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1.
Nat Immunol ; 25(5): 916-924, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38698238

RESUMO

B cells and T cells are important components of the adaptive immune system and mediate anticancer immunity. The T cell landscape in cancer is well characterized, but the contribution of B cells to anticancer immunosurveillance is less well explored. Here we show an integrative analysis of the B cell and T cell receptor repertoire from individuals with metastatic breast cancer and individuals with early breast cancer during neoadjuvant therapy. Using immune receptor, RNA and whole-exome sequencing, we show that both B cell and T cell responses seem to coevolve with the metastatic cancer genomes and mirror tumor mutational and neoantigen architecture. B cell clones associated with metastatic immunosurveillance and temporal persistence were more expanded and distinct from site-specific clones. B cell clonal immunosurveillance and temporal persistence are predictable from the clonal structure, with higher-centrality B cell antigen receptors more likely to be detected across multiple metastases or across time. This predictability was generalizable across other immune-mediated disorders. This work lays a foundation for prioritizing antibody sequences for therapeutic targeting in cancer.


Assuntos
Linfócitos B , Neoplasias da Mama , Vigilância Imunológica , Humanos , Feminino , Neoplasias da Mama/imunologia , Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Monitorização Imunológica , Sequenciamento do Exoma , Antígenos de Neoplasias/imunologia , Metástase Neoplásica , Células Clonais
2.
Nat Biomed Eng ; 8(3): 214-232, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37814006

RESUMO

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody-antigen interactions. The method, which we named 'deep screening', involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument's flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.


Assuntos
Anticorpos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anticorpos/genética , Anticorpos/metabolismo , Biblioteca Gênica , Fragmentos de Imunoglobulinas , Ribossomos/genética , Ribossomos/metabolismo
3.
ACS Cent Sci ; 8(8): 1182-1195, 2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36032770

RESUMO

The assembly of robust, modular biological components into complex functional systems is central to synthetic biology. Here, we apply modular "plug and play" design principles to a solid-phase protein display system that facilitates protein purification and functional assays. Specifically, we capture proteins on polyacrylamide hydrogel display beads (PHD beads) made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA), that react covalently with SNAP-/Halo-tag fusion proteins, respectively, in a specific, orthogonal, and stable fashion. Anchors, and thus proteins, are distributed throughout the entire bead volume, allowing attachment of ∼109 protein molecules per bead (⌀ 20 µm) -a higher density than achievable with commercial surface-modified beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either noncovalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher, and SnpTag), in mono- and multivalent display formats. Solid-phase protein binding and enzymatic assays are carried out, and incorporating the photocleavable protein PhoCl enables the controlled release of modules via visible-light irradiation for functional assays in solution. We utilize photocleavage for valency engineering of an anti-TRAIL-R1 scFv, enhancing its apoptosis-inducing potency ∼50-fold through pentamerization.

4.
MAbs ; 14(1): 2006123, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34923908

RESUMO

The increasing global occurrence of recalcitrant multi-drug resistant Klebsiella pneumoniae infections warrants the investigation of alternative therapy options, such as the use of monoclonal antibodies (mAbs). We used a target-agnostic phage display approach to K. pneumoniae bacteria lacking bulky, highly variable surface polysaccharides in order to isolate antibodies targeting conserved epitopes among clinically relevant strains. One antibody population contained a high proportion of unique carbohydrate binders, and biolayer interferometry revealed these antibodies bound to lipopolysaccharide (LPS). Antibodies that bound to O1 and O1/O2 LPS were identified. Antibodies were found to promote opsonophagocytic killing by human monocyte-derived macrophages and clearance of macrophage-associated bacteria when assessed using high-content imaging. One antibody, B39, was found to protect mice in a lethal model of K. pneumoniae pneumonia against both O1 and O2 strains when dosed therapeutically. High-content imaging, western blotting and fluorescence-activated cell sorting were used to determine binding to a collection of clinical K. pneumoniae O1 and O2 strains. The data suggests B39 binds to D-galactan-I and D-galactan-II of the LPS of O1 and O2 strains. Thus, we have discovered an mAb with novel binding and functional activity properties that is a promising candidate for development as a novel biotherapeutic for the treatment and prevention of K. pneumoniae infections.


Assuntos
Anticorpos Antibacterianos/imunologia , Epitopos/imunologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Animais , Farmacorresistência Bacteriana Múltipla/genética , Farmacorresistência Bacteriana Múltipla/imunologia , Epitopos/genética , Humanos , Infecções por Klebsiella/genética , Klebsiella pneumoniae/genética , Lipopolissacarídeos/genética , Camundongos , Opsonização
5.
Anal Chem ; 93(4): 2166-2173, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33397084

RESUMO

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (Kd) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.


Assuntos
Proteínas Fúngicas/química , Proteínas Imobilizadas/química , Microesferas , Peptídeos/química , Proteínas Recombinantes/química , Anticorpos de Cadeia Única/química , Ligação Proteica
6.
Proc Natl Acad Sci U S A ; 117(44): 27307-27318, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33067389

RESUMO

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.


Assuntos
Anticorpos/genética , Afinidade de Anticorpos/genética , Engenharia Genética/métodos , Afinidade de Anticorpos/imunologia , Evolução Molecular , Humanos , Mutação INDEL/genética , Região Variável de Imunoglobulina/genética , Mutagênese , Mutagênese Insercional/métodos , Deleção de Sequência
7.
Nat Commun ; 11(1): 1326, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165615

RESUMO

Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.


Assuntos
Fator H do Complemento/metabolismo , Trypanosoma/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Bovinos , Membrana Celular/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/química , Cricetinae , Cricetulus , Camundongos Endogâmicos BALB C , Parasitemia/sangue , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação para Cima
8.
Front Immunol ; 11: 605170, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33384691

RESUMO

Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 31 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients. Clonal expansion of the B cell population is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 1,254 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls or individuals following seasonal influenza vaccination. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralizing antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA, and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand patient responses.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/patologia , Receptores de Antígenos de Linfócitos B/genética , SARS-CoV-2/imunologia , Linfócitos B/imunologia , COVID-19/imunologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Nat Microbiol ; 4(12): 2074-2081, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636418

RESUMO

To maintain prolonged infection of mammals, African trypanosomes have evolved remarkable surface coats and a system of antigenic variation1. Within these coats are receptors for macromolecular nutrients such as transferrin2,3. These must be accessible to their ligands but must not confer susceptibility to immunoglobulin-mediated attack. Trypanosomes have a wide host range and their receptors must also bind ligands from diverse species. To understand how these requirements are achieved, in the context of transferrin uptake, we determined the structure of a Trypanosoma brucei transferrin receptor in complex with human transferrin, showing how this heterodimeric receptor presents a large asymmetric ligand-binding platform. The trypanosome genome contains a family of around 14 transferrin receptors4, which has been proposed to allow binding to transferrin from different mammalian hosts5,6. However, we find that a single receptor can bind transferrin from a broad range of mammals, indicating that receptor variation is unlikely to be necessary for promiscuity of host infection. In contrast, polymorphic sites and N-linked glycans are preferentially found in exposed positions on the receptor surface, not contacting transferrin, suggesting that transferrin receptor diversification is driven by a need for antigenic variation in the receptor to prolong survival in a host.


Assuntos
Interações Hospedeiro-Parasita/imunologia , Evasão da Resposta Imune , Receptores da Transferrina/química , Receptores da Transferrina/imunologia , Transferrina/metabolismo , Trypanosoma brucei brucei/imunologia , Variação Antigênica , Variação Genética , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Tripanossomíase Africana/imunologia
10.
Nat Commun ; 10(1): 2607, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197133

RESUMO

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Antineoplásicos/farmacologia , Desenho de Fármacos , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Repetição de Anquirina , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Histidina/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
PLoS Negl Trop Dis ; 13(5): e0007373, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120889

RESUMO

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antiprotozoários/administração & dosagem , Benzodiazepinas/administração & dosagem , Pirróis/administração & dosagem , Tripanossomíase Africana/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Antiprotozoários/química , Benzodiazepinas/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pirróis/química , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/parasitologia
12.
PLoS One ; 14(3): e0214545, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925190

RESUMO

Doxorubicin is a chemotherapeutic agent that is commonly used to treat a broad range of cancers. However, significant cardiotoxicity, associated with prolonged exposure to doxorubicin, limits its continued therapeutic use. One strategy to prevent the uptake of doxorubicin into cardiac cells is the encapsulation of the drug to prevent non-specific uptake and also to improve the drugs' pharmacokinetic properties. Although encapsulated forms of doxorubicin limit the cardiotoxicity observed, they are not without their own liabilities as an increased amount of drug is deposited in the skin where liposomal doxorubicin can cause palmar-plantar erythrodysesthesia. Exosomes are small endogenous extracellular vesicles, that transfer bioactive material from one cell to another, and are considered attractive drug delivery vehicles due to their natural origin. In this study, we generated doxorubicin-loaded exosomes and demonstrate their rapid cellular uptake and re-distribution of doxorubicin from endosomes to the cytoplasm and nucleus resulting in enhanced potency in a number of cultured and primary cell lines when compared to free doxorubicin and liposomal formulations of doxorubicin. In contrast to other delivery methods for doxorubicin, exosomes do not accumulate in the heart, thereby providing potential for limiting the cardiac side effects and improved therapeutic index.


Assuntos
Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Exossomos/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico , Linhagem Celular , Exossomos/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Cinética
13.
Sci Rep ; 8(1): 12079, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104686

RESUMO

Human T regulatory cells (T regs) express high levels of TNF receptor 2 (TNFR2). Ligation of TNFR2 with TNF, which can recognise both TNFR1 and TNFR2, or with a TNFR2-selective binding molecule, DARPin 18 (D18) activates canonical NF-κB signalling, assessed by IκBα degradation, and the magnitude of the response correlates with the level of TNFR2 expression. RNA-seq analysis of TNF- or D18-treated human T regs revealed that TNFR2 ligation induces transcription of NFKB2 and RELB, encoding proteins that form the non-canonical NF-κB transcription factor. In combination with IL2, D18 treatment is specific for T regs in (1) stabilising NF-κB-inducing kinase protein, the activator of non-canonical NF-κB signalling, (2) inducing translocation of RelB from cytosol to nucleus, (3) increasing cell cycle entry, and (4) increasing cell numbers. However, the regulatory function of the expanded T regs is unaltered. Inhibition of RelB nuclear translocation blocks the proliferative response. We conclude that ligation of TNFR2 by D18 enhances IL2-induced T regs proliferation and expansion in cell number through the non-canonical NF-κB pathway.


Assuntos
Regulação da Expressão Gênica/imunologia , Interleucina-2/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/genética , Linfócitos T Reguladores/imunologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/genética , Células Cultivadas , Voluntários Saudáveis , Humanos , Interleucina-2/imunologia , Inibidor de NF-kappaB alfa/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Transcrição RelB/metabolismo
14.
Nanoscale ; 10(29): 14230-14244, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30010165

RESUMO

Exosomes are extracellular vesicles that mediate cell-to-cell communication by transferring biological cargo, such as DNA, RNA and proteins. Through genetic engineering of exosome-producing cells or manipulation of purified exosomes, it is possible to load exosomes with therapeutic molecules and target them to specific cells via the display of targeting moieties on their surface. This provides an opportunity to exploit a naturally-occurring biological process for therapeutic purposes. In this study, we explored the potential of single chain variable fragments (scFv) as targeting domains to achieve delivery of exosomes to cells expressing a cognate antigen. We generated exosomes targeting the Her2 receptor and, by varying the affinity of the scFvs and the Her2 expression level on recipient cells, we determined that both a high-affinity anti-Her2-scFv (KD≤ 1 nM) and cells expressing a high level (≥106 copies per cell) of Her2 were optimally required to enable selective uptake. We also demonstrate that targeting exosomes to cells via a specific cell surface receptor can alter their intracellular trafficking route, providing opportunities to influence the efficiency of delivery and fate of intracellular cargo. These experiments provide solid data to support the wider application of exosomes displaying antibody fragments as vehicles for the targeted delivery of therapeutic molecules.


Assuntos
Exossomos/química , Receptor ErbB-2/química , Anticorpos de Cadeia Única/química , Linhagem Celular Tumoral , Células HEK293 , Humanos
16.
Nat Commun ; 8: 16111, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28706291

RESUMO

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Inhibition of Ras nucleotide exchange is a promising new approach but better understanding of this mechanism of action is needed. Here we describe an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of Ras. K27 binds preferentially to the inactive Ras GDP form with a Kd of 4 nM and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK, and slows the growth in soft agar of HCT116 cells. K27 is a potent, non-covalent inhibitor of nucleotide exchange, showing consistent effects across different isoforms of Ras, including wild-type and oncogenic mutant forms.


Assuntos
Anticorpos/química , Proteínas ras/antagonistas & inibidores , Repetição de Anquirina , Anticorpos/imunologia , Anticorpos/farmacologia , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Células HCT116 , Células HEK293 , Humanos , Estrutura Molecular , Terapia de Alvo Molecular , Proteínas ras/imunologia
17.
Drug Discov Today Technol ; 23: 83-90, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28647091

RESUMO

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.


Assuntos
Anticorpos/química , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos
18.
J Immunol ; 198(1): 528-537, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27881707

RESUMO

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.


Assuntos
Abatacepte/farmacologia , Desenho de Fármacos , Imunossupressores/farmacologia , Abatacepte/química , Animais , Antígeno B7-1/imunologia , Antígeno B7-2 , Estabilidade de Medicamentos , Humanos , Imunossupressores/química , Ligação Proteica/imunologia
19.
Sci Rep ; 6: 36391, 2016 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-27819305

RESUMO

Affinity panning of large libraries is a powerful tool to identify protein binders. However, panning rounds are followed by the tedious re-screening of the clones obtained to evaluate binders precisely. In a first application of Bead Surface Display (BeSD) we show successful in vitro affinity selections based on flow cytometric analysis that allows fine quantitative discrimination between binders. Subsequent consensus analysis of the resulting sequences enables identification of clones that bind tighter than those arising directly from the experimental selection output. This is demonstrated by evolution of an anti-Fas receptor single-chain variable fragment (scFv) that was improved 98-fold vs the parental clone. Four rounds of quantitative screening by fluorescence-activated cell sorting of an error-prone library based on fine discrimination between binders in BeSD were followed by analysis of 200 full-length output sequences that suggested a new consensus design with a Kd ∼140 pM. This approach shortens the time and effort to obtain high affinity reagents and its cell-free nature transcends limitations inherent in previous in vivo display systems.


Assuntos
Proteínas/metabolismo , Anticorpos de Cadeia Única/metabolismo , Técnicas de Visualização da Superfície Celular , Citometria de Fluxo , Humanos , Biblioteca de Peptídeos , Ligação Proteica
20.
Oncotarget ; 7(42): 68278-68291, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27626702

RESUMO

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.


Assuntos
Imunoterapia/métodos , Neoplasias/terapia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Células HEK293 , Humanos , Células Jurkat , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Fenótipo , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos
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