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

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

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

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)

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

(ASH 2014, Abstract 382, 2014).

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

Emphasize the co-stimulatory molecules importance

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

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

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

Clinical Data – Hematological Malignancy ALL CLL Chemo-refractory lymphoma

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

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

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

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

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

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

Maus et al. Blood 2015

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.