In conclusion, the addition of rituximab to CHOP chemotherapy, given for eight cycles to elderly patients with newly diagnosed

diffuse large-B-cell lymphoma, significantly increases the rate of complete response, decreases the rates of treatment failure and

relapse, and improves event-free and overall survival as compared with standard CHOP alone. These gains were achieved without a

significant increase in clinically significant toxic effects.

Trial Outcomes (10 Year Follow Up)

Blood (2010) 116:2040

Most related to underlying conditions prior to lymphoma diagnosis

No response

Patients who had a complete

remission and relapsed

median time to progression 4.8 years in R-CHOP arm

median time to progression 1.2 years in CHOP arm

Progression Free Survival Long Term: CHOP Versus R-CHOP

Disease Free Survival in Patients Who had Originally had a Complete Remission

median time to event not reached in R-CHOP arm

median time to event 3.4 years in CHOP arm

These findings confirm that the use of R-CHOP can improve patient outcomes in elderly DLBCL patients,

and that the beneficial effects are sustained over a long follow-up period.

Rituximab’s Success has Opened the Floodgates of Further Developments in Therapeutic Antibody

Development

Refinements on Rituximab

Antibodies against other targets for use in different cancers

FDA approved therapeutic antibodies circa 2006

Rituximab’s Success has Opened the Floodgates of Further Developments in Therapeutic Antibody

Development

Continued refinement

Rituximab CDR – complementarity determining region

Light chain 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 27A 27B 27C 27D 27E 27F 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 95A 95B 95C 95D 95E 95F 96 97 98 99 100 101 102 103 104 105 106 106A 107 108 109

L1: L24-L34L2: L50-L56L3: L89-L97

H1: H26-H35BH2: H50-H65H3: H95-H102

Sequence gazing (L1): • starts approximately at residue 24• residue before always a cysteine• residue after always a tryptophan Length 10 to 17 residues

Kabat System for Identifying Complementary Determining Regions of Immunoglobulins

Making a Humanized Antibody from Mouse Complementary Determining Regions

• Replace human with mouse CDR

• Expression vector have complete human light chain

• Replacement not by cloning but by oligonucleotide directed mutagenesis

Polymerase Chain Reaction

• Starting DNA is rapidly diluted out with each cycle of PCR

• After several cycles, newly synthesized DNA bounded by forward and reverse primers is the dominant DNA species

✓

✓

Using PCR for Deletion Mutagenesis

• Starting material: plasmid DNA containing a gene of interest

• Two primers forming the boundaries of the region that will be deleted –directing DNA synthesis away from the region that will be deleted

• PCR amplification of DNA excluding the region that will be deleted

• Ligation of PCR product, confirmation by DNA sequence analysis

PCR can also be Used for Substitution Mutagenesis

• Starting material: plasmid DNA containing a gene of interest

• Two primers abutting one another facing in opposite directions

• One or both primers have desired substitutions but retain sufficient complementarity to anneal to human sequences

• PCR amplification of DNA: DNA with altered sequence will eventually predominate

• Ligation of PCR product, confirmation by DNA sequence analysis

Making a Humanized Antibody from Mouse Complementary Determining Regions

Replace human with mouse CDRs

Three-Dimensional Structure of the Variable Regions of Heavy and Light Chains

Determining Dissociation Constants Using Surface Plasmon Resonance (Biacore Biosensors)

• gold layer has a characteristic angle of reflected light

• angle altered by binding interactions on the surface exposed to flow cell

flow cellimmobilized

Biacore Readout

On rate

Off rate

Three-Dimensional Structure of the Variable Regions of Heavy and Light Chains

Refining the Properties of Humanized Antibodies

EC50- the higher the value the worse the binding

Pharmacokinetics of Humanized Antibody

• Half life in humans 17-21 days

• No evidence of anti-drug antibodies

Humanized Antibody has Anti-Tumor Properties in a Pre-Clinical Model

Antibody raised to VEGF

VEGF: vascular endothelial growth factor

Targeting Angiogenesis in Cancer Chemo- and Immuno-Therapy

Folkman (1971, NEJM 285,1182) rather than target tumor cells themselves attack the tumor’s supply of nutrients and other factors needed for survival

• New capillary growth (for tumors) is even more vigorous and continuous than a similar outgrowth of capillary sprouts observed in fresh wounds or inflammation

• The growth of solid neoplasms is always accompanied by neovascularization

• Targeting normal cells supporting tumor cell growth may be more efficacious than targeting genetically unstable tumor cells

• Target for anti-angiogenesis therapy - VEGF

Angiogenesis

Growth of new blood vessels from exiting vasculature

scientific papers published on angiogenesis

1970

2

2009

>5,200

75,832

total hits Jan 9, 2015

Angiogenesis

Normal processes

• growth and regression according to metabolic needs

• pregnancy

• wound healing

pathogenic processes

• tumorigenesis

Angiogenesis differs from vasculogenesis which is the formation of blood vessels de novo from mesodermal cells

Angiogenesis Versus Vasculogenesis

Vascular Endothelial Growth Factor

gradients of VEGF are major players in new vessel directional growth during vasculogenesis and angiogenesis

Tip Cell Selection Tip Cell Navigation Stalk Elongation

Tip Cell Fusion Perfusion/Oxygenation Pericyte Stabilization

mature endothelial cell(phalanx cells)

tip cell, initiates new vessel growth

stalk cell, dividing, filling in behind tip

Overview of Angiogenesis

pericyte

What is causing parenchymal cells to produce VEGF?

HYPOXIA

1 2 3

Oxygen Sensing

Hydroxylation of Pro and Lys residues in collagen requires:

• Fe2+

• α-ketoglutarate

• vitamin C

• O2

prolyl hydroxylase

hypoxia inducible factor

HIF-Pro-OH HIF-P-OH

Ub

Ub

proteasome

transcription factor

ubiquitin ligase

Prolyl hydroxylase enzymes have been adapted to function as an oxygen sensing device

when oxygen is available, prolyl hydroxylase is active

HIF Activation by Hypoxia

histone acetyltransferase

activity

VEGF-A

Epo

Von-Hippel-Lindau tumor suppressor

• tumors require an oxygen supply – biallelic inactivation of VHL leads to a variety of tumors

• congenital mutations in VHL may also lead to polythycemia

cis-acting sequence – hypoxia response element

prolyl hydroxylaseE3 ubiquitin

ligase

erythropoietin a cytokine that promotes red cell production

under hypoxic conditions PHD has reduced activity

Vascular Endothelial Growth Factor (VEGF) Family

• 7 separate genes encoding VEGF family members – VEGF (A-F) and placental growth factor

• splice variants produced from individual genes further increasing diversity

• VEGF-A a prototype

• 165 amino acids• multiple isoforms expressed in different tissues

6 amino acids found at the C-terminus coming

from exon 8a or 8b determines whether factor is pro- or anti-

angiogenic

choice between exons

Differential Splicing Gives Rise to Pro- and Anti-Angiogenic Forms of VEGF-A

favored downstream of HIF activation

3’ proximal splice site

3’ distal splice site

basal expression or promoted by a signaling pathway opposing angiogenesis

exon 7

VEGF-A Structure

ER secretory pathway

VEGF functions as a dimer VEGF-A can bind to one of two receptors

The GAG heparin can influence

VEGF-A/receptor interactions

6 amino acids at the C-terminus determines whether NP-1 is engaged

and whether VEGF-A isoform is pro- or anti-angiogenic

Anti - SLTRKDPro - CDKPRR

major form expressed at basal levels

in many adult tissues

VEGF in purple and yellow

VEGF Receptor Signaling

VEGF receptor is a member of the receptor tyrosine kinase family

Ligand binding to extracellular domain induces intracellular kinase activity and subsequent downstream signaling pathways

neuropilin 1

C-terminus

C-terminus

not all of these signaling pathways occur in the same cell at the same time - context

VEGF Signaling to Endothelial Cells Promoting Angiogenesis: Migration of Tip Cells

before initiating any substantive movement toward VEGF gradient endothelial cells have to deal with

the basal laminaphenotypic change in endothelial

cells, enhanced expression of matrix degrading proteases and

enhanced migratory behavior

epithelial mesenchymal

tip cells undergo a

epithelial to mesenchymal transition to

initiate angiogenesis

Tip cells are highly motile, but non-proliferative; there is roughly one tip cell per spout

matrix-metalloproteases

Some of the Phenotypic Characteristics of Cells Involved in Angiogenesis are Shared by Metastatic

Tumor Cells

blood vessel lining

1 2

3

cells undergo a epithelial to

mesenchymal transition to

promote metastasis

broad front of migration

slender projections extending from leading edge of lamellipodia, driven

by actin polymerization

cytoskeletal rearrangements needed for directional migration regulated by small

GTPase Rho/Rac

integrins found at tips of filopodia help extensions

adhere and pull cells forward

matrix metalloproteases help tips move through barriers

integrins at trailing edge need to dissociate

Migratory Behavior of Tip Cells

gene expression needed for motility driven by

transcription factors like SRF

Endothelial Cells of the Stalk have Phenotypes Distinct from Endothelial Tip Cells and Quiescent

Endothelial Cells

stalk cells are proliferative, but

lack sprouts

Interaction of Dll4 with notch represses VEGFR2 expression in stalk cells

suppressing the formation of new

spouts

tip cells have sprouts but much

reduced proliferation

Dll = delta-like ligand

Notch can also repress expression

Tip Cell Fusion

How do we know which is an arterial and which is a

venous vessel?

Differentiating Arteries and Veins During Angiogenesis

Steps in Cancer Metastasis

blood vessel lining1 2

3

colon adenocarcinoma metastasized to the lung

central areas of necrosis caused by anoxia as tumor

outgrows blood supply

Metastases may Retain Characteristics of Original Tumor and also Require a Blood Supply as Tumors Grow

Normal Angiogenesis is a Careful Balance of Pro- and Anti-Angiogenic Stimuli

This is not the case for tumor vasculature where pro-angiogenic signals predominate

creating vasculature that is anything but normal

angiostatin – inhibitor of

angiogenesis

Angiogenesis as a Target of Cancer Chemotherapy

M. Judah Folkman major proponent of using this approach to treat cancer

bevacizumab – antibody that recognizes and binds VEGF

• used alone to treat glioblastoma refractory to other treatments

• used in combination with other drugs to metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer

Strategies for Anti-Angiogenesis Therapy

sorafenib – small molecule inhibitor of VEGFR-2 – tyrosine kinase inhibitor

Suffixes in drug development

mab – antibody-based therapy

nib – small molecule kinase inhibitors

(u)

humanized antibodies

A Notorious Angiogenesis Inhibitor

thalidomide lenalidomide

currently approved for use in multiple myeloma and myelodysplastic

syndromes

tumor vessels lack normal arterial venous hierarchy, and are leaky and often

insufficient

this can impede drug delivery

anti-angiogenic therapy can

normalize tumor vasculature and improve delivery of other drugs

Effects of Normalizing Tumor Vasculature in Anti-Angiogenesis Therapy

normalized vasculature may reduce metastasis

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%

Limitations of Anti-Angiogenic Therapy for Cancer

• Many tumors overcome effects of targeted anti-angiogenic therapies by up-regulating alternative pathways

other growth factors unrelated to VEGF can also promote angiogenesis, eg fibroblast derived growth factor and platelet derived growth factor

• Hypoxia may promote more invasive phenotypes in tumor cells