Fc-Independent Protection from SARS-CoV-2 Infection by Recombinant Human Monoclonal Antibodies

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.

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Fc-independent neutralization of SARS-CoV-2 by recombinant human monoclonal antibodies

10.1101/2021.05.15.443978 ◽

2021 ◽

Author(s):

Tal Noy-Porat ◽

Avishay Edri ◽

Ron Alcalay ◽

Efi Makdasi ◽

David Gur ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Human Monoclonal Antibodies ◽

Antibody Treatment ◽

Effector Cells ◽

Future Design ◽

Endogenous Immune Response ◽

The Complement System

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in vivo neutralization of SARS-CoV-2 is not yet clear. Delineating the role this process plays in antibody-mediated protection will have a great impact on the design of such therapeutics. Here, the Fc of two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, targeting distinct domains of the spike, was engineered to abrogate their Fc-dependent functions. The protective activity of these antibodies was tested against lethal SARS-CoV-2 infections in K18-hACE2 transgenic mice, both before or two days post-exposure in comparison to their original, Fc-active antibodies. Antibody treatment with both Fc-variants similarly rescued the mice from death, reduced viral load and prevented signs of morbidity. In addition, surviving animals developed a significant endogenous immune response towards the virus. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in antibody-mediated protection, which should aid in future design of effective antibody-based therapies.

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The Role of Membrane Bound Complement in the Aggregation of Mammalian Platelets by Collagen

10.1055/s-0039-1689456 ◽

1975 ◽

Author(s):

B. V. Chater

Keyword(s):

Electron Microscope ◽

Light Microscopy ◽

Complement System ◽

Membrane Bound ◽

The Complement System ◽

Adhesion Of Platelets ◽

Collagen Stimulation

It has been observed in dogs decomplemented with purified Cobra Venom Factor, that their platelets lose the ability to aggregate in response to collagen stimulation. Further investigation of the effect of CVF in vitro in man, dog and rabbit, and in vivo in dog, reveals that in each case CVF abolishes the collagen response of platelets, and that this effect is dose related. Resuspension of CVF inactivated platelets in plasma containing complement, results in a total return of sensitivity to collagen. Examination of CVF inactivated platelets with the electron microscope, fails to show any marked difference from control platelets. Serotonin granules are still present and the platelets retain a discoid appearance. Incubation of platelets with antibodies to Cl, C3 and C5, results in inhibition of the collagen response, this effect also being dose related. Light microscopy studies indicate that CVF does not affect the adhesion of platelets to collagen but appears to prevent subsequent aggregation. It is suggested that the complement system is involved in the induction of release by collagen, and that inhibition by CVF and anti-complement antibodies is the result of a blocking of the release reaction.

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A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

Journal of Virology ◽

10.1128/jvi.01126-21 ◽

2021 ◽

Author(s):

Kevin Chiem ◽

Desarey Morales Vasquez ◽

Jesus A. Silvas ◽

Jun-Gyu Park ◽

Michael S. Piepenbrink ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vaccine Efficacy ◽

Neutralizing Antibodies ◽

Viral Infections ◽

Rapid Assessment ◽

The United States ◽

Viral Fitness ◽

Human Monoclonal Antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, β) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro , as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of human monoclonal antibodies (hMAbs) with neutralizing activity against SARS-CoV-2, including variants of concern (VoC) in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

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Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00196.2012 ◽

2013 ◽

Vol 304 (12) ◽

pp. L863-L872 ◽

Cited By ~ 27

Author(s):

Daniel Rittirsch ◽

Michael A. Flierl ◽

Brian A. Nadeau ◽

Danielle E. Day ◽

Markus S. Huber-Lang ◽

...

Keyword(s):

Complement System ◽

Serine Proteases ◽

Neutralizing Antibody ◽

Dependent Manner ◽

Neutrophil Accumulation ◽

The Complement System ◽

Dose Dependent

Zonulin is a protein involved in the regulation of tight junctions (TJ) in epithelial or endothelial cells. Zonulin is known to affect TJ in gut epithelial cells, but little is known about its influences in other organs. Prehaptoglobin2 has been identified as zonulin and is related to serine proteases (MASPs, C1qrs) that activate the complement system. The current study focused on the role of zonulin in development of acute lung injury (ALI) in C57BL/6 male mice following intrapulmonary deposition of IgG immune complexes. A zonulin antagonist (AT-1001) and a related peptide with permeability agonist activities (AT-1002) were employed and given intratracheally or intravenously. Also, zonulin was blocked in lung with a neutralizing antibody. In a dose-dependent manner, AT-1001 or zonulin neutralizing antibody attenuated the intensity of ALI (as quantitated by albumin leak, neutrophil accumulation, and proinflammatory cytokines). A similar pattern was found using the bacterial lipopolysaccharide model of ALI. Using confocal microscopy on sections of injured lungs, staining patterns for TJ proteins were discontinuous, reduced, and fragmented. As expected, the leak of blood products into the alveolar space confirmed the passage of 3 and 20 kDa dextran, and albumin. In contrast to AT-1001, application of the zonulin agonist AT-1002 intensified ALI. Zonulin both in vitro and in vivo induced generation of complement C3a and C5a. Collectively, these data suggest that zonulin facilitates development of ALI both by enhancing albumin leak and complement activation as well as increased buildup of neutrophils and cytokines during development of ALI.

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A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

Nuklearmedizin ◽

10.1055/s-0038-1624353 ◽

1987 ◽

Vol 26 (01) ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

S. Selvaraj ◽

M. R. Suresh ◽

G. McLean ◽

D. Willans ◽

C. Turner ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Tumor Cell ◽

T Antigen ◽

Human Carcinomas ◽

Animal Tumor ◽

Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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Blocking the GITR-GITRL pathway to overcome resistance to therapy in sarcomatoid malignant pleural mesothelioma

Communications Biology ◽

10.1038/s42003-021-02430-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Meilin Chan ◽

Licun Wu ◽

Zhihong Yun ◽

Trevor D. McKee ◽

Michael Cabanero ◽

...

Keyword(s):

Tumor Growth ◽

Malignant Pleural Mesothelioma ◽

Neutralizing Antibodies ◽

Pleural Mesothelioma ◽

Spheroid Formation ◽

Resistance To Therapy ◽

Sarcomatoid Mesothelioma

AbstractMalignant pleural mesothelioma (MPM) is an aggressive neoplasm originating from the pleura. Non-epithelioid (biphasic and sarcomatoid) MPM are particularly resistant to therapy. We investigated the role of the GITR-GITRL pathway in mediating the resistance to therapy. We found that GITR and GITRL expressions were higher in the sarcomatoid cell line (CRL5946) than in non-sarcomatoid cell lines (CRL5915 and CRL5820), and that cisplatin and Cs-137 irradiation increased GITR and GITRL expressions on tumor cells. Transcriptome analysis demonstrated that the GITR-GITRL pathway was promoting tumor growth and inhibiting cell apoptosis. Furthermore, GITR+ and GITRL+ cells demonstrated increased spheroid formation in vitro and in vivo. Using patient derived xenografts (PDXs), we demonstrated that anti-GITR neutralizing antibodies attenuated tumor growth in sarcomatoid PDX mice. Tumor immunostaining demonstrated higher levels of GITR and GITRL expressions in non-epithelioid compared to epithelioid tumors. Among 73 patients uniformly treated with accelerated radiation therapy followed by surgery, the intensity of GITR expression after radiation negatively correlated with survival in non-epithelioid MPM patients. In conclusion, the GITR-GITRL pathway is an important mechanism of autocrine proliferation in sarcomatoid mesothelioma, associated with tumor stemness and resistance to therapy. Blocking the GITR-GITRL pathway could be a new therapeutic target for non-epithelioid mesothelioma.

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Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

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Anti-GD2 IgA kills tumors by neutrophils without antibody-associated pain in the preclinical treatment of high-risk neuroblastoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003163 ◽

2021 ◽

Vol 9 (10) ◽

pp. e003163

Author(s):

Mitchell Evers ◽

Marjolein Stip ◽

Kaylee Keller ◽

Hanneke Willemen ◽

Maaike Nederend ◽

...

Keyword(s):

High Risk ◽

Sensory Neurons ◽

Complement System ◽

Severe Pain ◽

Neuroblastoma Cells ◽

Antibody Therapy ◽

Preclinical Treatment ◽

The Complement System

BackgroundThe addition of monoclonal antibody therapy against GD2 to the treatment of high-risk neuroblastoma led to improved responses in patients. Nevertheless, administration of GD2 antibodies against neuroblastoma is associated with therapy-limiting neuropathic pain. This severe pain is evoked at least partially through complement activation on GD2-expressing sensory neurons.MethodsTo reduce pain while maintaining antitumor activity, we have reformatted the approved GD2 antibody ch14.18 into the IgA1 isotype. This novel reformatted IgA is unable to activate the complement system but efficiently activates leukocytes through the FcαRI (CD89).ResultsIgA GD2 did not activate the complement system in vitro nor induced pain in mice. Importantly, neutrophil-mediated killing of neuroblastoma cells is enhanced with IgA in comparison to IgG, resulting in efficient tumoricidal capacity of the antibody in vitro and in vivo.ConclusionsOur results indicate that employing IgA GD2 as a novel isotype has two major benefits: it halts antibody-induced excruciating pain and improves neutrophil-mediated lysis of neuroblastoma. Thus, we postulate that patients with high-risk neuroblastoma would strongly benefit from IgA GD2 therapy.

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DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES

10.1055/s-0038-1643865 ◽

1987 ◽

Author(s):

J Abbink ◽

J Nuijens ◽

C Huijbregts ◽

E Hack

Keyword(s):

Monoclonal Antibodies ◽

Longitudinal Studies ◽

Human Plasma ◽

Dextran Sulfate ◽

Optimal Conditions ◽

Α2 Macroglobulin ◽

In Vitro Activation

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.

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