When B Cells Go Bad: Infection, Inflammation and Chronic B Cell Stimulation Karen S. Anderson MD PhD Associate Professor, Biodesign Institute Arizona 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 >10 11 potential molecules

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

Protein Expression … 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 Screening Gene CloningBacterial plating DNA preparation Array Printing

p53 Antigen array Add patient serum

Human papilloma virus (HPV16): ~70% of oropharyngeal cancers Emerging epidemic in US and Europe Serologic Biomarkers for HPV+ Cancer HPV Genome: 8 ORFs Diagnosis Prognosis 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 Patients NCI/Early Detection Research Network Zicarelli Foundation