CCMB Antibody

What methodologies does CCMB use for virus culturing in antibody development?

CCMB has successfully cultured the novel Coronavirus from samples collected from COVID-19 patients using multiple cell lines. Their methodology involves:

• Isolation of infectious viruses by virologists headed by Dr. Krishnan H Harshan

• Culture of the virus in Vero cells and Vero E6 cells, which yield higher amounts of the virus

• Culture in human lung epithelial cells for more physiologically relevant models

• Development of assay systems that enable testing of potential therapeutic antibodies

This cultured virus serves six potential research applications: vaccine development, developing antibodies/antidotes, testing antibodies, drug screening, testing disinfectants, and validating instruments designed to neutralize the virus .

How does CCMB isolate antibodies from convalescent patients?

CCMB employs a sophisticated B-cell isolation approach:

• Collection of peripheral blood mononuclear cells (PBMCs) from convalescent patients

• Staining cells with fluorescently labeled prefusion stabilized spike (S) probes

• Isolation of single B cells that are positive for S-2P trimer and/or S1 probe using flow cytometry (FACSAria III cell sorter)

• Direct collection of these cells into PCR plates containing RNA preservation reagents (RNase OUT, First-Strand Buffer, dithiothreitol, and Igepal)

• Cloning of IgG genes from isolated B cells

• Production of monoclonal antibodies through cell culture

This methodology maximizes the chances of isolating potent neutralizing antibodies against viral pathogens.

What is the scientific rationale behind CCMB's fragment-based antibody therapy?

CCMB's approach to fragment-based antibody therapy addresses several limitations of conventional plasma therapy:

• Theoretical basis: While plasma-based passive immunity has shown efficacy in clinical trials, it faces limitations in the availability of human plasma samples

• Alternative strategy: Using horses or other higher animals to generate antibodies against SARS-COV2 viral antigens

• Processing methodology: The antibodies raised in horses using inactivated coronavirus are fractionated and purified to produce antibody fragments F(ab')2

• Mechanism of action: These fragments specifically neutralize coronavirus in patients, facilitating recovery

• Scale and accessibility: Horse-based immunoglobulins can be produced in large quantities, providing an economical alternative that can be made readily available to larger populations

What experimental techniques does CCMB use to evaluate antibody efficacy?

CCMB employs multiple techniques to assess antibody efficacy:

• Binding affinity assessment: Measuring the binding strength of antibodies to S protein and the Receptor Binding Domain (RBD)

• Pseudovirus neutralization assays: Testing neutralizing ability with pseudoviruses that express viral surface proteins

• Plaque reduction neutralizing test (PRNT): Quantifying the ability of antibodies to reduce viral infection in cell cultures

• In vivo efficacy testing: Examining therapeutic efficacy in transgenic (TG) mice challenged with a lethal dose of virus

How does CCMB's therapeutic antibody treatment for COVID-19 compare methodologically to plasma therapy?

CCMB researchers have established that therapeutic antibody treatment offers significant advantages over plasma therapy:

Scientists at CCMB believe that immunotherapy using antibody fragments is more effective and feasible than plasma therapy due to these advantages .

What methodological approaches does CCMB use to assess antibody efficacy against viral variants?

CCMB employs a multi-faceted approach to evaluate antibody effectiveness against viral variants:

• Viral genome sequencing: Identifying viral variants through next-generation sequencing (e.g., using TaqPath, ThermoScientific or COVIDseq, Illumina systems)

• In vitro neutralization assays:

  • Pseudovirus neutralization assays to assess neutralizing activity against specific variants

  • Comparison of neutralizing capacity between original strains and variants of concern

• Clinical parameters monitoring:

  • Time to resolution of symptoms

  • Changes in viral load (interpreted by Ct values)

  • Complete blood counts and inflammatory markers (ferritin, neutrophil counts, lymphocyte counts)

• Comparative analysis:

  • Analyzing the effectiveness of antibody cocktails (e.g., REGEN-COV) against variants like the delta variant (B.1.617.2)

  • Comparing outcomes between antibody therapy and standard of care treatments

For example, in a study on the REGEN-COV antibody cocktail against the delta variant, researchers found that the neutralizing activity against delta was comparable to its activity against the original Wuhan-Hu-1 strain, with significantly fewer patients remaining symptomatic by Day 7 in the antibody cocktail group (21.30% vs. 50.0%, p=0.0001) .

How does CCMB evaluate and optimize antibody developability characteristics?

CCMB and collaborating institutions use advanced molecular surface descriptors specifically designed for predicting antibody developability:

• Surface descriptor analysis: Evaluating antibody surface properties that affect developability

• Benchmarking correlations with experimentally determined biophysical properties:

  • Viscosity

  • Aggregation propensity

  • Hydrophobic interaction chromatography profiles

  • Human pharmacokinetic clearance

  • Heparin retention time

  • Polyspecificity

• Sensitivity assessment of surface descriptors to methodological variables:

  • Choice of interior dielectric constant

  • Different hydrophobicity scales

  • Various structure prediction methods

  • Conformational sampling impact

• Computational enhancement:

  • Averaging descriptor values over conformational distributions from molecular dynamics

  • Mitigating systematic shifts in descriptor distribution

  • Improving consistency across different structure prediction methods

• Risk flag development:

  • Based on benchmarking analysis, researchers propose six in silico developability risk flags

  • Assessment of effectiveness in predicting potential developability issues for case study molecules

What collaborative frameworks has CCMB established for antibody research?

CCMB has developed extensive collaborative networks to advance antibody research:

• Academic-Industry Partnerships:

  • Collaboration with Hyderabad Central University and Vins Bioproducts Ltd for fragment-based therapeutic antibody treatment against COVID-19

  • Partnership with Apollo Hospitals to jointly produce and commercialize dry swab rapid test kits

• Research Integration Framework:

  • UoH team (led by Dr. Nooruddin Khan) specializing in immunology, infection biology, and vaccinology

  • CCMB team (led by Dr. Krishnan Harinivas) focusing on molecular virology

  • Vins Bioproducts team (led by Dr. Krishna Mohan) focusing on bioprocessing and product development

• Technology Transfer Mechanism:

  • Framework for scaling up antibody technologies for commercial use through corporate biotech-pharma partnerships

  • Technology transfer readiness for other infectious diseases beyond COVID-19, including Tuberculosis, Dengue, and Malaria

• Advanced Partnerships:

  • Collaboration with Aganitha to apply Generative AI solutions for small molecule and antibody design

  • Framework agreement signed to translate CCMB's R&D findings into therapeutic candidates

  • Initial phase focusing on multiple targets for Malaria and TB

  • Design of research antibodies for furthering neurology investigations

How does CCMB integrate antibody development with mRNA vaccine technology?

CCMB has developed an integrated approach combining antibody research with mRNA vaccine technology:

• Platform development:

  • Creation of India's first mRNA vaccine technology, adapting technical knowledge from existing platforms

  • Integration of antibody research to enhance vaccine efficacy

• Immune response characterization:

  • Evaluation of anti-spike antibodies generated by the mRNA vaccine

  • Quantification of neutralizing capacity (>90% efficiency in preventing human ACE2 receptor binding to coronavirus)

• Preclinical validation methodology:

  • Challenge studies on hamsters at IISc Bangalore

  • Evaluation of efficacy against live virus infection

  • Assessment of antibody production and quality

• Translational research approach:

  • Adaptation of technology for multiple infectious diseases

  • Application of modular platform for rapid vaccine development

  • Integration with existing antibody therapeutics research

What methodologies does CCMB employ for antibody-mediated immunotherapy in cancer research?

CCMB and affiliated institutions have developed sophisticated approaches to antibody-mediated cancer immunotherapy:

• Immune checkpoint inhibition mechanisms:

  • Targeting CTLA-4, PD-1, and PD-L1 molecules that turn off normal immune responses

  • Blocking these molecules to reawaken the body's ability to recognize cancer cells

  • Leveraging antibodies to disrupt the immune checkpoint pathways

• Technical immunotherapy approaches:

  • Monoclonal antibodies directed at inhibitory immune checkpoints (e.g., anti-CTLA-4 antibody ipilimumab, anti-PD-1 antibodies nivolumab and pembrolizumab)

  • Administration protocols: ipilimumab every three weeks for four doses; nivolumab every two weeks; pembrolizumab every three weeks

  • Continuation criteria based on toxicity and disease progression evaluation

• Response assessment methodology:

  • Development of immune-mediated response criteria

  • Recommendation for continued treatment while patient remains stable

  • Close follow-up with repeat imaging after 4-6 weeks before concluding disease progression

  • Recognition and evaluation of pseudoprogression (apparent tumor size increase due to inflammatory response)

• Toxicity management protocols:

  • Identification of common toxicities: rash, diarrhea, elevated liver enzymes, endocrinopathies, pneumonitis

  • Prompt corticosteroid use (0.5 to 1mg/kg) for early grade 3 or prolonged grade 2 toxicities

How is CCMB applying antibody research to microRNA-based cancer diagnostics?

CCMB is integrating antibody array technology with microRNA research for comprehensive cancer diagnostics:

• Integrated methodology:

  • Extraction of proteins from invasive ductal carcinoma (IDC) samples of different stages and grades and their adjacent controls

  • Protein labeling with appropriate conjugates followed by hybridization with antibody arrays

  • Arrays consisting of 500 monoclonal antibodies related to oncogenes and pathways spotted on microscopic glass slides

  • Visualization and analysis according to manufacturer's protocols

• Statistical analysis approach:

  • Analysis of TLDA (TaqMan Low Density Array) data using StatMiner software

  • Consideration of differentially expressed miRNAs with FDR adjusted p-value < 0.05

  • Two-tailed statistical t-test for LNA arrays with p-values lower than 0.001

  • Heat map generation based on p < 0.001 cutoff

• Validation methodology:

  • Double-blind TaqMan individual assays in 100 IDC samples

  • MiRNA cluster analysis to decipher co-adaptation and functionality

  • Revelation of coordinated expression patterns

This integrated approach has identified distinct miRNA signatures that differentiate types, grades, and stages of invasive ductal carcinoma, providing potential biomarkers for robust classification and early detection of breast cancer.

What methodological advances has CCMB made in testing antibody effectiveness against SARS-CoV-2 variants?

CCMB has developed sophisticated methodologies to assess antibody effectiveness against viral variants:

• Virus culturing and variant isolation:

  • Culture of double mutant strain (B.1.617) and other variants of concern

  • Assessment of whether antibodies from vaccinated or recovered persons can neutralize these variants

• Variant profiling and comparison:

  • Identification of dominant variants in different geographical regions (UK variant in Punjab/Delhi; double mutant in Maharashtra/Telangana/Andhra Pradesh)

  • Monitoring variant spread efficiency through systematic genomic surveillance

• Historical evolution tracking:

  • Documentation of virus type evolution (e.g., A3i type initially prevalent in India, believed to travel from South East Asia, with 41% presence)

  • Transition to A2A virus becoming predominant, believed to have entered from Europe

• Scientific communication:

  • Development of protocols taking 10+ days for comprehensive variant assessment

  • Communication of findings to health authorities and public

How does CCMB approach the complexities of antibody-based therapies for infectious diseases beyond COVID-19?

CCMB has developed a transferable framework for antibody research across multiple infectious diseases:

• Cross-disease application methodology:

  • Adaptation of antibody research platforms for tuberculosis, dengue, and malaria

  • Leveraging modular technology with rapid turnaround times

  • Minimal modification requirements for targeting different pathogens

• Collaborative research structure:

  • Engagement with Aganitha to apply Generative AI for antibody design across disease areas

  • Initial phase targeting malaria and TB

  • Structure-based discovery of small molecule inhibitors targeting essential proteins of Mycobacterium tuberculosis

• Disease-specific approaches:

  • For malaria: Leveraging Generative AI capabilities for validated parasite drug targets

  • Development of nanobody binders for neurotransmitter receptors (GluD1)

  • Potential therapeutic applications for neurological disorders like Alzheimer's and epilepsy

• Translational research methodology:

  • Prediction, testing, and optimization of potential compounds

  • Interdisciplinary collaboration to address challenges like drug resistance

  • Development of frameworks for both therapeutic and diagnostic applications