A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcγRIIA and FcγIIIA genes

Miescher, Sylvia; Spycher, M. A.; Amstutz, Hanspeter; Haas, Masja de; Kleijer, Marion; Kalus, Ulrich; Radtke, H.; Hubsch, Alphonse; Andresen, Irmgard; Martin, Roland; Bichler, Johann

doi:10.1182/blood-2003-11-3929

Cited by 74 publications

(55 citation statements)

References 54 publications

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“…35 Macrophages of the spleen and liver are responsible for the clearance of IgG-opsonized blood cells in vivo, but remarkably little is known about expression levels of FcgRs on these macrophages. It has been proposed that mainly the low-affinity FcgRs are involved, 1 based only on circumstantial evidence, such as association studies with genetic polymorphisms in the low-affinity FcgRs 5,41 and in vivo blocking studies with specific FcgR MoAbs in limited series of patients. 42,43 If it is indeed the case that these macrophages have a predominant expression of low-affinity FcgRs, we may expect the difference between dimeric and monomeric IgG to be even greater than the difference we have found in monocyte-derived macrophages.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages

Nagelkerke

Dekkers²,

Kustiawan

et al. 2014

Blood

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Section: Discussionmentioning

confidence: 99%

Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages

Nagelkerke

Dekkers²,

Kustiawan

et al. 2014

Blood

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“…In our study we generated a panel of 17 CHO-mAb subclones expressing a humanized antibody against the Rhesus D antigen (RhD) (Asvadi et al, 2002;Miescher et al, 2004) and analyzed these high-and low-producer clones regarding their growth characteristics, mAb productivity, their molecular features, and their stability in long-term culture. These extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.…”

Section: Introductionmentioning

confidence: 99%

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Chusainow

Yang

Yeo

et al. 2008

Biotech & Bioengineering

278

223

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Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell À1 day À1 using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high-and lowproducer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.

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“…Although the SRBC Ags are poorly defined, it has nevertheless been suggested that epitope masking by the anti-SRBC Ab likely plays a major role in reducing the immune response to these cells (13)(14)(15). Although not universally agreed upon, the most commonly accepted theories to explain the AMIS effect with erythrocytes are 1) that the AMIS Ab is able to rapidly clear Ag-positive target cells before they can be recognized by the immune system (10-12, 16, 17), theoretically based on FcgRmediated phagocytosis of D + RBCs preventing B cells from recognizing the D Ag (10,11,17), or 2) that the AMIS Ab sterically hinders the ability of the immune response to see or detect the Ag (4,15,16,18). Other theories with direct experimental support have suggested that inhibition may occur through the inhibitory FcgRIIB (19), that inhibitory cytokines may be produced (20), that glycosylation of the AMIS Ab may play a role (21)(22)(23), and/or that Abmediated immune deviation (AMID) away from the Ag and directed toward the sensitizing Ab may be involved (5).…”

mentioning

confidence: 99%

Antibody-Mediated Immune Suppression of Erythrocyte Alloimmunization Can Occur Independently from Red Cell Clearance or Epitope Masking in a Murine Model

Stowell

Bernardo

et al. 2014

The Journal of Immunology

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Anti-D can prevent immunization to the RhD Ag on RBCs, a phenomenon commonly termed Ab-mediated immune suppression (AMIS). The most accepted theory to explain this effect has been the rapid clearance of RBCs. In mouse models using SRBC, these xenogeneic cells are always rapidly cleared even without Ab, and involvement of epitope masking of the SRBC Ags by the AMIS-inducing Ab (anti-SRBC) has been suggested. To address these hypotheses, we immunized mice with murine transgenic RBCs expressing the HOD Ag (hen egg lysozyme [HEL], in sequence with ovalbumin, and the human Duffy transmembrane protein) in the presence of polyclonal Abs or mAbs to the HOD molecule. The isotype, specificity, and ability to induce AMIS of these Abs were compared with accelerated clearance as well as steric hindrance of the HOD Ag. Mice made IgM and IgG reactive with the HEL portion of the molecule only. All six of the mAbs could inhibit the response. The HEL-specific Abs (4B7, IgG1; GD7, IgG2b; 2F4, IgG1) did not accelerate clearance of the HOD-RBCs and displayed partial epitope masking. The Duffy-specific Abs (MIMA 29, IgG2a; CBC-512, IgG1; K6, IgG1) all caused rapid clearance of HOD RBCs without steric hindrance. To our knowledge, this is the first demonstration of AMIS to erythrocytes in an all-murine model and shows that AMIS can occur in the absence of RBC clearance or epitope masking. The AMIS effect was also independent of IgG isotype and epitope specificity of the AMIS-inducing Ab.

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A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcγRIIA and FcγIIIA genes

Cited by 74 publications

References 54 publications

Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages

Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Antibody-Mediated Immune Suppression of Erythrocyte Alloimmunization Can Occur Independently from Red Cell Clearance or Epitope Masking in a Murine Model

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