Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo

Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.     

Immunocytochemical visualization of P185HER2 receptor using antibodies fused with dibarnase and conjugate of barstar with colloidal gold

The localization of the P185^HER2 transmembrane receptor in SKOV-3 and BT-474 cancer cells was studied by fluorescence, confocal, and electron immunomicroscopy. The P185^HER2 receptor is a marker of breast and ovarian tumors; it is also considered to be a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, firstly, to study the distribution of P185^HER2 in cancer cells, secondarily, to remove P185^HER2 from the cell surface and, thirdly, to eliminate target cells. In this study, for visualization P185^HER2 we propose an immunocytotoxic system, which consists of monoclonal miniantibody 4D5 scFv to the extracellular P185^HER2 domain fused with two molecules of barnase (cytotoxic RNAase from Bacillus amyloliquefaciens) and its specific inhibitor, barstar. Fluorescent microscopy showed that the module 4D5 scFc-dibarnase:barstar is efficient for identifying P185^HER2 on the surfaces of cancer cells. It was found by confocal microscopy that interaction with 4D5 scFc-dibarnase results in the internalization of P185^HER2. The localization of P185^HER2 in human ovarian carcinoma cells (SKOV-3) and breast carcinoma cells (BT-474) was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185^HER2 is distributed unequally on the cell surface with preferential localization on protrusions or close to their bases and at contacts between protrusions and the cell membrane. At 37°C, P185^HER2 is internalized through coated pits and vesicles and concentrates in endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in BT-474 cells.     

Application of fusion protein 4D5 scFv-dibarnase:barstar-gold complex for studying P185HER2 receptor distribution in human cancer cells

Overexpression of the P185^HER2 protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185^HER2 in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185^HER2 domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au.     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

Colocalization of the p185HER2 oncoprotein and integrin α6β4 in Calu-3 lung carcinoma cells

Anti-p185^HER2 monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185^HER2). To evaluate a possible interaction between p185^HER2 and adhesion molecules or their receptors, the polarity of p185^HER2 was tested in lung carcinoma cell line Calu-3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185^HER2 is concentrated on the baso-lateral membrane of cells and that it colocalizes with the integrin α6β4 at the cell-cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185^HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185^HER2 tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185^HER2 phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185^HER2. © 1994 Wiley-Liss, Inc.     

Biological studies and potential therapeutic applications of monoclonal antibodies and small molecules reactive with theneu/c-erbB-2 protein

The overexpression of the growth factor receptor p185 ^neu/c-erbB-2 has been observed in a number of human adenocarcinomas and is mechanistically linked to neoplastic growth. Monoclonal antibodies raised against extracellular domains of the p185 ^neu/c-erbB-2 receptor oncoprotein have been utilized to inhibit the pathway ofneu-induced tumor development. Our laboratory has demonstrated a direct effect of anti-p185 ^neu/c-erbB2 antibodies which requires receptor ligation. This induced aggregation causes the downmodulation of cell-surface expression and eventual degradation of p185 ^neu/c-erbB-2 protein. In cells transformed by theneu oncogene, the result of antibody-induced p185 ^neu/c-erbB-2 receptor modulation is the reversion of the malignant pheno-type. We are exploiting the direct efficacy of this monoclonal antibody by developing small molecules (peptides and organic mimietics) based on anti-p185 ^neu/c-erbB-2 antibody structure that can mediate similar receptor binding and biological effects.     

Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185^HER2 overexpression per se triggers p185^HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185^HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185^HER2 MAbs, PC12–HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185^HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185^HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185^HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity. J. Cell. Biochem. 67:316–326, 1997. © 1997 Wiley-Liss, Inc.     

Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments.

The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (delta G degrees = -13.6 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells.

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

Inactivation of p16INK4a in Primary Tumors and Cell Lines of Head and Neck Squamous Cell Carcinoma

Inactivation of the p16^INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5 CpG islands can serve as an alternative mechanism for the inactivation of the p16^INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16^INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16^INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.     

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.     

Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4⁺ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as melanoma-specific. Optimal demonstration of the lytic activity of CD4⁺ CTL required a 16-h incubation period and an effectortarget cell ratio of 401. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN) consistently augmented lysis by these CD4⁺ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4⁺ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFN treatment. Thus, IFN may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4⁺CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8⁺ counterparts.This work was supported by USPHS grant R01-CA 36233, and a grant from the Concern Foundation for Cancer Research.     

Expression of alien histocompatibility antigens on SJL/J tumors detected by cell-mediated and serological analyses

Summary Reticulum cell sarcoma (RCS) cells of SJL/J (H-2^s) mice have been shown to express antigens that are cross-reactive with allogeneic cells of the H-2^d and H-2^b haplotypes by cell-mediated cytotoxicity, antibody-mediated cytotoxicity, immunofluorescence, and quantitative absorption assays. These alien antigens have been detected on both spontaneous and in vivo- and in vitro-passaged RCS cells to varying degrees.The in vitro cell lines were able to stimulate a syngeneic cytotoxic T cell response detected in a 4-h ⁵¹Cr release assay. The cytotoxic cells reacted with in vitro RCS tumor targets but not with in vivo or spontaneous RCS tumors. Furthermore, the cytotoxic cells lysed H-2^d and to a lesser extent H-2^b target cells, but not H-2^k, H-2^p, or H-2^r cells. The cross-reactivity was also observed with SJL/J anti-BALB/c cytotoxic cells, which can lyse in vitro RCS targets effectively. The in vivo tumors were not stimulatory in cytotoxic responses and did not serve as targets.H-2^d specificities were also detected in cultured RCS tumor cells by cytotoxic antibody. Both allogeneic SJL/J anti-BALB/c, C57B1/6 anti-BALB/c sera reacted with RCS tumor cells and not normal SJL/J cells. Furthermore, monospecific D^d sera were also cytotoxic against RCS lines. The cytotoxic activity could be absorbed by BALB/c cells and RCS cells but not with normal SJL/J cells. The H-2^d specificities were also detected on the in vivo lines by indirect immunofluorescence. The majority (60%) of spontaneously arising tumors expressed either H-2^d or H-2^b allospecificities in the immunofluorescence assays. Although these antigens may not be inappropriate for the SJL/J strain, their differential expression on tumor cells may be significant in the etiology of the tumor.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA     

Accumulation of Mutant p53 Modulates the Growth, Clonogenicity, and Radiochemosensitivity of Malignant Glioma Cells Independent of Endogenous p53 Status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53^V143A in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53^V143A modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53^V143A inhibited the camptothecin-induced accumulation of p21^WAF1/CIP1 in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53^V143A. Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53^V143A. Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53^V143A-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

Remodeling domain interfaces to enhance heterodimer formation.

An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower. Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.