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© 2016, Rubicon Biotechnology, LLC ‐ ALL RIGHTS RESERVED.

Fv‐Hsp70 Technology for Myocardial Infarction

Richard Richieri – Chief Operations Officer

[email protected]

(949) 215‐2756


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Fv‐Hsp70: Rubicon’s Solution to Acute Injuries Rubicon’s Solution: Fv‐Hsp70 will target and deliver Hsp72 in therapeutic quantities directly into damaged cells to prevent apoptosis (programmed cell death following cellular injury).

Created at UCLA/VA, Rubicon’s clinical candidate, Fv‐Hsp70 is a recombinant fusion protein consisting of a targeting molecule (Fv) linked to an effector (Heat Shock Protein 72; Hsp72). The Fv is a single chain fragment from the 3E10 monoclonal antibody, which targets injured cells specifically. Hsp72 is a critical cellular protein which protects and refolds other proteins.

Fv‐Hsp70 can be recombinantly produced at large scale using conventional industrial techniques. The Fv moiety is an antibody fragment that specifically targets the endogenous DNA that is released from cells. The Hsp72 is a “heat shock protein” which helps with the proper folding of new proteins during their

synthesis. However, Hsp72 has multiple other roles including, during cell stress, Hsp72 binds to critical proteins that are damaged and become misfolded. It binds to these critical proteins and protects them from aggregation and cellular destruction, thereby inhibiting these processes. Hsp72 also has a dual role binding and inhibiting the activities of several key proteins involved in multiple apoptotic pathways; hence, the increased presence of this protein in a dying cell may rescue its viability.

The protective effects of endogenously produced heat shock protein Hsp72 are well known. Unfortunately, more than 3 hours are required by our cells to initiate production of the endogenous protein, and peak production does not occur until 72 hours after induction. Following a heart attack, stroke or other acute assaults, time is of the essence, as cell damage begins immediately. Our technology will deliver therapeutic amounts of Hsp72 directly into the stressed and damaged cells immediately. Our NIH supported studies, executed at Mount Sinai Medical Center in NYC, have proven Fv‐Hsp70 administration will preserve myocardium in a rabbit model of left coronary artery ischemia‐reperfusion injury. Furthermore, NIH supported studies have also shown Fv‐Hsp70’s effectiveness in stroke and acute lung injury.


Fv‐Hsp70 Program Exclusive License from the Department of Veterans Affairs

Patent Title “Antibody Mediated Transduction of Heat Shock Proteins into Living Cells”

Fv‐Hsp70 is a fusion protein comprised of two moieties designed to salvage damaged cells from dying

• Fv is a 237 amino acid single chain antibody fragment of the 3E10 antibody

• Hsp72 is a 641 amino acid protein normally found in human cells

Compelling animal data is already obtained in heart attack, stroke and acute lung injury models

Other applications being pursued


Published Data on Fv‐Hsp70 Mechanism of Action Fv is a single chain fragment from a murine antibody, known as 3E10, found serendipitously

Weisbart et. al., A conserved anti‐DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus Fv fragment bound to DNA released from damaged cells, Journal of Immunology, 1990

Fv fragment binds to DNA released from damaged cells

Fv bound to DNA enters the cell through the ENT2 channel

ENT2 channel is an energy independent channel which allows Fv to enter damaged cells

Hansen et. al., Intranuclear Transduction through a Nucleoside Salvage Pathway, Journal of Biological Chemistry, 2007

Fv antibody fragment is linked to the human protein Hsp72 (Heat Shock Protein 72)

Hsp72 (72kd) is a normal and substantial (3‐5%) constituent of intracellular protein

Hsp72 functions to protect other intracellular proteins from damage, preventing their degradation

Hsp72 is expressed upon cell injury, but requires up to 72 hours for peak expression

The therapeutic goal of Fv‐Hsp70 is the rapid delivery of Hsp72 into damaged cells to

immediately increase intracellular levels, bypassing long lag times for protein induction


The Problem In heart attacks or strokes, the heart or brain is damaged at the cellular level by the hypoxic stress caused by the ischemic event itself and, ironically, by the oxidative stress caused by restoration of blood flow following the commonly used reperfusion procedures (percutaneous coronary intervention (PCI) or thrombolytic therapy). This damage leads to worsening heart function and lower quality of life and increases the morbidity rate following the event.

1. Lack of blood flow from the acute ischemic event 2. Restoration of blood flow after reperfusion


Summary of Fv‐Hsp70 Results in Myocardial Infarction 1.5 million heart attacks occur annually in the US. Mechanical reperfusions by percutaneous coronary intervention (PCI) with stents are becoming more frequent, with over 1 million procedures performed annually. There are no pharmacologic therapies after PCI that reduce the heart cell damage that results from both the heart attack itself and the restoration of blood flow by PCI.

Rubicon completed a comprehensive animal efficacy study at Mount Sinai Medical Center in which a heart attack was experimentally induced in 30 rabbits by surgically ligating, or tying off, the left coronary artery, thereby causing a complete occlusion. This type of heart attack in humans is often fatal, often called a “widow maker.” The ligature was removed from the left coronary artery after 40 minutes, restoring blood flow after the heart attack was induced by the hypoxic event. Myocardial damage following release of the ligature parallels reperfusion in humans when relieving the occlusion with PCI and stent placement. After the experimental heart attack and reperfusion, thirty rabbits were divided into five cohorts of six animals and were treated either with one of three controls: 1) saline, 2) Fv alone or 3) Hsp72 alone; or they received one of two treatment arms of Fv‐Hsp70. Heart attack damage was assessed by three measures. Damage or infarct volume was visualized and quantitated by SPECT imaging. Heart function (Left Ventricular Ejection Fraction) was measured by cardiac ultrasound, as performed in humans. And cell death was further measured using a serum biomarker, Troponin I, commonly used in the clinic for cardiac patients. After study animals received Fv‐Hsp70 intravenously (immediately upon release of the ligature of the coronary artery), decrease in heart muscle damage was observed compared to control animals. Smaller infarct area was observed in SPECT imaging. Higher ejection fraction was observed. Smaller Troponin I release was observed. Controls receiving either Fv only or Hsp72 only showed no benefit compared to saline controls.


Fv‐Hsp70 Mechanism of Action (1) There is an abundant exogenous pool of DNA in areas of cell damage e.g. from an infarction or other acute assaults. Fv‐Hsp70 binds to the DNA (2) and is transported into stressed cells via the ENT2 channel (3). (4) Once internalized in the cell, the Hsp72 then carries out cell salvage activities by binding to critical proteins that are misfolded and helps the cell avoid apoptosis.

Fv‐Hsp70’s ability to directly deliver Hsp72 into cells has advantages over small‐molecule strategies designed to induce Hsp72 synthesis. For a small molecule approach, the need to evaluate unintended induction of other genes is critical. Add to that complication, induction of Hsp72 synthesis with a small molecule during cell stress requires a significant lag time to take effect, limiting its usefulness as a therapeutic agent. Some inducers require lag times varying from 8 to 24 hours in in vivo models, depending on the organ being targeted. Furthermore, induction of Hsp72 is attenuated with aging, dampening the effectiveness of the small molecule strategy in older individuals.


Fv‐Hsp70: Myocardial Infarction Experimental Model Our animal model exactly mimics a human heart attack

Human heart attacks occur when thrombus forms at the site of atherosclerotic plaque rupture, completely occluding flow beyond the thrombus

Heart muscle tissue begins to die immediately following occlusion

Patients presenting with a heart attack in the US often can take up to 40 minutes to get to a hospital or clinic, where they immediately undergo a catheterization procedure

Our animal model maintained a coronary occlusion for 40 minutes to simulate this event

Cardiac catheterization identifies the occluded coronary artery for subsequent intervention

Intervention is accomplished by opening the occluded artery by angioplasty and stent placement

Further heart muscle death is limited as blood flow is restored

Heart muscle already substantially damaged by the hypoxic injury is unlikely to recover

Further heart muscle injury occurs paradoxically as a result of re‐establishing flow during the intervention, the reperfusion injury


Experiments Conducted at Mount Sinai Medical Center Heart attack was experimentally induced in 30 rabbits (5 cohorts of six animals: saline, Fv alone, Hsp72 alone, and two treatment

arms of Fv‐Hsp70) by completely blocking the major heart artery, by surgically ligating, or tying off, to completely occlude the left coronary artery

Complete occlusion of the Left Anterior Descending artery results in a major heart attack, often fatal, hence this type of heart attack being called the “widow maker”

The ligature was removed from the Left Anterior Descending artery after 40 minutes, restoring blood flow after the heart attack was induced by the hypoxic event

Myocardial damage following release of the ligature parallels reperfusion injury in humans after relieving the occlusion with angioplasty and stent placement

In control animals not given Fv‐Hsp70, a large infarct volume was observed Heart attack damage was visualized by SPECT imagining Substantial loss of heart function (decrease in Ejection Fraction) was measured by cardiac ultrasound, as performed in humans Study animals received Fv‐Hsp70 intravenously immediately upon release of the ligature of the coronary artery, as would be

administered following an angioplasty and stent procedure in humans Decrease in heart muscle damage, the infarct size, was observed compared to control animals Improved heart function, substantially lower decrease in Ejection Fraction, was observed in animals treated with Fv‐Hsp70 Sham controls receiving Fv only showed no benefit compared to saline controls Sham controls receiving Hsp72 only showed no benefit compared to saline controls


Fv‐Hsp70: Myocardial Infarction Experimental Results


Fv‐Hsp70: Myocardial Infarction Experimental Results Ex‐Vivo Results

Results from tissue histology of the infarcted rabbit heart has shown significantly less cell death in the Fv‐Hsp70 treated rabbits versus the control groups.

A c‐myc tag incorporated into the Fv‐Hsp70 sequence was used to detect our drug.

Fv‐Hsp70 was found in the nuclei of many cardiomyocytes within the infarct zone (brown, see arrows). Compare these to the hematoxylin counterstained nuclei lacking Fv‐Hsp70 (blue).


Fv‐Hsp70 Data Summary for Myocardial Infarction 43% less cellular damage as assessed by SPECT in the Apex and Area at Risk

27% higher Ejection Fraction (a measure of heart function)

42% lower levels of Troponin I (a blood marker indicating heart damage)


Contact Richard Richieri – Chief Operations Officer

[email protected]

(949) 215‐2756

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Fv Hsp70 Technology for - rubiconbio.com€¦ · study animals received Fv‐Hsp70 intravenously (immediately upon release of the ligature of the coronary artery), decrease in heart