AntiCD 47 Immunotherapy Jennifer Stokes MS MTASCP Objectives
Anti-CD 47 Immunotherapy Jennifer Stokes MS, MT(ASCP)
Objectives • Describe the structure and function of the CD 47 molecule. • Summarize how anti-CD 47 immunotherapy functions. • Discuss the interference anti-CD 47 immunotherapy causes with routine immunohematology testing. 1
What is CD 47? • Cluster of Differentiation 47 (CD 47) was first known as Integrin-associated protein (IAP) – First identified in the 1980 s – 50 k. Da in size – 4 known isoforms From: Brown, E. J. , & Frazier, W. A. (2001). Integrin-associated protein (CD 47) and its ligands. Trends in Cell Biology, 11(3), 130– 135. 2
CD 47 Function • CD 47 is found on the surface of many cells in the body. • CD 47 has been described as a “don’t eat me” signal. • As cells age, the amount of CD 47 decreases until there is no longer enough CD 47 left on the cell surface to prevent phagocytosis • Researchers have discovered that almost every type of cancer cell expresses large amounts of CD 47 on their surface. This protects the cancer cells from destruction by the body’s immune system. 3
• CD 47 interacts with several ligands. – SIRPα (signal regulatory protein α) • CD 47 -SIRPα interaction produce the “do not eat me” signal – Integrin ανβ 3 (RGD receptor) – Integrin αIIbβ 3 (platelet fibrinogen receptor) – Integrin α 2β 1 (collagen receptor) – Thrombospondins (TSP-1 to TSP-5) – Cell Adhesion Peptibe (4 N 1 K) 4
CD 47 on Red Blood Cells • CD 47 is part of the Rh complex on the red cell surface. • Unlike CD 38 which is weakly expressed on red cells, CD 47 is highly expressed. • Expression levels of CD 47 differ with Rh phenotype and correlate with Rh. CE protein levels. 5 From: Velliquette, R. W. , Aeschlimann, J. , Kirkegaard, J. , Shakarian, G. , Lomas-Francis, C. , & Westhoff, C. (2019). Monoclonal anti-CD 47 interference in red cell and platelet testing. Transfusion, 59(2), 730– 737.
CD 47 on Platelets • CD 47 acts to regulate normal platelet turnover and FcγR-mediated clearance of Ig. G-sensitized platelets. • CD 47 was found to mediate a synergistic effect of soluble type I collagen and TSP-1 or 4 N 1 K, which enhance α 2β 1 integrin-mediated platelet activation or vascular smooth muscle cell chemotaxis. 6
Anti-CD 47 • Development of this immunotherapy is based on the fact that CD 47 regulates cell survival and death through its interaction with SIRPα on macrophages. • Several studies have shown that cancer cells can sustain or even increase CD 47 expression to escape clearance by the immune system. • Trials are underway using both anti-CD 47 monoclonal antibodies and CD 47 blocking fusion proteins to treat both hematologic malignancies and solid tumors. 7
How does Anti-CD 47 work? From: Velliquette, R. W. , Aeschlimann, J. , Kirkegaard, J. , Shakarian, G. , Lomas-Francis, C. , & Westhoff, C. (2019). Monoclonal anti-CD 47 interference in red cell and platelet testing. Transfusion, 59(2), 730– 737. 8
Anti-CD 47 Interference in Serologic Testing • Samples were received in a reference lab after patients were receiving Anti-CD 47 (Hu 5 F 9) immunotherapy. • Interference was detected in: – ABO typing – DAT – Antigen Typing – Antibody detection – Platelet Antibody Testing 9
Other Methods Used – Pretreatment • • • Papain Trypsin α-chymotrypsin 0. 2 M dithiothreitol (DTT) W. A. R. M. (warm autoantibody removal medium, Immucor) Ethylenediaminetetraacetate glycine acid (EGA) – Removes Ig. G coating the red cells • Gamma ELU-KIT II – Ficin-treated panels – Cord Red Cells – Rare phenotype Red Cells – Titration studies – Adsorption 10 None o ft metho he pretreatm ds ent the CD were able to 47 cl surface from the red eave cell
Plasma Studies • Patients were tested prior to administration of anti-CD 47 and found to be negative for atypical antibodies. • ABO typing • Antibody Detection • Titer 11
Red Cell Studies • DAT • Elution • EGA Treatment • Warm washing 12
Adsorptions • Cells used – Red cells (all treated with papain) • R 1 R 1 • R 2 R 2 • rr – Outdated apheresis platelets – Human platelet concentrate (HPC, Immucor) – PEG adsorption 13
Platelet Antibody Testing • Samples were tested using 2 Capture-P methods and Pak. Plus. • Both Capture-P methods were positive and Pak. Plus was negative in both samples tested. • The Capture-P methods use intact platelets or platelet membranes during testing which expresses CD 47. • Pak. Plus uses monoclonal captured or affinity purified glycoprotein molecules. 14
Anti-CD 47 vs Anti-CD 38 15 CD 47 Low level of RBC expression High level of RBC expression Epitope/antigen may shed No shedding of epitope/antigen Ig. G 1 subclass Ig. G 4 subclass No ABO interference present No D or extended typing interference Possible interference with D and extended typing Mitigated with DTT treated RBCs May be mitigated by use of Immucor Gamma monoclonal anti-Ig. G for IAT Can not be alloadsorbed May be alloadsorbed with RBCs or PLTs DAT negative or weak + due to antigen loss DAT negative or weak + due to blocking Eluate negative or weak + Eluate strongly positive Capture-P shows variable reactivity Capture-P positive Pak. Plus negative
• The use of monoclonal antibody therapy to treat various diseases is on the rise. • Interference with pre-transfusion testing may become more common. • Interference may be drug specific based on its binding profile. • CD 47 Immunotherapy causes some degree of anemia and thrombocytopenia in recipients, increasing the likelihood of transfusion. 16
Recommendations • Pre-therapy communication with the blood bank and baseline ABO and antibody screen. • Extended phenotype/genotype performed prior to initiation of therapy. • Consider antigen matched red cells for transfusion. • Have manufacturers develop neutralizing agents to remove the excess anti-CD 47 from the sample. 17
Clinical Trials • Hu 5 F 9 -G 4 (Forty Seven) • CC 9002 (Celgene) • SRF 231 (Surface Oncology) • ALX 148 (ALX Oncology) • TT 1 -621 (Trillium) 18
References • CD 47. (2019). Retrieved March 25, 2019, from https: //med. stanford. edu/stemcell/CD 47. html • Branch, D. R. (2019). Immunotherapy: the good, the bad, the ugly, and the really ugly. Transfusion, 59(2), 437– • • 19 440. Brown, E. J. , & Frazier, W. A. (2001). Integrin-associated protein (CD 47) and its ligands. Trends in Cell Biology, 11(3), 130– 135. Chaffin, J. (2019, February 12). 063 CE: Anti-CD 47 Testing Interference with Connie Westhoff -. Retrieved March 25, 2019, from https: //www. bbguy. org/2019/02/06/063/ National Cancer Institute. Clinical Trials Using Anti-CD 47 Monoclonal Antibody Hu 5 F 9 -G 4. Retrieved September 24, 2019 from https: //www. cancer. gov/about-cancer/treatment/clinical-trials/intervention/anti-cd 47 monoclonal-antibody-hu 5 f 9 -g 4 Isenberg, J. S. , Roberts, D. D. , & Frazier, W. A. (2008). CD 47. Arteriosclerosis, Thrombosis, and Vascular Biology, 28(4), 615– 621. https: //doi. org/10. 1161/atvbaha. 107. 158154 Lian, S. , Xie, R. , Ye, Y. , Xie, X. , Li, S. , Lu, Y. , . . . Jia, L. (2019). Simultaneous blocking of CD 47 and PD-L 1 increases innate and adaptive cancer immune responses and cytokine release. EBio. Medicine, . Retrieved from www. ebiomedicine. com Olsson, M. (2005). Platelet homeostasis is regulated by platelet expression of CD 47 under normal conditions and in passive immune thrombocytopenia. Blood, 105(9), 3577– 3582. https: //doi. org/10. 1182/blood-2004 -08 -2980 Reinhold, M. I. , Lindberg, F. P. , Plas, D. , Reynolds, S. , Peters, M. G. , & Brown, E. J. (1995). In vivo expression of alternatively spliced forms of integrin-associated protein (CD 47). Journal of Cell Science, 108, 3419– 3425. Velliquette, R. W. , Aeschlimann, J. , Kirkegaard, J. , Shakarian, G. , Lomas-Francis, C. , & Westhoff, C. (2019). Monoclonal anti-CD 47 interference in red cell and platelet testing. Transfusion, 59(2), 730– 737.
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