Structure/Function Analysis of Complement C3 and the Early Development of Modified C3

Proteins as a Possible Therapeutic

David C. Fritzinger, Ph.D.

Complement System •  Part of the Innate Immune System •  Ancient

•  Predates Adaptive Immune System •  Present in all vertebrates, some invertebrates •  Complete system present in bony fish, more primitive

organisms have more primitive complement system •  Recognizes �non-self� surfaces

•  Targets for phagocytosis •  Activation of complement can result in cell lysis •  Provides a link between the innate and adaptive immune

systems •  Consists of more than 30 proteins

•  Cell surface and solution phase

Complement Pathways

Regulation of Alternative Pathway: Fluid Phase Proteins

• Factor H•  Binds to C3b and prevents binding of factor B

•  Serves as cofactor for the cleavage of C3b by factor I

• Factor I•  Cleaves C3b when bound to fH

•  Cleavage products cannot form convertase

Change in structure between C3 and C3b

C3 C3b

Complement and Disease•  Complement is part of the host defense against disease

organisms.•  Under certain circumstances, inappropriate activation of

complement can cause, or be a contributing factor in certain diseases•  Ischemia/Reperfusion injuries

-  Stroke-  Myocardial infarction

•  Rheumatoid Arthritis•  Myasthenia gravis•  Others

Naja kaouthia

Similarity of CVF to C3 from Different Species

Comparison of the structures of C3 and Cobra Venom Factor (CVF)

Crystal Structures of C3b, C3c and CVF

C3b C3cCVF

Cub Domain Cub Domain

Alternative Pathway C3 Convertases

Formation C3b + Factor B

CVF + Factor B

Factor D

Factor D

Mg++

Mg++

C3bBb + Ba

CVFBb + Ba

Function C3 + H2O C3bBb or CVFBb C3b + C3a

Properties of C3b,Bb and CVF,Bb

C3b,Bb CVF,Bb

T1/2 at 37 oC 1.5 min. 7 hr.

Inactivation by Factors H and I Susceptible Resistant

C5 Convertase Activity - +

Site of Action Cell Surface Fluid phase

Implications of the stability of the CVF-containing Convertase

•  Because of the convertase stability and resistance to regulators of complement activity:•  CVF is able to exhaustively activate complement in vitro

and in vivo

•  This property has therapeutic implications, in that complement-mediated diseases could be treated by depleting complement.

In vivo complement depletion by CVF

Rat Human

Two parts to talk •  Preparation of a �Humanized CVF� (Substitution of CVF

sequences into human C3) •  Characterization of a number of hybrid proteins

•  Preclinical testing of one �Humanized CVF� •  Toxicity testing

•  Possible therapeutic in disease models

�Humanization” of CVF

•  CVF/cobra C3 substitutions demonstrated that the C-terminus of CVF β-chain (C3 α-chain) is important for C3 function and for functional differences between C3 and CVF

•  Prepared series of hybrid proteins in which portions of the C-terminus of the α-chain of human C3 replaced with homologous CVF sequences.

Human C3/CVF hybrids

Human C3

Complement Depletion

Factor B Cleavage

C3 Conversion

Measuring convertase decay by SPR !HC3-1550-4

HC3-1550 1491 RFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVRIEEQDGNDI 1560 |||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| HC3-1550-4 1491 RFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACETGVDYVYKTRLVRIEEQDGNDI 1560 ||||| | | | |.| .|||| |.| ... . ...|||||.|||||||.|.|||||||||| HC3-1496 1491 RFYHPDKGTGLLNKICIGNVCRCAGETCSSLNHQERIDVPLQIEKACETNVDYVYKTKLLRIEEQDGNDI 1560

(31.2 hr.)

(2.4 hr) (4.3 min)

(3.9 hr)

Ionic Interactions between C3b or CVF and factor B:

HC3-1550-4

HC3-1550 1491 RFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVRIEEQDGNDI 1560 |||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| HC3-1550-4 1491 RFYHPEKEDGKLNKLCRDELCRCAEENCFIQKSDDKVTLEERLDKACETGVDYVYKTRLVRIEEQDGNDI 1560 ||||| | | | |.| .|||| |.| ... . ...|||||.|||||||.|.|||||||||| HC3-1496 1491 RFYHPDKGTGLLNKICIGNVCRCAGETCSSLNHQERIDVPLQIEKACETNVDYVYKTKLLRIEEQDGNDI 1560

CVF,B (HC3-1550-4) C3b,Bb (HC3-1550)

Conclusions

•  Substitution of small portions of human C3 with homologous CVF sequences at the C-terminus of the C3 alpha-chain results in human C3 derivatives exhibiting CVF-like functions

•  Comparing activities of different hybrids allows one to predict the functions of sequences in C3/CVF

•  In several cases, predictions are supported by X-ray crystal structure and activity results

Preclinical testing of HC3-1496

•  InCode Biopharmaceutics, Inc.

•  Received Venture Capital funding from Avalon Ventures. •  Funding preclinical studies on HC3-1496

-  Toxicity testing

-  Efficacy in animal disease models

CVF replacements in hCVF (HC3-1496)

CVF is not very toxic

•  When injected in animals, it depletes complement, but is only somewhat toxic in large amounts

•  However, CVF activates C5 •  C5 activation results in C5a anaphylatoxin production

•  C5a rapidly inactivated by Carboxypeptidase N

-  Product is C5a-desArg (Missing C-terminal arginine)

-  C5a-desArg is still active in recruiting Neutraphils, which can cause lung damage

Does hCVF (HC3-1496) activate C5?

1) in vitro C5 activation 2) C5a ELISA

C5 α-chain

C5 α’-chain

C5 β-chain

Factor H

In vivo Complement Depletion by hCVF

In Rats In Primates

Pharmacokinetic study of hCVF in mice

Is hCVF toxic? In vivo Complement Depletion by hCVF: C3a and C5a production

C3a Production C5a Production

In vivo Complement Depletion by hCVF: Physiological Affects

Heart Rate Blood Pressure

In vivo Complement Depletion by HC3-1496: Physiological Affects

Mean Airway Pressure

hCVF in Animal Disease Models: Myocardial Ischemia/Reperfusion

Ejection Volume

*

hCVF in Animal Disease Models: Myocardial Ischemia/Reperfusion

C3b deposition PBS hCVF CVF

Control hCVF CVF

hCVF in animal disease models: Paroxysmal Nocturnal Hemoglobinuria

(PNH) Disease Model •  PNH is a rare disease

•  Clonal stem cell disorder-caused by PIG-A mutation

•  Affected cells generate little or no glycosylphosphatidylinositol (GPI)

•  Are unable to bind certain regulators of complement activation -  DAF (CD55)

-  CD59: interrupts formation of MAC

•  Affected cells more prone to be lysed by complement

•  Only current treatment is Eculizumab (Alexion�s Soliris) •  Humanized anti-C5 antibody

•  Prevents MAC formation

hCVF in animal disease models: Paroxysmal Nocturnal Hemoglobinuria

(PNH) Disease Model •  Experiment performed in vitro

•  Human serum samples treated with either hCVF, CVF, or PBS

•  Serum samples tested for remaining complement activity •  Samples blinded and sent to Mike Pangburn’s lab at

University of Texas Health Center in Tyler, TX •  Used FACS to measure amount of CD59 on cell surface

of erythrocytes from PNH patients

•  Cells treated with serum samples and recombinant factor H fragment which causes lysis of cells.

PNH Data

hCVF in animal disease models: Paroxysmal Nocturnal Hemoglobinuria

(PNH) Disease Model

% PNH cells lysed Serum treatment

% Complement depleted Patient 1 Patient 2 Patient 3 Patient 4 Average

Untreated Serum 0 84 87 95 91 89.3 hCVF 73 3 3 4 9 4.8 hCVF 100 0 11 7 0 4.5 CVF 100 0 0 0 0 0.0 No Serum NA 0 0 0 1 0.3

hCVF in animal disease models: Collagen induced Arthritis (CIA)

•  Mice immunized with chicken CII collagen. •  Two weeks later, a booster immunization was

given. •  Two groups

1) Untreated control. PBS injection.

2) Experimental 500 µg/kg HC3-1496 administered 6 days after booster immunization. 250 µg/kg HC3-1496 administered 5 days/week.

• Arthritis monitored by measuring diameter of hind paws, fore paws and ankles. Data on graph represents sum of measurements.

hCVF in animal disease models: Collagen induced Arthritis (CIA)

hCVF is less immunogenic than CVF

Mice injected with CVF Mice injected with hCVF

Other disease models •  AMD (Age-related Macular Degeneration) •  Myasthenia gravis •  Immunomodulation during recombinant Factor

VIII treatment of hemophilia •  Increasing effectiveness of mAb cancer

treatment: ADCC (Antibody-dependent cell mediated toxicity)

•  Ventilator Induced Lung Injury

Conclusions

•  Injection of hCVF into both monkeys and mice show the protein is essentially non-toxic

•  hCVF shown to be effective therapeutic in several disease models

•  hCVF has potential as a therapeutic in complement-mediated diseases

•  Like CVF, hCVF (HC3-1496) is capable of rapidly depleting complement, both in vitro and in vivo.

•  Unlike CVF, hCVF does not activate C5 •  Lessens chance of neutrophil activation and resulting tissue

damage •  hCVF also appears to be less immunogenis than CVF

Future work •  Two main areas of work.

•  Improve stability of proteins in vivo. -  It is most likely that hCVF is degraded by factors H and I -  Need to reduce binding of factor H to hybrid protein.

-  Crystal structure of C3b:fH complex. -  Have identified several residues in C3b important for factor H binding and

prepared clones to produce several of these proteins.

•  Reduce immunigenicity of proteins -  CVF substitution in HC3-1496 is only 167 amino acids: ~10% of protein. -  Of these 167 amino acids, about 40% are identical to human C3.

Therefore, we believe that immunogenicity should not be a problem. Moreover, we have shown results that suggest that hCVF has minimal immunogenicity in mice.

-  Have shown that changing one or several amino acids can have large effect on protein activity. -  It should be possible to design a hybrid protein with activity approximately

equal to HC3-1496 that only contains a few substitutions.

University of Texas HSC Michael Pangburn Viviana FerreiraCharles River Laboratories Stephen WilsonHarvard University Greg StahlUtrecht University Piet Gros Bert JanssenIncode Biopharmaceutics, Inc.

William St. JohnPaul Finnegan

University of Hawaii Cancer CenterCarl-Wilhelm VogelBrian HewMike ThorneJune LeeKatina Wong

Acknowledgements