Introduction

• It is use of immune cell populations with distinct biological properties to treat cancers, infections and other diseases.
• Normally, immune system recognizes malignant cells that keep generating, and kill them. This is called as tumor surveillance. Malignancy develops when immune system fails to recognize a malignant cell.
• T cells recognize antigenic tumor peptides presented by the major histocompatibility complex protein on the surface of antigen presenting cells. This requires engagement of T cell receptor with MHC protein. This also requires additional costimulatory signal by CD80/B7 protein that engages with CD28 on T cells.
• HSCT is also a type of cell therapy/ immunotherapy, in which immune cells capable of recognizing still surviving/ relapsing tumor cells are given to the patient.

Sources of cells

• Blood
• Bone marrow
• Adipose tissue
• Umbilical cord blood
• Fetal/ embryonal cell lines
• Induced pleuripotent stem cells

Engineering techniques applied to harvested cells

• Selection
• Depletion
• Expansion
• Genetic modification

Raw materials needed to produce cell products

• Culture media
• Sera
• Growth factors
• Cytokines
• “Feeder cells” that are used to support cell growth

Maximum aseptic precautions are needed, as terminal sterilization of product is not possible.

Center for biologicals evaluation and research is the regulatory agency.

Autologous cell products

• Less risk of immunological reactions
• Less bio-incompatibility
• Less risk of communicable disease transmission

Allogenic cell products

• Potential to treat hundreds of patients from single manufactured lot of cells

Cellular therapy products include

• Unmanipulated leucocytes
• Donor lymphocyte infusions
• Cytokine induced T cells
• Donor derived/ third party allogenic- virus specific cytotoxic T cells Ex: CMV specific, EBV specific cytotoxic T cells
• Lymphokine activated killer cells
• Tumor infiltrating lymphocytes (Harvested from tumor tissues and expanded in vivo with IL2)
• T regulatory cells
• Antigen specific T cells
• Mesenchymal cells
• Dendritic cells- They are antigen presenting cells that are sensitized in vitro to specific antigens. When infused they are able to recognise tumor/ infectious antigens and present them to T cells.
• NK cells
• Chimeric Antigen Receptor T cells (CAR-T cells)

CAR-T Cells:

• Normally cytotoxic T cells are MHC restricted and tumor cells down regulate MHC molecules. Both together result in failure of T cells to recognize tumor antigens.
• T cells from patient are isolated and are genetically modified ex-vivo with chimeric receptors that consists of an extracellular antigen recognition domain linked to an intracellular signaling domain. CAR-T cells combat tumor cells without any dependency on HLA antigens.
• Parts of Chimeric Antigen Receptors
  • Transmembrane domain
  • Intracellular signaling motif
  • Extracellular tumor specific antibody (which is involved in binding to malignant cell)
• Types of Chimeric Antigen Receptors:
  • First generation: Intracellular signaling domain contains only CD3 zeta intracellular domain of the T cell receptor. Hence T cell proliferation and cytokine production is not sufficient.
  • Second generation: They contain additional costimulatory signaling domains (such as CD 28 or 4-1BB), that result in enhanced persistence and proliferation once the cells are infused back into the patient.
  • Third generation: Both CD28 and 4-1BB are added.
  • Fourth generation: various cytokines such as IL-12 have been added to endo-domain of the second generation of CARs. Hence both T cells and NK cells are stimulated.
  • Fifth generation: Binding site for STAT3 transcription factor and IL-2 receptor are added to induce cytokine storm
• Chimeric antigen receptors are artificial T cell receptors which are MHC unrestricted.
• Chimeric antigen receptor is introduced into the cells through infection with lentivirus/ retrovirus that carried the gene encoding the CAR.
• Antitumor monoclonal antibody is attached to signaling domain of T cells.
• These cells bind to tumor cells in MHC unrestricted manner and then kill the tumor cells.
• For example, in case of Tisagenlecleucel, a self inactivating lentivius vector is used which contains a transgene coding for anti-CD19 receptor protein. This CAR (antibody fragment that recognizes CD19) is fused to CD8-alpha hinge and transmembrane region. Upon binding to CD19 bearing cancer cell, CAR activates intracellular signaling pathways, including 4-1BB and CD3 Zeta chain. This initiates T cell activation and target cell elimination. 4-1BB activation enhances expansion and persistence of Tisagenlecleucel. Hence, these CAR-T cells can be detected in the blood for many years.
• Ideally, CAR T cell should target a tumor restricted antigen to avoid toxicity that may result in an immune recognition against healthy tissues. Identifying such an antigen for myeloid malignancies is very difficult.
• Manufacturing CAR-T cells occurs at a central facility and this process is closely co-ordinated with treatment center.
• Actual steps include:
  • Collection of leukocytes from patient by non-mobalized leukapharesis.
  • Cryopreservation of collected cells and transport to manufacturing facility.
  • After thawing, T cells are separated from other cells in the harvest.
  • Separated T cells are activated by binding to anti-CD3/CD 28 antibody coated paramagnetic beads and transdused with the lentiviral vector containing the anti-CD19 CAR transgene.
  • These cells are cultured till adequate numbers of CAR-T cells are formed.
  • CAR-T cells are separated from beads, washed and then cryopreserved.
  • After quality check (purity, identity, sterility etc), cells are sent back to treatment center.
  • Approximate time from collection of cells and infusion of CAR-T cells is 4 weeks. Hence bridging chemotherapy may be needed in some cases. However, as compared to BMT, achieving CR is not necessary prior to CAR-T cell infusion.
  • Lymphodepleting chemotherapy (Fludarabine- 30mg/m2 IV daily for 4 doses and cyclophosphamide- 500mg/m2 IV daily for 2 doses) must be administered 2-14 days prior to CAR-T cell infusion (Except for those with profound lymphopenia. i.e. TLC of <1000/cmm).
• Side effects-
  • Cytokine release syndrome (CRS)
    • Occurs due to release of inflammatory cytokines from activated T cells.
    • Presents with high fever, tachycardia, and myalgia.
    • May result in shock, respiratory failure and multiple organ dysfunction.
    • Seen in 80-90% patients
    • Severe CRS is seen in 30% patients, especially those with high tumor burden and high CAR-T cell expansion.
    • Treated with
      • Anti IL-6 Receptor antibody: Tocilizumab
      • Corticosteroids- Used in severe cases
      • 3^rd line: Siltuximab, ATG, Alemtuzumab, Cyclophosphamide
    • Tocilizumab may be used as prophylaxis against CRS
  • CAR-T Cell related Encephalopathy syndrome (CRES)
    • Confusion, delirium, hallucinations, aphasia, and/or seizures
    • Occurs due to endothelial activation and disruption of the blood-brain barrier
    • Usually resolves within days
    • Siltuximab may be used for severe and persistent cases. Levetiracetam is used as prophylaxis against seizures.
  • B cell aplasia
    • Regular IVIg infusions are hence necessary (Once in 4 weeks)
    • Continue till CAR-T cells persist in the body
  • Infections
  • Persistent cytopenia
  • Tumor lysis syndrome
• Other limitations:
  • Antigen escape: Cancer cells without target antigen proliferate
  • On target- off tumor effects
  • Problems with CAR-T cell trafficking and tumor infiltration
  • Immunosuppressive microenvironment
• Follow up
  • Number of CAR-T cells can be monitored using flow cytometry or quantitative PCR for inserted gene.

Antigens which are being used as targets:

• CD19- (Tisagenlecleucel)- B- Acute lymphoblastic leukemia 
• CD 19 – (Axicabtagene ciloleucel, lisocabtagene maraleucel))- Large B cell lymphoma
• CD 38-  (idecabtagene vicleucel)- Multiple myeloma
• B Cell Maturation Antigen- BCMA- (bb2121)- Multiple myeloma
• CD20- (Brexucabtagene autoleucel) – Mantle cell lymphoma
• CD137- CLL 
• CD133 and CD33- AML
• CD30- Hodgkin lymphoma
• P21RAS- Acute leukemia
• BCR-ABL- CML
• PML/RAR- APML
• CD22- B Cell tumors
• EBV-LMP1- Hodgkin's lymphoma
• WT1- Acute leukemia

Companies producing CAR-T cells at present in India

• Immuneel (Bangalore)
• ImmunoACT (Mumbai)
• Aurigene (Hyderabad)
• Intas (Ahmedabad).

Mesenchymal stromal cells

• Spindle shaped cells which can differentiate into variety of mesenchymal cells
• Criteria to denote as MSCs
  • Plastic adherent when maintained in standard culture conditions
  • Must express CD105, CD73, CD90
  • Must lack- CD45, CD34, CD14, CD11b, CD79, CD19 and HLA-DR
• Sources: Bone marrow, adipose tissue, umbilical cord blood, placenta
• They lack HLA class II molecules, hence do not elicit immunological response.
• Life span- 6-9 months in recipient
• Inhibit proliferation of allogenic lymphocytes and B cells. Hence are useful in treatment of GVHD.
• They foster engraftment of stem cells. Hence can be used if CD34 count is less.
• Complication of  MSC therapy
  • Ectopic tissue formation- Bone, cartilage, adipose tissue etc.
  • Malignant transformation- Due to remarkable replicative capacity

Recent advances:

CD19 CAR T-Cell Therapy in Autoimmune Disease

In this study, 15 patients with severe autoimmune diseases, including systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis, received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy was evaluated up to 2 years after CAR T-cell infusion using various criteria for remission and disease activity. All patients achieved significant clinical responses, with SLE patients achieving remission according to DORIS criteria, myositis patients showing major clinical responses, and systemic sclerosis patients experiencing a decrease in disease activity index scores. Immunosuppressive therapy was discontinued in all patients, and adverse events, including cytokine release syndrome and infections, were generally manageable. 

https://doi.org/10.1056/NEJMoa2308917

Sequential CD7 CAR T-Cell Therapy and Allogeneic HSCT without GVHD Prophylaxis

This study investigates a novel treatment approach for relapsed or refractory CD7-positive leukemia or lymphoma. Ten patients received sequential CD7 chimeric antigen receptor (CAR) T-cell therapy followed by haploidentical hematopoietic stem-cell transplantation (HSCT). While one patient died from complications, the remaining patients achieved remission, with some experiencing grade 2 HSCT-associated acute GVHD. Overall, the sequential therapy was deemed safe and effective, offering promise as a treatment option for patients ineligible for conventional allogeneic HSCT.

https://doi.org/10.1056/NEJMoa2313812

Risk of Second Tumors and T-Cell Lymphoma after CAR T-Cell Therapy

A study reviewed 724 patients who received CAR T-cell therapy at a single institution since 2016 to assess the occurrence of second tumors. One patient developed a lethal T-cell lymphoma following axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma. The secondary T-cell lymphoma and initial B-cell lymphoma had distinct molecular and genomic profiles but were both Epstein–Barr virus-positive and linked to DNMT3A and TET2 mutant clonal hematopoiesis. No oncogenic retroviral integration was detected. 

https://doi.org/10.1056/NEJMoa2401361

CAR T-cell infusion for large B-cell lymphoma in complete remission

This study evaluated the outcomes of patients with relapsed or refractory large B-cell lymphomas (LBCL) who were in complete response (CR) before CD19 CAR T-cell (CAR-T) therapy. Among 134 patients from the CIBMTR registry with a median of 3 prior lines of therapy, the 2-year progression-free survival was 43.5%, and overall survival was 63.8%. Non-relapse mortality and relapse/progression rates at 2 years were 9.2% and 47.3%, respectively. The rates of severe cytokine release syndrome (CRS) and neurotoxicity (ICANS) were low at 2.2% and 8.2%. 

https://doi.org/10.1038/s41375-024-02242-6

Tocilizumab administration in cytokine release syndrome is associated with hypofibrinogenemia after CAR T-cell therapy for hematologic malignancies

In a study of 41 adults undergoing CAR T-cell therapy, hypofibrinogenemia was observed from CRS grade 1, with severe hypofibrinogenemia linked to severe CRS (≥ grade 3). Tocilizumab, used to manage CRS, was found to significantly increase the risk of hypofibrinogenemia, as it inhibits fibrinogen synthesis in response to CRS. This suggests that fibrinogen levels initially rise due to an interleukin-6-driven acute phase reaction but drop when tocilizumab is administered. These findings highlight the importance of monitoring fibrinogen levels closely in patients receiving tocilizumab for CRS.

https://doi.org/10.3324/haematol.2023.284564

Five-Year Follow-Up of Standard-of-Care Axicabtagene Ciloleucel for Large B-Cell Lymphoma: Results From the US Lymphoma CAR T Consortium

This study evaluated long-term outcomes of axicabtagene ciloleucel (axi-cel) therapy for relapsed/refractory large B-cell lymphoma. At 5 years, progression-free survival was 29%, and overall survival was 40%. Non-relapse mortality (NRM) reached 16.2%, with higher rates in patients over 60 due to infections and secondary malignant neoplasms (SMNs). SMNs were seen in 9% of patients, including therapy-related myeloid neoplasms and solid tumors. While axi-cel showed durable responses, late infections and SMNs remain significant survivorship concerns, particularly in older patients.

https://doi.org/10.1200/JCO.23.02786

BCMA-Targeted T-Cell–Engager Therapy for Autoimmune Disease

Teclistamab, a BCMA-targeted T-cell engager, was tested in four patients with severe autoimmune diseases resistant to multiple treatments. It demonstrated good safety with mild cytokine release syndrome and led to significant clinical improvements, including reduced disease activity and autoantibody levels. Teclistamab effectively depleted circulating B cells and plasma cells, suggesting its potential in treating autoimmune conditions where traditional therapies fail. 

https://doi.org/10.1056/NEJMc2408786

Manufacturing of primary CAR-NK cells in an automated system for the treatment of acute myeloid leukemia

The study focuses on the potential of natural killer (NK) cells, with their inherent killing capacity against acute myeloid leukemia (AML), as a treatment option. It addresses the challenge of generating chimeric antigen receptor (CAR)-modified NK cells, typically difficult and time-consuming, by using an automated system, the CliniMACS Prodigy® platform. The study demonstrates successful generation of high numbers of CD33-targeting CAR-NK cells using this platform, which exhibit similar phenotype and cytotoxicity to manually produced CAR-NK cells. 

https://doi.org/10.1038/s41409-023-02180-4

HA-1–targeted T-cell receptor T-cell therapy for recurrent leukemia after hematopoietic stem cell transplantation

A phase 1 clinical trial investigated T cell receptor (TCR)-engineered T cells targeting the hematopoietic-restricted minor histocompatibility antigen, HA-1, for treating or consolidating the treatment of recurrent or persistent leukemia and myeloid neoplasms after allogeneic hematopoietic stem cell transplantation (HCT). The trial assessed the feasibility and safety of HA-1 TCR-T cell administration. HA-1 TCR-T cells were successfully manufactured and administered to 9 HCT recipients following lymphodepleting chemotherapy. The infused T cells expanded and persisted in vivo, with no dose-limiting toxicities observed. While the study was not designed to evaluate efficacy, 4 patients achieved or maintained complete remissions, with one remaining in remission for over 2 years. Single-cell RNA sequencing of relapsed leukemia post-therapy revealed upregulated molecules linked to T-cell dysfunction and cancer survival. The results suggest HA-1 TCR-T therapy is feasible, safe, and shows initial efficacy signals.

https://doi.org/10.1182/blood.2024024105