STEM CELL BIOLOGY AND HEMATOPOIETIC GROWTH FACTORS David J. Kuter, MD, DPhil Hematology Program Director Massachusetts General Hospital Professor of Medicine Harvard Medical School Depend upon it, sir, when a man knows he is to be hanged in a fortnight, it concentrates his mind wonderfully

Samuel Johnson (Boswell’s Life of Johnson, 1791) GOALS

• Review general principles • Erythroid growth factors of hematopoietic growth – epoetin alpha factors – darbepoetin alpha

• Myeloid growth factors • Thrombopoietic growth – filgrastim (G-CSF) factors – tbo-filgrastim – romiplostim – filgrastim-sndz – eltrombopag – filgrastim-aafi – avatrombopag – pegfilgrastim – lusutrombopag – pegfilgrastim-jmdp – interleukin-11 – sargramostim (GM-CSF)

Hematopoietic Cytokines - 1

• Early acting (IL-3, IL-6, IL-11, GM-CSF, SCF) – Affect early maturational stages – Lineage non-specific – Locally acting - circulating levels not important • Late acting cytokines (EPO, G-CSF, M-CSF, TPO) – Affect late maturational stages/release of cells – Lineage specific – Circulating levels important – Prevent apoptosis • Act at very low concentrations • Work via specific cell surface receptors Hematopoietic Cytokines - 2 G-CSF, M-CSF, EPO, TPO

• Circulating level due to production and/or metabolism – EPO - mostly regulated at production level – TPO, M-CSF - primarily regulated via platelet or monocyte/macrophage clearance – G-CSF - regulated by production and clearance

• Circulating level inversely proportional to mass of terminally differentiated cell [Erythropoietin] inversely proportional to Hct

Erslev, Semin Hematol 1991;28:2 Hematopoietic Cytokines - 3 G-CSF, M-CSF, EPO, TPO

• Normal response altered by pathological states – Anemia of chronic disease -  EPO production – Acute bacterial infection -  G-CSF production – Liver failure –  TPO production

• Pharmacological administration also operates on an exponential dose-response curve Platelet response to TPO dose

From Harker et al Blood 1996;87:1833.

MYELOID GROWTH FACTORS G-CSF Filgrastim

Tbo-filgrastim Short Filgrastim-sndz Acting Filgrastim-aafi

Pegfilgrastim Long Pegfilgrastim-jmdp Acting

GM-CSF Sargramostim Granulocyte Colony Stimulating Factor - G-CSF G-CSF BIOCHEMISTRY AND PHYSIOLOGY

• Structure – 174 aa protein (MW=25,000) – glycosylated; critical S-S bridge – single gene - 17 q11.2-q21 • Synthesis – macrophages, monocytes, endothelial cells, fibroblasts – TNFα, IL-1, GM-CSF, IL-3, IL-4, IF-γ  production • Metabolism – mature neutrophils via G-CSF receptor – liver, kidney to lesser degree G-CSF BIOCHEMISTRY AND PHYSIOLOGY

• Circulating levels: – non-infected - inversely proportional to ANC, production , cleared by neutrophils – infected - production  normal - < 39 ng/L bacterial - 799 +/- 1501 viral - 58 +/- 34 mycoplasma - 60 +/- 33 Cyclic Neutropenia: Relationship of G-CSF levels to neutrophil count

Guerry et al Blood 1974;44:257 G-CSF BIOCHEMISTRY AND PHYSIOLOGY

• Circulating levels: – non-infected - inversely proportional to ANC, production , cleared by neutrophils – infected - production  normal - < 39 ng/L bacterial - 799 +/- 1501 viral - 58 +/- 34 mycoplasma - 60 +/- 33 G-CSF Biological Effects-1

 Demargination of neutrophils  Release of neutrophils from marrow reserves  Neutrophil survival

 Production of neutrophils: shortened maturation time slight L shift Dohle and toxic granulation G-CSF Biological Effects-2

 motility of neutrophils  skin window migration

 Neutrophil function (of nml and abn neutrophils) -  phagocytosis,  O2 generation  endocytosis,  FcRI  LAP,  Ab-dependent cellular cytotoxicity

 Mobilization of peripheral blood progenitor cells Multiple types of G-CSF: but they are all the same!!

Filgrastim (Neupogen) Tbo-filgrastim (Granix) Filgrastim-sndz (Zarzio) Filgrastim-aafi (Nivestym) Filgrastim (Neupogen): FDA-approved indications • Cancer patients receiving myelosuppressive chemotherapy • Cancer patients with acute myeloid leukemia receiving induction or consolidation chemotherapy • Cancer patients undergoing bone marrow transplantation • Patients undergoing autologous peripheral blood progenitor cell collection and therapy • Patients with severe chronic neutropenia • Patients having acute radiation syndrome Tbo-filgrastim (Granix) The “non-biosimilar” Filgrastim & tbo-filgrastim: same PK/PD

PK PD

H Lubenau et al. Int J Clin Pharmacology and Therapeutics 2009;47:275-282. Breast Cancer CT Patients*: tbo-filgrastim = filgrastim (Cycle 1)

A del Giglio et al. BMC Cancer 2008;8:332-339 *Docetaxel/doxorubicin Tbo-filgrastim: FDA-approved indications

• Cancer patients receiving myelosuppressive chemotherapy • Cancer patients with acute myeloid leukemia receiving induction or consolidation chemotherapy • Cancer patients undergoing bone marrow transplantation • Patients undergoing autologous peripheral blood progenitor cell collection and therapy • Patients with severe chronic neutropenia • Patients having acute radiation syndrome Filgrastim-sndz (Zarxio) The first US biosimilar HPLC Peptide Map Filgrastim-sndz vs filgrastim

F Sorgel at al. BioDrugs 2015;29:123-131. PD/PK analysis of filgrastim-sndz

PK PD Healthy volunteers

F Sorgel at al. BioDrugs 2015;29:123-131. Filgrastim-sndz vs filgrastim ANC (mean ± SD) cycle 1

Breast cancer patients: TAC

K. Blackwell et al. Ann Oncol 2015;26:1948-1953 Filgrastim-aafi (Nivestym) Another biosimilar Filgrastim-sndz, Filgrastim-aafi: FDA-approved indications • Cancer patients receiving myelosuppressive chemotherapy • Cancer patients with acute myeloid leukemia receiving induction or consolidation chemotherapy • Cancer patients undergoing bone marrow transplantation • Patients undergoing autologous peripheral blood progenitor cell collection and therapy • Patients with severe chronic neutropenia • Patients having acute radiation syndrome Using G-CSF Recombinant Products filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi

• Dosage: 5µg/kg/day (~ 230 µg/m2/day) – round down - remember log dose-response curve – much lower doses often adequate (0.4 µg/kg)

• Route: sq better than iv

• T1/2 = 3.5 h

• Clearance: neutrophils > kidneys

• Timing: 24 h post-chemo, stop 24 h before chemo – stop ANC > 10,000 (could stop at lower ANC) Hematological Response to G-CSF (filgrastim)

Morystyn et al, JCO 1989;7:1554 G-CSF (filgrastim) Side Effects

• Bone pain (15-39% on G vs 0-21% on placebo) • Exacerbation of pre-existing inflammatory conditions (eczema, psoriasis, vasculitis) • Rare allergic reactions at injection sites • Sweet’s syndrome (acute, febrile neutrophilic dermatosis) •  LDH, uric acid, alk phos, LAP • Antibody formation; none neutralizing • Rare: splenic rupture, ARDS, sickle cell crisis • Poverty Pegfilgrastim (Neulasta) Pegfilgrastim-jmdp (Fulphila) Pegfilgrastims - 1

• Protein structure is that of filgrastim – 20,000 dalton PEG moiety added at N-terminus – MW: 39,000

• Clearance - not renal – neutrophils/precursors - mostly (“self-regulating”)

• Dose: 6mg = 100 µg/kg

• T1/2=33h (varies from 15-80 h depending on ANC) – at least 10-fold greater T1/2 than filgrastim PK Studies in Healthy Humans Pegfilgrastims -2

• Product available: – 6 mg (0.6 mL) prefilled syringes – cost: $3254 (AWP) – filgrastim @ $288.88/d x 10 d = $2889

• Adverse events same as filgrastim • Equivalent to daily filgrastim in chemotherapy trials

• Timing: usually on Day 2 of CT cycle – DO NOT give in period 14 d before to 24 h after CT – Maybe an exception for GYN malignancies Same vs next day Pegfilgrastim in breast cancer patients on TAC (Kaufman, 2004)

Same Day 24 Hr Later (n=45) (n=45) Cycle 1, Gr 4 neutropenia 2.6 d 1.4 d Cycle 3, duration Gr 4 neutro 2.1 d 1.2 d All cycles, frequency FN 33% 11% All cycles, FN requiring Rx 22% 7% SAE 24% 11% All cycles, chemo on time 78% 85%

Burris HA et al. J Oncol Pract 2010;6:133-140 INDICATIONS FOR MYELOID GROWTH FACTORS

From NCCN Guidelines 2018

J Crawford, et al. JNCCN 2018 (In press) G-CSF, GM-CSF INDICATIONS IN ONCOLOGY

• 1o prophylaxis of FN if incidence > 20% • Consider if FN incidence > 10-20% • No 1o prophylaxis if FN incidence <10% • 2o prophylaxis of FN to keep dose intensity • After allogeneic or autologous stem cell txp • PBPC mobilization • MDS with neutropenia and recurrent infection • AML induction chemotherapy (>55 years old) Primary Prophylaxis of Neutropenia with Filgrastim

Crawford in K Welte Blood 1996;88:1907 G-CSF, GM-CSF INDICATIONS IN ONCOLOGY

• 1o prophylaxis of FN if incidence > 20% • Consider if FN incidence > 10-20% • No 1o prophylaxis if FN incidence <10% • 2o prophylaxis of FN to keep dose intensity • After allogeneic or autologous stem cell txp • PBPC mobilization • MDS with neutropenia and recurrent infection • AML induction chemotherapy (>55 years old)

J Crawford, et al. JNCCN 2018 (In press) NCCN SUMMARY – 1o Prophylaxis

J Crawford, et al. JNCCN 2018 (In press) G-CSF, GM-CSF INDICATIONS IN ONCOLOGY

Probably NOT for:

• Treating afebrile neutropenia (unless “high risk”) • Treating febrile neutropenia (unless “high risk”) • Increasing dose intensity (off protocol) • Concomitant XRT/chemotherapy • ? Pediatric patients? (Very limited data) Who is patient with “high risk”?

• Sepsis syndrome • Age >65 • Severe neutropenia (ANC < 100/mcl) • Neutropenia expected to last more than 10 days • Pneumonia • Invasive fungal infection • Other clinically documented infections • Hospitalized at the time of the fever • Prior episode of febrile neutropenia

J Crawford, et al. JNCCN 2018 (In press) G-CSF, GM-CSF Other Uses - 1

In symptomatic patients: • Drug-induced neutropenia - low dose (0.4-5 µg/kg) • Aplastic anemia, some patients • Hairy cell leukemia • ALL • Severe chronic neutropenia – idiopathic – cyclic – congenital (ie Kostmann’s) Treatment of chronic neutropenia with G-CSF

K Welte Blood 1996;88;1907 G-CSF, GM-CSF Contraindications

• Concomitant radiotherapy -  neutropenia

• Concomitant chemotherapy -  neutropenia

• “Priming” leukemia cells prior to or during chemotherapy -  survival rate

• ? Presence of myeloid blast cells ? What about the pegfilgrastims? Indications for the pegfilgrastims

• FDA approved for reducing the incidence of febrile neutropenia in patients with non-myeloid malignancies receiving myelosuppressive chemotherapy associated with a clinically significant incidence of febrile neutropenia

Two randomized, double-blind studies vs filgrastim in patients with metastatic breast cancer treated with doxorubicin and docetaxel every 21 days Pegfilgrastim = filgrastim

Pegfilgrastim Filgrastim

Cycle 1 - days of severe neutropenia 1.8 1.6 All cycles - febrile neutropenia 13% 20%

CT on schedule 96% 98%

MD Green et al. Annal Oncol 2003;14:29-35. ERYTHROID GROWTH FACTORS

EPOETIN ALPHA DARBEPOETIN ALPHA EPOETIN BETA (CKD only) Crystal Structure of rHuEPO Bound to its Receptor

rHuEPO

carbohydrate side chains • 3 N-linked (CHO) chains • Maximum 14 sialic acids

• MW ~ 30,400 daltons

• 40% carbohydrate Receptor 1 Receptor 2 Erythropoiesis

Bone marrow Sinusoidal Circulation wall

CFU-E Mature red blood cell Erythrocyte Pronormoblast (RBC)

Basophilic normoblast Reticulocyte

Polychromatic normoblast Orthochromatic normoblast

CFU-E = erythroid colony-forming unit. EPO Biochemistry and Physiology

• Synthesis – Produced by interstitial peritubular fibroblasts of kidney

• Circulating levels regulated by rate of production – Efficient renal sensor - cytochrome - hypoxia – TNFα, IL-1  production - anemia of chronic disease Molecular Regulation of EPO Production-HIFα

HIF = hypoxia inducible factor

Proline hydroxylase Correlation Between Hgb and Serum EPO Levels in Various Disease States

200 A – Hemolysis B – Iron deficiency C – Anemia D – Diabetes E – Cancer /mL) C F – Inflammation mU

( 100 G – Infection H – Alcoholism B A sEPO D E

G H F 0 5 10 15 Hgb (g/dL) Modified from Spivak JL. Semin Hematol 1993:30:2-11 Epoetin alpha (Epogen, Procrit, Retacrit*) FDA-approved Indications

For symptomatic individuals: • Chronic renal failure -  Hct,  quality of life • HIV infection - when EPO levels are < 500 mIU/ml • Cancer chemotherapy - platinum decreases EPO • Pre-operative anemia (Hgb 10-13) • (Anemia of chronic disease - EPO deficiency) • (Mobilization of Fe in hemochromatosis) • (Improved harvesting of autologous blood) • (Mobilize PBPCs) • (Neuroprotection – studies in progress)

*Retacrit is new (first) biosimilar epoetin alpha Epoetin alpha Use In Oncology - 1

• Fatigue present in 76% of cancer patients • Relationship of fatigue to anemia becoming clearer • EPO levels low in cancer (chemo, chronic disease) • EPO in cancer patients – decreases need for RBC transfusions – increases quality of life (QOL) – from Hgb 10 to 12 most QOL benefit, little effect > 13 • QOL improvement is related to Hgb rise • But concerns over increased disease progression, thrombosis, mortality Epoetin alpha Increases the Hgb and Decreases the Need for RBC Transfusions

Gabrilove JL et al. J Clin Onc 2001;19:2875-2882 Community-Based Studies of rHuEPO in Cancer-Related Anemia

70

65

60

55

50 Demetri 45 Glaspy 40

35

Overall HRQOL LASA Score (mm) Score LASA HRQOL Overall 30 7 8 9 10 11 12 13 14 Hb Level (g/dL) Demetri n = 181 551 818 680 472 345 221 114 Glaspy n = 118 497 812 931 515 286 278 125 Crawford J et al. Cancer. 2002;95:888-895. Erythroid Growth Factors

• Recombinant erythropoietin (epoetin alpha) • Hyperglycosylated, recombinant erythropoetin (darbepoetin alpha) • Epoetin beta (methoxy polyethylene glycol- epoetin beta [Mircera])-CKD only • Erythropoietin peptide mimetics • Erythropoietin non-peptide mimetics (oral) • Erythropoietin receptor modulators • HIF stabilizing agents (Proline hydroxylase inhibitors) Molecular Comparison of Epoetin alpha and Darbepoetin alfa

rHuEPO carbohydrate Darbepoetin alpha Additional carbohydrate side chains side chains

Receptor 1 Receptor 1 Receptor 2 Receptor 2

• 3 N-linked (CHO) chains • 5 N-linked (CHO) chains

• Maximum 14 sialic acids • Maximum 22 sialic acids

• MW ~ 30,400 daltons • MW ~ 37,100 daltons

• 40% carbohydrate • 51% carbohydrate Single-Dose Pharmacokinetics of Intravenous Darbepoetin alfa

100 darbepoetin alfa (oncology; 0.5 mcg/kg, n = 20)*1 darbepoetin alfa (dialysis; 0.5 mcg/kg, n = 11)2 rHuEPO (dialysis; 100 U/kg, n = 10)2 10

- corrected t1/2 = 38.8 hours

1

t1/2 = 25.3 hours

0.1 t1/2 = 8.5 hours serum concentration (ng/mL) Mean (SD) baseline

0.01 0 25 50 75 100 Time post intravenous injection (hours) *Oncology patients received 2.25 mcg/kg; data shown is normalized for 0.5 mcg/kg

1. Heatherington A, et al. Proc ASCO 2002. 2. Macdougall I, et al. J Am Soc Nephrol. 1999;10:2392–2395. Indication for darbepoetin alfa (Aranesp™)

• Darbepoetin alpha is indicated for the treatment of anemia associated with chronic renal failure (CRF), including patients on dialysis and patients not on dialysis.

• Darbepoetin alpha is indicated for the treatment of anemia in cancer patients receiving chemotherapy. REMEMBER importance of adequate iron stores!! Iron Stores Rapidly Decrease After Use of Erythroid Growth Factors

Rutherford CJ et al. Am J Med 1994;96:139-145 Iron Affects Response to EPO 40,000 QW Hgb Rise

M Auerbach et al. JCO 2004;22:1301-1307 EPO Side Effects – General

• Hypertension - renal failure patients

• Seizures - renal failure patients

• Allergic reactions – rare

• Antibody formation leading to pure red cell aplasia - European formulation; very rare US EPO Side Effects – Newer

• All patients – 1.52-fold increase in thrombosis

• Renal patients – increased CV complications and death at Hgb >11

• Cancer patient (breast, head-neck, cervical, lymphoma) on chemo – decreased survival if target Hgb >12

• Cancer patients (not receiving chemo) – decreased survival Meta-analysis (Evidence Level Ia) of epoetin-α/β or darbepoetin-α vs. placebo/RBC transfusion 91 RCTs (N = 20,102) • ESA reduce RBC transfusions (70 studies; N = 16,093) – RR 0.65 (0.62-0.68) • ESA reduced RBC transfusion by ~one unit (19 studies; N=4715) – Mean difference (MD) = -0.98 (-1.17 to -0.78) • Hematological response higher with ESA (31 studies; N=6413) – RR=3.93 (3.10-3.71) • Quality of life may be improved (suggestive data) • ESA had no effect on tumour progression (15 studies; N=5012) – RR=1.02 (0.98-1.06) • ESA increased on study mortality (70 studies; N=15,935) – HR=1.17 (1.06-1.29) • ESA suggestion of reduced OS (78 studies; N=19,003) – HR=1.05 (1.00-1.11) • ESA increased TECs (57 studies; N=15,498) – RR=1.52 (1.34-1.77) T Tonia et al. Cochrane Database Systemic Review 2012;CD003407 THROMBOPOIETIC GROWTH FACTORS IL-11 rhuTPO (TPIAO) Romiplostim Eltrombopag Avatrombopag TPO Receptor Agonists Lusutrombopag Human TPO Structure

Nonpolar W F L M A V I P Polar S T C Y N Q G Acidic D E Thrombin Basic R K H 191 H P L P D P L T V T P L G R T T R A N-Glycosylation E P S L 250 A R G S L S Q P V 40 S R C P G L 80 L A P P S S A H T Q L P 300 H D P T 29 C G P G L V R 310 T L I G L F 85 L A H 20 A P Q S P L 185 N S S V T V S 240 F K T D S A D R L M F P V G L 200 W E L L 90 V T P T G Q L L G G S K Q L S T S R Q L L E P P D T G S G G L S G L L 70 L L 260 T G F S V E 50 Q L W L T G 234 N R T R 10 V 319 N P S L A R R T V K L L T P L L K 176 N 1 D A T 100 L L G Q S L P E I P S P A P P A C L Y P P H P L G I M T F N I G E F I A L D E P P A R R V C 151 L A Q H L L R L N S N Q E T N T L F S L A K T S Q L L T 130 P Q Y P L 213 N T T Q H L 110 60 A 160 S D G 327 N Q 270 G Y Q L L T Q G G V G L K L L Y T V G R S 280 T S P S L P G H P V K A Q P P I R S S V 120 P T R L T Q E P S Q S M R T R G 332 G H T P D L L F 140 220 117 Thrombin TPO - Mechanism of Action

thrombopoietin thrombopoietin receptor

inactive receptor active receptor

Cell membrane SHC GRB2 RAS/RAF SOS P P

P JAK STAT P Cytoplasm MAPKK Anti-apoptosis pathways

p42/44

Signal Transduction

Increased platelet production Erythroid-burst forming units (E-BFU) Pluripotential Stem Cell Granulocyte-erythroid-macrophage-megakaryocyte colony- forming cells (GEMM-CFC)

Meg-CFC

EndomitosisPolyploid 2N 4N 8N 16N 32N 64N

Immature Megakaryocytes

Mature Megakaryocytes

Platelets Romiplostim - Platelet Response In Healthy Humans

Wang B et al. Clin Pharm Therapeutics. 2004;76:628-638. Thrombopoietin Physiology

DJ Kuter et al PNAS 1994;91:11104 Thrombopoietins

1st Generation 2nd Generation • rhuTPO • TPO receptor agonists • PEG-rhuMGDF – Peptide • romiplostim – Non-peptide small molecules • eltrombopag • avatrombopag • lusutrombopag • LGA-4665 • NIP-004, NIP-022 • butyzamide

• Anti-Mpl antibody Romiplostim (AMP2, AMG 531, Nplate, Romiplate)

N terminus • Fusion protein of Fc (M1) and TPO mimetic C7 peptides (peptibody) C10 • No sequence Fc Domain C42 homology with native CH2 (228 a.a.) TPO C102 • SQ administration C148 CH3 • Stimulates platelet C206 production by same 5 Glycine spacer Thrombopoietin receptor- mechanism as TPO binding domain (14 a.a.) 8 Glycine spacerIEGPTLRQWLAARA • Recycled by FcRn on Thrombopoietin receptor- binding domain (14 a.a.) C terminus endothelial cells (A269)

G Molineux et al. Ann NY Acad Sci

Eltrombopag (SB-497115, Promacta, Revolade)

O O H • Small molecule (MW=442) • Orally bioavailable • Once daily dosing • Stimulates megakaryocyte O H N H proliferation and N

H C O differentiation 3 • Increases platelet counts N N

C H • Not immunogenic 3

C H • Does not activate platelets 3 • Active in humans, chimps but no other species Avatrombopag (AKR-501, YM477, E5501, Doptelet) • Small molecule (MW=650) • C29H34Cl2N6O3S2 • Orally bioavailable • Once daily dosing • Stimulates MK proliferation and differentiation • ~3-fold more potent than eltrombopag • No food interactions • No effect on liver function • Active in humans, chimps but no other species Lusutrombopag (Mulpleta) • Small molecule (MW=592) • Orally bioavailable • Once daily dosing • Stimulates MK proliferation and differentiation • C29H32Cl2N2O5S • Active in humans, chimps but no other species TPO Receptor Agonists Have Different Mechanisms of Action

thrombopoietin Eltrombopag romiplostim receptor Avatrombopag Lusutrobopag

inactive receptor active receptor

Cell membrane SHC GRB2 RAS/RAF SOS P P

P JAK STAT P Cytoplasm MAPKK

p42/44

Signal Transduction

Increased platelet production Clinical Studies With TPO Receptor Agonists

ITP Hepatitis C Thrombocytopenia Aplastic Anemia Chronic liver disease Chemotherapy-induced Thrombocytopenia Other ITP ITP is a disorder of reduced platelet production - TPO Receptor MegakaryocyteLymphocyteAntiplatelet attacksundergoesTPO megakaryocytetoab theattacks apoptosis rescue Mk - Antiplatelet antibody - Antiplatelet lymphocyte - TPO Romiplostim ITP Trials – Platelet Count

D Kuter, J Bussel, et al. Lancet 2008;371:395-403. Romiplostim trials – key outcome parameters

100 p<0.0001 p<0.0001 90 87 83.1 80

70 59.5 60

50 Placebo 40 38 Romiplostim

30 Percentage of patients 21.7 20

10 7.1

0 Overall platelet response* Use of rescue medication** Discontinued or reduced concurrent therapy

*Weekly platelet count ≥ 50,000 on 4 or more weeks of study; **IVIG, anti-D, increased steroid dose, etc Kuter DJ et al. Lancet 2008;371:395–403 Romiplostim pivotal trials: summary of bleeding events

40 Placebo (n=41) 35 34% Romiplostim (n=84) 30 25 20 15% 15 12% Percentage 10 7% 5 0 Grade ≥2 Grade ≥3 • All grade ≥2 bleeding events occurred at platelet counts <50 x 109/L • All grade ≥3 bleeding events occurred at platelet counts <20 x 109/L • No clinically significant (severe, life-threatening, or fatal) bleeding events occurred at platelet counts >20 x 109/L • Bleeding events were captured as adverse events and were not a predefined endpoint in the pivotal trials

Kuter DJ et al. Lancet 2008;371:395–403; Gernsheimer T et al. J Thromb Haemost. 2010;8:1372–1382 Stability of Platelet Counts and Romiplostim Dose Over Time

10

8

6

4

2 Mean Dose (µg/kg)

0 1 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 200 208 216 224 232 240 n = 290 276 270 257 234 184 147 130 120 115 113 107 101 97 91 90 91 86 75 64 51 48 29 24 23 24 22 20 18 17

/L 300 9 250

200

150

100

50 Median (Q1,Median Q3)

Platelet Count x 10 0 1 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 200 208 216 224 232 240 Study Week n = 289 255 240 229 209 163 126 111 102 99 101 98 89 85 81 76 77 75 67 56 45 41 27 25 21 21 22 22 16 16 Note: data points with n < 5 not plotted Kuter et al Br J Haematol 2013; 161:411-423 TPO Receptor Agonists: ITP FDA-approved indications • Romiplostim – Treatment of thrombocytopenia in patients with chronic ITP who had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. • Eltrombopag: – Promacta is indicated for the treatment of thrombocytopenia in adult and pediatric patients 1 year and older with chronic ITP who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. • Avatrombopag: (pending) • Lusutrobopag: (pending) Hepatitis C Eltrombopag and viral cure – platelets

N Afdahl et al. Gastroenterology 2014;1465:442-452 Eltrombopag and viral cure – SVR

RVR, rapid virologic response EVR, early virologic response cEVR, complete EVR ETR, end of treatment response N Afdahl et al. Gastroenterology 2014;1465:442-452 SVR, sustained virologic response TPO Receptor Agonists: Hepatitis C FDA-approved indications • Romiplostim: none • Eltrombopag: – Promacta is indicated for the treatment of thrombocytopenia in patients with chronic hepatitis C to allow the initiation and maintenance of interferon-based therapy. Promacta should be used only in patients with chronic hepatitis C whose degree of thrombocytopenia prevents the initiation of interferon-based therapy or limits the ability to maintain interferon-based therapy. • Avatrombopag: none • Lusutrobopag: none Aplastic Anemia Some responders had increased bone marrow cellularity

MJ Olnes et al. NEJM 2012; 367:11-19 Eltrombopag in AA—larger pool

• 17/43 (40%) respond over 3-4 months – 14/17 improved more over longer time on eltrombopag • 7/17 major trilineage responses • 5/7 major responders stopped treatment after median of 29 (range 9-37) month • All 5 maintained counts a median of 13 (range: 1- 15) subsequent months • Responders all needed >100 mg/d • No thrombotic events or reticulin

R Desmond et al. Blood 2014:123:1818-1825 Eltrombopag in AA—larger pool (n=43) 7 trilineage responses

R Desmond et al. Blood 2014:123:1818-1825 TPO Receptor Agonists: Aplastic Anemia FDA-approved indications • Romiplostim – None • Eltrombopag: – Promacta is indicated for the treatment of patients with severe aplastic anemia who have had an insufficient response to immunosuppressive therapy. • Avatrombopag: – None • Lusutrobopag: – None Chronic Liver Disease needing procedure ADEPT – 1: Primary Endpoint

Mean Platelet Counts by Treatment Group and Visit Day

N Terrault et al. Gastroenterology 2018 (May 17) ADEPT – 1: Primary Endpoint

Proportion of Patients who did NOT Require Platelet Transfusion or any Rescue Procedure for Bleeding

N Terrault et al. Gastroenterology 2018 (May 17) TPO Receptor Agonists: CLD FDA-approved indications • Romiplostim – None • Eltrombopag: – None • Avatrombopag*: – Doptelet is indicated for the treatment of thrombocytopenia in adult patients with CLD who are scheduled to undergo a procedure. • Lusutrombopag*: – Mulpleta is a TPO receptor agonist indicated for the treatment of thrombocytopenia in adult patients with CLD who are scheduled to undergo a procedure N Terrault et al. Gastroenterology 2018 (May 17) *They mean minor procedures Chemotherapy Effect of PEG-rHuMGDF on Platelet Recovery After Chemotherapy

Fanucchi et al. N Engl J Med 1997;336:404 rhTPO Reduces Need for Platelet Transfusions in Nonmyeloablative Chemotherapy

80 Cycle 1: Chemotherapy 70 Cycle 2: Chemotherapy + rhTPO 60 P<0.002 50

40 P<0.013 P<0.006 30 20

10 P<0.002 0 Platelet count Platelet count Time to Patients nadir <20 × 109/L (d) platelet count requiring platelet (109 cells/L) >100 × 109/L (d) transfusions (%)

Vadhan-Raj. Ann Intern Med 2000;132:364 Case - CIT

Romiplostim Gemcitabine

5 FU XRT MGH summary-demographics

22 Solid tumors-metastatic/regionally advanced • GI: 11 • Esophageal: 2 • Lung: 2 • Breast: 2 • Other: 5

H Al-Samkari et al. Haematologica 2018;103:e169-e172 MGH summary-protocol

• Patients receiving cancer chemotherapy for solid tumors – Prior platelet count <75x109/L AND – Prior dose reduction OR – Prior dose delay >4 weeks • Romiplostim 2 µg/kg (usually 375 µg) weekly until completion of therapy • Dosed on same say as chemotherapy

H Al-Samkari et al. Haematologica 2018;103:e169-e172 Mean (+/- IQR) weekly platelet counts

H Al-Samkari et al. Haematologica 2018;103:e169-e172 Rapid response times for majority

• 17 Patients <75,000 at onset romiplostim – 10 >100,000 after 1 week dosing – 3 after 2-3 weeks dosing – 3 after 7-12 weeks dosing – 1 never >100,000 (marrow infiltrated with metastatic breast cancer)

H Al-Samkari et al. Haematologica 2018;103:e169-e172 Platelets in desired range for most subsequent chemotherapy cycles

• 83% of all weekly platelet counts >75,000

• 71% of all weekly platelet counts >100,000

H Al-Samkari et al. Haematologica 2018;103:e169-e172 Chemotherapy outcomes

Before On P romiplostim romiplostim Chemotherapy 17/18 (94%) 8/22 (36%) 0.0002 delay Duration delay, 4 (1-11) weeks 1 (1-4) week median (range) Dose reduction 14/18 (78%) 4/22 (18%) 0.0002

H Al-Samkari et al. Haematologica 2018;103:e169-e172 What about rHuTPO (TPIAO)?

• Widely used China for – ITP • Pregnancy ITP—very good data – Chemotherapy-induced thrombocytopenia • Ho-hum data Effect of rhTPO on platelet count

FDA filing 2014 Study 005 – outcomes

Placebo rhTPO P value Nadir plt (×109/L), mean (SD) 49 (35) 61 (51) 0.015 Highest recovery platelet count (×109/L), mean (SD) 153 (82) 261 (164) <0.001 Platelet count difference (×109/L), mean (SD) 104 (74) 199 (158) <0.001 Duration platelets <50×109/L post-CT (days), mean 4.7 (6.9) 3.1 (4.4) <0.05 (SD) Time for platelets to rise >75×109/L (days), mean 17.2 12.0 (9.7) <0.001 (SD) (10.9) Time for platelets to rise >100×109/L (days), mean 21.2 15.8 (9.7) <0.001 (SD) (10.6) Platelet transfusion events, mean (SD) 0.6 (1.3) 0.3 (0.7) <0.05 Platelet units transfused, mean (SD) 3.0 (6.6) 1.5 (4.4) <0.05

FDA filing 2014 Potential Adverse Consequence of Thrombopoietic Growth Factors

• Thrombocytosis • Autoantibody • Thrombosis formation • Stimulation of tumor • Stem cell depletion growth • Reduction in • Stimulation of threshold for platelet leukemia cell activation growth • Rebound worsening • Interactions with of thrombocytopenia other cytokines • Increased bone marrow reticulin