Abstract
While ABO/Rh(D) red blood cells (RBC)-matched transfusions are generally considered as safe, a significant risk of alloimmunization to non-A/B blood group antigens exists; especially in chronically transfused patients. Indeed, alloimmunization to non-A/B antigens can be so severe that RBC transfusion can no longer be safely administered without the risk of a potentially deadly immune haemolytic reaction. Currently, no satisfactory solutions exist either to prevent blood group alloimmunization or to cost-effectively treat patients with severe alloimmunization. To address this problem, we have pioneered the immunocamouflage of donor RBC. The immunocamouflaged (stealth) RBC is manufactured by the covalent grafting of biologically safe polymers to RBC membrane proteins. As a result of the grafted polymer, non-A/B blood group antigens are biophysically and immunologically masked. Of particular interest is the immunocamouflage of the Rh(D) antigen which could be used to improve blood inventory and transfusion safety. The polymer-modified RBCs are morphologically normal and, in mice, exhibit normal in vivo survival at immunoprotective grafting concentration. In this chapter, we explore both the biophysical and immunological consequences of the grafted polymers, explore the conditions in which they might be appropriately used, and describe the technology necessary to manufacture functional transfusable units of these cells within the clinical setting.
Keywords
- red blood cell
- immunocamouflage
- alloimmunization
- Rh(D)
- polymer
1. Introduction
The transfusion of red blood cells (RBC) remains the most common, and best tolerated, form of tissue transplantation. Indeed, an estimated 108 million units of whole blood (~49 million litres) are collected annually worldwide for processing and eventual transfusion [1]. In spite of this massive collection effort, the need for blood constantly exceeds availability due to a combination of collection, manufacturing, storage and, most important clinically, biological (i.e., immunological) issues. The biological challenges facing successful RBC transfusions are vastly underappreciated, largely because of the long history and ubiquity of blood transfusions in modern medicine. Indeed, the RBC is an immunological complex cell with 35 major blood group systems that give rise to over 300 unique antigens capable of eliciting an immune response. Moreover, this immunological complexity is further exacerbated by the finding that the non-A/B (often referred to as
Among the non-A/B blood groups, the Rh system, and in particular Rh(D), is considered to be the most immunogenic antigen. Indeed, the Rh(D) antigen is highly immunogenic and when Rh(D)+ blood is transfused into an Rh(D)− individual, there is a 50% risk for the development of anti-Rh(D) antibodies resulting in very high risk of a haemolytic transfusion upon a second Rh(D)+ transfusion. Consequent to its immunogenicity, Rh(D) is always determined simultaneously with ABO type and constitutes the ‘±’ found alongside the ABO phenotype. Consequent to its immunogenicity, Rh(D) poses a significant challenge to blood operators since Type O Rh(D)− (O−) blood is the universal donor cell. In Euro-centric populations, 6–7% of the population is O− making the maintenance of an adequate inventory of this universal donor blood problematic but possible. Indeed, in North America and Europe, virtually all blood service providers experience a chronic shortage of Type O− blood. However, in other geographic regions, especially Asia, Rh(D)− individuals are extremely rare. Indeed, in China, only 0.1–0.4% of the population, regardless of ABO type, is Rh(D)−, making the Rh(D)− individual (especially with the increasing influx of European tourists) an at-risk patient [4]. Thus, within transfusion medicine, Rh(D) remains a significant problem in terms of both supply and its clinical risk.
Despite the immunological complexity of the RBC, simple ABO/Rh(D) matching has been, typically, considered sufficient for most acute transfusion needs. However, even when ABO/Rh(D) are appropriately matched, transfusion reactions still occur as mismatched non-A/B antigens do carry some immunological risks to a patient. While the incidence of clinically noteworthy (i.e.,
Historically, various interventions have been used in an attempt to prevent transfusion reactions arising from alloimmunization. While ABO/Rh(D) typing has been used since the 1940s, the practice of phenotyping some of the more problematic non-A/B/Rh(D) antigens is still uncommon and likely underlies the high frequency of alloimmunization in chronically transfused patients. In studies on β thalassemia, up to 20% of these chronically transfused individuals demonstrate
2. Bioengineering the red blood cell
Currently, no satisfactory solutions exist to prevent or cost-effectively treat blood group alloimmunization or to improve the inventory of Rh(D)− blood. To address these unmet needs, the covalent grafting of biocompatible polymers to donor RBC has been proposed to
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F1.png)
Figure 1.
Comparison of mPEG, PEOZ and HPG. The repeating structures of mPEG and PEOZ are denoted by the shaded areas while HPG, consisting of repeating polyglycerols, functions more as a mass. The
Most commonly, the chemically activated polymers are covalently grafted to proteins at exposed lysine residues. As a result of the grafted polymer, donor blood group antigens are biophysically and immunologically masked while the modified RBC remaining biologically and functionally viable. To date, most studies have focused on mPEG as the polymer of choice due to its superior ability to both sterically and charge camouflage allogeneic RBC and its well-characterized, and safe, pharmacological profile. The basic chemical structure of mPEG is HO-(CH2CH2O)n-CH2CH2CH3. mPEG is of low toxicity and is US FDA approved for oral, intravenous, subcutaneous and intramuscular administration [29]. The mPEG polyether polymer is neutrally charged, available in an extraordinarily wide range of molecular weights, and is highly soluble in aqueous-based solutions making it very suitable for pharmacological use. In contrast, both the POZ (e.g., PEOZ) and HPG polymers are poorly soluble in aqueous solutions and only confer weak charge camouflage.
A large number of biological and biophysical studies have been done to characterize the effects of polymer grafting on immune recognition and
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F2.png)
Figure 2.
Biophysical mechanisms of immunocamouflage. Panels A and B: Prevention of plasma protein (e.g., immunoglobulins) interaction with the cell membrane is due to both steric exclusion (shaded areas induced by the polymers radius of gyration; RF: Flory radii is the root mean square of end-to-end length of the polymer chain) and surface charge camouflage. The effects of both short chain (Panel A) and long chain (Panel B) polymers on the immunocamouflage of surface proteins (X, Y, Z) are schematically shown. The steric effect is maximized when chains are grafted at higher density, that is, with small separation between the chains (d). Importantly, antibody-antigen interaction is, biophysically speaking, charge-mediated. Membrane surface charge camouflage is primarily driven by polymer-mediated extension of the shear plane (SP) toward a region of decreased surface potential (Surface Potential Gradient). In the absence of polymer, the inherent shear plane (SP) of a cell is typically located 1–3 nm above the surface. The extension of SP is proportional to the hydrodynamic thickness of the polymer layer, which in turn is governed by the RF of the grafted polymer. Thus, 20 kDa polymers (large RF; Panel B) provide improved charge camouflage over 2 kDa polymers (small RF; Panel A). Delta (Δ) is the difference in the surface potential at the shear plane of a particle modified with the short (∆1) versus the long polymer (∆2). The membrane proteins X, Y and Z denote blood group antigens extending different distances from the cell surface. Panel C: Not all proteins in the complex topology of the RBC are equally accessible to grafting by the activated polymer, due to either its location in the protein complex or the paucity of lysines (the grafting site of activated mPEG). For example, Rh(D) is deeply buried in the complex while Kell is easily accessible. Thus,
Indeed, one of the most promising prospects of RBC immunocamouflage is in both diminishing the risk of Rh(D) alloimmunization and safely increasing blood inventory during emergency situations or in circumstances where Rh(D)- blood is unavailable. As shown in Figure 3A, immune recognition and phagocytosis of anti-D (RhoGAM®; Rho(D) Immune Globulin (Human) RhoGAM Ultra-Filtered PLUS; Ortho Clinical Diagnostics)-opsonized Rh(D)+ RBC are blocked in a grafting concentration-dependent manner by the grafted mPEG polymer. Importantly, RhoGAM® is a highly purified and concentrated human-derived anti-D IgG antibody that is highly effective at RBC opsonization yielding Monocyte Index (MI) scores in the monocyte-monolayer assay (MMA) in the range of 60–100%. The RhoGAM® antibody is used clinically for the prevention of Rh immunization, including during and after pregnancy and other obstetrical conditions or incompatible transfusion of Rh-positive blood. However, RhoGAM® does not fully reflect the biological/clinical heterogeneity of anti-D alloantibodies arising in alloimmunized individuals. To assess the potential utility of mPEG-RBC in alloimmunized individuals, human-sourced anti-D alloantibodies (
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F3.png)
Figure 3.
Polymer size and grafting concentration governs the efficacy of D immunocamouflage and the inhibition of erythrophagocytosis. Panel A: Effect of polymer size and grafting concentration on MMA phagocytosis of RhoGAM-opsonized Rh(D)+ RBC. As shown, short chain polymers (2–10 kDa) were ineffective at inhibiting erythrophagocytosis. In contrast, membrane modification of Rh(D)+ RBC with both the 20 and 30 kDa mPEG showed a significant (
Importantly, at immunologically protective grafting concentrations, the grafted polymer does not affect RBC structure or function as evidenced by normal morphology, O2 uptake and delivery, cellular deformability or ion transport [30, 33, 34, 37, 41, 52, 55]. While virtually indistinguishable from unmodified cells in most aspects, one interesting difference was noted between unmodified and mPEG-modified RBC. Consequent to the charge camouflage of the RBC, the cell:cell interactions necessary for Rouleaux formation were abrogated (Figure 4). Because the grafted polymer camouflages the charge of the cell necessary for cell:cell interaction, Rouleaux formation is inhibited resulting in attenuation of RBC sedimentation and, physiologically, decreased low-shear viscosity (Figure 4). Importantly, the decrease in low-shear viscosity may make the use of stealth RBC highly suitable for patients with diseases characterized by RBC-vaso-occlusive events (e.g., sickle cell). Importantly, RBC PEGylation inhibits both non-antibody (e.g., sickle cell self-aggregation) and antibody-mediated aggregation events and donor mPEG-RBC can serve as an efficient chain-breaker in pro-aggregation states [33].
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F4.png)
Figure 4.
Biophysical consequences of RBC PEGylation. Panel A: Charge camouflage of RBC is readily accomplished by polymer grafting and is a function of both grafting concentration and polymer size. As shown, the electrophoretic mobility of the human RBC was completely abrogated by the 20 kDa-activated mPEG at very low grafting concentrations. Mobility of the unmodified human RBC was −1.18±0.12 (µm/s)/(V/cm). Per cent change in mobility was normalized to the mobility of unmodified RBC. Panel B: Polymer grafting prevents cell:cell interaction (Rouleaux formation) via steric and charge camouflage. The loss of Rouleaux formations leads to a dramatically decreased RBC sedimentation rate. Panel C: Low-shear viscosity of the PEG-RBC is significantly reduced in comparison to normal control cells. Control and PEGylated (5 kDa mPEG) RBCs were resuspended to a 40% hematocrit in autologous plasma. Viscosity was measured over a range of shear rates using a Contraves LS30 low-shear viscometer (Contraves AG, Zurich, Switzerland). Data derived from Armstrong et al. [
The grafting of immunologically ‘inert’ polymers to the membrane of allogeneic RBC effectively camouflages multiple non-ABO antigens from immune recognition. The combined actions of both steric and charge camouflage underlie the ability of the grafted polymer to camouflage allogeneic blood group antigens (immunocamouflage) from the recipient’s immune system. These immunocamouflaged (i.e., stealth) RBC may be an effective tool in both preventing and treating alloimmunization in the chronically transfused patient; the transfusion of individual patients with rare blood phenotypes; emergency situation or geographic locations (e.g., China) where Rh(D)− blood is unavailable. Moreover, immunocamouflaged RBCs are inexpensively and easily manufactured in the clinical setting.
3. Manufacturing the ‘stealth RBC’
The immunocamouflaged RBC is unlikely to be a
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F5.png)
Figure 5.
Homogeneity of polymer grafting is a critical concern. Extreme grafting levels yield mechanically unstable RBC, while minimally modified RBC retain significant immunologic recognition. To achieve improved grafting homogeneity, ‘manufacturing’ processes and devices have been developed by our lab. Panels B and C: Using the semi-automated devices described in this chapter, RBCs are uniformly modified by the activated polymer. The uniformity of grafting was documented using an mPEG polymer formulation containing 1% fluorescent 20 kDa mPEG. As shown, the PEGylated RBCs are all fluorescently labeled showing complete derivatization with the semi-automated devices described in this chapter. Modified from Wang et al. [
To achieve the homogeneity necessary for a clinical mPEG-RBC unit (Figure 5), two scalable devices (Figures 6 and 7) utilizing micro-mixing chambers (alternatively Y-connectors to induce turbulence and mixing) have been designed, constructed and validated to semi-automate the RBC derivatization process and minimize the risk of contamination. Both approaches can be done aseptically using modifications of existing blood bags and sterile docking devices. The
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F6.png)
Figure 6.
Schematic representation for the clinical PEGylation of a single blood unit using the
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F7.png)
Figure 7.
Schematic representation for the clinical PEGylation of a single blood unit using the
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F8.png)
Figure 8.
Assessing the efficacy of the derivatization reaction via the two-phase PEG-Dextran partitioning system. Panel A: Quantification of phase separation was done via hemoglobin concentration in the PEG layer. Results are expressed as mean ± standard error mean (SEM). Panel B: Representative photos of phase separation of 20 kDa mPEG-RBC over 0–2 mM grafting concentrations. Reflecting increased grafted polymer, the mPEG-RBCs show increased partitioning into the upper PEG layer. Photos were taken 20 min post mixing. Data derived from Bradley and Scott [
4. Evaluating the potential clinical utility of the stealth RBC
While multiple studies have demonstrated that RBC immunocamouflage can effectively block immune recognition of multiple blood group antigens, the diversity of alloantibodies produced to a single blood group antigen by humans is staggering. Hence, a crucial step in the clinical use of the stealth RBC should be evaluating the potential efficacy of the stealth cell in the individual patient. While one might assume that standard serological testing techniques would suffice, this is not the case for a variety of reasons. Primary among these reasons, and as shown in Figure 3C, is that the laboratory serological score does not correlate significantly with the MI value or the risk of an acute transfusion reaction. Indeed, multiple studies have demonstrated that the antiglobulin test poorly reflects the amount of IgG bound and is, at best, a very weak predictor of RBC phagocytosis [60–63]. This confounding finding is actually by design. Serological testing is ‘overly’ sensitive in order to detect miniscule amounts of bound antibody to assure the appropriate typing of an individual or to detect the presence of a potentially dangerous alloantibody. Another potential complication is that a large number of commercial testing protocols employ PEG (as either a listed or unlisted ingredient) as a component of the testing reagents. The reagent PEG will cause the mPEG-RBC to segregate as ‘PEG likes PEG’ (Figure 8B). Hence, other predictors of the potential clinical utility of the stealth RBC for an individual patient are needed.
Perhaps the most definitive testing approach for the potential clinical value of the stealth RBC is the monocyte-monolayer assay (MMA). The MMA assesses FcγR-mediated adherence and phagocytosis of alloantibody-opsonized donor RBC by monocytes and has been clinically correlated with
Schematically, the MMA is described in Figure 9 (and experimentally demonstrated in Figure 3). The MMA examines FcγR-mediated phagocytosis
![](http://cdnintech.com/media/chapter/55207/1512345123/media/F9.png)
Figure 9.
Individualized testing of the potential clinical utility of the stealth RBC. Schematic representation of the monocyte-monolayer assay (MMA). (A) Untreated RBC; (B) RBC treated with control (antigen matched) serum or plasma; (C) RBC treated with alloimmunized serum/plasma; and (D) PEGylated RBC treated with alloimmunized serum/plasma. Representative photomicrographs for panels C and D are shown in
By using the MMA, the potential efficacy of RBC PEGylation can be done on an individualized basis. The probable success of the stealth RBC transfusion can be further enhanced by serologically selecting (or even better MMA testing) the best possible matches from the donor RBC inventory so as to minimize the risks of additional complications. Once the serological or MMA testing of the donor blood unit has been done, the unit can be PEGylated as described then tested against the individual’s alloantibodies via the MMA prior to transfusion into the recipient. However, the identification of the donor unit(s), PEGylation and MMA testing does require 48–96 h lead time. Hence, identification of potential patients should be done as early as possible to assure the availability to source appropriate polymer stock, prepare the PEGylation device and identify and test possible donor units. Concurrent with the evaluation of the potential clinical value of the stealth RBC, the physician/transfusion service must also receive institutional and governmental approval for their use.
5. Institutional and governmental approval for patient use
Prior to the actual clinical use of the stealth RBC in a seriously ill patient, compassionate use approval must be obtained from both the hospital Research Ethics Board (REB; or equivalent) and the appropriate governmental agencies (e.g., in Canada, Health Canada). This is likely to be a physician-driven process done in conjunction with the hospital's transfusion service and/or blood provider. Key to these requests is the need to clearly cite the lack of, or very limited availability, of suitable donor RBC. Once institutional approval has been obtained, the hospital REB would likely lead the interaction with the appropriate governmental agency (e.g., Health Canada) regarding an Investigative New Drug (IND) submission. For compassionate use in a single patient who would be likely to die in the absence of a transfusion, a formal IND submission may or may not be necessary. These steps will, obviously, change from country to country.
One question likely to be raised by the REB is whether the proposed mPEG-dosing is safe. The answer to this important question is, at least in part, addressed by recent Phase I–III clinical trials of PEGylated human haemoglobin (PEG-Hb;
6. Conclusion
Grafting of immunologically ‘inert’ polymers to the membrane of allogeneic RBC can effectively camouflage non-ABO antigens from immune recognition. These immunocamouflaged (i.e., stealth) RBCs may be an effective tool in both preventing and treating alloimmunization in the chronically transfused patient; the transfusion of individual patients with rare blood phenotypes; for emergency situation or geographic locations (e.g., China) where RhD-negative blood is unavailable. Importantly, several characteristics of the immunocamouflaged RBC may also make them highly suitable in patients/diseases characterized by RBC-mediated vaso-occlusive events (e.g., sickle cell) consequent to the polymer-mediated reduction in low-shear viscosity. For the hospital or clinic, the immunocamouflaged RBCs are inexpensively and easily manufactured using commonly available equipment and existing blood bags. Moreover, the potential clinical utility of the stealth RBC can be evaluated for the individual patient using the clinically validated monocyte-monolayer assay in which antigen-mismatched RBCs are PEGylated and then opsonized with the patient’s own alloantibody.
Acknowledgments
The authors would like to thank the past laboratory members whose work as graduate students and postdoctoral researchers have been cited in this chapter: Dr Duncheng Wang, Dr Yevgeniya Le, Dr Dana Kyluik-Price, Dr Li Li, Dr Amanda Bradley, Dr Audrey Chen and Dr Kari Murad. This study was supported by grants from the Canadian Institutes of Health Research (Grant No. 123317; MDS), Canadian Blood Services (MDS) and Health Canada (MDS). The views expressed herein do not necessarily represent the view of the federal government of Canada. We thank the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the University of British Columbia Centre for Blood Research. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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