text stringlengths 1 5.46k |
|---|
Identity and homogeneity MS Used to confirm the identity of antibody samples by matching observed mass to sequence predicted mass. Mass confirmation within the expected mass error range of the mass spectrometer is desired to confirm molecular identity. The majority of antibodies produced through AbCellera's high-throug... |
Hydrophobicity and homogeneity aHIC Used to assess the hydrophobicity and heterogeneity of antibody samples. Higher levels of a single species (main peak) having shorter retention time indicative of low hydrophobicity is desired. Hydrophobicity is benchmarked against published antibodies in clinical development. The ma... |
Thermal stability nDSF Used to assess the thermal stability of antibody samples. Tm and Tagg data are provided, where Tm is the thermal stability as measured by intrinsic fluorescence, and Tagg is the thermal stability as measured by the onset of light scattering due to temperature-induced aggregation. Higher thermal s... |
Propensity for self-interaction AC-SINS Used to assess the propensity for antibodies to self-interact. Lower AC-SINS scores are desired. AC-SINS scores are benchmarked against published antibodies in clinical development. The majority of antibodies produced through AbCellera's high-throughput production platform have l... |
Polyreactivity BVP-ELISA Used to assess the propensity for antibodies to demonstrate nonspecific binding. Lower BVP scores are desired. BVP scores are benchmarked against published antibodies in clinical development. The majority of antibodies produced through AbCellera's high-throughput production platform have low BV... |
PROJECT PROPOSAL |
PARTNER AbbVie |
TARGET Pathological α-Synuclein (α-Syn) |
SCOPE The scope of this collaboration is to generate a best-in-class α-Syn antibody with excellent biophysical profiles as well as an ability to interfere with the cell-to-cell spreading of pathological α-Syn in vitro and in vivo models. The antibody is aimed to be developed as passive immunization therapy to treat pat... |
TARGET BACKGROUND α-Syn is a neuronal protein that regulates synaptic vesicle trafficking and neurotransmitter release. It is expressed in the central nervous system, but can also be found peripherally in heart, muscle, and bodily fluids including saliva, plasma, and cerebrospinal fluid. |
α-Syn is an intrinsically disordered protein. In disease states (also called synucleinopathies) α-Syn misfolds to seed larger aggregates in disease-specific conformations referred to as strains. These give rise to higher order structures including oligomers, proto-fibrils, fibrils, and finally make up Lewy bodies. Lewy... |
TARGET SEQUENCE Uniprot ID P37840 (140 amino acids) |
Homology of human α-Syn to other species: Organism % identity Cynomolgus (Macaca fascicularis) 98.6% Mouse (Mus musculus) 95% Rat (Rattus norvegicus) 95% Alpaca (Vicugna pacos) 92.1% Rabbit (Oryctolagus cuniculus) 89.26% |
Sequence alignment: [Image placeholder] |
Homology of human α-Syn to human paralogues: Paralogue % identity β-Syn 60.96% γ-Syn 55.12% |
Sequence alignment: [Image placeholder] |
PROTEIN STRUCTURE α-Syn is an intrinsically disordered monomer. The monomer contains a non-amyloid component (NAC), a hydrophobic domain within α-Syn required for fibril formation. Pathological α-Syn forms larger aggregates that are heterogenous. |
DESIRED THERAPEUTIC ANTIBODY PROFILE 1. Binding to human α-Syn aggregates (required)* 2. Binding to human α-Syn monomer (required)* 3. No cross-reactivity to human β-Syn or γ-Syn (required)* 4. Sub nM apparent affinity binding to α-Syn aggregates (required) 5. >1000 fold selective for aggregate over monomer (desired) a... |
6. Binding to α-Syn independently of post-translational modifications (desired) 7. Cross-reactivity to cyno α-Syn (98% seq id) monomer (desired)* a. 90% of human binding affinity 8. Cross-reactivity to mouse α-Syn monomer (desired) a. 90% of human binding affinity 9. C-terminal binding to α-Syn (desired) 10. Preferred ... |
*To be assessed during primary screening pending assay development. Remaining properties to be assessed during secondary screening only provided reagent compatibility and QC within validation assay format. |
IMMUNIZATIONS AND SAMPLES Samples for screening will include spleen, lymph nodes, and/or bone marrow from wild-type mice, KO mice (pending availability), humanized mice immunized, rats* and / or rabbits* immunized with AbbVie's proprietary PD-templated human α-Syn aggregates (PD-PFF). |
*Pending reagent production at Abbvie, rats and rabbit cohorts will be staggered after the mice to allow extra time for antigen production (estimated eight weeks). |
SINGLE-CELL SCREENING ASSAYS To identify antibodies binding to human aggregate α-Syn, two antibody selection assays are proposed for evaluation: a multiplexed bead-based cross reactivity assay and an unconjugated antigen assay. Feasibility of these assays will be tested during assay development. * |
1. Multiplexed bead-based cross reactivity assay to allow for selection of antibodies that bind to human α-Syn aggregates, human and cyno α-Syn monomers and human α-Syn 40aa peptide of interest, and which do not bind to human β-Syn and γ-Syn. |
[Image placeholder] |
2. Unconjugated antigen assay to allow for selection of antibodies that bind to human α-Syn aggregates without the need to conjugate α-Syn aggregates to beads or fluorophores. |
*Assay development pending reagent availability, reagent suspension and compatibility with AbCellera's screening instrumentation and workflow. Assay development includes an assessment of reagent conjugation to beads or fluorophores, and/or amenability of specialized secondary detection on our screening devices. |
ACTIVITIES PREFERRED STAGE ADVANCEMENT CRITERIA |
RESEARCH ACTIVITIES STAGE 1 ACTIVITIES |
Reagent and Assay Set-up (All activities to be performed by AbCellera) Transfer or sourcing/generation of reagents |
AbbVie to transfer internal reagents and AbCellera to procure all commercially available and custom peptides for assay setup and immunization. |
Prior to initiation of Stage 1 activities, extensive QC of aggregate reagents will be required, including assessment of appropriate buffers for suspension, amenability of reagents to AbCellera's immunization and screening instrumentation and workflow |
AbCellera to provide updates to AbbVie on reagent QC and (if applicable) potential mitigation strategies will be discussed in joint project team meetings. |
Assay Development (Assay development is being performed at AbCellera in collaboration with AbbVie) |
Assessment of reagent conjugation to beads for microfluidic screening |
No aggregation (etc) of assay reagents in assay format, and confirmation of specific target engagement with control antibodies. In case full multiplexing of all peptides is not enabled, alternative routes will be decided by the JGC. |
Direct labelling or custom secondary detection of aggregates in soluble format for primary screening |
Confirmation of required labelling ratio of detection label on aggregates by standard characterization techniques. |
Assay development for SPR-based assays (epitope binning (monomer only) and kinetics on monomer and multimeric a-syn) |
Reproducible assay performance on monomeric and multimeric α-Syn with tool antibodies. In case that multimeric a-Syn SPR does not achieve sufficient assay performance the JGC decides on the alternative methods or SPR setups that enable reliable kinetic characterizations of mAb binding to aggregated a-Syn |
Test screen from antibody secreting cells to assess antigen-specific frequency |
Stage advancement will be initiated when all tiered assays are validated for antibody screening |
Immunization (All activities to be performed by AbCellera) ● Immunization of up to 20 rodents using soluble protein antigen ● Five (5) Alloy ● Five (5) Trianni ● Five (5) wild-type mice ● Five (5) α-Syn KO mice (pending availability of KO mice) ● Five (5) wild-type rats* ● Two (2) wild-type rabbits* ● Titre measurement... |
Desired that at least two (2) animals with highest titre from each cohort be selected for single-cell screening. If equal titre values are distributed across animal groups, take top responders from each strain (up to eight animals). Animal selection distribution to be discussed within the AbCellera & AbbVie joint proje... |
STAGE 2 ACTIVITIES |
Antibody Discovery (All activities to be performed by AbCellera) ● Single-cell screening of up to eight (8) selected animals and/or tissues ● Recovery of up to 192 single cells per screen ● Sequencing of antibody heavy/light chain sequences from recovered cells ● Bioinformatic analysis to determine high confidence pair... |
Isolate & recover up to 192 hits with desired binding profiles from each selected animal. AbCellera to provide clarification on animal source of B cells of interest. Chemical liabilities that will be assessed include cysteine modifications and potential motifs for deamidation, oxidation, cleavage and N-linked glycosyla... |
STAGE 3 ACTIVITIES |
Antibody Validation and Characterization (All activities to be performed by AbCellera) ● Small-scale expression and purification performed on up to 184 project antibodies using AbCellera's high-throughput production platform |
Generation of up to hundreds of microgram quantities of antibody for agreed upon validation and characterization assay. |
● Purity and integrity analysis on a subset of project antibodies using microcapillary electrophoresis (ce-SDS) ● Antibody identity and heterogeneity analyzed using mass spectrometry (intact mass) ● Aggregation evaluated for a subset of antibodies using dynamic light scattering (DLS) ● Aggregation and heterogeneity eva... |
Full QC data package including purity and integrity analysis on antibodies using microcapillary electrophoresis, intact MS, DLS, aSEC. Data in range with typical material quality generated by AbCellera's high-throughput production platform. Sample quality and analytical properties suitable for validation and HT physico... |
● Stability evaluated for a subset of antibodies using nanoscale differential scanning fluorimetry (nDSF) ● Heterogeneity and hydrophobicity evaluated on a subset of antibodies using hydrophobic interaction chromatography (aHIC) |
Evaluation of recombinant antibody stability and hydrophobicity as specified in Appendix 2. |
● Self-association evaluated on a subset of antibodies using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) ● Polyreactivity evaluated on a subset of antibodies using baculovirus particle (BVP)-ELISA |
Evaluation of self-association and polyreactivity as specified in Appendix 2. |
● Kinetics, affinity, and specificity on monomeric and aggregated α-Syn evaluated for a subset of antibodies using a biosensor-based binding assay (SPR) |
ka, kd and KD data will be provided (1:1 Langmuir fit with monomeric α-Syn) or apparent ka, kd and KD data obtained (avidity data for aggregated α-Syn are force-fit to a 1:1 Langmuir model to provide estimates of relative binding between antibodies). Preferred antibodies will be identified as demonstrating selective bi... |
● Epitope binning performed using α-Syn monomers for a subset of antibodies using a biosensor-based assay, pending successful assay development |
Epitope bin and community data will be provided to demonstrate epitope diversity of discovered antibodies. Preference for broad epitope coverage with at least five (5) unique epitope bins and framework diversity with at least 10 unique clonal families. |
● In silico immunogenicity prediction performed on a subset of antibodies using EpiVax ISRPRI® platform or alternative platform to be decided at the JGC |
Immunogenicity predictor score to be comparable to benchmark control with known no/low immunogenicity. |
● Antibody binding properties confirmed using the same assay format as used during initial screening ● Non-specific binding validation to be performed on α-Syn knockout cell line ● Validation data delivered as an electronic report and static visualizations of antibody binding properties overlaid onto sequence data from... |
Evaluation of recombinant mAbs in direct binding assays with preference for <30% attrition per binding profiles listed under "therapeutic antibody requirements." If attrition is high, next steps to be discussed at the JGC. |
AbbVie will complete functional characterization and validation studies and notify AbCellera when activities are complete |
ESTIMATED TIMELINES *Estimated timelines do not include optional activities |
STAGE 1 Assay Set-up & Immunization Timeline does not include reagent generation, acquisition & QC: 6-12 weeks per cohort 14-20 weeks total *Due to staggered cohorts |
STAGE 2 Antibody Discovery: 8-10 weeks per cohort 16-20 weeks total* *Due to staggered cohorts |
STAGE 3 Antibody Validation & Characterization: 16-20 weeks |
TOTAL: 46-60 weeks |
DELIVERABLES Stage 1 Deliverables 1. Representative images from in vitro binding of AbbVie-provided and commercial control antibodies to antigen conjugated on beads and/or soluble antigen captured on beads on AbCellera's screening device. 2. Flow cytometry analysis of titre assessments from immunized animals. 3. Antige... |
Stage 2 Deliverables 1. Antigen-specific frequencies of recovered hits and representative images of binding observed from single-cell screens. 2. High confidence paired heavy and light chain sequences from single-cell screens. 3. Bioinformatic analysis of sequences, plus binding specificity assessments from single-cell... |
Stage 3 Deliverables 1. Bead-binding validation data delivered as an electronic report and static visualizations of antibody binding properties overlaid onto sequence data from Stage 2, as generated by AbCellera's custom software Celium™. 2. Analysis of results from biosensor-based assays (kinetics and epitope binning)... |
APPENDIX 1: REAGENTS & MATERIALS ADDITIONAL REAGENTS MAY BE ADDED AT A LATER STAGE |
Reagent Source Amount required |
Human SNCA knockout 293-T cells Abcam ab255433 N/A |
AbbVie proprietary PD-templated human α-Syn aggregates (PD-PFF) No tag AbbVie For immunization: 7 mg for 20 mice, 2 mg for 5 rats and 1.5 mg for 2 rabbits (total 11 mg including overage) |
AbbVie proprietary PD-templated human α-Syn aggregates (PD-PFF) Avi tag AbbVie 2 mg for assay development, screening, validation and characterization |
AbbVie proprietary synthetic human α-Syn fibrils ("PFF) Avi tag AbbVie 5 mg for assay development, screening, validation and characterization |
Human α-Syn monomers Avi tag AbbVie 2 mg for assay development, screening, validation and characterization |
Full length ps129 monomer Proteos RP-004 2 mg for assay development, screening, validation and characterization |
Cyno α-Syn monomers LS Bio LS-G23140-20 1 mg for assay development, screening, and validation |
Mouse α-Syn monomers Stressmarq SPR-323 1 mg for assay development, screening, and validation |
Human β- and γ-Syn Abcam ab48853 Abcam ab48712 1 mg for assay development, screening, and validation |
Post-translationally modified human α-Syn peptides [Acetylation (for N-term antibodies only), phospho and nitratrosylatedY125, phosphorylated and nitrosylatedS129] AbCellera recommends that the sequence to contain Cys start for tagging and bead conjugation New England Peptides Ordered by AbCellera, AbbVie to provide ex... |
Unmodified human α-Syn peptide for 40aa region of interest AbCellera recommends that the sequence to contain Cys start for tagging and bead conjugation New England Peptides Ordered by AbCellera, AbbVie to provide exact sequence 1 mg for assay development, screening, and validation |
Control antibody: Anti-human/cyno/mouse α-Syn Abcam ab214033 ab138501 0.5 mg for assay development, screening, and validation |
Control antibody: Anti-human β-Syn Abnova H00006620-M07 0.5 mg for assay development, screening, and validation |
Control antibody: Anti-human γ-Syn MAB5745 0.5 mg for assay development, screening, and validation |
Control antibody: Assay Control Ab A AbbVie 0.5 mg for assay development, screening, and validation |
α-Syn KO mice C57BL/6JOlaHsd Envigo Item# 5706F 5 |
Alloy mice AbCellera 5 |
Trianni mice AbCellera 5 |
Wild type mice (BALB/c) Charles River 5 |
Rats Charles River 5 |
Rabbits Charles River 2 |
APPENDIX 2: HIGH-THROUGHPUT ANTIBODY ASSESSMENT ANALYTICAL AND PHYSICOCHEMICAL ASSAY DATA AND DESIRED PROFILES TO INFORM SELECTION OF LEAD ANTIBODY PANEL WITH LOW DEVELOPABILITY RISK |
Physicochemical Property Method of Analysis Desired Results |
Purity and integrity CE-SDS Used to assess purity and integrity of antibody material and suitability for use in functional assessment or other assays. Higher purity is desired. Purity levels of >95% purity are desired for most downstream assays. The majority of antibodies produced through AbCellera's high-throughput pr... |
Hydrodynamic properties and aggregation DLS Used to assess the hydrodynamic properties and aggregation state of antibody material and suitability for use in functional assessment or other assays. Hydrodynamic radius values in the range of ~5.2-6.3 nm with low levels of detectable aggregation are desired. The majority o... |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.