Highly active engineered IgG3 antibodies against SARS-CoV-2

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

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Idiotypic self binding of a dominant germline idiotype (T15). Autobody activity is affected by antibody valency.

Journal of Experimental Medicine ◽

10.1084/jem.165.5.1332 ◽

1987 ◽

Vol 165 (5) ◽

pp. 1332-1343 ◽

Cited By ~ 25

Author(s):

C Y Kang ◽

H L Cheng ◽

S Rudikoff ◽

H Kohler

Keyword(s):

Sequence Analysis ◽

Binding Activity ◽

The Self ◽

Antigen Binding ◽

Simultaneous Presence ◽

Cdna Sequencing ◽

Antigen Binding Site ◽

Binding Antibody ◽

Polymeric State ◽

Germline Sequence

We have previously described (1-3) an IgM antibody that binds to PC, expresses the T15 idiotype, and binds also to itself or T15 if insolubilized. Because of the simultaneous presence of complementary idiotopes and paratopes this type of antibody has been termed autobody. The self binding involves the antigen-binding site because the F(ab')2 fragment of T15, PC, and no other haptens inhibit the self binding. DNA sequence analysis of 11E7-1 using primer extension cDNA sequencing showed that the variable sequences of H and L chains of 11E7-1 are identical to the germline sequence of the prototype T15 idiotype. Furthermore, monomeric and dimeric T15 IgA were shown to bind to insolubilized T15 and other T15+ antibodies including 11E7-1. Thus, the self-binding activity is an inherent property of the T15 germline sequence. The self binding is highly dependent on the polymeric state of the binding antibody since the IgM pentamer of 11E7-1 is about three fold more effective than the T15 dimer and 50 times more than the T15 monomer. These data suggest that the self-binding activity of a germline-encoded idiotype may play an important role in the biology of its expression, and more specifically, may be responsible for the establishment of its dominant expression.

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Glycan modulation and sulfoengineering of anti–HIV-1 monoclonal antibody PG9 in plants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1509090112 ◽

2015 ◽

Vol 112 (41) ◽

pp. 12675-12680 ◽

Cited By ~ 25

Author(s):

Andreas Loos ◽

Johannes S. Gach ◽

Thomas Hackl ◽

Daniel Maresch ◽

Theresa Henkel ◽

...

Keyword(s):

Mammalian Cells ◽

Great Promise ◽

Antigen Binding ◽

In Planta ◽

Antigen Binding Site ◽

Effector Functions ◽

Aids Therapy ◽

Anti Hiv ◽

Hiv 1 ◽

Made In

Broadly neutralizing anti–HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells (CHOPG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for CHOPG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti–HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.

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Faculty Opinions recommendation of Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1158476.618775 ◽

2009 ◽

Author(s):

Peter Colman ◽

Douglas Fairlie

Keyword(s):

Binding Site ◽

Antigen Binding ◽

Antigen Binding Site

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Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system

Scientific Reports ◽

10.1038/s41598-020-74105-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hadrien Peyret ◽

Daniel Ponndorf ◽

Yulia Meshcheriakova ◽

Jake Richardson ◽

George P. Lomonossoff

Keyword(s):

Hepatitis B ◽

Cost Saving ◽

Capsid Protein ◽

Recombinant Proteins ◽

Nicotiana Benthamiana ◽

Vaccine Development ◽

Binding Partner ◽

Gfp Fluorescence ◽

Display Systems

Abstract Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

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A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

Scientific Reports ◽

10.1038/s41598-021-92225-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hitomi Nakamura ◽

Moeka Yoshikawa ◽

Naoko Oda-Ueda ◽

Tadashi Ueda ◽

Takatoshi Ohkuri

Keyword(s):

Thermal Stability ◽

Pichia Pastoris ◽

Protein Stability ◽

Disulfide Bond ◽

Binding Activity ◽

Comprehensive Analysis ◽

The Other ◽

Constant Domain ◽

Intermolecular Disulfide Bond ◽

Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

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The mycoplasma surface proteins MIB and MIP promote the dissociation of the antibody-antigen interaction

Science Advances ◽

10.1126/sciadv.abf2403 ◽

2021 ◽

Vol 7 (10) ◽

pp. eabf2403

Author(s):

Pierre Nottelet ◽

Laure Bataille ◽

Geraldine Gourgues ◽

Robin Anger ◽

Carole Lartigue ◽

...

Keyword(s):

Surface Proteins ◽

Mycoplasma Species ◽

Antigen Binding ◽

Antigen Binding Site ◽

Fab Fragment ◽

Cryo Electron Microscopy ◽

Antigen Complex ◽

Antigen Interaction ◽

Immunoglobulin Binding

Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

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Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

Journal of Experimental Medicine ◽

10.1084/jem.164.5.1809 ◽

1986 ◽

Vol 164 (5) ◽

pp. 1809-1814 ◽

Cited By ~ 32

Author(s):

V Agnello ◽

J L Barnes

Keyword(s):

Rheumatoid Factor ◽

Primary Structure ◽

Binding Activity ◽

Light Chains ◽

Heat Denaturation ◽

Antigen Binding ◽

Rheumatoid Factors ◽

Simple Method ◽

Heat Labile

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

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Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen

Molecular Immunology ◽

10.1016/j.molimm.2016.07.004 ◽

2016 ◽

Vol 76 ◽

pp. 123-133 ◽

Cited By ~ 1

Author(s):

Mary H. Foster ◽

Elizabeth S. Buckley ◽

Benny J. Chen ◽

Kwan-Ki Hwang ◽

Amy G. Clark

Keyword(s):

Basement Membrane ◽

Binding Site ◽

Antigen Binding ◽

Antigen Binding Site ◽

Structural Motifs ◽

Basement Membrane Collagen ◽

Membrane Collagen

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A shared idiotype among human anti-Ro/SSA autoantibodies.

Journal of Experimental Medicine ◽

10.1084/jem.169.5.1583 ◽

1989 ◽

Vol 169 (5) ◽

pp. 1583-1588 ◽

Cited By ~ 5

Author(s):

K K Gaither ◽

J B Harley

Keyword(s):

Binding Site ◽

Heart Block ◽

Congenital Heart ◽

Congenital Heart Block ◽

Antigen Binding ◽

Normal Donor ◽

Neonatal Lupus ◽

Antigen Binding Site ◽

Fine Specificity ◽

Low Level

Idiotypes and antiidiotypes are thought to be important immune regulators and have provided clues for the origin and pathogenicity of autoantibodies. Many lupus and Sjögren's syndrome patients, as well as most neonatal lupus infants with congenital heart block or dermatitis, have antibodies to the ribonucleoprotein Ro/SSA, which is one of a group of RNA-protein autoantigens commonly found in human lupus sera. To characterize the fine specificity of anti-Ro/SSA antibodies, a rabbit antidiotypic serum was prepared against polyclonal affinity purified anti-Ro/SSA F(ab')2. The resulting antiidiotype, anti-Id-Rol, is specific for the F(ab')2 fraction of the anti-Ro/SSA immunogen and its binding to anti-Ro/SSA is inhibited by purified Ro/SSA. These data indicate that the Id-Rol epitope on anti-Ro/SSA is associated with the antigen binding site of these same antibodies. The Id-Rol idiotype was present by ELISA in 3 of 12 additional anti-Ro/SSA preparations from precipitin-positive donor sera and in anti-Ro/SSA from one normal donor with low level antibody. This is the first shared idiotype to be found in the human autoantibodies binding to this RNA-protein antigen. Idiotypic differences between anti-Ro/SSA autoantibodies have the potential to explain the variation in pathologic associations found in individuals who develop this autoantibody specificity.

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