Marta Castroviejo Bermejo Universidad Autónoma de ...€¦ · 3'LTR (truncated) CD19 BBζ EF-1α RRE Truncated env cPPT/CTS Truncated gag/pol 5'LTR R region pELPS 19-BB-ζ 11556

CART CELLS: A PROMISING IMMUNOTHERAPY FOR CHRONIC LYMPHOCYTIC LEUKEMIA

Marta Castroviejo Bermejo Universidad Autónoma de Barcelona. Bellaterra, 2014

REFERENCES: [1] Chen S, Mellman I. Oncology Meets Immunology: The Cancer-‐Immunity cycle. Immunity 2013; 39: 1-‐10. [2] Kindt TJ, Goldsby RA, Osborne BA. Inmunología de Kuby. 6ª ed. México: McGraw Hill, 2007: 525-‐545. [3] Ruella M, Kalos M. AdopUve Immunotherapy for Cancer. Immunological Reviews, 2013 [4] Rozovski U, Hazan-‐Havely I, KeaUng M, Estrov Z. Personalized medicine in CLL: Current status and future perspecUves. Cancer Le4ers, 2013. [5] Husebekk A, Fellowes V, Read EJ, Williams J, Petrus MJ, Gress RE, Fowler DH. SelecUon and expansion of T cells from untreated paUents with CLL: source of cells for immune reconsUtuUon? Cytotherapy, 2000. 2:187-‐193. [6] Barret DM, Singh N, Porter DL, Grupp SA, June CH. Chimeric AnUgen Receptor Therapy for Cancer. Annual Review of Medicine, 2014. 65: 10.1-‐10.15. [7] Liu L, Sun M, Wang Z. AdopUve T-‐cell therapy of B-‐cell malignancies: convenUonal and physiological chimeric anUgen receptors. Cancer Le4ers, 2013. 316: 1-‐5. [8] Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric AnUgen Receptor-‐Modified T Cells in Chronic Lymphoid Leukemia. The New England Journal of Medicine, 2011. 365: 725-‐733.

There are clear evidences about the essential role of the immune system in the control of cancer. An effective response needs some steps collectively known as “Cancer-Immunity Cycle”. However, tumors have immune system evasion mechanisms that can break this cycle. Thus, it is interesting to think about the possibility of act on the immune system to develop an effective anticancer response. In cancer treatment, immunotherapy is a promising field that has undergone a renaissance in recent years. Chronic lymphocytic leukemia (CLL) is the most common hematologic disease in Western Hemisphere and remains incurable, like most hematological malignancies. This leukemia is characterized by gradual accumulation of mature B-lymphocytes that co-express CD5, CD23 and CD19. CD19 is an exclusive antigen of B cells, which is an important advantage for therapy.

Immunotherapy for cancer has undergone a renaissance in recent years. Genetic modification can provide to lymphocytes specificity against tumors and thus overcome evasion mechanisms. The usage of CART-19 cells is a promising example with many encouraging results. Although there is much room for improvement, the immunotherapy based on engineered T cells can be a big part of the future for cancer treatment.

Cancer antigenpresentation

Priming and activation(APCs - T cells)

Trafficking of T cellsto the tumor

Infiltration of T cells into the tumors

Recognition of cancercells by T cells

Killing of cancer cells

Release of cancer antigens(cancer cell death)

Lymph node

Bloodvessel

Tumor

Figure 1: Cancer Immunity cycle. The cycle can be divided into seven steps, starting with the release of antigens from the cancer cell and ending with the killing of cancer cells. Abbreviations: APC, antigen presenting cells; CTLs, cytotoxic T lymphocytes. Adapted from reference [1].

ZAP70 ZAP70

CD3ς

CD CD28 28

VL

VH

VL

VH

3rd generation

Ligand binding domainScFv

Signaling and co-stimulation domains

CD3ς

41BB 41BB

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VL

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VL

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ZAP70 ZAP70

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VL

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CD3ς CD3ς

2nd generation1st generation

CARs have a single-chain antibody fragment (scFv), expressed in tandem with signaling elements derived from the T cell receptor (essentially CD3ζ) and co-stimulatory domains such as 4-IBB and CD28.

CAR therapy is similar to an autologous bone marrow transplantation procedure. T cells are collected from the patient, cultured and genetically modified using lentiviral vectors. During this process, the patient receives lymphocyte-depleting chemotherapy in order to create an environment that supports the homeostatic expansion of T-cells and improves their effector function. Then, CART-19 cells are infused to the patient. These cells will recognize CD19+ tumor cells (without the need for antigen presentation by HLA), and kill them. In culture of T cells, CD28, CD137 (4-IBB) and CD3 co-stimulation can improve the replicative capacity.

CART19 cells eradicate tumor

Adoptive cell transfer (ACT)

therapy

Anti-CD3

4-IBBL

Anti-CD28

CD3

4-IBB

CD28

BEAD

T CELL

•  Optimal tumor antigens and factors associated with expansion and persistence in vivo. •  Non-viral gene transfer technologies è minimal T cells manipulation ex vivo. •  Combinatorial strategies: ACT and agents that impact on tumor biology è high response •  Issues about scale, automation, commercialization and intellectual property.

ê Cellular therapy, still immature, will revolutionize cancer treatment.

INTRODUCTION

IMMUNOTHERAPY: ADOPTIVE CELL TRANSFER WITH CART-19 CELLS

CONCLUSIONS

Figure 2: Schematic structure of chimeric antigen receptors (CARs). The structures of first-, second- and third-generation CARs are shown.

Figure 3: (A) Scheme of ACT procedure. (B) Scheme of lentiviral vector. Abbreviations: sinLTR, self-inactivating long terminal repeat; RRE, rev responsive element; P, promoter; ψ, packaging signal.

Figure 4: Scheme of artificial antigen presenting cells (AAPCs) or beads for T-cell stimulation in culture.

CLINICAL TRIALS USING CART-CELLS

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T h e n e w e ngl a nd j o u r na l o f m e dic i n e

n engl j med 365;8 nejm.org august 25, 2011730

an approach that may have some advantages over the use of retroviral vectors.12

In previous trials of chimeric antigen receptor T cells, objective tumor responses have been mod-est, and in vivo proliferation of modified cells has not been sustained.13-15 We developed a second-generation chimeric antigen receptor designed to

address this limitation by incorporation of the CD137 (4-1BB) signaling domain, on the basis of our preclinical observation that this molecule promoted the persistence of antigen-specific and antigen-nonspecific chimeric antigen receptor T-cells.5,6 Brentjens and colleagues reported pre-liminary results of a clinical trial of CD19-targeted

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TNF-α Interleukin-6 Interferon-γ

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The New England Journal of Medicine Downloaded from nejm.org at CRAI UNIVERSITAT DE BARCELONA on June 5, 2012. For personal use only. No other uses without permission.

Copyright © 2011 Massachusetts Medical Society. All rights reserved.



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A Lentiviral Vector B Serum Creatinine, Uric Acid, and LDH

C Bone Marrow–Biopsy Specimens

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Figure 3. Expansion and Persistence of Chimeric Antigen Receptor T Cells In Vivo.

Genomic DNA (gDNA) was isolated from samples of the patient’s whole blood (Panel A) and bone marrow aspirates (Panel B) collected at serial time points before and after chimeric antigen receptor T-cell infusion and used for quantitative real-time polymerase-chain-reaction (PCR) analysis. As assessed on the basis of transgenic DNA and the percentage of lymphocytes expressing CAR19, the chimeric antigen receptor T cells expanded to levels that were more than 1000 times as high as initial engraftment levels in the peripheral blood and bone marrow. Peak levels of chimeric antigen receptor T cells were temporally correlated with the tumor lysis syndrome. A blood sample ob-tained on day 0 and a bone marrow sample obtained on day −1 had no PCR signal at baseline. Flow-cytometric analysis of bone marrow aspirates at baseline (Panel C) shows predominant infiltration with CD19+CD5+ cells that were clonal, as assessed by means of immu-noglobulin kappa light-chain staining, with a paucity of T cells. On day 31 after infusion, CD5+ T cells were present, and no normal or malignant B cells were detected. The numbers indicate the relative frequency of cells in each quadrant. Both the x axis and the y axis show a log10 scale. The gating strategy involved an initial gating on CD19+ and CD5+ cells in the boxes on the left, and the subsequent identification of immunoglobulin kappa and lambda expression on the CD19+CD5+ subset (boxes on the right).





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NEGATIVE ASPECTS

Figure 6A: Serum creaUnine, uric acid, and lactate dehydrogenase (LDH) levels from day 1 to day 28 ajer the CART-‐19 cell infusion. Reference [8].

Figure 6B: Flow-‐cytometric analysis of bone marrow aspirates on day 31 ajer infusion shows CD5+ T cells were present, and no normal or malignant B cells were detected.. Reference [8].

POSITIVE ASPECTS

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Amp R


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1 Mo of Treatment

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0.4 94.8

1.33.5



0.2 0.3

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A"

B"

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/Cy5.5

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"

Evidence of immune response: Cytokine increase + CART cells infiltration Specific immune response against CD19 è Leukemia cells elimination

Figure 5: (A) InducUon of the immune response in bone marrow. The cytokines TNF-‐α, interleukin-‐6, interferon-‐γ, chemokine CXCL9, and soluble interleukin-‐2 receptor were measured in supernatant fluids of marrow aspirates at various days before and ajer CART-‐19 cell infusion. The increases in levels of interleukin-‐6, interferon-‐γ, CXCL9, and soluble interleukin-‐2 receptor coincided with the tumor lysis syndrome (5a), peak chimeric anUgen receptor T-‐cell infiltraUon, and eradicaUon of leukemic infiltrate (B). (B) Bone marrow biopsy specimens for 3 days ajer chemotherapy and 23 days and 6 months ajer CART19-‐cell infusion (hematoxylin and eosin). Reference [8].

CAR

Culture and ex vivo genetic modification

of T cells

Cancer cell

CD19

PerforinGranzymes

CART-19 cell

FUTURE DIRECTIONS

CHIMERIC ANTIGEN RECEPTOR (CAR)

Total white cells

Lymphocyte-depletingchemotherapy

T cellsExpansion

Beads or AAPCs

CART-19 cells

Ex vivo cell processing

Transduction with lentivirus vector encoding CAR gene

VL VHCo-stimulatory domainT-cell activation domain

Hinge and transmembrane

region

sinLTRP

sinLTR

RREΨ

A

B

Documents

Marta Castroviejo Bermejo Universidad Autónoma de ...€¦ · 3'LTR (truncated) CD19 BBζ EF-1α RRE Truncated env cPPT/CTS Truncated gag/pol 5'LTR R region pELPS 19-BB-ζ 11556