Caplacizumab, a potential treatment for TTP

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Clinical outcomes






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Can Caplacizumab be a potential first drug therapy for Thrombotic Thrombocytopenic Purpura?


Name: Eugene Acquah
Supervisor: Professor Gary Stephens





I confirm that this is my own work and the use of materials from other sources has been properly and fully acknowledged.
Signature: Date: / /



Table of Contents

Abstract2

1. Introduction3
1.1 Thrombotic thrombocytopenic purpura: A Brief History3
1.2 Plasma Exchange6
1.3 Ablynx nanobody(Caplacizumab)8
1.4 Project12
2. Methods13
2.1 Selection Process14
2.2 Data extraction15
2.3 Statistics15
3. Results17
3.1 TITAN Trial17
3.1.2 Vesely et al.17
3.1.3 Chaturvedi et al.18
3.1.4 Zhan et al.19
3.1.5 Coppo et al.19
4. Discussion22
5. Conclusion31
Bibliography33
Appendix 137




ABSTRACT
Background: Thrombotic thrombocytopenic purpura (TTP) is a blood disorder which presents with severe thrombocytopenia, shistocytes (irregularly shaped red blood cells) which serve as an indication of microangiopathic hemolytic anemia, aggregation of platelets leading to organ damage due to tissue ischaemia. TTP is a very rare disorder with a prevalence between 4-11 cases per million being reported in numerous papers. Plasma exchange (PE) is a procedure which essentially involves filtering the blood: it has reduced the mortality rate of TTP. A novel nanobody developed by Ablynx – Caplacizumab works by binding to von Willebrand Factor (vWF) thereby preventing platelet aggregation.
Methods: The following databases were used in searching for literature on TTP; PubMed, Google Scholar, Science Direct and Web of Science. Keywords such as "Plasma exchange and clinical outcome" were used.
Results: The results from the trial showed that there was a statistically significant difference in days to platelet normalisation between treatment arm and the placebo. Clinical outcome was also superior in the treatment group with fewer exacerbations and 0 deaths being reported. Results from the other studies faired differently when compared to TITAN treatment group. Due to the different inclusion criteria for each trial and the number of patients recruited, it would not be appropriate to make deductions from the data available. Overall, the results from the TITAN trial indicates that when caplacizumab is used in conjunction with PE, the clinical outcomes are much better when compared to plasma exchange on its own.

INTRODUCTION
1.1 Thrombotic Thrombocytopenic Purpura: Brief History
The first description of Thrombotic Thrombocytopenic Purpura is detailed in an account presented by Dr Eli Moschcowitz to the New York Pathological Society in 1924 (J. Evan Sadler 2008). In this account that he presented, a 16 year old girl in good health was taken ill abruptly and presented with the following symptoms; frail arms, fever, pain when moving wrist and elbow and finally, pallor. An initial 10 days passed without any improvements in her symptoms after which she was taken into hospital for admission. Her symptoms deteriorated further. Within the period of 4 days, she succumbed to facial paralysis and coma which led to her subsequent death the following day. Examination of the body revealed the presence of blood clots in arterioles and other blood vessels of various organs i.e. heart, spleen, liver and kidney). Unfortunately, Dr Moschcowitz gave no account of platelet count or the presence of shistocytes, however, the findings from the autopsy led to the recognition of this case as the first reported incidence of idiopathic thrombotic thrombocytopenic purpura. Following this, TTP was recognised by a group of five symptoms with variable presentations in each individual patient: this pentad consisted of fever, microangiopathic haemolytic anaemia (MAHA), thrombocytopenia, neurological symptoms and renal function (Scully et al. 2014). However, the distinctive pathological feature of TTP was the presence of platelet aggregates in many small vessels of numerous organs (Scully et al. 2014).
During those times, the outcome of TTP was almost certainly death. The mortality rate surpassed 90% with the length of hospital admission at an average of 2 weeks before expiration (J. Evan Sadler 2008). Reports of plasma exchange or plasma infusion as treatment of acute TTP surfaced between 1964 and the 1980s. One of the most notable reports was presented by Jefferson D Upshaw in regards to a female patient with MAHA and thrombocytopenia which was resolved post-treatment with "plasma containing blood products" (Kremer Hovinga & Lämmle 2012). A comparison of the effectiveness between plasma exchange and plasma infusion was carried out in a randomized trial. The trial established plasma exchange as the treatment of choice with 78% of patients surviving after 6 months compared to 63% of those on plasma infusion (Vesely et al. 2012). Introduction of plasma exchange saw a significant increase in survival rate from a mere 10% to above 90% (Furlan & Lämmle 2001). However, this is not the case reported in every patient. Treatment fails in a small proportion of patients with a resistant form of TTP and for that reason, they continually require plasma exchange (Kremer Hovinga & Lämmle 2012).
Moake et al published a paper in 1982 (J. Evan Sadler 2008) which identified and characterized the underlying pathogenesis of TTP. They reported the presence of large multimers of vonWillebrand Factor (vWF) found in plasma samples obtained from 4 different patients. The unusually high large vWF multimers became known as ultra large vonWillebrand Factor (UL-vWF) (Sarig 2014). UL-vWF was found to be present only in patients with acquired or congenital TTP but absent in healthy individuals. This led to the proposition that TTP patients lacked an enzyme that cleaves UL-vWF thus preventing platelet aggregation (J. Evan Sadler 2008). It wasn't until 1996 that Tsai and Furlan (J. Evan Sadler 2008) identified this enzyme. The preceding years led to the purification and cloning of the vWF-cleaving enzyme. This protease was named ADAMTS13 (a disintegrin and metalloprotease with thrombospondin repeats) - the number 13 denotes its position in the metalloprotease family. The location of the gene which codes for ADAMTS13 was mapped to chromosome 9q34 (Levy et al. 2001; Zheng et al. 2001): a mutation of this gene is responsible for hereditary TTP. Knowledge of the role of ADMATS13 was acquired in the years following identification but a major problem is how to apply the knowledge in clinical settings. The Figure below illustrates the normal activities of ADAMTS13 protein and effects of its deficiency.

Figure 1_ (A) ADAMTS13 enzymes found in the plasma of normal people bind to and cleaves ULVWF multimers which are produced and secreted in long strands by the endothelial cells. (B) There is high probability of strands of ULVWF multimers being attached firmly to endothelial cells by P-selectin molecules. The way in which the ADAMTS13 enzyme functions is to attach itself to exposed domains thereby cleaving the ULVWF proteolytically. VWF (smaller forms) resulting from the cleaving process are able to circulate the blood without the risk of adhesion or aggregate formation. (C) TTP patients have an absence or are severely deficient in ADAMTS13 activity. This leads to ULVWF multimers remaining uncleaved thus inducing platelet adhesion and aggregation. Mutations in ADAMTS13 gene (results in congenital TTP) or acquired flaws can both cause TTP. (Wyrick-Glatzel 2004)

1.2 Plasma Exchange
Plasma exchange (aka plasmapheresis) is a method by which blood is purified via the removal of blood components such as autoantibodies, immune complexes, endotoxins and immunoglobulins (Basic-Jukic et al. 2005). Abel, Rowntree, and Turne coined the term "apheresis" from the Greek word "aphairesis", which means to divide into constituent or distinct elements. They employed this term to describe a process then known as manual plasma exchange (Winters 2012). The term apheresis is not exclusive to plasma exchange: it is now used widely in describing other therapeutic methods of segregating, blood components (Winters 2012). Of all the 'apheresis' procedures in use, plasma exchange or therapeutic plasma exchange (TPE) is the most frequently used procedure. TPE requires the removal of large quantities of plasma and without replacement of the lost fluid, the patient could develop vasomotor collapse as a result of hypovolemia (Zbigniew M. et al. 2013). Although the usage of the term plasmapheresis and plasma exchange are intertwined, it is worth noting that there are differences in the procedures. For that reason, the term plasma exchange shall be used solely henceforth.
It is believed that, TPE removes and replaces substances in the blood that contribute to the pathogenesis of TTP and other blood related disorders. However, this attribute of TPE does not paint the full picture when it comes to the responses shown in certain disorders. It has also been reported that, TPE could have potential effects on immunomodulation other than effects on Ig.
The removal and exchange of plasma can be done in two ways: separating different components from the plasma based on size or their densities (Winters 2012). For this reason, there are two types of devices used in TPE which are categorized based on their separation technique. These devices use filters or centrifugation respectively. Centrifugation devices use centrifugal force to separate components of the blood. The heavier (most dense) components such RBCs (red blood cells), fall to the bottom of the tube whereas the lighter elements such as plasma, layers at the top. In between the two extremes are granulocytes, lymphocytes and platelets (Winters 2012). The plasma acquired from this process is no longer required therefore, it is discarded: the remaining components are returned to the patient in addition with replacement fluid. The recommended amount of plasma volume that can be exchanged safely without further complications is 1-1.5 plasma volumes. Beyond this volume, there will be no benefit to the patient but rather an increase in the risk of complications associated with TPE. TPE is an indiscriminate process therefore it removes both normal and disease causing plasma components. There are downsides to this. For example, laboratory testing can provide false negative results for infections, autoantibodies, alloantibodies, activity of enzyme and coagulation factors. As mentioned above, fluid replacement is a major requirement in TPE. Human Albumin at a strength of 4%-5% in saline solution is the first choice fluid. Albumin is highly priced compared to plasma - this is seen as a disadvantage. The positive effect of albumin on hypovolemia makes it an essential component of fluid replacement. It achieves this positive effect as a result of its hyper oncotic properties (Winters 2012).
Access to the vascular system is required in order to carry out TPE and for this reason, it is a requirement for all patients if possible to have an entry point which will provide access to a central venous line for an effective treatment. Reports in literature have no backing for this perception. As of now, the standard therapy for treating acquired TTP is plasma exchange. There are a number of different types of plasma available: 24-h plasma, cryoprecipitate-poor plasma and the most commonly used, fresh-frozen plasma (FFP) (Gail A. Rock et al. 1991)Fresh Frozen Plasma is plasma which has been acquired from a donor and kept under freezing conditions in order to preserve it and keep it fresh. Once acquired, regulations require the plasma to be frozen within eight hours thus preserving the coagulation factors which are liable to change (Chaffin n.d.). There are risks associated with the administration of FFP especially allergic reactions such as anaphylaxis, haemolysis and transfusion-related acute lung injury (Duguid et al. 2004). Cryoprecipitate-poor plasma is plasma derived from thawed FFP. Once thawed, the surplus plasma is separated from the precipitate and discarded off. The remaining plasma is then known as Cryoprecipitate poor plasma (Cryo-Poor plasma). It is usually stored at -18 degrees and thawed between 1-6 degrees prior to use 24-h plasma is essentially FFP but with one major difference: 24-h plasma is kept in the freezer for 24 hours as compared to 8 hours for FPP. It is however considered to be clinically equivalent to FFP but with slight differences in the Factor V and Factor VIII levels (Duguid et al. 2004).
The overall mechanism of plasma exchange is the replenishment of ADAMTS13, the removal of multimers of UL-vWF and any autoantibodies against ADMATS13.
1.3 Nanobody
A nanobody can be considered to be a smaller form of an antibody with enhanced stability and specificity. Antibodies consist of two heavy chains, two light chains and a hypervariable region which gives rise to many different types of antibodies. In the case of nanobodies, the light chains are absent: it consist of only heavy chains with a single variable domain (VHH) and two constant domains known as CH2 and CH3 (refer to Figure 2a. for a comparison between a nanobody and an antibody). Nanobody technology came into existence when it was discovered that camels and llamas poses functioning antibodies deprived of light chains. Nanobodies have properties of conventional antibodies in addition to characteristics of small molecule drugs. The following are the properties (Ablynx.com, 2015) of nanobodies:
low intrinsic toxicity
high specificity and affinity for targets
high stability
other possible routes of administration other than injection
VHH12-15kDaunique formatting flexibility (up to penta-velant)speed and ease of generating multi-specificsnano- to picomolar affinitiesfavourable biophysical properties (Tm, solubility, viscosity)tackling intractable targetsmultiple administration routesmanufacturing in microbial cellsAblynx's NanobodyVHH12-15kDaunique formatting flexibility (up to penta-velant)speed and ease of generating multi-specificsnano- to picomolar affinitiesfavourable biophysical properties (Tm, solubility, viscosity)tackling intractable targetsmultiple administration routesmanufacturing in microbial cellsAblynx's Nanobody Ease of manufacturing.
VHH
12-15kDa
unique formatting flexibility (up to penta-velant)
speed and ease of generating multi-specifics
nano- to picomolar affinities
favourable biophysical properties (Tm, solubility, viscosity)
tackling intractable targets
multiple administration routes
manufacturing in microbial cells
Ablynx's Nanobody
VHH
12-15kDa
unique formatting flexibility (up to penta-velant)
speed and ease of generating multi-specifics
nano- to picomolar affinities
favourable biophysical properties (Tm, solubility, viscosity)
tackling intractable targets
multiple administration routes
manufacturing in microbial cells
Ablynx's Nanobody





Figure 2a.

Ablynx NV is a company specialising in the development of nanobodies. This Belgium based biopharmaceutical company have produced a nanobody, caplacizumab (ALX-0081) for use as treatment of TTP. They currently have two formulations undergoing clinical tests: one suitable for intravenous (I.V) and the other for subcutaneous (s.c) administration (Holz 2012). ALX-0081 is believed to work by targeting the domains in vWf specifically the A1 domains thus preventing an interaction between GP1b receptor glycoprotein located on platelets and vWf. In so doing, it prevents the aggregation of platelets (see figure 2b.). At the moment, there are several marketed anti-thrombotic drugs available on the market - ALX-0081 showed similar effectiveness and a relatable safety profile as the marketed drugs (Gurevitz et al. 1998). During Phase I clinical trials, the safety and tolerability of ALX-0081 was evaluated via randomised, double-blind, placebo-controlled studies which involved administering single or multiple doses via i.v. injections/infusions or s.c. injections. The results of this trial showed good tolerability of ALX-0081 in the absence of notable complications or bleeding events (Holz 2012) After completion of the Phase I trials, ALX-0081 underwent a study which examined the efficacy and safety in conjunction with TPE. The design of the TITAN trial (Phase II) took the format of a single-blind, randomised, placebo-controlled trial involving participants from different countries. A summary of the study format is presented in the Fig 3. (Holz 2012). The criteria by which participants were recruited into the study are numerous therefore, only the major exclusion criteria will be listed. These are bone marrow transplant, known congenital TTP, severe active infections or disseminated intravascular coagulation (Holz 2012). The qualified subjects were then divided into the placebo group and treatment group in a 1:1 ratio. The treatment group received the standard of care (plasma exchange) and ALX-0081 whereas the placebo group only received the standard of care. The patients were monitored from the first day of treatment up until 12 months after the last dose. The overall aim of the TITAN trial was to determine the effect ALX-0081 will have in association with TPE in improving the outcome of acute TTP (Holz 2012).




Figure 2b. (Ablynx 2014). anti-vWF Nanobody (Caplacizumab) binds to the A1 domain on ULvWF and prevents an interaction between the platelets and the ULvWF. This ensures that aggregates of ULvWF and platelets do not form.

Main inclusion criterium:Patients with acquired TTP, requiring PE.Main exclusion criteria:Severe infection/sepsisPregnancyBone marrow transplant.Disseminated intravascular coagulationKnown congenital TTP.Main inclusion criterium:Patients with acquired TTP, requiring PE.Main exclusion criteria:Severe infection/sepsisPregnancyBone marrow transplant.Disseminated intravascular coagulationKnown congenital TTP.

Main inclusion criterium:

Patients with acquired TTP, requiring PE.

Main exclusion criteria:

Severe infection/sepsis
Pregnancy
Bone marrow transplant.
Disseminated intravascular coagulation
Known congenital TTP.

Main inclusion criterium:

Patients with acquired TTP, requiring PE.

Main exclusion criteria:

Severe infection/sepsis
Pregnancy
Bone marrow transplant.
Disseminated intravascular coagulation
Known congenital TTP.











Figure 3. Summary of TITAN Trial (Holz 2012). The initial participant projection was set at 110 patients. After recruiting 75 patients, admission was stopped in order to aid fast processing of trial data.


1.4 Project
"Caplacizumab; Potential first drug Therapy for Thrombotic Thrombocytopenic Purpura or in conjunction with Plasma Exchange?" is the title of my project. As the title briefly explains, my project is based solely on providing an answer to the question being asked. My approach to answering this question is based on published literature, data collection and analysis of the data.
This project will involve a great deal of focused and directed web searches on databases such as PubMed, Google Scholar, ClinicalTrials.gov, web of science and Science Direct. Information retrieved from these sources will then be used as an aid in answering the question as stated above. Finally the aim of this project is to compare and contrast the efficacy of Caplacizumab and Plasma Exchange (PEX) and the prospects of it being either a standalone therapy or in conjunction with Plasma Exchange.











METHODS
Thrombotic Thrombocytopenic Purpura (TTP) is a very complex disease with knowledge regarding its pathogenesis still not fully understood. In recent years, there has been an explosion of interest in this rare blood disorder. This explosion of interest has led to an exponential growth in the number of research papers and other sources of information being made available. Majority of research on TTP are based on understanding the complexity of the pathogenesis, how to improve diagnosis and better management of the condition.
In order to fully answer my research question, it was essential for me to be aware of the various channels of scientific information available. In modern times, the internet is becoming a significant avenue for information but however, due to the ease of accessibility of this medium, great care must be taken in selecting relevant and accurate information. My Methodology was to systematically search online for publications relating to TTP on the numerous databases available. Databases such as PubMed, Web of Science, Science Direct, and Google Scholar were all used. Out of all the previously listed databases, PubMed was the most frequently accessed. PubMed is part of the Entrez information retrieval system which was first made available to the public in January of 1996 (Nlm.nih.gov, 2015). This database is under the care of the National Centre for Biotechnology Information (NCBI) at the Nation Library of Medicine (NLM), which is itself, the largest library biomedical sciences in the world (Nlm.nih.gov, 2015). PubMed offers the user a range of services which make it easier and faster when searching for specific topics. From access to MEDLINE (a database in fields of medicine, veterinary medicine, dentistry and nursing) to spell check features: PubMed is fully equipped as an aid to literature search. Web of Science was another important database heavily used. It was developed based on the ideas of Dr. Eugene Garfield. Web of Science offers a comprehensive search tool which enables the user to make subject specific searches. This database is managed and controlled by Thomson Reuters and it is currently the leading source of research data. The other sources as mentioned above were also essential to this project.
2.1 Selection Process
Before beginning the process of searching, a list of four main search areas was made; TTP, ADMATS13, platelet count and Plasma Exchange. The search process included articles published between 1920 and 2015. Web of Science provides the capability of searching based on more than one field of keyword. I utilized this function by using a string of keywords whilst searching for articles on TTP. The keywords used were as follows: "pathogenesis, epidemiology, diagnosis, and treatment, management, acquired and congenital". Other keywords used in the search were, ADAMTS13 activity, platelet normalisation, von Willebrand factor, plasma exchange. Information regarding the nanobody caplacizumab was derived solely from the manufacturer Ablynx. Data from the TITAN trial (a phase II trial) was also derived from this company. Due to the infancy of this nanobody and its clinical trial, there was scarcity of other sources of data regarding its clinical effectiveness. Only articles written in the English language were assessed for their suitability from all the databases. The initial process of selection involved identifying the title and ensuring its appropriateness and careful reading of the abstract. Articles were selected based on their germaneness to the topic at hand. Some relevant articles which were identified could not be assessed therefore, were not included.

2.1.2 Data Extraction
Data extracted from the selected articles were mainly based on ADAMTS13 activity and the clinical outcomes of plasma exchange and other treatments. The sample sizes used in each of the studies were different and for this reason, most or all of the data required normalisation in order to accurately compare the results. Normalisation was achieved by converting the raw data into percentages. In order to provide a more visual representation of the collected data, a boxplot and a stacked bar chart were produced. A variety of tables were produced indicating the clinical and demographic presentations of participants in the trial.
2.1.3 Statistics
In order to make accurate comparisons between clinical outcomes from the different treatment groups in each study, a statistical test was needed. The test used was a Z test; it calculates the difference between two population proportions thus determining whether they differ significantly in a particular category i.e. response to treatment or exacerbation. A null hypothesis is then formulated for each of the following clinical outcomes. An example null hypothesis would be, there is no significant difference between patients who had a response from the treatment group and the placebo group. There are two results derived from the Z test; the Z-score and p-value. The Z-score is the test statistic whereas the p-value gives a probability of a statistical observation which can then be assessed using a normal distribution calculator (Stattrek.com, 2015). Calculating the Z test with the results available would not have been possible without the help of a Z test calculator (Socscistatistics.com, 2015). The calculation is based on the equation below.
p1- p2-0p 1- p(1n1+ 1n2)
Where:
p1 = proportion from the first population
p2 = proportion from the second population
n (1, 2) = total number of people from each population.
The requirements of this test are; a sample from each of the groups for comparison and categorical data. For this reason, the Z-test is the most suitable test for the purpose of making comparisons between clinical outcomes from different groups.










RESULTS
3.1 TITAN Trial
A total of 75 patients were recruited into the TITAN trial. Out of the 75 participants, 36 were randomly allocated into the treatment group and the remaining 39 were placed in the placebo group. The overall mean age of patients in both the treatment group and the placebo group was 41.6 ± 12.9. There were 31 males and 44 females working out as a percentage of 41% and 59% respectively. 88% of the participants were Caucasian and the remaining 12% were black. The mean baseline BMI was 29.1 ± 7.7. Out of the 75 patients, only 68% of them experienced an initial episode of TTP. ADAMTS13 activity < 10% was recorded in 28 patients and activity 10% was present in 2: ADAMTS13 activity was not recorded in 6 patients. 3 patients from the treatment group experienced exacerbations as compared to the placebo arm. 29 achieved complete remission in treatment group and 18 in placebo group. 13 patients from both treatment groups experienced relapse one month after treatment. There were 0 deaths in the treatment group and 2 in the placebo group (Ablynx 2014). Table 1 summarizes the demographics of the patients along with clinical features.
3.1.2 Vesely et al
This study looks at 142 patients diagnosed with TTP who were on the register of the Oklahoma Blood Institute (OBI). The treatment standard was the daily exchange of plasma using either FFP or cryosupernatant plasma. The average age of the 142 patients was 47.3 years and 69% of them were female. The racial distribution were as follows: white 71%; African American 19%; Native American 6% and other races 4%). They presented with an average platelet count of 13.25 x 109/L and 1270.3 U/L for LDH levels. 25 out of 142 had