Research papers by Daniel Perez-Witzke
Journal of Immunological Methods, 2021
Interfering with signalling pathways by targeting cell surface proteins has become an important s... more Interfering with signalling pathways by targeting cell surface proteins has become an important strategy in the development of novel therapeutic agents. Notably, interfering with cytokine signalling revolutionised the treatment of chronic diseases. Cytokines can induce a range of effects that are not always accounted for in assays detecting cytokine binding to cell surface receptors and/or proximal signalling interference. Hence, robust assays are needed to characterise the activity of potential drug candidates targeting such effects. We chose interleukin-7 (IL7) as a cytokine model due to its long-term effect on T-cells. In this report we describe the development and refinement of an in vitro assay for measuring the long-term effect of IL7, more specifically on CD4+ T-cells, while the assay could be adapted to look at CD8+ T-cells. PBMCs and/or purified CD4+ T-cells stained with VPD450 (cell cycle dye) were expanded for 5 days using the mitogen Phytohemagglutinin and/or CD3/CD28 agonists. This resulted in cell proliferation (VPD450 dilution) and activation-induced cell death (7-AAD uptake) which was rescued by the addition of IL7, resulting in cell survival over a further 5 days. JAK-inhibitor (Tofactinib) or a blocking anti-IL7Rα antibody (clone R34.34) abolished cell survival suggesting antagonism, while another antibody (clone A019D5) displayed an agonist effect. These results were confirmed at the proximal signalling level using an IL7/STAT5-luciferase reporter assay. This novel assay for a biological long term effect may be useful for the characterisation of potential therapeutic drugs targeting the IL7/IL7R in CD4+ T-cells.
Immunoglobulin (Ig)E-mediated allergy and certain autoimmune diseases are characterized by the pr... more Immunoglobulin (Ig)E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T-helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule CTLA-4 with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70-kDa polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than IgE's. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε – but not IgE – induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, thus blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of the lymphocyte proliferation in human PBMC samples stimulated in vitro with Con A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.
Presentation at meetings by Daniel Perez-Witzke
Background
The immune system in RA is thought to be ageing about 20-30 older than what is normal... more Background
The immune system in RA is thought to be ageing about 20-30 older than what is normally observed in health. This is illustrated by dysfunctions at several levels from thymic output of naïve cells to telomere shortening, lack of ability to provide help, regulation, excessive cytokine response and more. IL-7 is a cytokine that regulates some of these functions. We have shown that reduced levels of IL-7 are related to some of these dysfunctions. The aim of this short-term project was to develop an assay that would allow us to analyse the effect of IL-7 on T-cells. However, as IL-7 affect survival rather than proliferation, the assay requires a lot of optimisation also because T-cell responses are highly variable even between healthy controls.
Materials and Methods
The assay was optimised for its activation phase with PHA (1 or 2 or 3 %) in PBMCs. The second phase was the induction of IL-7 mediated survival and how long after (10, 12 or 13 days), the survival effect could be best measured. The IL-7 dependent effect on both CD4 and CD8 T-cells in the presence of 7AAD was assessed using flow cytometry. Finally, using the best conditions determined by these optimisation phases, a cell cycle die (VPD450) was used to analyse the progression of proliferation and then survival effect of IL-7. A STAT-5 inhibitor was used as control of the IL-7 effects.
Results
The optimisation of the assay requires 3 preparation steps. First, we optimised the amount of PHA to be used to stimulate cells so that activation induced cell death was measurable but not overwhelming. We compared PBMC activation in 3 donors and decided on PHA 1% which is less consistent in the proliferation index but allow cells better cell survival. After 5 days cells we compared cell continued growth with and without a ficoll step which was introduced to eliminate the dying PBMCs. This additional step was particularly successful in increasing data reproducibility later. Flow cytometry at day 10-12-13 showed a particular protective survival effect of IL-7 on CD4+T-cells but lesson effect on CD8+T-cells, which continue proliferating and then died massively at day 13. Finally, the introduction of the VPD450 cell cycle analysis dye in the experiment allowed to clearly demonstrate an accumulation of CD4+T-cells in G4 generation at day 10 in the presence of IL-7 while all other conditions (control, STAT-5/inh and IL-7+STAT-5/Inh) showed a blockade in proliferation at G3 with similar results at day 5, 7 and 10, and a major apoptosis death detected by Annexin-V and 7AAD staining.
Conclusions
This short project run by 2 ERASMUS students, provides the basis for a functional test allowing the analysis of IL-7 mediated effect on CD4+T-cells from RA patients in the near future.
Una proteína de fusión CTLA4Fcε, con posible acción inmunomoduladora de las reacciones de hiperse... more Una proteína de fusión CTLA4Fcε, con posible acción inmunomoduladora de las reacciones de hipersensibilidad tipo I, fue usada en este trabajo para evaluar su efecto en la unión de la IgE a sus receptores de alta y baja afinidad, FcεRI y FcεRII/CD23, respectivamente. Para alcanzar este objetivo, se realizaron ensayos de competencia entre la IgE y CTLA4Fcε por la unión a FcεRI; utilizando células RBL-SX38, un transfectoma de basófilo de rata que expresa FcεRI humano. Mediante citometría de flujo se determinó que usando una concentración de CTLA4Fcε cuatro veces superior a la de IgE, se logra un bloqueo del 51% de la unión de IgE. Por otra parte, se estudió la unión de la IgE y de CTLA4Fcε a FcεRII/CD23 en la membrana de células RPMI-8866, una línea linfoblastoide B humana que además expresa las moléculas receptoras de CTLA4, CD80 y CD86. Se determinó mediante análisis por citometría de flujo que la unión de IgE a CD23, en el rango de concentraciones empleado, exhibe un comportamiento concentración dependiente. Sin embargo, la unión de CTLA4Fcε a CD23 mostró un comportamiento diferente; en el mismo rango de concentraciones, no se observó correlación alguna entre la concentración de CTLA4Fcɛ y el valor de intensidad media de fluorescencia (IMF), manteniéndose los valores siempre cercanos a la máxima IMF, sugiriendo una unión más estable. Cuando el mismo experimento se realizó bloqueando las moléculas CD80/CD86 con anticuerpos monoclonales, CTLA4Fcɛ se comportó como la IgE. Estos resultados sugieren que la estabilidad de la unión de CTLA4Fcε a CD23 puede ser consecuencia de la asociación simultánea a CD80/CD86, un efecto que podría tener consecuencias directas en eventos clave para el desarrollo de hipersensibilidad tipo I, como son el cambio de clase de Ig y la presentación antigénica por parte de células B a los linfocitos T alérgeno-específicos.
En alergias y algunas formas de autoinmunidad es común la predominancia de la respuesta TH2 y la ... more En alergias y algunas formas de autoinmunidad es común la predominancia de la respuesta TH2 y la producción de IgE alérgeno especifica o autoreactiva. Un blanco terapéutico atractivo en estos casos es la inhibición de la producción de la forma soluble de la molécula CD23 (sCD23), un factor de proliferación de linfocitos B que coopera en el cambio de clase a IgE. Una proteína de fusión CTLA4Fce podría alcanzar este objetivo. En este trabajo se purificó y caracterizó una proteína de fusión CTLA4Fcε. También se evaluó in vitro su efecto sobre la producción de sCD23. Mediante western-blot se determinó que CTLA4Fcε es un homodímero de subunidades ecCTLA4-(Gly)5-Cε2-Cε3-Cε4 (condiciones reductoras 80kDa y no reductoras 160kDa). Mediante citometría de flujo y utilizando anticuerpos fluoromarcados bloqueantes se confirmó la unión de CTLA4Fcε a los receptores FcεRI en células CHO-3D10, y CD23, CD80 y CD86 en células RPMI‐8866. Mediante ELISA se estimó la concentración de sCD23 en sobrenadantes de cultivo de células RPMI-8866 realizados en presencia de CTLA4Fcε ó IgE. La producción de sCD23 se incrementó de manera inversamente proporcional a la concentración de IgE [no tratamiento: 100% (DS=6.62); 4μg/ml IgE: 96.14% (DS=8.09;P<0.001) / 0.5μg/ml IgE: 122.4% (DS=4.77;P>0.05)], confirmando la regulación de la producción de sCD23 por la concentración de IgE. Contrariamente, CTLA4Fcε inhibió la escisión de CD23 de manera directamente proporcional a la concentración [0.5μg/ml CTLA4Fcε: 83.7% (DS=1.78;P<0.001)/ 4μg/ml CTLA4Fcε: 62.8% (DS=1.67;P<0.001)]. El efecto inhibitorio de CTLA4Fcε puede ser consecuencia de la asociación simultánea de CTLA4Fcε con CD80/CD86 y CD23, resultando en un impedimento estérico para la actividad de la metaloproteasa ADAM10 (anclando CD23 a la membrana). Una posible consecuencia in vivo de la inhibición de la producción de sCD23 es la disminución de la producción de anticuerpos tipo IgE y la liberación de otros factores de crecimiento de células B como IL-6.
Conference Presentations by Daniel Perez-Witzke
Conference: Festival of Biologics, 2022
Not every antibody can be combined to produce well-behaved multispecifics. The valency and geomet... more Not every antibody can be combined to produce well-behaved multispecifics. The valency and geometry of each design can determine the production, target engagement and ultimately the requisite biological functions. In this case study, we selected two established antibody therapeutics, trastuzumab and a humanized OKT3, to produce 17 different bispecific formats to compare the feasibility of each format.
Conference: 27th ESACT Meeting, 2022
Absolute Antibody Ltd has a catalogue of 11,000+ mAbs for Analysis of individual vector variants ... more Absolute Antibody Ltd has a catalogue of 11,000+ mAbs for Analysis of individual vector variants production in their high-throughput, intensive, transient gene expression platform. To improve yields, endogenous and novel highly active synthetic vector components were screened one-factor-at-atime, before testing in combination with three commercial mAbs. A transient OriP/EBNA system was then implemented. Aiming to further improve expression via vector retention and replication. Using this generic, universally applicable genetic vector technology, a 7-fold increase in expression compared to the existing industry-standard vector was achieved.
Polyclonal antibodies are widely used across research and diagnostics, and whilst they offer a ra... more Polyclonal antibodies are widely used across research and diagnostics, and whilst they offer a range of advantages such as excellent performance across different methods, they suffer from high batch-to-batch variability and require the continued use of animals for their production. Here we report the de novo sequencing of monoclonal antibodies from a polyclonal goat antibody sample by mass-spectrometry, followed by recombinant expression and testing. To our knowledge this is the first report of a successful conversion of a goat polyclonal to a monoclonal using just antibody protein as a template. Wider adoption of this approach will have profound impact on the research reagents and diagnostics industry, and also shows great potential for the development of therapeutics from existing polyclonal antibodies.
Annals of the Rheumatic Diseases, 2019
Introduction. IL-7R is a heterodimer constituted by the IL-7R alpha (a) chain (CD127) and the com... more Introduction. IL-7R is a heterodimer constituted by the IL-7R alpha (a) chain (CD127) and the common gamma (g) chain (CD132). IL-7 binding to IL-7R expressed on CD4+ T cells induces a survival signal. The IL-7/IL-7R signalling axis has been validated as a therapeutic target for treatment of both T-cell driven autoimmune diseases (AIDs)1 and T Acute Lymphoblastic Leukaemia (T-ALL).2 Affimers are small and stable artificial proteins which bind with nanomolar affinities to human proteins and can block protein-protein interactions.3 They are becoming widespread owing to their stability, ease of production and versatility.
Objectives. Identify Affimers that recognise the IL-7Ra and inhibits the IL-7 signalling cascade. This may result in an attractive approach for the treatment of both T-cell driven autoimmune diseases and T-ALL.
Methods. The type-II Affimer library (1010) was interrogated by Phage display using fully glycosylated human IL-7Ra ectodomain (ECD). PhageELISA and DNA sequencing were used to either obtain or elucidate the unique binders (Affimers), respectively. Affimers were produced as His-Tagged proteins (~13 kDa) in E. coli and purified using IMAC. Affimer binding to IL-7Ra was confirmed by pull-down assays (soluble) and Flow cytometry (membrane). An IL-7 reporter assay using HEK-IL7R (HEK293 cells stably transfected with the IL-7R) was developed and the biological effect of the Affimers was elucidated.
Results. We have screened an Affimer library using human ECD-IL7Ra and after three consecutive panning rounds, 20 Affimers were raised as shown by PhageELISA and DNA sequencing. From these, 17 were able to pull-down the soluble ECD-IL7Ra and 7 stained specifically HEK-IL7R cells (by flow cytometry using anti-His Tag Abs). Finally, we have identified 3 Affimers (1, 42 and 96) that showed inhibition of the IL-7 signalling cascade on HEK-IL7R cells.
Conclusions. Our work demonstrates the possibility of screening an Affimer library for a cytokine-receptor target, and selecting specific binders, some of which showed the desired antagonist activity of the cytokine signalling cascade. IL-7 itself is a validated target, so this work offers an alternative to antibody-mediated protein interference. With further biological validation including animal models, it may even offer a novel therapeutic tool for AIDs and T-ALL.
Papers by Daniel Perez-Witzke
Company case study, 2022
Circulating in blood is a multitude of biologically important antibodies. These pools of polyclon... more Circulating in blood is a multitude of biologically important antibodies. These pools of polyclonal antibodies (pAb) are invaluable sources for drug discovery against various diseases, and for the development of robust immunoreagents for diagnostics, and research. However, accessing the richness of clinically relevant pAb remains difficult due to batch-to-batch variability, and shortcomings in available cell-based discovery. Herein, we present the first report of the de novo sequencing of monoclonal antibodies (mAbs) from a goat pAb without cell or nucleic material input. The decoded mAb sequences were recombinantly expressed and analyzed with enzyme-linked immunosorbent assays (ELISA). The discovered antibodies faithfully recapitulated the specific binding affinity of the polyclonal antibody. Key Takeaways ▪ pAb have been highly sought after due to their biophysical properties and high affinity to a target ▪ It has been difficult to completely capture pAb properties without compromising sensitivity and/or reproducibility ▪ Protein sequencing of pAbs, when combined with recombinant expression, can yield a collection of rmAbs that perform as well or better than the original pAb ▪ Full sequence coverage and correct chain pairing of mAbs directly from a protein sample is now possible RAPID INSIGHTS | CASE STUDY
Company application note, 2022
Absolute Antibody recently expanded its recombinant antibody expression offerings with the additi... more Absolute Antibody recently expanded its recombinant antibody expression offerings with the addition of the CHXpress™ transient CHO expression service. The addition builds upon our established HEXpress™ platform, enabling us to offer serum-free mammalian transient expression in both HEK and CHO cells to better serve clients through their full antibody development pathway.
We still recommend our cost-effective HEK expression platform for early-stage antibody development, as well as all reagent and diagnostics antibodies, but transient CHO production at milligram-to-gram scales is particularly useful for bridging the gap to stable CHO production, or for tackling challenging constructs. It enables therapeutic antibody developers to quickly manufacture and screen a group of antibodies before taking the time to generate stable cell lines for their most promising candidates. While stable cell line generation typically takes six months to a year, our transient CHO platform can produce high quality recombinant antibodies in engineered formats within a month.
In this application note, we review the quality and batch-to-batch reproducibility of the recombinant antibodies manufactured with the CHXpress™ transient CHO platform. Data demonstrate that the platform produces high quality, fully glycosylated human IgG antibodies with high consistency between batches, as well as antibodies in engineered formats with reliably robust production yields and expected binding activity.
Uploads
Research papers by Daniel Perez-Witzke
Presentation at meetings by Daniel Perez-Witzke
The immune system in RA is thought to be ageing about 20-30 older than what is normally observed in health. This is illustrated by dysfunctions at several levels from thymic output of naïve cells to telomere shortening, lack of ability to provide help, regulation, excessive cytokine response and more. IL-7 is a cytokine that regulates some of these functions. We have shown that reduced levels of IL-7 are related to some of these dysfunctions. The aim of this short-term project was to develop an assay that would allow us to analyse the effect of IL-7 on T-cells. However, as IL-7 affect survival rather than proliferation, the assay requires a lot of optimisation also because T-cell responses are highly variable even between healthy controls.
Materials and Methods
The assay was optimised for its activation phase with PHA (1 or 2 or 3 %) in PBMCs. The second phase was the induction of IL-7 mediated survival and how long after (10, 12 or 13 days), the survival effect could be best measured. The IL-7 dependent effect on both CD4 and CD8 T-cells in the presence of 7AAD was assessed using flow cytometry. Finally, using the best conditions determined by these optimisation phases, a cell cycle die (VPD450) was used to analyse the progression of proliferation and then survival effect of IL-7. A STAT-5 inhibitor was used as control of the IL-7 effects.
Results
The optimisation of the assay requires 3 preparation steps. First, we optimised the amount of PHA to be used to stimulate cells so that activation induced cell death was measurable but not overwhelming. We compared PBMC activation in 3 donors and decided on PHA 1% which is less consistent in the proliferation index but allow cells better cell survival. After 5 days cells we compared cell continued growth with and without a ficoll step which was introduced to eliminate the dying PBMCs. This additional step was particularly successful in increasing data reproducibility later. Flow cytometry at day 10-12-13 showed a particular protective survival effect of IL-7 on CD4+T-cells but lesson effect on CD8+T-cells, which continue proliferating and then died massively at day 13. Finally, the introduction of the VPD450 cell cycle analysis dye in the experiment allowed to clearly demonstrate an accumulation of CD4+T-cells in G4 generation at day 10 in the presence of IL-7 while all other conditions (control, STAT-5/inh and IL-7+STAT-5/Inh) showed a blockade in proliferation at G3 with similar results at day 5, 7 and 10, and a major apoptosis death detected by Annexin-V and 7AAD staining.
Conclusions
This short project run by 2 ERASMUS students, provides the basis for a functional test allowing the analysis of IL-7 mediated effect on CD4+T-cells from RA patients in the near future.
Conference Presentations by Daniel Perez-Witzke
Objectives. Identify Affimers that recognise the IL-7Ra and inhibits the IL-7 signalling cascade. This may result in an attractive approach for the treatment of both T-cell driven autoimmune diseases and T-ALL.
Methods. The type-II Affimer library (1010) was interrogated by Phage display using fully glycosylated human IL-7Ra ectodomain (ECD). PhageELISA and DNA sequencing were used to either obtain or elucidate the unique binders (Affimers), respectively. Affimers were produced as His-Tagged proteins (~13 kDa) in E. coli and purified using IMAC. Affimer binding to IL-7Ra was confirmed by pull-down assays (soluble) and Flow cytometry (membrane). An IL-7 reporter assay using HEK-IL7R (HEK293 cells stably transfected with the IL-7R) was developed and the biological effect of the Affimers was elucidated.
Results. We have screened an Affimer library using human ECD-IL7Ra and after three consecutive panning rounds, 20 Affimers were raised as shown by PhageELISA and DNA sequencing. From these, 17 were able to pull-down the soluble ECD-IL7Ra and 7 stained specifically HEK-IL7R cells (by flow cytometry using anti-His Tag Abs). Finally, we have identified 3 Affimers (1, 42 and 96) that showed inhibition of the IL-7 signalling cascade on HEK-IL7R cells.
Conclusions. Our work demonstrates the possibility of screening an Affimer library for a cytokine-receptor target, and selecting specific binders, some of which showed the desired antagonist activity of the cytokine signalling cascade. IL-7 itself is a validated target, so this work offers an alternative to antibody-mediated protein interference. With further biological validation including animal models, it may even offer a novel therapeutic tool for AIDs and T-ALL.
Papers by Daniel Perez-Witzke
We still recommend our cost-effective HEK expression platform for early-stage antibody development, as well as all reagent and diagnostics antibodies, but transient CHO production at milligram-to-gram scales is particularly useful for bridging the gap to stable CHO production, or for tackling challenging constructs. It enables therapeutic antibody developers to quickly manufacture and screen a group of antibodies before taking the time to generate stable cell lines for their most promising candidates. While stable cell line generation typically takes six months to a year, our transient CHO platform can produce high quality recombinant antibodies in engineered formats within a month.
In this application note, we review the quality and batch-to-batch reproducibility of the recombinant antibodies manufactured with the CHXpress™ transient CHO platform. Data demonstrate that the platform produces high quality, fully glycosylated human IgG antibodies with high consistency between batches, as well as antibodies in engineered formats with reliably robust production yields and expected binding activity.
The immune system in RA is thought to be ageing about 20-30 older than what is normally observed in health. This is illustrated by dysfunctions at several levels from thymic output of naïve cells to telomere shortening, lack of ability to provide help, regulation, excessive cytokine response and more. IL-7 is a cytokine that regulates some of these functions. We have shown that reduced levels of IL-7 are related to some of these dysfunctions. The aim of this short-term project was to develop an assay that would allow us to analyse the effect of IL-7 on T-cells. However, as IL-7 affect survival rather than proliferation, the assay requires a lot of optimisation also because T-cell responses are highly variable even between healthy controls.
Materials and Methods
The assay was optimised for its activation phase with PHA (1 or 2 or 3 %) in PBMCs. The second phase was the induction of IL-7 mediated survival and how long after (10, 12 or 13 days), the survival effect could be best measured. The IL-7 dependent effect on both CD4 and CD8 T-cells in the presence of 7AAD was assessed using flow cytometry. Finally, using the best conditions determined by these optimisation phases, a cell cycle die (VPD450) was used to analyse the progression of proliferation and then survival effect of IL-7. A STAT-5 inhibitor was used as control of the IL-7 effects.
Results
The optimisation of the assay requires 3 preparation steps. First, we optimised the amount of PHA to be used to stimulate cells so that activation induced cell death was measurable but not overwhelming. We compared PBMC activation in 3 donors and decided on PHA 1% which is less consistent in the proliferation index but allow cells better cell survival. After 5 days cells we compared cell continued growth with and without a ficoll step which was introduced to eliminate the dying PBMCs. This additional step was particularly successful in increasing data reproducibility later. Flow cytometry at day 10-12-13 showed a particular protective survival effect of IL-7 on CD4+T-cells but lesson effect on CD8+T-cells, which continue proliferating and then died massively at day 13. Finally, the introduction of the VPD450 cell cycle analysis dye in the experiment allowed to clearly demonstrate an accumulation of CD4+T-cells in G4 generation at day 10 in the presence of IL-7 while all other conditions (control, STAT-5/inh and IL-7+STAT-5/Inh) showed a blockade in proliferation at G3 with similar results at day 5, 7 and 10, and a major apoptosis death detected by Annexin-V and 7AAD staining.
Conclusions
This short project run by 2 ERASMUS students, provides the basis for a functional test allowing the analysis of IL-7 mediated effect on CD4+T-cells from RA patients in the near future.
Objectives. Identify Affimers that recognise the IL-7Ra and inhibits the IL-7 signalling cascade. This may result in an attractive approach for the treatment of both T-cell driven autoimmune diseases and T-ALL.
Methods. The type-II Affimer library (1010) was interrogated by Phage display using fully glycosylated human IL-7Ra ectodomain (ECD). PhageELISA and DNA sequencing were used to either obtain or elucidate the unique binders (Affimers), respectively. Affimers were produced as His-Tagged proteins (~13 kDa) in E. coli and purified using IMAC. Affimer binding to IL-7Ra was confirmed by pull-down assays (soluble) and Flow cytometry (membrane). An IL-7 reporter assay using HEK-IL7R (HEK293 cells stably transfected with the IL-7R) was developed and the biological effect of the Affimers was elucidated.
Results. We have screened an Affimer library using human ECD-IL7Ra and after three consecutive panning rounds, 20 Affimers were raised as shown by PhageELISA and DNA sequencing. From these, 17 were able to pull-down the soluble ECD-IL7Ra and 7 stained specifically HEK-IL7R cells (by flow cytometry using anti-His Tag Abs). Finally, we have identified 3 Affimers (1, 42 and 96) that showed inhibition of the IL-7 signalling cascade on HEK-IL7R cells.
Conclusions. Our work demonstrates the possibility of screening an Affimer library for a cytokine-receptor target, and selecting specific binders, some of which showed the desired antagonist activity of the cytokine signalling cascade. IL-7 itself is a validated target, so this work offers an alternative to antibody-mediated protein interference. With further biological validation including animal models, it may even offer a novel therapeutic tool for AIDs and T-ALL.
We still recommend our cost-effective HEK expression platform for early-stage antibody development, as well as all reagent and diagnostics antibodies, but transient CHO production at milligram-to-gram scales is particularly useful for bridging the gap to stable CHO production, or for tackling challenging constructs. It enables therapeutic antibody developers to quickly manufacture and screen a group of antibodies before taking the time to generate stable cell lines for their most promising candidates. While stable cell line generation typically takes six months to a year, our transient CHO platform can produce high quality recombinant antibodies in engineered formats within a month.
In this application note, we review the quality and batch-to-batch reproducibility of the recombinant antibodies manufactured with the CHXpress™ transient CHO platform. Data demonstrate that the platform produces high quality, fully glycosylated human IgG antibodies with high consistency between batches, as well as antibodies in engineered formats with reliably robust production yields and expected binding activity.