RESUMO
A major challenge in adoptive T cell immunotherapy is the discovery of natural T cell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human T cell line at high throughput. We applied TCR-Engine to successfully engineer synthetic TCRs for increased potency and specificity to a clinically relevant tumor-associated antigen (MAGE-A3) and validated their translational potential through multiple in vitro and in vivo assessments of safety and efficacy. Thus, TCR-Engine represents a valuable technology for engineering of safe and potent synthetic TCRs for immunotherapy applications.
Assuntos
Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T , Antígenos de Neoplasias , Humanos , Imunoterapia , PeptídeosRESUMO
In recent years, chimeric antigen receptor (CAR) T cell cancer immunotherapies have advanced substantially in the clinic. However, challenges related to safety persist; one major concern occurs when CARs trigger a response to antigen present on healthy cells (on-target, off-tumor response). A strategy to ameliorate this relies on the complex relationship between receptor affinity and signaling, such that one can engineer a CAR that is only activated by tumor cells expressing high antigen levels. Here, we developed a CAR T cell display platform with stable genomic expression and rapid functional screening based on interleukin-2 signaling. Starting with a CAR with high affinity toward its target antigen, we combined CRISPR-Cas9 genome editing and deep mutational scanning to generate a library of antigen-binding domain variants. This library was subjected to multiple rounds of selection based on either antigen binding or cell signaling. Deep sequencing of the resulting libraries and a comparative analysis revealed the enrichment and depletion of specific variants from which we selected CARs that were selectively activated by tumor cells based on antigen expression levels. Our platform demonstrates how directed evolution based on functional screening and deep sequencing-guided selection can be combined to enhance the selectivity and safety of CARs.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Animais , Afinidade de Anticorpos , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Técnicas de Cocultura , Feminino , Edição de Genes/métodos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Células MCF-7 , Camundongos , Receptor ErbB-2/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/imunologiaRESUMO
IL-17-producing γδ T (γδT-17) cells have proved to be an important early source of IL-17 in many inflammatory settings and are emerging as an important participant in protumor immune responses. Considering that their peripheral activation depends largely on innate signals rather than TCR ligation, it is important to understand what mechanisms exist to curb unwanted activation. Expression of the high-affinity IL-2R on γδT-17 cells prompted us to investigate a role for this cytokine. We found γδT-17 cells to be enriched, not depleted, in IL-2-deficient mice. The absence of IL-2 also resulted in higher IL-17 production and the emergence of IL-17+IFN-γ+ double producers. Furthermore, the addition of IL-2 to in vitro cultures of sorted γδT-17 cells was able to moderate IL-17 and affect differentiation into polyfunctional cytokine-producing cells. Interestingly, the Vγ6+ subset was more susceptible to the effects of IL-2 than Vγ4+ γδT-17 cells. We also found that unlike other γδ T cells, γδT-17 cells do not produce IL-2, but express Blimp-1, a known transcriptional repressor of IL-2. Although IL-2 was able to induce robust proliferation of γδT-17 cells, it did not sustain viability, negatively impacting their survival via downregulation of the IL-7R. Taken together, these data indicate that IL-2 can augment the γδT-17 response in favor of short-lived effectors with limited plasticity, particularly in the presence of IL-1ß and IL-23. In this way, IL-2 may act to curtail the innate-like response of γδT-17 cells upon arrival of IL-2-producing adaptive immune cells at the site of inflammation.
Assuntos
Interleucina-17/biossíntese , Interleucina-2/imunologia , Interleucina-2/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Citometria de Fluxo , Inflamação , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-17/imunologia , Interleucina-2/deficiência , Interleucina-2/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptores de Interleucina-7/genética , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The best-understood mechanisms for achieving antibody self/non-self discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgM(low) IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2). In a Hy10 antibody transgenic model where anergic B cells respond to a biophysically defined lysozyme epitope displayed on both foreign and self-antigens, cell transfers revealed that anergic IgM(low) IgD+ B cells form twice as many GC progeny as naïve IgM(hi) IgD+ counterparts. Their GC progeny were rapidly selected for CDR2 mutations that blocked 72% of antigen-binding sites with N-linked glycan, decreased affinity 100-fold, and then cleared the binding sites of blocking glycan. These results provide evidence for a mechanism to acquire self/non-self discrimination by somatic mutation away from self-reactivity, and reveal how varying the efficiency of N-glycosylation provides a mechanism to modulate antibody avidity.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Anergia Clonal/imunologia , Centro Germinativo/imunologia , Região Variável de Imunoglobulina/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autoanticorpos/genética , Feminino , Glicosilação , Humanos , Imunoglobulina D/genética , Imunoglobulina D/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
Monoclonal antibodies are a growing class of targeted cancer therapeutics, characterized by exquisite specificity, long serum half-life, high affinity and immune effector functions. In this review, we outline key advances in the field with a particular focus on recent and emerging classes of engineered antibody therapeutic candidates, discuss molecular structure and mechanisms of action and provide updates on clinical development and practice.
Assuntos
Anticorpos Monoclonais , Neoplasias , Humanos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologia , Neoplasias/tratamento farmacológico , RadioimunoterapiaRESUMO
Characterization of COVID-19 antibodies has largely focused on memory B cells; however, it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in resolving SARS-CoV-2 infection. Little is known about the specificity of plasma cells, largely because plasma cells lack surface antibody expression, thereby complicating their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and mammalian display to interrogate the specificity of plasma cells from 16 convalescent patients. Single-cell sequencing allows us to profile antibody repertoire features and identify expanded clonal lineages. Mammalian display screening is used to reveal that 43 antibodies (of 132 candidates) derived from expanded plasma cell lineages are specific to SARS-CoV-2 antigens, including antibodies with high affinity to the SARS-CoV-2 receptor-binding domain (RBD) that exhibit potent neutralization and broad binding to the RBD of SARS-CoV-2 variants (of concern/interest).
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Plasmócitos/metabolismo , SARS-CoV-2/imunologia , Análise de Célula Única/métodos , Animais , Anticorpos Antivirais/isolamento & purificação , COVID-19/imunologia , COVID-19/prevenção & controle , Células Cultivadas , Estudos de Coortes , Biblioteca Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mamíferos , Testes de Neutralização , Biblioteca de Peptídeos , Plasmócitos/químicaRESUMO
COVID-19 disease outcome is highly dependent on adaptive immunity from T and B lymphocytes, which play a critical role in the control, clearance and long-term protection against SARS-CoV-2. To date, there is limited knowledge on the composition of the T and B cell immune receptor repertoires [T cell receptors (TCRs) and B cell receptors (BCRs)] and transcriptomes in convalescent COVID-19 patients of different age groups. Here, we utilize single-cell sequencing (scSeq) of lymphocyte immune repertoires and transcriptomes to quantitatively profile the adaptive immune response in COVID-19 patients of varying age. We discovered highly expanded T and B cells in multiple patients, with the most expanded clonotypes coming from the effector CD8+ T cell population. Highly expanded CD8+ and CD4+ T cell clones show elevated markers of cytotoxicity (CD8: PRF1, GZMH, GNLY; CD4: GZMA), whereas clonally expanded B cells show markers of transition into the plasma cell state and activation across patients. By comparing young and old convalescent COVID-19 patients (mean ages = 31 and 66.8 years, respectively), we found that clonally expanded B cells in young patients were predominantly of the IgA isotype and their BCRs had incurred higher levels of somatic hypermutation than elderly patients. In conclusion, our scSeq analysis defines the adaptive immune repertoire and transcriptome in convalescent COVID-19 patients and shows important age-related differences implicated in immunity against SARS-CoV-2.
Assuntos
Envelhecimento/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , SARS-CoV-2/fisiologia , Imunidade Adaptativa , Adulto , Idoso , Células Cultivadas , Convalescença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Análise de Célula Única , Transcriptoma , Adulto JovemRESUMO
High-throughput single-cell sequencing (scSeq) technologies are revolutionizing the ability to molecularly profile B and T lymphocytes by offering the opportunity to simultaneously obtain information on adaptive immune receptor repertoires (VDJ repertoires) and transcriptomes. An integrated quantification of immune repertoire parameters, such as germline gene usage, clonal expansion, somatic hypermutation and transcriptional states opens up new possibilities for the high-resolution analysis of lymphocytes and the inference of antigen-specificity. While multiple tools now exist to investigate gene expression profiles from scSeq of transcriptomes, there is a lack of software dedicated to single-cell immune repertoires. Here, we present Platypus, an open-source software platform providing a user-friendly interface to investigate B-cell receptor and T-cell receptor repertoires from scSeq experiments. Platypus provides a framework to automate and ease the analysis of single-cell immune repertoires while also incorporating transcriptional information involving unsupervised clustering, gene expression and gene ontology. To showcase the capabilities of Platypus, we use it to analyze and visualize single-cell immune repertoires and transcriptomes from B and T cells from convalescent COVID-19 patients, revealing unique insight into the repertoire features and transcriptional profiles of clonally expanded lymphocytes. Platypus will expedite progress by facilitating the analysis of single-cell immune repertoire and transcriptome sequencing.
RESUMO
The therapeutic development of monoclonal antibodies requires robust and reliable methods for their recombinant expression and characterization. In this context, an increasingly important aspect in the antibody development process is to determine the contribution of Fc-mediated immune effector functions to therapeutic activity. Here we describe steps for the cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions, based on mutation of Fc-gamma receptor and complement-binding sites. The resulting antibody preparations contain low levels of endotoxin and are suitable for testing in animal models of disease.
Assuntos
Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Linhagem Celular , Cromatografia de Afinidade , DNA/metabolismo , Vetores Genéticos/metabolismo , Humanos , Imunoglobulina G/química , Região Variável de Imunoglobulina/química , Plasmídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells. Antibody constructs are designed by combining antibody variable domains, generated by phage display or derived from human/humanized monoclonals, with constant regions. The constructs are then expressed from mammalian vectors, secreted into culture media, purified by affinity chromatography and characterized by biolayer interferometry. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin-free antibody reagents suitable for testing in animal models within a 3-week time frame.
Assuntos
Anticorpos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Animais , Anticorpos/metabolismo , Células Cultivadas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida/métodos , Vetores Genéticos , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Interferometria/métodos , Mamíferos , Proteínas Recombinantes/metabolismoRESUMO
Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).
Assuntos
Antígenos/sangue , Linfócitos B/imunologia , Cromatografia de Afinidade/métodos , Epitopos , Análise de Sequência de Proteína/métodos , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Interferometria , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Interleukin-2 (IL-2) is an established therapeutic agent used for cancer immunotherapy. Since treatment efficacy is mediated by CD8+ and NK cell activity at the tumour site, considerable efforts have focused on generating variants that expand these subsets systemically, as exemplified by IL-2/antibody complexes and 'superkines'. Here we describe a novel determinant of antitumour activity using fusion proteins consisting of IL-2 and the antibody fragment crystallizable (Fc) region. Generation of long-lived IL-2-Fc variants in which CD25 binding is abolished through mutation effectively prevents unwanted activation of CD25+ regulatory T-cells (Tregs) and results in strong expansion of CD25- cytotoxic subsets. Surprisingly, however, such variants are less effective than wild-type IL-2-Fc in mediating tumour rejection. Instead, we report that efficacy is crucially dependent on depletion of Tregs through Fc-mediated immune effector functions. Our results underpin an unexpected mechanism of action and provide important guidance for the development of next generation IL-2 therapeutics.
Assuntos
Antineoplásicos/farmacologia , Fragmentos de Imunoglobulinas/imunologia , Imunoterapia , Ativação Linfocitária , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Feminino , Imunoglobulina G/imunologia , Memória Imunológica , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/citologia , Subpopulações de Linfócitos/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutagênese , Neoplasias/terapia , Proteínas Recombinantes/metabolismo , Baço/citologia , Baço/metabolismoRESUMO
The archetypal T cell-dependent antigen is sheep red blood cells (SRBCs), which have defined much of what we know about humoral immunity. Early studies using solubilized or sonicated SRBCs argued that the intact structure of SRBCs was important for optimal antibody responses. However, the reason for the requirement of intact SRBCs for the response to polyvalent protein antigen remained unknown. Here, we report that the immune response to SRBCs is driven by cytosolic recognition of SRBC RNA through the RIG-I-like receptor (RLR)-mitochondrial anti-viral signaling adaptor (MAVS) pathway. Following the uptake of SRBCs by antigen-presenting cells, the MAVS signaling complex governs the differentiation of both T follicular cells and antibody-producing B cells. Importantly, the involvement of the RLR-MAVS pathway precedes that of endosomal Toll-like receptor pathways, yet both are required for optimal effect.
Assuntos
Eritrócitos/imunologia , RNA/imunologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Poli I-C/farmacologia , Ovinos , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMO
The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.
RESUMO
The first candidates from the promising class of small non-antibody protein scaffolds are now moving into clinical development and practice. Challenges remain, and scaffolds will need to be further tailored toward applications where they provide real advantages over established therapeutics to succeed in a rapidly evolving drug development landscape.