1M-CAgD-US-3006 February 2018

Understanding the Complement Cascade and Its Role inCold Agglutinin Disease

2M-CAgD-US-3006M-CAgD-US-3006 February 2018

Instructions

This information is provided as an educational resource for healthcare providers. It is not intended to be a substitute for review of the underlying reference materials and scientific literature. Healthcare providers should make all treatment decisions based on their medical judgement and the clinical profile of the individual patient. The following document cannot be copied, modified, used or distributed without the express written consent of Bioverativ.

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Pathogenesis of Cold Agglutinin Disease

LPD, lymphoproliferative disorder.Randen U, et al. Haematologica. 2014;99(3):497-504.

• Cold agglutinin–

associated

lymphoproliferative bone

marrow disorder

• A distinct type of LPD

• Cold agglutinin–

associated

lymphoproliferative bone

marrow disorder

• A distinct type of LPD

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Pathogenesis of Cold Agglutinin Disease

Hemolysis is entirely classical complement pathway dependentHemolysis is entirely classical complement pathway dependentC, complement; CA, cold agglutinin.Berentsen S. Br J Haematol. 2011;153(3):309-317.

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Pathogenesis: Therapeutic Implications Hemolysis is entirely classical

complement pathway dependent2

Therapeutic implications

Distinct clonal B-cell bone marrow disorder1

Therapeutic implications

C, complement; Ig, immunoglobulin; INH, inhibitor.1. Randen U, et al. Haematologica. 2014;99(3):497-504. 2. Berentsen S. Blood. 2014;123(26):4010-4012.

BIVV009BIVV009 is under

development and is not approved for the treatment of

cold agglutinin disease

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The Complement System: A Family of Circulating Proteins in the Blood Responsible for Immune Surveillance1

Classical Lectin Alternative

C, complement protein; MBL, mannan-binding lectin.1. Murphy K, et al. Janeway’s Immunobiology. 8th ed. New York: Garland Science; 2012.

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The Complement System: A Family of Circulating Proteins in the Blood Responsible for Immune Surveillance1

Classical Lectin Alternative

C, complement protein; MBL, mannan-binding lectin.1. Murphy K, et al. Janeway’s Immunobiology. 8th ed. New York: Garland Science; 2012.

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The C1 Complex Is the Activation Mechanism of the Classical Complement Pathway1

C1 complex activates the classical complement pathway:

1. The pattern recognition molecule C1q binds items such as immune complexes, apoptotic cells, etc

2. C1q undergoes a conformational change following binding to a target, resulting in C1r autoactivation

3. Activated C1r cleaves and activates C1s

4. Active C1s cleaves C4 and C2, forming the CP/LP C3 convertase

C, complement protein; CP, classical pathway; LP, lectin pathway; MBL, mannan-binding lectin.1. Murphy K, et al. Janeway’s Immunobiology. 8th ed. New York: Garland Science; 2012.

Classical Lectin Alternative

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The Classical Pathway Activates Upon Binding to Antibodies: A Bridge Between the Innate and Adaptive Immune Systems

C, complement protein; IgG, immunoglobulin G.1. Murphy K, et al. Janeway’s Immunobiology. 8th ed. New York: Garland Science; 2012.

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Cold Agglutinin Disease

Overview:• Autoimmune hemolytic anemia1

• Prevalence1,2: ~1/60,000–1/100,000; incidence3,4: ~1/300,000–1/1,000,000 • ~5000 patients in the United States• Patients are often elderly2 and present with anemia, fatigue, hemoglobinuria, and

acrocyanosis4

Etiology: • Primary (idiopathic)1

• Secondary: caused by an underlying condition (eg, infection, malignancy)• Majority of patients have detectable clonal B cells responsible for the production

of an autoantibody that binds to RBC <37°C (cold)5

Clinical symptoms: • Frequent need for transfusions1

• Chronic anemia with related symptoms (decreased quality of life)4

• Agglutination-associated symptoms4

• Thromboembolic complications4 CA activation in the classical pathway6

C, complement protein; CA, cold agglutinin; RBC, red blood cell.1. Berentsen S, et al. Haematologica. 2006;91(4):460–466. 2. Gertz MA. Hematol Am Soc Hematol Educ Program.2006;19–23. 3. Sokol RJ, et al. Br Med J. 1981;282:2023–2027. 4. Mullins M, et al. Blood Adv. 2017;1(13):839–848. 5. Bass GF, et al. Autoimmunity Rev. 2014;13(4-5):560–564. 6. Shi J, et al. Blood. 2014;26(123):4015–4022.

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Compared with healthy individuals, patients with cold agglutinin disease have lower levels of C4 and C2 but not C5, suggesting that the classical pathway is consumed in these patients, but the terminal pathway is not activated (ie, C5 cleavage)1

Patients With Cold Agglutinin Disease Are Hypocomplementemic for Upstream Classical Pathway Substrates

55

2.51

C5

Healthy Cold agglutinin disease

µg/m

L(m

ean

±SE

)

7.520

µg/m

L(m

ean

±SE

)

C2

Healthy Cold agglutinin disease

55207.5

12.5

b400

55

7.5

1

C4

Healthy Cold agglutinin disease

µg/m

L(m

ean

±SE

) b

a55207.5

12.5

C1s

Healthy Cold agglutinin disease

µg/m

L(m

ean

±SE

)

C, complement protein; SE, standard error. aP<0.01; bP<0.001.1. Shi J, et al. Blood. 2014;26(123):4015–4022. 2. Murphy K, et al. Janeway’s Immunobiology. 8th ed. New York: Garland Science; 2012.

Classical2 Lectin2 Alternative2

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Understanding the Role of Complement in Mediating RBC Destruction in Cold Agglutinin Disease1

Patient Sample

Complement-coated RBC

Flow cytometry Lysis+ Complement

Normal RBC+

RBC coated with antibodies

FL1-A, fluorescence intensity; FSC-A, forward scatter; RBC, red blood cell.1. Jager U, et al. ASH 2015; Poster.

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Cold Agglutinin Titers Correlate With the Amount of Complement Deposited on the RBC1

N = 40 samples

Cold agglutinin titer (1:X)12 150 1,800 22,000

55

7.5

1

0.15

C3

frag

men

t–po

sitiv

e R

BC

(%) r = 0.76

P<0.0001

C, complement protein; RBC, red blood cell.1. Shi J, et al. Blood. 2014;26(123):4015–4022.

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C3b Is an “Eat Me” Signal to Cells of the Immune System

MacrophageC3b-coated cells

C, complement protein.1. Wouters D, Zeerleder S. Haematologica. 2015;100(11):1388–1395.

PhagocytosisC3b is deposited on the target cell’s membrane, promoting its phagocytosis1

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An In Vitro Proxy for Extravascular Hemolysis

Normal RBC

+

RBC coated with complement

+ Complement

± inhibitor

+ Green dye

+Phagocytosis

RBC, red blood cell.

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C1s Inhibition Prevents Phagocytosis of RBCs Exposed to Cold Agglutinin Disease Autoantibodies1

No serum +100 µg/mL TNT003

+100 µg/mL IgG2a

25% NHS 10 mM EDTA

Phag

ocyt

osis

(mea

n %

±SE

) C3 fragm

ent–positive RB

C(%

control)

C3 fragment–positive RBCPhagocytosis 150

100

50

0

125

75

25

150

100

50

25

0

125

75

C, complement protein; IgG, immunoglobulin G; mAb, monoclonal antibody; NHS, normal human serum; RBC, red blood cell; SE, standard error. 1. Shi J, et al. Blood. 2014;26(123):4015–4022.

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C1s Inhibition Prevents Complement Dependent Hemolysis Driven by Cold Agglutinin Disease Autoantibodies

Thus, an upstream classical pathway inhibitor would prevent both extravascular and intravascular hemolysis1

Antibody concentration (µg/mL)

C, complement protein; IgG, immunoglobulin G; SE, standard error.1. Shi J, et al. Blood. 2014;26(123):4015–4022.

IgG2a

TNT003

120

100

80

60

40

20

00.01 1 100

Col

d ag

glut

inin

dis

ease

au

toan

tibod

y–m

edia

ted

hem

olys

is (m

ean

% ±

SE)

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C1s Inhibitors Prevent Anaphylatoxin Production Generated by Cold Agglutinin Disease Autoantibodies

[Ab] g/mL

C5a

(% In

hibi

tion)

0.01 1 1000

50

100

0.01 1 1000

50

100

Anaphylatoxin2

C3a

inhi

bitio

n (m

ean

% ±

SE)

C5a

inhi

bitio

n (m

ean

% ±

SE)

C, complement protein; IgG, immunoglobulin G; MBL, mannan-binding lectin; SE, standard error. 1. Murphy K, et al. Janeway’s Immunobiology. 8th ed. New York: Garland Science; 2012. 2. Shi J, et al. Blood. 2014;26(123):4015–4022.

Classical1 Lectin1 Alternative1 IgG2aTNT003

IgG2aTNT003

IgG2aTNT003

C4a

inhi

bitio

n(m

ean

% ±

SE)

Antibody concentration (µg/mL)

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Summary

Cold agglutinin disease is an autoimmune hemolytic anemia1

Cold agglutinins bind to the RBC and activate the classical complement pathway, leading to opsonin (C3b) deposition on the RBC surface and, in some cases, direct cellular lysis2,3

Opsonin-coated RBCs travel to the liver where they are phagocytosed2,3

Classical pathway inhibitors that target upstream of C3 and prevent opsonin deposition rescue cells from being phagocytosed or lysed by complement, specifically demonstrating the role of the classical pathway in mediating cold agglutinin–driven complement activation4

Classical pathway inhibitors, therefore, could potentially be efficacious in the treatment of cold agglutinin disease4

C, complement protein; RBC, red blood cell.1. Berentsen S, et al. Haematologica. 2006;91(4):460–466. 2. Stone MJ. Blood. 2010;116(17):3119–3120. 3. Berentsen S. Clin Lymphoma Myeloma. 2009;9(1):110-112. 4. Shi J, et al. Blood. 2014;26(123):4015–4022.