Imlifidase: a new interferent on serum protein electrophoresis looking as a rare plasma cell dyscrasia

To the Editor,

Serum protein electrophoresis (SPE) represents the most employed screening test in detecting and monitoring monoclonal components (MC). Any observable abnormality suggestive of monoclonal gammopathy requires capillary zone immunosubtraction electrophoresis (ISE) or agarose gel electrophoresis immunofixation (IFE) to confirm and to identify the immunoglobulin (Ig) heavy and light chains.

To date, several substances are known to interfere with the accuracy and reliability of SPE results. Some of them are endogenous, therefore intrinsic to the sample, such as hyperbilirubinemia, hyperlipidemia, fibrinogen, or free hemoglobin in hemolyzed samples. Some others are exogenous, such as contrast agents, plasma expanders gelatin-based, antibiotics, antimycotics, or, as is increasingly the case, therapeutic monoclonal antibodies that may alter SPE, simulating the presence of true monoclonal proteins [1].

This document reports the discovery of an interference on SPE, due to a new pharmaceutical agent called imlifidase. This drug works by causing an alteration of the immune system that simulates the presence of a rare hematological disease called gamma-heavy chain disease (γ-HCD) on SPE, as well as on ISE and IFE.

The discovery of the new interference started from a routine analysis of a 49-year-old man admitted to the Kidney and Pancreas Transplantation Unit of Padua University-Hospital whose SPE, performed on capillary electrophoresis (Capillarys 3 Tera, Sebia, France) evidenced a qualitative and quantitative abnormality in the beta-2 globulin fraction and hypogammaglobulinemia (Figure 1A). Both findings suggested the presence of MC in the beta-2 zone.

Figure 1:

Details of the beta and the gamma-zones from serum protein electrophoresis (SPE), immunosubtraction test (ISE), and immunofixation test (IFE) on days +1, +7 and +14 after imlifidase infusion. The “monoclonal protein” in the beta-2 zone was present in ISE and IFE on days +1 and + 7: a lower intensity after one week was due to the exhaustion of the drug’s action and to the renal clearance of Fab and Fc fragments produced. On Day +14, the “monoclonal protein” completely disappeared.

In the ISE (Immunotyping test Capillary 3 Tera, Sebia) the abnormal area disappeared only on the anti-gamma heavy chains pattern (Figure 1B). No immunosubtraction was observed neither in the anti-kappa nor in the anti-lambda light chains patterns, as well as in the gamma-globulin zone incubated with anti-gamma chain (Figure 1B).

The IFE on agarose gel (Hydrasys, Sebia) revealed the presence of a narrow, monoclonal-looking band in the beta-2 globulin zone immunofixed only by anti-gamma heavy chains antiserum, not accompanied by a similar band neither in the anti-kappa nor in the anti-lambda region. Moreover, there was a total absence of IgG in the gamma-globulin area (Figure 1C).

SPE and IFE of the patient infused with imlifidase were very similar to those of patients with heavy chain disease, specifically γ-HCD [2]: little differences in IFE are related to the width of monoclonal band, larger in γ-HCD subjects, and to the presence of some complete IgG in the gamma–zone, not detectable in the patient studied (Figure 1C).

HCDs are rare diseases characterized by the proliferation of a single B-lymphocyte clone that produces a monoclonal immunoglobulin composed of truncated heavy chains without corresponding light chains. Based on the heavy chain isotype, they can be divided into three forms: α-HCD, the most common, γ-HCD, and µ-HCD [3].

γ-HCD can occur in people affected by mature-B lymphoid neoplasms, usually with plasmacytoid differentiation (i.e. chronic lymphocytic leukemia, non-Hodgkin lymphoma, plasma-cell disorders), or in people with history of autoimmune disorder (i.e. autoimmune hemolytic anemia, immune thrombocytopenia, rheumatoid arthritis, Sjogren syndrome, systemic lupus erythematosus) [3].

It can affect mainly adults, with a slight women predominance and a median age of 65 years [4]. Clinical presentation can be heterogeneous: the main signs and symptoms are lymphadenopathy (34 %), hepatomegaly (4 %), splenomegaly (30 %), bone marrow involvement (30 %). B symptoms are common, but patients can also be asymptomatic [2].

In γ-HCD, truncated heavy chains usually migrate in the beta region of capillary zone electrophoresis (60–86 % of cases) and ISE shows a subtraction only by the anti-gamma heavy chains antiserum. However, IFE is the only definitive procedure to demonstrate the existence of the heavy-chain MC [3], 4].

In this case, the presence of an interference was hypothesized because the clinical characteristics of γ-HCD did not seem compatible with the patient’s history. Indeed, a further consultation with the clinicians revealed that the patient underwent a second kidney transplant and, for this reason, received an infusion of imlifidase the day before the blood sampling.

Imlifidase is a new immunosuppressant drug used in transfusion medicine and organ transplantation, particularly to treat highly sensitized patients with high levels of anti-HLA (human leukocyte antigen) antibodies [5].

Its active component is IdeS (IgG-degrading enzyme of S. pyogenes), a pepsin-like cysteine protease of 35kDA discovered by Von Pawel in 2002 and produced by S. pyogenes to evade the adaptive immune response during Streptococcal infection [6]. IdeS works by cleaving the heavy chains of all subclasses of IgG antibodies generating one F(ab’)₂ and one Fc fragment [5], 6].

In particular, it cleaves the disulfide bridge in the hinge region between Gly-236 and Gly-237 of IgG with a high degree of specificity. This region of the Fc fragment represents the binding site for the Fcγ receptor in the Fc–complex [7]. For this reason, the Fc-dependent effector functions, which are responsible for immune reactions, particularly antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), are eliminated [5].

Imlifidase was approved by EMA (European Medicines Agency) in 2020 and by AIFA (Italian Medicines Agency) in 2022 for the desensitization treatment of hyperimmune adult patients undergoing kidney transplantation with positive crossmatch against an available deceased donor, where it cleavages the anti-HLA antibodies developed against the donor (donor-specific antibodies-DSAs) [5]. Recently, the use in lung transplantation has also been liberalized [8]. Moreover, due to the high substrate specificity, exclusively towards IgG, well correlated with the lack of toxicity of the enzyme, it is considered a therapeutic potential for the treatment of several autoimmune conditions characterized by the presence of pathogenic IgG (i.e. Guillain–Barré syndrome, Anti-glomerular basement membrane disease) and to overcome the presence of neutralizing antibodies in adenoviral vector gene therapy [5], 9].

Imlifidase works quickly since it cleaves IgG within a few hours after infusion. At the same time, its action is transient since the drug, as well as Fc and Fab fragments, has renal clearance within a few days and does not inhibit the synthesis of new antibodies. Approximately two weeks after drug administration, endogenous IgG concentration normalizes and IgG becomes visible even in SPE, as was highlighted by Milhes et al. in 2023 [5], 10].

This rapid action is particularly beneficial in transplant cases, as it reduces the risk of organ rejection by eliminating potentially harmful antibodies [5], 10].

This kinetics of action is well reflected in the clinical case described. SPE performed one week after the infusion did not highlights qualitative and quantitative abnormality in the beta-2 globulin zone (Figure 1D). However, ISE (Figure 1E) and mostly IFE (Figure 1F) still revealed the presence of the “monoclonal component”, reduced in intensity due to exhaustion of the drug’s action and renal clearance of the fragments produced. On the contrary, SPE performed 2 weeks later showed a polyclonal hypergammaglobulinemia, also due to double infusion of IgG (IGIV) (Figure 1G), and the disappearance of the abnormality in the beta-2 globulin zone on ISE (Figure 1H) and IFE test (Figure 1I).

These qualitative findings were confirmed by quantitative measurements of IgG (Optilite, The Binding Site, Great Britain) (Figure 2).

Figure 2:

Trend of IgG concentrations of patient’s serum samples in relation to drug treatment. IgG concentrations began to decrease as early as 3 h after imlifidase infusion and remained low for up to the following 8 days. Subsequently, IgG concentrations started to increase due to both neosynthesis and the double infusion of IgG (IVIG).

In conclusion, imlifidase is a new exogenous substance that interferes with SPE, ISE and IFE in a new way. It is not interfering in itself, but causing, and this is the target effect, the generation of truncated IgG immunoglobulins. Therefore, both γ-HCD and the interference caused by imlifidase are characterized by the presence of γ-chain fragments, however, in the first case, they result from overproduction by the clonal population, while in the second one they are generated by the drug through its mechanism of action on the designed target.

Understanding how different substances can interfere with electrophoresis SPE is vital for accurate diagnosis and monitoring.

Collaboration between laboratory specialists and clinicians is essential to achieve the appropriateness of the request and to guarantee the accurate interpretation of results avoiding misdiagnoses.