Research and Development

We are actively developing innovative approaches to deliver affordable, safe, and effective CAR T-cell therapies, aiming to significantly reduce treatment costs while maintaining the highest standards of quality and efficacy.

Mission: develop a second-generation autologous human anti-CD19 CAR-T product (“huAnti-CAR19”) using a lentiviral vector platform that is safe, effective and materially lower in cost to expand access.

Value proposition: a clinically optimized CAR construct + streamlined autologous manufacturing (process and QC innovations, local decentralized manufacturing options, and supply-chain engineering) to lower per-patient cost while meeting global regulatory safety standards.

Novelty and Advancement over Current State-of-the-Art

Current State-of-the-Art (Problem):

USFDA-approved anti-CD19 CAR-T cell therapies — Breyanzi, Tecartus, Yescarta, and Kymriah utilize the FMC63-derived anti-CD19 CAR construct incorporating CD28/4-1BB-CD3ζ signaling domains. These constructs feature only one cation-π interaction with the human CD19 antigen, enabling recognition of CD19 isoforms 1–7 and 9.However, CD19 isoforms 8 and 10 escape recognition, leading to antigen escape–mediated relapse in treated patients.

Similarly ,an Indian ImmunoACT NexCAR19, featuring two cation–π interactions, improves recognition to cover isoforms 1–7 and 9–10, but still fails to target isoform 8, leaving room for antigen escape.

Proposed Solution — LYSINE BIOTECH Innovation:

The Indian Lysine Biotech huanti-CD19 CAR-CD27/4-1BB-CD3ζ construct introduces six distinct cation-π interactions, enabling comprehensive recognition of all ten human CD19 antigen isoforms (1–10).

Novel Features:

Pan-isoform targeting: No CD19 antigen isoform escape.

Enhanced binding affinity and specificity toward human CD19 antigen.

Reduced relapse potential due to elimination of antigen escape variants.

Improved therapeutic efficacy and persistence through incorporation of CD3zeta Single ITAM Activating T cell and CD27, and 4-1BB costimulatory domains.

Intellectual Property: Lysine Biotech Private Limited, USPTO Published Patent No.: 8/897,292 (USPTO Application No.: US 20250250316 A1) and Indian Patent Granted No.: 567126 (Application Number:.202341064804).

Goal

The chimeric antigen receptor (CAR) single-chain variable fragment (ScFv) contains aromatic residues—such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W)—within its heavy and light chain variable regions. These aromatic residues can engage in cation–π interactions with positively charged amino acids, such as lysine (Lys, K) or arginine (Arg, R), present on the target CD19 antigen.

These cation–π interactions contribute to the stabilization of the CAR–antigen interface, thereby enhancing the binding affinity, specificity, and overall therapeutic efficacy of the CAR-T cell.

Provide a modular CAR-T concept against CD19 that separates antigen recognition, co-stimulatory signals and CD3ζ signalling to improve control, tuning and safety

Project name: LysCAR19 (CD19-targeted T cell therapy)

Purpose: Develop a next-generation CAR T therapy that targets CD19-positive B-cell malignancies while improving persistence and safety through modular co-stimulation and optimized activation.

Mechanism of Enhancement — Cation–π Interaction:

Cation–π interactions are non-covalent forces between a positively charged residue (like lysine or arginine) and an aromatic ring (like phenylalanine, tyrosine, or tryptophan).

By introducing lysine residues strategically near aromatic residues in the binder interface, the molecule can form cation–π interactions that:

Stabilize the antigen–antibody complex,

Increase binding affinity (lower dissociation constant),

Enhance specificity by reducing off-target interactions,

Improve structural rigidity, aiding stability under physiological conditions.

Therapeutic Implications

The engineered huanti-CAR19 binder could improve:

Affinity for the CD19 epitope on B cells → better CAR-T cell targeting.

Specificity → fewer off-target toxicities.

Efficacy → stronger and more durable anti-tumor response.

Stability and half-life → better pharmacokinetics.

Design highlights

Target: CD19, a validated marker on many B-cell cancers.

Targeting head (scFv): A humanized CD19-binding domain that recognizes the cancer cell surface antigen.

Surface display & orientation: A secretion leader and optimized hinge position the binding head for reliable antigen engagement.

Signaling strategy: The receptor transmits two complementary co-stimulatory signals (CD27 and 4-1BB) plus a tuned CD3ζ activation module. This combination aims to:

Increase T cell survival and long-term persistence (4-1BB, CD27),

Maintain potent tumor killing when antigen is present (CD3ζ),

Reduce premature exhaustion and improve durable responses by balancing activation and co-stimulation.

Linkers & architecture: Flexible linkers are used to preserve domain function and reduce misfolding; transmembrane and hinge choices are optimized for surface stability.

Why this approach?

Durability: 4-1BB and CD27 are chosen to encourage memory formation and long-term persistence.

Balanced activity: Optimized CD3ζ and co-stimulation aim to produce strong tumor clearance without excessive early exhaustion.

Translational focus: Humanized components reduce immune rejection risk; modular architecture supports future tuning or safety switches.

Safety & control

Built-in strategies include consideration of clinically accepted safety switches, expression control, and careful scFv selection to minimize off-target binding. Preclinical safety testing and GMP manufacturing are planned prior to any clinical work.

Development plan

Finalize non-actionable design and in-silico assessments (immunogenicity, manufacturability).

Preclinical functional and safety evaluations in controlled labs (expression, activity, specificity, tonic signaling).

Process development for GMP manufacture; regulatory engagement and toxicology studies.

Clinical translation with staged safety-focused studies.

High-level risks & mitigations

Tonic signaling/exhaustion: Mitigate via CD3ζ tuning and scFv screening.

Immunogenicity: Use humanized domains; monitor immune responses.

Manufacturing challenges: Early CMC planning and partner with experienced GMP manufacturers.

High-level conceptual overview

Separation of functions: Safer lentiviral systems separate the vector (transfer plasmid carrying the transgene and cis-elements) from packaging functions (Gag/Pol) and regulatory/accessory proteins (Rev, Tat) on separate plasmids so no single plasmid can produce replication-competent virus.

Packaging vs transfer elements: The transfer vector contains the transgene flanked by cis-acting sequences required for packaging and integration (e.g., LTRs, packaging signal), while packaging plasmids provide structural and enzymatic proteins in trans.

Pseudotyping: A heterologous envelope glycoprotein (commonly VSV-G) is used to broaden tropism and stabilize particles — conceptually this is a separate plasmid encoding the envelope protein used only during vector production.

Regulatory/accessory proteins: Proteins like Rev and Tat regulate RNA export/transcription in lentiviruses; some systems supply these from separate plasmids or replace functions with heterologous elements to improve safety.

Safety-by-design: Modern (3rd-gen/4th-gen) vector designs reduce risk by using self-inactivating (SIN) LTRs, splitting components across plasmids, removing accessory genes, and minimizing sequence overlap to lower recombination risk.

Important safety, ethical, and compliance notes

Work with viral vectors requires institutional biosafety committee (IBC) approval, appropriate training, and suitable containment (e.g., BSL-2 or higher depending on vector and use).

Follow national and institutional regulations, export controls, and donor/patient consent rules if working with clinical material.

Use validated, reputable sources and reagents (commercial kits or repositories) and perform work only in accredited laboratories.

Phase 1: Proof of Concept (POC)

Goal: Validate the scientific feasibility of the CAR-T construct and lentiviral vector system.

Key Activities:

Design and cloning of CAR constructs.

Small-scale lentivirus production and T-cell transduction.

In vitro functional testing (cytotoxicity, cytokine release).

In vivo validation (mouse xenograft models).

Phase 2: Establishing Adherent 293T Master Cell Bank (MCB)

Goal: Create a stable, traceable, and well-characterized producer cell line for lentivirus production.

Key Activities:

Culture optimization for adherent 293T cells.

reparation of MCB under controlled conditions.

Cell line characterization (sterility, identity, mycoplasma, adventitious agents).

Phase 3: Pre-Clinical Trial (PCT) – Efficacy and Safety

Goal: Demonstrate safety, potency, and preliminary efficacy in preclinical animal models.

Key Activities:

GLP-compliant toxicology and biodistribution studies.

Dose optimization studies.

Off-target and persistence analysis.

Phase 4: Plasmid DNA GMP Production

Goal: Manufacture high-quality, GMP-grade plasmids for lentiviral vector generation.

Key Activities:

Production of transfer, packaging, and envelope plasmids.

QC testing (purity, supercoiled ratio, endotoxin levels, sequence verification).

Phase 5: Lentiviral Vector GMP Production

Goal: Manufacture clinical-grade lentivirus for CAR-T transduction.

Key Activities:

Scale-up of lentivirus production using MCB.

Purification and concentration of lentiviral vector.

Potency, sterility, and safety testing under GMP.

Phase 6: CAR-T Cell GMP Manufacturing

Goal: Establish a reproducible, GMP-compliant CAR-T cell manufacturing process.

Key Activities:

T-cell isolation, activation, transduction, and expansion.

Batch release testing (viability, transduction efficiency, potency, sterility).

Cryopreservation and storage protocols.

Phase 7: Clinical Partnership

Goal: Form strategic alliances to support clinical development and regulatory engagement.

Key Activities:

Partner with clinical research organizations (CROs) and clinical centers.

Prepare IND/CTA submission packages.

Develop regulatory and safety monitoring strategies.

Phase 8: Clinical Trial (CT)

Goal: Demonstrate safety and efficacy in human subjects through a phased clinical trial program.

Key Activities:

Phase I: Safety and dose-escalation study.

Phase II: Expanded efficacy and optimization.

Phase III: Large-scale multicenter validation.

Data collection, statistical analysis, and regulatory reporting.

Phase 9: Business Strategy

Goal: Build a sustainable business and commercialization roadmap.

Key Activities:

IP protection and licensing strategy.

Market access and reimbursement planning.

Fundraising and investor relations.

Phase 10: Commercial Manufacturing Collaboration

Goal: Scale up production for market supply and global distribution.

Key Activities:

Partner with CDMOs for large-scale GMP production.

Establish supply chain, QA/QC systems, and tech transfer.

Regulatory approval and launch readiness.

Read More About Products R&D Pipline

3.A patent is published in the USA (USPTO) and granted in India

1)Patent Title: Design and Composition of HuAnti-CD19 Chimeric Antigen Receptor Targeting B-Cell Malignancies Thereof

U.S. Patent No.: 8,897,292, U.S. Patent Application Number: US 2025/0250316 A1, Publication Date: August 7, 2025.Global Dossier.

2).Patent Title: Design and Composition of HuAnti-CD19 Chimeric Antigen Receptor Targeting B-Cell Malignancies Thereof

Indian Patent No.: 567126, Indian Patent Application Number: 202341064804, Date of Issue: June 3, 2025.ipindia.gov.in.