Pharmaceutical Biotechnology PHT 426 “Monoclonal Antibodies & Hematopoietic Growth Factors” Dr. Mohammad Alsenaidy Department of Pharmaceutics College.

Pharmaceutical BiotechnologyPHT 426

“Monoclonal Antibodies & Hematopoietic Growth Factors”

Dr. Mohammad Alsenaidy

Department of Pharmaceutics

College of Pharmacy

King Saud University

Office: AA 101

[email protected]

Previously on PHT 426!!

Insulin

History Chemical description Formulation Pharmaceutical concerns

Introduction to Proteins

Purification of Proteins

Characterization of Proteins

Protein’s Instabilities

Formulation

Examples of Protein

therapeutics

Top nine categories of biological drugs

Milstein and Kohler

• The exciting field of therapeutic monoclonal antibodies (MABs) had its origins as Milstein and Koehler presented their murine hybridoma technology in 1975.

• This technology provides a reproducible method for producing monoclonal antibodies with unique target selectivity in almost unlimited quantities.

• First therapeutic MAB got market authorization from the FDA in 1986 (Orthoclone OKT3).

Monoclonal Antibodies“History and Background”

• To date, more than 30 MABs and MAB derivatives including fusion proteins and MAB fragments are available for different therapies. • Oncology. • Inflammation. • Transplantation.

• MABs are generally very safe drugs because of their target selectivity, thus avoiding unnecessary exposure to and consequently activity in nontarget organs.

• This is particularly apparent in the field of oncology, where MABs like rituximab, trastuzumab, and bevacizumab can offer a more favorable level of efficacy/safety ratios compared to common chemotherapeutic treatment regimens for some hematologic and solid tumors.

Monoclonal Antibodies

The Journal of Experimental Medicine 2012, 209 (13), 2367-2381

Monoclonal Antibodies

Monoclonal Antibodies two major effector functions

A

B

Monoclonal Antibodies

CANCER

INFLAMMATION

Cancer

Rituximab• Rituximab, the first MAB approved for the treatment of cancer, was approved for use in 1997.

• It is a chimeric murine/human MAB directed against the CD20 antigen found on nearly all B lymphocytes.

• Used to treat B cell non-Hodgkin lymphoma.

• CD20 is involved with cell cycle initiation, regulation, and differentiation.

• Rituximab is thought to mediate death of CD20-positive tumor cells via antibody dependent cell-mediated cytotoxicity (ADCC) mechanism.

Mechanism 1: Cluster of differentiation ( CD ) targeted

Rituximab Mechanism

BevacizumabMechanism 2: Vascular endothelial growth factor ( VEGF ) targeted

• Bevacizumab is a humanized MAB that acts by binding and neutralizing the Vascular endothelial growth factor-A isoform (VEGF-A). • The depletion of VEGF downregulates the VEGF/VEGF receptor pathway, resulting in inhibition of new vessel formation and induction of a more normal tumor vasculature pattern leading to decreased vascular permeability.• Bevacizumab was first approved by the FDA in 2004 for the treatment of metastatic colorectal cancer and has since received approvals for the treatment of metastatic renal cell cancer, nonsquamous non-small cell lung cancer, and glioblastoma multiforme.

Cancer

Bevacizumab Mechanism

Trastuzumab

Mechanism 3: Epidermal growth factor receptor ( EGFR ) targeted

• Trastuzumab is a humanized MAB that selectively binds to the extracellular domain of the human Her-2 receptor with high affinity.• Her-2 receptor (tyrosine kinase) plays a critical role in cellular growth and is frequently up-regulated in numerous cancers, including breast carcinoma (2 x 106 vs 2 x 104).• Trastuzumab is indicated for the treatment of Her-2- positive breast cancer (20-30 %) in the adjuvant and the metastatic setting and is also indicated for the treatment of Her-2-positive gastric cancer.• This receptor-antibody interaction blocks downstream signaling pathway as well as flag the tumor cells for destruction by immune cells (ADCC).

Cancer

Trastuzumab Mechanism

Etanercept

Inflammation (Rheumatic Arthritis)

Adalimumab Certolizumab pegol GolimumabInfliximab

• Etanercept is a dimeric fusion protein consisting of the human TNFα receptor linked to the Fc portion of human IgG1. It is the first anti-TNFα biologic agent approved for arthritide indication.

• Infliximab is a chimeric IgG1 monoclonal antibody containing ~25 % mouse sequence and ~75 % human sequence.

• Certolizumab pegol is a Fab antibody fragment linked to polyethylene glycol that enhances solubility and prolongs elimination half-life.

• Adalimumab and golimumab are two fully human IgG1 monoclonal antibodies.

Hematopoietic Growth Factors

• Hematopoiesis is an intricate, well-regulated, and homeostatic multistep process that allows immature precursor cells in the bone marrow to proliferate, differentiate, mature, and become functional blood cells that transport oxygen and carbon dioxide; contribute to host immunity; and facilitate blood clotting.

ERYTHROPOIESIS-STIMULATING AGENTS

• Erythropoietin (EPO) is a 30.4 kD glycoprotein hormone secreted by the kidneys in response to tissue hypoxia, which stimulates red blood cell (RBC) production.• EPO requires glycosylation to regulate erythrocyte production by activating the EPO receptor (EPOR) and stimulating the proliferation and differentiation of erythrocytic progenitors in the bone marrow, which leads to reticulocytosis and an increase in the number of erythrocytes and the concentration of hemoglobin in the blood.• Epoetin alfa (Epogen ®), the first commercial form of recombinant human erythropoietin (rHuEPO) marketed in the USA, EU, Japan, and China, and epoetin beta (Recormon ® , NeoRecormon ® ) marketed outside of the USA are both expressed in Chinese hamster ovary cells. Both have the same 165 amino acid sequence, which is identical to human EPO, and contain two disulfide bonds and three N-linked and one O-linked sialic acid-containing carbohydrate chains and lead to the same biological effects as endogenous EPO.

• Darbepoetin alfa (Aranesp ® ) is a hyperglycosylated erythropoietin analog with five amino acid changes and two additional N-linked carbohydrate chains, which has the same mechanism of action as EPO.• However, darbepoetin alfa has a threefold increased serum half-life and increased in vivo potency, allowing for more convenient modes of administration, including extended dosing intervals.

ERYTHROPOIESIS-STIMULATING AGENTS

Blood doping!!!

• HSA was used in EPREX® as a stabilizer until 1998.• HSA was then removed and replaced with PS-80.• A significant increase in Pure Red Cell Aplasia (PRCA) cases were reported after the formulation change.• Patients develop anti-erythropoietin antibodies, which neutralize any administered epoetin and also endogenous erythropoietin, leading to a depletion of erythroid precursor cells in the bone marrow.• Why?• Theory 1: PS-80, a surfactant that tends to form micelles, might have promoted the formation of epoetin-containing micelles that resembled foreign pathogens, thereby unleashing the breakdown of immune self-tolerance. • Theory 2: PS-80 might have reacted with the uncoated rubber stoppers of the prefilled syringes to produce leachates that could act as adjuvants, provoking an antibody response.

The EPREX® Story

MYELOID HEMATOPOIETIC GROWTH FACTORS

Granulocyte Colony-Stimulating Factor (G-CSF)

• G-CSF has 174 amino acids and is produced in monocytes, fibroblasts, endothelial cells, and bone marrow stromal cells.• Important factor in Neutrophils production pathway.• Products:• Filgrastim (non-glycosylated G-CSF).• Lenograstim (glycosylated G-CSF, 25% more bioactive compared to non-glycosylated).• Pegfilgrastim (20 kDa PEG molecule is covalently bound to the N-terminal methionine

residue).• Filgrastim, Lenograstim, and Pegfilgrastim are indicated for the prevention and treatment of chemotherapy induced febrile neutropenia in cancer patients receiving chemotherapy and hematopoiesis after bone marrow transplantation, among others.• Administering these drugs reduce infection-related morbidity in cancer patients.