The Latest in PNH: New Treatment Options and Dealing with Side Effects

Camille Abboud, MD Washington University School of Medicine in St Louis Disclosures for Camille Abboud, MD

Employee Washington University Research support Novartis, Pfizer, Astrazeneca, Merck, Selvita , Actinium Consultant Cardinal Health, Seattle Genetics, Incyte, Nkarta, Tetraphase Equity ownership: BMS, Abbvie, Merck, Gilead, Abbott labs Immediate family member

Speakers Bureau Jazz (Vyxeos) Scientific Advisory Board Seattle Genetics, Jazz, Nkarta,Dova Honoraria Cardinal Health, Jazz PNH Diagnosis and Characteristics • PNH incidence is 2-5 per million in the USA. • The FLAER in conjunction with monoclonals antibodies on granulocytes and monocytes has more sensitivity and specificity. • Robert A. Brodsky: How I treat PNH, Blood June 25: 113;6522-6527. • Clinical manifestations: hemoglobinuria, episodic hemolysis, marrow hypoplasia, thromboembolic complications, acute and chronic renal insufficiency, dysphagia and male impotence, infections while ANC is low or steroids are used, viral or bacterial infections also can precipitate acute hemolysis, usually splenic enlargement but rarely can be associated with atrophy and hyposplenism

Fluorescein-labeled proaerolysin variant (FLAER) Is used by flow cytometry to diagnose PNH. CD59 is expressed on all cell lineages. Because of rare genetic deficiencies for GPI Variants at least two monoclonals are utilized To make the diagnosis.

FIGURE 32.3 Complement-mediated lysis of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes. The hemolysis of PNH is caused by aberrant regulation of the alternative pathway of complement (APC). The APC is a component of the innate immune system. Unlike the classical pathway of complement that requires a recognition factor such as antibody to activate the pathway, the APC is continuously active. Therefore safeguards have evolved to protect host cells against APC-mediated injury. In the case of erythrocytes, two glycosyl phosphatidylinositol-anchored proteins (GPI-APs), CD55 and CD59, serve this function. Two enzymatic convertases amplify the activity of the APC (top panel). The C3 convertase consists of activated C3 (C3b), activated factor B Bb, the enzymatic subunit of the complexes that is proteolytically activated by factor D, (a trace plasma protein that may be activated by one of the mannose-binding lectin-associated serine proteases) and factor P (formerly called properdin). Factor P stabilizes the C3 convertase, allowing each convertase to activate many molecules of C3, and in the process, generate the weak anaphylatoxin, C3a. The C5 convertase is similar in structure to the C3 convertase except that two molecules of C3b are required to position C5 for cleavage by activated factor B (Bb). Many molecules of C5 are cleaved by the C5 convertase, and this process generates many molecules of the potent anaphylatoxin and neutrophil chemoattractant, C5a. Activated C5 (C5b) is the nidus for formation of the membrane attack complex (MAC) of complement consisting of C5b, C6, C7, C8, and multiple molecules of C9. The MAC inserts into the lipid bilayer of the cell, forming a transmembrane torus that results in osmotic lysis. CD55 (DAF) blocks the formation and stability of both the C3 and C5 convertases, whereas CD59 (MIRL) blocks the formation of the cytolytic MAC, primarily by inhibiting binding and multiplicity of C9. There is also evidence that CD59 participates in regulation of the C3/C5 convertase. Eculizumab is a humanized monoclonal anti-C5 antibody that prevents activation of C5 by the C5 convertase. Consequently the MAC cannot form (and C5a is not generated), accounting for the inhibition of the intravascular hemolysis of PNH. However, eculizumab does not inhibit formation of the C3 convertase, accounting for the opsonization by activation and degradation products of C3 observed in patients with PNH treated with eculizumab. Normal red blood cells (RBCs) are protected against APC- mediated injury (black crosses represent APC C3 and C5 convertase formation and yellow stars represent MAC formation) by CD55 (blue ovals) and CD59 (green ovals) (bottom panel). PNH cells lacking the complement inhibitory proteins CD55 and CD59 undergo complement-mediated lysis, releasing cellular contents including hemoglobin (red circles) and lactate dehydrogenase (LDH) into the plasma. FIGURE 1 | Complement activation on PNH erythrocytes. (A) PNH erythrocytes in absence of anti- complement treatment. The complement system may activate due to different triggers through the alternative, classical and mannose/lectin pathway. Spontaneous C3 tick-over continuously generates low- grade activation of the alternative pathway in the fluid phase and possible binding of activated C3 fragments on erythrocytes. Due to the lack of CD55, this leads on PNH erythrocytes to the generation of C3 convertase, with further generation of C3b, which eventually leads to the assembly of C5 convertase. Then, the terminal pathway of the complement cascade is activated, with the generation of the MAC, eventually leading to lysis of PNH erythrocytes lacking CD59

Risitano et al. Next-Generation Anti-complement Treatment for PNH, Frontiers in Immunology June 2019, Vol 10, Article 1157. www.frontiersin.org

Glycosyl Phosphatidylinositol-Anchored Proteins Deficient in PNH

Protein Complement Regulatory Proteins CD55 (decay accelerating factor, Anti-DAF) on all blood cells CD59 (membrane inhibitor of reactive lysis, Anti-MIRL) on all blood cells Proteins With Immunologic Significance CD58 (lymphocyte function antigen-3) on all blood cells CD16b (Fc receptor gamma IIIb, CD16b) on granulocytes and Natural Killer cells CD14 (endotoxin binding protein) on granulocytes, monocytes and macrophages Receptors CD87 (urokinase plasminogen activator receptor) on monocytes and granulocytes Folate receptor (on myeloid and erythroid cells) Cellular prion protein (on resting platelets) For complete list see Maeda K et al, Text PNH from Bench to Beside, Tokyo, Japan, Springer 2017, 11-54. Glycosyl Phosphatidylinositol-Anchored Proteins Deficient in PNH

Protein Enzymes Leukocyte alkaline phosphatase Acetylcholinesterase 5’-ectonucleotidase Miscellaneous Proteins CD24, (on B lymphocytes and granulocytes) involved in B cell differentiation CD48 (on all leukocytes : mast cells, monocytes and granulocytes) is an adhesion molecule CD52 (Campath-1) (on lymphocytes, monocytes and neutrophils) CD66c CD66b (formerly CD67) CD90 (Thy-1) CD108 (John-Milton-Hagen, JMH-bearing protein), Semaphorin SEM7A, receptor for malaria parasite Plasmodium falciparum, present on activated lymphocytes and erythrocytes CD157 (on mature monocytes) is involved in adhesion and transmigration of monocytes Presenting features in 80 patients with PNH

Signs and Symptoms Number of Patients (%) Anemia 28 (35) Hemoglobinuria 21 (26) Hemorrhagic signs and symptoms 14 (18) Aplastic anemia 10 (13) Gastrointestinal symptoms 8 (10) Hemolytic anemia and jaundice 7 (9) Iron deficiency anemia 5 (6) Thrombosis or embolism 5 (6) Infections 4 (5) Neurologic signs and symptoms 3 (4) From Dacie JV, Lewis SM, Paroxysmal nocturnal haemoglobinuria: clinical manifestations, nature of the disease. Sem Haematol 1972:5:3-24. Chronic Hemolysis in PNH: QOL, organ dysfunction, thrombosis, iron toxicity, infections …

How I treat paroxysmal nocturnal hemoglobinuria

Robert A. Brodsky, How I treat paroxysmal nocturnal hemoglobinuria, Blood, 2009, Figure 3

Risk Factors Affecting Survival Before Eculizumab Availability

Factor Relative Risk of Disease-Related Mortality (95% P value Confidence Interval) Development of thrombosis 10.2 (6.0-17) <0.0001 Progressive pancytopenia 5.5 (2.8-11) <0.0001 MDS or AML 19.1 (7.3-50) <0.001 Age over 55 4.0 (2.4-6.9) <0.0001 More than 1 treatment 2.1 (1.3-3.6) <0.003 Thrombocytopenia at diagnosis 2.2 (1.3-3.8) <0.003 Aplastic anemia before PNH 0.32 (0.14-0.72) <0.023 Thrombosis and PNH: major cause of morbidity and much improved after Eculizumab therapy Balance between inflammation and infections, vascular endothelial activation and hemolysis Pathogenesis under study see below Propensity towards thrombosis roughly proportional to the size of the PNH clone Risk is higher in Caucasian and African American patient s than in patients of Asian/Pacific Island or Hispanic ancestry even when adjusted for clone size Patients at high risk are candidates for anticoagulation unless on eculizumab if the clone is > 50% GPI-AP deficient granulocytes Patients with PNH who have experienced a thromboembolic event should remain anticoagulated indefinitely even if on eculizumab treatment Sites of thrombosis that often occur in PNH: Hepatic vein (Budd-Chiari syndrome), Superior mesenteric vein, portal vein, cerebral vein (sagittal sinus thrombosis) and dermal veins. Therapeutic terminal complement inhibition using eculizumab has revolutionized the therapy and prognosis in PNH

Survival among patients with paroxysmal nocturnal hemoglobinuria (PNH) on eculizumab therapy compared with survival before the eculizumab era. First published in Blood by Kelly and colleagues,101 reused under general permission. Copyright: Blood, the Journal of the American Society of Hematology Factors Influencing Hemostasis and thrombosis in PNH Patients

Development of Novel Anti-Complement Treatment for PNH: Time for Proximal Complement Inhibition? • Patients response to eculizumab is heterogenous and no more than a third normalize their hemoglobin. Some patients require RBC transfusions. • Factors at play include: marrow dysfunction,residual intravascular hemolysis, and emergence of C3-mediated extravascular hemolysis. • Despite novel anti-C5 reagents with longer half life and also SC administration there is still a need to inhibit the proximal complent activation pathway. • Risitano AM et al: Frontiers in Immunology June 2019, Vol 10; 1157, pp 1-24.

Complement activation cascade illustrating targets for therapeutic modulation in PNH

Dimitrios C. Mastellos, et al: Expanding complement therapeutics for the treatment of PNH, Semin Hematol 2018;55(3);167-175.

Complement cascade & targets for therapeutic modulation in PNH. Novel agents may result in deeper inhibition of C5 leading to better control of hemolysis than eculizumab Neutralizing activity of anti-C5 antibody on C5 with non synonymous SNPs: SKY59 (Roche,Chugai) inhibits all C5 variants while Eculizumab did not inhibit R885H C5.

Fukuzawa T., Sampei Z, Haraya K., et al: Long lasting neutralization of C5 by SKY59, a novel recycling antibody, is a potential therapy for complement-mediated diseases. Sci Rep, 2017:7:1080 Novel small molecule C5 inhibitors may rewrite the future for PNH and Complement Mediated Hemolytic Anemias / Diseases

Nature Chemical Biology Vol 15, July 2019: 666-668

Highlights of BBMT manuscript

• All patients attained successful donor engraftment. No relapses seen. • Cumulative incidences of aGVHD grades II to IV and grades III to IV were 15.91% and 2.27% respectively. • Cumulative incidences of cGVHD and moderate to severe cGVHD were 26.73 and 9.7% respectively. • The 3 year overall survival probability was 90.4% • The 3 year GVHD-free, failure-free survival probability was 85.6%

Advances in Allogeneic Stem Cell Transplantation in PNH / AA Patients • Both myeloablative as well as reduced intensity SCT have been carried out. Sources of stem cells are marrow, or peripheral blood derived stem cells. Indicated if clonal evolution to MDS or AML. • ATG or PT-CY are added in MUDs and Haplo identical procedures. • Eltrombopag has been the mainstay of AA patients and has been used with G-CSF to decrease graft failure and improve count recovery. • Eculizumab has been used safely in PNH patients undergoing alloSCT. • Novel agents now being tested to decrease acute and chronic GVHD JAK 2 inhibitors, actively being studied in Hematologic Malignancies Conclusions

• Novel agents have longer half life and allow patients to self administer anti C5 agents at home. • The proximal pathway of complement activation may yield better overall results alone or when combined with Eculizumab. • Risk(s) of serious infections will likely increase and physicians and patients have to be aware of the need for prophylaxis and/or prompt antibiotic therapy • Eculizumab can display incomplete terminal pathway inhibition in PNH patients during infections (pharmacodynamic breakthrough). Literature Cited • Wintrobe’s Clinical Hematology, Chapter 32, 783-810, 2019. • Berentsen S et al, Novel insights into the treatment of complement-mediated hemolytic anemia, Ther Adv Hematol, 2019;Vol 10, 1-20. • Lee J W, et al, Ravulizumab (ALXN1210) vs eculizumab in adult patients with PNH naïve to complement inhibitors: the 301 study. Blood 2019;133(6):530-539. • Jelinek T et al, Effectiveness of eculizumab in patients with paroxysmal nocturnal hemoglobinuria (PNH) with or without aplastic anemia in the International PNH Registry. AJH, October 18 2018. • Griffin M, et al, Significant hemolysis is not required for thrombosis in paroxysmal nocturnal hemoglobinuria. Haematologica 2019:104:e94-e95. • Harder MJ et al, Different levels of incomplete terminal pathway inhibition by eculizumab and clinical response of PNH patients. Frontiers in Immunology July 2019;vol 10: Article 1639. • Lee S-E et al, Outcomes of allogeneic stem cell transplantation in patients with PNH with or without aplastic anemia. Eur J Haematol 2017;99:336-343. • Mei M et al, Post-Allogeneic hematopoietic stem cell transplantation eculizumab as prophylaxis against hemolysis and thrombosis for patients with hematologic disorders associated with PNH clones. Biol Blood Marrow Transplant 25 (2019;e183-e185. https://bookshelf.vitalsource.com/#/books/9781496367112/cfi /6/104!/4/2/32/2/2@0:92.4