Biotechnology: Lecture 8 - Drugging innate immunity – Complement Therapeutics

Give an overview of the complement system pathway

1. C4: C3 (passive) → C3a + C3b (active)

2. C3b targets surface of pathogens → conformational change → attracts other complement proteins → cascade → C3 convertase

   • opsonization: labels for phagocytosis

   • C3 convertase → C5 convertase recruits more proteins → membrane attack complex → membrane C5b9 pores (lysis)

3. C3 convertase activates more C3 proteins (amplification)

4. Activation of C3a + C5a → anaphylatoxins

5. C3a activates immune cells (chemotaxis)

Give an overview of the classical complement system pathway

1. (IgG + IgM bind to pathogen) binds to C1

2. C1 binds → conformational channge → activation

3. C1 cleves C4 + C2 → C3 convertase

4. Activates the complement system

Give an overview of the mannose-binding lectin (MBL) complement system pathway

1. MBL/ ficolin detects carbohydrates on surface of pathogens

2. conformational change → C4 + C2 → C3 convertase

3. Activates the complement system

Give an overview of the alternative complement system pathway

1. spontaneous hydrolysis: C3 → C3b

2. distinct C3 convertase

3. Activates the complement system

Natural complement regulators

1. C1 inhibitor: Inhibits C1r and C1s, blocks MASP

2. CR1: degradation of C3b and C4b (classical & alternative)

3. MCP: promoting cleavage of C3b and C4b by Factor I

4. DAF: Accelerates decay of C3 and C5 convertases

5. CD59: Blocks MAC formation by inhibiting C9 polymerization

6. C4bp: inactivate C4b, dissociates C3 convertases

7. No regulation: MAC on own cells

Tissue damage in autoimmune disease

• deposition of autoimmunoglobulin and immune complexes in affected organs

• Vicious cycle’: complement activation and inflammation → tissue damage → C activation

• complement not always cause of disease, exacerbates condition and sustains proinflammatory cycle

Physiological roles of complement

• Direct killing of invading bacteria, infected cells

• Initiation/enhancement of inflammation at sites of injury or infection

• Cell activation: triggering of oxidase response and other cell activation events

• Enhancement of phagocytosis

• Immune complex handling

• B cell activation, adaptive immunity

considerations when designing anti-complement therapeutics

Point of inhibition

• C3?

• C5? (Start of the terminal pathway, pre-C5a)

• MAC formation?

Abundance of Target proteins

• Most abundant: C3 (~1mg/ml in serum)

• Acute phase response: don’t know how much protein to target

Long term systemic complement inhibition

• bacterial infections?

• immune complex disease?

Cost of generating biological reagents

• short half-life, more expensive

• bacterial expression vs. eukaryotic

What are some successful anti-complement therapeutics

1. sCR1: Avant Therapeutics

2. Anti-C5: Alexion Pharmaceuticals

3. APL-2: Apellis

Types of anti-complement therapeutics

• Biological reagents based on mammalian (human) naturally occurring regulators (e.g. sCR1/TP10)

• Antibody based therapeutics (e.g. anti-C5, Soliris)

• Small molecule inhibitors/ naturally occurring compounds (e.g. Compstatin)

Generating optimized recombinant proteins

1. Commercial Synthetic Construct

2. mRNA is isolated and reverse transcribed into cDNA

3. Plasmid clone library

• Inserted into plasmids using restriction enzymes (Nhe I and Not I).

• Recombinant plasmids are used to transform bacteria or transfect mammalian cells for protein production

Transfection of Mammalian Cells ( CHO, HEK, etc)

1. Transfection: introduce plasmid DNA

2. Initial Expression: cells begin to express the recombinant protein

3. Antibiotic Selection (Hygromycin): hygromycin resistance = sucessful transfection

4. Colony Isolation (Limiting Dilution): each well contains cells derived from a single colony

5. Screening (ELISA & Dot Blot/Western Blot): measures the amount and confirms identity

6. Clone Selection and Expansion: highest expressing clone is chosen

7. Protein Purification: Ion exchange/ Affinity chromatography, Gel filtration

1st generation recombinant complement inhibitor: sCR1 (TP10)

• soluble form of Complement Receptor 1 (CR1)

• Inhibits complement activation by accelerating decay of C3 and C5 convertases

• Human clinical trials: myocardial infarction, ARDS (acute

• respiratory distress syndrome), transplantation

• Ischaemia-reperfusion (many tissues), rats/mice

• Experimental demyelination (MS), rats

• Experimental myaesthenia gravis, rats

• Xenotransplantation (pig-primate)

• Antigen arthritis (rats)

2nd generation recombinant complement inhibitor

• modifying protein function to become more drug like

  • enhancing potency, specificity, half-life, and clinical efficacy

• Adding Fc domains increases serum half life

• APT070 in arthritis: anchors to cell membranes and inhibits C3 convertases

Ways to extend half life

• Fuse to antibody

• PEGylation: modification of lysine residues in the protein with polyethylene glycol (PEG)

  • increases size: reduces renal clearance and proteolysis

  • increases solubility

• Albumin-binding domains (Streptococcal protein G)

• Half-life doesn't always matter

Tailed reagent

Location at membrane

Systemic inhibition

Advantages of monoclonal antibodies

• increase half life

• ‘natural’ product, body is tolerant to them

• (target C5) does not affect opsonisation

• humanise mouse mAbs

describe structure of Eculizumab (Zuber et al, 2012)

• humanised mAb

• derived from the murine antihuman C5 antibody

• CDR = mouse anti-human C5 IgG

• FR = human germline

• hinge region =.human IgG2 (does not bind Fc receptors)

• CH2–CH3 = human IgG4 (unable to activate complement)

• modifications minimize immunogenicity and prevent pro-inflammatory responses mediated by the IgG Fc portion

describe function of Eculizumab

• Binds to C5

• blocks C5 convertase from binding (steric hindrance)

• cannot be ceaved into C5a and C5b (MAC)

• cleared from system

what alteraction were made to Ravalizumab?

• changed 2 amino acids in CDR to loosen binding

  • Tyr - 27 - His

  • Ser - 57 - His

• when pH changed from 6 to 7, antibody releases from C5

• Ag degraded but Ab can be recirculated

• changed 2 amino acids in Fc to enhance binding

  • Tyr - 27 - His

  • Ser - 57 - His

• half life = >30 days (longer than Eculizumab)

• extended dosing interval

Recombinant anti-C5: scFv and minibodies

(Marzari et al, 2002)

• C5 cleavage site of C5 = common target for neutralizing human mAbs: in vitro and in vivo

• isolation of an anti-C5 scFv Ab from a human phage display library

• Ab inhibits activation of C5 and the terminal complement complex implicated in cell and tissue damage

• Engineered into a minibody, has enhanced binding due to dimerisation

Problems with C5 inhibitors

• No terminal pathway = risk of infection with encapsulated bacteria

  • Neisseria species

• immunized patients with polyvalent meningococcal vaccine (2 weeks before)

• Children treated with eculizumab should also be vaccinated against Streptococcus pneumoniae and Haemophilus influenzae type b

• Extravascular Haemolysis = anaemia

Small molecule inhibitors: Compstatin and derivatives (Lambris et al)

• All based on a ~13-residue cyclic peptide,

• binds to C3 and inhibits the cleavage of C3 to C3a and C3b

• highly potent and selective C3 inhibitor

Compstatin Phage display

• Target = C3b

• Also checked for ability to block lytic attack (sheep red blood cells)

How does Compstatin function (MAstellos et al, 2015)

• binds to C3+C3b and prevents convertase interacting

  • binds between MG4 and MG5

• stops complement activation

• small peptide = cheap to make

• half life is an issue

Compstatin Derivatives

• Compstatin derivatives have been shown to be safe and effective in a series of ex vivo and in vivo experiments

• many diseases are targeted.

• But serum half-life was initially poor.

• Apellis attached a PEG moiety to 2 copies of APL-1 = APL-2 (pegcetacoplan)

  • increase half life

  • Clinically, the most successful derivative to date

• druggable: interactions are fairly low affinity so rely on avidity

Novel uses for Compstatin (Cp40)

Toothpaste: block gum disease and tooth decay

Natural reagents from other species: Ticks

• Ectoparasites: feed on blood

• Complement-inhibitory activities identified in salivary glands of soft tick (Ornithodoros moubata)

• Recombinant protein (OMCI) inhibits terminal pathway of human complement at C5 stage

• Inhibits in many species: easier in lab

• Short half life; resynthesis of C5 by liver is restoring complement levels

  quickly

Other small molecule inhibitors

• 3D53: a macrocyclic compound, binds the C5aR

  • potent, selective, orally active

  • potent inhibition in vivo in many rat models of human disease: neutropenia/sepsis, arthritis, immune-complex dermal inflammation, and ischemia-reperfusion injury

• K76COOH: partially oxidized derivative of natural terpenoid K-76

  • inhibits production of C5a

• FUT-175: synthetic broad-specificity serine protease inhibitor.

  • Potently inhibits both coagulation and complement proteinases inhibits C1s, factor D

• BCX-1470: serine protease inhibitor, inhibits factor D, C1s

• Selective inhibition of the membrane attack complex of complement by low molecular weight components of the aurin tricarboxylic acid synthetic complex

LN023 – Factor B

• Oral drug x2 a day

• reversible FB blocker

• Alternate pathway specific – blocking C3/C5 convertase

• protect against rheumatoid arthritis, C3G and PNH

The C3 GOF mouse model of aHUS

Idorsia Pharmaceutics Ltd: hC5a R antagonist

Blocks C5 receptor - stops its signalling immune cells

Protected mice on diet with it

reduction of MAC

what starts the Alternative pathway of complement activation?

spontaneous hydrolysis

name two cell membrane bound regulators of complement

DAF (CD55) Decay Accelerating Factor: Disrupts C3/C5 convertases

CD59 (Protectin): Inhibits the Membrane Attack Complex (MAC)

name the first licenced terminal pathway blocking monoclonal antibody

Eculizumab, 2007

what type of molecule is compstatin and how was it found

• Small molecule inhibitor

• Phage display screening