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ALLOGENIC CAR-T CELL THERAPY
2
ALLOGENIC CAR-T CELL THERAPY
TREATMENT OF CHRONIC MYELOID LEUKAEMIA
Raheem, Kainat, Sidra
PROPOSAL
WHAT IS
CHRONIC MYELOID LEUKAEMIA?
- Type of cancer that affects WBC.
- Genetic change in the production of myeloid cells.
- Inability to produce lymphocytes.
- Abnormal gene created: BCR-ABL.
- Partial maturation of cells.
- Unable to fight infections well.
- Symptoms include:
- Tiredness
- Night Sweats
- Weight Loss
- Pale Skin
- Bruising
TARGET AUDIENCE
TARGET AUDIENCE
- Over 829 new cases of CML every year in the UK.
- 224 deaths every year.
- Diagnosis occurs:
- 23% in 75 y.o >
- 77% in 60-74 y.o >
- Total amount current suffers is estimated to be around 15,000~
- No current FDA CAR-T cell therapy available for CML.
(Source: hematologyadvisor.com)
CAR-T CELL THERAPY
CAR-T CELL THERAPY
- New efficacious treatment for cancer patients suffering from leukaemia and lymphoma.
- Genetically modifying T cell.
- CAR – directed at antigens on target tumour cells.
- The GM CAR T cells undergo ex vivo amplification.
- Cells reintroduced into patient.
- Can even kill tumour cells in advanced stages of the disease.
(Source: mesothelioma.com)
DIFFERENCES IN THERAPY
AUTOLOGOUS VS ALLOGENIC
- Autologous - Cells are taken from the patients, processed and returned to same patient.
- Allogeneic - Cells are taken from a donor and given to a patient with similar genetics.
- Present CAR-T cell therapies are autologous.
- Autologous CAR-T cell therapy is very expensive as it is catered t each individual.
DIAGRAM OF AUTOLOGOUS CAR-T CELL THERAPY
(Source: touchoncology.com)
EXPANSION
EXPANSION
- We are using T cells as our biological platform.
- Using a viral vector to transport CAR gene into the T cells.
- Mostly use viruses as vectors.
- High availability of different viruses with different characteristics.
- Virus vectors: Retroviruses (E.g. Lentiviruses), Adenoviruses, and Adeno-associated viruses.
- Sleeping beauty transposon system as an alternative.
AUTOLOGUS
CAR-T CELL THERAPY
CURRENT LIMITATIONS
(Souce: nature.com)
- Cells are derived from the individual and therefore the specificity of the treatment means high cost - £230,000+
- Time consuming process.
- Delay in treatment can be problematic to patients with profilerating diseases.
- Quality of the starting T cells from patient maybe questionable as the patient is most likely recieving lymphodepleting chemotherapy and/or radiotherapy.
- Incorporation into hospitals due to singularity of treamtent.
(Souce: nature.com)
HISTORY OF CAR-T CELL DEVELOPMENT
CURRENT TIMELINE
(Source: s3.amazonaws.com)
CURRENT CLINCIAL TRIALS
(ALLOGENIC CAR-T CELLS AGAINST LEUKEMIA/LYMPHOMA)
(Source: clinicaltrials.gov)
PRODUCTION
COSTS
PRODUCTION
Stem Cell Laboratory: £50,000
Lab Running Costs: £6000
Lab Equipment: £3000 (incl. Cell Washer)
WAVE Bioreactor: £5000
200x T225cm Flasks: £28,000
TOTAL COSTS: £92,000
DOSES
- 500 Doses will be created.
- One dose is one IV infusion.
- CD22 (B Cell Lymphocytes) expression ≥ 90%
- Weight Range: 76 - 80 KG
- Adminstration: 7.6 x 10^6 CAR-T Positive T-Cells.
- 28-Day recovery period within the hospital.
- Current stem cell transplant costs £150,000 on NHS.
- Autologous CAR-T cell therapy is significantly more:
- YESCARTA - £322,000~
- KYRMIAH - £410,000
(Figures adjusted from $ to £)
- Competition: Juno, Novartis, Cellectis & Kite Pharma.
- Current ongoing trials for allogenic CAR-T cell therapy by Cellectis.
- By 2026, CAR-T cells will compromise the largest share of oncology drug sales.
- We are asking for £300,000 over a 3 year period.
- If we get 90% of T-cells extracted from the Donor, the estimated yield should be about 70%.
- Yield is affected by the health of the donor.
YIELD
SCALING
- 200 Donors (5L of CAR-T Cells Made From Each Donor)
- 200x T225 cm Flasks
- Continous-flow manfacturing production.
- No need to increase reactor size, but flask size can be increased.
OUR PRICE: £10,000
MANFACTURING CHALLENGES:
GMO
Viral Vector: CTL019 (Tisagenlecleucel)
LENTIVIRAL VECTORS
- Binds to CD19 on B Cells.
- CD3-Zeta signalling domain causes T Cell activation.
- Increases profileration of CAR-T cells within patient.
- A type of Retrovirus that intergrate themselves into the genome of the host.
- T Cells are incubated using the Lentiviral Vector which encodes the Chimeric Antigen Receptor.
- Inserts RNA.
- RNA is then reverse-transcribed into DNA.
- Cells divide and grow in the bioreactor.
- Translated to express on the surface of T cells in the patient.
- Safer intergration profile instead of gammaretroviral vectors (prone to mutagenesis).
(Source: fda.gov)
DIAGRAM: CTL019 Targeting B+ Cells to cause a cytokine reaction and the desctruction of tumour cell
(Source: fda.gov)
UPSTREAM PROCRESSING
UPSTREAM PROCRESSING
STEP #1: Leukapheresis – remove lymphocytes/immune cells from blood.
STEP #2: Leukocytes washed out in cell washer
Lymphocytes are separated by counterflow centrifugal elutriation.
STEP #3: Product of apheresis cultured in Wave bioreactor with APCs and a viral vector encoding CAR gene.
STEP #4: Alternative device: CliniMACS Prodigy
Nutrients needed for growth are oxygen and growth factors such as IL-2
STEP #5: 9-11 days – car t cell culture expanded to 5 L.
STEP #6: Magnetic beads removed
STEP #7: CAR T cell culture is concentrated using cell washer
STEP #8: Concentrated CAR T cells collected
(For Autologous - CAR T cells are returned back to patient alongside chemotherapy)
(Source: nanbiosis.es)
UPSTREAM PROCRESS FLOWCHART
GOOD MANFACTURING PRACTICE
ETHICAL PRACTICE
- Manufacturing of viral vector in Grade A Laminar Flow Cabinets.
- CAR T cell manufacturing done in GMP approved clean room facility.
- Reduce chances of cross contamination by using disposable equipment.
- Apheresis is a closed system – reduces chances of contamination.
- Product yield is affected by donor health
- GMP regulations require expensive and complex systems.
- Must be fully equipped with: Facilities, Systems, Environmental Monitoring Systems, Manufacturing Equipment, & analytical equipment.
- Should have detailed records of all equipment and reagents used.
- Staff must be skilled and have knowledge of the GMP requirements.
- Unidirectional flow is best.
SOURCES
REFERENCES:
CAR-T CELL THERAPY: https://www.cancerresearchuk.org/about-cancer/cancer-in-general/treatment/immunotherapy/types/CAR-T-cell-therapy
PHASE 1 STUDY OF UCART22: https://doi.org/10.1182/blood-2020-138594
TRANSPON BASED PRODUCTION OF CAR-T CELLS: https://jitc.bmj.com/content/10/9/e005189
GMO STRATEGIES TO ENHANCE CAR-T CELL PERSISTENCE FOR PATIENTS WITH SOLID TUMOURS: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384227/
SCALABLE MANFACTURING OF CAR-T CELLS FOR IMMUNOTHERAPY: https://doi.org/10.1158/2643-3230.BCD-21-0084
ALLOGENIC CAR-T CELLS: AN ALTERNATIVE TO OVERCOME CHALLENGES: https://www.frontiersin.org/articles/10.3389/fimmu.2021.640082/full
LIMITATIONS OF CAR-T CELL THERAPY: https://www.nature.com/articles/s41408-021-00459-7
LENTIVIRAL VECTORS: https://www.creative-biogene.com/support/Lentiviral-Vectors-the-Application-for-CAR-T-Therapies
THANK YOU FOR LISTENING TO OUR PRESENTATION
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DOWNSTREAM PROCESSING
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PLAN
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TIMELINE
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