when b cells go bad: infection, inflammation and chronic b cell stimulation

When B Cells Go Bad: Infection, Inflammation and Chronic B

Cell StimulationKaren S. Anderson MD PhD

Associate Professor, Biodesign InstituteArizona State University

Mayo Clinic Arizona

Conflicts of Interest• I serve on the advisory board and have received consultant

fees and stock options with Provista Dx.

• No off-label clinical diagnostics or therapeutics will be discussed.

W. Michael Kuehl & P. Leif Bergsagel, 2002

Monoclonal Gammopathy of Undetermined Signficance

• 3.2% of adults over the age of 50; 6.6% of adults over age 80• Premalignant disease that can transition to myeloma; average

2-15 years, rate 1% per year• Over 90% of patients with MM have a premalignant plasma

cell disorder• 50% Ig translocations; 50% hyperdiploid

– Errors in switch recombination and somatic hypermutation of the B cell

• Likely due to an abnormal response to antigenic stimulation

Chronic Infections and Antibodies

• EBV, HHV-8, HCV, (CMV), H. pylori are associated with B cell lymphoma and B cell chronic lymphocytic leukemia– what about myeloma?

• Ig in HCV-positive myeloma patients can target the virus

• ~20% of MGUS and myeloma Abs target infectious antigens (ASH 2013)

• Would treatment of chronic infections also prevent or treat MGUS?

Chronic Infections not Associated with MGUS

Bida and Rajkumar Mayo Clin Proc 2009

Moir and Fauci, Nat Rev Immunol 2009

How B cells Proliferate in response to infection

Antibody structure:

Ligands (antigens)

• What is the specificity of antibodies?• What is the diversity of antibodies?

When Ig genes rearrange, there are >1011 potential molecules

To facilitate research, DNASU stores over 162,000 plasmids and distributes these to researchers in 37 states and 38 countries

ProteinExpression

…

1. Print Plasmids

2. Express & capture proteins

Nucleic Acid-Programmable Protein Array (NAPPA)

Replicate arrays of candidate proteins

Find antibodies in patients’ blood

• Proteomics: the study of all of the proteins in the human body• We use plasmids to make over 10,000 human proteins• By putting these plasmids on a glass slide (NAPPA) we can make and study over 10,000

proteins at one time

NAPPA Array Production and ScreeningGene Cloning Bacterial plating

DNA preparation

Array Printing

p53

Antigen array

Add patient serum

Human papilloma virus (HPV16): ~70% of oropharyngeal cancersEmerging epidemic in US and Europe

Serologic Biomarkers for HPV+ Cancer

HPV Genome: 8 ORFsDiagnosis Prognosis

0.00

0.25

0.50

0.75

1.00

Pro

port

ion

surv

ivin

g

13 9 6 4 3 1NE2 negative84 75 66 59 33 10NE2 positive

Number at risk

0 12 24 36 48 60Follow-up time (months)

NE2 positiveNE2 negative

Kaplan-Meier survival estimates

NE2, p<0.001

Identification of the Targets of Antibodies

• Protein microarrays are now used to test >10,000 proteins for antibody targets

• Large gene collections can be leveraged for rapid protein display• We are developing pathogen-specific arrays to identify

antibodies in blood

• We need to measure the antibody IMMUNOME to understand the pathogenesis of MGUS

What is the diversity of Antibodies?Can we detect specific Ig rearrangements?

DNA Origami nanostructure design

Hao Yan and Joe Blattman, Biodesign

Overall strategy to obtain linked RNA sequences from single cells.

Conclusions

Antibodies have extraordinary sequence diversity

There are emerging technologies for quantitating that diversity

By linking single-cell RNA capture with next-gen sequencing, we may be able to: Identify early events (?pre-MGUS) of loss of diversity Rapidly generate patient-specific probes for molecular detection of

rearrangements

Acknowledgements

Mayo Oncology Don Northfelt Doug Lake Barb Pockaj Michael Barrett

ASU Biodesign Institute Joseph Blattman Hao Yan Josh LaBaer Ji Qiu Garrick Wallstrom Laura Gonzalez Jin Park Fernanda Festa

Anderson Lab Julia Cheng Ting Li Rizwan Alam Benjamin Katchman Krishna Sundaresan Diego Chowell Shay Ferdosi Hans Frykman I.Purushothaman Fernando Hernandez Robert Brown Alison Goulder Jack Resnik Peter Chang

Our PatientsNCI/Early Detection Research NetworkZicarelli Foundation