Lecture-14 CAR-T Cells for personalised immunotherapy.pptx

The importance of T cells as master immune cells and the outlines of CAR-T cell engineering design & development using gene construct in lentivirus are familiarized.
Types of hematological cancers approved by FDA for curing using CAR-T cells targeting their respective surface markers will are shown.
The Platforms developed to prepare living drugs i.e., tailored immune cells are depicted.
Indian based firms in CAR-T cells production are highlighted.

1. Lecture 14 PERSONALIZED IMMUNOTHERAPY USING CAR-T CELLS Prof. Sreerama Krupanidhi Email: [email protected] 9/10/2024 Prof. Sreerama Krupanidhi 1

2. Focus To appreciate the biology & importance of T cells as master immune cells. To outline the preparation of CAR gene construct using lentivirus. To visualize CAR-T Cell engineering design & development. To review CAR-T Cells curing blood cancers. To appraise the living drugs (cell therapy) made using autologous T cells. 9/10/2024 Prof. Sreerama Krupanidhi 2

3. Learning outcomes The importance of T cells as master immune cells will be known. The outlines of CAR-T cell engineering design & development using gene construct in lentivirus will be familiarized. Types of hematological cancers approved by FDA for curing using CAR-T cells targeting their respective surface markers will be appreciated. The Platforms developed to prepare living drugs i.e., tailored immune cells will be acquainted. Indian based firms in CAR-T cells production will be known 9/10/2024 Prof. Sreerama Krupanidhi 3

4. How do CAR-T cells Target Tumor cell? CAR s are genetically encoded engineered fusion molecules namely ScFv (small chain fragment variable) that can reprogram the specificity of peripheral blood polyclonal T-cells against a selected cell surface target of cancer cell . CARs are endowed with the specific tumor antigen binding domain s from a primed B cell of a tumor targeting antibody and with T cell signaling domain s to allow specifically targeted ScFv redirected T cell activation . 9/10/2024 Prof. Sreerama Krupanidhi 4

5. Immunotherapy to treat blood and solid tumors 9/10/2024 Prof. Sreerama Krupanidhi 5 9/10/2024 Prof. Sreerama Krupanidhi 5 MULTIPLE MYELOMA CELLS (plasma cells in bone marrow) 6. CAR-T cells BCMA, CD19 4. Bispecific abs BCMA/CD3 2. Monoclonal Abs CD38, SLAMF7 1. Vaccines Conditioned Dendritic Cell/TILs infusion 5. Immune Checkpoint Inhibitors PD1, PDL1, CTLA4 3. Ab drug conjugates BCMA

6. Immune cells Macrophage Fibroblasts Endothelial cells Stem cells Hematopoiesis Mast cell Eosinophil NK cell LAK cell Cytotoxic T cell T helper cell B cell Plasma cells Antibodies Neutrophil Inflammation 9/10/2024 Prof. Sreerama Krupanidhi 6

7. Thymic Cell 9/10/2024 Prof. Sreerama Krupanidhi 7

8. T-Cell Receptor Complex 9/10/2024 Prof. Sreerama Krupanidhi 8

9. Cell Surface Antigens as Possible Targets for CAR-T Cell Therapy CAR-T cells targeting CD19 and BCMA (CD 269) have been approved by the FDA for remission from B-Cell Lymphoma and Multiple Myeloma . https://doi.org/10.1007/s43152-024-00055-4 ( Khan, T.H. et al) 9/10/2024 Prof. Sreerama Krupanidhi 9

10. CAR-T Targets 20 Different CAR-T targets in clinical trials Jonshon and June Cell Resarch 2017 B-cell related surface antigens 9/10/2024 Prof. Sreerama Krupanidhi 10

11. Hematological biomarkers as predictors of response to immunotherapy CDs Function Cell Type of cancer CD 19 Dominant signaling component B-Cell Acute Lymphocytic leukemia B cell lymphoma Leukemia Non-Hodgkin Lymphoma CD 33 Transmembrane receptor Myeloid lineage Myeloid leukemia CD5 TCR inhibitory Molecule T-Cell T cell acute lymphoblastic lymphoma, T-non-Hodgkin lymphoma CD 123 Hematopoietic progenitor cell differentiation and proliferation Hematopoietic blast cells Acute Myeloblastic Leukemia Leukemia BCMA CD269 Maturation Antigen B Cell Multiple Myeloma https://doi.org/10.1186/s13046-018-0817-0 ( Michellie,H et al) 9/10/2024 Prof. Sreerama Krupanidhi 11

12. Engineering of Chimeric antigen: Lentivirus Biology 9/10/2024 Prof. Sreerama Krupanidhi 12

13. Lentiviral Genome

14. Lentivirus genome organization Lentiviral vectors are a type of viral vector that are used in gene therapy to modify, insert, or delete genes in organisms. They are derived from the human immunodeficiency virus type-1 (HIV-1) and are a popular method for stably modifying mammalian cells. Essential genes : gag; pol; tat; rev. env (SU, TM) Accessory genes : vif ; vpr ; vpu ; nef . MA : Matrix; CA : Capsid; NC : Nucleo Capsid; PR: Viral protease; RT : Reverse Transcriptase; IN : Integrase 9/10/2024 Prof. Sreerama Krupanidhi 14

15. Third generation lentiviral vector with a construct gene of interest Third-generation lentiviral vector is with: 1. Tat independent. 2. Composed of two separate packaging plasmids: ( i ) one encoding gag and pol (ii) another encoding rev. 3. An additional plasmid encodes the envelope protein , derived from the VSV-G. 4. Plasmid encoding the gene of interest contains lentiviral LTR sequences that have been altered to be self-inactivating ( SIN) to prevent recombination. (LTR long terminal repeat, VSV vesicular stomatitis virus) Overview of large-scale vector manufacturing: Manufacture of a lentiviral vector begins with the culture of a packaging cell line in a facility that uses Good Manufacturing Practices. 9/10/2024 Prof. Sreerama Krupanidhi 15

16. CAR-T C ell R eceptor Design CAR, chimeric antigen receptor; mAb, monoclonal antibody; TAA, tumor -associated antibody; TCR, T -cell receptor. Ref: Sadelain M, et al. Nat Rev Cancer. 2003;3:35-45. 9/10/2024 Prof. Sreerama Krupanidhi 16

17. CAR Gene Construct Transduction to T-C ells Ref: Sadelain M, et al. Nat Rev Cancer. 2003;3:35-45. Brentjens RJ, et al. Nat Med. 2003;9:279-286 9/10/2024 Prof. Sreerama Krupanidhi 17

18. G enerations of CAR s with four domains ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a role in initiating T-cell responses 9/10/2024 Prof. Sreerama Krupanidhi 18 an antigen recognition domain (targeting moiety) a hinge/spacer a transmembrane element a signaling endo-domain

19. Autologous CAR-T cell preparation Patient’s blood collection T-Cell Isolation Activation Engineer Lentivirus with ScFv binding domain of IgG Transduction Car-T cell Manufacturing Procedure takes 3 months 9/10/2024 Prof. Sreerama Krupanidhi 19

20. Patient readiness for CAR T cell infusion Patient Screening Bridging Therapy Lymphodepletion Infusion One time Remission within two weeks No Relapse even up to five years 9/10/2024 Prof. Sreerama Krupanidhi 20

21. Outlines of CAR-T Cell Manufacturing & Infusion into patients CAR-T cell process begins with the patient's blood being drawn. leukapheresis, Specifically, T-cells, an essential part of the immune system, are then separated from the blood. The T-cells are then transferred to a specialized laboratory, where they are engineered using recombinant lentiviral vectors to produce specific chimeric antigen receptors (CARs) on their surface. They are allowed to grow in either static or dynamic culture, cryopreserved. While the modified T-cells grow in the laboratory, the patients receives chemotherapy to suppress their immune system. These CAR-T cells are then re-injected into the patient’s bloodstream. The new CARs latch on to an antigen on tumor cells – significantly increasing their ability to effectively hunt down and destroy cancerous cells. This creates a more efficient setting for the CAR-T cells to do their job and destroy malignant cells. 1. Collection of WBCs Including T cells from patient 2. Separation of T cells 3 . Genetic Manipulation of T cells using l entivirus 6 . CAR T cells Infused into the patient Obtain Engineered T cells With Chimeric Receptor 4. Proliferation of Engineered T cells 5. Patient treated for Chemotherapy prior to infusion of CAR T cells 9/10/2024 Prof. Sreerama Krupanidhi 21

22. Chronology of CAR-T Cell Adoption for Cancer Treatment In 1987, an Israeli immunologist, Zelig Eshhar , created the first “chimeric antigen receptor,” an engineered receptor that does not exist in nature. The DNA encoding the ScFV portion of the receptor was implanted in the T cells enabling the m fight and kill cancer. In the year 2010 the first successful cancer treatment with CAR-T was given to an advanced follicular lymphoma patient and was reported by the lab of Steven Rosenberg, M.D., Ph.D., C hief of the Surgery Branch in NCI’s Center for Cancer Research. On August 30, 2017, tisagenlecleucel ( Kymriah) was the first CAR - T cell i m m u n o t h e ra p y app r o v e d b y the FD A . It was a pp r ov e d for c hi l d ren a n d young adults aged 25 who relapsed or were not responding to therapy for acute lymphoblastic leukemia (ALL). 9/10/2024 Prof. Sreerama Krupanidhi 22

23. PROF. ZELIG ESHHAR, AN ISRAELI IMMUNOLOGIST AT THE WEIZMANN INSTITUTE OF SCIENCE, IS A PIONEER OF THE CAR-T CELL APPROACH AND ITS APPLICATION FOR CANCER THERAPY - 1987 “Our biggest satisfaction was that CAR-T therapy was approved by the FDA and now patients benefit by getting the treatment all over the world,” Prof. Eshhar remarked. 9/10/2024 Prof. Sreerama Krupanidhi 23

24. Approved CAR-T Cells (living drugs) for Immuno Therapies CAR-T Cell Product Name and FDA Approved Date Target Antigen Lisocabtagene maraleucel ) – 2021 ( Breyanzi ) CD19 Idecabtagene vicleucel ) – 2021 ( Abecma ) BCMA Ciltacabtagene autoleucel ): 2 heavy chains for 2 epitopes - 2022 ( Carvykti ) BCMA Brexucabtagene autoleucel -2021 (Kite Pharma) CD19 Mantle Cell Lymphoma (MCL) NexCAR19 ( Talicabtagene autoleucel ) 2023 Idea sprouted from IITB/TATA CD19 Afamitresgene autoleucel ( Tecelra ) (2024) ( Adaptimmune ) Metastatic synovial sarcoma, a type of soft tissue sarcoma Axicabtagene ciloleucel ( Yescarta ) (2017) ( Kite Pharma) Diffuse large B-cell lymphoma 9/10/2024 Prof. Sreerama Krupanidhi 24

25. Firms involved in CAR-T cells manufacturing and cost of treatment Firm Types of blood cancers targeted Cell surface marker targeted Cost of the preparation of drug & treatment Breyanzi Bristol-Myers Squibb Diffuse large B-cell lymphoma ( DLBCL ) High grade B-cell lymphoma ( HGBCL ) Primary mediastinal large B-cell lymphoma ( PMBCL ) Follicular lymphoma grade 3B ( FL3B ) CD19 Lisocabtagene maraleucel $4,10,000  ABECMA Celgene Corporation Multiple myeloma BCMA Idecabtagene vicleucel $5,45,000 CARVYKTI Novartis Multiple myeloma BCMA Ciltacabtagene autoleucel $5,04,344 NexCAR19 ImmunoAct (IITB/TATA) B cell Lymphoma Leukemia CD19 Talicabtagene autoleucel Rs. 40 lakhs (<$500 ) 9/10/2024 Prof. Sreerama Krupanidhi 25

26. Types of Blood Cancers approved for CAR-T Cell Therapy Non-Hodgkin Lymphoma (NHL): B-cell lymphoma and follicular lymphoma. Multiple Myeloma (MM) : Bone marrow cancer, plasma cells. Hodgkin Lymphoma (HL) : B cell lymphoma. Men are more likely to develop HL than women. Reed–Sternberg cells originate from mature B cells. Acute Lymphoblastic Leukemia (ALL) : A form of blood and bone marrow malignancy, excessive production of immature lymphocytes called lymphoblasts . Acute Myeloid Leukemia (AML): Immature white blood cells called myeloblasts . It affects older adults most frequently. 9/10/2024 Prof. Sreerama Krupanidhi 26

27. ImmunoAct ImmunoACT , an IIT-Bombay spin-off incubated at SINE ( Society for innovation and entrepreneurship ) , is India's first gene-modified cell therapy company, fully integrated from R&D to Manufacturing and Commercialization. The firm’s goal is bringing innovative, affordable and accessible cell & gene therapies to the masses, to improve overall survival outcomes in diseases with a clinically unmet need. CAR-T cell therapies, beginning with NexCAR19 ( Talicabtagene autoleucel ) are personalized for each patient and are specifically designed to be more familiar to the human immune system, limiting their side-effects and rendering them safer. NexCAR19 ( ImmunoACT ) costs approximately  Rs 40 lakh   for CAR T-cell therapy in India. The firm is working to reduce the cost to Rs.15 lakhs in near future. 9/10/2024 Prof. Sreerama Krupanidhi 27

28. Partner hospitals in India with ImmunoAct for CAR-T therapies ( NexCAR19 ) Patients can contact the following mobile for any enquiry and guidance related to CAR-T cell Therapy and treatment: WhatsApp/ call: 8882424372 (Satyughealthcare.com) 9/10/2024 Prof. Sreerama Krupanidhi 28

29. How will a patient receive NexCAR19? Patient blood cells will be sent to a manufacturing center to make tailored NexCAR19. Before patient gets NexCAR19, he/she will get 3 days of chemotherapy to prepare the body. When NexCAR19 is ready, patient healthcare provider will give it to patient through a catheter placed into vein (intravenous infusion). The infusion usually takes less than 30 minutes. Patient will be monitored while receiving treatment daily for at least 7 days for patients with MCL and at least 14 days for patients with ALL after the infusion. Patient should plan to stay close to the location where he/she received treatment for at least 4 weeks after getting NexCAR19. Healthcare provider will help patient with any side effects that may occur. Healthcare provider will discharge patient if anticipated side effects are under control. 9/10/2024 Prof. Sreerama Krupanidhi 29

30. ClineMax Prodigy 9/10/2024 Prof. Sreerama Krupanidhi 30 Two processes for the CliniMACS Prodigy accommodate requirements of modern transduction-based CAR and TCR engineered T cell manufacturing.

31. Cocoon Lonza 9/10/2024 Prof. Sreerama Krupanidhi 31 The Cocoon® Platform is a functionally closed, highly customizable and scalable integrated cell. 2. The Cocoon® Platform to perform protocols including isolation, cell selection, activation, transduction/transfection, expansion and harvest in an automated system. 3. Protocols are performed within the functionally closed cassette, based on pre-set programming. Each cassette: Is functionally closed, adaptable, disposable Is process-specific for adherent/suspension cells Has an integrated cold chamber for internalizing process reagents and consumables Is appropriate for lentiviral and gamma-retroviral transduction processes as well as non-viral transfection with Lonza's 4D-Nucleofector™ LV Unit Aseptic processing and sampling through weldable tubing or SPIROSTM medical-grade connections for maintaining a closed environment

32. Cytiva Sefia 9/10/2024 Prof. Sreerama Krupanidhi 32 Tailored cells manufacturing platform Automate cell therapy manufacturing with the following: ( i ) Sefia Select™ system to automate cell isolation, harvest, and formulation steps (ii) Sefia ™ expansion system to automate cell activation, transduction, and cell expansion steps.

33. Indian firms manufacturing CAR-T cells 9/10/2024 Prof. Sreerama Krupanidhi 33 Firm Sponsors Product ImmunoAct Dr. Purwar - IITB/TATA NexCAR19 (First Indian Product) Immuneel Therapeutics Pvt. Ltd Kiran Mazumdar Shaw-Biocon Varnimcabtagene Product yet to get approved by FDA CMC –Vellore, Dr. Mathews Automated closed system using the GMF Miltenyi CliniMACS Prodigy (25-47 fold T cell expansion) VELCART , Anti CD 19 CAR-T cells Aurigene Oncology Reddy’s lab, Hyderabad, Under clinical phases (as of 9/2024)

34. Benefits and drawbacks of CAR T cell therapy Benefits Remission within two weeks No relapse even up to 5 years No host vs . graft rejection due to autologous engineered T cell Drawbacks Procedure is Expensive-Costs 4-5 lakh US dollars Three months waiting period (including screening and Bridging therapy) for initiating infusion Yet to reach people of all corners Not all insurance companies accept the medical claim under CAR-T therapy. 9/10/2024 Prof. Sreerama Krupanidhi 34

35. Summary Outlines of the Design and development of CAR-T cells using patients’ T Cells and Lentivirus gene construct are shown. Lymphodepleting chemotherapy: A short course of chemotherapy administered to deplete existing T-cells prior to the CAR–T cell therapy. The purpose of this chemotherapy cycle is to suppress host immune system slightly so that it does not reject the CAR-T cells once they are infused. Tailored CAR-T cell manufacturing using automated platforms viz., ClineMax Prodigy, Cocoon Lonza and Cytiva Sefia are shown. Admit in Hospital as in-Patient to monitor, Follow-up Health care. Find remission within Two weeks of infusion Names of the CAR-T cell living drugs: (1) Talicabtagene autoleucel ; (2) Ciltacabtagene autoleucel ; (3) Idecabtagene vicleucel and (4) Lisocabtagene maraleucel 9/10/2024 Prof. Sreerama Krupanidhi 35

36. Acknowledgements 9/10/2024 We acknowledge the online resources and public domains for the Preparation of the content to develop teaching material and for the dissemination of knowledge. https://www.pharmafocusasia.com/articles/car-t-cell-therapy-and-nexcar19 Prof. Sreerama Krupanidhi 36 Life Saving Service conceived and rendered by Dr. Purwar , who was Dr. Dwivedi’s graduate advisor at IIT Bombay and is founder and CEO of ImmunoACT , said in a statement. “Now our patients in India and [other] countries with limited resources will have access to this lifesaving drug at an affordable cost.”

37. Thank you 9/10/2024 37 Prof. Sreerama Krupanidhi