scholarly journals Design Principles for Bispecific IgGs, Opportunities and Pitfalls of Artificial Disulfide Bonds

Antibodies ◽  
2018 ◽  
Vol 7 (3) ◽  
pp. 27 ◽  
Author(s):  
Lilach Vaks ◽  
Dana Litvak-Greenfeld ◽  
Stav Dror ◽  
Galia Matatov ◽  
Limor Nahary ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Heavy Chain ◽  
Light Chain ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Optimal Solution ◽  
Bispecific Antibodies ◽  
Variable Domains ◽  
Correct Pairing ◽  
Recombinant Fragments

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many of them are small recombinant fragments, their utility could be limited. For some therapeutic applications, full-size IgGs may be the optimal format. Two challenges should be met to make bispecific IgGs; one is that each heavy chain will only pair with the heavy chain of the second specificity and that homodimerization be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and not with the light chain of the second specificity. The first solution to the first criterion (knobs into holes, KIH) was presented in 1996 by Paul Carter’s group from Genentech. Additional solutions were presented later on. However, until recently, out of >120 published bsAb formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested. We present a solution for the second challenge—correct pairing of heavy and light chains of bispecific IgGs; an engineered (artificial) disulfide bond between the antibodies’ variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL. We name antibodies produced according to this design “BIClonals”. Bispecific IgGs where the artificial disulfide bond is placed in the CH1-CL interface are also presented. Briefly, we found that an artificial disulfide bond between VH position 44 to VL position 100 provides for effective and correct H–L chain pairing while also preventing the formation of wrong H–L chain pairs. When the artificial disulfide bond links the CH1 with the CL domain, effective H–L chain pairing also occurs, but in some cases, wrong H–L pairing is not totally prevented. We conclude that H–L chain pairing seems to be driven by VH–VL interfacial interactions that differ between different antibodies, hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs, making it necessary to carefully evaluate the optimal solution for each new antibody.

Relative Affinity of Plasmin for the α1-Macroglobulin, Cl Inactivator and α1-Antitrypsin Inhibitors

1975 ◽  
Author(s):  
Peter C. Harpel
Keyword(s):  
Complex Formation ◽  
Protease Inhibitors ◽  
Disulfide Bond ◽  
Light Chain ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Molar Ratio ◽  
Molar Ratios ◽  
Relative Affinity ◽  
Quantitative Contribution

The quantitative contribution of three major plasma protease inhibitors in binding plasmin has been studied. Mixtures of plasmin and each of the purified inhibitors were analyzed by SDS-acrylamide gel electrophoresis. Plasmin remained bound to its inhibitors in the presence of SDS and urea. A 1 : 1 molar ratio for complex formation was established, and treatment of the complexes with a disulfide bond reducing agent showed that the light chain of plasmin contained the binding sites for both CĪ inactivator and α-antitrysin. Limited degradation of all three inhibitors by plasmin was observed, and the altered inhibitor remained complexed to the enzyme. The competitive binding of 125I plasmin to mixtures of these inhibitors was followed by sucrose density ultracentrifugation and by SDS-gel electrophoresis. In mixtures containing physiologic molar ratios of enzyme and inhibitors, over 80% of the bound plasmin was complexed to the α2-macroglobufui (α2M). No evidence for an exchange of plasmin between the inhibitors was obtained.

scholarly journals Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies

2017 ◽  
Vol 26 (10) ◽  
pp. 2021-2038 ◽  
Author(s):  
K. J. Froning ◽  
A. Leaver-Fay ◽  
X. Wu ◽  
S. Phan ◽  
L. Gao ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Computational Design ◽  
Bispecific Antibodies

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

Disulfide Bond Introduction for General Stabilization of Immunoglobulin Heavy-Chain Variable Domains

2008 ◽  
Vol 377 (2) ◽  
pp. 478-488 ◽  
Author(s):  
Dirk Saerens ◽  
Katja Conrath ◽  
Jochen Govaert ◽  
Serge Muyldermans
Keyword(s):  
Disulfide Bond ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Variable Domains

scholarly journals Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin.

1985 ◽  
Vol 100 (4) ◽  
pp. 1016-1023 ◽  
Author(s):  
G Peltz ◽  
J A Spudich ◽  
P Parham
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Molecule ◽  
Dictyostelium Myosin ◽  
Antigenic Sites ◽  
Myosin Filaments ◽  
A Site

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.

scholarly journals Bradykinin regulates the expression of kininogen binding sites on endothelial cells

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2936-2946 ◽  
Author(s):  
JM Zini ◽  
AH Schmaier ◽  
DB Cines
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Single Class ◽  
Protein Kinase C Inhibitors ◽  
Molar Excess

The vasoactive compound bradykinin (BK) is liberated by proteolytic cleavage from high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Expression of kininogens on cell surface receptors may affect the delivery of BK at sites of inflammation. Therefore, we investigated whether BK itself alters the expression of binding sites for its parent molecules, HK and LK, on the surface of cultured human umbilical vein endothelial cells (HUVEC). 125I-LK and 125I-HK each bind to a single class of sites on HUVEC in reactions that are saturable, reversible, and zinc-dependent (Bmax = 9.7 +/- 0.2 x 10(5) sites/cell; kd = 43.3 +/- 8 nmol/L; n = 5 and Bmax = 10.3 +/- 0.4 x 10(5) sites/cell; kd = 40.3 +/- 0.9 nmol/L; n = 3 for LK and HK, respectively). HK and LK compete for the same binding site (Ki = 19.4 +/- 5 nmol/L HK v 125I-LK; Ki = 24.5 +/- 4 nmol/L LK v 125I-HK, n = 3). Moreover, 50-fold molar excess light chain of HK inhibits 125I-LK binding 51% and 50-fold molar excess LK and the heavy chain of HK inhibit 125I-light chain of HK binding 92% and 76%, respectively. Preincubation of HUVEC with BK produces a transient, concentration- dependent increase in the binding of HK and LK, reaching a maximum 3 to 4 hours after addition of BK (46% increase over control for HK; 57% increase over control for LK; P < .005 for each ligand). Des-Arg9- bradykinin, a B1 receptor agonist, increases kininogen binding to the same extent as BK; the upregulation of kininogen binding sites by BK is partially blocked by a B1 but not by a B2 receptor antagonist. The protein kinase C inhibitors (PKC), sphingosine and H7, completely block the induction of HK receptors by BK. Phorbol 12-myristate 13-acetate (PMA), which also activates PKC, stimulates the binding of HK and the purified light chain of HK to HUVEC as well. However, unlike HK and its light chain, binding of LK and the heavy chain of HK are increased by PMA only in the presence of added calcium ion. These studies show that BK upregulates a common binding site for HK, LK, and each chain of HK on HUVEC. Induction of kininogen receptors on endothelial cells by BK may modulate the generation of this vasoactive compound at sites of vascular injury.

ROLE OF DISULFIDE BONDS NEAR THE CALCIUM BINDING SITES IN FIBRINOGEN

1987 ◽  
Author(s):  
R Procyk ◽  
B Blomback
Keyword(s):  
Calcium Chloride ◽  
Disulfide Bond ◽  
Calcium Ions ◽  
Binding Sites ◽  
Calcium Binding ◽  
Disulfide Bonds ◽  
Terminal Region ◽  
Clotting Time ◽  
Calcium Binding Sites

Incubation of fibrinogen with 0.5 mM dithiothreitol in the presence of .20 mM calcium chloride cleaved disulfide bonds located at: the N-terminal end of the Aα-chain (either Aα28-Aα28 or Aα45-γ23), the C-terminal end of the Aα-chain (Aα442-Aα472) and the N-terminal end of the γ-chain (either of the symmetrical γ8, γ9 disulfides or the Aα45-γ23 disulfide bond). In the absence of calcium ions two additional disulfides, γ326-γ339, and one in the N-terminal end of the γ-chain were reduced.Plasmin digestion of the reduced fibrinogens in buffers containing calcium chloride produced fragments D and E, except that smaller fragments of D were generated from the fibrinogen in which the γ326-γ339 disulfide bonds were reduced and alkylated. In these samples calcium did not protect the C-terminal end of the γ-chain from extensive digestion.Addition of thrombin to partially reduced and alkylated fibrinogen prepared in the presence of calcium gave a clotting time similar to control unreduced fibrinogen. However, when the γ326-γ339 disulfide bonds and another γ-chain disulfide bond most likely in the N-terminal region were cleaved in reduced fibrinogen prepared in the absence of calcium, the thrombin clotting time was extremely prolonged. Apparently the disulfide bonded structure supported by γ326-γ339 was important both for binding of calcium and also for normal clotting.

THE INTERACTION BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
Joost A Koedam ◽  
Rob J Hamer ◽  
Nel H Beeser-Visser ◽  
Etienne Jap Tjoen San ◽  
Kees Schippers ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Disulfide Bonds ◽  
Factor Xa ◽  
Exchange Chromatography ◽  
Solid Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) circulates in plasma as a non-covalent complex with von Willebrand factor (VWF), a large multimeric adhesive glycoprotein. VWF serves as a carrier for FVIII and is thought to stabilize FVIII. The interaction between the two proteins was studied by binding purified human 125I-FVIII to VWF which was coated on a solid matrix. Experiments employing isolated heavy and light chains of FVIII and monoclonal antibodies indicated that binding occurred through the carboxyterminal 80kDa light chain of factor VIII. Treatment of VWF-bound 125I-FVIII with thrombin resulted in the release of a light chain-derived 70kDa fragment and a heavy chain-derived 50kDa fragment. A 42kDa heavy chain-derived fragment was found in the fraction which remained bound to VWF. Treatment with factor Xa (FXa) resulted in the release of 63, 50, 45, and 42kDa fragments. No phospholipids were required for proteolysis of FVIII by either of these enzymes. In solution, the activation of FVIII by FXa, but not by thrombin, was inhibited by VWF. Neither activation, nor cleavage or release from VWF were observed when FVIII was incubated with factor IXa. Activation of FVIII was parallelled by its release from VWF. We conclude that the thrombin-activated form of FVIII consists of a complex between the 70kDa and 50kDa fragments. Inactivation of FVIII by activated protein C (APC) was inhibited when FVIII was complexed to VWF. This protective effect of VWF was abolished upon activation of FVIII and its subsequent release from VWF.In order to locate the binding site for FVIII on the VWF molecule, we digested VWF with Staphylococcal V8 protease (Sp). Digestion products were isolated with Mono Q ion-exchange chromatography and identified as Spl (39 kDa), SpII dimers (220 kDa) and Spill dimers (a triplet ranging from 210-280 kDa) by their molecular weight and chromatographic behaviour (J.-P. Girma et al.. Biochemistry 1986, 25:3156-3163). Purified VWF or digestion products were spotted on nitrocellulose paper, followed by blocking with an albumin solution. Binding of FVIII was studied by incubating the filters with 125I-FVIII, followed by autoradiography. Fifty ng of VWF was sufficient in order to detect FVIII binding. No binding was observed to partially reduced dimeric undigested VWF. Of the isolated digestion products, only the SpIII dimer was able to bind 125I-FVIII. After Western blotting of VWF-fragments from SDS-polyacrylamide gels, 125I-FVIII bound only to the bands which represented SpIII. Therefore, the domain on VWF responsible for the binding of FVIII seems to be located on its aminoterminal SpIII fragment. The integrity of internal disulfide bonds and dimerisation of VWF are required for FVIII binding.

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