In vitro and in vivo modifications of recombinant and human IgG antibodies

Abstract Abstract 2373 Poster Board II-350 Background: CLL cells are not highly proliferative, but are resistant to apoptosis and thus exhibit prolonged survival and accumulation in vivo. Exogenous soluble BLyS (B-Lymphocyte Stimulator) and APRIL (A PRoliferation-Inducing Ligand) bind to the TACI receptor and have been shown to protect CLL cells from apoptosis in vitro. Nurse-like cells derived in vitro from CLL patients express high levels of APRIL and BLyS and thereby support CLL cell survival. Inhibition of both APRIL and BLyS (but not BLyS alone) significantly reduces CLL viability in vitro, indicating an important role for APRIL in this setting. Atacicept is a soluble receptor fusion protein comprised of the extracellular domain of TACI and the Fc (modified) portion of a human IgG. It binds and neutralizes both APRIL and BLyS. We present the final results of a phase I study of atacicept. Methods: This is an open-label, dose-escalation trial to assess the safety, pharmacokinetics and biological effects of atacicept dosed intravenously once weekly for 5 weeks to patients with refractory or relapsed CLL. Eligible patients have been enrolled in sequential cohorts of 3 to receive atacicept at doses of 1, 4, 10, 15, 20 or 27 mg/kg (max. dose proposed due to high protein load). Patients who demonstrated at least stable disease after the first cycle (5 weeks treatment, 3 weeks follow-up) were allowed to receive up to 4 additional treatment cycles depending on response. PK was assessed after the 1st, 2nd, 3rd, and 5th dose in cycle 1. The biological activity assessment comprised peripheral leukocyte counts, lymphocyte subpopulation counts (by flow cytometric analysis) and lymph node size (assessed by CT scan). Response has been assessed using NCI-WG criteria at the end of cycle 1 and at regular intervals thereafter. Results: Twenty-one CLL patients with an average number of 7 prior treatments have been treated with atacicept for at least one cycle. Before starting Atacicept treatment, most patients had rapidly increasing leukocyte counts. No dose-limiting toxicity has been reported and no SAE related to study drug has been observed. One case of mild nausea is the only drug-related toxicity reported to date. Five of nine patients treated with 10, 15 or 20 mg/kg experienced a stabilization of their disease during the treatment period. At the dose of 27 mg/kg, one of six patients experienced a partial response (PR), which has lasted for 12 months. Five of six patients treated with 27 mg/kg experienced a stabilization of their disease during the treatment period. At lower dose levels (1 and 4 mg/kg) all patients demonstrated progressive disease. Conclusions: Treatment with atacicept was well tolerated. The multiple cases of disease stabilization and one PR seen at higher dose levels in this heavily pre-treated patient population suggest that atacicept might interfere with CLL cell proliferation and survival in vivo. Disclosures: Off Label Use: Atacicept is a soluble receptor fusion protein comprised of the extracellular domain of TACI and the Fc (modified) portion of a human IgG. It binds and neutralizes the proteins APRIL and BLyS which are suspected to drive disease progression in CLL patients. Gianella-Borradori:Merck Serono: Employment.

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Inhibition of in Vitro Growth of Plasmodium falciparum by Purified Antimalarial Human IgG Antibodies.

Scandinavian Journal of Immunology ◽

10.1111/j.1365-3083.1983.tb00893.x ◽

1983 ◽

Vol 18 (6) ◽

pp. 567-571 ◽

Cited By ~ 18

Author(s):

S. JEPSEN

Keyword(s):

Plasmodium Falciparum ◽

In Vitro Growth ◽

Igg Antibodies ◽

Human Igg

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Abstract 456: Antiphospholipid Antibodies Promote Platelet Activation and Thrombosis by Inhibition of Platelet eNOS

Circulation ◽

10.1161/circ.116.suppl_16.ii_76-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Craig Morrell ◽

AnneMarie Swaim ◽

Tanika Martin ◽

Guillermina Girardi ◽

Jane E Salmon ◽

...

Keyword(s):

Platelet Activation ◽

Antiphospholipid Antibodies ◽

Thrombus Formation ◽

Ldl Receptor ◽

Wild Type ◽

Human Igg ◽

Increased Risk ◽

Receptor Family

The antiphospholipid syndrome (APS) is an autoimmune systemic disorder characterized by the persistent presence of antiphospholipid antibodies (aPL Ab) and increased risk of thrombosis, coronary artery disease and myocardial infarction. Although platelets are known direct targets of aPL Ab action, the molecular basis of aPL Ab actions on platelets remains unclear. Platelet endothelial NO synthase (eNOS) is a key regulator of platelet function, with NO causing blunted activation. We therefore determined whether aPL Ab modulate platelet eNOS. Normal human IgG (NH IgG) and human IgG containing polyclonal aPL Ab were obtained from healthy individuals and APS patients, respectively, and purified using protein G-Sepharose chromatography. Using both human and mouse platelets, we found that aPL Ab increased agonist-induced platelet activation whereas NH IgG did not. In contrast to the enhanced activation by aPL Ab in platelets from wild-type mice, aPL Ab had no effect on platelets isolated from eNOS null mice. Pre-treatment of platelets with aPL Ab also inhibited insulin-mediated eNOS stimulation as evidenced by diminished cGMP production and DAF2 fluorescence. Receptor associated protein (RAP), an antagonist of ligand binding to members of the LDL receptor family, blocked aPL Ab-induced increases in platelet activation. RAP also prevented aPL Ab-mediated antagonism of platelet eNOS, indicating that aPL Ab signal through the platelet ApoER2â ϵ™ (LRP8) to attenuate eNOS activity. Furthermore, using intravital microscopy of the mouse mesenteric circulation, we demonstrated that platelets from wild-type mice treated with aPL Ab have increased rolling on a stimulated endothelium and a decreased time to thrombus formation in vivo versus platelets treated with NH IgG. In contrast, aPL Ab did not alter the in vivo function of platelets from eNOS null mice. These cumulative in vitro and in vivo findings demonstrate that aPL Ab antagonism of platelet eNOS through LDL receptor family member binding underlies aPL Ab-mediated thrombosis.

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ENHANCEMENT OF THE ACTIVITY OF THYROID-STIMULATING ANTIBODIES BY ANTI-HUMAN IgG ANTIBODIES IN VITRO

Clinical Endocrinology ◽

10.1111/j.1365-2265.1989.tb01256.x ◽

1989 ◽

Vol 31 (3) ◽

pp. 325-334 ◽

Cited By ~ 1

Author(s):

T. SAITO ◽

H. SHIMURA ◽

T. ENDO ◽

T. ONAYA

Keyword(s):

Igg Antibodies ◽

Human Igg ◽

Thyroid Stimulating Antibodies

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Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

10.1101/2020.12.20.423708 ◽

2020 ◽

Author(s):

William N. Voss ◽

Yixuan J. Hou ◽

Nicole V. Johnson ◽

Jin Eyun Kim ◽

George Delidakis ◽

...

Keyword(s):

Interfacial Area ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Cell Cloning ◽

Effector Functions ◽

Ighv Gene ◽

Plasma Response

SUMMARYAlthough humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

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Protein-based RBD-C-tag COVID-19 Vaccination Candidate Elicits Protection Activity against SARS-COV-2 Variant Infection

10.1101/2021.08.17.456704 ◽

2021 ◽

Author(s):

Jaganathan Subramani ◽

Namir Shaabani ◽

Dhwani Shetty ◽

Haiwa Wu ◽

Sunkuk Kwon ◽

...

Keyword(s):

South Africa ◽

T Cells ◽

Aluminum Phosphate ◽

Spike Protein ◽

Igg Antibodies ◽

Th1 Polarization ◽

Protection Activity ◽

Virus Strains

ABSTRACTThe identification of a vaccination candidate against COVID-19 providing protecting activity against emerging SARS-COV-2 variants remains challenging. Here, we report protection activity against a spectrum of SARS-COV-2 and variants by immunization with protein-based recombinant RBD-C-tag administered with aluminum-phosphate adjuvant intramuscularly. Immunization of C57BL/6 mice with RBD-C-tag resulted in the in vivo production of IgG antibodies recognizing the immune-critical spike protein of the SARS-COV-2 virus as well as the SARS-COV-2 variants alpha (“United Kingdom”), beta (“South Africa”), gamma (“Brazil/Japan”), and delta (“India”) as well as wt-spike protein. RBD-C-tag immunization led to a desired Th1 polarization of CD4 T cells producing IFNγ. Importantly, RBD-C-tag immunization educated IgG production delivers antibodies that exert neutralizing activity against the highly transmissible SARS-COV-2 virus strains “Washington”, “South Africa” (beta), and “India” (delta) as determined by conservative infection protection experiments in vitro. Hence, the protein-based recombinant RBD-C-tag is considered a promising vaccination candidate against COVID-19 and a broad range of emerging SARS-COV-2 virus variants.

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Publisher Correction: In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies

Nature Communications ◽

10.1038/s41467-018-07480-8 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Andreas H. Laustsen ◽

Aneesh Karatt-Vellatt ◽

Edward W. Masters ◽

Ana Silvia Arias ◽

Urska Pus ◽

...

Keyword(s):

Igg Antibodies ◽

Human Igg

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In Vivo Crystallization of Human IgG in the Endoplasmic Reticulum of Engineered Chinese Hamster Ovary (CHO) Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m110.204362 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19917-19931 ◽

Cited By ~ 43

Author(s):

Haruki Hasegawa ◽

John Wendling ◽

Feng He ◽

Egor Trilisky ◽

Riki Stevenson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Synthesis ◽

Cho Cells ◽

Membrane Integrity ◽

Chinese Hamster ◽

High Concentration ◽

Human Igg ◽

Secretory Capacity

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.

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