The immunoregulatory and anti-infective properties of normal circulating polyclonal Abs have been exploited for the therapeutic purposes in the form of IVIG as well as several hyperimmune globulins. Current knowledge on the therapeutic use of normal Igs is based on the discoveries made by several pioneers of the field. In this paper, we review the evolution of IVIG over the years. More importantly, the process started as an s.c. replacement in γ globulin–deficient patients, underwent metamorphosis into i.m. Ig, was followed by IVIG, and is now back to s.c. forms. Following successful use of IVIG in immune thrombocytopenic purpura, there has been an explosion in the therapeutic applications of IVIG in diverse autoimmune and inflammatory conditions. In addition to clinically approved pathological conditions, IVIG has been used as an off-label drug in more than 100 different indications. The current worldwide consumption of IVIG is over 100 tons per year.
More than 100 y of investigation into the structure and function of Abs (or Igs) has not only served to emphasize the complex nature of these glycoproteins but also their therapeutic exploitation. Currently, five classes of Abs have been identified: IgG, IgM, IgA, IgE, and IgD. Igs have two major functions: first, as surface receptors for Ags and allowing cell signaling and activation; second, binding and neutralizing different Ags. Individual Ig can bind to limited and defined set of ligands. However, Igs as a population can bind to a nearly indefinite set of various Ags.
The discovery of Abs was made by German physician E. von Behring, considered one of the “greatest benefactors of humanity” (British Medical Journal, 1929). During the 1880s, about the time when bacteria that cause diphtheria and tetanus were isolated, E. von Behring showed that fresh serum from white rats could kill anthrax bacilli in vitro. More precisely, in the 1890s E. von Behring and S. Kitasato immunized guinea pigs and rabbits with a killed tetanus or diphtheria broth cultures, and demonstrated that sera of immunized animals contain an antitoxin factor that could neutralize activity of the toxin and may be passively transferred to nonimmune animals (1).
The clinical application of this discovery was unraveled in 1894 when P.P.E. Roux successfully treated 300 children at the Hôpital des Enfants-Malades in Paris, using antidiphtheria serum (2). Soon, antitoxin was commercially available. P. Ehrlich showed that temperature could alter the toxin–antitoxin reaction and established a standard by which the antitoxin content in the sera could be measured. This work formed the basis for all future standardizations of sera (3). These immunological studies allowed Ehrlich to formulate the side-chain theory of immunity. Ehrlich coined the name side chains for the various different receptors present in all cells. He proposed that these receptors have a unique structure and work like gatekeepers for the cell by allowing substances that match the structure of the side chains to enter the cell (4). P. Ehrlich also contributed to the identification of chemical substances that have special affinities for pathogens as antitoxins. These chemical substances that Ehrlich nicknamed “magic bullets” bind to the toxins to which they are specifically related and neutralize them. A Nobel Prize was bestowed upon P. Ehrlich in 1908 for his studies on antitoxin substances, now famously known as Abs (5).
The work of P. Ehrlich led to the emergence and widespread use of serotherapy to achieve protective immunity either through use of serum or its active components, Abs. Serotherapy constitutes the most important form of passive immunity and was made possible based on deeper biochemical understanding of the Ag–Ab complex. J.R. Marrack’s work on the Ag–Ab reaction and its interpretation was another important addition to the field. He showed that the precipitate formed by diphtheria toxin and antitoxin is mostly antitoxin, and that antitoxin is composed of serum globulin. Quantitative tests showed that increasing the amounts of Ag results in an enhancement in the proportion of Ag in the complexes. Marrack further proposed that the relationship between Ag and Ab is similar to that of molecules in a crystal (crystal-lattice model) (6). Immune complexes were linked not by true chemical valence, but rather by short-range forces acting on the molecule. This theory explained immune precipitation as the attraction of polar groups of the globulin to each other, enabling the formation of an aggregate.
The increased use of Abs as therapies led to the identification of side effects. One of the first adverse effects described was the Arthus reaction (7). Arthus repeatedly injected horse serum s.c. into rabbits. After several injections, he found that there was swelling and that serum was not absorbed as quickly. Further injections eventually led to gangrene. Today we know that an Arthus reaction involves in situ formation of Ag–Ab complexes following intradermal injection of an Ag. If an individual is previously sensitized, an Arthus reaction occurs as a type III hypersensitivity reaction. The Arthus reaction manifests as local vascular inflammation due to deposition of IgG immune complexes in the blood vessels of the dermis. Activation of complement and formation of C5a and C3a recruits leukocytes and promotes degranulation of mast cells (8, 9). Additional harmful effects of Abs became evident when Landsteiner discovered blood group systems (10, 11).
Normal Igs as therapeutic molecules: conception of i.m. Ig followed by i.v. Ig
M. Heidelberger and E.A. Kabat were the first to explain specificity and diversity of Ag-binding site (12). Studies on the electrophoretic pattern of serum proteins by A. Tiselius and E.A. Kabat showed that Abs migrate in the γ globulin fraction. Studies by R. Porter and G.M. Edelman in 1972 revealed the primary and secondary structures of Abs.
The journey of naturally occurring Igs as therapeutic molecules started in 1952 when O.C. Bruton identified a clinical condition with the absence of γ globulin (now known as btk-deficient or B cell–deficient patients) and recurrent bacterial infections. More importantly, the journey started as an s.c. replacement in a γ globulin–deficient patient, subsequently underwent metamorphosis into i.m. Ig (IMIG), followed by IVIG, and now back to s.c. Ig (SCIG).
Bruton demonstrated apparent control of infection by s.c. injection of γ globulin in a 8-y-old boy who had experienced several episodes of clinical sepsis (13). Although Bruton used a 16% SCIG preparation for his first patient, his contemporaries used an IMIG mainly to overcome presumed shortcomings of the s.c. administration route to deliver Ig at higher doses and its need for procedural expertise.
During the early 1950s, the British Medical Research Council recommended a dose of 100 mg/kg per month of IgG for immunodeficient patients (14) as opposed to 50 mg/kg per week, which was by then the standard practice. This recommendation was proposed because of the marked discomfort experienced by the patients receiving weekly dose of Igs via the i.m. route, and was based on the proof of concept that the 100 mg/kg per month dosage schedule increases serum IgG similar to the 50 mg/kg per week dose.
Further, Janeway proposed 300 mg/kg as a loading dose to effectively prevent invasive bacterial infections (15). These developments posed new challenges, as i.m. injections were extremely painful. Also, the bioavailability of IgG was erratic by the i.m. route and IgG bioavailability by the i.m. route was determined by the rate of absorption, injection site vasculature, and proteolysis. To administer the prescribed dose, IMIG had to be injected at multiple sites.
Patients receiving IMIG developed sterile abscesses, fibrosis, hematoma, and sciatic nerve injury at the injection sites. These risks were amplified in malnourished children, particularly in those with an immunodeficiency. The serum levels of IgG did not rise in the 24 h post–i.m. administration and the clinical benefits were seen in <50% of patients receiving these injections. Besides these limitations, mercury, the preservative used in the IMIG preparation, posed a risk of toxicity at higher doses. All these factors led to the exploration of an alternative route of administration, i.e., IVIG.
Initial phases of IVIG development
The basic methodologies for the preparation of human Igs for i.v. use originated from Cohn et al. (16). They developed the techniques of plasma protein separation based on low temperatures and use of low concentrations of ethanol, acidic pH, and lesser ionic strength. This approach was further refined by J.L. Oncley, who further subfractionated fraction III of Cohn’s method. This refinement led to an increase in the yield of IgG from the plasma (17). A major drawback of these preparations was the occurrence of a serious anaphylactoid reaction upon i.v. administration. These reactions were attributed to the formation of IgG aggregates during the storage and presence of certain proteases in the serum. The IgG aggregates have a propensity to activate complement cascade by mimicking the structure of an Ag–Ab complex.
To overcome IgG aggregate-induced adverse effects, various enzymatic cleavage and chemical treatment of Igs was attempted. The first attempt was treating the collected Igs with pepsin, which resulted in IVIG preparation comprising F(ab′)2 fragments. Although the resulting product retained the ability to form Ag–Ab complexes, it had a major disadvantage in the form of its short biological half-life (1–2 d), and hence was impractical for the use in patients with primary immunodeficiencies (PID). Plasmin that cleaves IgG immediately before disulphide bonds of Fc was found useful in generating the monovalent Fab fragment. But all IgG molecules were not uniformly degraded by this process, leading to a heterogenous mixture of intact and fragmented Igs (18). β-Propiolactone treatment (19), sulphonation, and alkylation helped to minimize the infusion reaction caused by complement activation. Although these modifications could partially inhibit complement activation, the process was still incomplete.
Ig preparations derived from enzymatic cleavage and chemical treatment had altered Abs that were rapidly scavenged by the reticuloendothelial system. Moreover, these Abs had a reduced opsonizing capacity that made bacterial clearance ineffective.
Evolution of current IVIG preparations
During the early 1980s, IVIG became available as a commercial preparation. Collected from the large pools of human plasma, IgG was concentrated by cold ethanol fractionation (16). Although the process had a purified end product, few clinically significant impurities like prekallikrein, activated coagulation factors, complement proteins, and IgA and IgM were present in minute quantities (20).
The first-generation IVIG preparations were 5% lyophilized products that needed reconstitution with distilled water or normal saline. With advancing technology, 10% liquid formulations with various stabilizing agents were developed for i.v. administration (21).
Common stabilizers used in the earlier generations of IVIG were sugar derivatives like sucrose, glucose, maltose, d-sorbitol, and manitol. Use of sugars as stabilizers posed three major problems. The most dreaded complication was onset of renal dysfunction and acute renal failure. Although sucrose-containing IVIG had the highest propensity to cause acute renal failure (70–90% of patients) (22), other sugars also posed a significant risk (23). Additional drawbacks were the very high osmolality of products and fluctuations in blood sugar levels in diabetes mellitus patients (21).
Stabilizers currently used in IVIG are nonessential amino acids like glycine and l- proline. As compared with sucrose, amino acid stabilizers were less effective in preventing IgG dimerization. This problem was solved by reducing the pH of IVIG to 4.1–6. This acidic product could be stored in liquid form for a prolonged duration. The product was demonstrated to be safe despite the nonphysiological pH, and did not cause any alteration in acid base homeostasis in the patients upon i.v. infusion.
Amino acid–stabilized IVIG are not directly nephrotoxic, but have the potential to induce autoimmune hemolysis. These preparations are safe in patients with diabetes. Proline is contraindicated in patients with hyperprolinemia as it can cause oxidative stress–mediated neurotoxicity (24). Given their safety profile and reasonable efficacy, amino acids are the stabilizers of choice in the modern day IVIG preparations.
Caprylic acid is a short-chain (C-8) saturated fatty acid. The idea of using caprylic acid to purify IVIG was conceived based on work showing that α and β globulins but not γ globulins form insoluble precipitates upon treatment with a short-chain fatty acid (C6–C12) (25). The addition of caprylate under vigorous stirring results in a product enriched in IgG. The purity of the IgG depends on the pH (4.5 is desirable), the quantity of caprylic acid, and the molarity of the buffer used. The procedure was further refined by Lebing et al. (26) by combining two caprylate precipitation steps with two anionic exchange processes.
Variations in the processing of IVIG products and the geographical location of plasma donors might influence the efficacy of IVIG in PID (27).
Viral inactivation during IVIG preparation.
IVIG fractionated out of the cold ethanol fractionation was presumed to be safe. This perception was challenged for the first time in 1983, when an experimental IVIG transmitted non-A, non-B hepatitis (later known as hepatitis C) (28). Subsequently, a few reports of HIV and non-A non-B hepatitis transmission by some IVIG products (29) led to reconsideration and regulation of viral safety norms in IVIG manufacturing (30). Currently, it is mandatory to use a minimum of two complementary methods of viral inactivation, of which at least one step must target the elimination of nonenveloped viruses.
At present, all blood products including IVIG are rendered virus free by following three principles (31–33): care when selecting donors, optimum screening of collected products for known infective agents, and use of virus inactivation methods like fractionation, and physical and chemical treatment including caprylation and nanofiltration.
A U turn: back to SCIG for PID
Following the initial enthusiasm, SCIG was gradually phased out of clinical practice by IMIG. In the 1980s, with the advent of commercial IVIG, usage of SCIG further declined. However, during the early 1990s, infusion pumps allowed rapid administration of Igs and hence the use of SCIG was reviewed (34).
SCIG has various advantages over IVIG. Administration of SCIG does not require venous access and is not associated with systemic side effects. Therefore, premedication with corticosteroids and antihistamines is not essential. Moreover, the convenience of SCIG injection by self-administration has resulted in a better quality of life for PID patients and less absence from work.
The volume of Ig that can be administered by s.c. route is a challenge. To overcome the drawbacks of multiple site injections and the limitations of volume per infusion, a 20% IgG product containing l-proline as a stabilizing agent (Hizentra) was introduced in 2010 (35). In 2016, based on a phase II/III study in PID (36), the glycine-stabilized 20% IgG product Cuvitru was approved by the Food and Drug Administration. These products are only for s.c. use and have the advantage of delivering a higher dose of Ig in a lower volume. The stabilizing agents prevent aggregation of IgG molecules during storage, which translates to fewer systemic side effects (37).
The anatomical structure of the site of s.c. administration is the major limiting factor for large-volume administration of Igs. The space is occupied by s.c. fat and the collagen matrix constituted mainly by the high m.w. copolymer of glucuronic acid and N-acetyl glucosamine-hyaluronan. Modification of this space using enzymes like recombinant human hyaluronidase (rHuPH20) facilitated larger infusion volumes per site. Externally administered hyaluronidase has short half-life and hence s.c. concentration of hyaluronic acid is restored within 48 h by local fibroblasts.
IVIG as an immunotherapeutic molecule for autoimmune and inflammatory diseases
P. Imbach observed that periodic substitutive IVIG treatment led to a slight platelet increase in a boy with hypogammaglobulinemia-associated infections and thrombocytopenia due to Wiskott–Aldrich syndrome. A subsequent pilot study in 12 consecutive immune thrombocytopenic purpura (ITP) children without hypogammaglobulinemia also showed consistent improvement in platelet counts (40).
Following successful use of IVIG in ITP, the therapeutic applications of IVIG were expanded to other autoimmune and inflammatory conditions. Randomized trials have established the therapeutic benefits of high-dose IVIG (1–2 g/kg) in the treatment of several autoimmune diseases (41–45). Newer indications are continuously explored, and IVIG is currently used in over 100 different diseases in an off-label manner (46) including epilepsy, autism, acute brain injury, pyoderma gangrenousum, and others. The current worldwide consumption of IVIG for autoimmune diseases and PID is over 100 tons per year (47).
Data from four randomized clinical trials involving 34 patients formed the basis for approval of IVIG therapy in multifocal motor neuropathy (48). IVIG significantly improved muscle strength in patients compared with placebo. In myasthenia gravis, three different clinical trials involving a total 303 patients demonstrated therapeutic efficacy of IVIG (2 g/kg) (49, 50). Currently, the IVIG indication in myasthenia gravis is limited to either exacerbated or worsened clinical conditions.
Seven randomized clinical trials enrolling 287 patients provided evidence for the clinical efficacy of IVIG (2 g/kg) as a first-line therapy in chronic inflammatory demyelinating polyneuropathy (51, 52). The efficacy of IVIG, however, was transient and hence multiple infusions of IVIG were proposed. In case of Guillain–Barré syndrome, at least three randomized clinical trials comprising up to 536 patients established the utility of IVIG therapy (400 mg/kg, 5 d) in improving motor functions (53). IVIG therapy, however, should be initiated within 2 wk of the onset of clinical symptoms.
A meta-analysis of 13 trials involving 646 patients validated IVIG therapy in ITP at 1 g/kg for two consecutive days. IVIG therapy enhanced the platelet count in the patients and showed nearly 80% efficacy (54, 55). Similarly, several studies comprising over 1000 children established high-dose IVIG therapy as a first-line therapy in Kawasaki disease, and to reduce coronary artery abnormalities.
In addition to the above-mentioned diseases, randomized trials have demonstrated the usefulness of IVIG in the prevention of kidney graft rejection and in autoimmune mucocutaneous blistering diseases such as bullous pemphigoid and pemphigus vulgaris or pemphigus foliaceus (56–59). In dermatomyositis, the landmark clinical trial of 1993 was subsequently confirmed by many open studies (60).
In other autoimmune and inflammatory diseases, IVIG efficacy has been reported in open-label studies and randomized clinical trials are lacking. Some of these pathologies include stiff-person syndrome, toxic epidermal necrolysis, Stevens–Johnson syndrome, autoimmune uveitis, birdshot chorioretinopathy, antineutrophil cytoplasmic Ab vasculitis, autoimmune hemolytic anemia, systemic lupus erythematosus, catastrophic antiphospholipid syndrome, streptococcal or staphylococcal sepsis and toxic shock syndrome, systemic juvenile idiopathic arthritis, autoimmune congenital heart block, B19 parvovirus erythroblastopenia, acquired von Willebrand syndrome, and others (41).
The successful immunotherapeutic use of IVIG in autoimmune diseases also led to the exploration of cellular and molecular mechanisms of action. Autoimmune and inflammatory diseases are characterized by aberrant activation of the cells of innate and adaptive immune compartments and release of inflammatory mediators. The current data suggest that IVIG targets each and every arm of the immune system, culminating in inhibition of inflammatory cells while reciprocally enhancing immune regulators.
Based on the data from ITP, it was believed initially that IVIG exerts a beneficial effect in autoimmune patients via the saturation of Fc receptors on phagocytes (61). The remission in autoantibody-mediated conditions led to the proposition of neutralization of pathogenic autoantibodies by anti-idiotypic Abs against idiotypes expressed by autoantibodies and by inhibiting autoantibody production by binding to autoreactive B lymphocytes (62). Since then, several mechanisms of IVIG have been reported (Fig. 1), including:
Binding of IVIG to C3b and C4b fragments of complement, thereby inhibiting their tissue deposition as well as generation of the C5 convertase, and hampering the subsequent formation of membrane attack complex (63).
Ability of F(ab′)2 and Fc portions of IgG to inhibit lymphocyte proliferative responses and modulation of inflammatory cytokines (42).
Effect on enhancement of glucocorticoid receptor-binding affinity, subsequent enhanced glucocorticoid sensitivity, and synergistic suppression of lymphocyte activation when combined with glucocorticoids (72).
Adverse effects of IVIG therapy
Overall, IVIG has a very good safety profile and mild to moderate adverse reactions to high-dose IVIG therapy might be seen in nearly 30% of patients (41). Some of the common adverse reactions include fever, headache, chills, nausea, hypotension, and muscle cramps. Temporary cessation of the IVIG infusion, slow infusion, and use of general anti-inflammatory agents can control these events. Switching to SCIG is another viable alternative. Anaphylaxis can occur in IgA-deficient patients and use of low IgA-containing IVIG preparations is recommended in such cases.
Other effects include meningeal inflammation and aseptic meningitis due to the release of inflammatory cytokines and neutrophil activation by IgG Abs within IVIG that mimic antineutrophil cytoplasmic Abs; intravascular hemolysis due to the presence of anti-A or anti-B isoagglutinins or less commonly anti-D or anti-K Abs; thromboembolism because of contamination of IVIG with clotting factors and formation of platelet-leukocyte aggregates; and renal complications due to osmotic injury that could be prevented by using non–sugar-stabilized IVIG.
Possible contamination of IVIG with infectious agents like viruses and prions always exists. However, viral inactivation steps, caprylation, and nanofiltration aid in the safety profile of IVIG.
It was a long journey of more than 100 y from the discovery of Igs to the widespread use of polyclonal normal IgG in several human disorders. The benefits of IVIG therapy in PIDs also encouraged its potential application in various infectious diseases, particularly in patients with diminished immunity (93, 94). Although clinical trials have confirmed therapeutic utility of IVIG in many autoimmune diseases, such trials are lacking for the majority of them. As nearly 60–70% of current IVIG supply is used for off-label purposes, prioritization of IVIG for PID and established clinical indication are important.
The success of IVIG in clinics also inspired the exploration of the anti-inflammatory properties of other subclasses of normal Igs, particularly IgA and IgM. Preclinical evaluation of pooled IgA and IgM (analogous to IVIG) provided evidence that they also target inflammation and hence merit further clinical evaluation (95–97). Similarly, identification of sialic acid linkages of Fc fragment as one of the mediators of anti-inflammatory action of IVIG led to conception of sialic acid linkage–containing recombinant Fc fragments (98) and derivation of IVIG with controlled tetra-Fc sialylation (99). Although sialic acid–dependent mechanisms were not reproduced by other laboratories (100), those reports provide good evidence that fundamental immunology research could lead to newer immunotherapeutic tools (101).
We thank all colleagues and collaborators who contributed to the IVIG field.
This work was supported by INSERM and research grants from CSL Behring (Switzerland and France) and Laboratoire Français du Fractionnement et des Biotechnologies, France.
J.B. and S.V.K. are supported by research grants from CSL Behring (Switzerland and France), and Laboratoire Français du Fractionnement et des Biotechnologies, France. The other authors have no financial conflicts of interest.