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Haematology module 1 > Assessmnet: ACE910 > Flashcards

Assessmnet: ACE910 Flashcards

1h of questions on ACE910 paper under exam conditions

1
Q

Describe the structure of protein C

A
  • Vitamin K-dependent serine protease zymogen
  • Conserved domain structure with FVII, FX, FIX
  • 1 Serine protease domain
  • 2 EGF like domains
  • 1 GLA domain
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2
Q

Describe the structure of FX (or FIX)

A
  • Serine protease domain
  • 2 EGF like domains
  • 1 GLA domain
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3
Q

In figure 6a, what reasons may explain the unexpected peak in the 0.3mg/kg group D-dimer?

A
  • The authors stated the anomaly was due to a blood collection artefact as none of the other coagulation markers were raised.
  • The authors also stated that all subjects were healthy.
  • Possible other causes would include pneumonia, vasculitis and unstable angina
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4
Q

Describe the pathophysiology of Haemophilia A

A
  • Factor VIII deficiency caused by mutation on X chromosome.
  • Recessive inheritance pattern
  • It affects the intrinsic pathway of the coagulation cascade
  • FVIII is unable to provide its cofactor function to FIX, so effective conversion of FX to FXa is impaired, leading to impaired generation of thrombin.
  • Increased risk of bleeding results from impaired thrombin generation
  • Severity of bleeding and risk is inversely proportional to plasma FVIII levels
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5
Q

What is a bispecific antibody and how does it work? How does it work in this study?

A
  • A bispecific antibody can recognise two different types of antigens.
  • It is made up of two different antigen binding fragments.
  • Variable domains in each fragment are engineered to bind to 2 distinct antigens simultaneously
  • The purpose of binding to 2 different antigens is usually to bring them together to potentiate an interaction
  • In this study, ACE910 has only a partial cofactor activity.
  • ACE910 doesn’t stabilise the FIXa active site or promote phospholipid binding, unlike FVIIIa
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6
Q

What are some of the advantages and disadvantages of this new ACE910 therapy?

A
  • Advantages include high bioavailability, SC administration (as opposed to IV) and reduced dosing frequency.
  • Also, ACE910 is unlikely to result in antibody formation that cross-reacts with FVIII, due to it’s different molecular structure.
  • ACE910 is unlikely to cause hypersensitivity reactions as the ADAs detected in the study were not IgE
  • One disadvantage is that ACE910, may increase risk of VTE and microangiopathy.
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7
Q

In figure 7, why did the development of ADA antibodies in the white participant affect the efficacy of ACE910? Would this affect the efficacy of ACE910 over time after multiple injections (Graphs show efficacy after 1 dose).

A
  • ADA antibodies were made in response to the foreign antigen of ACE910, so it was specifically targeted for the drug.
  • The antibodies in circulation must bind to and neutralise the effect of ACE910, either by binding to it’s active site (The fab regions) or by changing it’s conformation.
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8
Q

How is FVIII activated?

A
  • Thrombin activates FVIII into FVIIIa
  • FVIII interacts with exosite 1 and 2 on thrombin
  • Thrombin cleaves FVIII at multiple sites: R372 (Requires both exosites) and R1689 requires only exosite I
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9
Q

Why was a stepwise dose-escalation scheme used?

A
  • To identify and mitigate risk of investigational drugs given to humans for the first time
  • To make the impact of possible adverse reactions as small as possible as smaller doses are used.
  • Such adverse events may include thrombotic events relating to hypercoagulabilty (e.g DVT and PE)
  • Another adverse event that the dose-escalation scheme accounts for is the formation of allo-antibodies against the investigational compound.
  • The risk of anaphylaxis in reaction to foreign antigens is reduced when starting in smaller doses.
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10
Q

What disease might mimic mild haemophilia A?

A
  • Type 2 vWD: This is caused by a mutation in D’-D3 domain
  • FVIII can no longer be stabilised.
  • Therefore, the half-life of FVIII will be reduced, causing an increased bleeding tendency which is similar to that of mild haemophilia A
  • Also, type 3 vWD: Those lacking vWF can’t support factor VIII, (FVIII normally remains bound to vWF in circulation) so it is also low.
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11
Q

What are the limitations of current treatments that indicate the need for new therapies?

A
  • FVIII replacement therapy has a short half-life and involves frequent injections
  • IV administration also makes for greater inconvenience
  • Frequent injections place a burden on the life of the patient
  • FVIII replacement therapy requires dosing adjustment to maintain a FVIII:C>1%
  • In FVIII replacement therapy, some patients develop FVIII inhibitors in response to therapy
  • A new treatment with an easier administration route, longer half-life, less immunogenicity and more predictable pharmacokinetics is required to improve treatment and quality of life in haemophilia A patients
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12
Q

How is APC formed in response to coagulation?

A
  • Initial thrombin generation occurs
  • TM-FIIa complex forms around platelet plug (Thrombomodulin and thrombin)
  • PC-EPCR complex forms (Protein C and Endothelial Protein C Receptor)
  • Then above two complexes are brought together
  • APC formed and released from phospholipid membrane of endothelium.
  • Protein S acts as a non-enzymatic co factor (APC is fundamentally reliant on this).
  • APC proteolytically cleaves FVa and FVIIIa
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13
Q

Even though APTT approaches normal range with the ACE910, why is the maximum thrombin peak not reached? (Figures 4B, 5C and 5D)

A
  • possibly due to the fact that ACE910 doesn’t requirer activation before exerting it’s pharmacological effect
  • So ACE910 may accelerate the coagulation earlier, leading to APTT being higher than the APTT the equivalent activity of FVIII would produce.
  • So, therefore, the maximum thrombin peak may not have been reached since the dosage was simply not high enough.
  • Another possibility: ACE910 somehow interferes with the TFPI pathway, speeding up coagulation, but not affecting the overall thrombin generation as much.
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14
Q

Describe the current treatments for haemophilia A

A
  • Prophylactic intravenous FVIII agents, either recombinant ro plasma derived.
  • Desmopressin is an on-demand treatment for prolonged bleeding
  • Treatment for developers of ihibitors: ‘bypassing agents’ such as recombinant activated FVIII (rFVIIIa) and activated prothrombin complex concentrate (aPCC)
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15
Q

Suggest the possibilities for why one participant (Japanese) already had antidrug antibodies even before starting the therapy

A
  • Patient may have had a pre-existing autoimmune condition, where antibodies had already developed that can bind to any region or domain structure present on ACE910. The fact ACE910 is derived from humanised IgG supports this idea.
  • May be due to normal variation of endogenous antibodies and chance
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16
Q

How are bispecific antibodies produced to allow them to bind FIX and FX without neutralising their effect?

A

Anti-FIXa antibodies and anti-FX antibodies were produced.
-Variable heavy chains were fused with human IgG
-Fc heterodimerisation engineering brought the two binding regions together to form a humanised bispecific antibody.
//Look up: domain structures of FIX and FX

17
Q

What is the structure of ACE910?

A
  • ACE910 is a humanised bispecific monoclonal antibody
  • Made of Fc (crystallisable fragment) and Fab (antigen binding fragment)
  • The Fab is made of 2 light chain and 2 heavy chain, which are further divided into constant and variable domains.
  • Each arm of the Fab has a separate distinct variable region
  • It functions by bridging activated FIX (FIX) and FX (FXa) to restore function of missing FVIII
  • ACE910 has no structural homology to FVIII, so it is not expected to induce FVIII inhibitors or be affected by the presence of inhibitors.
18
Q

Explain figures 4a and 5a

A
  • Fig 4a represents normal plasma, and shows slightly shorted APTT with ACE910 administration due to mimicking activity of FVIII.
  • However, since there were no clinical findings or laboratory tests that indicated hypercoagulabilty (Figure 6), the change was not considered a clinically relevant coagulation abnormality.
19
Q

Why does this bispecific antibody have a long half-life with respect to it’s pharmacokinetics and pharmacodynamics? How is ACE910 removed from the circulation?

A
  • Amino-acid substituting in the engineering process of the antibody reduced it’s isoelectric point
  • This involves placing negatively charged amino acid residues next to positively charged clusters, to neutralise the overall charge.
  • Previous versions of the drug may have had positively charged clusters of amino acid residues that would have increased non-specific binding to the ECM, which would increase clearance of the molecule.
20
Q

Using the data in table 1, why wouldn’t the results be representative of what you would see in real clinical practice?

A
  • Haemophilia is seen more commonly in caucasians whereas the main patient population in this study was Japanese
  • The study used healthy participants who did not have haemophilia, their FVIII was just neutralised which does not replicate true FVIII deficiency
  • The age of subjects was about 30, no children of elderly were included in the study. In clinical practice, the range of age of patients would be expected to be wider.
21
Q

In the coagulation system, what is the domain structure of FVIII?

A
  • Polypeptide of 2332 amino acids
  • Composed of five globular domains and contains one Ca2+ and two Cu2+ ions
  • Made up of A1, a1, A2, a2, B, a3, A3, C1 and C2 domains.
  • The A domain is involved in protein-protein interactions.
  • The A2 domain binds to factor IXa heavy chain
  • The B domain is heavily N-glycosylated, enhances cellular transport but impairs transcription of FVIII. It is not necessary for the main function of FVIII.
  • The C domain is involved in phospholipid binding.
  • The a domains are small acidic peptides involved in protein binding and thrombin cleavage.
  • The a3 domain contains the vWF binding site
  • Structurally very similar to FV, suggesting they are the result of a duplication event
22
Q

Describe and explain figure 7

A
  • One white participant developed functional ADAs after exposure to ACE910, which caused a reduced ACE910 concentration, reduced APTT correction and reduced thrombin generation (Figure 7D, 7E, 7F).
  • The efficacy of ACE910 was significantly diminished, suggesting that patients who develop anti-ACE910 antibodies may not respond effectively to ACE910.
  • The data shows that the effect of ACE910 was diminished 56 days after administration. This could either be due to a delayed response and latent production of ADAs that neutralise ACE910, or that the ADAs increased ACE910 clearance while unaffecting it’s function.
23
Q

What are the main problems of factor replacement therapy?

A 
IV administration
Inhibitor Development
Short half-life
Variation of Pharmacokinetics
Patient adherence
24
Q

What is the function of FVIII in coagulation?

A
  • FVIII acts as a co-factor to amplify the proteolysis of FX by FIXa
  • FVIII is activated into FVIIIa by thrombin
  • FVIIIa forms the intrinsic tenase complex in combination with FIXa, which is more efficient than FIXa alone in converting FX into FXa
  • This leads to increased thrombin generation burst in the coagulation cascade.
25
Q

Compare and describe figures 4a and 5a

A
  • Figure 4a shows that when ACE910 was given to healhty subjects without FVIII neutralization, APTT was shortened slightly, in a dose-dependent manner.
  • Over time, APTT returned to ~30s, perhaps due to drop in ACE910 concentration (supported by fig 2A)
  • Figure 5a shows APTT when endogenous FVIII activity was depleted ex vivo by addition of anti-FVIII neutralising antibodies, in order to mimic the FVIII deficiency seen in haemophilia A patients, thus the placebo shows prolonged APTT of ~120s compared to the placebo of ~30s in figure 4a.
  • ACE910 shortened APTT (and increased peak height of TG) in a dose-dependent manner
  • In both figures, ACE910 demonstrates a long PD response, as long as 24 weeks.
  • In figure 5a, at a dose of 1mg/kg, the APTT reached the level equivalent to the normal plasma with endogenous FVIII activity of fig 4a (~30 seconds).
26
Q

How is FVIII inactivated (or removed from the circulation)?

A
  • FVIII is inactivated by the activated protein C pathway
  • Thrombin can act as an anticoagulant after interaction with thrombomodulin
  • Thrombin activates protein C, by cleavage at Arg169
  • Protein C, in complex with protein S (cofactor), can inactivate FVa and FVIIIa by proteolysis (Arg336 more so than Arg562 in FVIII)
  • Protein S enhances inactivation ~3fold
  • In addition to inactivation by APC, FVIIIa can spontaneously dissociate.
  • In activated FVIIIa, the A1 and A2 domains are only held together by weak (ionic) bonds, making it very unstable.

Haematology module 1 (2 decks)

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