1. Maude et al. BLOOD, 25 JUNE 2015 x VOLUME 125, NUMBER 26

2. Objectives
Review B and T cell receptors and rationale behind the CAR T cell
Review the engineering of CARs
Discuss the clinical results of CARs in hematology malignancy
Complications and future directions of CAR T cells

6. Why CAR’s? Best of both worlds of the immune system B cell specificity
T cell cytotoxicity without presentation
Form of Adoptive T cell therapy
Synthetically engineered receptors designed to overcome immune tolerance / tumor evasion
Targets surface molecules in their native confirmation
Engage target independent of antigen presenting cell (APC) and MHC complex

7. Immune Evasion/Tolerance
Tumors decrease T cell response
Down regulate MHC I, II
Impair Antigen processing
Down regulation of Co-stimulatory molecules (CD28), Increase PD1 , Increase T regulatory cells
Tolerance
Unresponsiveness to Ag+ despite exposure to lymphocytes (Anergy)

8. NEED TO T CELL DEPLETE BEFORE YOU ADD NEW T CELLS CARS to decrease antigen Load, decrease tumor burden
conditioning regmines Cy/Flu then CAR T cells

10. (ASH 2014, Abstract 382, 2014).

12. Maus et al. Blood 2013 First Gen leads to anergy
Second and Third increase replication and proliferation of T cells
Unclear which type to co-stimulatory molecules are superior
Maus et al. Blood 2013

13. Emphasize the co-stimulatory molecules importance

14. Ideal CAR Target… Tumor specific
Universally expressed on only tumor cells
Cell surface molecule
CD 19
Found on B cell malignant cells (NHL, CLL, ALL, etc)
Expressed on early B cells but NOT stem cells

15. Complications of CAR T cells
Cytokine release syndrome (CRS)
Typically within 5 days and CRP best predictor
Exponential T cell proliferation leads to IL2, IL6, IFN
Can lead to macrophage activation syndrome and shock / organ failure
Treated with IL6 monoclonal antibodies (Tocilizumab)
Steroids are second line
IFNy activates macrophages  MAH
Steroids are lymphotoxic
Maude et al. Blood May 2015

16. Complications of CAR T cells
B Cell aplasia
Immunoglobulin replacement required to keep Ig > 500
Encephalopathy
6/30 patients in CTL019 ALL study
Unclear pathogenesis
Self limiting
No long term complications
CAR T cells in CSF in all patients
IFNy activates macrophages  MAH
Steroids are lymphotoxic
Maude et al. Blood May 2015

17. Clinical Data – Hematological Malignancy
ALL
CLL
Chemo-refractory lymphoma

20. Event-free survival and overall survival did not differ significantly between the patients who had previously undergone stem-cell transplantation and those who had not under- gone stem-cell transplantation (P=0.21 for event- free survival and P=0.24 for overall survival)
Maude et al. NEJM 2014

21. Results / Complications
27/30 had CR (90%)
6 month EFS 67%
6 month OS 78%
All patients had Cytokine release syndrome
27% had severe CRS
13 patients had neurologic deficits ranging from delirium to encephalopathy
6 patients had severe aphasia, confusion, delirium, seizures and hallucinations
B cell aplasia
No circulating B cells by flow cytometry in all patients that persisted for up to one year
Maude et al. NEJM 2014

24. omplete remissions (CRs) of chemotherapy-refractory large-cell lympho- mas in patients receiving anti-CD19 chimeric antigen receptor T cells. (A) Positron emission tomography (PET)/computed tomography (CT) scans show CR of chemotherapy- refractory primary mediastinal B-cell lym- phoma (PMBCL) in patient No. 2. (B) PET/CT scans demonstrate CR of lymphoma in patient No. 8 who had chemotherapy- refractory PMBCL with extensive liver in- volvement. (C) PET/CT images show CR of diffuse large B-cell lymphoma, not other- wise specified, in patient No. 14, who had extensive splenic lymphom
Kochenderfer et al. JCO 2015

25. Results DLBCL (n=7) PMBCL (n=4) CLL (n=4) 4 CR 2 PR 1 SD 2 CR
1 Not evaluated
CLL (n=4)
3 CR
1 PR
Kochenderfer et al. JCO 2015

28. Lentiviral vector 1.5 x 105 cells/kg Refractory CLL Pentostatin day -4
lentiviral vector used to infect
T cells from the patient. A pseudotyped, clinical-grade lentiviral vector of vesicular stomatitis virus protein G (pELPs 19-BB-z) directing expression of anti-CD19 scFv derived from FMC63 murine monoclonal antibody, human CD8α hinge and transmembrane domain, and human 4-1BB and CD3ζ signaling domains was produced. Details of the CAR19 transgene, at the bottom of the panel, show the major functional elements. The figure is not to scale. 3′LTR denotes 3′ long terminal repeat; 5′LTR, 5′ long terminal repeat; Amp R, ampicil- lin resistance gene; Bovine GH Poly A, bovine growth hormone with polyadenylation tail; cPPT/CTS, central polypurine tract with central termination sequence; EF-1α, elongation factor 1-alpha; env, envelope; gag, group-specific antigen; pol, HIV gene encoding poly- merase and reverse transcriptase; R, repeat; RRE, rev response element; scFv, single-chain variable frag- ment; TM, transmembrane; and WPRE, woodchuck hepatitis virus post-transcriptional regulatory element
Porter et al. NEJM 2011

29. C shows bone marrow–biopsy specimens ob- tained 3 days after chemotherapy (day –1, before CART19-cell infusion) and 23 days and 6 months after CART19-cell infusion (hematoxylin and eosin). The baseline specimen shows hypercellular bone marrow (60%) with trilineage hematopoiesis, infiltrated by pre- dominantly interstitial aggregates of small, mature lymphocytes that account for 40% of total cellularity. The specimen obtained on day 23 shows residual lym- phoid aggregates (10%) that were negative for chronic lymphoid leukemia (CLL), with a mixture of T cells and CD5-negative B cells. The specimen obtained 6 months after infusion shows trilineage hematopoiesis, without lymphoid aggregates and continued absence of CLL. Panel D shows contrast-enhanced CT scans obtained before the patient was enrolled in the study and 31 days and 104 days after the first infusion. The preinfusion CT scan reveals 1-to-3-cm bilateral masses. Regression of axillary lymphadenopathy occurred within 1 month after infusion and was sustained. Arrows highlight vari- ous enlarged lymph nodes before therapy and lymph- node responses on comparable CT scans after therapy
Porter et al. NEJM 2011

31. Maus et al. Blood 2015

32. Challenges of CAR T cells
Feasibility ($, Institution, etc.)
Training physicians
Relapse of CD19+ or CD19- cells
Defining best CAR
retrovirus vs. lentivirus
CD28 or 4-IBB as co-stimulatory molecule
Lentiviruses can deliver a significant amount of viral RNA into the DNA of the host cell and have the unique ability among retroviruses of being able to infect non-dividing cells, so they are one of the most efficient methods of a gene delivery vector.