Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

Abstract

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/ CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.

Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR

“…The parental clones used as templates for mutagenesis ( Table 1 ) were identified using a microfluidics-based single cell antibody discovery platform. 21 , 43 – 45 Briefly, fully human transgenic mice (Trianni, San Francisco, CA) were injected with soluble antigen. 31 Millions of B cells were run through the microfluidic system, generating yeast scFv libraries of natively paired Ig heavy and light chains.…”

Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers

“…In practice, AM is induced through administration of some dose of attenuated Ag, often mixed with adjuvants and other additives that have both immune-stimulatory effect and facilitate retention of Ag for longer periods of time ( Asensio et al, 2019 ; HogenEsch et al, 2018 ; Awate et al, 2013 ; Coffman et al, 2010 ). Whilst the adjuvant and additives define the nature of the immune response ( Coffman et al, 2010 ), Ag dose is a major variable in AM ( Eisen, 2014 ; Foote and Eisen, 1995 ; Kang et al, 2015 ).…”

How IvIg Can Mitigate Covid-19 Disease: A Symmetrical Immune Network Model

“…For example, the proposed symmetrical network involving antibody and anti-Id antibodies, produced by regulatory Bidregs could be studied at a monoclonality level. Monoclonal antibodies rescued by single cell immunoglobulin extraction (29) would support the hypothesis and allow functional and structural studies of the regulatory network.…”