Báo cáo khoa học: Differential binding of human immunoagents and Herceptin to the ErbB2 receptor ppt

Differential binding of human immunoagents and Herceptin to the ErbB2 receptor Fulvia Troise 1, *, Valeria Cafaro 1, *, Concetta Giancola 2 , Giuseppe D’Alessio 1 and Claudia De Lorenzo 1 1 Dipartimento di Biologia Strutturale e Funzionale, Universita ` di Napoli Federico II, Italy 2 Dipartimento di Chimica, Universita ` di Napoli Federico II, Italy ErbB2 (HER2 ⁄ neu) is a proto-oncogene of the erbB family of tyrosine kinase receptors [1]. It encodes a 185 kDa transmembrane protein, which comprises an extracellular domain (ECD) and an intracellular tyro- sine kinase activity. Although no natural ligand has been identified for this receptor, it has been ascer- tained that its overexpression is associated with various carcinomas, in particular with human breast cancer [2]. As ErbB2 overexpression is involved in the progression of the malignancy, and is a sign of a poor prognosis, ErbB2 is a valid target of therapeutic inter- vention. However, when ErbB2 is overexpressed, not all of the ErbB2-ECD protein is embedded in the membrane of malignant cells; a fraction of ErbB2-ECD is proteo- lytically removed from the receptor [3] and shed as a soluble protein in the sera of breast cancer patients [4]. Herceptin [5], a humanized anti-ErbB2 IgG1, has been proven to be an essential tool in the immunother- apy of breast carcinoma. However, some ErbB2-posi- Keywords binding affinity; ErbB2; herceptin; immunoRNase; immunotherapy Correspondence C. De Lorenzo, Dipartimento di Biologia Strutturale e Funzionale, Universita ` di Napoli Federico II, Via Cinthia, 80126 Naples, Italy Fax: +39081679159 Tel: +39081679158 E-mail: cladelor@unina.it *These authors contributed equally to this work (Received 9 June 2008, revised 29 July 2008, accepted 1 August 2008) doi:10.1111/j.1742-4658.2008.06625.x Overexpression of the ErbB2 receptor is associated with the progression of breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized antibody directed to the ErbB2 receptor, has been proven to be effective in the immunotherapy of breast cancer. However, it can result in cardiotoxicity, and a large fraction of breast cancer patients are resistant to Herceptin treat- ment. We have engineered three novel, fully human, anti-ErbB2 immuno- agents: Erbicin, a human single-chain antibody fragment; ERB-hRNase, a human immunoRNase composed of Erbicin fused to a human RNase; ERB-hcAb, a human ‘compact’ antibody in which two Erbicin molecules are fused to the Fc fragment of a human IgG1. Both ERB-hRNase and ERB-hcAb strongly inhibit the growth of ErbB2-positive cells in vivo. The interactions of the Erbicin-derived immunoagents and Herceptin with the extracellular domain of ErbB2 (ErbB2-ECD) were investigated for the first time by three different methods. Erbicin-derived immunoagents bind soluble extracellular domain with a lower affinity than that measured for the native antigen on tumour cells. Herceptin, by contrast, shows a higher affinity for soluble ErbB2-ECD. Accordingly, ErbB2-ECD abolished the in vitro anti- tumour activity of Herceptin, with no effect on that of Erbicin-derived immu- noagents. These results suggest that the fraction of immunoagent neutralized by free extracellular domain shed into the bloodstream is much higher for Herceptin than for Erbicin-derived immunoagents, which therefore may be used at lower therapeutic doses than those employed for Herceptin. Abbreviations EDIA, Erbicin-derived immunoagent; ErbB2-ECD, extracellular domain of ErbB2 receptor; ERB-hcAb, human compact antibody against ErbB2 receptor; ERB-hRNase, human anti-ErbB2 immunoRNase with Erbicin fused to human pancreatic RNase; ITC, isothermal titration calorimetry; RU, response unit; scFv, single-chain antibody fragment; SPR, surface plasmon resonance. FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4967 tive carcinomas are resistant to the inhibitory effect on growth of Herceptin [6], and, in other patients, the resistance of malignant cells is induced at a later stage in treatment [7]. Furthermore, it has been found that Herceptin can lead to cardiotoxicity in a significant fraction of treated patients [8,9]. An alternative approach to the use of Herceptin in immunotherapy has been promoted, based on the administration of Herceptin combined with other antibodies directed to the ErbB2 receptor [10,11]. A prerequisite for this strategy is that the latter antibodies are directed to epi- topes on ErbB2-ECD different from that recognized by Herceptin. Based on these considerations, we instituted a search for novel immunoagents directed to epitopes different from that recognized by Herceptin, with no cardiotoxic side-effects and able to fulfil the therapeutic need of Herceptin-unresponsive patients. This led us to the production of a novel, fully human, anti-ErbB2 single- chain antibody fragment (scFv), isolated from a large phage display library through a double selection strat- egy performed on live cells. This scFv, named Erbicin [12], specifically binds to ErbB2-positive cells, inhibits receptor autophosphorylation and is internalized by target cells. Erbicin was used to construct human anti- ErbB2 immunoagents by two different strategies. The first was based on Erbicin fused to an RNase, i.e. a pro-toxin, as RNase becomes toxic only when Erbicin promotes its internalization in target cells. An immun- oRNase, denoted as ERB-hRNase (Erbicin-human- RNase), was produced by the fusion of Erbicin to human pancreatic RNase [13]. The second strategy aimed to produce a therapeutic reagent with an increased half-life, prolonged tumour retention and an ability to recruit host effector func- tions. Erbicin was thus fused to the Fc region from a human IgG1 to obtain an immunoglobulin-like anti- body version [14,15]. The engineered antibody was denoted as ERB-hcAb (human anti-ErbB2-compact antibody) because of its ‘compact’ size (100 kDa) com- pared with the full size (155 kDa) of a natural IgG. Both Erbicin-derived immunoagents (EDIAs) were found to selectively and strongly inhibit the growth of ErbB2-positive cells, both in vitro and in vivo. How- ever, to define and implement the antitumour potential of Erbicin and EDIAs, we deemed it essential to study their interaction with ErbB2. To determine and evalu- ate quantitatively their affinity for ErbB2, we used recombinant ErbB2-ECD as a homogeneous, soluble antigen. With this aim, three different analytical meth- ods were employed: ELISA, surface plasmon reso- nance (SPR) and isothermal titration calorimetry (ITC). The results obtained with Erbicin and EDIAs were compared with the results obtained with Hercep- tin. Furthermore, we determined and compared the affinity values of Herceptin and EDIAs for the free ECD structured within the whole receptor molecule, natively inserted into the cell membrane, with the val- ues measured using isolated ECD. We found that EDIAs bound soluble ECD with an affinity lower than that of Herceptin. However, the novel EDIAs bound ErbB2 exposed on the cell surface with a higher affinity than that of Herceptin [13,14]. These results indicate that the fraction of immunoagent neutralized by free ECD shed into the bloodstream, and hence lost to immunotherapy, could be much higher for Herceptin than for the novel immunoagents. Results Production and characterization of ErbB2-ECD The cDNA coding for ErbB2-ECD was stably trans- fected in the 293 cell line. The encoded protein was expressed as a secretion product in the culture med- ium, as revealed by western blotting (Fig. 1A) and immunoprecipitation analyses performed (see Experi- mental procedures) with ERB-hcAb or Herceptin as anti-ErbB2 agent (see Fig. 1B). The final yield of ErbB2-ECD, purified by affinity chromatography (see Experimental procedures), was 12 mgÆL )1 of medium. The protein was analysed by SDS-PAGE, followed by Coomassie staining and western blotting with Her- ceptin or ERB-hcAb (Fig. 1C). Its molecular size was about 80 kDa, as expected. Analysis by ELISA of the interactions of EDIAs and Herceptin with soluble ErbB2-ECD ELISA sandwich assays were performed to determine the ability of Erbicin and EDIAs to recognize soluble ErbB2-ECD. Herceptin fixed on the microplate was used to capture ErbB2-ECD, which, in turn, could inter- act with the anti-ErbB2 immunoagents. The affinity of ERB-hcAb or Herceptin for ErbB2-ECD was measured by ELISA on ECD directly coated to the wells. The results are given in Table 1 as apparent binding constants, measured from the binding curves as the concentrations corresponding to half-maximal sat- uration. The values obtained (50 nm for Erbicin, 30 nm for ERB-hRNase and 7 nm for ERB-hcAb) were found to be higher than those obtained with ErbB2 embedded in ErbB2-positive cells [13,14]. Thus, these data indi- cate that the immunoagents have a higher affinity for ErbB2-ECD when it is inserted in the cell membrane. Binding of human immunoagents to ErbB2 F. Troise et al. 4968 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS Interestingly, the lower binding affinity of EDIAs for soluble ErbB2-ECD was not shared by Herceptin, which displayed a high affinity for soluble ErbB2-ECD (0.1 nm), about 50-fold higher than that determined when Herceptin was tested with ErbB2-ECD expressed on live cells (see Table 1). These findings can be explained by the fact that parent Erbicin was selected from a phage library using ErbB2-ECD inserted into ErbB2-positive cells, whereas, for the isolation of Herceptin, free, soluble ECD was used [16]. ELISA sandwich assays with Herceptin as a capture agent have been performed to confirm that Erbicin and the novel immunoconjugates recognize, on ErbB2-ECD, an epitope different from that selected by Herceptin, as reported previously [17]. However, this type of assay was carried out for Erbicin and the immunoRNase only, as the peroxi- dase-conjugated anti-His IgG1 capable of revealing scFv and Erb-hRNase is unaffected by the presence of Herceptin; it was not performed with Erb-hcAb, as the anti-human secondary IgG serum fraction, used for its detection, could not discriminate between Erb-hcAb and Herceptin. Thus, for Erb-hcAb and Herceptin, the assays were performed on ECD directly immobilized on the plate. We then tested whether soluble ErbB2-ECD could affect the binding of anti-ErbB2 immunoagents to ErbB2-positive cells by performing ELISA with ERB-hcAb or Herceptin in the absence or presence of free ECD. Each antibody was tested at increasing concentrations, with soluble ECD added either in equimolar amounts or in a 10-fold molar excess to the number of receptor molecules on the cell membrane [18]. As a control, parallel assays were carried out in the absence of ErbB2-ECD. As shown in Fig. 2A, the binding curves obtained for ERB-hcAb in the absence or presence of soluble ECD were found to be superimposable. This finding suggests that the binding ability of ERB-hcAb to ErbB2-positive cells is unaffected by the presence of soluble ECD. In contrast, the binding of Herceptin to ErbB2-positive cells (Fig. 2B) was strongly reduced by ECD used at a 1 : 1 ratio with the receptor number, and fully inhibited with a 10-fold molar excess of ECD. These results, in line with those described above on the high affinity of Herceptin for soluble ECD, indicate that, for Herceptin, there is a favourable com- petition of soluble ErbB2-ECD over ECD on the cell membrane, whereas there is no detectable competition in the case of ERB-hcAb. Effects of soluble ErbB2-ECD on the cytotoxicity of ERB-hcAb and Herceptin On the basis of the results discussed above, the antitumour effects of ERB-hcAb and Herceptin on 1 A B C 34 ERB-hcAb (100 kDa) 1 Herceptin (155 kDa) 2 80 kDa ECD (80 kDa) ECD (80 kDa) 134 80 kDa 2 2 Fig. 1. Detection of ErbB2-ECD expression. (A) Western blotting analyses from conditioned medium of transfected 293 cells, with Herceptin as primary antibody followed by horseradish peroxidase- conjugated anti-human (Fc-specific) IgG serum fraction. Lane 1, negative control (medium from non-transfected 293 cells); lanes 2–4, conditioned medium produced by various selected clones. (B) Immunoprecipitation analyses of ErbB2-ECD from 293 cell condi- tioned medium with ERB-hcAb (lane 1) or Herceptin (lane 2). (C) SDS-PAGE analyses of purified ErbB2-ECD. Lane 1, molecular weight standards; lane 2, ErbB2-ECD eluted from immunoaffinity chromatography stained with Coomassie blue; lanes 3 and 4, wes- tern blot analyses of the sample in lane 2 using ERB-hcAb and Her- ceptin, respectively, as anti-ErbB2-ECD immunoagents. Table 1. Relative affinity of Erbicin, EDIAs and Herceptin for solu- ble ErbB2-ECD, as measured by ELISAs. Previous data [13,14] obtained with ErbB2-positive cells are also shown. K D (apparent) (nM) ErbB2-ECD ErbB2-positive cells Erbicin 50 5 ERB-hRNase 30 4.5 ERB-hcAb 7 1 Herceptin 0.1 5 F. Troise et al. Binding of human immunoagents to ErbB2 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4969 ErbB2-positive cells were tested in the absence or pres- ence of soluble ErbB2-ECD. Antibodies were incu- bated with soluble ECD, added at a concentration of 20 nm (eight-fold molar excess over antibodies), which was chosen on the basis of ELISA conditions in which Herceptin binding to ErbB2-positive cells was fully inhibited (Fig. 2B). As shown in Fig. 2C, ERB-hcAb inhibited the growth of SKBR3 cells similarly in the absence or presence of soluble ErbB2-ECD. In contrast, soluble ErbB2-ECD completely abolished the antitumour activity of Herceptin. These results indicate that, in the presence of soluble ECD, ERB-hcAb preserves its cytotoxic power on ErbB2-positive cells, whereas Herceptin does not exert cytotoxic activity because of its high affinity for solu- ble ECD. ECD is capable of neutralizing antibody binding to the cells, in agreement with previously reported data [19]. Analyses by SPR of the interactions of EDIAs and Herceptin with ErbB2-ECD To compare the binding properties of Erbicin, EDIAs and Herceptin with ErbB2-ECD using a direct meth- odology based on physicochemical principles, SPR analyses were carried out. The experimental system consisted of ErbB2-ECD (a monovalent ligand) cova- lently immobilized on the chip surface, with monova- lent (Erbicin or ERB-hRNase) or bivalent (Herceptin or ERB-hcAb) analytes injected and flowing over the sensor chip. The kinetic constants for monovalent Erbicin and ERB-hRNase were obtained by fitting the curves with a 1 : 1 interaction model. Similar binding curves were recorded for these immunoagents (see Fig. 3A,B), with almost identical association rate constants, but slightly different dissociation rate constants (see Table 2). Erbicin, with a k d value of 6.16 · 10 )3 s )1 , dissociated from ErbB2-ECD 1.5 times faster than did ERB-hRNase (k d = 4.12 · 10 )3 s )1 ). This indicated a higher stability for the ERB-hRNase ⁄ ErbB2-ECD complex with respect to the Erbicin ⁄ ErbB2-ECD com- plex, with equilibrium K D values of 46.7 and 27.2 nm, respectively. The significant difference in K D values could be clearly ascribed to the lower dissociation rate constant measured for the ERB-hRNase ⁄ ErbB2-ECD complex. It should be noted that the data were in very good agreement with those reported above from the ELISA experiments (see Table 1). The possibility was considered that the higher stabil- ity of the ERB-hRNase ⁄ ErbB2-ECD complex might be caused by aspecific electrostatic interactions between the positively charged RNase linked in the immunoconjugate and the negatively charged carb- oxymethyl-dextran matrix of the SPR chip. Thus, the kinetic analyses of the ERB-hRNase ⁄ ErbB2-ECD complex were repeated in the presence of soluble carboxymethyl-dextran as an added quencher. How- ever, identical constants were measured for the 1.5 2 A B C 0 0.5 1 0 4 8 1012141618 2 2.5 Protein concentration (nM) 0 0.5 1 1.5 Absorbance (450 nm) Absorbance (450 nm) 40 0246810 Protein concentration (nM) 10 20 30 0 Cell growth inhibition (%) Control Herceptin Herceptin + ECD ERB-hcAb ERB-hcAb + ECD 26 Fig. 2. Effects of soluble ErbB2-ECD on the binding and cytotoxic- ity of ERB-hcAb and Herceptin. Binding curves of ERB-hcAb (A) and Herceptin (B) to SKBR3 cells obtained by ELISA performed in the absence (open symbols) or presence (filled symbols) of soluble ECD. Soluble ECD was added at a ratio of 1 : 1 (filled squares) or 10 : 1 (filled circles) to the number of receptor molecules on the cell membrane. (C) Antitumour activity of ERB-hcAb and Herceptin on SKBR3 cells determined in the absence or presence of soluble ECD. Binding of human immunoagents to ErbB2 F. Troise et al. 4970 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS ERB-hRNase ⁄ ErbB2-ECD complex in the presence or absence of soluble carboxymethyl-dextran (Table 2). This indicates that the higher stability of the ERB-hRNase ⁄ ErbB2-ECD complex is not caused by simple coulombic interactions with the non-immune moiety, but by its specific structural features which 120 60 Response (RU) 80 40 0 40 20 0 0 100 200 300 400 500 Response (RU) 160 80 A B C D E F 60 0 100 200 300 400 500 Time (s) Time (s) Response (RU) 120 40 80 0 40 20 0 0 100 200 300 400 500 Response (RU) 6 7 100 120 0 100 200 300 400 500 450 550 300 350 400 Time (s) Time (s) 0 1 2 3 4 5 0 20 40 60 80 100 120 R eq (RU) R eq /ERB-hcAb (RU/n M) 0 20 40 60 80 R eq (RU) -50 50 150 250 350 200 250 300 350 R eq (RU) R eq /Herceptin (RU/n M) 0 50 100 150 200 250 R eq (RU) 0 50 100 150 200 250 300 350 400 [ERB-hcAb] (nM) 0 50 100 150 200 250 300 350 400 [Herceptin] (nM) Fig. 3. Determination by SPR of the binding between anti-ErbB2 immunoagents and ErbB2-ECD. Representative sensorgrams (jagged grey lines) recorded for Erbicin (A), ERB-hRNase (B), ERB-hcAb (C) and Herceptin (D). Smooth black lines represent the global fits of the sensor- grams to a 1 : 1 bimolecular interaction model. Erbicin was passed across the surface (500 RU of ErbB2-ECD) at concentrations of 10.9– 350 n M (A) and ERB-hRNase at concentrations of 8.4–269 nM (B). Soluble ErbB2-ECD was passed over ERB-hcAb (density, 202 ± 4 RU) or Herceptin (density, 1151 ± 5 RU), each captured by Protein A, at concentrations of 14.6–470 n M (C) and 22.7–728 nM (D). Binding isotherms of ERB-hcAb (E) and Herceptin (F) to immobilized ErbB2-ECD (500 RU). The equilibrium binding data (R eq ) were measured directly on the sensorgrams obtained by subsequent injections of analytes, and represent the mean of two determinations. The analysed concentrations were 7.6–356.6 n M for ERB-hcAb (E) and 0.5–361 nM for Herceptin (F). The equilibrium binding data were fitted to a two-site binding hyper- bola (R 2 = 0.994 and R 2 = 0.999 for ERB-hcAb and Herceptin, respectively). The insets show the Scatchard analysis of the binding data. The calculated constants from these plots (K D1 and K D2 ) are listed in Table 2. Table 2. Affinity and rate constants for ErbB2-ECD ⁄ ligand interactions determined by SPR. k a (M )1 Æs )1 ) a K d (s )1 ) a K D (nM) a K D1 (nM) b K D2 (nM) c Erbicin (1.33 ± 0.13) · 10 5 (6.16 ± 0.42) · 10 )3 46.7 ± 5.5 ERB-hRNase (1.50 ± 0.18) · 10 5 (4.12 ± 0.84) · 10 )3 27.2 ± 2.5 24 ERB-hRNase d 1.61 · 10 5 4.47 · 10 )3 27.8 ERB-hcAb (1.77 ± 0.13) · 10 4 (4.35 ± 0.09) · 10 )4 24.7 ± 2.4 31 5.6 Herceptin (7.25 ± 2.41) · 10 3 (6.50 ± 1.12) · 10 )5 9.4 ± 1.5 8.9 0.1 a The reported constants are average values obtained from three independent analyses using different biosensors, sample preparations and ligand densities on the flow cell surfaces. The equilibrium dissociation constants (K D ) were calculated from the relationship: K D = k d ⁄ k a . b Equilibrium dissociation constants for the 1 : 1 complexes, calculated from the Scatchard plot analyses. c Apparent affinity constants for the bivalent complexes, calculated from the Scatchard plot analyses. d Reported data were measured in the presence of soluble carboxym- ethyl-dextran added to the sample at a final concentration of 5 mgÆmL )1 . F. Troise et al. Binding of human immunoagents to ErbB2 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4971 lead to tighter binding in the complex, a result also obtained for the other Erbicin-derived immunoconju- gate, ERB-hcAb (see below). We then studied, by equilibrium SPR analyses, the interactions of bivalent Herceptin and ERB-hcAb, each endowed with two identical antigen binding sites, with immobilized ErbB2-ECD. In this experimental approach, the analytes were expected to have the potential to bind either to a single site or bivalently. The ratio between the monovalent and bivalent complexes would be dependent on the relative concentrations of ligand and analyte flowing over the surface. Equilibrium binding responses (R eq ) were determined directly at increasing analyte concentrations. The anal- yses of the equilibrium binding data were carried out using Scatchard plots with the binding models described by Junghans [20] to count receptors and other cell surface molecules with bivalent analytes (IgG). The binding isotherms and corresponding Scat- chard plots are shown in Fig. 3. Biphasic Scatchard plots were obtained, which are consistent with the bivalent binding model. The constants calculated from these plots, listed in Table 2, highlight the different binding behaviour as a function of analyte concentration. At higher antibody concentrations (about 45–360 nm for ERB-hcAb and 100–360 nm for Herceptin), the K D1 constants were 31 and 8.9 nm for ERB-hcAb and Herceptin, respectively. The maximum binding responses (B max1 ), which are proportional to the moles of bound antibody, were 116 and 336 response units (RU) for ERB-hcAb and Herceptin, respectively. At lower analyte concentra- tions (about 8–45 nm for ERB-hcAb and 0.5–10 nm for Herceptin), the K D2 constants were 5.6 and 0.1 nm for ERB-hcAb and Herceptin, respectively, and the maximum binding responses (B max2 ) were 76 and 298 RU for ERB-hcAb and Herceptin, respectively. These data can be interpreted by surmising that, at low concentrations, the antibody can simultaneously bind two receptor molecules. This reflects the high affinity of the antibody for the receptor, as marked by a low K D2 constant. It should be noted (see Table 2) that the K D2 values are virtually identical to the appar- ent binding constants determined by ELISA (7 and 0.1 nm for ERB-hcAb and Herceptin, respectively, compared with 5.6 and 0.1 nm, respectively). Indeed, the antibody concentration range explored by ELISA was very similar to that examined by SPR. At high antibody concentrations, the crowding of antibody molecules on the immobilized ErbB2-ECD renders it difficult to obtain simultaneous binding of antibody to two receptor molecules, and the binding is mainly monovalent; this is reflected in the low affinity with a higher K D1 constant. The analysis of the maximum binding values of the bivalent analytes, calculated from the Scatchard plots at low and high analyte concentrations (see below), supports these hypotheses. If bivalent binding is achieved, the maximum binding response (B max2 ) should be one-half of the maximum binding response expected for monovalent binding (B max1 ). For ERB-hcAb, B max2 (76 RU) was indeed about one-half of the B max1 value (116 RU), in good agreement with the above-mentioned hypothesis. Furthermore, as a control experiment, a Scatchard plot analysis of the equilibrium binding data of ERB-hRNase was carried out (data not shown). This immunoagent is a monovalent analyte with a molec- ular weight of 46 kDa, about half of the ERB-hcAb molecular weight (100 kDa), and showed binding to immobilized ECD in a 1 : 1 ratio. In this case, the expected B max value should be simi- lar to B max2 determined for ERB-hcAb bivalent bind- ing. The calculated B max value for ERB-hRNase was found to be 84 RU, very similar to the value calcu- lated for the bivalent binding of ERB-hcAb (B max2 = 76 RU), in line with the hypothesis formu- lated above. The K D value calculated from the Scat- chard plot for ERB-hRNase was 24 nm (Table 2), in very good agreement with that determined by SPR kinetic experiments (27.2 nm). However, the B max2 value for the bivalent binding of Herceptin (298 RU) was higher than the expected one- half value of B max1 (1 ⁄ 2B max1 = 168 RU). This differ- ence could be ascribed to the lack of equilibrium response data at very low Herceptin concentrations (< 0.5 nm), required to accurately define the Scat- chard plot for bivalent binding. These results, in line with those predicted using the binding models described by Junghans [20], indicate that bivalent binding typically dominates over mono- valent binding up to very high antibody concentra- tions. To further test this hypothesis, different ECD densi- ties were immobilized on the chip surface for analysis of the antibody affinity at equilibrium. However, when a low ECD density (260–340 RU) was used, it was not possible to record the equilibrium responses at anti- body concentrations close to the K D value; when a higher ECD density (1500–1800 RU) was used, equi- librium responses were recorded, but the maximum antibody binding values (900–1300 RU) were too high to be reliable. It may be of interest that the equilibrium binding analyses carried out by SPR may be applied to deter- Binding of human immunoagents to ErbB2 F. Troise et al. 4972 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS mine both affinity constants: the equilibrium dissocia- tion constant (K D1 ) for the 1 : 1 complex, an intrinsic property of the binding site, and the apparent affinity constant (K D2 ) for the bivalent complex, dependent on steric features. It has been reported that, in mammary carcinomas, in which ErbB2 is overexpressed, the antitumour action of Herceptin can be neutralized in part by bind- ing to soluble ErbB2-ECD, which is proteolytically cleaved and shed into the patients’ sera [19]. Given the interest in the binding properties of anti-ErbB2 immunoagents (ERB-hcAb and Herceptin) to soluble ErbB2-ECD, a different SPR system was implemented by performing assays on the antibodies trapped on the sensor chip (see Experimental procedures), with soluble ErbB2-ECD freely passed over the chip. The kinetic constants for association (k a ) and disso- ciation (k d ) were determined. Figure 3C,D shows the binding curves used to determine k a and k d (see Table 2) for the complexes of ERB-hcAb or Herceptin with ErbB2-ECD. These analyses highlight the differ- ent kinetic behaviour for the two antibodies. Herceptin binds ErbB2-ECD with a relatively low value of k a (7.25 · 10 3 m )1 Æs )1 ), about three-fold lower than the value determined for the ERB-hcAb ⁄ ErbB2-ECD com- plex (1.77 · 10 4 m )1 Æs )1 ). With regard to the dissocia- tion step, the Herceptin ⁄ ErbB2-ECD complex was found to be much more stable, with a k d value of 6.5 · 10 )5 s )1 , about one order of magnitude lower than the k d value of the ERB-hcAb ⁄ ErbB2-ECD complex (4.35 · 10 )4 s )1 ). The calculated equilibrium dissociation constants (K D ) for Herceptin ⁄ ErbB2-ECD and ERB-hcAb ⁄ ErbB2-ECD 1 : 1 complexes were 9.4 and 24.7 nm per binding site, respectively. Interestingly, these K D values were very similar to the K D1 values determined by equilibrium SPR analyses at high antibody concentrations, when only a single antigen binding site interacts with ErbB2-ECD (see Table 2). The kinetic constants for the association (k a ) and dissociation (k d ) phases, determined by SPR, are clo- sely correlated with the bivalent affinity enhancements (avidities) relative to the monovalent interactions (intrinsic affinities). The avidity is usually considered as a measure of the resistance of antibody ⁄ antigen complexes to dissociation after dilution [21]. As the stability of immunocomplexes is mainly the result of a low dissociation rate constant value (k d ), it could be predicted that the increase in affinity of bivalent complexes in comparison with mono- valent complexes should be inversely proportional to the dissociation rate constant value (k d ). Therefore, the Herceptin ⁄ ErbB2-ECD complex with a dissociation rate constant (k d ) of 6.5 · 10 )5 s )1 , about one order of mag- nitude lower than the k d value of the ERB-hcAb ⁄ ErbB2-ECD complex (4.35 · 10 )4 s )1 ), is expected to exhibit a larger increase in affinity when bivalent binding is allowed. This is in line with the findings that the apparent affinity constant for the Herceptin ⁄ ErbB2-ECD bivalent complex (K D2 = 0.1 nm) is 100- fold lower than the equilibrium dissociation constant (K D1 = 9.6 nm) for the monovalent complex, whereas the apparent affinity constant for the ERB-hcAb ⁄ ErbB2-ECD bivalent complex (K D2 = 5.6 nm) is only five-fold lower than the equilibrium dissociation con- stant (K D1 = 24.7 nm) for the monovalent complex. Together, these data confirm once again that Herceptin binds to soluble ErbB2-ECD with a higher affinity than does ERB-hcAb, so that, in vivo, the frac- tion of Herceptin strongly sequestered into this immu- nocomplex may not be available for interactions with cell-embedded ErbB2. Analyses by ITC of the interactions of soluble EDIAs and Herceptin with soluble ErbB2-ECD Given the intriguing results obtained by studying the binding of free, soluble ErbB2-ECD to anti-ErbB2 immunoagents, we studied these interactions by ITC, another analytical tool firmly based on physicochemical principles. Using this methodology, a ligand is gradually titrated against a macromolecule, thus evolving or tak- ing up measurable heat. In the ITC experimental set-up, both the macromolecules, in our case ErbB2-ECD and the immunoagents, are free in solution. Figure 4 shows the results of calorimetric titrations for the interactions of the immunoagents (Erbicin, EDIAs, Herceptin) with ErbB2-ECD. Exothermic heat pulses were observed after each injection of immuno- agent into the ErbB2-ECD solution (see insets in Fig. 4). The integration of the heat produced per injec- tion as a function of time, and conversion to per mole of immunoagents, gave the corresponding binding iso- therms (see Fig. 4). The data, plotted as a function of the molar ratio, indicate a binding stoichiometry of 1 : 1 for ERB-hRNase and Erbicin and of 0.5 : 1 for ERB-hcAb and Herceptin, i.e. each identical antigen binding site binds one molecule of ErbB2-ECD. The binding constants (K b ; converted for comparison to K D ) and enthalpy changes (D b H°), obtained by stan- dard equations, led to the thermodynamic parameters summarized in Table 3. Their inspection reveals K D values close to those obtained with ELISA and SPR (see Tables 1, 2) for the binding of monovalent Erbicin and ERB-hRNase. With regard to the complexes with bivalent ERB-hcAb and Herceptin, the K D values were F. Troise et al. Binding of human immunoagents to ErbB2 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4973 higher than those obtained with ELISA, but close to those determined by SPR, based on either the kinetic constants or equilibrium measurements analysed using the Scatchard equation. In particular, they were close to the K D1 values calculated at high ligand concentra- tions when monovalent binding prevails (see Table 2). As shown by the D b H° values in Table 3, binding is driven by a favourable binding enthalpy, but opposed by an unfavourable binding entropy change, D b S°. It is of interest that, of the studied systems, Erbicin shows the lowest affinity for ErbB2-ECD, in agree- ment with ELISA and SPR results, and the interaction is characterized by the lowest enthalpy change. This indicates a lower number of non-covalent interactions on binding of EDIAs (ERB-hcAb and ERB-hRNase), as also revealed by ELISA and SPR (see above). However, the unfavourable D b S° value recorded for these interactions indicates a greater dec- rease in conformational stability on complex formation. When an attempt was made to study the interactions of the immunoagents with live cells, i.e. with ErbB2 inserted on the cell membrane, it was verified that the experimental system could, in principle, be used, with stoichiometric values identical to those obtained with ErbB2-ECD and immunoagents in solution. However, surprising results were found: the D b H° values were about 100-fold higher than those measured with solu- ble receptor and ligand, and the binding constants were about 1000-fold higher. To verify whether the very high D b H° and affinity constants could be related to events occurring on inter- nalization of the immunoagents, the experiments were –100 –50 0 A B C D 0 2000 4000 6000 8000 10 000 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 –350 –300 –250 –200 –150 Time (s) Power (µJ·s –1 ) 0.15 0.20 0.25 –60 –30 0 [Erbicin]/[ECD] [ERB-hRNase]/[ECD] 2000 4000 6000 8000 10 000 –0.15 –0.10 –0.05 0.00 0.05 0.10 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 –150 –120 –90 Time (s) Power (µJ·s –1 ) Power (µJ·s –1 ) –100 –80 –60 –40 –20 0 0.6 0.9 1.2 1.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 –180 –160 –140 –120 [ERB-hcAb]/[ECD] 0 0 2000 4000 6000 8000 10 000 –0.3 0.0 0.3 Time (s) –120 –100 –80 –60 –40 –20 0.3 0.6 0.9 1.2 1.5 Power (µJ·s –1 ) kJ·mol –1 kJ·mol –1 kJ·mol –1 kJ·mol –1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 –160 –140 [Herceptin]/[ECD] 0 2000 4000 6000 8000 10 000 –0.3 0.0 Time (s) Fig. 4. Determination by ITC of the binding interactions between anti-ErbB2 immunoagents and ErbB2-ECD (ECD). Binding isotherms are shown for the titration of ErbB2-ECD with Erbicin (A), ERB-hRNase (B), ERB-hcAb (C) and Herceptin (D). The raw data are shown in the insets. Table 3. Thermodynamic parameters of Erbicin, EDIAs and Herceptin for soluble ErbB2-ECD by ITC assays. nK b ⁄ 10 7 (M )1 ) K D (nM) D b H° (kJÆmol )1 ) TD b S° (kJÆmol )1 ) D b G° (kJÆmol )1 ) Erbicin ⁄ ErbB2-ECD 1.0 1.3 ± 0.3 77 ± 19 )139 ± 8 )260 ± 4 )44 ± 11 ERB-hRNase ⁄ ErbB2-ECD 1.0 4.8 ± 1.2 21 ± 5 )300 ± 18 )95 ± 20 )40 ± 10 ERB-hcAb ⁄ ErbB2-ECD 0.5 2.2 ± 0.6 45 ± 11 )175 ± 11 )133 ± 16 )48 ± 11 Herceptin ⁄ ErbB2-ECD 0.5 8.4 ± 2.1 12 ± 3 )150 ± 6 )105 ± 5 )45 ± 11 Binding of human immunoagents to ErbB2 F. Troise et al. 4974 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS repeated using, as immunoagent, an anti-ErbB2 mono- clonal which was not internalized (anti-ErbB2 N28), or by testing cells poisoned to inhibit endocytosis. Very similar values were obtained. This indicates that the surprising values are not a result of the internalization process. As an alternative, we concluded that the interactions of anti-ErbB2 immunoagents with ErbB2 on live cells could not be interpreted as simple ligand ⁄ receptor interactions. It has been anticipated in recent reports that ligand binding to cell receptors may trigger higher order events in the membrane of targeted cells. In these events, directly stimulated receptors and other seemingly unrelated receptors and effectors are engaged in the formation of complex networks and receptor mosaics [22], and may induce membrane bending and remodelling [23]. An ErbB signalling net- work was proposed [24] after an analysis at the sys- tems level, with ErbB2 as an amplifier of the network [25]. Thus antibody binding, which mimics ligand binding, may set off events beyond binding, which are demonstrated by a higher binding affinity and a high heat production. Discussion The novel antitumour immunoagents Erbicin, ERB- hcAb and ERB-hRNase have most, if not all, the features that make an immunoagent a valid, precious tool for anticancer immunotherapy: (a) they are all of human origin, which strongly decreases, if not elimi- nates, the risks of an immune response; (b) they are directed to a cell receptor, such as ErbB2, which is minimally present in non-malignant cells, but overex- pressed in many carcinomas, especially in breast cancer cells; (c) they selectively kill ErbB2-positive cells, both in vitro and in vivo; (d) their size, smaller than that of immunoglobulins, should favour penetration in solid tumours; however, in the case of ERB-hcAb and ERB- hRNase, it should also allow for a prolonged half-life in the bloodstream. Binding to a cell-embedded tumour-associated anti- gen is the first key step in the mechanism of antitu- mour immunoagents. Thus, we directed our attention to studying the binding properties of the novel EDIAs, as well as Herceptin, an immunoagent successfully employed in the therapy of breast cancer. Further- more, the availability of soluble ErbB2-ECD enabled us to describe, for the first time, the binding of these immunoagents to the isolated, free ECD of ErbB2. In addition, for the first time, the binding study was con- ducted not only using a semiquantitative methodology, such as that based on ELISA, previously used to measure Herceptin binding [14], but also using quanti- tative methods based on physicochemical principles, such as SPR and ITC. The main results of this study can be summarized as follows. 1. For the first time, extensive and conclusive infor- mation is reported on the relative affinity and binding kinetics of the EDIAs and Herceptin for soluble or cell-linked ErbB2. 2. The results were validated by the use of three independent methodologies, ELISA, SPR and ITC, which gave coherent results. 3. The binding of Erbicin to ErbB2-ECD was found to be enhanced and stabilized by the linking of Erbicin scFv to either an RNase or the Fc antibody fragment, as in ERB- hRNase and ERB-hcAb, respectively. This was revealed by the higher binding affinity of the Erbicin immunoconjugates with respect to that of free Erbicin scFv. 4. The novel EDIAs display a binding affinity towards soluble ErbB2-ECD which is lower than that measured for ECD embedded in the membrane of ErbB2-positive cells. Herceptin, by contrast, shows a higher affinity for soluble ErbB2-ECD. Furthermore, binding of ERB-hcAb to cancer cells and its antitu- mour activity are not affected by soluble ECD, whereas the same properties of Herceptin are strongly inhibited. As soluble ECD is proteolytically released from the surface of ErbB2-overexpressing cancer cells, and is detected in the serum of patients with advanced breast cancer, a fraction of Herceptin is neutralized in these patients by serum ECD, and hence its cell-direc- ted antitumour action is reduced [19]. It has been reported that free, soluble ECD can induce resistance to the growth inhibitory activity of anti-ErbB2 anti- bodies [26], can neutralize their activity and affect their pharmacokinetics, thus leading to resistance to immu- notherapy [27]. However, the use of immunoagents with a low affinity for soluble ECD, such as the Erbi- cin-based immunoagents, will allow for lower thera- peutic doses to be used compared with those needed for Herceptin-based therapy. 5. A binding study car- ried out by ITC on anti-ErbB2 immunoagents tested directly on live cells revealed that the association of the immunoagents with the receptor inserted into live cells cannot be interpreted as a simple ligand ⁄ receptor interaction. Antibody binding, just like ligand binding, triggers higher order events which engage other mem- brane receptors and effectors in the formation of com- plex networks and receptor mosaics. These data strongly imply that ITC on live cells and high-affinity antibodies to ErbB2, a critical receptor for which no specific ligand has yet been found, could be employed in a systems biology approach to unravel the physio- logical significance of the cell receptor. F. Troise et al. Binding of human immunoagents to ErbB2 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS 4975 Experimental procedures Cell lines and antibodies The 293 cell line (human embryonic kidney) was cultured in DMEM (Gibco Life Technologies, Paisley, UK). The SKBR3 cell line (human breast cancer) was cultured in RPMI 1640 (Gibco Life Technologies). The media were supplemented with 10% heat-inactivated fetal bovine serum, 50 UÆmL )1 penicillin and 50 lgÆmL )1 streptomycin (all from Gibco Life Technologies). All the cell lines were obtained from the American Type Culture Collection and cultured at 37 °Cina5%CO 2 atmosphere. The antibodies used were as follows: Herceptin (Genen- tech, South San Francisco, CA, USA); horseradish peroxi- dase-conjugated anti-His IgG1 (Qiagen, Valencia, CA, USA); horseradish peroxidase-conjugated goat anti-human affinity-isolated IgG1 (Fc-specific; Sigma, St Louis, MO, USA). Erbicin, ERB-hRNase and ERB-hcAb were pre- pared as described previously [12–14]. The anti-ErbB2 N28 monoclonal was a generous gift from Michael Sela (Weiz- man Institute of Science, Rehovot, Israel). Production of ErbB2-ECD ErbB2-ECD, the extracellular domain of ErbB2 (residues 1–624), was stably expressed and secreted by the 293 cell line. The culture medium of 293 cells, before and after transfection, was analysed: (a) by 8% SDS-PAGE and wes- tern blotting with Herceptin followed by horseradish perox- idase-conjugated anti-human (Fc-specific) IgG serum fraction; (b) by immunoprecipitation assays carried out by the incubation of 10 mL aliquots of 293 cell conditioned medium with 10 lgÆmL )1 of Herceptin or ERB-hcAb in NaCl ⁄ P i for 3 h at 4 °C. The immune complexes were then collected by adsorption to protein A-Agarose (Sigma) for 1 h at 4 °C. After washing with NaCl ⁄ P i , the proteins were released by boiling in loading buffer [28], and run using 8% SDS-PAGE, followed by immunoblotting assays as described above. Protein purification ErbB2-ECD, secreted by transfected 293 cells, was purified from the culture medium by immunoaffinity chromatogra- phy with the AKTA Purifier system (GE Healthcare, Amer- sham Bioscience AB, Uppsala, Sweden). The affinity column was prepared by coupling 8 mg of Herceptin to 1.5 g of CNBr-activated Sepharose 4B Fast Flow (GE Healthcare). The antibody was immobilized to agarose via a secondary amine according to the manufacturer’s instruc- tions. The resulting 4 mL column was loaded with 10 mL of 10-fold concentrated conditioned medium, washed with three volumes of 10 mm Tris ⁄ HCl, pH 7.4 and eluted with 50 mm glycine pH 3.0 containing 1 m NaCl. The collected fractions were immediately neutralized with a 1 : 10 volume of 1 m Tris ⁄ HCl pH 8.0. The purity of the preparation was evaluated by 8% SDS- PAGE, followed by Coomassie staining or western blotting analyses with either Herceptin or ERB-hcAb as primary antibody, followed by horseradish peroxidase-conjugated anti-human IgG1 (Fc-specific) mAb. ELISA The affinity of Erbicin or ERB-hRNase for soluble ErbB2-ECD was measured by an ELISA sandwich assay. A 96-well plate was coated with 5 lgÆmL )1 of Herceptin in NaCl ⁄ P i (Sigma), kept overnight at 4 °C and blocked for 1 h at 37 °C with 5% BSA (Sigma) in NaCl ⁄ P i .To the plate, rinsed with NaCl ⁄ P i , a solution of purified ErbB2-ECD in NaCl ⁄ P i (5 lgÆmL )1 ) was added. After 1 h at room temperature, the plate was washed, and increasing concentrations of purified ERB-hRNase or Erbicin (50– 500 nm) were added in ELISA buffer (NaCl ⁄ P I –BSA 1%) in triplicate wells, and incubated for 2 h at room tempera- ture with a blank control of NaCl ⁄ P i . After rinsing with NaCl ⁄ P i , an anti-His horseradish peroxidase-conjugated IgG1 was added in ELISA buffer. After 1 h at room temperature, the plate was rinsed with NaCl ⁄ P i , and bound immunoagents were detected using 3,3¢,5,5¢-tetramethyl- benzidine as a substrate (Sigma). The product was measured at 450 nm using a microplate reader (Multilabel Counter Victor 3, Perkin Elmer, Cologno Monzese, Italy). The affinity of ERB-hcAb or Herceptin antibodies for ErbB2-ECD was measured as follows. A 96-well plate was coated with 5 lgÆmL )1 of purified ECD in NaCl ⁄ P i and left overnight at 4 °C. After blocking as above, increasing concentrations of ERB-hcAb (10–60 nm ) or Herceptin (0.1–10 nm) were added in ELISA buffer for 2 h at room temperature. The plate was rinsed with NaCl ⁄ P i and an anti-human (Fc-specific) horseradish peroxidase-conjugated IgG serum fraction was incubated for 1 h, and detected as described above. The reported affinity values are the means of at least three determinations (standard deviation, £ 5%). ELISAs with ErbB2-positive cells were carried out on SKBR3 cells as described previously [14]. ERB-hcAb (1–16 nm) or Herceptin (1–8 nm) was tested in the presence or absence of soluble ErbB2-ECD, added either in equimo- lar amounts or in a 10-fold molar excess to the ErbB2 receptor number on SKBR3 cells [18]. Cytotoxicity assays ErbB2-positive cells were treated as described previously [14] with ERB-hcAb or Herceptin at concentrations of 2.5 nm in the absence or presence of soluble ErbB2-ECD (20 nm). Cell growth inhibition was reported as the percent- age of cell survival reduction induced by the treatment with Binding of human immunoagents to ErbB2 F. Troise et al. 4976 FEBS Journal 275 (2008) 4967–4979 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... 4977 Binding of human immunoagents to ErbB2 F Troise et al 2.5 · 105 cells, corresponding to 0.83 pmol of ErbB2 receptor [18], were diluted to 2 mL in buffer and introduced into the sample compartment of the instrument The immunoagents were added by successive injections as above, up to a total of 1.66 pmol The data were treated as described above for the experiments with soluble proteins In the experiments... flow rate of 30 lLÆmin)1, and association and dissociation phases were recorded for 200 and 300–600 s, respectively The sensor surface was regenerated by 30 s injections of 10 mm glycine–HCl, pH 2.2, at the end of each binding cycle To measure the binding properties of Erbicin and ERBhRNase to the extracellular domain of ErbB2, ECD was immobilized onto the surface of sensor chip CM5 using the standard... of 2 mL The concentration of ErbB2- ECD in the cell was about 30 lm, and the immunoagent (Erbicin, ERB-hRNase, ERB-hcAb, Herceptin) concentration in the syringe was about 3 lm For each titration, 10 lL aliquots of immunoagents in NaCl ⁄ Pi solution were injected into the ErbB2- ECD solution in NaCl ⁄ Pi at 400 s intervals Binding curves involved the addition of 25 injections The heat of dilution of the. .. dilution of the immunoagents into the solvent was measured in a separate experiment The data were integrated, corrected for the heats of dilution, normalized for concentration and analysed assuming a model based on a single set of identical independent binding sites, using the Bindwork software supplied with the instrument, which provided the stoichiometry of binding (n ligand : protein), the change in... ethanolamine hydrochloride and HBS-EP running buffer were purchased from Biacore AB Soluble carboxymethyldextran sodium salt was obtained from Fluka (Buchs SG, Switzerland) and Protein A from Staphylococcus aureus was purchased from GE Healthcare To investigate the binding properties of Herceptin and ERB-hcAb to the soluble ECD of the ErbB2 receptor, a capture method was chosen Herceptin and ERB-hcAb were captured... fitting data to the 1 : 1 Langmuir binding model Values of v2 for the fits were £ 0.8, indicating good fits The equilibrium dissoci- Binding of human immunoagents to ErbB2 ation constants (KD) were calculated from the values of the association rate constant ka and dissociation rate constant kd, according to the thermodynamic relationship KD = kd ⁄ ka Standard deviations were obtained from three independent... preparations and ligand densities on the flow cell surfaces Surface plasmon resonance was also employed to carry out equilibrium binding analyses of Herceptin and ERB-hcAb, as bivalent analytes, and ERB-hRNase, as a monovalent analyte, to ErbB2- ECD For this purpose, Herceptin, ERB-hcAb or ERB-hRNase was passed over ErbB2- ECD (500 RU) immobilized on a CM5 sensor chip [17] Increasing concentrations of Herceptin. .. second set of binding curves was recorded for ERB-hRNase in the presence of soluble carboxymethyl-dextran at a final concentration of 5 mgÆmL)1 The rate constants of the interactions described above were calculated by non-linear analysis of the association and dissociation curves using SPR kinetic evaluation software (package BIAevaluation 3.2, Biacore AB), fitting data to the 1 : 1 Langmuir binding model... antibody plus Binding of human immunoagents to ErbB2 28 29 30 31 32 cisplatin in patients with HER2 ⁄ neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment J Clin Oncol 16, 2659–2671 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 Karlsson R, Michaelsson A & Mattsson L (1991) Kinetic analysis of monoclonal... Typically, 500 RU of ErbB2- ECD were immobilized onto the sensor surface Binding curves were recorded by injecting Erbicin (5–700 nm) or ERB-hRNase (3–700 nm) over the immobilized ErbB2- ECD at a constant flow rate of 30 lLÆmin)1 Association and dissociation phases were recorded for 200 and 300 s, respectively At the end of each detection, the sensor surface was regenerated by injecting 10 lL of 10 mm NaOH . Healthcare. To investigate the binding properties of Herceptin and ERB-hcAb to the soluble ECD of the ErbB2 receptor, a capture method was chosen. Herceptin and. the case of ERB-hcAb. Effects of soluble ErbB2- ECD on the cytotoxicity of ERB-hcAb and Herceptin On the basis of the results discussed above, the antitumour