The addition of bevacizumab to oxaliplatin, fluorouracil, and leucovorin improves survival duration for patients with previously treated metastatic colorectal cancer.

A Bevacizumab “Success” Story

J. Clin. Onc. (2007) 25, 1539-1544

829 patients, metastatic colon cancer, treated with standard therapy or standard therapy + bevacizumab

The median duration of survival for the group treated with FOLFOX4 and bevacizumab was 12.9 months compared with 10.8 months for the group treated with FOLFOX4 alone (corresponding hazard ratio for death 0.75; P .0011), and 10.2 months for those treated with bevacizumab alone.

1 year survival SC+B, 56%; SC, 43%

Biological Agents Targeting the Same Molecule

Chimeric Antibody Fully Human Antibody

Soluble TNF receptor, normally involved in

down regulating inflammatory responses

Fc region only present for stability in serum

Intercept: obstruct (someone or something) so as to prevent them from continuing to a

destination

So what is Etanercept Intercepting?

TNF, tumor necrosis factor

• Name derived from an experiment performed in 1896 demonstrating that bacterial extracts could shrink tumors

• Extracts contained bacterial lipopolysaccharide which stimulated inflammation and induced TNF

Goal: Develop Drugs to Treat Chronic Inflammatory Diseases

Rheumatoid Arthritis

• ~ 1% of population

• average age of onset 40 years

• female/male 3:1

• joint erosion within 2 years of diagnosis, disabilities after 5 years

• average medical costs $23,00/year

• chronic pain, stiffness of joints; tiredness and lethargy

Tumor Necrosis Factor (TNF): A Cytokine Target for Treating Rheumatoid Arthritis

Anti-TNF

Early studies suggested TNF might be a master regulator of the inflammatory process

Encouraging Data from First Phase I Trail for Infliximab

CRP: C reactive protein, a marker for inflammation

Phase II Trial Indicates Infliximab Synergizes with Methotrexate

Loss of response correlates with patient antibodies to Infliximab

Immunosuppressive effects of methotrexate likely reduce patients ability to raise antibodies to Infleximab

HUMA: human anti-mouse antibodies

In the setting of Crohn’s disease ~ 60% of patients raise antibodies to Infliximab by the 5th injection

Crohn’s Disease and Ulcerative Colitis are Other Chronic Inflammatory Diseases Where Anti-TNF

Therapy is Indicated

HUMA Responses to Different Anti-TNF Drugs

Infliximab – 51, 21, and 7% of patients raised antibodies in response to 1, 3, and 10 mg/kg dosing

Fewer antibodies and higher doses of drug suggest tolerance mechanisms may come into play

Adalimumab – less immunogenic 6% of patients raised antibodies at the lowest dose

Etanercept – less immunogenic 2% of patients raised antibodies at the standard dose

Antibodies will always have novel sequences in their antigen binding regions – antibodies against these regions are called anti-idiotype antibodies

Success in Treating Chronic Inflammatory Diseases: At What Cost?

Serious infections have happened in people taking HUMIRA. These serious infections include tuberculosis (TB) and infections caused by viruses, fungi, or bacteria that have spread throughout the body. Some people have died from these infections. Your doctor should test you for TB before starting HUMIRA, and check you closely for signs and symptoms of TB during treatment with HUMIRA. If your doctor feels you are at risk, you may be treated with medicine for TB.

Cancer. For children and adults taking TNF blockers, including HUMIRA, the chance of getting lymphoma or other cancers may increase. There have been cases of unusual cancers in children, teenagers, and young adults using TNF blockers. Some people have developed a rare type of cancer called hepatosplenic T-cell lymphoma. This type of cancer often results in death. If using TNF blockers including HUMIRA, your chance of getting two types of skin cancer (basal cell and squamous cell) may increase. These types are generally not life-threatening if treated; tell your doctor if you have a bump or open sore that doesn’t heal.

Approaches to Isolation of Fully Human Antibodies (Adalimumab): Phage Display

Each individual phage particle embodies both phenotype (expressed antibody) and genotype (the gene encoding the antibody)

Select for a phage particle that binds to a particular antigen and you immediately have the gene encoding the antibody responsible for binding

Fusion protein produced between antibody fragment and gene 3 protein

Antibody Fragments Used in Phage Display

Single Chain Fv

Single Domain Ab

A Fully Human Production Platform

binding to antigen

randomized library of tri-nucleotides

A Semi-Synthetic Human Diversity Library

VH VL scFv

human framework human framework human framework

A Semi-Synthetic Human Platform

7H and 7L consensus frameworks, randomized in VH CDR3, initial library 2 x 109 variants

Adalimumab: A Fully Human Monoclonal Antibody????

Fully Human Antibodies: Transgenic Mice

• Human heavy (1.3 mg) and κ light chain (1.8 mb) on chromosomes 14 and 2, respectively

• Heavy: 95 V, 30 D, 6 J, and the constant region

• Light: 76 V, 65J, and the constant region

Expression of Human Immunoglobulins by Xenomouse (II) Mice

B lymphocyte marker

wild-type mice knockout mice Xenomouse (II) mice

Xenomouse (II) is Capable of Producing High Affinity Fully Human Antibodies

Is Ofatumumab Better than Rituximab?

Ofatumumab is a fully human antibody produced in transgenic mice immunized with CD20

• UK-based GlaxoSmithKline and Danish biotech firm Genmab have reported results from a Phase III trial (ORCHARRD) of ofatumumab (Arzerra) plus chemotherapy versus rituximab plus chemotherapy to treat patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL)

• The trial did not meet its primary endpoint as there was no statistically significant difference in progression free survival (PFS) between the treatment arms.

• According to the firms, there were no differences in adverse events (AEs) leading to treatment discontinuation, Grade >3 AEs, severe adverse events (SAEs), or fatal SAEs between the treatment arms.

• "Based on today's results, we are unlikely to move forward with a regulatory filing," Winkel said.

Allora, Is There Anything Our There that can Beat Rituximab in a Head to Head Trial?

Enter Obinutuzumab, a glyco-engineered humanized antibody

• Virtually identical Kd to CD20 as rituximab

• Type I antibodies cluster CD20 in lipid rafts and show a bias toward CDCC

• Type II antibodies show a bias toward more direct effects

Differences reside in mechanisms of target destruction

Antibodies are Glycoproteins and Glycosylation can Influence Effector Function

Antibodies are subject to N-linked glycosylation at residue 297 of the Fc region

Glycoproteins

N-linked glycosylation – oligosaccharides linked to Asn residues in the sequence context

Asn-X-Ser or Asn-X-Thr

Proteins are initially modified by a 14 residues core oligosaccharide

Dolichol is a lipid

CH2

C ONSugar

N-glycosidic bond

Hunusual high energy phosphate

ester linkage provides energy for forming N-glycosidic bond

Cotranslational Addition of the Core Oligosaccharide

oligosaccharide transferase

targeting signal

cytoplasm

rough ER membrane

ER lumen

complex

high mannose

Asn-X-SerAsn-X-Thr

Processing the Core Oligosaccharide

Glucose

Mannose

N-acetyl glucosamine

Fucose

Galactose

Sialic acid

High mannose class switches from trimming to addition

Complex class switches from trimming to addition

Cell Death Assays: Annexin Staining

Phosphatidylserine (PS) normally found on cytoplasmic surface of plasma membrane

During intermediate stages of apoptosis PS is translocated to the outer surface of the plasma membrane

Externalization of PS makes it available for binding to fluorescent PS-binding proteins (Annexins)