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

Kelley, Robert F.; O'Connell, Mark P.; Carter, Paul; Presta, Leonard G.; Covarrubias, M.A. Laredo; Snedecor, Brad; Bourell, James H.; Vetterlein, David

doi:10.1021/bi00139a003

Cited by 97 publications

(65 citation statements)

References 32 publications

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“…This is in agreement with earlier results (42) and with the functional affinities determined for the monoclonal antibody humAb4D5-8 (24) and the 4D5 Fab fragment (70). In all of these measurements the soluble recombinant HER-2-ECD was used, whereas the RIA experiments were performed on whole cells.…”

Section: Comparison Of Binding Kinetics By Surface Plasmonsupporting

confidence: 92%

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

Kubetzko

Balic

Waibel

et al. 2006

Journal of Biological Chemistry

111

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A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185 HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

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Section: Comparison Of Binding Kinetics By Surface Plasmonsupporting

confidence: 92%

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

Kubetzko

Balic

Waibel

et al. 2006

Journal of Biological Chemistry

111

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“…, which were previously reported for various antigen-antibody associations (28,29,35,37,38,40,41,(43)(44)(45). In general, the negative ⌬Cp values for protein folding and protein-ligand association are proportional to the reduction in water-accessible nonpolar surface areas of the molecules, and related to the contribution of hydrophobic effect to molecular association (13)(14)(15)(16)51).…”

supporting

confidence: 61%

“…Recently this method has been applied to the quantitative thermodynamic analyses of antibody binding to various types of antigens, e.g. haptens (28 -31), oligosaccharides (32)(33)(34), and proteins (35)(36)(37)(38)(39)(40)(41)(42)(43)(44). However, no calorimetric studies have been reported on the antigen-antibody interaction in affinity maturation.…”

mentioning

confidence: 99%

The Affinity Maturation of Anti-4-hydroxy-3-nitrophenylacetyl Mouse Monoclonal Antibody

Torigoe¹,

Nakayama

Imazato

et al. 1995

Journal of Biological Chemistry

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To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45°C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.In affinity maturation of the immune response, the average affinity of the immunized serum generally increases with time after immunization (1). To investigate the relationship between the primary sequence diversity of antibodies and the progressive change in binding affinity, extensive analyses of antibodies have been carried out by using several haptens, 4-hydroxy-3-nitrophenylacetyl (NP) 1 (2-5), p-azophenylarsonate (6), phosphorylcholine (7), and 2-phenyl-5-oxazolone (8).A series of anti-NP mouse monoclonal IgG antibodies used in the present study were produced by the immune response of C57BL/6 mice against NP coupled to T cell-dependent carrier, chicken ␥-globulin (9, 10). The variable regions of the primary response anti-NP antibodies show low affinity for NP and carry few, if any, somatic mutations (2, 4), whereas those of the secondary response anti-NP antibodies usually exhibit increased affinity for NP and are somatically mutated (5). The secondary response antibodies are divided into two groups by carrying or lacking a somatic Trp 3 Leu exchange at position 33 in the variable region of the heavy chain (11,12). In the present study we compare N1G9, a primary response anti-NP antibody, with 3B44 and 3B62, which are secondary response anti-NP antibodies with and without Trp 3 Leu exchange, respectively. Thermodynamic aspect of antigen-antibody association is essential in order to understand the mechanism of the high affinity and specificity of antigen-antibody interaction. Thermodynamic parameters such as Gibbs free energy change, ⌬G, enthalpy change, ⌬H, entropy change, ⌬S, and heat capacity change, ⌬Cp, can provide useful information to identify fundamental forces involved in the antigen-antibody interaction. For instance, the magnitude of ⌬Cp is usually related to the contribution of the ...

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“…Production and purification of rhuMAbHER2, rhuMAbHER2.Fab, and NRG1 177-244 -poly-L-lysine conjugates We used a humanized recombinant HER2 Ab (rhuMAbHER2), 36 its Fab product (rhuMAbHER2.Fab), 37 or the EGF-like domain of NRG1 (NRG1 177-244 ) 38 as the basis for our gene transfer vehicles. These agents allowed us to characterize the utility of targeting the HER2 molecule in a monovalent versus bivalent fashion (rhuMAbHER2.Fab versus rhuMAbHER2) or NRG1 binding sites (HER3 homodimers, HER4 homodimers, and HER2/HER3 or HER2/HER4 heterodimers).…”

Section: Resultsmentioning

confidence: 99%

“…The humanized HER2 Ab (rhuMAbHER2), 36 the Fab of this Ab (rhuMAbHER2.Fab), 37 and the EGF-like domain of NRG1 (NRG1 177-244 ) 38 have been described previously. Poly-L-lysine polymers (pLYS) of different chain lengths (158 and 251 lysines) were obtained from Sigma (St. Louis, Mo).…”

Section: Preparation Of Poly-l-lysine Dna Complexesmentioning

confidence: 99%

Neuregulin receptor-mediated gene transfer by human epidermal growth factor receptor 2-targeted antibodies and neuregulin-1

Kern

Wakita²,

Sliwkowski³

1999

Cancer Gene Ther

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

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

Cited by 97 publications

References 32 publications

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

The Affinity Maturation of Anti-4-hydroxy-3-nitrophenylacetyl Mouse Monoclonal Antibody

Neuregulin receptor-mediated gene transfer by human epidermal growth factor receptor 2-targeted antibodies and neuregulin-1

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