Ancient Lamprey VLR Antibodies as Tumor Diagnostic and Tumor Targeting Reagents Open Access

Nakahara, Hirotomo (2017)

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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


Part I: Finding Inspiration in the Immune System

Vaccination 1

Serum 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 18

Chapter 2: Chronic Lymphocytic Leukemia Monitoring with a Lamprey Idiotope-Specific Antibody 31

Abstract 32 Introduction 33

Materials and Methods 35

Results and Discussion 39 Figures and Legends 43 Supplementary Materials and Methods 49 Supplementary Figures and Legends 53 References 63

Chapter 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 93

Chapter 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 109

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. 60

Table S2: Detection of CLL recurrence by quantitative real-time PCR 62

CHAPTER 3 Figure 1: Flow cytometric analysis of monoclonal VLR-C8 reactivity. 82

Figure 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. 86

Figure 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. 106

Table 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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