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Treating stroke with Affibody molecules targeting the inflammatory mediator HMGB1

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

Executive Summary:
The scope of the COUNTERSTROKE project was to generate a candidate drug targeting the inflammatory mediator High mobility group box 1 protein (HMGB1) to be used in treatment of stroke. The candidate drug should be validated in animal models, characterized and optimized for subsequent clinical development.
Stroke remains a leading cause of death and disability throughout the world. Within EU, more than 500 000 persons die of stroke each year. Stroke occurs as a consequence of hemorrhagic insult or artery occlusion due to underlying cardiovascular disease. Pivotal in beneficial treatment of stroke is instant intervention. Blood clot dissolution using recombinant tissue plasminogen activator is the only pharmacological treatment demonstrated to limit neurological damage in stroke, but is only effective for patients who present within 3 hours after stroke onset. Thus, there is an unmet need for an efficacious therapy that can be administered within and beyond 3 hours to achieve neuroprotection.
HMGB1, released during the cerebral ischemic event and the subsequent neuro-inflammation, is a well-characterized mediator of inflammation. Beneficial effects of blocking HMGB1 is proven in preclinical stroke studies. The candidate drug to be developed should be an Affibody molecule (AFB) binding to and neutralizing HMGB1. AFB molecules are engineered proteins significantly smaller than antibodies and therefore having favorable biodistribution properties, and a history of being efficient and non-toxic in clinical trials.

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 favourable 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. The outline of the COUNTERSTROKE project plan is shown in Figure 1.

Figure 1: Outline of the COUNTERSTROKE project plan

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 HMGB1 target molecules followed by a validation based on liquid chromatography and tandem mass spectrometry technology.
3. Affibody molecule generation
4. Functional validation
5. Evaluation in in vitro cellular assays and in vivo disease model.
6. The capacity of the selected drug candidate to penetrate the blood-brain-barrier.
7. The therapeutic efficacy of the Affibody molecules in in vivo stroke models.
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.

Additionally, over-arching objectives of the COUNTERSTROKE project has been to supply an efficient management structure (included in WP9) and to form a structure for dissemination and IPR handling (included in WP10).

Ethical permits required for studies performed within the consortium are in place and reported in the periodic reports. All studies have been performed in compliance with the approved ethical permits.

Project Results:
The Counterstroke project has mainly progressed according to the initially described project plan and thus the description of main S&T results below will follow the chronological order of the included work packages (see Figure 1).
The project has been greatly impacted by a case of scientific misconduct performed by our partner Daniel J Antoine, University of Liverpool. This was revealed in April 2018 which has made it difficult to perform corrective measures. This will further be discussed in section scientific misconduct by consortia partner below.

3.1 Generation of targets (HMGB1 variants)
Work package 1 leader: HMGBiotech srl, lead beneficiary number 3.

In order to generate the necessary target proteins for the generation of HMGB1-specific AFBs, the Counterstroke project was initiated by the production and expression of HMGB1 protein variants. These were successfully cloned (by partner 1) and produced (by partner 3).

3.2 Validation of HMGB1 target proteins
Work package 2 leader: The University of Liverpool (UofL), lead beneficiary number 4.

Produced HMGB1 target proteins were characterized by bioanalytical assessments in order to define purity, structure, redox status and functional activities. Partner UNISR (partner 5) evaluated the functional activities of the produced full-length proteins and found them to be able to induce migration of fibroblasts (full-length all-thiol HMGB1) and cytokine production in macrophages (full-length disulfide HMGB1).
In conjunction with the revealed scientific misconduct by the partner at UofL, partner 4, in April 2018, the raw data for the first deliverable within this work package was revisited. UofLs internal investigation then revealed that they can find no records of any analysis being performed at all with regard to WP2. As a consequence, the first deliverable was rejected, rewritten and re-approved in September 2018.

The impact of this scientific misconduct for the Counterstroke project is manageable as the results regarding functionality and purity are correct. The continuous work based on the use of the produced proteins is still valid.

3.3 Generation and characterization of Affibody molecules (AFBs)
Work package 3 leader: Affibody AB, lead beneficiary number 2.

The next step within the Counterstroke project was to generate primary AFBs against different HMGB1 variants, using the recombinant proteins produced within the project.

3.4 Functional validation of Affibody molecules
Work package 4 leader: UNISR, lead beneficiary number 5.

This part of the Counterstroke work was initiated in parallel with work package 3, the generation of Affibody molecules. The objective was to validate the inhibitory functionality of the generated Affibody molecules in cell-based in vitro assays.

3.5 Functional validation of Affibody molecules
Work package 5 leader: University of Liverpool, lead beneficiary number 4.

To further confirm that selected Affibody molecules had the ability to inhibit HMGB1-
mediated inflammation in vivo, the Affibody molecules selected in WP04 were tested in vivo.

3.6 The ability of AFBs to penetrate the Blood-brain barrier
Work package 6, lead beneficiary Karolinska Institutet (partner 1)

This part of the Counterstroke work was performed in order to ensure that an Affibody molecule, specifically the nominated candidate drug, can penetrate the blood-brain-barrier after an ischemic stroke.

3.7 Functional studies in experimental stroke
Work package 7; lead beneficiary Charité (partner 6)

The objective of this work was to determine ameliorative effects of treatment with Affibody molecules targeting HMGB1 on different preclinical ischemic stroke models.

By using two different ischemic stroke models and two different treatment protocols in each model, the effects on functional outcome, lesion size and BBB leakage by the nominated candidate drug were evaluated. We could not demonstrate any ameliorative effects on functional outcome and lesion size. Thus, HMGB1 blockage by AFB as treatment for stroke was not successful.

The negative results above could have several explanations. The two most likely are either the inability of the selected Affibody molecules in the selected doses to neutralize HMGB1 released during stroke and the second is the redundancy in the inflammatory mechanisms activated after a stroke. In that scenario, neutralization of HMGB1 alone is not sufficient to impact inflammation ensuing ischemic stroke.

3.8 Candidate drug evaluation
Work package 8; lead beneficiary Affibody AB (partner 2)

In the drug evaluation part of COUNTERSTROKE, the objective was to generate a well characterized Affibody candidate drug (CD) for further preclinical and clinical development. We set out:
1. To assess potential immunogenicity of studied Affibody molecules
2. If needed, to de-immunize the Affibody molecules of interest by protein engineering
3. To nominate one Affibody candidate drug
4. To produce the Affibody candidate drug in large scale
5. To perform initial toxicology studies with one optimized Affibody candidate drug

Potential Impact:
The Counterstroke project – an EU funded effort to develop new stroke therapies
Stroke and other conditions related to a decreased oxygen supply to the brain are the third most common cause of death in Europe. One out of three stroke incidents lead to permanent damage, and stroke is a major cause of adult disability. Within EU, more than 500 000 persons die of stroke each year. Of those surviving stroke, 50% are left with physical or cognitive impairment and the total annual cost of stroke was estimated at Euro 27 billion at the initiation of the COUNTERSTROKE project.
The COUNTERSTROKE consortium, which consists of six European research institutions and companies, has successfully collaborated in research for developing new therapeutics in the field of stroke. A key success factor for the Counterstroke project has been the network of specialists within the consortium. Experts in the fundamental biology of HMGB1 and its role in stroke, industrial partners that have brought the biopharmaceutical into preclinical and clinical development and clinicians focused on improving the care of stroke patients.
During the twenty years HMGB1 has been studied, it has become evident that HMGB1 plays an important role in several inflammatory conditions, including stroke, and that a blockade of HMGB1 could reduce the symptoms. The overall goal of the COUNTERSTROKE project has been to generate a Candidate Drug molecule for further preclinical and clinical development. The consortium has effectively developed Affibody® molecules specific for HMGB1 that potentially can be beneficial not only to stroke patients but also to individuals suffering from chronic diseases such as rheumatism and lupus and also acute inflammatory conditions such as sepsis. Affibody® molecules are a new class of biopharmaceuticals which mimics antibodies with regards to selectivity and strong binding to the target protein with the advantage that they are significantly smaller and are inherently inert, meaning that they do not have any functions that involve activation of the body's immune system. In order to not jeopardize the possibilities for further exploitation of the project results and ultimately the benefit to patients, the COUNTERSTROKE project has intentionally delayed any publications sufficiently for obtaining a solid foundation for patent applications.
The COUNTERSROKE project has successfully generated Affibody® molecules specific for HMGB1, however, more research is needed to clarify the mode of action and full efficacy of the candidate drug before entering clinical trials. The project consortium members will therefore continue the collaboration beyond the COUNTERSTROKE project to generate data towards this important goal.

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