RESUMO
Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) possess mutations that prevent antibody therapeutics from maintaining antiviral binding and neutralizing efficacy. Monoclonal antibodies (mAbs) shown to neutralize Wuhan-Hu-1 SARS-CoV-2 (ancestral) strain have reduced potency against newer variants. Plasma-derived polyclonal hyperimmune drugs have improved neutralization breadth compared with mAbs, but lower titers against SARS-CoV-2 require higher dosages for treatment. We previously developed a highly diverse, recombinant polyclonal antibody therapeutic anti-SARS-CoV-2 immunoglobulin hyperimmune (rCIG). rCIG was compared with plasma-derived or mAb standards and showed improved neutralization of SARS-CoV-2 across World Health Organization variants; however, its potency was reduced against some variants relative to ancestral, particularly omicron. Omicron-specific antibody sequences were enriched from yeast expressing rCIG-scFv and exhibited increased binding and neutralization to omicron BA.2 while maintaining ancestral strain binding and neutralization. Polyclonal antibody libraries such as rCIG can be utilized to develop antibody therapeutics against present and future SARS-CoV-2 threats.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/genética , Anticorpos Monoclonais/uso terapêutico , Antivirais , Saccharomyces cerevisiae , Anticorpos Neutralizantes/uso terapêutico , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais/uso terapêuticoRESUMO
PURPOSE: Most individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT. METHODS: We sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT. RESULTS: Compared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations). CONCLUSION: Antibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.
Assuntos
Imunoglobulina G , Leucócitos Mononucleares , Humanos , Imunoglobulina M , Sequência de Bases , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.
RESUMO
Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.
Assuntos
Linfócitos B/imunologia , COVID-19/terapia , Globulinas/biossíntese , SARS-CoV-2/imunologia , Animais , Anticorpos Antivirais/imunologia , Células CHO , Cricetulus , Ensaio de Imunoadsorção Enzimática , Globulinas/imunologia , Humanos , Imunização Passiva , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Zika virus/imunologia , Soroterapia para COVID-19RESUMO
T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.
Assuntos
Antígenos/análise , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/citologia , Engenharia Celular , Biblioteca Gênica , Humanos , Células Jurkat , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Linfócitos T/imunologia , Vírus/imunologiaRESUMO
To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism.
RESUMO
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.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Diversidade de Anticorpos , Imunização/métodos , Microfluídica/métodos , Animais , Anticorpos Monoclonais Humanizados/imunologia , Linfócitos B/imunologia , Antígeno CTLA-4/imunologia , Regiões Determinantes de Complementaridade/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas , Camundongos , Camundongos Transgênicos , Biblioteca de Peptídeos , Receptor de Morte Celular Programada 1/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.
Assuntos
Linfócitos B/imunologia , Biblioteca Gênica , Cadeias Leves de Imunoglobulina , Subunidade alfa de Receptor de Interleucina-21 , Anticorpos de Cadeia Única , Animais , Humanos , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Subunidade alfa de Receptor de Interleucina-21/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-21/imunologia , Camundongos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
Affinity-matured, functional anti-pathogen antibodies are present at low frequencies in natural human repertoires. These antibodies are often excellent candidates for therapeutic monoclonal antibodies. However, mining natural human antibody repertoires is a challenge. In this study, we demonstrate a new method that uses microfluidics, yeast display, and deep sequencing to identify 247 natively paired anti-pathogen single-chain variable fragments (scFvs), which were initially as rare as 1 in 100,000 in the human repertoires. Influenza A vaccination increased the frequency of influenza A antigen-binding scFv within the peripheral B cell repertoire from <0.1% in non-vaccinated donors to 0.3-0.4% in vaccinated donors, whereas pneumococcus vaccination did not increase the frequency of antigen-binding scFv. However, the pneumococcus scFv binders from the vaccinated library had higher heavy and light chain Replacement/Silent mutation (R/S) ratios, a measure of affinity maturation, than the pneumococcus binders from the corresponding non-vaccinated library. Thus, pneumococcus vaccination may increase the frequency of affinity-matured antibodies in human repertoires. We synthesized 10 anti-influenza A and nine anti-pneumococcus full-length antibodies that were highly abundant among antigen-binding scFv. All 10 anti-influenza A antibodies bound the appropriate antigen at KD<10 nM and neutralized virus in cellular assays. All nine anti-pneumococcus full-length antibodies bound at least one polysaccharide serotype, and 71% of the anti-pneumococcus antibodies that we tested were functional in cell killing assays. Our approach has future application in a variety of fields, including the development of therapeutic antibodies for emerging viral diseases, autoimmune disorders, and cancer.
Assuntos
Anti-Infecciosos/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Genômica/métodos , Microfluídica/métodos , Sequência de Aminoácidos , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/metabolismo , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/imunologia , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/imunologiaRESUMO
Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with KD < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Genômica/métodos , Microfluídica/métodos , Receptor de Morte Celular Programada 1/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Regiões Determinantes de Complementaridade/metabolismo , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas , Camundongos , Biblioteca de Peptídeos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging.
Assuntos
Aminoácidos/genética , Proteínas do Capsídeo/genética , Levivirus/genética , Montagem de VírusRESUMO
Nature uses protein compartmentalization to great effect for control over enzymatic pathways, and the strategy has great promise for synthetic biology. In particular, encapsulation in nanometer-sized containers to create nanoreactors has the potential to elicit interesting, unexplored effects resulting from deviations from well-understood bulk processes. Self-assembled protein shells for encapsulation are especially desirable for their uniform structures and ease of perturbation through genetic mutation. Here, we use the MS2 capsid, a well-defined porous 27 nm protein shell, as an enzymatic nanoreactor to explore pore-structure effects on substrate and product flux during the catalyzed reaction. Our results suggest that the shell can influence the enzymatic reaction based on charge repulsion between small molecules and point mutations around the pore structure. These findings also lend support to the hypothesis that protein compartments modulate the transport of small molecules and thus influence metabolic reactions and catalysis in vitro.