PROS by the Stony Brook University 2022 iGEM Team

Proposed Implementation

We envision that our project, PROS, will be a long-term treatment therapy for patients with protein S deficiency, with the potential to act as a short-term therapeutic for patients suffering from related disorders including COVID-19 and traumatic brain injury (TBI). To achieve this, we aimed to produce recombinant human protein S using genetically engineered E.coli and SF9 insect cells. This protein will be identical to human protein S, and can be directly administered to patients intravenously.

Additionally, we did extensive research into the safety and efficacy of administration of recombinant protein S in order to make sure our therapeutic will be effective and feasible. Our ultimate goal is to ensure safe delivery into the human bloodstream. We recognize that challenges still exist that need to be solved for our product.

Description of PROS Therapy

PROS (Recombinant Human Protein S) will be manufactured in E.coli and SF9 cells using genetic engineering techniques. The future manufacturing process for PROS will include processing steps designed to reduce the risk of viral transmission. Further safety implementation would include virus clearance studies.

PROS is to be used for both pediatric and adult patients with congenital protein S deficiency for the prevention and treatment of venous thrombosis and purpura fulminans. It can also be administered to pregnant women, patients suffering from severe COVID-19, traumatic brain injury (TBI), and other disorders that result in either inherited or acquired protein S deficiency, either in the long-term or short-term, as recommended by a doctor.

Treatment can be initiated under the supervision of an experienced physician. The dose, administration frequency, and duration of treatment with PROS will depend on a variety of factors including the patient's age, their clinical condition, and the levels of protein S and protein S activity in the bloodstream. The dose regimen will be adjusted to the pharmacokinetic profile for each individual patient.

More detailed information about the PROS injectable therapy, including dosage information, administration and delivery, clinical trials, health and safety precautions and considerations, etc. can be found below. We also separated this information into a handout that can be further improved upon and provided to healthcare personnel and patients using this therapy. The document is linked at the bottom of this page. Finally, we created an AUTOCAD injectable design of our potential product.

PROS: A Novel Therapy for the Administration of Protein S, Created by the 2022 Stony Brook University iGEM Team

Overview

PROS (Recombinant Human Protein S) will be manufactured in E.coli and SF9 cells using genetic engineering techniques. The manufacturing process for PROS includes processing steps designed to reduce the risk of viral transmission. Further safety implementation would include virus clearance studies.

This section proposes a potential detailed prescription plan and method of PROS therapy that can be altered and changed as our project continues and new insights are revealed. The information is purely hypothetical, and is based on past research and similar treatments. Beyond the competition, we plan on further purifying and characterizing our protein, creating the product, and designing and carrying out clinical trials to test our proposed dosages and the therapy overall.

PROS [Protein S Concentrate (Human)] Lyophilized Powder for Solution for Injection

1. INDICATIONS AND USAGE

PROS, Protein S Concentrate (Human), is an anticoagulant indicated for neonates, pediatric and adult patients with severe congenital Protein S deficiency for the prevention and treatment of venous thrombosis and purpura fulminans. It can also be used for various disorders associated with a decline in protein S levels such as severe COVID-19 and TBI.

2. DOSE

Initiate treatment with PROS under the supervision of a physician experienced in replacement therapy with coagulation factors/inhibitors where monitoring of protein S activity is feasible.

The dose, administration frequency, and duration of treatment with PROS depends on a variety of factors which include the severity of the protein S deficiency, the patient’s age, the clinical condition of the patient, and the patient’s plasma level of protein S.

Adjust the dose regimen according to the pharmacokinetic profile for each individual patient. [See DOSAGE AND ADMINISTRATION: Protein S Activity Monitoring].

Table 1 provides a potential PROS dosing schedule for acute episodes, short-term prophylaxis and long-term prophylaxis.

	Initial Dose	Subsequent 3 Doses	Maintenance dose
Acute Episodes, Short-term Prophylaxis	100-120 IU/kg	60-80 IU/kg Every 6 hours	45-60 IU/kg every 6 hours or 12 hours
Long-term Prophylaxis	N/A	N/A	45-60 IU/kg every 12 hours

*Dosing would be adjusted based on future clinical trials
**Adjust the dose according to the pharmacokinetic profile for each individual patient. Continue PROS until desired anticoagulation is achieved.

Table 1: PROS Dosing Schedule for Acute Episodes, Short-term Prophylaxis and Long- term Prophylaxis

These doses are the same as the dose recommended for use by the protein C injectable, CEPROTIN. Trials for the PROS injectable have not started, so it is unsure if these same dosages will be adequate for the PROS injectable. However, because protein S is very similar in terms of activity and quantity in the blood, if drug trials were to commence, initial trials may want to attempt dosage levels similar to CEPROTIN.

Determine protein S recovery and half-life with an initial dose of 100-120 IU/kg in patients receiving treatment for acute episodes and short-term prophylaxis.
Adjust the dose to maintain a target peak protein S activity of 100 %.
Continue the patient on the same dose after resolution of the acute episode to maintain trough protein S activity level above 25% for the duration of treatment.
Patients receiving prophylactic administration of PROS may warrant higher peak protein
S activity levels in situations of an increased risk of thrombosis (such as infection, trauma, or surgical intervention). Therefore it is recommended to maintain trough protein S activity levels above 25%

Protein S Activity Monitoring

Determine the patient’s protein S plasma level before and during treatment with PROS using a commercially available assay
Certain clinical conditions, such as acute thrombosis, purpura fulminans, and skin necrosis, may shorten the half-life of PROS.
In case of an acute thrombotic event, immediately measure protein S activity before the next injection until the patient is stable and monitor the protein S levels to maintain the trough protein S level above 25%.

3. PREPARATION

Reconstitution: Use Aseptic Technique

1. Bring the PROS (powder) and Sterile Water for Injection, USP(diluent) to room temperature.

Remove caps from the PROS and diluent vials
Cleanse stoppers with germicidal solution, and allow them to dry before use.
Remove protective covering from one end of the double-ended transfer needle and insert the exposed needle through the center of the diluent vial stopper.
Remove protective covering from the other end of the double-ended transfer needle. Invert diluent vial over the upright PROS vial; then rapidly insert the free end of the needle through the PROS vial stopper at its center. The vacuum in the vial will draw in the diluent.
Disconnect the two vials by removing the needle from the diluent vial stopper. Then, remove the transfer needle from the PROS vial. Gently swirl the vial until all powder is dissolved. Be sure that PROS is completely dissolved; otherwise, active materials will be removed by the filter needle.

4. ADMINISTRATION

Administration: Use Aseptic Technique
Doctors usually visually inspect CEPROTIN for particulate matter and discoloration before administration. After reconstitution, the solution should be colorless to slightly yellowish and clear to slightly opalescent, and free of visible particles. We anticipate the same criterion for PROS. The solution should not be met if it does not meet these criteria.

Administer PROS at room temperature not more than 3 hours after reconstitution.

Attach the filter needle to a sterile, disposable syringe and drawback the plunger to admit air into the syringe.
Insert the filter needle into the vial of reconstituted PROS.
Inject air into the vial and then withdraw the reconstituted PROS into the syringe.
Remove and discard the filter needle in a hard-walled Sharps container for proper disposal. Use filter needles to filter the contents of a single vial of PROS only.
Attach a suitable needle or infusion set with winged adapter, and inject intravenously as instructed below under Administration by Infusion.

Administration by Infusion
Administer PROS at a maximum injection rate of 2 mL per minute except for children with a bodyweight of < 10 kg, where the injection rate should not exceed a rate of 0.2 mL/kg/minute.

5. DOSAGE FORMS AND STRENGTHS

PROS will be available in single-dose vials that contain nominally 500 or 1000 International Units (IU) human protein S and is reconstituted with 5 mL and 10 mL of Sterile Water for Injection respectively, to provide a single dose of human Protein S at a concentration of 100 IU/mL.

PROS, when reconstituted with the appropriate volume of diluent, contains the following excipients: 8 mg/mL human albumin, 4.4 mg/mL trisodium citrate dihydrate, and 8.8 mg/mL sodium chloride.

6. CONTRAINDICATIONS

None predicted.

7. WARNINGS AND PRECAUTIONS

Clinical trials will need to be performed in order to generate a safety profile for PROS therapy. Based on the engineering and design process, there is currently no anticipated risk of transmissible infectious agents or hypersensitivity. To fully understand adverse reactions, the possible presence of inhibiting antibodies, as well as bleeding episodes that may occur as a result of concurrent anticoagulant medication or tissue plasminogen activator.

8. DRUG INTERACTIONS

We are currently not planning on any formal drug interaction studies, but these could be conducted to develop an increased understanding of PROS.

9. USE IN SPECIFIC POPULATIONS

Pregnancy

Risk Summary
There is no data to suggest PROS use in pregnant women would inform a drug-associated risk, but this would have to be further studied. Animal reproduction studies could be conducted to this extent. It is also not known whether PROS can cause fetal harm when administered to a pregnant woman or can affect reproduction capacity. PROS can be studied for use during labor and delivery. In the U.S. general population, the estimated background risk of major birth defects and miscarriages in clinically recognized pregnancies is 2-4% and 15-20%, respectively, regardless of drug exposure.

Lactation

Risk Summary
There is no information regarding the presence of protein S in human milk, the effect on the breastfed infant, or the effects on milk production. PROS has not been studied for use in nursing mothers. The developmental and health benefits of breastfeeding should be considered along with the mother’s clinical need for PROS and any potential adverse effects on the breastfed child from PROS or from the underlying maternal condition.

Pediatric Use

Clinical studies would be designed specifically so that neonatal and pediatric subjects will be enrolled during the prospective and retrospective studies. There is no data done to understand how administration of protein S would affect children specifically.

Geriatric Use

Clinical studies of PROS would include sufficient numbers of subjects aged 65 and over to determine whether they respond differently from younger subjects.

10. DESCRIPTION

PROS [Protein S Concentrate (Human)] is manufactured from bacterial and insect cells purified by a combination of filtration and chromatographic procedures. The manufacturing process for PROS would include processing steps designed to reduce the risk of viral transmission.

11. CLINICAL PHARMACOLOGY

Mechanism of Action

Protein S functions as an anticoagulant by directly inhibiting procoagulants, such as Factor Xa (FXa), FVa, and FIXa, while also serving as a cofactor for anticoagulants such as Activated Protein C and Tissue Factor Pathway Inhibitor. Protein C is the precursor of a vitamin K-dependent anticoagulant glycoprotein (serine protease) that is synthesized in the liver. It is converted by the thrombin/thrombomodulin-complex on the endothelial cell surface to activate Protein C (APC). APC is a serine protease with potent anticoagulant effects, especially in the presence of its cofactor protein S. APC exerts its effect by the inactivation of the activated forms of factors V and VIII, which leads to a decrease in thrombin formation. APC has also been shown to have profibrinolytic effects.

This pathway provides a natural mechanism for control of the coagulation system and prevention of excessive procoagulant responses to activating stimuli. A complete absence of protein S is not compatible with life. A severe deficiency of this anticoagulant protein causes a defect in the control mechanism and leads to unchecked coagulation activation, resulting in thrombin generation and intravascular clot formation with thrombosis.

Pharmacodynamics

In clinical studies, the intravenous administration of PROS will be monitored to see if it demonstrates a temporary increase, within approximately half an hour of administration, in plasma levels of APC. Replacement of protein S in protein S-deficient patients is expected to control or, if given prophylactically, to prevent thrombotic complications.

Pharmacokinetics

We will analyze pharmacokinetic results for asymptomatic and symptomatic subjects with protein S deficiency.

The protein S plasma activity will be measured by chromogenic and/or clotting assay. The maximum plasma concentrations (Smax) and area under the plasma concentration-time curve (AUC) will be analyzed, and the median incremental recovery will be calculated. The median half-lives, based on non-compartmental methods, will also be recorded. A formal study or analysis can be further done to evaluate the effect of covariates such as race and gender on the pharmacokinetics of PROS.

The pharmacokinetic profile in pediatric patients will also be formally assessed to see if the pharmacokinetics of PROS may be different between very young children and adults. This will help when establishing a dosing regimen for children. Doses should be individualized based upon protein S activity levels.

12. NONCLINICAL TOXICOLOGY

Carcinogenesis, Mutagenesis, Impairment of Fertility

Protein S contained in PROS is a normal constituent of human plasma and acts like endogenous protein S. Studies in heterologous species to evaluate carcinogenicity, reproductive toxicology and developmental toxicology can be performed. In order to determine if PROS will demonstrate mutagenic potential, the Salmonella Typhimurium reverse mutation assay (Ames test), will be used.

Animal Toxicology and/or Pharmacology

Safety Pharmacology
Cardio-respiratory studies can be performed in dogs to evaluate mean arterial pressure, cardiac output, systemic vascular resistance, heart rate, QT interval changes, pulmonary artery pressure, respiratory rate and respiratory minute volume to see if there are adverse effects at a maximum dose of 500 IU/kg. Anaphylactoid reactions can be determined by measurement of bronchospastic activity in guinea pigs, in order to see if there are adverse effects at the maximum dose of 300 IU/kg. Thrombogenic potential can be evaluated in rabbits using the Wessler stasis model to observe adverse effects at 200 IU/kg. Overall, safety pharmacology studies evaluating cardio-respiratory function, acute dose anaphylactoid potential and thrombogenicity will be utilized to demonstrate the risk of adverse effects in a range of doses from 1.6 to 4.2 times the maximum single human dosage per kilogram body weight.

Acute Dose Toxicity
Toxicity testing in rats and mice following single dosing of 2000 IU/kg or 1500 IU/kg, respectively, can also be done to observe adverse clinical effects or gross pathology at 14 days post dosing.

Repeated Dose Toxicity
Studies can also be potentially conducted to evaluate repeated-dose toxicity in animals. The long-term toxicity potential of PROS following repeated dosing in animals is currently unknown.

Local Tolerance Testing
Investigation of the route of injection tolerance can be used to demonstrate whether PROS will result in any local reactions after intravenous, intra-arterial injections of 500 IU/kg (5 mL) and paravenous injections of 100 IU/kg (1 mL) in rabbits.

Citrate Toxicity
Studies done in in mice evaluating 1000 IU vials reconstituted with 10 mL vehicle followed by dosing at 30 mL/kg (132 mg/kg TCD) and 60 mL/kg (264 mg/kg TCD), can be done to observe if there are signs of citrate toxicity (dyspnea, slowed movement, hemoperitoneum, lung and thymus hemorrhage and renal pelvis dilation).

13. CLINICAL STUDIES

We would conduct a multi-center, open-label, non-randomized study, in order to evaluate the the safety and efficacy of PROS in subjects with severe congenital protein S deficiency for the (on-demand) treatment of acute thrombotic episodes, such as purpura fulminans (PF), warfarin-induced skin necrosis (WISN) and other thromboembolic events, and for short-term or long-term prophylaxis. We would also test the administration of this therapy in patients with severe COVID-19 and TBI.

Retrospective Analysis

A retrospective study to capture dosing information and treatment outcome data in subjects with severe congenital protein S deficiency who were treated with PROS under an emergency use would also be conducted. The treatment outcome for these episodes would be rated in all cases.

14. HOW IT IS SUPPLIED/STORAGE AND HANDLING

PROS, Protein S Concentrate (Human), would be supplied as a sterile, white or cream colored, lyophilized powder for IV injection. It would have a pH between 6.7 and 7.3, suitable for the bloodstream, and an osmolality not lower than 240 mosmol/kg, also suitable for the bloodstream We would adjust so that one International Unit (IU) of protein S corresponds to the amidolytically measured activity of protein S in 1 mL of normal plasma. The potency (IU) would be determined using a chromogenic substrate method referenced against the World Health Organization (WHO) International Standard.

PROS would be available in single-dose vials that contain the following nominal product strengths:

500 IU per vial
1000 IU per vial

The actual potency will be printed on the vial label.

One package of PROS would contain one glass vial of PROS powder, one glass vial of Sterile Water for Injection, USP, one transfer needle, one filter needle, one full prescribing physician insert and one patient package insert.

PROS, packaged for sale, should be stable for 3 years when stored refrigerated at 2°C–8°C (36F- 46F). It should not freeze in order to prevent damage to the diluent vial. The vial should be stored in the original carton to protect it from light. The reconstituted solution should be used within 3 hours of reconstitution. We would recommend not using the product beyond the expiration date.

15. PATIENT COUNSELING INFORMATION

We would advise the patient to read the FDA-approved patient labeling, and that doctors should inform patients of the possible warnings and precautions (which have not yet been fully established).

AUTOCAD Injectable Design Model