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Periodic Report Summary 3 - COUNTERSTROKE (Treating stroke with Affibody molecules targeting the inflammatory mediator HMGB1)

Project Context and Objectives:
The great majority of stroke patients in Europe receive no specific therapy as they present more than 3 hours after insult onset. After this time point secondary neuroinflammation caused by the ischemic tissue damage results in additional loss of neurons. The objective of the COUNTERSTROKE project is to develop a new efficient therapy for treatment of stroke based on specific blocking of a well-characterized mediator of neuroinflammation, High Mobility Group Box 1 protein (HMGB1), which is released during the cerebral ischemic event and the subsequent sterile neuroinflammation. A uniquely wide therapeutic time window for successful HMGB1 targeting therapy has previously been demonstrated in several animal disease models, including experimental stroke. The therapeutic agent to be developed within COUNTERSTROKE consists of a specific Affibody molecule binding to and thereby neutralizing HMGB1. Affibody molecules are a class of engineered proteins acting as antibody mimetics, but they are significantly smaller than antibodies and therefore have favorable biodistribution properties. Affibody molecules have been used and tested in the clinic with regard to imaging with excellent results and no evidence of toxic adverse events. The scope of COUNTERSTROKE is to generate a candidate drug characterized and optimized for subsequent clinical development. For this purpose, COUNTERSTROKE has brought together 2 industrial partners and 5 expert groups from 4 European countries with world-renowned competence in the relevant fields, and with a strong commitment for collaborative work (see below). The participants possess complementary expertise and cutting-edge technology that are crucial for the development of a new therapeutic approach for cardiovascular diseases (CVDs). To achieve our objectives, we are undertaking an integrated programme of research focused on understanding key aspects of neuroinflammation and stroke and the industrial partners will translate this new knowledge into a novel therapeutic. Our programme is based on recent important and directly relevant know-hows generated in the laboratories of the participants on new pathways and the pathology in stroke and on relevant cutting-edge technology employed by these laboratories. COUNTERSTROKE has been specifically designed to provide key answers to the F P - 7 call HEALTH.2013.2.4.2-1: Discovery research to reveal novel targets for CVD treatment.

An amendment of the original general agreement has been approved for COUNTERSTROKE. The original structure of the Counterstroke project can be viewed as three tracks running in parallel. In comparison to many other projects with one defined target, Counterstroke started off with three defined targets, hence the three tracks. The difficulties encountered with the C-tail target slowed the project down during period one but as that track was abandoned it has not affect ed work with the remaining two tracks during period 2. Furthermore, as the two targets for the remaining two tracks (A-box and B-box) have been workable and the A-box target track this far has resulted in drug candidates with in vivo efficacy, the overall goal for the Counterstroke project is still achievable within the stipulated project time frame. Work on the B-box as a drug target has been halted as the A box track has resulted in drug candidates with excellent in vivo efficacy; the overall goal for the Counterstroke project. Additional tasks in the amendment were added as they provide additional information on the neutralizing features of candidate. The Project steering committee thus approached our EU scientific officer and proposed to amend the General Agreement with these changes. The amendment was approved as of 23d of May 2016..

The specific objectives as listed in Annex I are stated below. Objectives 3-6 are addressed in
this reporting period. Objective 7 is partially addressed in this reporting period.

Project objectives:

1. The overall aim is to develop one HMGB1-blocking Affibody molecule for successful treatment of stroke. The identification of such a therapeutic molecule in animal models as a future candidate drug is highly relevant for later clinical studies, since the HMGB1 molecule is 99% identical in mammals.

2. The initial step in our project will be to generate truncated HMGB1 and full length HMGB1 target molecules (A box, B box and C-terminal domains) followed by a validation based on liquid chromatography and tandem mass spectrometry technology.

3. Affibody molecule generation will be performed using phage display selection of primary leads and characterization using HMGB1-binding ELISA assays. Affibody molecules will be selected for their performance in binding in competition assays with two different, well characterized neutralizing anti-HMGB1 mAbs or with the TLR4/MD2 receptor complex.

4. Functional validation of Affibody molecules that are selected in the HMGB1-competition ELISA systems will primarily be performed in cell culture systems. Cell-based assays will be used to study the capacity of selected Affibody molecules to inhibit TNF release from HMGB1 - activated macrophages or to inhibit HMGB1-induced cell migration.

5. Affibody molecules demonstrating good functionality in the in vitro cellular assays will then be studied in vivo in a highly HMGB1-dependent, rapid, animal disease model of drug-induced liver toxicity and inflammation (DILI) (Antoine DI, et al. Mol Med. 2010; 16:479) Antoine DI, et al.20 12. I Hepatol. 56:1 070). The readout will be effects of selected Affibody molecules on survival and tissue damage in acetaminophen-induced intoxication in mice.

6. The capacity of selected drug candidates to penetrate the blood-brain-barrier will be studied with labeled Affibody molecules injected systemically into either healthy or stroke-induced mice or rats.

7. The therapeutic efficacy in stroke of selected Affibody molecules will be subsequently studied in three different stroke models performed in two independent laboratories. Functional (behavioral outcome), MRI imaging and brain histology assessments will be performed as readout methods in the MCAO mouse model (Gertz K. et al. 2012. Brain 135(Pt6): 1964; Katchanov I et al. 2003. Brain Pathol. 13:452;). Longitudinal brain MRI imaging and histological assessments will be studied in a novel rat stroke model (Amberg F, et al. 2012. Stroke. 43:2437).

8. The candidate drug that will be defined in these studies will be further evaluated in immunogenicity tests in silica and in human sera. Protein engineering steps will be undertaken to de-immunize, if needed. Characterization of the candidate molecule in preparation for human studies will be performed using production scale-up by peptide synthesis and toxicology studies in one animal species.

Project Results:
Successful achievements in period 1
The COUNTERSTROKE project was initiated 1st October 2013 and has thus been ongoing
for 36 months. The main achievements in period 1 (month 1-18) were:

• Successful cloning and production of the required recombinant HMGB1 protein variants
• Successful cloning and production of high affinity Affibody molecules specific for 2 of the 3 defined HMGB1 target epitopes.
• Successful definition of Affibody molecules with HMGB1 neutralising properties
verified by in vitro cellular assays.
• Successful definition of Affibody molecules with HMGB1 neutralising properties verified in the in vivo model of acetaminophen-induced liver toxicity.

The main achievements in period 2 of COUNTERSTROKE are:
• Successful affinity improvement of previously defined Affibody molecules by the process of formatting.
• Successful definition of improved HMGB1 neutralising activity of formatted Affibody molecules verified in the in vivo model of acetoaminophen-induced liver toxicity.

Successful achievements in period 3

• Demonstration that the radiolabeled candidate drug could pass through the blood brain barrier after systemic administration and enter the area of ischemic stroke.
• Based on available data, the PSC (TC 17/12/12) has nominated an Affibody candidate drug (CD), PEP26152/ABY-047 for further development.

Potential Impact:
The expected final result of the COUNTERSTROKE project is the generation of a candidate drug characterized and optimized for subsequent clinical development as treatment of stroke. The candidate drug shall be an Affibody molecule with high affinity and specificity for HMGB1 with demonstrated efficacy in experimental models of stroke. Following completion of the COUNTERSTROKE project, the main objective of exploitation is to progress the Candidate Drug through further preclinical and clinical development, and, if successful in pivotal clinical trials, to be followed by product registration and routine availability to stroke patients. Stroke is the second most common cause of death and the leading cause of disability in Europe. The total number of stroke deaths in the 27 European Union member countries is 508,000 per year European Heart Network, Brussels 2008) and the annual cost for stroke in this area is estimated at €27 billion (70% direct and 30% indirect costs). Reducing stroke mortality and long-term disability by evidence- based acute treatment is thus a major necessity. Fifteen years after its approval, recombinant tissue plasminogen activator remains the only pharmacological therapy approved for acute treatment of ischemic stroke. Because of strict inclusion criteria and safety concerns, the drug is administered to fewer than 10% of diagnosed patients. Given these facts, there remains a dire need and enormous opportunity for innovative therapies that can safely and more effectively treat a greater proportion of acute stroke patients. The concept of the COUNTERSTROKE project, i.e. to counteract neuroinflammation and reduce damage from acute ischemic cerebral infarction, warrants detailed study as the likelihood of success is high, and the pay-off in terms of reduced patient suffering and improved clinical outcomes is enormous. The project public website can be found at: See also: projects/counterstroke_en.html
List of Websites:

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