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CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applicationsAnke Steinmetz3, Fran?ois Vallée3, Christian Beil1, Christian Lange1, Nicolas Baurin3,4, Jochen Beninga1, Cécile Capdevila2, Carsten Corvey1, Alain Dupuy3, Paul Ferrari2, Alexey Rak3, Peter Wonerow1, Jochen Kruip1, Vincent Mikol3, Ercole Rao1Supplementary MaterialsAuthor affiliations1Sanofi-Aventis Deutschland GmbH, R&D, Global Biotherapeutics, Industriepark Hoechst, 65926 Frankfurt am Main, Germany2Sanofi R&D, Global Biotherapeutics, Centre de Recherche Vitry-sur-Seine, 94403 Vitry-sur-Seine Cedex, France3Sanofi R&D, LGCR, Centre de Recherche Vitry-sur-Seine, 94403 Vitry-sur-Seine Cedex, France4currently: Sanofi Pasteur, Dengue Unit, 69007 Lyon, FranceTable of contentsPageItem2Table of contents4Alphabetical list of abbreviations7Initial CODV-Fab prototypes8CODV-Ig binding to membrane receptors10Table 1: The CODV format maintains antigen affinities at the level of the parental antibodies without positional effect.14Table 2: The CODV format is universally applicable. 15Table 3: The CODV format preserves full Fc functionality in comparison to control IgG1.16Tables 4a & b: CODV architecture results in compact stable Igs18Table 5: Data Collection and refinement statistics20Figure 1: The three-dimensional modelling strategy efficiently supports the design of fully functional, universally applicable CODV-Ig constructs.21Figure 2: Antigen-binding to CODV-Ig is independent without positional, allosteric, or cooperative effects.22Figure 3: Sequence of CODV-FabIL4 x IL13 and IL423Figure 4: IL4 epitope and paratope24Figure 5: The paratopes of CODV-Ig are directed in opposite directions thus the format allows to accommodate a large variety of antigen sizes. 25Figure 6: The cross-over architecture is self-supporting and allows for limited reorientation of Fv and Fc1 domains. 26Figure 7: The CODV format binds FcRn and serum half-life extension by endocytic salvage is expected when IgG frameworks are employed. 27Figure 8: The CODV format binds Fc?Rs and thereby has the potential to activate ADCC and ADCP.28Figure 9: CODV-Ig architecture is compatible with hexamerization required for complement-dependent cytotoxicity.29Figure 10: CODV architecture retains intrinsic functionalities of natural IgGs such as ADCC and CDC. 31Materials & Methods45ReferencesAlphabetical list of abbreviations??TCR – α and β chains that are part of the T cell receptor complexADCC – antibody-dependent cell-mediated cytotoxicityADCP – antibody-dependent cell-mediated phagocytosisCD19 – Cluster of Differentiation 19, B-lymphocyte antigen CD19CD3? – Cluster of Differentiation 3 epsilon, T-cell surface glycoprotein CD3 epsilon chain, part of the T cell receptor complexCDC – complement-dependent cytotoxicityCDR1/2/3 – first/second/third complementarity-determining region of VL or VHCH1/2/3 – first/second/third constant domain of the heavy chain of an IgCHO cells – Chinese hamster ovary cellsCL – constant domain of the light chain of an Ig, pairs with CH1 CODV-Ig – cross-over dual variable Ig-like proteinsCODV-Fab – Fab-equivalent of CODV-IgDSF – differential scanning fluorimetryDVD-Ig – dual-variable-domain IgEGFR – epidermal growth factor receptorFab – antigen-binding fragment of an antibodyFc1/2/3 – a constant fragment of an antibody composed of (CL+CH1)/(CH2)2/(CH3)2FcR – Fc-binding immunoglobulin receptorFc?R1 – FcR Fc-gamma receptor 1, Cluster of Differentiation 64 (CD64)FcRn – neonatal FcRFv – the variable fragment of an antibodyG4S – linker with amino acid sequence glycine-glycine-glycine-glycine-serineHC / LC – heavy/light chain of an immunoglobulin HER2 – human epidermal growth factor receptor 2Ig – immunoglobulinIGF1R – insulin-like growth factor 1 receptorIL1?/4/12/13/23 – interleukin 1?/4/12/13/23L1/2/3/4 – first/second/third/fourth linker of CODV-Ig and CODV-Fabna – not applicableNALM-6 – cell line of pre-B cell leukaemiand – not determinednm – not measurednp – not producednr – not reportedRFU – relative-fluorescence-unitsrmsd – root mean square deviationSEC – size exclusion chromatographySPR – surface plasmon resonanceTNF? – tumor necrosis factor-alphaVH – variable domain of the heavy chain of an IgVL – variable domain of the light chain of an IgInitial CODV-Fab prototypesCODV prototypes are developed using parental monoclonal antibodies directed against interleukins 4 and 13 (IL4 and IL13, respectively). The format is based on the novel concept of asymmetrically introducing FvIL4 as an insertion domain on either heavy or light chain between the FvIL13 and Fc1 domains of IgGIL13 and N-terminally extending the other IgG chain. Thus, the insertion domain assigned to one of the chains must be introduced in a manner maintaining proper domain dimerization of both Fvs. Initial evaluation of putative arrangements of domains FvIL13 and FvIL4 suggests that G4S or (G4S)2 linkers for L1 and L3, and no residues for L2 and L4 are suitable. All possible constructs with respect to linker lengths and order of FvIL13 and FvIL4 domains are produced with expected molecular weight, very little aggregation is observed, and the purified proteins are stable in solution over time. However, none of these proteins shows any affinity for either of the antigens in surface plasmon resonance (SPR) experiments that evince antigen binding by the parent Fab fragments. CODV-Ig binding to membrane receptorsHere we expose observations in the modelling of the CODV-IgHER2 x HER3/HER2/HER3 complexes that convince us that the modelled CODV-IgHER2 x HER3 should indeed inhibit HER2/HER3 signalling. We also comment on the stoichiometries of CODV-Ig binding to membrane-located targets and steric considerations possibly impacting biological outcome.We construct homology models of CODV-IgHER2 x HER3/HER2/HER3 complexes including the transmembrane helices of HER2 and HER3 to gain insight into CODV-Ig binding membrane-located targets. As described in Methods (Supplements, pages 29-45), the templates of the transmembrane helices are carefully placed at the end of the composition of the master template to allow for construction of the membrane model that correctly surrounds these helices. In a first attempt a pair of properly dimerized helices is placed as closely as possible to the C-termini of the extracellular domains of HER2 and HER3 to construct a model of CODV-Ig binding to the active HER2/HER3 complex. However, the N-termini of the transmembrane helices cannot be simultaneous placed in sufficient proximity of the C-termini to which they have to be respectively connected to obtain a model that retains the extracellular domains correctly folded. Therefore, the template helical pair is placed closely enough to the C-terminus of the extracellular domain of HER2 to properly connect one helix. The second helix is then translated in the imaginary membrane plane to the appropriate vicinity of the C-terminus of the extracellular domain of HER3. Subsequently, the master template is assembled, the homology model of the complex constructed, and an explicit membrane model included in the preparation of the molecular dynamics simulation. During the simulation the transmembrane helices stay in the lipid bilayer as they should and they do not dimerize. Furthermore, no tendency is observed that could suggest that the helices would approach each other in much longer simulations while the antibody remains bound to both targets. This observation confirms the self-supporting architecture of the CODV architecture. The situation is qualitatively the same in models which engage one Fab-arm with HER2 and the other with HER3. Therefore we anticipate that the modelled CODV-IgHER2 x HER3 inhibits HER2/HER3 signalling.The three dimensional models of the CODV-IgHER2 x HER3/HER2/HER3 complex show that CODV-IgHER2 x HER3 should easily bind two membrane receptors concomitantly. Simultaneous engagement of four membrane receptors on the same cell might require a minimum of cellular plasticity. Alternatively, it might be necessary to target different HER2 or HER3 epitopes in case concomitant binding of four membrane receptors is desired. In a more general perspective, steric constraints in vivo may have an important impact on the efficiency of the biologic response not only as far as antigen engagement is concerned but also for concomitant binding of target and Fc receptors or complement components in ADCC or CDC as we already observed in the T-cell dependent cytotoxicity of CODV- and DVD-Fabs. Table 1: The CODV format maintains antigen affinities at the level of the parental antibodies without positional effect. The table indicates protein format, order of targeted antigens, CODV format type, and lengths of all-glycine linkers. Data on protein production yield, aggregation propensity, and antigen affinities are reported for all four CODV types with all-Glycine linkers targeting IL4 and IL13. Yield ranges between 0.1 and 61 mg/L culture medium with aggregation between 2 and 93 % with medians of 4 mg/L and 12 %, and mean values of 9.4 mg/L and 21.5 %, respectively. The parental antibodies IgGIL4 and IgGIL13 bind their antigens with a KD of about 8 and 80 pM, respectively. The KD values of the CODV-Ig constructs are observed between 1 and 80 pM with a median of 6 pM and a mean value of 7 pM for IL4 binding and between 9 and 265 pM with a median of 60 pM and a mean value of 64 pM for IL13 binding. Abbreviations: na = not applicable, nm = not measured, np = not produced, nr = not reportedTable 2: The CODV format is universally applicable. The table indicates protein format, targeted antigens, CODV format type, and linker sequences. L1 and L3 of CODV-Ig correspond to LL and LH of DVD-Ig, respectively; Fv2-Fc transitions of DVD-Ig do not require linker insertions that would correspond to L2 and L4 of CODV-Ig. Data on protein production yield, aggregation propensity, and antigen affinities as well as activities in cellular assays are reported and compared for constructs of CODV- and DVD-Ig format targeting various antigens. Abbreviations: na = not applicable, nm = not measured, np = not produced, nr = not reported.Table 3: The CODV format preserves full Fc functionality in comparison to control IgG1. The affinities of IgG1IL4 and CODV-IgIL4 x IL13 to human, cynomolgus monkey, and murine FcRn and human Fc?R1 are determined by SPR. Abbreviations: nd = not determined Tables 4 a (above) and b (below): CODV architecture results in compact stable Igs. Melting temperatures of CODV constructs directed against IL4 and IL13 are slightly reduced compared to the parental antibodies. Notably, only one melting temperature is observed in CODV-Igs suggesting that melting of Fab- and Fc domains occurs concomitantly. We consider this observation as a hallmark of the circular self-contained architecture that functions as a self-supporting truss. Antigen order and linker composition do not affect thermal stability significantly, however linker lengths affects melting temperatures slightly in the examples shown. Melting temperatures are determined by differential scanning fluorimetry (DSF). * FvIGF1R from robatumumab; ** FvIGF1R from cixutumumabTable 5: Data Collection and refinement statistics Refinement of apo-CODV-FabIL4 x IL13 resulted in higher R-factors compared to the structure of the complex due to slight disorder in the position of the Fc domain. This observation is underlined by locally increased B-factors. a Values in parenthesis refer to statistics in the highest bin. b Rmerge = ∑hkl∑i|Ii(hkl) - <I(hkl)>| / ∑hkl∑iIi(hkl) where Ii(hkl) is the intensity of an observation and <I(hkl)> the mean value of its unique reflection. c Rfactor = ∑h |Fo(h) –Fc(h)| / ∑h Fo(h) where Fo and Fc are observed and calculated structure factor amplitudes, respectively. d Rfree is calculated with 5% of the data excluded from refinement. e Root mean square deviation from ideal values. f Categories defined by MOLPROBITY.Figure 1: The three-dimensional modelling strategy efficiently supports the design of fully functional and universally applicable CODV-Ig constructs. This flow-chart illustrates the modelling steps in the design of CODV-Igs described in detail in Materials & Methods. Figure 2: Antigen-binding to CODV-Ig is independent without positional, allosteric or cooperative effects. Overlay of SPR sensograms of detailed kinetic analysis of CODV-IgIL4 x IL13 by injecting both antigens simultaneously, IL4 followed by IL13, IL13 followed by IL4, and baseline (magenta, grey, blue, and red lines, respectively). In these SPR experiments a consecutive co-injection of IL4 and IL13 at 3nM and 25nM, respectively, or vice versa results in the same binding signal than the injection of a mixture of IL4 and IL13 at 3nM and 25nM, respectively. This indicates that saturation of the first binding domain with antigen does not alter binding of the antigen to the second binding domain. Therefore, we conclude that positional, allosteric, or cooperative effects are absent in CODV format. Figure 3: Sequences of CODV-FabIL4 x IL13 and IL4 in one-letter code. Violet, magenta, green, yellow, and blue indicate IL4, FvIL4, FvIL13, Fc1, and linker regions; dark and light shades differentiate heavy and light chains, respectively. Letters in grey represent residues not constructed in the crystal structure of CODV-FabIL4 x IL13/IL4 due to insufficient definition in the electron density maps. Figure 4: IL4 epitope and paratope. (a) 2-D scheme of CODV-FabIL4 x IL13/IL4, (b) epitope, and (c) paratope of IL4, both depicted by solvent accessible surfaces in colour coding as in Figure 3. Residues with atoms within 3.4 ? of the epitopes and paratopes are highlighted in grey, darker shades indicate shorter distances.70358017743IL13IL4FvIL13FvIL4IL13IL4FvIL13FvIL42550160265430L1L13382010343354L4L4Figure 5: The paratopes of CODV-Ig are directed in opposite directions, thus the format allows to accommodate a large spectrum of antigen sizes. Top view of the model of CODV-FabIL4 x IL13/IL4/IL13 shows that the distance between the epitopes is ~65 ?. The model is obtained by superposition of the crystal structures of CODV-FabIL4 x IL13/IL4 (5FHX.pdb) and FabIL13/IL13 (in-house) and colour coded as in Figure 3.Figure 6: The cross-over architecture is self-supporting and allows for limited reorientation of Fv and Fc1 domains. Superposition of the apo and complexed CODV-FabIL4 x IL13 structures: the superposition shows that the Fc1 domain is slightly reoriented in the apo and complexed structures as highlighted by the grey arrows (right). The root-mean square deviation (rmsd) of the ?-carbon traces of domains Fv1, Fv2, and Fc superimposed each alone is 0.6, 0.8, and 3.8 ?, respectively. Superimposing domains Fv1 and Fv2 simultaneously results in an??-carbon rmsd of 2.9 ?. Fixing this superposition gives rise to a rmsd of 12.7 ? for the ?-carbon trace of domain Fc. The apo structure is represented by a grey ribbon, the complexed structure by a coloured ribbon with colour coding as in Figure 3. The top view to the left includes a semi-transparent, coloured representation of the solvent accessible surface area of CODV-FabIL4 x IL13. It illustrates that the same relative orientation of FvIL4 and FvIL13 is observed in both crystal structures. The side view to the right is rotated by 90° around the horizontal axis in the xy-plane compared to the top view to the left. Figure 7: The CODV format binds FcRn and serum half-life extension by endocytic salvage is expected when corresponding IgG frameworks are employed. The model of the FcRn/CODV-IgIL4 x IL13/IL4/IL13 complex is obtained by the superposition of the crystal structure of the FcRn/Fc complex on the final CODV-Ig model. FcRn binding to CODV-Ig is not sterically affected by the additional Fv domain compared to IgG and the FcRn binding mode should be unchanged. Antigens and FcRn could bind simultaneously in relevant situations. The bispecific antibody is depicted by its solvent accessible surface coloured in magenta, green, orange, and blue for FvIL4, FvIL13, Fc regions, and linkers, respectively. Dark and light shades differentiate heavy and light chains. IL4, IL13, and FcRn are shown in violet, green, and grey ribbon representation, respectively.Figure 8: The CODV format binds Fc?Rs and thereby has the potential to activate ADCC and ADCP. The model of the Fc?R3/CODV-IgIL4 x IL13/IL4/IL13 complex is obtained by the superposition of the crystal structure of the Fc?R3/Fc complex on the final CODV-Ig model. Less steric hindrance is observed between Fc?R3 and the Fab region of CODV-Ig than in the equivalent superposition on IgG1 (not shown).The conformational flexibility of the CH1-CH2 transitions of CODV-Ig and IgG are probably alike and the Fc?R binding mode should be preserved. Neither the additional Fv nor antigen binding per se appears to interfere with Fc?R association. Surface and ribbon representations of the bispecific antibody, IL4, and IL13 are as in Figure 5. Fc?R is depicted by a grey ribbon representation.Figure 9: CODV-Ig architecture is compatible with hexamerization required for complement-dependent cytotoxicity by IgG. The CODV-IgIL4 x IL13/IL4/IL13 hexamer is modelled on the crystallographic IgG1 hexamer that is competent for complement activation. To the left is shown the top view of the hexamer, to the right a side view of two adjacent bispecific antibodies of the hexamer that illustrates the placement of the Fab-arms either above, in, or below the plane defined by the Fc domains. The bispecific antibody is depicted in surface representation; to highlight the possibility of concomitant antigen-binding IL4 and IL13 are represented by ribbon diagrams; colour coding is applied as in Figure 5. The lines in free-hand style delimit one CODV-Ig antigen complex. Figure 10: CODV architecture retains intrinsic functionalities of natural IgGs such as ADCC or CDC. Immunoglobulins are tested on CHO cells as target cells which express membrane-bound TNF?. Effector cells of the ADCC assay are Jurkat cells which express Fc?IIIR and activate a NFAT driven luciferase reporter upon antibody-binding and cross-linking due to target cell engagement. CDC activity is measured by LDH release into the supernatant as surrogate for : ADCC activity of IgGTNF? and CODV-IgIL12/23 x TNF? is measured by activation of the Jurkat luciferase reporter translating antibody-dependent cellular cytotoxicity in the presence and absence of TNF? (orange line and tilted square, redline and square, respectively, with dotted or dashed lines and open symbols indicating presence of TNF?). ADCC activity is induced by IgGTNF? and CODV-IgIL12/23 x TNF?. Their ADCC activity is completely inhibited by soluble TNF? demonstrating that the TNF? binding site is necessary to make effector-target cell contact. Bottom: CDC of IgGTNF? and CODV-IgIL12/23 x TNF? is evinced by pre-incubation of CHO cells with antibodies and 6 h incubation of target cells with complement in 1:10 dilution followed by LDH detection in the supernatant (orange line and tilted square, redline and square, respectively). Complement mediated lysis depends on both, a functional anti-TNF? binding site and functional Fc domains: neither CODV-IgIL4 x IL13 nor CODV-FabIL12/23 x TNF? show CDC activity in this assay because they either lack binding to cell membrane targets or do not fix complement complexes (green line and triangle, blue dotted line and square, respectively). 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Gabriele</AUTHOR><AUTHOR>Shao, Lily</AUTHOR><AUTHOR>Birtalan, Sara</AUTHOR><AUTHOR>Sidhu, Sachdev S.</AUTHOR><AUTHOR>Eigenbrot, Charles</AUTHOR></AUTHORS><ISBN>0022-2836 DO - sequence diversity</KEYWORD><KEYWORD>binary phage library</KEYWORD><KEYWORD>HER2</KEYWORD><KEYWORD>receptor oligomer</KEYWORD><KEYWORD>X-ray structure</KEYWORD></KEYWORDS></MDL></Cite></EndNote>16, 1N8Z ADDIN EN.CITE <EndNote><Cite><Author>Cho</Author><Year>2003</Year><RecNum>175</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>175</REFNUM><YEAR>2003</YEAR><AUTHORS><AUTHOR>Cho, Hyun-Soo</AUTHOR><AUTHOR>Mason, Karen</AUTHOR><AUTHOR>Ramyar, Kasra X.</AUTHOR><AUTHOR>Stanley, Ann Marie</AUTHOR><AUTHOR>Gabelli, Sandra B.</AUTHOR><AUTHOR>Denney, Dan W.,</AUTHOR><AUTHOR>Leahy, Daniel J.</AUTHOR></AUTHORS><TITLE>Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab</TITLE><ALTERNATE_TITLE>Nature</ALTERNATE_TITLE><DATE>2003/02/13/print</DATE><VOLUME>421</VOLUME><NUMBER>6924</NUMBER><PAGES>756-760</PAGES><ISBN>0028-0836</ISBN><URL> (HER2/Fab of “tryptophan-rich antibody”, HER2/Fab of tastuzumab), 3P11 ADDIN EN.CITE <EndNote><Cite><Author>Schaefer</Author><Year>2011</Year><RecNum>176</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>176</REFNUM><AUTHORS><AUTHOR>Schaefer, Gabriele</AUTHOR><AUTHOR>Haber, Lauric</AUTHOR><AUTHOR>Crocker, Lisa&#xA0;M.</AUTHOR><AUTHOR>Shia, Steven</AUTHOR><AUTHOR>Shao, Lily</AUTHOR><AUTHOR>Dowbenko, Donald</AUTHOR><AUTHOR>Totpal, Klara</AUTHOR><AUTHOR>Wong, Anne</AUTHOR><AUTHOR>Lee, Chingwei&#xA0;V.</AUTHOR><AUTHOR>Stawicki, Scott</AUTHOR><AUTHOR>Clark, Robyn</AUTHOR><AUTHOR>Fields, Carter</AUTHOR><AUTHOR>Lewis Phillips, Gail&#xA0;D.</AUTHOR><AUTHOR>Prell, Rodney&#xA0;A.</AUTHOR><AUTHOR>Danilenko, Dimitry&#xA0;M.</AUTHOR><AUTHOR>Franke, Yvonne</AUTHOR><AUTHOR>Stephan, Jean-Philippe</AUTHOR><AUTHOR>Hwang, Jiyoung</AUTHOR><AUTHOR>Wu, Yan</AUTHOR><AUTHOR>Bostrom, Jenny</AUTHOR><AUTHOR>Sliwkowski, Mark&#xA0;X.</AUTHOR><AUTHOR>Fuh, Germaine</AUTHOR><AUTHOR>Eigenbrot, Charles</AUTHOR></AUTHORS><YEAR>2011</YEAR><TITLE>A Two-in-One Antibody against HER3 and EGFR Has Superior Inhibitory Activity Compared with Monospecific Antibodies</TITLE><SECONDARY_TITLE>Cancer Cell</SECONDARY_TITLE><VOLUME>20</VOLUME><NUMBER>4</NUMBER><PAGES>472-486</PAGES><DATE>2011/10/18/</DATE><ISBN>1535-6108 DO - , 4LEO ADDIN EN.CITE <EndNote><Cite><Author>Mirschberger</Author><Year>2013</Year><RecNum>177</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>177</REFNUM><YEAR>2013</YEAR><AUTHORS><AUTHOR>Mirschberger, Christian</AUTHOR><AUTHOR>Schiller, Christian B.</AUTHOR><AUTHOR>Schr&#xC3;&#xA4;ml, Michael</AUTHOR><AUTHOR>Dimoudis, Nikolaos</AUTHOR><AUTHOR>Friess, Thomas</AUTHOR><AUTHOR>Gerdes, Christian A.</AUTHOR><AUTHOR>Reiff, Ulrike</AUTHOR><AUTHOR>Lifke, Valeria</AUTHOR><AUTHOR>Hoelzlwimmer, Gabriele</AUTHOR><AUTHOR>Kolm, Irene</AUTHOR><AUTHOR>Hopfner, Karl-Peter</AUTHOR><AUTHOR>Niederfellner, Gerhard</AUTHOR><AUTHOR>Bossenmaier, Birgit</AUTHOR></AUTHORS><TITLE>RG7116, a Therapeutic Antibody That Binds the Inactive HER3 Receptor and Is Optimized for Immune Effector Activation</TITLE><DATE>2013/08/15</DATE><SECONDARY_TITLE>Cancer Research</SECONDARY_TITLE><PAGES>5183-5194</PAGES><VOLUME>73</VOLUME><NUMBER>16</NUMBER><URL> (HER3/Fab of antibodies DL11 or RG7116, respectively), 2KS1 ADDIN EN.CITE <EndNote><Cite><Author>Mineev</Author><Year>2010</Year><RecNum>24</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>24</REFNUM><YEAR>2010</YEAR><TITLE>Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases</TITLE><SECONDARY_TITLE>Journal of Molecular Biology</SECONDARY_TITLE><VOLUME>400</VOLUME><NUMBER>2</NUMBER><PAGES>231-243</PAGES><DATE>2010/7/9/</DATE><AUTHORS><AUTHOR>Mineev, Konstantin S.</AUTHOR><AUTHOR>Bocharov, Eduard V.</AUTHOR><AUTHOR>Pustovalova, Yulia E.</AUTHOR><AUTHOR>Bocharova, Olga V.</AUTHOR><AUTHOR>Chupin, Vladimir V.</AUTHOR><AUTHOR>Arseniev, Alexander S.</AUTHOR></AUTHORS><ISBN>0022-2836 DO - tyrosine kinases</KEYWORD><KEYWORD>transmembrane domain</KEYWORD><KEYWORD>dimerization</KEYWORD><KEYWORD>structure</KEYWORD><KEYWORD>NMR</KEYWORD></KEYWORDS></MDL></Cite></EndNote>20, 2L9U ADDIN EN.CITE <EndNote><Cite><Author>Mineev</Author><Year>2011</Year><RecNum>178</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>178</REFNUM><AUTHORS><AUTHOR>Mineev, K.S.</AUTHOR><AUTHOR>Khabibullina, N.F.</AUTHOR><AUTHOR>Lyukmanova, E.N.</AUTHOR><AUTHOR>Dolgikh, D.A.</AUTHOR><AUTHOR>Kirpichnikov, M.P.</AUTHOR><AUTHOR>Arseniev, A.S.</AUTHOR></AUTHORS><YEAR>2011</YEAR><TITLE>Spatial structure and dimer-monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles</TITLE><SECONDARY_TITLE>Biochimica et Biophysica Acta (BBA) - Biomembranes</SECONDARY_TITLE><VOLUME>1808</VOLUME><NUMBER>8</NUMBER><PAGES>2081-2088</PAGES><DATE>2011/8//</DATE><ISBN>0005-2736 DO - tyrosine kinases</KEYWORD><KEYWORD>Transmembrane domain</KEYWORD><KEYWORD>Dimerization</KEYWORD><KEYWORD>Structure</KEYWORD><KEYWORD>NMR</KEYWORD><KEYWORD>Dimerization constant</KEYWORD></KEYWORDS><URL> (references for HER2 and HER3 transmembrane domains), and an in-house crystal structure of a complex of FabIL13/IL13. In-house sequences and sequences with UniProt identifiers P05112 ADDIN EN.CITE <EndNote><Cite><Author>Yokota</Author><Year>1986</Year><RecNum>179</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>179</REFNUM><YEAR>1986</YEAR><AUTHORS><AUTHOR>Yokota, T</AUTHOR><AUTHOR>Otsuka, T</AUTHOR><AUTHOR>Mosmann, T</AUTHOR><AUTHOR>Banchereau, J</AUTHOR><AUTHOR>DeFrance, T</AUTHOR><AUTHOR>Blanchard, D</AUTHOR><AUTHOR>De Vries, J E</AUTHOR><AUTHOR>Lee, F</AUTHOR><AUTHOR>Arai, K</AUTHOR></AUTHORS><TITLE>Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities</TITLE><DATE>1986/08/01</DATE><SECONDARY_TITLE>Proceedings of the National Academy of Sciences</SECONDARY_TITLE><PAGES>5894-5898</PAGES><VOLUME>83</VOLUME><NUMBER>16</NUMBER><URL>, P35225 ADDIN EN.CITE <EndNote><Cite><Author>Minty</Author><Year>1993</Year><RecNum>180</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>180</REFNUM><YEAR>1993</YEAR><AUTHORS><AUTHOR>Minty, A.</AUTHOR><AUTHOR>Chalon, P.</AUTHOR><AUTHOR>Derocq, J.-M.</AUTHOR><AUTHOR>Dumont, X.</AUTHOR><AUTHOR>Guillemot, J.-C.</AUTHOR><AUTHOR>Kaghad, M.</AUTHOR><AUTHOR>Labit, C.</AUTHOR><AUTHOR>Leplatois, P.</AUTHOR><AUTHOR>Liauzun, P.</AUTHOR><AUTHOR>Miloux, B.</AUTHOR><AUTHOR>Minty, C.</AUTHOR><AUTHOR>Casellas, P.</AUTHOR><AUTHOR>Loison, G.</AUTHOR><AUTHOR>Lupker, J.</AUTHOR><AUTHOR>Shire, D.</AUTHOR><AUTHOR>Ferrara, P.</AUTHOR><AUTHOR>Caput, D.</AUTHOR></AUTHORS><TITLE>lnterleukin-13 is a new human lymphokine regulating inflammatory and immune responses</TITLE><ALTERNATE_TITLE>Nature</ALTERNATE_TITLE><DATE>1993/03/18/print</DATE><VOLUME>362</VOLUME><NUMBER>6417</NUMBER><PAGES>248-250</PAGES><URL>, P04626 ADDIN EN.CITE <EndNote><Cite><Author>Yamamoto</Author><Year>1986</Year><RecNum>181</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>181</REFNUM><AUTHORS><AUTHOR>Yamamoto, Tadashi</AUTHOR><AUTHOR>Ikawa, Shuntaro</AUTHOR><AUTHOR>Akiyama, Tetsu</AUTHOR><AUTHOR>Semba, Kentaro</AUTHOR><AUTHOR>Nomura, Nobuo</AUTHOR><AUTHOR>Miyajima, Nobuyuki</AUTHOR><AUTHOR>Saito, Toshiyuki</AUTHOR><AUTHOR>Toyoshima, Kumao</AUTHOR></AUTHORS><YEAR>1986</YEAR><TITLE>Similarity of protein encoded by the human c-erb-B-2 gene to epidermal growth factor receptor</TITLE><SECONDARY_TITLE>Nature</SECONDARY_TITLE><VOLUME>319</VOLUME><NUMBER>6050</NUMBER><PAGES>230-234</PAGES><DATE>1986/01/16/print</DATE><TYPE_OF_WORK>10.1038/319230a0</TYPE_OF_WORK><ALTERNATE_TITLE>Nature</ALTERNATE_TITLE><URL>, and P21860 ADDIN EN.CITE <EndNote><Cite><Author>Kraus</Author><Year>1989</Year><RecNum>182</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>182</REFNUM><YEAR>1989</YEAR><AUTHORS><AUTHOR>Kraus, M H</AUTHOR><AUTHOR>Issing, W</AUTHOR><AUTHOR>Miki, T</AUTHOR><AUTHOR>Popescu, N C</AUTHOR><AUTHOR>Aaronson, S A</AUTHOR></AUTHORS><TITLE>Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors</TITLE><DATE>1989/12/01</DATE><SECONDARY_TITLE>Proceedings of the National Academy of Sciences</SECONDARY_TITLE><PAGES>9193-9197</PAGES><VOLUME>86</VOLUME><NUMBER>23</NUMBER><URL> are used to construct CODV homology models or reconstruct complete models of IL4, IL13, and the extracellular and transmembrane domains of HER2 and HER3, respectively.Homology models, CODV-Fab, and CODV-Ig models are constructed, loop and side chain conformations are optimized, and the models are minimized with program Prime according to standard procedures unless indicated otherwise. The general outline of CODV homology model construction is given here. Details referring to individual models are found in dedicated sections below. Sequences are edited in fasta format. Reference structures are superimposed and the fragments that are to be replaced or in duplicate are deleted. Conserved fragments are renumbered, assigned consistent chain identifiers, and merged in a master template while retaining all relevant sugar moieties and ions of the parental structures. Models of the chains of the final complexes including sugar moieties and ions are constructed separately and merged in a final model. All final models are subjected to loop and side chain optimization, and global minimization. Selected final models are explored by molecular dynamics simulations using programs Impact or Desmond.Protein-protein docking of FvIL13 and FvIL4 is carried out with program ZDock employing standard settings in software package Discovery Studio ADDIN EN.CITE <EndNote><Cite><Author>Chen</Author><Year>2002</Year><RecNum>183</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>183</REFNUM><YEAR>2002</YEAR><AUTHORS><AUTHOR>Chen, Rong</AUTHOR><AUTHOR>Weng, Zhiping</AUTHOR></AUTHORS><TITLE>Docking unbound proteins using shape complementarity, desolvation, and electrostatics</TITLE><SECONDARY_TITLE>Proteins: Structure, Function, and Bioinformatics</SECONDARY_TITLE><ALTERNATE_TITLE>Proteins</ALTERNATE_TITLE><VOLUME>47</VOLUME><NUMBER>3</NUMBER><PUBLISHER>Wiley Subscription Services, Inc., A Wiley Company</PUBLISHER><ISBN>1097-0134</ISBN><URL> DO - 10.1002/prot.10092</URL><PAGES>281-294</PAGES></MDL></Cite><Cite><Author>Pierce</Author><Year>2011</Year><RecNum>184</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>184</REFNUM><YEAR>2011</YEAR><TITLE>Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library</TITLE><AUTHORS><AUTHOR>Pierce, Brian G.</AUTHOR><AUTHOR>Hourai, Yuichiro</AUTHOR><AUTHOR>Weng, Zhiping</AUTHOR></AUTHORS><DATE>2011/09/19 &lt;p&gt;Computational prediction of the 3D structures of molecular interactions is a challenging area, often requiring significant computational resources to produce structural predictions with atomic-level accuracy. This can be particularly burdensome when modeling large sets of interactions, macromolecular assemblies, or interactions between flexible proteins. We previously developed a protein docking program, ZDOCK, which uses a fast Fourier transform to perform a 3D search of the spatial degrees of freedom between two molecules. By utilizing a pairwise statistical potential in the ZDOCK scoring function, there were notable gains in docking accuracy over previous versions, but this improvement in accuracy came at a substantial computational cost. In this study, we incorporated a recently developed 3D convolution library into ZDOCK, and additionally modified ZDOCK to dynamically orient the input proteins for more efficient convolution. These modifications resulted in an average of over 8.5-fold improvement in running time when tested on 176 cases in a newly released protein docking benchmark, as well as substantially less memory usage, with no loss in docking accuracy. We also applied these improvements to a previous version of ZDOCK that uses a simpler non-pairwise atomic potential, yielding an average speed improvement of over 5-fold on the docking benchmark, while maintaining predictive success. This permits the utilization of ZDOCK for more intensive tasks such as docking flexible molecules and modeling of interactomes, and can be run more readily by those with limited computational resources.&lt;/p&gt;</DATE><SECONDARY_TITLE>PLoS ONE</SECONDARY_TITLE><ALTERNATE_TITLE>PLoS ONE</ALTERNATE_TITLE><VOLUME>6</VOLUME><NUMBER>9</NUMBER><URL> EP -</PAGES><PUBLISHER>Public Library of Science</PUBLISHER><TYPE_OF_WORK>doi:10.1371/journal.pone.0024657</TYPE_OF_WORK></MDL></Cite><Cite><Author>Accelrys</Author><Year>2015</Year><RecNum>185</RecNum><MDL><REFERENCE_TYPE>16</REFERENCE_TYPE><REFNUM>185</REFNUM><AUTHORS><AUTHOR>Accelrys</AUTHOR></AUTHORS><YEAR>2015</YEAR><TITLE>Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0, San Diego: Accelrys Software Inc., 2013.</TITLE><DATE>February 3, 2015</DATE><URL>. 2000 unrefined poses with 99 cluster centres and 478 singletons of the first screening round are exported for analysis in Maestro Package. The 99 cluster centres and the 478 singletons are visually inspected. Complexes of FabIL13/IL13 and FvIL4/IL4 are superimposed on each pose to reveal the possibility of proper N/C-terminal connectivity between Fvs and Fc, absence of steric interference at the paratopes potentially leading to loss in antigen binding, and to estimate linker lengths. Selected poses are merged with the structure of FabIL13/IL13 to construct homology models of CODV-FabIL4 x IL13 with minimal, intermediate, and maximal linker lengths. Selected models of CODV-FabIL4 x IL13 with minimal and intermediate linker lengths are checked for structural stability by molecular dynamics simulations at 298.15 K, 2 fs time steps, 100 or 500 ps trajectory, Surface Generalized Born implicit solvent model, and standard settings in program Impact. Neither structural instability nor any other structural problems are revealed by the molecular dynamics simulations.The final model of CODV-IgIL4 x IL13/IL4/IL13 is built by homology as outlined above using pdb entries 1HZH, 5FHX, and the in-house crystal structure of FabIL13/IL13. The final model is checked for structural stability by a molecular dynamics simulation at 300 K for 5 ns trajectory applying an explicit solvent model with 250714 SPC water molecules, neutralization by 14 chlorine ions, NVT ensemble, and recommended standard settings including pre-equilibration in program Desmond. Standard simulation quality analysis confirms successful pre-equilibration and production simulation. No structural problems are revealed by the molecular dynamics simulation. The complexes of FcRn/Fc, Fc?R/Fc, protein A/Fc, or protein A/Fab are superimposed on the Fc or Fv regions of the final CODV-IgIL4 x IL13/IL4/IL13 model and analysed. The crystallographic hexamer of 1HZH is generated with software COOT ADDIN EN.CITE <EndNote><Cite><Author>Emsley</Author><Year>2010</Year><RecNum>186</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>186</REFNUM><YEAR>2010</YEAR><AUTHORS><AUTHOR>Emsley, P.,</AUTHOR><AUTHOR>Lohkamp, B.,</AUTHOR><AUTHOR>Scott, W. G.,</AUTHOR><AUTHOR>Cowtan, K.,</AUTHOR></AUTHORS><DATE>2010/04/01</DATE><TITLE>Features and development of Coot</TITLE><PAGES>486-501</PAGES><NUMBER>4</NUMBER><VOLUME>66</VOLUME><URL> building</KEYWORD></KEYWORDS><SECONDARY_TITLE>Acta Crystallographica Section D</SECONDARY_TITLE><ALTERNATE_TITLE>Acta Cryst. D</ALTERNATE_TITLE><PUBLISHER>International Union of Crystallography</PUBLISHER><ISBN>0907-4449</ISBN></MDL></Cite></EndNote>29. The hexamer units are exported to superimpose CODV-IgIL4 x IL13/IL4/IL13 on domains Fc2-Fc3 of IgG1gp120 and carry out analysis in Maestro.Homology models of CODV-IgHER2 x HER3/HER2/HER3 are built as outlined above using crystal structures 3P11, 4LEO, 3N85 or 1N8Z, and the final CODV-IgIL4 x IL13/IL4/IL13 model. Chains A of 2KS1 and 2L9U are manually placed to optimize linker distances to HER2 and HER3 while maintaining proper relative orientations for modelling of the cellular membrane. They are included in the master template. Thereby the models of HER2 and HER3 include extracellular and transmembrane domain residues 23 to 648 and 27 to 676, respectively. The final model of CODV-IgHER2 x HER3/HER2/HER3 based on 3N85 is checked for structural stability by a molecular dynamics simulation for 8.5 ns at 300 K applying explicit solvent and membrane models, NP?T ensemble, force field OPLS2.1, and recommended standard settings in program Desmond. The system is set up with 329139 SPC water and 1346 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) lipid molecules and neutralized with 40 sodium ions. It is pre-equilibrated by a standard protein-membrane equilibration protocol provided by Schr?dinger. Standard simulation quality analysis confirms successful pre-equilibration and production simulation. No structural problems are revealed by the molecular dynamics simulation. The model of frame 523 at 5.021 ns simulation is low in potential energy and therefore selected for representation in Figure 5.Protein production, biochemical, and biophysical characterization of CODV constructsCloning of CODV-Ig and CODV-FabsAll DNA constructs encoding CODV-Ig and CODV-Fab are generated by gene synthesis at Geneart (Regensburg, Germany). VL and CL domains (IGKC, GenBank Accession code Q502W4) are fused by digestion with restriction endonucleases ApaLI and BsiWI and subsequently ligated into the ApaLI/BsiWI sites of episomal expression vector pFF to create the mammalian expression plasmid of the light chains ADDIN EN.CITE <EndNote><Cite><Author>Durocher</Author><Year>2002</Year><RecNum>17</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>17</REFNUM><AUTHORS><AUTHOR>Durocher, Yves</AUTHOR><AUTHOR>Perret, Sylvie</AUTHOR><AUTHOR>Kamen, Amine</AUTHOR></AUTHORS><YEAR>2002</YEAR><TITLE>High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells</TITLE><SECONDARY_TITLE>Nucleic Acids Research</SECONDARY_TITLE><VOLUME>30</VOLUME><NUMBER>2</NUMBER><PAGES>e9</PAGES><DATE>January 15, 2002</DATE><ALTERNATE_TITLE>Nucleic Acids Research</ALTERNATE_TITLE><CALL_NUMBER>10.1093/nar/30.2.e9</CALL_NUMBER><URL>. VH domains are fused to the "Ted" variant of the human constant heavy chain (IGHG1, GenBank Accession No. 569F4), or alternatively, to a CH1 domain from the human constant IGHG1 harbouring C-terminally five amino acid sequence “DKTHT” from the hinge region followed by six histidines in order to create a bispecific Fab. Next, the VH domain is digested with ApaLI and ApaI and then fused to the IGHG1 or His-tagged CH1 domain, respectively, by ligation into the ApaLI/ApaI sites of the episomal expression vector pFF to create the mammalian expression plasmids for expression of the heavy chains (IgG1 or Fab, respectively).Protein production & purificationThe expression plasmids encoding the heavy and light chains of the corresponding constructs are propagated in E. coli DH5a cells. Plasmids used for transfection are prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit. HEK 293-FS cells growing in Freestyle Medium (Invitrogen) are transfected with indicated LC and HC plasmids using 293fectin (Invitrogen) transfection reagent following recommendations by the manufacturer. Cells are cultivated at 37°C in a Kuhner ISF1-X shaking incubator at 110 rpm with 8 % CO2. After 7 days of cultivation cells are removed by centrifugation, 10 % (Vol/Vol) 1 M Tris HCl pH 8.0 is added and the supernatant is filtered via a 0.2 ?M bottle top filter to remove particles. CODV-IgG1 constructs are purified by affinity chromatography on Protein A columns (HiTrap Protein A HP Columns, GE Life Sciences). After elution from the column with 0.1 M Citrate pH 3.0, the CODV-IgG1 constructs are desalted using HiPrep 26/10 Desalting Columns, formulated in PBS (Gibco 14190-136). Bispecific CODV-Fab constructs are purified by HisTrap High Performance columns (GE Healthcare, Cat. No.: 17-5248-02). After elution from the column (Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) the protein containing fractions are pooled and desalted using HiPrep 26/10 Desalting Columns, formulated in PBS (Gibco 14190-136). To separate CODV-IgG or CODV-Fab monomers from aggregates a high resolution fractionation step in PBS (Gibco 14190-136) is performed using a HiLoad Superdex 200 26/60 320 ml column (GE Healthcare Cat. No.: 29-9893-36). Monomeric fractions are pooled and concentrated up to 1 mg/ml, using Vivaspin 20 centrifugation columns (VS2002 Sartorius Stedim biotech) and filtered using a 0.22 ?m membrane (Millex? Syringe Filters SLGV033RS). Protein concentrations are determined by measurement of absorbance at 280 nm wavelength. Each batch is analysed by SDS-PAGE under reducing and non-reducing conditions to determine purity and molecular weights of each subunit and of the monomer.IL4 is expressed in baculovirus in insect cells and purified by affinity chromatography via its His-tag followed by size exclusion chromatography on superdex 200 according to the same purification protocol described for the CODV-Fab. To prepare samples for crystallization purified partners were pooled with an excess of 1.5 moles of IL4 for one mole of CODV-FabIL4 x IL13. IL4 excess is removed by size exclusion chromatography (SEC) on superdex 200 equilibrated with phosphate buffer saline. Fractions corresponding to complex CODV-FabIL4 x IL13/IL4 are pooled and used for crystallization trials.Human, cynomolgus monkey, and murine FcRn are produced as follows: The expression plasmids encoding extracellular domain of FCGRT and full length β2 microglobulin are generated using pFF vectors as described above. HEK 293-FS cells are transiently transfected using 293Fectin. This co-transfection leads to expression and secretion of a soluble heterodimer which is the functional FcRn. Cell cultures are centrifuged and supernatants are purified on IgG Sepharose 6FF? (GE Healthcare) with loading and washing at pH 6.0 and elution at pH 8.0. Purified FcRn is conditioned in 50 mM MES pH 6.0.Analytical size-exclusion chromatographyAnalytical SEC is performed using an ?KTA explorer 10 (GE Healthcare) equipped with a TSKgel G3000SWXL column (7.8 mm x 30 cm) and TSKgel SWXL guard column (Tosoh Bioscience). The analysis is run at 1 ml/min using a buffer composed of 250 mM NaCl, 100 mM Na-phosphate pH 6.7 with detection at 280 nm wavelength. 30 ?l of protein sample at a concentration of 0.5 to 1 mg/ml) are applied onto the column. For estimation of the molecular size the column is calibrated using a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation is performed using UNICORN software v5.11.Binding kinetics by surface plasmon resonance (SPR)The reagents recombinant human IL13 and IL4 are purchased from Chemicon (USA). Recombinant human TNFα is purchased from Sigma Aldrich (H8916-10?g). Recombinant human IL1β (201-LB/CF), recombinant human IL23 (1290-IL/CF), recombinant human EGFR (344 ER), recombinant human HER2 (1129-ER-50), and Fc?R1 (1257-FC-050) are purchased from R&D Systems. Detailed kinetic binding analysis of purified proteins by SPR is performed on a Biacore 3000 (GE Healthcare) instrument. The capture assay uses a species specific antibody (human-Fc specific MAB 1302, Chemicon) or the human Fab capture kit (GE Healthcare) for capture and orientation of IgG, CODV-Ig, Fab or CODV-Fab, respectively. The capture antibody is immobilized via primary amine groups (11000 RU) on a research grade CM5 chip (GE Healthcare) by standard procedures. The analysed antibody is captured at a flow rate of 10 ?l/min with an adjusted RU value that would result in maximal analyte binding of 30 RU. Binding kinetics are measured against targeted antigen or Fc?R over a concentration range between 0 to 25 nM in HBS EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005 % Surfactant P20) at a flow rate of 30 ?l/min. Chip surfaces are regenerated with 10 mM glycine pH 2.5. Kinetic parameters are analysed and calculated in the BIAevaluation program package v4.1 using a flow cell without captured antibody as reference. To investigate additive binding of both antigens, a SPR experiment is performed with CODV-Ig binding IL4 and IL13 using a co-injection of both antigens in three separate analysis cycles. The co-injection was done with 3.125 nM IL4/25 nM IL13 and vice versa and with a 1:1 mixture of 3,125 nM IL4 and 25 nM IL13. A co-injection of HBS-EP buffer was done as a reference. The resulting binding levels are compared. Binding affinity to FcRn is studied using Biacore3000. FcRn is covalently attached to a CM5 sensor chip by amine reactive coupling to 300 RU. A dilution series of CODV-Ig or isotype control IgG1IL4 from 0 to 800 nM in 150mM NaCl, 50mM Na-phosphate, 0.005% surfactant P-20 at pH 6 is flowed over reference and sample flow cells. KD values are determined using the steady state binding model in BIAevaluation software.To determine the binding affinity of CODV-Ig to Fc?R1 a His-tag capture chip surface (His capture Kit, GE Healthcare) is used. The receptor is captured to 50 RU and CODV-Ig or isotype control IgG1IL4 is flowed over the surface in a dilution series of 0 to 100 nM. Binding affinity is determined using the 1:1 Langmuir model in the BIAevaluation software. Binding affinities of CODV-Igs, CODV-Fabs, and DVD-Igs directed against EGFR and HER2 are measured using a Proteon XPR36 protein interaction array system (Biorad). The antigens are immobilized by amine reactive coupling on GLC sensor chips (Biorad). Dilution series of the bispecific antibody variants in PBSET buffer (Biorad) are analysed parallel in one-shot kinetics mode with double referencing. Data are analysed using Proteon Manager Software v3.0 (Biorad) with either Langmuir 1:1 model with mass transfer (for CODV-Igs and CODV-Fabs) or bivalent analyte model (DVD-Igs and IgGs).Thermostability studiesDSF conditions 1: Samples are diluted in D-PBS buffer (Invitrogen) to a final concentration of 0.2 ?g/?l including a 4x concentrated solution of SYPRO-Orange dye (Invitrogen, 5000x stock in DMSO) in D-PBS in white semi-skirt 96-well plates (BIORAD). All measurements are done in duplicate using a MyiQ2 real time PCR instrument (BIORAD) at a heating rate of 1°C/min (one data point per minute collected). Negative first derivative curves (-d(RFU)/dT) of the melting curves are generated in the iQ5 Software v2.1 (BIORAD). Data are then exported into Excel for Tm determination and graphical display of the data. DSF conditions 2: DSF studies of CODV-Igs and CODV-Fabs are carried out in PBS buffer at a protein concentration of 0.9 mg/mL, ten-fold excess of sypro orange dye purchased from Invitrogen on a CFX 96 BioRad instrument. A heating rate of 0.2°C/12 sec in the range of 20 to 90°C is applied (5 data points per minutes collected). The results are analysed with standard BioRad CFX Manager software. Cellular assays monitoring IL4, IL13, or TNF? activityActivities of bispecfic antibodies or derivatives against cytokines IL4, IL13, or TNF? are determined in commercially available HEK-Blue IL4/IL13 reporter cells or HEK-Blue TNF? reporter cells (both InvivoGen). HEK-Blue IL4/IL13 cells are designed to monitor the activation of the STAT6 pathway by IL4 or IL13. Stimulation of the cells with either cytokine results in production of the reporter gene secreted embryonic alkaline phosphatase (SEAP) which can be measured in the culture supernatant with QUANTI-Blue assay. To test antibody activities against IL4, IL13, or TNF?, the cytokines are pre-incubated for 1 hour with different concentrations of the antibodies and added to 50.000 HEK-Blue reporter cells. Cytokine-mediated induction of SEAP is measured after 24 hours incubation in the cell culture supernant with QUANTI-Blue assay as recommended by the vendor (InvivoGen).Protein crystal structure determinationX-ray diffraction quality crystals are obtained using iterative robotically assisted microseed matrix screening technique ADDIN EN.CITE <EndNote><Cite><Author>D&apos;Arcy</Author><Year>2007</Year><RecNum>18</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>18</REFNUM><AUTHORS><AUTHOR>D&apos;Arcy, Allan, </AUTHOR><AUTHOR>Villard, Frederic, </AUTHOR><AUTHOR>Marsh, May,</AUTHOR></AUTHORS><YEAR>2007</YEAR><TITLE>An automated microseed matrix-screening method for protein crystallization</TITLE><SECONDARY_TITLE>Acta Crystallographica Section D</SECONDARY_TITLE><VOLUME>63</VOLUME><NUMBER>4</NUMBER><PAGES>550-554</PAGES><ISBN>0907-4449</ISBN><ACCESSION_NUMBER>doi:10.1107/S0907444907007652</ACCESSION_NUMBER><KEYWORDS><KEYWORD>crystallization</KEYWORD><KEYWORD>seeding</KEYWORD><KEYWORD>nucleation</KEYWORD><KEYWORD>screening</KEYWORD></KEYWORDS><URL>. CODV-FabIL13 x IL4/IL4 protein complex is crystallized at 15 mg/mL by vapour diffusion method at 20°C in hanging drop geometry under 25% PEG3k, 0.1 M sodium acetate at pH 4.6 or 20% PEG3350, 0.2 M potassium acetate conditions. CODV-FabIL13 x IL4 is crystallized under the same conditions with 17.5% PEG 2k MME and 0.1 M Hepes pH 7.5. The resulting crystals are cryoprotected with 25% (v/v) glycerol and flash frozen in liquid nitrogen for x-ray data collection at 100K. Data are collected at beamlines Proxima-1, Soleil, Saint-Aubin and ID-29, ESRF, Grenoble, both in France. They are processed and scaled with software packages GLOBALPHASING ADDIN EN.CITE <EndNote><Cite><Author>Bricogne G.</Author><Year>2011</Year><RecNum>193</RecNum><MDL><REFERENCE_TYPE>6</REFERENCE_TYPE><REFNUM>193</REFNUM><AUTHORS><AUTHOR>Bricogne G., Blanc E., Brandl M., Flensburg C., Keller P., Paciorek W.,</AUTHOR><AUTHOR>Roversi P, Sharff A., Smart O.S., Vonrhein C., Womack T.O.</AUTHOR></AUTHORS><YEAR>2011</YEAR><TITLE>BUSTER version X.Y.Z.</TITLE><AUTHOR_ADDRESS>Cambridge, United Kingdom: Global Phasing Ltd.</AUTHOR_ADDRESS></MDL></Cite></EndNote>32 and AIMLESS ADDIN EN.CITE <EndNote><Cite><Author>Winn</Author><Year>2011</Year><RecNum>192</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>192</REFNUM><YEAR>2011</YEAR><AUTHORS><AUTHOR>Winn, Martyn D.,</AUTHOR><AUTHOR>Ballard, Charles C.,</AUTHOR><AUTHOR>Cowtan, Kevin D.,</AUTHOR><AUTHOR>Dodson, Eleanor J.,</AUTHOR><AUTHOR>Emsley, Paul,</AUTHOR><AUTHOR>Evans, Phil R.,</AUTHOR><AUTHOR>Keegan, Ronan M.,</AUTHOR><AUTHOR>Krissinel, Eugene B.,</AUTHOR><AUTHOR>Leslie, Andrew G. W.,</AUTHOR><AUTHOR>McCoy, Airlie,</AUTHOR><AUTHOR>McNicholas, Stuart J.,</AUTHOR><AUTHOR>Murshudov, Garib N.,</AUTHOR><AUTHOR>Pannu, Navraj S.,</AUTHOR><AUTHOR>Potterton, Elizabeth A.,</AUTHOR><AUTHOR>Powell, Harold R.,</AUTHOR><AUTHOR>Read, Randy J.,</AUTHOR><AUTHOR>Vagin, Alexei,</AUTHOR><AUTHOR>Wilson, Keith S.,</AUTHOR></AUTHORS><DATE>2011/04/01</DATE><TITLE>Overview of the CCP4 suite and current developments</TITLE><PAGES>235-242</PAGES><NUMBER>4</NUMBER><VOLUME>67</VOLUME><URL> crystallography</KEYWORD><KEYWORD>software</KEYWORD><KEYWORD>collaboration</KEYWORD><KEYWORD>automation</KEYWORD><KEYWORD>macromolecular structure determination</KEYWORD></KEYWORDS><SECONDARY_TITLE>Acta Crystallographica Section D</SECONDARY_TITLE><ALTERNATE_TITLE>Acta Cryst. D</ALTERNATE_TITLE><PUBLISHER>International Union of Crystallography</PUBLISHER><ISBN>0907-4449</ISBN></MDL></Cite></EndNote>33. The structure of the complex is solved by molecular replacement using program PHASER of software package CCP4 ADDIN EN.CITE <EndNote><Cite><Author>Winn</Author><Year>2011</Year><RecNum>192</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>192</REFNUM><YEAR>2011</YEAR><AUTHORS><AUTHOR>Winn, Martyn D.,</AUTHOR><AUTHOR>Ballard, Charles C.,</AUTHOR><AUTHOR>Cowtan, Kevin D.,</AUTHOR><AUTHOR>Dodson, Eleanor J.,</AUTHOR><AUTHOR>Emsley, Paul,</AUTHOR><AUTHOR>Evans, Phil R.,</AUTHOR><AUTHOR>Keegan, Ronan M.,</AUTHOR><AUTHOR>Krissinel, Eugene B.,</AUTHOR><AUTHOR>Leslie, Andrew G. W.,</AUTHOR><AUTHOR>McCoy, Airlie,</AUTHOR><AUTHOR>McNicholas, Stuart J.,</AUTHOR><AUTHOR>Murshudov, Garib N.,</AUTHOR><AUTHOR>Pannu, Navraj S.,</AUTHOR><AUTHOR>Potterton, Elizabeth A.,</AUTHOR><AUTHOR>Powell, Harold R.,</AUTHOR><AUTHOR>Read, Randy J.,</AUTHOR><AUTHOR>Vagin, Alexei,</AUTHOR><AUTHOR>Wilson, Keith S.,</AUTHOR></AUTHORS><DATE>2011/04/01</DATE><TITLE>Overview of the CCP4 suite and current developments</TITLE><PAGES>235-242</PAGES><NUMBER>4</NUMBER><VOLUME>67</VOLUME><URL> crystallography</KEYWORD><KEYWORD>software</KEYWORD><KEYWORD>collaboration</KEYWORD><KEYWORD>automation</KEYWORD><KEYWORD>macromolecular structure determination</KEYWORD></KEYWORDS><SECONDARY_TITLE>Acta Crystallographica Section D</SECONDARY_TITLE><ALTERNATE_TITLE>Acta Cryst. D</ALTERNATE_TITLE><PUBLISHER>International Union of Crystallography</PUBLISHER><ISBN>0907-4449</ISBN></MDL></Cite></EndNote>33 and five search models for FvIL13, CH1IL13 and CLIL13 from an in-house structure, IL4 (1HIK.pdb ADDIN EN.CITE <EndNote><Cite><Author>M&#xFC;ller</Author><Year>1995</Year><RecNum>187</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>187</REFNUM><AUTHORS><AUTHOR>M&#xFC;ller, Thomas</AUTHOR><AUTHOR>Oehlenschl&#xE4;ger, Frank</AUTHOR><AUTHOR>Buehner, Manfred</AUTHOR></AUTHORS><YEAR>1995</YEAR><TITLE>Human Interleukin-4 and Variant R88Q: Phasing X-ray Diffraction Data by Molecular Replacement Using X-ray and Nuclear Magnetic Resonance Models</TITLE><SECONDARY_TITLE>Journal of Molecular Biology</SECONDARY_TITLE><VOLUME>247</VOLUME><NUMBER>2</NUMBER><PAGES>360-372</PAGES><DATE>1995/3/24/</DATE><ISBN>0022-2836 DO - mutants</KEYWORD><KEYWORD>NMR structure</KEYWORD><KEYWORD>X-ray structure</KEYWORD><KEYWORD>molecular replacement</KEYWORD></KEYWORDS><URL>), and one homology model of FvIL4 built with Ab Modeler in software package MOE ADDIN EN.CITE <EndNote><Cite><Author>Chemical_Computing_Group_Inc.</Author><Year>2015</Year><RecNum>188</RecNum><MDL><REFERENCE_TYPE>6</REFERENCE_TYPE><REFNUM>188</REFNUM><AUTHORS><AUTHOR>Chemical_Computing_Group_Inc.</AUTHOR></AUTHORS><YEAR>2015</YEAR><TITLE>Molecular Operating Environment (MOE), 2013.08</TITLE><AUTHOR_ADDRESS>Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7</AUTHOR_ADDRESS></MDL></Cite></EndNote>35. Linkers were built manually using software Coot ADDIN EN.CITE <EndNote><Cite><Author>Emsley</Author><Year>2010</Year><RecNum>186</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>186</REFNUM><YEAR>2010</YEAR><AUTHORS><AUTHOR>Emsley, P.,</AUTHOR><AUTHOR>Lohkamp, B.,</AUTHOR><AUTHOR>Scott, W. G.,</AUTHOR><AUTHOR>Cowtan, K.,</AUTHOR></AUTHORS><DATE>2010/04/01</DATE><TITLE>Features and development of Coot</TITLE><PAGES>486-501</PAGES><NUMBER>4</NUMBER><VOLUME>66</VOLUME><URL> building</KEYWORD></KEYWORDS><SECONDARY_TITLE>Acta Crystallographica Section D</SECONDARY_TITLE><ALTERNATE_TITLE>Acta Cryst. D</ALTERNATE_TITLE><PUBLISHER>International Union of Crystallography</PUBLISHER><ISBN>0907-4449</ISBN></MDL></Cite><Cite><Author>Emsley</Author><Year>2004</Year><RecNum>249</RecNum><MDL><REFERENCE_TYPE>0</REFERENCE_TYPE><REFNUM>249</REFNUM><YEAR>2004</YEAR><AUTHORS><AUTHOR>Emsley, Paul,</AUTHOR><AUTHOR>Cowtan, Kevin,</AUTHOR></AUTHORS><DATE>2004/12/01</DATE><TITLE>Coot: model-building tools for molecular graphics</TITLE><PAGES>2126-2132</PAGES><NUMBER>12</NUMBER><VOLUME>60</VOLUME><URL> structure analysis</KEYWORD><KEYWORD>model building</KEYWORD><KEYWORD>map fitting</KEYWORD><KEYWORD>molecular graphics</KEYWORD></KEYWORDS><SECONDARY_TITLE>Acta Crystallographica Section D</SECONDARY_TITLE><ALTERNATE_TITLE>Acta Cryst. D</ALTERNATE_TITLE><PUBLISHER>International Union of Crystallography</PUBLISHER><ISBN>0907-4449</ISBN></MDL></Cite></EndNote>29,36 and the overall structure was further refined using program Autobuster of software package GLOBALPHASING. The apo-structure is solved by molecular replacement with three probes for FvIL4, FvIL13, and Fc derived from the complex structure, refined, and rebuild with the same software (data collection and refinement statistics in Supplements Table 4).Biological functional proof of conceptIsolation of human T-lymphocytesPeripheral blood mononuclear cells (PBMCs) are isolated from 200 ml peripheral blood of healthy donors treated with EDTA by Ficoll density centrifugation. 15 ml Histopaque (Sigma-Aldrich) is preloaded on a 50 ml Leucosep-Tube (Greiner bio-one). Blood is diluted with autoMACS Rinsing Buffer + 1% BSA (Miltenyi Biotec) and loaded on the membrane of a total of ten prepared tubes. Tubes are centrifuged without brake for 10 min at 1000 x g. PBMCs are collected and washed three times with autoMACS Rinsing Buffer + 1% BSA. Finally, PBMCs are resuspended in autoMACS Running Buffer (Miltenyi Biotec) for isolation of T lymphocytes by autoMACSpro technology using the Pan T Cell isolation Kit (Miltenyi Biotec) according to manufacturer’s instructions. Purity of separated T cells is analysed by MACSQuant flow cytometer using the human 7-Color Immunophenotyping Kit (Miltenyi Biotec).Flow cytometry based cytotoxic assayT-cell engaging effect of bispecific antibodies is analysed by a flow cytometry based cytotoxic assay. CD19 positive target cells (Nalm6) are stained for 15 min at 37°C with 1 ?M CFSE in 1 ml RPMI + GlutaMAX I (Gibco) per 107 cells. Afterwards, cells are washed twice and resuspended in RPMI + GlutaMAX I + 10% FCS (Invitrogen). 2.5 x 104 Target cells are seeded in 96-well U-bottom suspension culture plates (Greiner bio-one) in 50 ?l medium per well. Isolated T-cells are resuspended in RPMI + GlutaMAX I + 10% FCS and are added at 10:1 effector-to-target ratio in 50 ?l per well to the target cells. 5 ?l of each serial dilution of bispecific antibody in PBS (Invitrogen) are added to the cells at indicated final concentration for the assay. The assay is incubated for 20 h at 37°C in 5% CO2. To detect dead target cells, cells are stained with 7-AAD: 5 ?g/ml 7-AAD diluted in Stain Buffer with FBS (BD Pharmingen) are added to each well and incubated for 15 min at 4°C in the dark. CFSE and 7-AAD double positive cells are measured using LSRII (BD) flow cytometer, respectively. Further data analyses are performed using FlowJo software (Tree Star, Inc.).ADCC assay The ADCC assay is performed using the ADCC Reporter Bioassay Core Kit (Promega) according to the manufacturer’s recommendations. In brief, Jurkat Fc?IIIa NFAT luc reporter cells (Promega) are used as effector cells and incubated with membrane-bound human TNF? expressing Chinese hamster ovary (CHO) cells (Clean Cells; CHO-TNF clone MC2-1B10) as target cells in an effector to target ratio of 2.5:1 and 10:1. The target cells are pre-incubated with antibodies for 1 h followed by addition of ready-to-use effector cells and incubation at 37°C, 5% CO2. After 6 hours incubation Bio-Glo Luciferase Assay Reagent is added and luciferase activity is measured in a Perkin Elmer Victor Light 1420 luminescence counter in relative-fluorescence-units (RFU). For competition assays the antibodies are pre-incubated for 30 min with 1?g/ml recombinant human TNF? (R&D Systems) before addition to the target cells. CDC assay CHO cells expressing membrane bound TNF? are seeded into a 96-well plate (20.000 cells/well in 200?l) and incubated overnight at 37°C, 5% CO2. Cultures are changed to medium without fetal calf serum; cells are pre-incubated with antibodies for 1 h followed by addition of human complement containing serum (Sigma S1764; 1:10, 1:100, or 1:1000 dilutions). LDH release into supernatant is measured as surrogate for cytolysis after 6 hours of additional incubation by using a commercially available LDH detection kit (Roche) according to manufacturer’s recommendations. References ADDIN EN.REFLIST 1.Madhavi Sastry, G., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. Journal of Computer-Aided Molecular Design 27, 221-234 (2013).2.Jacobson, M. P., Friesner, R. A., Xiang, Z. & Honig, B. On the Role of the Crystal Environment in Determining Protein Side-chain Conformations. Journal of Molecular Biology 320, 597-608 (2002).3.Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins: Structure, Function, and Bioinformatics 55, 351-367 (2004).4.Guo, Z. et al. Probing the a-Helical Structural Stability of Stapled p53 Peptides: Molecular Dynamics Simulations and Analysis. Chemical Biology & Drug Design 75, 348-359 (2010).5.Schr?dinger. Schr?dinger Release 2014-4: Maestro, version 10.0, Schr?dinger, LLC, New York, NY, 2014. (2015).6.Saphire, E. O. et al. Crystal Structure of a Neutralizing Human IgG Against HIV-1: A Template for Vaccine Design. Science 293, 1155-1159 (2001).7.Wlodaver, A., Pavlovsky, A. & Gustchina, A. Crystal structure of human recombinant interleukin-4 at 2.25 ? resolution. FEBS Letters 309, 59-64 (1992).8.Evans, E. J. et al. Crystal structure of a soluble CD28-Fab complex. 6, 271-279 (2005).9.Ito, S. et al. Crystal Structure of the Antigen-Binding Fragment of Apoptosis-Inducing Mouse Anti-Human Fas Monoclonal Antibody HFE7A. Journal of Biochemistry 131, 137-143 (2002).10.Burmeister, W. P., Huber, A. H. & Bjorkman, P. J. Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372, 379-383 (1994).11.Sondermann, P., Huber, R., Oosthuizen, V. & Jacob, U. The 3.2-? crystal structure of the human IgG1 Fc fragment-FcgRIII complex. Nature 406, 267-273 (2000).12.Deisenhofer, J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-.ANG. resolution. Biochemistry 20, 2361-2370 (1981).13.Deis, L. N. et al. Suppression of conformational heterogeneity at a protein-protein interface. Proceedings of the National Academy of Sciences 112, 9028-9033 (2015).14.Donaldson, J. M. et al. Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies. Proceedings of the National Academy of Sciences 110, 17456-17461 (2013).15.Graille, M. et al. 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