RESUMO
Bispecific antibodies continue to represent a growth area for antibody therapeutics, with roughly a third of molecules in clinical development being T-cell engagers that use an anti-CD3 binding arm. CD3 antibodies possessing cross-reactivity with cynomolgus monkey typically recognize a highly electronegative linear epitope at the extreme N-terminus of CD3 epsilon (CD3ε). Such antibodies have high isoelectric points and display problematic polyreactivity (correlated with poor pharmacokinetics for monospecific antibodies). Using insights from the crystal structure of anti-Hu/Cy CD3 antibody ADI-26906 in complex with CD3ε and antibody engineering using a yeast-based platform, we have derived high-affinity CD3 antibody variants with very low polyreactivity and significantly improved biophysical developability. Comparison of these variants with CD3 antibodies in the clinic (as part of bi- or multi-specifics) shows that affinity for CD3 is correlated with polyreactivity. Our engineered CD3 antibodies break this correlation, forming a broad affinity range with no to low polyreactivity. Such antibodies will enable bispecifics with improved pharmacokinetic and safety profiles and suggest engineering solutions that will benefit the large and growing sector of T-cell engagers.
Assuntos
Anticorpos Biespecíficos , Animais , Macaca fascicularis , Linfócitos T , Complexo CD3 , Muromonab-CD3RESUMO
The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Sítios de Ligação , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , COVID-19/prevenção & controle , COVID-19/terapia , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Epitopos/imunologia , Humanos , Imunização Passiva , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos Endogâmicos BALB C , Domínios Proteicos , Engenharia de Proteínas , Receptores de Coronavírus/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/terapia , Glicoproteína da Espícula de Coronavírus/metabolismo , Soroterapia para COVID-19RESUMO
The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.
RESUMO
Respiratory syncytial virus (RSV) is a seasonal virus that infects the lungs and airways of 64 million children and adults every year. It is a major cause of acute lower respiratory tract infection and is associated with significant morbidity and mortality. Despite the large medical and economic burden, treatment options for RSV-associated bronchiolitis and pneumonia are limited and mainly consist of supportive care. This publication covers the medicinal chemistry efforts resulting in the identification of JNJ-53718678, an orally bioavailable RSV inhibitor that was shown to be efficacious in a phase 2a challenge study in healthy adult subjects and that is currently being evaluated in hospitalized infants and adults. Cocrystal structures of several new derivatives helped in rationalizing some of the structure-activity relationship (SAR) trends observed.
Assuntos
Antivirais/química , Descoberta de Drogas/métodos , Imidazolidinas/química , Indóis/química , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Inibidores de Proteínas Virais de Fusão/química , Administração Oral , Antivirais/administração & dosagem , Cristalografia por Raios X/métodos , Células HeLa , Humanos , Imidazolidinas/administração & dosagem , Indóis/administração & dosagem , Estrutura Secundária de Proteína , Vírus Sincicial Respiratório Humano/fisiologia , Inibidores de Proteínas Virais de Fusão/administração & dosagemRESUMO
The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.
Assuntos
Antígenos Virais/química , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Prolina/química , Conformação Proteica , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genéticaRESUMO
Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full advantage of this method, the value of ISO is demonstrated by using it to successfully crystallize a panel of six diverse proteins. The results suggest that ISO is an effective method to obtain macromolecular crystals, particularly for proteins that crystallize under a narrow range of precipitant concentrations.
Assuntos
Cristalização/métodos , Cristalização/normas , Ensaios de Triagem em Larga Escala/normas , Proteínas/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease in young children and elderly people. Although the virus was isolated in 1955, an effective RSV vaccine has not been developed, and the only licensed intervention is passive immunoprophylaxis of high-risk infants with a humanized monoclonal antibody. During the past 5 years, however, there has been substantial progress in our understanding of the structure and function of the RSV glycoproteins and their interactions with host cell factors that mediate entry. This period has coincided with renewed interest in developing effective interventions, including the isolation of potent monoclonal antibodies and small molecules and the design of novel vaccine candidates. In this Review, we summarize the recent findings that have begun to elucidate RSV entry mechanisms, describe progress on the development of new interventions and conclude with a perspective on gaps in our knowledge that require further investigation.
Assuntos
Antivirais/farmacologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/fisiologia , Internalização do Vírus/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Ensaios Clínicos como Assunto , Humanos , Infecções por Vírus Respiratório Sincicial/terapia , Bibliotecas de Moléculas Pequenas/farmacologia , Vacinas ViraisRESUMO
A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Especificidade de Anticorpos , Antivirais/uso terapêutico , Cristalografia por Raios X , Células HEK293 , Humanos , Palivizumab/uso terapêutico , Ligação Proteica , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/uso terapêutico , Ressonância de Plasmônio de SuperfícieRESUMO
Human metapneumovirus (hMPV) is a frequent cause of bronchiolitis in young children. Its F glycoprotein mediates virus-cell membrane fusion and is the primary target of neutralizing antibodies. The inability to produce recombinant hMPV F glycoprotein in the metastable pre-fusion conformation has hindered structural and immunological studies. Here, we engineer a pre-fusion-stabilized hMPV F ectodomain and determine its crystal structure to 2.6 Å resolution. This structure reveals molecular determinants of strain-dependent acid-induced fusion, as well as insights into refolding from pre- to post-fusion conformations. A dense glycan shield at the apex of pre-fusion hMPV F suggests that antibodies against this site may not be elicited by host immune responses, which is confirmed by depletion studies of human immunoglobulins and by mouse immunizations. This is a major difference with pre-fusion F from human respiratory syncytial virus (hRSV), and collectively our results should facilitate development of effective hMPV vaccine candidates.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulinas Intravenosas/imunologia , Metapneumovirus/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Chlorocebus aethiops , Cristalografia por Raios X , Feminino , Metapneumovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Engenharia de Proteínas , Redobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.
Assuntos
Imidazolidinas/metabolismo , Indóis/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Inibidores de Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Epiteliais , Humanos , Imidazolidinas/farmacologia , Imidazolidinas/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Estrutura Molecular , Pneumonia Viral/tratamento farmacológico , Ratos , Mucosa Respiratória/citologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/metabolismo , Ovinos , Relação Estrutura-Atividade , Células Vero , Inibidores de Proteínas Virais de Fusão/farmacologia , Inibidores de Proteínas Virais de Fusão/uso terapêuticoRESUMO
Two new bispecific T-cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3ε binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3ε on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3, but not huNKG2D-OKT3, is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T-cell cytokine production in comparison with B2-OKT3. No T-cell pretreatment was required for IFNγ production upon coculture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T-cell compartment was the primary source of IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density-dependent production of IFNγ from both CD4+ and CD8+ T cells. There was 2-fold more IFNγ produced per CD8+ T cell and 5-fold greater percentage of CD8+ T cells producing IFNγ compared with CD4+ T cells. In addition, both BiTEs elicited significant antitumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust antitumor activity of these NKG2D ligand-binding bispecific proteins and support their further development for clinical use. Mol Cancer Ther; 16(7); 1335-46. ©2017 AACR.
Assuntos
Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias/tratamento farmacológico , Anticorpos de Cadeia Única/administração & dosagem , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Humanos , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias/genética , Neoplasias/imunologia , Ligação Proteica , Anticorpos de Cadeia Única/imunologia , Linfócitos T/imunologiaRESUMO
LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas de Bactérias/imunologia , Leucocidinas/imunologia , Animais , Proteínas de Bactérias/química , Dimerização , Humanos , Leucocidinas/químicaRESUMO
Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.
Assuntos
Antivirais/farmacologia , Modelos Moleculares , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Proteínas Virais de Fusão/antagonistas & inibidores , Antivirais/química , Bioensaio , Colorimetria , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective HIV-1 vaccine. However, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. Here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of HIV envelope variants. Because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer HIV spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. We report for the first time the display of a designed SOSIP gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. These efforts represent an initial and critical step toward the ability to rapidly engineer HIV-1 envelope immunogens via directed evolution.
Assuntos
Anticorpos/química , Evolução Molecular Direcionada , HIV-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Vacinas contra a AIDS/imunologia , Anticorpos/imunologia , Biblioteca Gênica , Glicosilação , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only â¼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Proteínas Hemolisinas/imunologia , Imunoglobulina G/imunologia , Leucocidinas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/química , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Proteínas de Bactérias/química , Linhagem Celular , Proteínas Hemolisinas/química , Humanos , Imunoglobulina G/química , Leucocidinas/química , Coelhos , Staphylococcus aureus/químicaRESUMO
The methylotropic yeast Pichia pastoris has been used for more than two decades to successfully produce a large number of recombinant proteins. Currently, a wide variety of auxotrophic and drug based selection markers are employed to screen for clones expressing the protein of interest. For most proteins an increased copy number of the integrated plasmid results in higher levels of expression, but these multi-copy integrants can be unstable due to the propensity of P. pastoris for homologous recombination. Here we describe a multi-copy selection system based on ade1 and ade2 auxotrophic parent strains and the respective attenuated markers with truncated promoter regions. We show that for all four proteins we tested, the use of the attenuated markers leads to increased protein expression when compared with selection based on the full strength markers. The fact that the adenine auxotrophic strains grow more slowly than the complemented counterparts essentially ensures the stability of multi-copy integration. At the same time, the accumulation of a red dye in the auxotrophic strains also provides an easy, color-based selection for transformants with multiple copies.