Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

PROTEIN REPLACEMENT THERAPIES

József Tőzsér

Molecular Therapies- Lectures 5-6

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

• Replacement of missing or abnormal proteins (e.g.

Insulin)

• Influencing signal transduction pathways (e.g.

interferons)

• Supplementation of growth hormones (e.g. PDGF)

• Influencing the haemostatic system (e.g. tPA)

• Degradation of molecules with enzymes (e.g.

Asparaginase

• Use of protein vaccines (e.g. recombinant HPV vaccine)

Protein-based therapies

• Insulin (diabetes)

• Albumin (hypoalbuminaemia)

• Lactate (lactose intolerance)

• Factor VIII. (haemophilia)

• Factor IX. (haemophilia)

• Protein C (protein C deficiency)

• Beta-glucocerebrosidase (Gaucher’s disease)

Examples of proteins replaced in therapy

• Humulin (insulin)

• Novolin (insulin)

• Flexbumin 25% (albumin)

• Lactaid (lactose)

• ReFacto (F VIII)

• BeneFix (F IX)

• Ceprotin (aktivált protein C)

Examples of protein therapeutic products

Therapeutic proteins

• The first therapeutic protein use was the utilization of cow pox „protein vaccine" against smallpox by Jenner (1796)

• The first therapeutic protein used was insulin by Banting and Best (1922)

• More than 200 peptide or protein has been approved in USA for use in therapy

• Therapeutic proteins may come from different sources

Therapeutic proteins – sources

Protein Original source

• Albumin Human blood

• Insulin Pig, bovine pancreas

• Factor VIII Human blood

• Factor IX Human blood

• Kalcitonin Salmon

• Anti-venom Horse, dunkey blood

• β-glucorerebrosidase Human placenta

Therapeutic proteins – sources

• Natural sourse is usually sparse and expensive

• It is difficult to satisfy demands

• Hard to isolate the product

• May lead to immune intolerance (e.g. in case of animan proteins)

• Potential viral and pathogen contaminations

• Nowadays most of the protein drugs are made by recombinant techniques

• Cheaper, safer, unlimited source

Insulin, the first protein replacement drug

• In January 1922, Banting and Best used first insulin to treat a 14-years-old patient named Leonard Thomson

• He became more ill as the consequence of the injection, but his blood glucose level decreased, therefore the improvement of the preparation

technique was decided

• 6 weeks later a better extract was able to decrease the blood glucose level from 520 mg/dL to 120 mg/dL within 24 hours.

• Leonard lived for additional 13 years, he died of pneumonisa at the age 27

Structure of insulin

Two polypeptid chains, A.chain:

21 aminoacid residues B-chain:

30 aminoacid residues

Chains are held together by a disulfide bridge

Insulin gene is located at chromosome 11

Protein therapeutics – from blood

• The human body contains approx. 6 liters of blood

• 60-70% of blood is plasma, 8-9%- proteins. Therefore it is an important protein source

• Human plasma contains about 10,000 different proteins

• About 20 proteins make up the 99% of the total protein content of plasma

• Annually several million liters of outdated

transfusion plasma is genereated, therefore it is an excellent and relatively abundant protein source

Examples of blood protein products

• Factor VIII (correction of haemophilia)

• Factor IX (correction of haemophilia)

• Albumin (correction of hypoalbuminaemia)

• Intravenous IgG (in infections)

• Antithrombin III (correction of coagulation disorder)

• Alpha I-PI (correction of emphysema)

Cohn fractionation

• Cohn fractionation was originally developed in 1946.

• In Cohn fractionation plasma proteins are selectively precipitated by using ethanol, salt, temperature

change.

• Separation of the fractions is achieved by centrifugation.

Recombinant technologies

• They were developed in the 1970s and 1980s

• Paul Berg (1973): discovery of restriction enzymes

• Herbert Boyer (1978): cloning of the human insulin gene into E. coli - Genentech

• Fundamentally two major approaches – Expressin in isolated cells

– Expression in transgenic plants or animals

All components of the intrinsic pathway are in the bloodstream.

Extrinsic pathway is initiated by the TF that is normally excloded from the Bloodstream.

Cascade (avalange) model.

VII +

TF, Ca²⁺

VIIa TF, Ca²⁺

IXa, VIII PL Ca²⁺

X ^Xa

V PL Ca²⁺

prothrombin thrombin

fibrinogen fibrin

crosslinked fibrin

XIII

XIIIa + Ca²⁺

Extrinsic pathway

Intrinsic pathway

XIa XI Ca²⁺

IX

Common pathway

Haemophilias A and B

• Hemophilias A and B are caused by deficiencies in factors VIII or IX, respectively

• Affect ~1 in 5,000 males in Hemophilia A, ~1 in 30,000 males in Hemophilia B

• Inherited as a recessive X-linked trait in both cases (Mother would be an unaffected carrier)

• Treated by administration of factor VIII or factor IX concentrates

• Recombinant factor VIII or IX

• Gene therapy trials

Haemophilia A – gene therapy

• Only FVIII level is  1 % of normal causes severe

symptoms (spontaneous bleeding into joints, vital organs), therefore even low levels of proteins are beneficial

• Tight control of FVIII production is not required

• Broad therapeutic index minimizes the risk of overdose

• Delivery to bloodstream does not require expression in the liver

• Domain B is not required for function, in its absence the expression levels are higher