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. 2017 May 30;16(2):200–208. doi: 10.2450/2017.0176-16

Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies

Dylana Díaz-Solano 1, Jaheli Fuenmayor 1, Ramon F Montaño 1,✉
PMCID: PMC5839618  PMID: 28686149

Abstract

Background

Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex.

Materials and methods

Recombinant polymeric anti-G IgG1 (IgG1μtp) and IgG3 (IgG3μtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays.

Results

The recombinant IgG1μtp and IgG3μtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D.

Discussion

The enhanced opsonic properties of the polymeric anti-G IgG1μtp and IgG3μtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

Keywords: Rh blood group, erythrocytes, opsonin, phagocytosis, recombinant antibody

Introduction

Human monoclonal antibodies against the Rhesus blood group (Rh or CD240) have been used as tools for the identification and elucidation of the molecular characteristics of the proteins that carry these red blood cell (RBC)-specific alloantigens and as reagents for blood classification1,2. Monoclonal antibodies specific for the D protein of the Rh complex (monoclonal anti-D) have additional potential in the prophylaxis of haemolytic disease of the foetus and newborn due to RhD incompatibility (HDFN-RhD)3, although the source of prophylactic anti-D currently available is the polyclonal immunoglobulin fraction (anti-D Ig) isolated from the plasma of human donors.

Since its introduction in the 1960s, the prophylaxis of HDFN-RhD with anti-D Ig has shown to be safe and effective4,5. Currently, the plasma used for preparing prophylactic anti-D Ig comes from hyperimmunised RhD-negative males. Ethical concerns have been raised about this practice and the associated risks of immunisation against other alloantigens and transmission of blood-borne pathogens during repeated inoculation of heterologous RBC6. Additionally, restrictions have been applied to the use of human plasma collected in certain European countries, especially the United Kingdom, for the production of plasma derivatives because of the still uncertain risk of transmission of the agent causing variant Creutzfeldt-Jacob disease7–9. The development of new, non-plasma-derived sources of prophylactic anti-D is, therefore, desirable.

Numerous monoclonal and recombinant IgM, IgG1, and IgG3 anti-D have been produced10–12. Some have been successfully developed as blood-typing reagents13. Others have been used for academic or clinical laboratory research14,15, while only a few of them have been evaluated, either as preparations of a single or a mixture (“blend”) of two monoclonal anti-D, to replace the prophylactic plasma-derived polyclonal anti-D Ig3,16. Despite important efforts invested in the development of a monoclonal-derived anti-D with prophylactic properties similar to the polyclonal ones, no such preparation is yet available.

The polyclonal antibodies found in prophylactic anti-D Ig belong to the IgG1 and IgG3 subclasses17–19. As the material is produced from large pools of plasma, it contains a varied collection of anti-D specificities. These mixtures of polyclonal IgG1/IgG3 anti-D do not normally mediate the direct agglutination of RhD-positive RBC in vitro, but they do facilitate the efficient clearance of RhD-positive RBC in vivo, and are deemed to be responsible for the RhD-specific immunosuppression observed during HDFN-RhD prophylaxis. This phenomenon of specific immunosuppression is not completely understood yet20, but the interaction of the Fc portion of the IgG anti-D molecules with Fc-gamma receptors (FcγR) on the surface of phagocytic cells21 seems to be of utmost importance for both a rapid clearance of foetal RhD-positive RBC and the specific suppression of the maternal anti-D antibody response in vivo.

Previously, we genetically fused the 18-amino acid tailpiece from the human Ig μ heavy (H) chain (μtp) to the carboxy-terminus of the Ig gamma 3 (γ3) H-chain of an antibody specific for the Rh “G” antigen (Rh12)22, and produced a recombinant anti-G IgM-like IgG3μtp23. This anti-G IgG3μtp is assembled and secreted as polymers capable of mediating the direct agglutination of RhD-positive RBC23. Here, we extend that work and describe the preparation of a polymeric anti-G IgG1μtp having the same specificity as the polymeric IgG3μtp previously reported. We took advantage of these preparations to examine the potential of polymeric anti-Rh IgGμtp as antibodies for possible prophylactic use. We studied both polymers in terms of their capacity to promote the phagocytosis and complement-mediated lysis of RhD-positive RBC, and compared them with their monomeric IgG versions and with prophylactic, plasma-derived polyclonal anti-D Ig. The results obtained may contribute to the development of a prophylactic polymeric anti-D product.

Materials and methods

Materials

The following proteins were used for comparisons in this study: recombinant monomeric anti-G IgG1 and IgG323, monoclonal anti-A IgM24, a blend of monoclonal anti-D IgG/IgM (Wiener Lab., Rosario, Sta. Fe, Argentina), human IgM (I-8260; Sigma. USA), and a prophylactic human polyclonal anti-D IgG (RhoGAM. Lot RGF226A1. Ortho-Clinical Diagnostics, Raritan, NJ, USA).

Six-week old Balb/c mice from the animal facility of the Venezuelan Institute of Scientific Investigation (IVIC), and venous blood samples from healthy human volunteers were obtained and used following procedures approved by the animal and human research ethics committees of IVIC, respectively.

Construction and expression of the γ1μtp anti-G H-chain vector

Details of the H and light (L) chain variable domains present in the recombinant Ig molecules studied, and of the original monoclonal antibody from which they were cloned out, have been reported elsewhere23,25. Standard molecular biology and recombinant DNA techniques were used to build a plasmid-based expression vector, here designated pAN-CMV/VHANTI-RhG/γ1μtp, which encodes the γ1μtp anti-G H-chain. The procedure followed is detailed in the Online Supplementary Content.

The cell line TAZZ, a transfectoma that secretes RhG-specific human Ig lambda (λ) L-chains23, was used as the recipient of pAN-CMV/VHANTI-RhG/γ1μtp. After transfection (see details in the Online Supplementary Content), the cells in culture were selected in the presence of 1 mg/mL G418 (Clontech, Mount View, CA, USA). Surviving clones were screened for IgG production using a human IgG-specific sandwich enzyme-linked immunosorbent assay (Sigma, St. Louis, MO, USA). To select a working clone, the IgG-containing supernatants were screened using the microhaemagglutination assay outlined below.

Production, purification, and structural characterisation of the anti-G IgGμtp

A select transfectoma actively secreting anti-G IgG1μtp was grown in roller bottles in Iscove’s modified Dulbecco’s medium supplemented with 2 mM L-glutamine and 10% low-IgG foetal calf serum (PAN-Biotech GmbH, Aidenbach, Germany). A cell line producing anti-G IgG3μtp23 was also grown under similar conditions. The antibodies present in both culture supernatants were purified by affinity chromatography using protein G (Amersham, Piscataway, NJ, USA). The purified antibodies were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were visualised using AgNO3 or Coomasie blue staining. Samples of recombinant monomeric anti-G IgG1 and IgG323 and commercial IgM were included as molecular weight standards.

Direct haemagglutination

A previously described micro-haemagglutination assay23 was used to evaluate the ability of the anti-G IgG1μtp to bind RhD-positive (phenotype DccEe) RBC (see details in the Online Supplementary Content). Sedimentation of RBC was visually assessed in microtitre plates. The wells containing RBC that sedimented tightly at the bottom of the plate were read as negative; those with RBC forming a spread, carpet-like structure covering a wide area of the well were read as positive.

Erythrophagocytosis

The opsonic capacity of the polymeric anti-G IgGμtp molecules was studied by sensitising group O RhD-positive RBC with them and subsequently exposing the sensitised RBC to phagocytic cells obtained from the peritoneal cavity of Balb/c mice. Erythrophagocytosis was assessed qualitatively using light microscopy, and quantitatively by a colorimetric assay26,27. The colorimetric assay measured the amount of ingested RBC as the haemoglobin content in phagocytic cells. The haemoglobin released after the lysis of the phagocytic cells was estimated in an enzymatic-chromogenic reaction, through the pseudo-peroxidase activity of the haemoglobin molecule on the substrate 2–7 diaminofluorene (DAF).

Thioglycolate-elicited, mouse peritoneal macrophages were obtained as described elsewhere28 and used as phagocytic cells (effectors). Group O RhD-positive RBC (Rh phenotype DccEe) were used as targets and sensitised with the different opsonins at a concentration of 3 μg/mL (see details in the Online Supplementary Content). The sensitised RBC were suspended in RPMI medium, and dispensed into the plates containing effector cells (effector-to-target ratio=1:5). Effector cells incubated with non-sensitised RBC served as a negative control. The plates were incubated at 37 °C for 3 hours and subsequently examined using a phase-contrast invertoscope (IM Zeiss, Oberkochen, Germany). Afterwards, the non-phagocytosed RBC were lysed and washed off, as described in the Online Supplementary Content. The remaining adhered cells (macrophages containing phagocytosed RBC) were lysed and the haemoglobin content estimated by colorimetry (see details in the Online Supplementary Content). The optical density values obtained were converted to number of ingested RBC following the procedure explained in the Online Supplementary Content. Finally, the phagocytosis was expressed as percentages, assuming the average value of six samples - each containing 106 lysed RBC - as 100% phagocytosis.

Antibody-dependent complement haemolysis

A previously described colorimetric-enzymatic microassay29 based on the quantification of the haemoglobin released from lysed RBC was used to study the ability of the polymeric antibodies to mediate the lysis of RBC due to complement activation. Group A RhD-positive (Rh phenotype DCcEe) or O RhD-positive (Rh phenotype DccEe) target RBC were sensitised with purified polymeric anti-G at 1.2 μg/mL, or with control antibody as explained in the Online Supplementary Content. Recombinant monomeric anti-G IgG1 or IgG323 (12 μg/mL), monoclonal anti-A IgM24 (used as a supernatant diluted at 1:4,096), plasma-derived polyclonal anti-D (6 μg/mL), and a commercial anti-D IgG/IgM blend (used at a 1:1,000 dilution) were used as controls. Samples prepared with non-sensitised RBCs were used as negative controls. After incubation at 37 °C for 1 hour, autologous serum was added as a source of complement and further incubation at 37 °C was allowed for 3 hours. To estimate the degree of haemolysis, the haemoglobin present in the supernatants was quantified by colorimetry using the DAF reagent (see the Online Supplementary Content for details). The optical density values obtained were transformed to percentages of lysis using the mean optical density value of a set of samples in which the RBC were treated with distilled water as 100% lysis.

Determination of complement activation by membrane deposition of C3d

The ability of the recombinant polymeric anti-G antibodies to activate complement was also studied using an antibody-dependent complement activation assay based on the deposition of C3d on the membrane of target RBC (see details in the Online Supplementary Content). An agglutinating anti-C3d reagent (Municipal Blood Bank, Caracas, Venezuela) was used for this purpose. Haemagglutination was determined macroscopically, as well as using light microscopy. Monomeric anti-G IgG3, monoclonal anti-A IgM, plasma-derived polyclonal anti-D (RhoGAM), and a monoclonal anti-D IgG/IgM blend were used as controls. Non-sensitised RBC, with or without anti-C3d, were used as negative controls.

Statistical analysis

Data in the figures are expressed as mean ± standard deviation of proportions calculated from three independent experiments performed in triplicate. Significant differences were estimated using one-way ANOVA tests as implemented in GraphPad Prism (version 3.02; GraphPad Software, San Diego, CA, USA). The proportions were Arcsin-transformed prior to statistical analysis to ensure normality of residuals, and p-value thresholds for significance were adjusted using Bonferroni’s correction30.

Results

Expression and structural characterisation of the anti-G IgG1μtp

The introduction of the vector encoding the Ig γ1μtp H-chain into the anti-G Ig L(λ) chain-producing cells allowed the generation of stably-transfected clones producing anti-G IgG1μtp. Culture supernatants from one actively secreting clone and from the anti-G IgG3μtp secreting cells we have previously produced23 were processed to purify the anti-G IgG1μtp and IgG3μtp.

The SDS-PAGE analysis under non-reducing conditions of the purified anti-G IgG1μtp showed multiple bands ranging in size from 150,000 to 1,000,000 Da (Figure 1A, lane 2). The anti-G IgG3μtp showed a similar electrophoretic migration profile (Figure 1A, lane 4). These migration patterns indicate that the IgGμtp proteins are secreted as mixed populations of oligomeric molecules, from dimers to hexamers, but also as monomers. In contrast, the recombinant anti-G IgG1 and IgG3 migrated as single bands at 150,000 and 170,000 Da, respectively (Figure 1A, lanes 1 and 3), and the human IgM as a fully-assembled polymer of 1,000,000 Da (Figure 1A, lane 5). The SDS-PAGE analysis under reducing conditions showed the γ1μtp and γ3μtp H-chains (Figure 1B, lanes 2 and 4) having a slightly higher molecular weight than their γ1/γ3 H-chain counterparts (Figure 1B, lanes 1 and 3), a difference that was expected due to the presence of 18 amino acid residues corresponding to the μ tailpiece. The small difference in size between γμtp vs γ H-chains also supports the notion that the presence of the μ tailpiece causes the occurrence of higher molecular weight species when the electrophoretic analysis is performed under non-reducing conditions.

Figure 1.

Figure 1

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Anti-G IgGμtp molecules form polymers.

The SDS-PAGE analysis of the recombinant anti-G antibodies was performed using purified proteins. Samples were analysed under (A) non-reducing and (B) reducing conditions, and stained with (A) AgNO3 or (B) Coomasie blue. Lane 1: monomeric IgG1; lane 2: polymeric IgG1μtp; lane 3: monomeric IgG3; lane 4: polymeric IgG3μtp; lane 5: commercial IgM. Positions of the molecular weight protein standards are indicated. “H” and “L” refer to Ig heavy and light chains, respectively. Ig: immunoglobulin.

The recombinant polymeric anti-G antibodies mediate direct agglutination of RhD-positive red blood cells

The incubation of group O RhD-positive RBC (phenotype DccEe) with purified polymeric anti-G IgG1μtp at a concentration of 40 μg/mL led to a positive direct agglutination reaction (Figure 2, Row C, C1). A positive agglutination reaction was also observed with RhD-positive RBC exposed to the polymeric anti-G IgG3μtp at a concentration of 20 μg/mL (Figure 2, Row D, D1), confirming our previous report23. In this respect, the polymeric anti-G molecules behaved as the commercial anti-D IgM used as a control (Figure 2, P). Serial double dilutions of the polymeric anti-G IgG1μtp or IgG3μtp (Figure 2, C2–C11 and D2–D11) eventually led to negative reactions (Figure 2, C9–C11 and D9–D11). No direct haemagglutination was observed upon incubation of RhD-positive RBC with monomeric anti-G IgG1 (starting at a concentration of 400 μg/mL) or IgG3 (starting at a concentration of 80 μg/mL) (Figure 2, rows A and B, respectively). Thus, polymerisation endowed the anti-G IgG1μtp with the capacity of directly agglutinating RhD-positive RBC, suggesting that it leads to an increased avidity of the antibody.

Figure 2.

Figure 2

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Polymeric anti-G IgGμtp antibodies induce direct haemagglutination.

Aliquots of 30 μL of a 2% v/v suspension of group O RhD-positive RBC were mixed and incubated in a U-bottomed 96-well microplate with 50 μL of serial double-dilutions (from undiluted [column 1] up to a 1:1,024 dilution [column 11]) of purified anti-G antibodies: monomeric IgG1 (row A) or IgG3 (row B); or polymeric IgG1μtp (row C) or IgG3μtp (row D). The pattern of spontaneous sedimentation was visually assessed as a measure of haemagglutination. A commercial monoclonal anti-D IgG/IgM blend (P: positive) and plasma-derived anti-D Ig (N: negative) were used as controls. Ig: immunoglobulin.

The recombinant polymeric anti-G antibodies mediate enhanced phagocytosis of RhD-positive red blood cells

Light microscopy studies showed that opsonisation of RhD-positive RBC (phenotype DccEe) with the polymeric anti-G IgGs led to abundant erythrophagocytosis (Figure 3E,F). Interestingly, the engulfment of RBC was substantially greater when phagocytic cells were exposed to polymeric IgGμtp-opsonised RBC, as compared to monomeric IgG-opsonised RBC (Figure 3C,D), or to plasma-derived polyclonal anti-D-opsonised RBC (Figure 3B). Quantification of RBC ingestion (Figure 4) confirmed the observations from light microscopy. The polymeric anti-G antibodies induced the erythrophagocytosis of approximately 80% of the target RBC, compared with monomeric anti-G IgG1 (~4%) or IgG3 (<20%), or with plasma-derived polyclonal anti-D (~40%). Even though plasma-derived polyclonal anti-D induced RBC phagocytosis at a level below that of the monoclonal polymeric anti-G IgG1μtp or IgG3μtp, it was clearly a better opsonin than monoclonal monomeric anti-G IgG1 or IgG3. No significant differences were observed between the extent of phagocytosis mediated by the polymeric anti-G IgG1μtp and the IgG3μtp, or between mixtures containing different proportions (50–50% and 75–25%) of polymeric anti-G IgG1μtp and IgG3μtp. All mixtures consistently showed an additive effect. The opsonic activity of the mixtures of polymeric molecules was also significantly greater than that of the plasma-derived polyclonal anti-D.

Figure 3.

Figure 3

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Microscopic evaluation of the erythrophagocytosis induced by polymeric anti-G IgGμtp antibodies.

Group O RhD-positive RBC (targets) were sensitised with the different opsonins at a concentration of 3 μg/mL and added to monolayers of thioglycolate-elicited mouse peritoneal macrophages (effectors) at a 5 to 1 target-to-effector ratio. The target and effector cells were examined by light microscopy after incubation at 37 °C for 3 hours. Frame “A” shows a representative image of the control condition (phagocytic cells exposed to unsensitised RBC). The other frames contain representative images taken from samples in which the RBC were sensitised with (B) plasma-derived polyclonal anti-D, (C) monomeric anti-G IgG1, (D) monomeric anti-G IgG3, (E) polymeric anti-G IgG1μtp, or (F) polymeric anti-G IgG3μtp. The images shown (x200) are representative of three independent experiments, in which each condition was tested in triplicate. Ig: immunoglobulin; RBC: red blood cell.

Figure 4.

Figure 4

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Polymeric anti-G IgGμtp antibodies are better opsonins than plasma-derived polyclonal anti-D.

Phagocytic cells and sensitised group O Rh-positive RBC were mixed and incubated as indicated in the legend of Figure 3. After lysing and washing off the non-phagocytosed erythrocytes, the remaining cells (macrophages containing ingested RBC) were lysed and the haemoglobin released was quantified at 630 nm. A standard curve of haemoglobin versus optical density was used to equate absorbance values to RBC numbers, which were converted to a percentage of phagocytosis. Data shown are the arithmetic mean ± standard deviation of three independent experiments, each performed in triplicate. “IgG1μtp=IgG3μtp” means a blend containing equal amounts of each polymeric antibody; “IgG1μtp>IgG3μtp” means a blend containing 75% polymeric IgG1μtp and 25% polymeric IgG3μtp. Statistically significant differences were calculated after Bonferroni’s correction (n= 16) and are indicated with one (p<0.0001) or two asterisks (p<0.001). Ig: immunoglobulin; RBC: red blood cell.

The recombinant polymeric anti-G antibodies do not trigger antibody-dependent, complement-mediated lysis of RhD-positive red blood cells

Anti-A IgM efficiently induced the complement-mediated lysis of group A RhD-positive RBC, whereas the polymeric anti-G IgG were inactive and caused no detectable haemolysis. In repeated assays using group A RhD-positive or O RhD-positive RBC, no anti-G IgGμtp-mediated haemolysis was detected (Figure 5). The group A RhD-positive RBC used in these assays were phenotyped as “Cc”, which means that they should have an increased number of “G” antigenic sites compared to the group O RhD-positive RBC used, which were typed as “cc”. Despite this, the polymeric anti-G antibodies were unable to induce the complement-mediated haemolysis of these group A RhD-positive RBC.

Figure 5.

Figure 5

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Polymeric anti-G IgGμtp antibodies do not induce complement-mediated haemolysis.

Group A RhD-positive (empty bars) or O RhD-positive (black-filled bars) RBC were sensitised with the antibodies indicated in the figure, and then mixed and incubated with autologous plasma as a source of complement. The haemolysis was quantified and normalised to a percentage as detailed in the text and in the online supplementary document. Data shown are the arithmetic mean ± standard deviation of three independent experiments, each performed in triplicate. “IgG1μtp=IgG3μtp” and “IgG1μtp>IgG3μtp” mean the same as indicated in the legend of Figure 4. Statistically significant differences were calculated after Bonferroni’s correction (n= 16) and are indicated with one asterisk (p<0.0001). RBC: red blood cell.

We also examined whether the polymeric anti-G antibodies promoted earlier events preceding complement-mediated haemolysis, such as the deposition of C3d on the RBC membrane and production of C5a. Neither C5a release, as determined by a commercial enzyme-linked immunosorbent assay (data not shown), nor deposition of C3d fragments on RBC were observed (Figure 6D,E). Instead, we only found C5a release (data not shown) and anti-C3d-mediated haemagglutination (Figure 6B) when anti-A IgM was used to sensitise group A RhD-positive RBC.

Figure 6.

Figure 6

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Polymeric anti-G IgGμtp antibodies do not induce C3d deposition on the membrane of RhD-positive RBC.

Group A RhD-positive RBC were suspended in HBS-BSA buffer containing (A) no antibody, (B) monoclonal anti-A IgM (a culture supernatant diluted at 1:262,144), (C) monomeric anti-G IgG3 (80 ng/mL), (D) polymeric anti-G IgG1μtp (9 ng/mL), or (E) polymeric anti-G IgG3μtp (9 ng/mL). The sensitised RBC were then incubated with autologous plasma, and finally with an IgM anti-C3d agglutinating reagent. The images shown (X 200) are representative of three independent experiments, in which each condition was tested in triplicate. RBC: red blood cell; HBS-BSA: hepes-buffered saline containing 0.1% bovine serum albumin.

Collectively, these results suggest that polymeric anti-G IgG1μtp and IgG3μtp do not activate complement or promote any lytic activity on target RBC.

Discussion

In this work, the 18-amino acid tailpiece from the Ig μ chain (μtp) was genetically joined to the carboxy-terminus of a human Ig γ1 H-chain specific for the G antigen of the Rh alloantigenic system, and an IgM-like IgG1μtp anti-G antibody was produced. Electrophoretic analyses determined that, as observed with a previously reported anti-G IgG3μtp23, the anti-G IgG1μtp molecules assemble and are secreted as polymers of variable sizes including pentamers and hexamers, as well as monomers. These results are consistent with those from previous studies in which IgGμtp antibodies with other specificities were also simultaneously secreted as polymers and monomers31–33.

The addition of μtp conferred enhanced functional properties to the polymeric anti-G IgG1 μtp and IgG3μtp. For example, they effectively cross-linked and directly agglutinated RhD-positive RBC, an ability not found in the monomeric versions. Hence, the addition of μtp endowed these molecules with a greater capacity to bind RhD-positive RBC. To rule out the possibility that this greater RBC binding capacity was due to the recognition, by the polymeric anti-G IgG, of “G” antigenic determinants on the Rh CE polypeptide, the RBC were selected in such a way that c (but not C) erythrocytes were used in the agglutination assays reported. According to the results presented here, the polymeric anti-G IgG1μtp and IgG3μtp perform in vitro similarly to the IgM anti-D reagents commonly used in blood typing.

In addition, polymeric anti-G IgG1μtp and IgG3μtp elicited much more phagocytosis of RhD-positive RBC than did the monomeric recombinant versions. The presence of “contaminant” monomeric antibody does not seem to have impaired the enhanced performance of the polymers in vitro. More importantly, in comparable erythrophagocytosis assays, the monoclonal polymeric anti-G IgGμtp outperformed the polyclonal, plasma-derived anti-D Ig as well. Interestingly, however, the polyclonal plasma-derived anti-D Ig was clearly a better opsonin than recombinant, monoclonal, monomeric anti-G IgG1 or IgG3, suggesting that having a broader range of specificities favours opsonisation. Collectively, these results also indicate that the enhanced opsonic activity of the polymeric IgGμtp molecules may have practical interest for the development of a monoclonal alternative to prophylactic anti-D Ig. It is well recognised that fast clearance of foetal RBC from the mother’s circulation is a key factor in preventing Rh alloimmunisation20. Fast RBC clearance in this setting is dependent on erythrophagocytosis by reticuloendothelial cells of the spleen, which is proficiently promoted by plasma-derived anti-D Ig, but poorly mediated by most monomeric monoclonal anti-D IgG tested so far3,16,34–37. The way novel molecules such as the polymeric IgGμtp described here - but with anti-D specificity - will behave if administered to RhD-negative mothers to foster the clearance from circulation of RhD-positive RBC is still unknown. It will be important to study whether a polymeric anti-D IgGμtp may have greater opsonic activity in vivo and would mediate faster splenic clearance of RhD-positive RBC than plasma-derived prophylactic anti-D Ig.

Altogether, the results from the agglutination and phagocytosis assays suggest that IgG polymerisation through the addition of μtp leads to an increased probability of a single IgG antibody molecule simultaneously interacting with two or more Rh molecules, located either in the same or in different RBC, but also to an increased probability of two or more Fc portions from the same polymeric IgG molecule interacting with two or more FcγR on phagocytic cells.

A comprehensive perspective on the in vivo properties of such novel molecules should consider other aspects such as persistence in circulation, the capacity to mediate antibody-dependent cellular cytotoxicity and RhD-specific immunosuppression, and safety as well. With regard to safety, polymeric IgG1μtp and IgG3μtp directed against antigens other than Rh have been documented to mediate enhanced complement activation as compared to their equivalent monomeric molecules31. This would be undesirable for a polymeric anti-D IgGμtp since the prophylactic anti-D Ig does not activate complement38. However, our in vitro results indicate that the polymeric anti-G IgG1μtp and IgG3μtp do not induce complement-mediated haemolysis or even complement fixation. In fact, the polymeric anti-G IgGμtp antibodies behaved as monomeric anti-G IgG or plasma-derived polyclonal anti-D Ig in this respect, none of which was able to induce complement-mediated haemolysis.

Conclusions

The polymeric anti-G IgG1μtp and IgG3μtp evaluated in this study are the first of their kind to be produced in vitro. Our results indicate that they are, individually or as a blend, more potent opsonins than the plasma-derived polyclonal anti-D Ig. This enhanced potency was also evident as an increased avidity for antigen and, conveniently, does not lead to antibody-mediated complement activation. Recombinant molecules such as those described here (but with anti-D specificity) may have clinical value as a replacement of the plasma-derived polyclonal anti-D Ig currently used to prevent the HDFN caused by anti-D.

Supplementary Information

Acknowledgements

We thank C. Cruz (Municipal Blood Bank, Caracas, Venezuela) for providing the anti-C3d reagent and for phenotyping the red blood cells used in this research. We also thank members of the IVIC community for their help: Dr. E. Romano for technical advice; Dr. B. Guerrero, Dr. P. Taylor, M. Romano† and the photography department for technical assistance; and Dr. K. Rodríguez-Clark and V. Hermann for comments on earlier versions of this manuscript.

Footnotes

Funding

This work was partially funded by the Venezuelan Institute of Scientific Investigation (IVIC) and the National Science and Technology Fund (FONACIT) of Venezuela. DD-S was supported by a scholarship for postgraduate studies from IVIC.

Authorship contributions

DD-S performed the experiments and analysed the data. RFM conceived the study and participated in its design and coordination and in the analyses of the experimental results. JF critically commented on the study design and performed part of the statistical analyses. DD-S and RFM prepared the figures and wrote the paper. All Authors contributed to the final version of the manuscript.

The Authors declare no conflicts of interest.

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