formulation and application

PART 1: INTRODUCTION AND LEARNING OBJECTIVES

Section 1: Learning Objectives (Page 2)

By the end of this lecture, students will be able to:

• Review the structure of an antibody.

• Discuss the concept of production of monoclonal antibodies.

• Appraise history and development of monoclonal antibodies.

• Outline the applications of monoclonal antibodies.

Section 2: References (Page 3)

PART 2: PROTEIN CHARACTERISTICS AND ANTIBODY STRUCTURE

Section 3: Protein Characteristics (Page 4)

• All antibodies are proteins, thus they suffer from the same stability issues as proteins.

• Mutations and denaturation can affect their:

  • Stability

  • Potency

  • Function

• Impurities arising from manufacturing of proteins can affect their stability.

• Precipitation of proteins can cause immunogenicity (the body mounts an immune response against the therapeutic antibody).

Section 4: Structure of Immunoglobulin (Antibody) (Page 5)

• Antibodies can target antigens and so can be used as targeted drugs in their own right.

• They are highly specific for cell surface markers.

Image Description (Page 5): A diagram of the classic Y-shaped antibody structure showing:

• Heavy chains (two identical) – constant and variable regions

• Light chains (two identical) – constant and variable regions

• Antigen-binding site (Fab region) – variable regions at the tips

• Fc region (crystallisable fragment) – constant region of heavy chains, responsible for effector functions (e.g., binding to immune cells, complement activation)

PART 3: MONOCLONAL ANTIBODIES – DEFINITION AND TYPES

Section 5: Monoclonal Antibodies vs. Polyclonal Antibodies (Page 6)

Section 6: mAb – Types and Nomenclature (Page 7)

Problems with Murine Antibodies:

• Reduced stimulation of cytotoxicity

• Formation of immune complexes after repeated administration

• Allergic reactions

• Anaphylactic shock

Advantage of Chimeric/Humanised Antibodies:

• Reduced immunogenicity – less likely to be recognised as foreign.

• Increased serum half-life – longer duration of action in the body.

PART 4: PRODUCTION OF MONOCLONAL ANTIBODIES

Section 7: Monoclonal Antibody Production – The Hybridoma Technology (Page 8)

Image Description (Page 8): A diagram of the hybridoma production process showing:

1. Immunisation: A mouse is immunised with the target antigen.

2. Spleen Cell Harvest: Spleen cells (B lymphocytes) are harvested from the immunised mouse.

3. Cell Fusion: Spleen cells are fused with myeloma cells (immortal cancer cells) using polyethylene glycol (PEG) to create hybridomas.

4. Selection in HAT Medium: Cells are cultured in HAT medium (hypoxanthine, aminopterin, thymidine).

   • Myeloma cells alone cannot survive in HAT medium because they lack the enzyme HGPRT (hypoxanthine-guanine phosphoribosyl transferase) needed for DNA synthesis via the salvage pathway.

   • Unfused B cells die because they cannot proliferate indefinitely (no telomerase).

   • Only hybridomas survive because they acquire immortality from the myeloma cells and the HGPRT enzyme from the B cells.

5. Screening and Cloning: Hybridomas are screened for antibody production and cloned to produce a single cell line producing the desired monoclonal antibody.

6. Production: Large-scale production in culture or in mouse ascites fluid.

Section 8: History and Development of Monoclonal Antibodies (Page 9)

PART 5: APPLICATIONS OF MONOCLONAL ANTIBODIES

Section 9: Applications Overview (Page 10)

Section 10: Examples of Monoclonal Antibodies and Fusion Proteins (Page 11)

Section 11: Diagnostic Applications – Radiolabelled Antibodies (Page 12)

11.1. Anti-Carcinoembryonic Antigen (CEA) Antibody:

• Labelled with technetium-99m (⁹⁹ᵐTc) .

• Used for imaging patients with metastatic colorectal carcinoma.

11.2. Nofetumomab:

• Murine monoclonal antibody coupled with ⁹⁹ᵐTc.

• Used for diagnosis to determine the extent and stage of disease in patients with small cell lung cancer.

Image Description (Page 12): Likely shows a gamma camera image or a diagram of a radiolabelled antibody binding to a tumour.

Section 12: Antibodies for Autoimmune Disorders – Targeting T Cells (Page 13)

Antibodies can bind to various receptors on T cells, blocking T cell activation and suppressing the immune response.

Image Description (Page 13): A diagram showing the interaction between T cells and antigen-presenting cells, with the blocking effects of these antibodies indicated.

Section 13: Anti-TNF-α Antibodies – Adalimumab and Infliximab (Page 14)

• Mechanism: Bind to TNF-α and prevent its binding to its receptor on inflammatory cells.

• Result: Suppression of release of inflammatory cytokines (IL-1, IL-6, etc.), reducing inflammation.

Section 14: Trastuzumab (Herceptin®) – Anti-HER2 for Breast Cancer (Page 15)

14.1. Mechanism:

• HER2 (Human Epidermal Growth Factor Receptor 2) is overexpressed in about 20-25% of breast cancers.

• Trastuzumab is a humanised monoclonal antibody that binds to the HER2 receptor.

• It prevents binding of natural ligands (growth factors) and downregulates the receptor.

• Approved for: Metastatic breast cancer with overexpressed HER2/neu (oncogene).

14.2. Formulation (Page 16):

• Dosage Form: Powder for concentrate for solution for infusion (lyophilised).

• Appearance: White to pale yellow lyophilised powder.

• Excipients:

  • L-histidine hydrochloride monohydrate – buffering agent.

  • L-histidine – buffering agent, cryoprotectant.

  • α,α-Trehalose dihydrate – stabilising agent, cryoprotectant.

  • Polysorbate 20 – stabilising agent (prevents aggregation and adsorption).

Image Description (Page 16): Likely shows a vial of Herceptin lyophilised powder and the reconstituted solution.

Section 15: Review Question (Page 17)

Which ONE of the following statements concerning antibodies is FALSE?

Correct Answer: E

PART 6: MECHANISMS OF ACTION AGAINST TUMOURS

Section 16: Mechanisms of mAb Action Against Tumours (Page 18)

Section 17: ADEPT – Antibody-Directed Enzyme Prodrug Therapy (Page 19)

• Definition: A two-step strategy involving the application of cancer-associated monoclonal antibodies linked to a drug-activating enzyme.

• Process:

  1. Antibody-enzyme conjugate is administered and allowed to localise to the tumour.

  2. A non-toxic prodrug is then administered systemically.

  3. The enzyme at the tumour site converts the prodrug into a toxic drug.

  4. The cytotoxic effect is targeted at malignant cells, sparing normal tissues.

Image Description (Page 19): A diagram showing the basic principle of ADEPT:

• Step 1: Antibody-enzyme conjugate binds to tumour cell.

• Step 2: Prodrug administered.

• Step 3: Enzyme activates prodrug to active drug at the tumour site.

Section 18: Immunoliposomes (Page 20)

• Definition: Antibody-conjugated liposomes.

• Principle: Antibodies can be used as targeting groups which are conjugated to other drugs or carrier systems such as liposomes.

• Function: Liposomes can carry drugs or therapeutic nucleotides (e.g., siRNA) and then be conjugated with monoclonal antibodies to achieve targeted delivery to specific cells (e.g., cancer cells).

PART 7: ADVERSE EFFECTS OF MONOCLONAL ANTIBODIES

Section 19: Adverse Effects of mAb (Page 21)

PART 8: SUMMARY

Section 20: Summary (Page 22)

• Monoclonal antibody therapy has revolutionised the treatment of cancer and other diseases.

• Clinical success of antibody therapy is dependent on understanding the effects of antibody therapy on the pathophysiology of the body.

• Antigen-specific mAbs can be produced against any type of antigen; hence, there is potential for vast diagnostic applications.

SUMMARY TABLE: TYPES OF MONOCLONAL ANTIBODIES