Monoclonal antibody therapy: What compatibility testing labs need to know

Utilizing immunotherapy to treat hematologic malignancies and solid tumors is becoming more commonplace. This article reviews the effects of monoclonal antibody (mAb) drugs on red blood cells and pretransfusion testing, as well as available methods used to minimize adverse effects. A widely used term for cell markers in immunophenotyping is Cluster of Differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD). CDs are cell surface molecules expressed on leukocytes and other cells pertinent to the immune system. The purpose of this nomenclature is to provide standardization as each antigen is assigned a unique CD number that correlates to the antibody which bind to its cognate antigen. More than 400 molecules have already been described that are being used as targets for cell immunophenotyping.¹ Particularly, a number of CD molecules expressed on tumor cells are being studied in order to develop more precise, targeted treatment for cancers. At the time of this publication, the two most frequently encountered therapeutic monoclonal antibodies seen in blood banks are anti-CD38 and anti-CD47.

In 2015, Darzalex (Daratumumab or “Dara”) became the first human CD38 mAb approved by the FDA for the treatment of relapsed or refractory multiple myeloma in patients who have received at least three prior treatments.⁵ Other anti-CD38 agents, such as MOR202, are in trials. In June 2019, the FDA approved a combination of Darzalex-Revlimid-dexamethasone to treat newly diagnosed multiple myeloma (MM) patients with who failed to qualify for autologous stem cell transplant.² Isatuximab (SAR-650984) is another CD38 IgG1 mAb in clinical trials for the treatment of patients with MM. It received orphan designation for relapsed/refractory multiple myeloma by the FDA in the second quarter of 2019.³ 

CD47 mAbs are currently undergoing clinical trials for multiple solid and hematologic malignancies, (e.g., Hu5F9-G4, CC-90002, SFR231) and in 2018 the FDA granted SFR231 orphan drug designation for MM.⁴ Refer to Figure 1 of a bone marrow aspirate with plasma cells in MM.  

CD38 and CD47 overview

CD38 is a type II transmembrane glycoprotein and found in low levels on the surface of many types of cells including B and T lymphocytes, natural killer (NK) cells, plasma cells, platelets, and red blood cells (RBCs). It functions as a receptor and adhesion molecule, as well as a manufacturer and hydrolyzer of an intracellular calcium ion-activating messenger. CD38 is highly expressed on plasma cells in MM; this makes it a desirable target for MM therapy. The binding of CD38 mAb to the CD38 receptor inhibits the growth and proliferation of MM cells and induces tumor cell death or apoptosis through a variety of immunological mechanisms. This includes Fc-dependent immune-effector mechanisms, direct apoptotic activity, and immunomodulatory effects by the elimination of CD38-expressing regulatory T, regulatory B, and myeloid-derived suppressor cells.^6,7 (Figure 2) 

 Expressed on virtually all tissue and cell types including RBCs and platelets, CD47 has many functions including cell migration, cell adhesion, and T cell function. CD47  is an integrin-associated transmembrane protein (IAP) that has high affinity for thrombospondin (TSP) and signal-regulatory protein alpha (SIRPα) on the macrophage membrane that is directly involved in the prevention of phagocytosis by macrophages.⁸ This “don’t eat me” signal protects transfused RBCs and platelets from rapid elimination by macrophages in the spleen. CD47 is also a transmembrane protein and part of the Rh complex. The expression of CD47 is affected by Rh phenotype; rr (dce/dce) having the highest expressions, compared to D-positive, and Rhnull having the least. Studies demonstrate that CD47 expression decreases as RBCs age, so it additionally functions as a “eat me” signal and considered a marker of senescence.^9,10 

A cell surface protein called calreticulin (CRT) appears to be expressed on malignant cells, but not on most normal cells. As cancer cells are dying, (e.g., during chemotherapy treatment) CRT is upregulated and emits “danger” signals (part of the warning system known as damage-associated molecular patterns or DAMPs). CRT interacts with pattern recognition receptors (PRRs) expressed on myeloid and lymphoid cells, such as low-density lipoprotein receptor-related protein 1 (LRP-1), to promote phagocytosis. Also highly expressed on cancer cells and in opposition, the CD47-SIRPα pathway impedes macrophage-mediated destruction. As a therapeutic human monoclonal IgG4 antibody, anti‐CD47 (e.g., Hu5F9‐G4) blocks interaction of CD47-SIRPα, allowing the pro-phagocytic calreticulin/LRP-1 collaboration to facilitate the “eat me” signal. As a consequence, treating patients with anti-CD47 paves the way for clearing malignant cells. Refer to Figure 3 for proposed mechanism of action of CD47‐ targeted anti‐tumor

therapy.^11-13

Although new monoclonal antibodies show promise for improved outcomes, patients receiving these treatments for MM and other hematological diseases present many challenges for both technologists performing pretransfusion compatibility testing and physicians that interpret the clinical significance of the results.

Interference in blood bank testing when samples contain anti-CD38

After treatment with anti-CD38 commences, immediate spin reactions are unaffected, but residual immunotherapeutic antibody in the patient’s plasma cross-reacts with RBCs. This causes unexpected, generally weak, panreactive serologic reactions when utilizing the indirect antiglobulin test (IAT) (e.g., antibody screens, antibody identification panels, phenotype tests, and crossmatches). Interference is seen with all media (e.g., saline, LISS [low-ionic-strength solution], and PeG [polyethelene glycol]), by all common test methods (tube, gel, and solid phase), and can persist for six months following treatment.^14,15 The autocontrol (AC) and direct antiglobulin test (DAT) may be positive or negative (most often), and results frequently resemble an antibody to a high incidence antigen or a warm autoantibody. Adsorptions using untreated or chemically treated cells are not effective.

Mitigation of anti-CD38 interference

The goal of any blood bank technologist when faced with panreactivity due to mAb treatment is to ensure clinically significant alloantibodies are not underlying the drug antibody. The most practical and inexpensive method employed to eliminate the interference of anti-CD38 is pretreating reagent red cells with dithiothreitol (DTT). DTT is a reducing agent that disrupts disulfide bonds formed between cysteine residues. The CD38 molecule (also on RBCs) has five disulfide bonds, so when a patient’s plasma is tested with reagent red cells treated with DTT, reactivity from anti-CD38 is abolished. It is important to keep in mind that Kell, Knops, Dombrock, Lutheran, Yt (formerly Cartwright), and JMH blood group system antigens, as well as the LWa antigen, are destroyed by DTT, which cannot be excluded during antibody identification.

Generally used by large reference laboratories, the patient’s plasma may be tested with a panel of cord RBCs that have been extensively phenotyped.¹⁶ Cord RBCs have very little CD38, if expressed at all. Recombinant soluble human CD38 or anti-daratumumab idiotype can be utilized to eliminate interference, but is not widely available at this time.¹⁴ 

For patients with a negative antibody screen using DTT-treated RBCs, an electronic or immediate-spin crossmatch with ABO/RhD compatible units may be performed.¹⁵ Units negative for the K (KEL1) antigen should be issued if the patient is negative for this antigen, or the antigen typing is unknown. For patients with known alloantibodies, antigen negative RBCs must be provided. This can be accomplished by providing phenotype or genotype matched units. Indirect antiglobulin crossmatches will be incompatible and local policies regarding ordering physician signature must be followed before transfusing blood products. Refer to Figure 4 for summary of anti-CD38 interference and mitigation strategies.⁷

Interference in blood bank testing when samples contain anti-CD47

Once treatment with anti-CD47 begins, false positive reactions can be seen in all phases of testing (immediate spin, 37°C, and IAT), with all media (e.g., saline, LISS, and PeG), and with all methods of IAT testing (i.e., tube, gel, solid phase). ABO discrepancies occur due to false positive results in the reverse typing (all types except for O), and false positive results in the forward typing due to spontaneous agglutination of patient RBCs. Because of the high level of CD47 on RBCs, these reactions may mimic those seen with IgM antibodies. Cord RBCs are also reactive in all phases. DATs may be falsely negative due to a “blocking effect” caused by high levels of antibody present, but eluates are strongly positive. False negative phenotyping test results can occur due to RBCs heavily coated by anti-CD47.

Mitigation of anti-CD47 interference

The most practical and cost- effective method currently available to eliminate the interference of anti-CD47 is to utilize an anti-IgG reagent that does not bind to IgG4, such as anti-IgG (Murine Monoclonal) Gamma-clone (Hu5F9-G4 and CC-90002 are monoclonal IgG4 anti-CD47). Occasionally, even using this method, it may still demonstrate a weak reactivity because of carryover to the IAT phase of testing. Allogenic adsorption or adsorption using pooled platelets or papain treated RBCs generally require four adsorptions.¹¹ 

What compatibility testing labs and clinicians need to know

Without a medication history when samples are submitted for work-up, regardless of the presentation, a blood bank technologist may be confounded and unknowingly waste precious time and resources chasing “ghosts.” With this said, it is very important that clinicians and other healthcare providers understand that pretransfusion testing, including an extended phenotype should be performed before initiating treatment (either serology or predictive genotype for D, C, c, E, e, K, Jka, Jkb, Fya, Fyb, S, and s antigens). In addition, a complete, accurate history (e.g., diagnosis, medications, transfusions, transplants, and pregnancies) must be provided any time pretransfusion testing and/or antibody identification is required. This information can be instrumental in helping the blood bank technologist provide the safest donor red blood cells for transfusion. As with any other emergent acute, life-threatening circumstance, group O-negative or O-positive RBCs can be transfused with physician approval, following  institutional protocols.  

Similar to interfering in RBC antibody detection, plasma containing anti-CD38 or anti-CD47 may also affect detection of antibodies to HLA Class I and platelet antigens, as well as interfere with platelet crossmatch testing. CD47 is weakly expressed or absent from the rare Rhnull cells.^8,17 Clinically significant alloantibodies may be masked by the presence of monoclonal antibodies. More extensive testing beyond standard methods can lead to delays in providing RBCs for transfusion. Hospitals must communicate to the compatibility testing laboratory when patients are expected to begin monoclonal antibody therapies.

In conclusion

Rapid development and use of monoclonal antibodies continue to be investigated for clinical applications, such as acute lymphoblastic leukemia (ALL), lymphomas, and acute myeloid leukemia (AML), as well as in solid tumors. Unfortunately, interference of such immunotherapies on pretransfusion testing results is often missed or insufficiently investigated. The impact on blood bank testing is only recognized when patients require a blood transfusion, yet clinically significant alloantibodies fail to be eliminated using routine test methods. Blood bank technologists and other healthcare providers must stay vigilant to remain up-to-date on published literature, workshops, and other educational venues for best practices to mitigate mAb interference during such testing.

REFERENCES

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