Ancient Lamprey VLR Antibodies as Tumor Diagnostic and Tumor Targeting Reagents Public
Nakahara, Hirotomo (2017)
Published
Abstract
Jawless vertebrates (lamprey and hagfish) possess an unusual adaptive immune system that lacks conventional Ig/TCR genes used by all other vertebrate species for antigen recognition. Instead, jawless vertebrates use leucine-rich repeats (LRR) to generate three types of variable lymphocyte receptors (VLR): VLRA and VLRB found on T-like cells, and VLRB found on B-like cells. In response to immunization, VLRB cells proliferate and differentiate into VLRB antibody-secreting plasmacytes. The potential VLRB antibody repertoire is estimated to be greater than 1014 unique VLR clones, which are generated through a gene conversion-like process that replaces the non-coding segments within the incomplete germline VLR gene with randomly selected sequence diverse LRR subunits. Given the 500 million years of evolution separating jawless vertebrates from all other vertebrates, VLR should be able to access novel epitopes that are forbidden to conventional Ig due to self-tolerance.
In search of novel tumor-specific epitopes, we immunized lampreys
with B cell leukemia clones from patients with B cell chronic
lymphocytic leukemia (CLL) or mouse BCL1 leukemia to generate
recombinant monoclonal VLRB antibody libraries, which were then
screened for tumor-specificity. From the CLL-immunized library, we
identified an antibody, VLR39, which was specific for the donor CLL
cells and recognized the heavy chain variable region (VH)
complementarity determining region 3 (CDR3) of the B-cell receptor
(BCR). Using this antibody to monitor the CLL donor after
chemoimmunotherapy-induced remission, we detected the recurrence of
the leukemic clone before significant increase in lymphocyte count
or CD5+ B cells. From the BCL1-immunized library, we identified an
antibody, VLR-C8, which was specific for the BCL1 clones and also
found to recognize the VH/VL CDR3 of the BCR.
Lamprey antibodies exhibit exquisite specificity for a protein
epitopes, which in this case was the signature VH/VL CDR3 sequence
of B cell leukemia clones, and offer a rapid strategy for
generating anti-idiotype antibodies for early detection of leukemia
recurrence and may potentially be used as a tumor targeting
reagent.
Table of Contents
TABLE OF CONTENTS ITEM PAGE CHAPTER 1: Introduction 1
Part I: Finding Inspiration in the Immune System
Vaccination 1Serum Therapy 2
Monoclonal Antibodies 3
Part II: Improving on Nature
Limitations of the IgG Format 5
Engineered Ig Domains 6
Non-Ig Protein Scaffolds 8
Part III: The Agnathan Adaptive Immune System
Non-Ig Adaptive Immune System 9
Variable Lymphocyte Receptors 10
VLRB protein and structure 11
Antigen-specificity of VLRB antibodies 12
The biotechnology niche occupied by VLRB 15
References 18Chapter 2: Chronic Lymphocytic Leukemia Monitoring with a Lamprey Idiotope-Specific Antibody 31
Abstract 32 Introduction 33Materials and Methods 35
Results and Discussion 39 Figures and Legends 43 Supplementary Materials and Methods 49 Supplementary Figures and Legends 53 References 63Chapter 3: Recognition of a mouse BCL1 leukemia BCR idiotope by a lamprey antibody 67
Abstract 68 Introduction 69 Materials and Methods 72 Results and Discussion 77 Figures and Legends 82 Supplementary Materials and Methods 89 References 93Chapter 4: Discussion and Future Directions 97
Tumor antigen discovery via VLRB antibodies 97 Clinical applications of anti-Id VLRB antibodies 100
Tumor-targeting VLRB antibodies 103 Figures and Legends 106 References 109LIST OF FIGURES CHAPTER/FIGURE PAGE CHAPTER 2
Figure 1: Flow cytometric analysis of monoclonal VLR39 reactivity. 43
Figure 2: ELISA and flow cytometric analysis of VLR39 binding to donor CLL Igs. 44
Figure 3: Monitoring for CLL recurrence. 46
Figure 4: B cell and absolute lymphocyte count of patient after treatment. 48
Figure S1: Flow cytometric analysis of monoclonal VLR39 reactivity with different cell types and cell lines. 53
Table S1: CLL VH gene families and HCDR3 sequences 55
Figure S2: Flow cytometric analysis of monoclonal VLR39 reactivity with CLL cells with different VH gene sets. 57
Figure S3: CLL VH gene sequence analysis. 58
Figure S4: IGHV-D-J sequence alignment of VLR39 sorted cells. 60Table S2: Detection of CLL recurrence by quantitative real-time PCR 62
CHAPTER 3 Figure 1: Flow cytometric analysis of monoclonal VLR-C8 reactivity. 82Figure 2: Flow cytometric analysis of VLR-C8 binding to BCL1-3B3 cells after surface Ig modulation. 84
Figure 3: Amino acid sequence alignment of Ig heavy and light chains comprising the hybrid scFv mutants. 85
Figure 4: VLR-C8 binding to BCL1, CLL, and hybrid scFv. 86Figure 5: Flow cytometric analysis of VLR-C8 expressed as a monomer and Fc-fusion protein. 87
CHAPTER 4 Figure 1: Flow cytometric analysis of plasma from CLL immunized lamprey. 106Table 1: Screening results of monoclonal VLRB antibodies from CLL immunized lampreys. 107
Figure 2: Flow cytometric analysis of anti-Id VLRB antibodies in monomeric form. 108
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