.

In a relatively short period of time, monoclonal antibodies have entered the mainstream of cancer therapy. Their first use was as antagonists of oncogenic receptor tyrosine kinases, but today monoclonal antibodies have emerged as long-sought vehicles for the targeted delivery of potent chemotherapeutic agents and as powerful tools to manipulate anticancer immune responses. With ever more promising results from the clinic, the future will likely see continued growth in the discovery and development of therapeutic antibodies and their derivatives.

Schwann cells are one of the principal components of the peripheral nervous system. They play a crucial role in nerve regeneration and can be used clinically in the repair of injured nerves. We have established serum-free, defined culture conditions that rapidly expand adult human Schwann cells without fibroblast growth. We find that Gas6, a ligand for the Axl and Rse/Tyro3 receptor protein tyrosine kinase family, stimulates human Schwann cell growth, increasing both cell number and thymidine incorporation. Gas6 has synergistic effects with the other known human Schwann cell mitogens, heregulin/glial growth factor and forskolin. Addition of Gas6 causes phosphorylation of Axl and Rse/Tyro3 simultaneously and results in ERK-2 activation. A combination of Gas6 with heregulin and forskolin, on a defined background, supports maximal Schwann cell proliferation, while preserving the typical Schwann cell morphology and expression of the Schwann cell markers S-100, glial fibrillary acidic protein, and low-affinity nerve growth factor receptor. Gas6 mRNA is present in both spinal motor neurons and large neurons of the dorsal root ganglia, and neural injury has been reported to upregulate Rse/Axl in the schwann cell. This is the first demonstration of a potentially important biological role for the human Gas6/Rse-Axl system.

Heregulin beta 1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor alpha, or amphiregulin, heregulin beta 1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-independent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin beta 1, whereas heregulin beta 1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells.

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.

The ability of sensory axons to stimulate Schwann cell proliferation by contact was established in the 1970s. Although the mitogen responsible for this proliferation has been localized to the axon surface and biochemically characterized, it has yet to be identified. Recently a family of proteins known as heregulins (HRGs) has been isolated, characterized, and shown to interact with a number of class 1 receptor tyrosine kinases, including the erbB2, erbB3, and erbB4 gene products. These factors include glial growth factor, a Schwann cell mitogen. We have tested the effects of antibodies against components of this system (HRG beta 1 and p185erbB2) in cocultures of rat sensory neurons and human (or rat) Schwann cells to elucidate the role of these proteins in axon-induced Schwann cell proliferation. 2C4, a monoclonal antibody specific for the human p185erbB2 receptor tyrosine kinase, bound to the surface of human Schwann cells and reduced human Schwann cell incorporation of [3H]thymidine by > 90% compared with untreated controls in this coculture system. This antibody had no effect on rat Schwann cell incorporation of [3H]thymidine under similar conditions. A polyclonal antibody raised against HRG beta 1 reduced human and rat Schwann cell incorporation of [3H]thymidine by nearly 80% and up to 49%, respectively, relative to controls. These results imply that a HRG, or a HRG-like molecule, is a component of the axonal mitogen. This mitogen is presented to Schwann cells by axons and induces proliferation through an interaction that involves p185erbB2 on Schwann cells.

The use of Schwann cell (SC) autotransplantation to influence neural repair in humans is dependent upon identifying mitogens that will effectively expand human Schwann cells (SCs) in culture. The recent purification and molecular cloning of glial growth factor (GGF), a potent mitogen for rat Schwann cells, has led to the recognition that a family of proteins (GGF/HRG/NDF/ARIA) are alternatively spliced products of a single gene. The heregulins (HRGs) have been characterized with respect to their influence on human breast cancer cell lines; here we examined whether the HRGs have mitogenic activity for human SCs. Using DNA synthesis assays and serial passaging of cells in culture, we demonstrate that HRG is an effective mitogen for human SCs and that, in the presence of agents that elevate cAMP, it is possible to expand these cells over multiple passages without overwhelming fibroblast contamination. One putative target for this family of proteins is p185erbB2, and EGF-like receptor tyrosine kinase that is encoded by the erbB2 protooncogene. In this report we also demonstrate that the erbB2/3/4 messages as well as the erbB2/3 receptor proteins are present within cultured human SCs. The addition of HRG to human SCs results in tyrosine phosphorylation of a 185 kDa protein. In the presence of stimulatory concentrations of HRG, a blocking monoclonal antibody (2C4) to p185erbB2 is capable of significantly inhibiting phosphorylation of a 185 kDa protein as well as the subsequent incorporation of 3H-thymidine within the human SC. These latter results implicate an important role for p185erbB2 in mediating the mitogenic response of human SCs to HRGs.

ErbB3 is a member of the epidermal growth factor (EGF) receptor subfamily of receptor tyrosine kinases and is believed to be a receptor for an unknown ligand. We have tested the possibility that heregulin, a growth factor possessing an EGF-like domain, is a ligand for ErbB3. We have found that the iodinated recombinant EGF-like domain of heregulin-beta 1 (125I-rHRG beta 1(177-244) bound specifically to insect cell-expressed bovine ErbB3 with a dissociation constant of 0.85 nM. Moreover, 125I-rHRG beta 1(177-244) bound to NIH3T3 fibroblasts stably transfected with bovine erbB3 with a dissociation constant of 60 pM, but did not bind to parental cells. 125I-rHRG beta 1(177-244) could be chemically cross-linked to a 170-180 kDa protein in erbB3-transfected fibroblasts, and the cross-linked product could be immunoprecipitated with antibodies specific for ErbB3. Finally, rHRG beta 1 stimulated the tyrosine phosphorylation of both ErbB3 and endogenous p185erbB2/neu in transfectants but not in parental cells. We conclude that ErbB3 is a receptor for HRG and is capable of mediating HRG-stimulated tyrosine phosphorylation of itself and p185erbB2/neu in cells that express both receptors.

The conversion of human proinsulin to insulin occurs only in specialized cells which contain the appropriate processing enzymes. To allow proinsulin processing to occur in a wide variety of cell types, we engineered human proinsulin to be cleaved in the constitutive secretory pathway. Using site-directed mutagenesis, we have introduced furin consensus cleavage sequences (Arg-X-Lys-Arg) into the human proinsulin cDNA. These mutations allowed for efficient proteolytic maturation of human proinsulin to insulin within cells containing only a constitutive pathway of secretion. Additionally, a naturally occurring mutation (histidine B10 to aspartic acid) yields a form of human insulin which accumulates 10- to over 100-fold more mature insulin when compared to the mutants lacking this change. Engineering furin-specific cleavage sites into each junction of the human proinsulin cDNA results in the secretion of peptides that display the expected molecular weights for the A and B chains of insulin. The accumulation of mature, processed, human insulin was measured in the supernatants by radioimmunoassay, and the bioactivity of this molecule was measured by its ability to stimulate autophosphorylation of the insulin receptor. Our results suggest that any cell type might be engineered to produce mature, active, and stable insulin in the constitutive pathway of secretion.

MEHD7945A is a novel dual-action monoclonal antibody in which each of the two antigen-binding fragments is capable of binding to EGFR and HER3 with high affinity. It is being evaluated as a potential therapy for human cancer. The purpose of these studies was to characterize the pharmacokinetics (PK) of MEHD7945A in mouse and monkey and predict its human PK and efficacious dose.PK of MEHD7945A was determined in SCID beige mice and cynomolgus monkeys after administration of single intravenous doses. Human PK profiles were projected from monkey PK profiles using a species-invariant time method, and human population PK parameters were estimated using a nonlinear, two-compartment model comprising specific (target-mediated) and nonspecific clearance pathways. The antitumor efficacy in mice bearing human tumor xenografts was used in conjunction with human PK projections to estimate human efficacious doses.The total clearance of MEHD7945A decreased with increase in dose in both mouse and monkey. The nonspecific clearance in monkey was estimated to be 14 mL/day/kg. The predicted nonspecific clearance range in humans was 6-10 mL/day/kg. Doses of 8-12 mg/kg administered every 2 weeks in humans were predicted to achieve exposure of 300 day μg/mL per week to match the efficacious exposure observed in xenograft models.The PK of MEHD7945A was nonlinear in mouse and monkey in the dose range tested. The nonspecific clearance in monkey was approximately twofold higher than typical humanized IgG1 antibodies. The projected human efficacious dose and dose regimen appear to be achievable in patients.

SummaryExtensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.

Epidermal growth factor receptor (EGFR) and HER3 each form heterodimers with HER2 and have independently been implicated as key coreceptors that drive HER2-amplified breast cancer. Some studies suggest a dominant role for EGFR, a notion of renewed interest given the development of dual HER2/EGFR small-molecule inhibitors. Other studies point to HER3 as the primary coreceptor. To clarify the relative contributions of EGFR and HER3 to HER2 signaling, we studied receptor knockdown via small interfering RNA technology across a panel of six HER2-overexpressing cell lines. Interestingly, HER3 was as critical as HER2 for maintaining cell proliferation in most cell lines, whereas EGFR was dispensable. Induction of HER3 knockdown in the HER2-overexpressing BT474M1 cell line was found to inhibit growth in three-dimensional culture and induce rapid tumor regression of in vivo xenografts. Furthermore, preferential phosphorylation of HER3, but not EGFR, was observed in HER2-amplified breast cancer tissues. Given these data suggesting HER3 as an important therapeutic target, we examined the activity of pertuzumab, a HER2 antibody that inhibits HER3 signaling by blocking ligand-induced HER2/HER3 heterodimerization. Pertuzumab inhibited ligand-dependent morphogenesis in three-dimensional culture and induced tumor regression in the heregulin-dependent MDA-MB-175 xenograft model. Importantly, these activities of pertuzumab were distinct from those of trastuzumab, a monoclonal antibody currently used for treatment of HER2-amplified breast cancer patients. Our data suggest that inhibition of HER3 may be more clinically relevant than inhibition of EGFR in HER2-amplified breast cancer and also suggest that adding pertuzumab to trastuzumab may augment therapeutic benefit by blocking HER2/HER3 signaling. [Cancer Res 2008;68(14):5878–87]

We show that two commonly occurring epidermal growth factor receptor (EGFR) somatic mutations, L858R and an in-frame deletion mutant, Del(746-750), exhibit distinct enzymatic properties relative to wild-type EGFR and are differentially sensitive to erlotinib. Kinetic analysis of the purified intracellular domains of EGFR L858R and EGFR Del(746-750) reveals that both mutants are active but exhibit a higher KM for ATP and a lower Ki for erlotinib relative to wild-type receptor. When expressed in NR6 cells, a cell line that does not express EGFR or other ErbB receptors, both mutations are ligand dependent for receptor activation, can activate downstream EGFR signaling pathways, and promote cell cycle progression. As expected from the kinetic analysis, the EGFR Del(746-752) is more sensitive to erlotinib inhibition than the EGFR L858R mutant. Further characterization shows that these mutations promote ligand-dependent and anchorage-independent growth, and cells harboring these mutant receptors form tumors in immunocompromised mice. Analysis of tumor lysates reveals that the tumorigenicity of the mutant EGFR cell lines may be due to a differential pattern of mutant EGFR autophosphorylation as compared with wild-type receptor. Significant inhibition of tumor growth, in mice harboring wild-type EGFR receptors, is only observed at doses of erlotinib approaching the maximum tolerated dose for the mouse. In contrast, the growth of mutant tumors is inhibited by erlotinib treatment at approximately one third the maximum tolerated dose. These findings suggest that EGFR somatic mutations directly influence both erlotinib sensitivity and cellular transformation. (Cancer Res 2006; 66(16): 8163-71)

Some Abs are more efficacious after being cross-linked to form dimers or multimers, presumably as a result of binding to and clustering more surface target to either amplify or diversify cellular signaling. To improve the therapeutic potency of these types of Abs, we designed and generated Abs that express tandem Fab repeats with the aim of mimicking cross-linked Abs. The versatile design of the system enables the creation of a series of multivalent human IgG Ab forms including tetravalent IgG1, tetravalent F(ab′)2, and linear Fab multimers with either three or four consecutively linked Fabs. The multimerized Abs target the cell surface receptors HER2, death receptor 5, and CD20, and are more efficacious than their parent mAbs in triggering antitumor cellular responses, indicating they could be useful both as reagents for study as well as novel therapeutics.

The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-A resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, TarcevaTM). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization.

Our understanding of the normal signaling mechanisms and functions of human epidermal growth factor receptor 2 (HER2) and other members of the HER family, namely epidermal growth factor receptor, HER3, and HER4, is growing rapidly. Activation of these receptors results in a diverse array of signals through the formation of homodimeric and heterodimeric receptor complexes; HER2 is the preferred dimerization partner for the other HERs. These oligomeric receptor complexes activate distinct signaling pathways, such as the Ras-MAPK and PI3-kinase pathways. These, in turn, affect various cellular processes. Recent gene deletion experiments in mice point to an important role for HER2 in cardiac and neural development, and evidence from other studies indicates that HER2 is involved in normal breast growth and development. Thus, HER2 is a key component of a complex signaling network that plays a critical role in the regulation of tissue development, growth, and differentiation.

Antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells. Anti-CD138 ADCs show promising preclinical efficacy for the treatment of multiple myeloma; however, the safety aspects of targeting CD138 have yet to be explored.

The human epidermal growth factor receptors 2, 3, and 4 (HER2, HER3, and HER4, respectively) are frequently overexpressed in many human cancers, and therefore may be potential targets for receptor-mediated gene transfer. To evaluate this possibility, we constructed a series of HER-targeted gene transfer vehicles by covalently linking poly-L-lysine polymers (pLYS) to the epidermal growth factor-like domain of the HER ligand neuregulin-1 (NRG1(177-244)), a HER2 antibody (Ab), and the Fab fragment of the HER2 Ab. In vitro, pLYS modification of NRG1(177-244) decreased the affinity of the ligand for HER3 or HER4 homodimer receptors by 6- to 7-fold. DNA loading of the pLYS-modified NRG1(177-244) had a minimal additional affect on the affinity of the complex for its receptor. In cell lines engineered to solely express HER2, HER3, or HER4, each vehicle correctly targeted the receptors; the NRG1(177-244) construct transferred a luciferase gene only into cells expressing HER4, whereas the HER2 Ab and Fab constructs transferred the reporter gene only into cells expressing HER2. The most efficient gene transfer occurred using the intact HER2 Ab as a gene transfer vehicle, whereas the Fab fragment of the HER2 Ab was the least efficient, and NRG1(177-244) was intermediate. These studies suggest that the NRG receptor or HER2, a component of the receptor, can be pursued as targets for gene transfer.

5575 Background: Oncogenic signaling through the epidermal growth factor receptor family is one of the most frequent alterations found in human epithelial cancers. These receptor tyrosine kinases mediate their effects via high-level co-expression and homo- and heterodimerization events that drive tumor growth, metastasis, and survival. Extensive preclinical studies suggested that some cell lines depend on oncogenic autocrine signaling through HER3 (Wilson et al.). This phenotype was particularly prominent in cell lines derived from SCCHN and was strongly correlated with high HRG expression. Interestingly, two patients with SCCHN tumors that expressed high levels of HRG in our phase Ia trial (abstract #95245) of MEHD7954A, a dual-action human IgG1 antibody that blocks ligand binding to EGFR and HER3 (Schaefer et al.) had partial responses. To further explore the hypothesis that high-level HRG expression defines a sub-population of SCCHN that may be sensitive to agents targeting HER3, and to identify other potential target indications for the development of MEHD7954A, we evaluated the expression of HRG in large cohorts of multiple solid tumor indications. Methods: HER3 and HRG expression was analyzed by qRT-PCR in 648 formalin-fixed paraffin embedded primary tumor samples from patients with NSCLC, SCCHN, melanoma, CRC and triple-negative breast cancer. Results: SCCHN-derived tumor samples had the highest levels of HRG expression, exhibiting a bimodal distribution in SCCHN – a pattern that is clearly distinct when compared to other tumor types. These data suggest that high HRG levels and potentially HER3-dependent autocrine signaling occur more frequently in SCCHN than in other tumors. Further we investigated whether overexpression of HRG in SCCHN correlated with stage and disease outcome. Updated results from these extended studies will be presented. Conclusions: SCCHN tumors exhibit bimodal expression of HRG, suggesting that HRG expression levels may be useful in identifying a subset of patients most likely to benefit from inhibition of HER3 activity. Antitumor activity in such patients has been observed in a phase I study of MEHD7954A (abstract #95245).

Dysregulation of the epidermal growth factor receptor family (EGFR, HER2, HER3, HER4) by mutation and/or overexpression plays an important role in tumorigenesis, and targeted agents directed against two members of the HER/ErbB family, epidermal growth factor receptor (EGFR/HER1), and HER2/ErbB2, are used in the treatment of cancer. Extensive crosstalk seen among these receptors implies that blocking signaling of more than one receptor may be more effective in inhibiting tumor growth and circumventing resistance mechanisms than targeting individual receptors. In particular, HER3 is considered a key mediator of resistance to many targeted agents. We generated a “two-in-one” antibody, MEHD7945A, that binds to EGFR and HER3 with high affinity, inhibits receptor function and is more broadly efficacious in various tumor types when compared to monospecific anti-EGFR or anti-HER3 antibodies. Given the ability of HER3 to potently activate the PI3K survival pathway, we investigated if antagonizing ligand-dependent HER signaling with MEHD7945A in the presence of chemotherapy augments cytotoxicity. We calculated combination index values the effects of combining MEHD7945A with commonly used chemotherapeutic agents in NSCLC and colorectal cell lines in vitro and in vivo. The NSCLC lines NCI-H292, NCI-H1666, NCI-H358 and HCC827 were treated with MEHD7945A plus gemcitabine over a wide range of drug concentrations. Cell proliferation data were analyzed using CalcuSyn software and all combination index values were Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1212. doi:1538-7445.AM2012-1212

Antibody drug conjugates (ADCs) are attractive targeted chemo-therapeutic molecules as they combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity. The successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency and mode of linker-drug conjugation to the antibody. Recently, we have developed antibodies with cysteine substitutions (THIOMABs) at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., Nature Biotech., 26, 925-932, 2008). These THIOMABs can then be derivatized through the engineered cysteine thiol group to obtain ADCs with uniform stoichiometry (∼2 drugs per antibody). Studies with multiple antibodies against different antigens have shown that THIOMAB drug conjugates (TDCs) are as efficacious as conventional conjugates in xenograft models and are tolerated at higher doses in relevant preclinical models. To further understand improved therapeutic activity and in vivo metabolism of TDCs, we have engineered TDCs with drug attachment at different parts of the antibody (light chain-Fab, heavy chain-Fab and heavy chain-Fc). TDCs produced by THIOMAB technology provided an unique advantage over conventional ADCs to answer several unresolved questions in ADC therapeutics due to their homogeneity and site-specific conjugation to cytotoxic drugs. We will discuss novel findings that were observed with these TDC varaints and the influence of conjugation site in modulating stability and biological activity of antibody drug conjugates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4396.

Publisher Summary This chapter discusses the assay of enzyme-catalyzed oxygen-dependent disulfide bond formation. Enzyme-catalyzed oxidation of thiols to disulfides with oxygen as the electron acceptor is mechanistically different from metal ion-catalyzed oxidation. Thus, sulfhydryl oxidase-catalyzed thiol oxidation results in the production of stoichiometric amounts of H2O2. Two methods are presented in this chapter for assay of sulfhydryl oxidase activity and determination of H2O2 in the presence of thiols. The first method described in the chapter is oxygen electrode-catalase-coupled system where one measures the reduction in the stoichiometry of O2 consumption in the presence of catalase. To confirm the generation of H2O2 as a stoichiometric product of enzyme-catalyzed thiol oxidation, the Clark electrode can be used in conjunction with catalase. The other method measures H2O2 from the horseradish peroxidase-catalyzed oxidation of a dye. The utility of these procedures has been demonstrated in both general assays and in mechanistic studies.

The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10−2 min−1. This provides a t 1 2 of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10−2 min−1 and 7 × 10−3 min−1 for uncatalyzed autoxidation and 4 × 10−2 min−1 and 1.1 × 10−2 min−1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.