Adoptive cellular therapy for chronic lymphocytic leukemia and B cell malignancies. CARs and more

Accepted Manuscript Adoptive cellular therapy for chronic lymphocytic leukemia and b cell malignancies. Cars and more Januario E. Castro, M.D., Thomas J. Kipps, M.D., Ph.D. PII:

S1521-6926(16)30013-5

DOI:

10.1016/j.beha.2016.08.011

Reference:

YBEHA 929

To appear in:

Best Practice & Research Clinical Haematology

Received Date: 8 August 2016 Accepted Date: 8 August 2016

Please cite this article as: Castro JE, Kipps TJ, Adoptive cellular therapy for chronic lymphocytic leukemia and b cell malignancies. Cars and more, Best Practice & Research Clinical Haematology (2016), doi: 10.1016/j.beha.2016.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ADOPTIVE CELLULAR THERAPY FOR CHRONIC LYMPHOCYTIC LEUKEMIA AND

1 Moores

UCSD Cancer Center, University of California,

San Diego, La Jolla, CA, USA. Research Consortium

Corresponding Author:

EP

Januario E. Castro, M.D.

TE D

2CLL

AC C

Moores UCSD Cancer Center

University of California, San Diego 3855 Health Sciences Drive La Jolla, CA 92093-0820

Phone: +1 (858) 822-6600 1

SC

M AN U

Januario E. Castro, M.D.1,2, Thomas J. Kipps, M.D., Ph.D. 1,2

RI PT

B CELL MALIGNANCIES. CARS AND MORE

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Email: [email protected]

2

ACCEPTED MANUSCRIPT

A. ABSTRACT

RI PT

Treatment of patients with chronic lymphocytic leukemia and other B cell malignancies is evolving very rapidly. We have observed the rapid transition during the last couple of years, from chemo-immunotherapy based treatments to oral targeted therapies based on B cell receptor signaling and Bcl-2 inhibitors and the use of second generation

SC

glyco-engineered antibodies.

M AN U

The next wave of revolution in treatment for this conditions is approaching and it will be based on strategies that harness the power of the immune system to fight cancer. In the center of this biotechnological revolution is cellular engineering, the field that had made possible to redirect the immune system effector cells to achieve a more effective and targeted adoptive cellular therapy.

TE D

In this chapter we will review the historical context of these scientific developments, the most recent basic and clinical research in the field and some opinions regarding the

EP

future of adoptive cellular therapy in CLL and B cell malignancies.

AC C

A. KEYWORDS: CLL, B cell malignancy, adoptive cellular therapy, cellular engineering.

3

ACCEPTED MANUSCRIPT

A. INTRODUCTION: Adoptive cellular therapy for cancer and other diseases is rapidly becoming one of

RI PT

the most actively studied and awaited treatment alternatives. Traditionally, transfer of allogeneic cells and the replacement of the immune system using allogeneic hematopoietic stem cell transplant (HSCT) has been the only therapy that has proven to be curative for advance hematological malignancies including relapse highlights the remarkable

SC

refractory leukemia and lymphoma. This clinical observation

power that the immunological graft has against the tumor cells, the so called “graft vs.

M AN U

tumor effect”.[1]

However, allogeneic-HSCT is a procedure that entitles significant risk of adverse events including infection, graft versus host disease (GVHD) and death. Hence, the search for a more specific, and hopefully, less toxic adoptive cellular therapy approach using

TE D

immune system cells that are redirected towards the target of interest. This is accomplished by harnessing modern molecular biology and cellular engineering techniques.

In this

chapter we highlight the most recent and relevant basic science and clinical trials using

EP

adoptive cellular therapy, specifically chimeric antigen receptor (CAR) expressing cells.

AC C

A. A BRIEF HISTORY OF CARS Cancer can be defined as an immunodeficiency disease and the ubiquitous presence

of cancer in humans is a testament to the surveillance failure of our immune system. Therefore, strategies that repair such immunodeficiency should in fact be the premiere options for curative therapy in cancer. 4

ACCEPTED MANUSCRIPT

One of the initial attempts to unravel the power of the immune system against cancer came from the studies performed by William Bradley Coley in the 1890s while working at the New York Cancer Hospital (now Memorial Sloan-Kettering Cancer Center).

RI PT

He used a pool or dead bacteria to elicit an immune inflammatory reaction in cancer patients. Surprising some of his patients responded with cancer remission supporting the notion that activation of the immune system could offer a promising therapeutic option in

SC

cancer.[2]

The first formal description of Adoptive T-cell transfer was made by Billingham et The term was used to highlight the potential of this strategy to overcome the need

M AN U

al.[3]

for antigen driven activation and expansion of T cells. Additional evidence came in the late 1960s when the first non-twin allo-HSCT was performed opening the clinical era of adoptive cell therapies.[4] Since then, we learned to recognize the “good” and “bad” properties of the immune system, from unparalleled anti-cancer effect to the potential

TE D

lethal consequences of GVHD. Precisely, this lack of “immunological accuracy” seeing in alloHSCT prompted the search for more targeted and redirected strategies using the adoptive transfer of engineered immune cells.

EP

In the late 1980 and 1990 seminal studies demonstrated the feasibility of

AC C

redirecting the T cells and inducing cellular activation and expansion independently of HLA and T-cell receptor (TCR) engagement. Gross and Goverman independently described [5,6] the concept of introducing genetic material into a T cell with the goal to express antibodies with specific recognition patterns. Additional efforts demonstrated that chimeric receptors

5

ACCEPTED MANUSCRIPT

encoding surface molecules linked to signaling domains had the ability to replicate the function of the TCR. [7,8] The first results of human clinical trials using chimeric antigen receptor T cells

RI PT

(CAR-T cell) technology were conducted from 1997-2000 in patients with HIV infection and provided the prove of concept and expansion platform to other diseases such as cancer. [9,10] The initial cancer specific CAR studies were reported in 2006 and described the use

SC

of genetically modified T cells in renal cell carcinoma and ovarian cancer [11,12]. Following these seminal publications there has been a flow of studies in the field that have set up the

M AN U

stage for the rapid translation from the bench to the bed side and the likelihood of an FDA approval for CAR-T cell. [13-18] Finally, after decades of broken promises, immunotherapy for cancer entered main street achieving the status of 2013 "breakthrough of the year" by Science.[19]

TE D

Toxicities are one of the main limiting factors for wide applicability of CAR-T cell medicated immunotherapy. Those adverse events include neurotoxicity and cytokine release syndrome (CRS). The pathogenesis of those are not fully understood but significant effort has been devoted to describe the clinical course of these complications and

EP

management options with the goal to enhance the safety of the treated patients. [20-22]

AC C

Several questions remain to be elucidated. For example, the definition of anti-tumor specificity requirements, diversity of antigens that need to be targeted (one or more in the same tumor), how to control bystander toxicity in healthy tissues / organs, what is the best source of the effector transferred T cells – autologous, allogeneic, T cells from peripheral blood or tumor-infiltrating lymphocytes (TILs). What is better to use TCR or single chain 6

ACCEPTED MANUSCRIPT

antibodies to redirect CARs.

The list can be more comprehensive, never the less, resolving

these questions will be critical for the development of adoptive cellular therapy. Despite these significant obstacles the use of adoptive cell immunotherapy in cancer

RI PT

represents a real opportunity to treat patients with refractory disease to conventional therapies. It is very encouraging that initial clinical evidence suggests that in some patients CAR-T cell mediated therapy can induce prolonged remissions, whether or not this

SC

translates into a cure will need to be assessed in larger clinical trials.[22-25]

M AN U

A. THE CAR FACTORY

Conceptually, the CAR design allows the gene-modified cell (T cell, NK cell or other effector immune cells) to acquire a new target specificity. Additional features include builtin stimulation signals such as costimulatory molecules, cytokine production, or cell activation modulatory signals [26,27].

TE D

Genetically engineered immunoreceptors used in CARs have minimum five elements: (1) Target-binding domain composed typically of a polypeptide sequence of the light and heavy chains from a single chain antibody (scFV), this provides specificity and is

EP

responsible to redirect the immune cell. Other receptor–ligand molecules can replace this domain provided that they have sufficient affinity.

Examples of those include, protein-

AC C

protein affinity constructs designed using HIV gp120 binding CD4: ξ [28], cytokine-cytokine receptor (e.g., IL3-IL-13Rα),[29] receptor-ligand constructs (e.g., CD27-CD70),[30] or a pattern-recognition receptor such as Dectin-1 for targeting foreign carbohydrates such as βglucan on Aspergillus; [31] (2) Hinge domain that provides flexibility to the target-binding domain allowing better affinity, (3) Transmembrane domain, (4) A primary signaling 7

ACCEPTED MANUSCRIPT

domain typically from the T-cell receptor (ξ chain) and (5) Additional costimulatory domains that stabilize and amplify the activation signal, enhance proliferation and persistence (examples of those are CD27, CD28, CD134 - OX40, CD137- 41BB, CD244, or

RI PT

ICOS).

The cytoplasmic activation, in turn, induces cytokine secretion (including IL-2 and others), proliferation and expansion of CAR T cells, which results in cytotoxicity of the

SC

target cell. Use of target-binding domain rather than typical TCR binding components confers several advantages, affinity and avidity are much higher, CARs recognition is not

M AN U

TCR and MHC-restricted. Therefore, it is not susceptible to tumor escape mechanisms such as HLA loss or altered antigen processing mechanisms.[32] However, the main disadvantage of CARs is the inability to recognize intracellular molecules.

One alternative

to bypass this obstacle is to engineer target-binding domains that recognize peptides / antigens presented at the MHC groove. A prove of concept of this strategy has been shown

TE D

using antibodies that exclusively recognize MHC presented peptides derived from the intracellular protein WT1.[33,34]

EP

A. CARS & TRUCKS , LOOKING FOR THE PERFECT MODEL The so called first generation CARs are those designed with constructs that express

AC C

one stimulatory molecule while those with more than one additional costimulatory molecule are called second and third generation. The incorporation of a single costimulatory molecule in addition to the TCR-ξ chain (second generation CAR) enhances persistence, expansion and other T-cell functions.[27]

In addition to signal 1 and 2, T-cells

typically require a third signal to achieve and sustain full activation. This third signal is 8

ACCEPTED MANUSCRIPT

mediated through the common γ-chain cytokine receptor and a coordinated delivery of certain cytokines activates this receptor enhancing CAR-T cell functions. 18–20 A new version of CARs, “the fourth generation”, responds to the need to modulate

RI PT

the tumor microenvironment in order to overcome tumoral antigen expression heterogeneity and resistance to CAR mediated cytotoxicity. This situation may be overcome by IL-2 or other cytokines that are released by the CAR-T cell to induce changes in the

SC

tumor microenvironment, and enhance the recruitment of other effector cells of the immune system including macrophages, dendritic cells and native T-cells.[35] The CAR-T

M AN U

cell carries this payload that is released upon activation, hence the name TRUCKs (T cells Redirected for Universal Cytokine Killing). TRUCK - T cells could be well applied in fields beyond cancer therapy including viral infections, auto-immune diseases or metabolic disorders.

TE D

A. THE CAR ASSEMBLY LINE AND T CELLS EXPANSION Transfer of the genetic material encoding the CAR into the target cell can be achieved by vectors or by physical / mechanical procedures. The most commonly used

EP

vectors are gamma-retrovirus or lentivirus, which have the ability to integrate into the host cell genome eliciting low immunogenicity that allows a permanent genomic integration and

AC C

expression. Notoriously, lentiviral vectors can integrate into non-dividing cells, are less immunogenic and can deliver larger DNA sequences [36]. Despite concern about insertional mutagenesis following retroviral transduction of hematopoietic stem cells, long-term follow-up has demonstrated the safety of these vectors in T cells [24].

9

ACCEPTED MANUSCRIPT

Examples of non-vector mediated transfer include the transposon/transposase system (Sleeping Beauty)[37], electroporation of plasmids and in vitro transcribed mRNA among others.[38] Compared with non-vector mediated transfer, the disadvantages of viral

requirements for long term follow-up of patients after therapy.

RI PT

approaches are the expense, expertise required for production, and the intense regulatory

Once gene transfer takes place the next challenge is to generate large quantities of

SC

CAR-T cells that meet the rigorous Good Manufacturing Practice (GMP), which are required for infusion into patients. The first step of T cell activation and expansion is achieved using

M AN U

anti-CD3 (OKT3 clone) plus anti-CD28 antibodies, anti-CD3/CD28 beads or artificial antigen-presenting cells.[39-41] The final step requires addition of cytokines such as IL-2, IL-7, IL-12, IL-15, or IL-21.[27,41,42] Typically, the expansion process takes 2-6 weeks. Overall, one big challenge for the applicability of CAR technology is the number of

TE D

variables that need to be adjusted to generate a product with reproducible characteristics. Ultimately, rigorous quality controls are required to monitor closely these biological products manufactured under GMP conditions.

EP

A. CARS ROAD TEST: TRAFFICKING, HOMING AND IN VIVO ACTIVITY After infusion of CAR-T cells they must travel to the tumor site and engage the target

AC C

cancer cells in order to exert their activity. In that quest for their cognate antigen, these engineered cells need to overcome several obstacles that include among others: (1) Follow the appropriate homing signaling via cytokines and chemokines present in the blood; (2) Conquer immunosuppressive signals from the cancer cell and tumor microenvironment

10

ACCEPTED MANUSCRIPT

and; (3) Provide long term persistence and survival in order to maintain effective immunosurveillance against residual cancer cells. Chemotherapy or radiation therapy conditioning regimens prior to infusion of CAR-

RI PT

T cells have been shown to induce host / patient lymphodepletion that is critical for trafficking, homing, engraftment, proliferation and persistence of the infused CAR-T cells. This process is mediated potentially by an inflammatory reaction of lymphoid and cancer

SC

tissues as well as by depleting immune cells that otherwise will complete for resources such as cytokines / chemokines.[26,43-45]

M AN U

CAR-T cell distribution and expansion has been studied using tracking methodologies. For the most part it appears that T cells are distributed throughout highly perfused organs such as the lung, liver and spleen.[46,47] After this, CAR-T cells migrate towards tissues directed primarily by the specificity of the target-binding domain in search

TE D

for their cognate antigen. It is there where biodistribution and off-target effects can generate significant toxicity.[48]

Recruitment of the infused T cell and expansion in the

tumor microenvironment depends on conditions such as microcirculation, expression of the target antigen and the presence of immunosuppressive checkpoint molecules among

EP

others.[49-51] In addition, expansion proliferation and persistence of T cells could also be Those can be more

AC C

impacted by the development of immune based rejection.[52]

notorious when murine scFv molecules are used and they could be mitigated by development of human base constructs with lower immunogenic potential. After initial expansion of the CAR-T cell it is important to maintain tumor

surveillance and persistence of memory type CAR-T cells is fundamental for that purpose. 11

ACCEPTED MANUSCRIPT

Certain cytokines such as IL-7 and IL-15 play important roles in T-cell expansion and persistence of memory T cells without increasing regulatory T-cell numbers.[53]

It is

unknown what is the duration of persistence of CAR-T cells that will provide superior

RI PT

clinical outcome. However, it is important to note that memory CAR-T cells can have a lifespan of many years and long term follow up of patient with HIV and hematological malignancies has shown that those engineered cells can be detected over 10 years after

SC

infusion [15,24].

New emerging technologies using co-transduction / transfection of bispecific target-

M AN U

binding or cytokine domains into the CAR-T cells will potentially improve these steps of trafficking, homing and persistence [54-56].

B. CELLULAR IMMUNOTHERAPY FOR B-CELL LYMPHOMA AND LEUKEMIA In the late 1990s, our team at the University of California San Diego (UCSD)

TE D

conducted the first studies of cellular therapy applied to patients with chronic lymphocytic leukemia (CLL) using intravenous infusions of autologous CLL leukemia cells transduced ex vivo with an adenovirus vector expressing chimeric (mouse/human) CD154 (Ad-CD154)

EP

[57-59]. The goal of those studies was to evaluate immune anti-leukemia responses and clinical outcomes.

AC C

Transduction of CLL B cells with Ad-CD154 induce leukemia cells to express

immune costimulatory molecules, thereby enhancing their capacity to present antigens to autologous T lymphocytes.[59] Eleven patients received a single infusion of autologous CLL cells transduced ex vivo with Ad-CD154.[58]

Nearly all treated patients exhibited

increased serum levels of IL-12 and IFN-γ, enhanced expression of immune costimulatory 12

ACCEPTED MANUSCRIPT

molecules on bystander leukemia cells, increased absolute numbers of blood T cells, and reduced blood leukemia cell counts and lymph node size. After additional infusions of AdCD154–transduced cells, patients showed disease stabilization, with delayed disease

additional therapy 4 years after treatment.[57]

RI PT

progression and the need for further treatment. Two of the treated patients did not require

On subsequent studies we tested an adenovirus vector expressing a membrane CLL (2 previously

SC

stable humanized homolog of CD154 (Ad-ISF35).[60] Patients with

untreated and 7 with relapse / refractory disease) received dose escalation administration

M AN U

of autologous leukemia cells transduced with Ad-ISF35. Similarly to what was observed with Ad-CD154, the infusions were well tolerated and clinical benefit was observed in the majority of patients including subjects with high risk CLL that have 17p deletion.

Years

later, we investigated the administration of Ad-ISF35 via direct intratumoral administration in patients with CLL. 15 patients with CLL received direct intranodal injection of Ad-ISF35

TE D

with a single ultrasound guided injection of 1 to 30 x 1010 Ad-ISF35 viral particles in 4 different dose cohorts. Injections were well-tolerated with some patients developing local swelling, erythema and “flu-like” symptoms. Ad-ISF35 intranodal injection resulted in

EP

significant reductions in blood leukemia cell counts, lymphadenopathy and splenomegaly in the majority of patients.

Although there was no evidence for dissemination of Ad-ISF35

AC C

beyond the injected lymph node, direct intranodal injection of Ad-ISF35 induced peripheral blood CLL cells to express death receptors, pro-apoptotic proteins, and immune costimulatory molecules suggesting the presence of a "bystander" and systemic effect.[61]

13

ACCEPTED MANUSCRIPT

These studies using transduced autologous CLL cells with homologs of CD154 showed the potential to elicit anti-leukemia immune response in CLL patients. Moreover, the anti-leukemia effect was associated with antibody production against a leukemia-

RI PT

associated surface antigen, which we identified as ROR1.[62] ROR1 is an oncofetal surface antigen and survival-signaling receptor that binds Wnt5a inducing activation of NF-kappaB. We conclude that patients treated with Ad-CD154 transduced CLL cells have a significant

other antigens that contribute to leukemia cell survival.

SC

immune stimulation that induced a break in immune tolerance to ROR1 and potentially

M AN U

Our current efforts are focused to develop CAR-T cells that express a scFv against ROR1 with the goal to engineered T cells redirected specifically against the leukemia cells sparing the normal B cell lymphocytes from cytotoxicity.[63] Using the Sleeping Beauty transposons system, we constructed 2nd generation ROR1-specific CARs signaling through CD3ξ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137).

TE D

After transfection, we selected and expanded T cells expressing CARs through co-culture with gamma-irradiated activating and propagating artificial APC cells (AaPC), which coexpressed ROR1 and co-stimulatory molecules. Such T cells produced interferon-gamma

EP

and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. We

AC C

anticipate that current and future clinical trials will help us to investigate the ability of ROR1-CAR-T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire in patients with CLL and other ROR1+ malignancies (NCT02194374).

14

ACCEPTED MANUSCRIPT

The first formal clinical study using CAR-T cells in hematological malignancies was reported in patients with indolent lymphoma that received T cells DNA-electroporated with a construct that expressed an anti-CD20 target-binding domain [13]. Most patient

RI PT

achieve stable disease and toxicities were manageable and cells persisted for 9 weeks. This prove of concept study inspired a rapid development of the field and since 2010 the number of publications has been expanding exponentially (Table 1 – Completed clinical trials).

SC

Different subtypes of B cell malignancies have been treated with CAR-T cells to date, including acute and chronic lymphocytic leukemia as well as low and high grade lymphoma.

M AN U

Collectively, this has enhanced our experience with different genetic transfer methods, construct designs, costimulatory molecules different T cell expansion protocols, cell doses and lymphodepletion regimens [14,43,64-72].

The following are important conclusion from those studies: (A) lymphodepletion is

TE D

critical for engraftment and expansion (Cyclophosphamide with or without fludarabine most commonly used regimen; (B) second generation construct that carry at least one additional costimulatory molecule, are more potent; (C) ALL is probably the most responsive disease; (D) adverse events can be significant and include CRS and

EP

neurotoxicity; (E) There was no dose-response relationship and no correlation of response

AC C

with initial bulk of disease. (F) CAR-T cells can penetrate the CNS and this could be responsible at least in part to the neurotoxicity observed on these patients; (G) CRS is mediated at least in part by macrophage activation; (H) allogeneic donor cells conserve their proliferative and cytotoxic potential and do not appear to induce higher levels of toxicity associated with GVHD [18,25,73,74] . 15

ACCEPTED MANUSCRIPT

Despite the excellent initial response observed in patients with B-ALL after antiCD19 CAR-T cell therapy, it is concerning the observation that some patients relapse with blast that are CD19 negative. This suggest the development of immune surveillance scape

RI PT

and indicates that additional treatment alternatives including treatment with CAR-T cells that target different molecular receptors will be required for complete eradication of residual disease.

SC

To solve this problem of immune scape, other molecular targets have been explored in pre-clinical models for the treatment of B-cell malignancies - CD22, CD23, ROR1.[63,75-

M AN U

77] As expected, there are potential advantages and disadvantages of each model, the level of expression of the target molecule in each pathological subtype, variability of expression within the tumor, expression in healthy cells / tissues, and potential off-target effects would be some of the limiting factors that will affect the development of these strategies.

TE D

A. THE CARS OF THE FUTURE

The technological advances in cellular engineering are moving at a fast pace and more and more new CAR models are been tested in vitro and animal models awaiting the

EP

opportunity to be launched into clinical trials. In the next section we describe some examples of those forefront platforms:

AC C

B. Combinatorial Antigen Recognition CARs: Current T-cell engineering approaches redirect patient T cells to tumors by

transducing them with one antigen-specific receptor. However, using this strategy T cells are transduced with two CARs, one that provides suboptimal activation upon binding of one 16

ACCEPTED MANUSCRIPT

antigen and a chimeric costimulatory receptor (CCR) that recognizes a second antigen. When both CARs are engaged the activation signal is amplified. This increases the specificity of CAR activation and overcome the need for a true tumor-specific antigen expressed in the

PSCA with good result in vitro. [78]

SC

B. Inhibitory Signaling CARs:

RI PT

cell of interest. So far this has been tested using the prostate tumor antigens PSMA and

This technology takes advantage of the negative feedback loops that regulate

M AN U

cellular signaling particularly in T cells. Using this design, two CARs are introduced in the T cell. The first one is the activating chimeric receptor and the second expresses an inhibitory component or iCAR using molecules such as CTLA-4- or PD-1. The activation and expansion of these iCARs are modulated by the balance provided by the activation / inhibitory signal allowing for a safer and potentially less toxic antigen recognition, proliferation and

TE D

cytotoxic effect. iCARs provide a dynamic, self-regulating safety switch that could prevent consequences of inadequate T cell specificity.

EP

B. Off-the-shelf (OTS) - CARs:

This is probably one of the most promising strategies that is currently in OTS–CARs respond to multiple challenges currently posed by the clinical

AC C

development.

expansion and application of adoptive cellular therapy using cellular engineering. One of those is the unparalleled logistics involved in the process of production of the patient’s product, the rigorous quality control involved in the process and probably the most important, the time that this process takes before the cellular product is delivered to the 17

ACCEPTED MANUSCRIPT

patient. The ideal OTS-CAR should meet at least some of the following requirements: A cellular product that can be prepared and cryopreserved in advance. The source of the immune cells ideally should be healthy donors previously screened for certain

RI PT

characteristics. A biological product that is already characterized with lots that are predetermined based on cellular characteristics, activity and other quality control tests. A product that has been produced in a centralized manufacturing facility meeting standards

SC

that allow reproducibility and comparability from batch to batch. A product that is ready to be shipped whenever is needed minimizing waiting time for the patient. A biological from

M AN U

an “universal” donor, which most likely will represent cells manipulated through gene editing techniques to remove unwanted alloreactivity mediated by endogenous TCRs, MHC and / or minor histocompatibility antigens (MHA).

An example of the clinical application of these OTS-CARs was recently reported on a pediatric patient with ALL that was treated on a single patient protocol under

TE D

compassionate use.[79] In this report allogeneic HLA mismatched donor T cells were transduced using a third generation self-inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both

EP

CD34 and CD20 epitopes.

AC C

Alloreactivity and the risk of lethal GVHD was mitigated using Transcription Activator-Like Effector Nucleases (TALEN)s that allowed gene editing of endogenous TCR and CD52 (rendering the cells insensitive to alemtuzumab-anti CD52 antibody, which was used in vivo as conditioning agent). This universal CAR19 (UCART19) cell bank has been

18

ACCEPTED MANUSCRIPT

characterized in detail, including sterility, molecular and cytometric analyses and human/murine functional studies. The patient treated was an infant with B cell ALL refractory after allo-HSCT. As part

RI PT

of the cytoreductive chemotherapy regimen the patient received alemtuzumab prior to infusion of UCART19 cells. The patient tolerated well the T cell infusion without any significant toxicity and no manifestations of CRS.

The patient showed a good response

SC

with molecular complete response, reconstitution of donor chimerism and detectable UCART19 persistence. This example represents the first-in-man application of TALEN

M AN U

engineered cells and provides proof of concept for OTS-CAR T cell applications that currently are undergoing testing in early phase clinical trials (NCT02808442) (Table 2 – Active clinical trials). A. SUMMARY

TE D

The applications of adopted cellular therapy are expanding exponentially bringing exciting therapeutic alternatives to patients that in many opportunities are condemned to succumb to cancer progression.

Moreover, the expectation is that CAR-T cell based

EP

immunotherapy will expand beyond oncology into areas such as infectious diseases, autoimmunity, immune deficiency to name a few.

AC C

The CAR design in itself possesses a big challenge and how to assemble the most

efficient and appropriate CAR for each medical condition will require significant amount of basic research and ultimately evaluation in human subjects.

19

ACCEPTED MANUSCRIPT

Optimization of gene transfer methods and large scale production and expansion of engineered T cells will be required to meet future demands of these new treatments. This will require the development of specialized facilities, implementation and optimization of

RI PT

standard operating procedures and the training of expert technicians in the field. Several pharmaceutical companies had stablished strategic partnerships with academic institutions in an effort to lead this effort.[80]

SC

Selection of the optimal targets suitable for adoptive immunotherapy on each specific disease. It is likely that the development of adoptive immunotherapy will need to

M AN U

parallel the redundancy observed in the normal immune system[81]. Most likely, we will need to engineered cells that express more than one CAR in order to provide that immunological redundancy, or infuse a mixture of engineered T cells with different CAR targets / specificities. Even better, more than one type of effector cell we will require to design the “perfect immunological cocktail” using redirected-engineered T and NK cells,

TE D

macrophages, dendritic cells, etc. Most likely, “One size will not fit all”… and consequently, we will need to develop tailored immune reconstitution protocols based on adoptive cellular therapy for each specific disease.

EP

How to make these therapies available world-wide? Availability of this new

AC C

immunotherapy treatments is going to be limited and initially accessible only to patients in large specialized centers. Broadening the coverage of adoptive cellular therapy will require the development of simplified protocols, use of more effective and safer versions of CARs, and most likely the availability of universal OTS-CARs that can guarantees easier logistics and shorter times for release and shipment of the cellular product. 20

ACCEPTED MANUSCRIPT

Once approved by the FDA, the cost of these novel adoptive cellular therapies will become one of the most important limiting factors for its wider use. The financial aspects of drug cost and coverage will create limitations due to variability in accessibility to health

RI PT

insurance and other social and economic variables of each patient.

What is the preferred source of cells for adoptive immunotherapy? This is another important question. The majority of the studies published so far have been conducted using

SC

autologous cells. This has the advantage that the cells are available readily available and that HLA matching is not required. However, whether those autologous cells from cancer

M AN U

patients are optimal for T-cell engineering is unknown. Many cancer patients are elderly and their immune system may be debilitated due to illness, prior therapy and the inherent biology of their disease.[82] Contrary to that, the prospect of immunologically healthier allogeneic T cells from younger donors appear to be very enticing, particularly when significant barriers regarding HLA matching can be minimized selecting haploidentical or

TE D

mismatch unrelated donor cells that are engineered using genetic editing techniques.[79] Very likely, those genetic editing tools will make possible to render adoptive cellular immunotherapy and OTS tool much more like “regular drugs”, available for immediate

EP

administration whenever they are needed. In fact, OTS cellular therapy could solve some of the major obstacles related to immediacy, logistics and quality consistency required to

AC C

expand in large scale adoptive cellular immunotherapy. Overall, adoptive immunotherapy using CAR-T cells represent a tremendous

advancement toward effective cancer therapy. Patients in desperate need for alternative treatments have seen the benefit of this strategy and that is probably the most important 21

ACCEPTED MANUSCRIPT

accomplishment achieved by CAR-T cells so far. Definitely, the road ahead looks promising for CARs and other adoptive cellular engineering based therapies, the upcoming challenges associated with the use of this new technologies are related to the understanding of what

RI PT

are the optimal ways to use these powerful weapons increasing their efficacy while

AC C

EP

TE D

M AN U

SC

maintaining patient safety.

22

ACCEPTED MANUSCRIPT

A. PRACTICE POINTS •

New developments in Adoptive cellular therapy, particularly cellular engineering

•

RI PT

and CAR-T cells are revolutionizing the way we treat patients with cancer. Additional clinical trials will be required to corroborate the encouraging observations •

CAR-T cells have associated adverse events including neurotoxicity and cytokine

SC

release syndrome and appropriate management of those symptoms is required for

A. RESEARCH AGENDA •

M AN U

the safety of patients.

The design of optimal engineered cells for adoptive cellular therapy will require

•

TE D

significant research and evaluation of different strategies.

Clinical protocols are needed to assess the real potential of “Universal” donor CAR-T

EP

cells using Off-the-shelf products.

AC C

A. CONFLICT OF INTEREST Dr. Kipps declares no relevant conflicts of interest in relation to this manuscript. Dr. Castro receives clinical trial research support from Kite Pharmaceuticals.

23

ACCEPTED MANUSCRIPT

1.

RI PT

A. REFERENCES:

Thomas ED. Allogeneic bone marrow transplantation for blood cell disorders. Birth Defects Orig Artic Ser 1982;18:361-369.

Decker WK, Safdar A. Bioimmunoadjuvants for the treatment of neoplastic and

SC

2.

2009;20:271-281. 3.

M AN U

infectious disease: Coley's legacy revisited. Cytokine Growth Factor Rev

Billingham RE, Brent L, Medawar PB. Quantitative studies on tissue transplantation immunity. II. The origin, strength and duration of actively and adoptively acquired immunity. . Volume 143. Proc. R. Soc. London Ser. B 1954. p 58-80. Buckner CD, Epstein RB, Rudolph RH, et al. Allogeneic marrow engraftment

TE D

4.

following whole body irradiation in a patient with leukemia. Blood 1970;35:741-

5.

EP

750.

Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric

AC C

molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 1989;86:10024-10028.

6.

Goverman J, Gomez SM, Segesman KD, et al. Chimeric immunoglobulin-T cell

receptor proteins form functional receptors: implications for T cell receptor

complex formation and activation. Cell 1990;60:929-939. 25

ACCEPTED MANUSCRIPT

7.

Letourneur F, Klausner RD. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins. Proc Natl Acad Sci U S A 1991;88:8905-

8.

RI PT

8909. Irving BA, Weiss A. The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 1991;64:891-901.

Deeks SG, Wagner B, Anton PA, et al. A phase II randomized study of HIV-specific T-

SC

9.

M AN U

cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Mol Ther 2002;5:788-797. 10.

Mitsuyasu RT, Anton PA, Deeks SG, et al. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood

11.

TE D

2000;96:785-793.

Lamers CH, Sleijfer S, Vulto AG, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase

Kershaw MH, Westwood JA, Parker LL, et al. A phase I study on adoptive

AC C

12.

EP

IX: first clinical experience. J Clin Oncol 2006;24:e20-22.

immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res

2006;12:6106-6115.

26

ACCEPTED MANUSCRIPT

13.

Till BG, Jensen MC, Wang J, et al. Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-

14.

RI PT

specific T cells. Blood 2008;112:2261-2271. Jensen MC, Popplewell L, Cooper LJ, et al. Antitransgene rejection responses

contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans. Biol Blood Marrow

Porter DL, Levine BL, Kalos M, et al. Chimeric antigen receptor-modified T cells in

M AN U

15.

SC

Transplant 2010;16:1245-1256.

chronic lymphoid leukemia. N Engl J Med 2011;365:725-733. 16.

Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19

17.

TE D

chimeric-antigen-receptor-transduced T cells. Blood 2012;119:2709-2720. Brentjens RJ, Riviere I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or

18.

EP

chemotherapy refractory B-cell leukemias. Blood 2011. Davila ML, Riviere I, Wang X, et al. Efficacy and toxicity management of 19-28z CAR

AC C

T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014;6:224ra225.

19.

Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science

2013;342:1432-1433.

27

ACCEPTED MANUSCRIPT

20.

Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368:1509-1518. Teachey DT, Lacey SF, Shaw PA, et al. Identification of Predictive Biomarkers for

RI PT

21.

Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia. Cancer Discov 2016;6:664-679. 22.

Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al. T cells expressing CD19

SC

chimeric antigen receptors for acute lymphoblastic leukaemia in children and young

23.

M AN U

adults: a phase 1 dose-escalation trial. Lancet 2015;385:517-528.

Maude SL, Teachey DT, Porter DL, et al. CD19-targeted chimeric antigen receptor Tcell therapy for acute lymphoblastic leukemia. Blood 2015;125:4017-4023.

24.

Scholler J, Brady TL, Binder-Scholl G, et al. Decade-long safety and function of

TE D

retroviral-modified chimeric antigen receptor T cells. Sci Transl Med 2012;4:132ra153. 25.

Feldman SA, Assadipour Y, Kriley I, et al. Adoptive Cell Therapy--Tumor-Infiltrating

EP

Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors. Semin Oncol 2015;42:626-639.

Gill S, June CH. Going viral: chimeric antigen receptor T-cell therapy for

AC C

26.

hematological malignancies. Immunol Rev 2015;263:68-89.

27.

Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for

human cancer. Science 2015;348:62-68. 28

ACCEPTED MANUSCRIPT

28.

Romeo C, Seed B. Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 1991;64:1037-1046. Brown CE, Starr R, Aguilar B, et al. Stem-like tumor-initiating cells isolated from

RI PT

29.

IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells. Clin Cancer Res 2012;18:2199-2209. 30.

Shaffer DR, Savoldo B, Yi Z, et al. T cells redirected against CD70 for the

Kumaresan PR, Manuri PR, Albert ND, et al. Bioengineering T cells to target

M AN U

31.

SC

immunotherapy of CD70-positive malignancies. Blood 2011;117:4304-4314.

carbohydrate to treat opportunistic fungal infection. Proc Natl Acad Sci U S A 2014;111:10660-10665. 32.

Spranger S. Mechanisms of tumor escape in the context of the T-cell-inflamed and

33.

TE D

the non-T-cell-inflamed tumor microenvironment. Int Immunol 2016;28:383-391. Dao T, Yan S, Veomett N, et al. Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 2013;5:176ra133. Houen G. Peptide Antibodies: Past, Present, and Future. Methods Mol Biol

EP

34.

AC C

2015;1348:1-6. 35.

Chmielewski M, Abken H. TRUCKs: the fourth generation of CARs. Expert Opin Biol

Ther 2015;15:1145-1154.

36.

Oldham RA, Berinstein EM, Medin JA. Lentiviral vectors in cancer immunotherapy.

Immunotherapy 2015;7:271-284. 29

ACCEPTED MANUSCRIPT

37.

Singh H, Moyes JS, Huls MH, et al. Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor. Cancer

38.

RI PT

Gene Ther 2015;22:95-100. Jones CH, Hill A, Chen M, et al. Contemporary approaches for nonviral gene therapy. Discov Med 2015;19:447-454. 39.

Rushworth D, Jena B, Olivares S, et al. Universal artificial antigen presenting cells to

40.

M AN U

specificity. J Immunother 2014;37:204-213.

SC

selectively propagate T cells expressing chimeric antigen receptor independent of

Cooper LJ. Moving from tinkering in the garage to assembly line production: the manufacture of genetically modified T cells expressing chimeric antigen receptors (CARs) comes on line. Cancer Gene Ther 2015;22:64-66.

Levine BL, June CH. Perspective: assembly line immunotherapy. Nature 2013;498:S17.

42.

TE D

41.

Klebanoff CA, Scott CD, Leonardi AJ, et al. Memory T cell-driven differentiation of

43.

EP

naive cells impairs adoptive immunotherapy. J Clin Invest 2016;126:318-334. Oluwole OO, Davila ML. At The Bedside: Clinical review of chimeric antigen receptor

AC C

(CAR) T cell therapy for B cell malignancies. J Leukoc Biol 2016.

44.

Davila ML, Brentjens R, Wang X, et al. How do CARs work?: Early insights from

recent clinical studies targeting CD19. Oncoimmunology 2012;1:1577-1583.

30

ACCEPTED MANUSCRIPT

45.

Brentjens RJ, Rivière I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or

46.

RI PT

chemotherapy refractory B-cell leukemias. Blood 2011;118:4817-4828. Dobrenkov K, Olszewska M, Likar Y, et al. Monitoring the efficacy of adoptively transferred prostate cancer-targeted human T lymphocytes with PET and bioluminescence imaging. J Nucl Med 2008;49:1162-1170.

Parente-Pereira AC, Burnet J, Ellison D, et al. Trafficking of CAR-engineered human T

SC

47.

Immunol 2011;31:710-718. 48.

M AN U

cells following regional or systemic adoptive transfer in SCID beige mice. J Clin

Brudno JN, Kochenderfer JN. Toxicities of chimeric antigen receptor T cells: recognition and management. Blood 2016;127:3321-3330. Cherkassky L, Morello A, Villena-Vargas J, et al. Human CAR T cells with cell-intrinsic

TE D

49.

PD-1 checkpoint blockade resist tumor-mediated inhibition. J Clin Invest 2016;126:3130-3144.

Beavis PA, Slaney CY, Kershaw MH, et al. Reprogramming the tumor

EP

50.

microenvironment to enhance adoptive cellular therapy. Semin Immunol 2015. Najjar AM, Manuri PR, Olivares S, et al. Imaging of Sleeping Beauty-Modified CD19-

AC C

51.

Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission

Tomography. Mol Imaging Biol 2016.

31

ACCEPTED MANUSCRIPT

52.

Davis JL, Theoret MR, Zheng Z, et al. Development of human anti-murine T-cell receptor antibodies in both responding and nonresponding patients enrolled in TCR

53.

RI PT

gene therapy trials. Clin Cancer Res 2010;16:5852-5861. Barrett DM, Singh N, Liu X, et al. Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy. Cytotherapy 2014;16:619-630.

Magnani CF, Turazzi N, Benedicenti F, et al. Immunotherapy of acute leukemia by

SC

54.

M AN U

chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform. Oncotarget 2016. 55.

Liu X, Barrett DM, Jiang S, et al. Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice. Blood Cancer J

56.

TE D

2016;6:e430.

Riet T, Holzinger A, Dörrie J, et al. Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy. Methods Mol Biol

57.

EP

2013;969:187-201.

Castro JE, Cantwell MJ, Prussak CE, et al. Long-term follow up of chronic lymphocytic

AC C

leukemia patients treated with CD40-ligand (CD154) gene therapy. Blood 2003;102:Abstract 1790.

58.

Wierda WG, Cantwell MJ, Woods SJ, et al. CD40-ligand (CD154) gene therapy for

chronic lymphocytic leukemia. Blood 2000;96:2917-2924.

32

ACCEPTED MANUSCRIPT

59.

Kato K, Cantwell MJ, Sharma S, et al. Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells. Journal of

60.

RI PT

Clinical Investigation 1998;101:1133-1141. Wierda WG, Castro JE, Aguillon R, et al. A phase I study of immune gene therapy for patients with CLL using a membrane-stable, humanized CD154. Leukemia 2010;24:1893-1900.

Castro JE, Sandoval-Sus JD, Melo-Cardenas J, et al. Phase I study of intranodal direct

SC

61.

M AN U

injection of adenovirus encoding recombinant CD40-ligand (Ad-ISF35) in patients with chronic lymphocytic leukemia. J Clin Oncol 2009;27:suppl; abstr 3003. 62.

Fukuda T, Chen L, Endo T, et al. Antisera induced by infusions of autologous AdCD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for

63.

TE D

Wnt5a. Proc Natl Acad Sci U S A 2008;105:3047-3052.

Deniger DC, Yu J, Huls MH, et al. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into

64.

EP

Diverse Memory T-Cell Populations. PLoS One 2015;10:e0128151. Savoldo B, Ramos CA, Liu E, et al. CD28 costimulation improves expansion and

AC C

persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 2011;121:1822-1826.

65.

Porter DL, Levine BL, Kalos M, et al. Chimeric Antigen Receptor–Modified T Cells in

Chronic Lymphoid Leukemia. New England Journal of Medicine 2011;365:725-733.

33

ACCEPTED MANUSCRIPT

66.

Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced

67.

RI PT

leukemia. Sci Transl Med 2011;3:95ra73. Chapuis AG, Ragnarsson GB, Nguyen HN, et al. Transferred WT1-Reactive CD8+ T Cells Can Mediate Antileukemic Activity and Persist in Post-Transplant Patients. Science Translational Medicine 2013;5:174ra127-174ra127.

Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained

SC

68.

69.

M AN U

remissions in leukemia. N Engl J Med 2014;371:1507-1517.

Kochenderfer JN, Dudley ME, Carpenter RO, et al. Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation. Blood 2013;122:4129-4139.

Ritchie DS, Neeson PJ, Khot A, et al. Persistence and efficacy of second generation

TE D

70.

CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther 2013;21:2122-2129.

Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of B-lineage cells and

EP

71.

regression of lymphoma in a patient treated with autologous T cells genetically

AC C

engineered to recognize CD19. Blood 2010;116:4099-4102. 72.

Till BG, Jensen MC, Wang J, et al. CD20-specific adoptive immunotherapy for

lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood 2012;119:3940-3950.

34

ACCEPTED MANUSCRIPT

73.

Cruz CR, Micklethwaite KP, Savoldo B, et al. Infusion of donor-derived CD19redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic

74.

RI PT

stem cell transplant: a phase 1 study. Blood 2013;122:2965-2973. Brudno JN, Somerville RP, Shi V, et al. Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of B-Cell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-

Haso W, Lee DW, Shah NN, et al. Anti-CD22-chimeric antigen receptors targeting B-

M AN U

75.

SC

Versus-Host Disease. J Clin Oncol 2016.

cell precursor acute lymphoblastic leukemia. Blood 2013;121:1165-1174. 76.

Giordano Attianese GM, Marin V, Hoyos V, et al. In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric

77.

TE D

antigen receptor. Blood 2011;117:4736-4745.

Berger C, Sommermeyer D, Hudecek M, et al. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer Immunol Res 2015;3:206-

78.

EP

216.

Kloss CC, Condomines M, Cartellieri M, et al. Combinatorial antigen recognition with

AC C

balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 2013;31:71-75.

79.

Qasim W, Amrolia PJ, Samarasinghe S, et al. First Clinical Application of Talen

Engineered Universal CAR19 T Cells in B-ALL. Blood 2015;126:2046 (S).

35

ACCEPTED MANUSCRIPT

80.

Brewer V. 2015 The CAR-T Cell-Race.


scientist.com/?articles.view/articleNo/42462/title/The-CAR-T-Cell-Race/>. Casadevall A, Pirofski LA. Exploiting the redundancy in the immune system:

RI PT

81.

vaccines can mediate protection by eliciting 'unnatural' immunity. J Exp Med 2003;197:1401-1404. 82.

Frumento G, Piazza T, Di Carlo E, et al. Targeting tumor-related immunosuppression

SC

for cancer immunotherapy. Endocr Metab Immune Disord Drug Targets

AC C

EP

TE D

M AN U

2006;6:233-237.

36

ACCEPTED MANUSCRIPT

Table 1. Completed clinical trials using adoptive transferred of engineered cells including CAR-T cells – Hematological

37

AC C

EP

TE D

M AN U

SC

RI PT

Malignancies

ACCEPTED MANUSCRIPT

67

Chapuis et al. Davila et al. 18 Brentjens et al. Maude et al. 68 Grupp et al. 20

17

73

Cruz et al.

2015

14

2015 2015

21 14

ALL or NHL NHL

2014

11

Leukemia

2014 2013 2014 2013 2013

Kochenderfer et al. Ritchie et al. 70 Kochenderfer et al. Till et al. 72

69

16

2013 2013 2012 2012

16 25 5 4 4 15 4 7 3

B-Cell Malignancies CLL

ALL ALL ALL CLL NHL AML CLL / NHL NHL

3 17

Brentjens et al.

2011

Savoldo et al.

66

2011 64

Kochenderfer et al. Jensen et al. Till et al.

13

14

71

None FLU/CY, PC, Benda FLU/CY FLU/CY Per discretion of treating physician CY Per discretion of treating physician

Gene Transfer

Target

Gamma retrovirus

CD19

1-10 x 10

CD19

0.14-11 x 10

CD19 CD19

1-3 x 10 6 0.3-5.0 x 10

Lentivirus Retrovirus Gamma retrovirus WT1-specific donorderived CD8+ cytotoxic TRetrovirus

3

CLL

WT1

3 x 10

CD19

0.8-21 x 10

FLU/CY FLU/CY FLU/CY CY

Retrovirus Gamma retrovirus Gamma retrovirus Electroporation

CD19 LeY CD19 CD20

None

Gamma retrovirus

CD19

1.2-3.0 x 10

CY

Gamma retrovirus

CD19

0.4-1.0 x 10

3.2 x 10

10

/m 2

6

CD19

7

6

NHL

None

Retrovirus

CD19

1 2 2 7

Lymphoma DLBCL FL NHL

FLU/CY

Retrovirus

CD19

FLU

Retrovirus

CD19

FLU or CY

Electroporation

CD20

1.46 x 10

2 x 10

1 x 10

7

/m

5

2

Anti-CD19 CAR T cells persisted for 14-19 months in some patients

NA 36

0

0

88

NA

Anti-CD19 CAR T cells. ALL pediatric or young adults (1-30 years) Anti-CD19 CAR T cells. Duration of ongoing CR responses (9-22 months) HLA A*0201-restricted WT1-specific donor-derived CD8+ cytotoxic T-cell clones were administered post-HCT. Anti-CD19 CAR T cells. Relpased/refractory ALL adults. One patient received less than the study dose

NA 0 0 27 25 43 33

7

0

0

7

0

25

100

0

0

0

100 100 0 29

0 0 0 14

- 2 x 10

2 x 10

9

8

/m 2

7

- 1.6 x 10

1-3 x 10

8

28

67 36

75 0 53 25 43 0

9

/m

2

8

/m 2

/m 2 - 3.3 x 10

9

Comments

/m 2

Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor

29

99

8

- 1.1 x 10

1-5 x 10 6 1.4-9.2 x 10 6 0.3-4.0 x 10 6 1 x 10 8 /m 2 - 3.3 x 10

CD19

2010

2008

6

6

Lentivirus

Lentivirus

8

/m 2 - 3.3 x 10

CD19

2011

2010

9

3.3 x 10

6

Retrovirus

Per discretion of treating physician

Response Rates CR (%) PR (%) 30 10

Cell dose/kg

None

CLL 4

Kalos et al.

Conditioning Regimen

RI PT

16

Disease

20

SC

Kalos et al. 66 Porter et al. 15 Lee et al. 22 Kochenderfer et al.

No. Patients

2016

M AN U

Year

74

TE D

References Brudno et al.

Anti-CD19 CAR T cells. 25 pediatric patients and 5 adult patients Retroviral FMC63 anti-CD19 scFv – CD28-CD3 Retroviral FMC63 anti-CD19 scFv-CD28-CD3 Anti-LeY CAR T cells persisted up to 10 months. Anti-CD19 CAR T cells. CAR T cells persisted < 3 months. Ani-CD20 CAR T cells. CAR T cells persisted 9-12 months. Anti-CD19 CAR T cells. This trial included a CAR T cell dose escalation and also compared responses in patients treated with or without conditioning chemotherapy before CAR T cell infusion. 2 ) is the lowest amount of Anti-CD19 CAR T cells. The CY dose (up to 3 g/m conditioning treatment among the published trials evaluating CD19-targeted CAR T cells for NHL. Patients were given a single course of chemotherapy during the week before infusion Retroviral FMC63 anti-CD19 scFv-CD3f and anti-CD19 scFv – CD28-CD3. CAR persistance for 6 weeks. First use of anti-CD19 CAR Retroviral FMC63 anti-CD19 scFv-CD3 with thymidine kinase suicide gene Anti-CD20 CAR T cells. CAR T cells persisted 5-9 weeks

38

AC C

EP

MCL= Mantle cell lymphoma; FL= Follicular lymphoma; DLCL= Diffuse large cell lymphoma; CLL= Chronic lymphocytic leukemia; NHL= Non-hodgkins lymphoma; ALL= Acute lymphoblastic leukemia; CR= Complete remission; PR= Partial remission; CY= Cyclophosphamide; FLU/CY= Fludarabine/Cyclophosphamide; PC= Pentostatin cyclophosphamide; Benda= Bendamustine; WT1= Wilms tumor antigen 1; LeY= Lewis Y antigen; NA= Not available.

ACCEPTED MANUSCRIPT

Target CD19

Center MSKCC

B-cell malignancies

Retrovirus

CD19

BCM

B-cell malignancies B-cell lymphoma B-ALL CLL B-ALL B-cell malignancies B-cell malignancies CD19+ ALL CLL/SLL Aggressive B-NHL, relapsed/refractory B-ALL B-cell malignancies B-cell malignancies B-cell NHL B-ALL B-ALL B-cell malignancies MCL B-cell malignancies B-cell NHL B-cell malignancies B-cell malignancies B-cell malignancies B-cell lymphoma B-cell lymphoma B-cell malignancies B-cell malignancies CD30+ Lymphoma (CARCD30) HL / NHL (CART CD30) B-cell malignancy or myeloma CLL / SLL

Retrovirus Retrovirus Retrovirus Retrovirus Retrovirus NA NA NA Lentivirus Retrovirus Retrovirus NA Lentivirus Lentivirus Lentivirus Lentivirus Retrovirus NA Retrovirus Retrovirus NA NA NA NA NA NA NA EBV CTLs EBV CTLs Retrovirus NA

ALL

Lentivirus

39

CD19

Clinicaltrials.gov ID Clinicaltrials.gov Status NCT00466531 Recruiting NCT00586391 Active, not recruiting NCT00608270 Active, not recruiting NCT00840853 Active, not recruiting NCT00924326 Active, not recruiting NCT00968760 Active, not recruiting NCT01044069 Recruiting Upfront therapy NCT01416974 Active, not recruiting After AlloHSCT, viral co-specificity NCT01430390 Recruiting After AlloHSCT NCT01497184 Active, not recruiting NCT01593696 Active, not recruiting EGFR+ construct (may allow deletion) NCT01683279 Active, not recruiting 2 dose level comparison NCT01747486 Recruiting After autologous SCT NCT01840566 Active, not recruiting NCT01860937 Recruiting NCT01864889 Recruiting NCT01865617 Recruiting NCT02030834 Recruiting EGFR+ construct (may allow deletion) NCT02028455 Recruiting NCT02030847 Recruiting After AlloHSCT NCT02050347 Recruiting NCT02081937 Recruiting NCT02132624 Recruiting NCT02134262 Recruiting Sequential CAR-T Bridging HSCT NCT02846584 Recruiting NCT02782351 Recruiting NCT02659943 Recruiting NCT02547948 Recruiting NCT02247609 Recruiting NCT02710149 Recruiting NCT02794961 Recruiting EBV CTLs Expressing CD30 Chimeric Receptors NCT01192464 Active, not recruiting NCT01316146 Active, not recruiting NCT00881920 Recruiting NCT02194374 Active, not recruiting

TE D

M AN U

NCI MDACC MSKCC MSKCC MSKCC MDACC NCI Seattle Children’s ACCUP MSKCC MSKCC Beijing FHCRC Penn Seattle Children’s ACCUP BCM Beijing Sweden Japan China China NCI China Peking University China China BCM UNCLCCC BCM MDACC / UCSD UCL, Great Ormond Hospital -UK

EP

CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD19 CD20 CD22 CD30 CD30 Kappa light chain ROR1

Comments Dose-escalation With ipilimumab Dose escalation After AlloHCT, viral co-specificity With IL-2 With or without IL-2

SC

Gene Transfer Retrovirus

AC C

Disease CLL

RI PT

Table 2. Ongoing clinical trials using adoptive transferred of engineered cells including CAR-T cells – Hematological

UCART19 -Universal donor CAR-T cells

NCT02808442

MCL= Mantle cell lymphoma; CLL= Chronic lymphocytic leukemia; SLL= Small lymphocytic lymphoma; HL= Hodgkin's lymphoma; NHL= Non-hodgkin's lymphoma; ALL= Acute lymphoblastic leukemia; AlloHSCT= Allogeneic hematopoietic stem cell transplantation; EGFR= Epidermal Growth Factor Receptor; EBV CTLs= EBV specific Cytotoxic T Lymphocytes; UNCLCCC= UNC Lineberger Comprehensive Cancer Center; MDACC= MD Anderson Cancer Center; NCI= National Cancer Institute; MSKCC= Memorial Sloan Kettering Cancer Center; BCM= Baylor College of Medicine; ACCUP= Abramson Cancer Center of the University of Pennsylvania.UCL=University College of London. UCSD=University of California-San Diego

Recruiting

ACCEPTED MANUSCRIPT

40

AC C

EP

TE D

M AN U

SC

RI PT

Malignancies

ACCEPTED MANUSCRIPT

Disease

Gene Transfer

Target

Cell dose/kg

20

B-Cell Malignancies

None

Gamma retrovirus

CD19

1-10 x 10

2015

14

CLL

FLU/CY, PC, Benda

Lentivirus

CD19

0.14-11 x 108

Lee et al. [4]

2015

21

ALL or NHL

FLU/CY

Retrovirus

CD19

Kochenderfer et al.[5]

2015

14

NHL

FLU/CY

Leukemia

Per discretion of treating physician

2014

Davila et al.[7]

2014

11

16 Brentjens et al. [8]

2013

Maude et al.[9]

2014

25

Grupp et al.[10]

2013

5

Cruz et al.[11]

2013

Kochenderfer et al. [12]

WT1

6

0.3-5.0 x 6 10 9

3.3 x 10 /m - 3.3 x 10 2 10 /m

CY

Retrovirus

CD19

3 x 10

ALL

Per discretion of treating physician

Lentivirus

CD19

0.8-21 x 10

ALL

4

CLL

2013

15

Ritchie et al. [13]

2013

Kochenderfer et al. [14] Till et al. [15]

None

Retrovirus

CD19

3.2 x 107 8 1.1 x 10

NHL

FLU/CY

Retrovirus

CD19

1-5 x 10

4

AML

FLU/CY

2012

7

CLL / NHL

2012

3

NHL

2011

AC C

3

FLU/CY

Gamma retrovirus Gamma retrovirus

LeY CD19

10

Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor

29

28

Anti-CD19 CAR T cells persisted for 14-19 months in some patients

67

NA

36

36

0

0

HLA A*0201-restricted WT1-specific donor-derived CD8+ cytotoxic T-cell clones were administered post-HCT.

88

NA

Anti-CD19 CAR T cells. Relpased/refractory ALL adults. One patient received less than the study dose

99

NA

Anti-CD19 CAR T cells. 25 pediatric patients and 5 adult patients

75

0

0

0

53

27

25

25

43

43

0

33

2

6

6

6

1.4-9.2 x 6 10 0.3-4.0 x 6 10 8 2 1 x 10 /m 9 2 3.3 x 10 /m

CY

Electroporation

CD20

None

Gamma retrovirus

CD19

1.2-3.0 x 7 10

0

0

CY

Gamma retrovirus

CD19

0.4-1.0 x 7 10

0

25

CLL

4

Comments

30

RI PT

6

ALL

4

Brentjens et al.[16]

CD19

1-3 x 10

SC

Chapuis et al. [6]

Gamma retrovirus WT1-specific donor-derived CD8+ cytotoxic T-cell

M AN U

Porter et al. [3]

TE D

Kalos et al. [2]

2016

EP

Brudno et al. [1]

Year

Response Rates CR PR (%) (%)

Conditioning Regimen

References

No. Patients

Anti-CD19 CAR T cells. ALL pediatric or young adults (1-30 years) Anti-CD19 CAR T cells. Duration of ongoing CR responses (9-22 months)

Retroviral FMC63 anti-CD19 scFv – CD28-CD3 Retroviral FMC63 anti-CD19 scFvCD28-CD3 Anti-LeY CAR T cells persisted up to 10 months. Anti-CD19 CAR T cells. CAR T cells persisted < 3 months. Ani-CD20 CAR T cells. CAR T cells persisted 9-12 months. Anti-CD19 CAR T cells. This trial included a CAR T cell dose escalation and also compared responses in patients treated with or without conditioning chemotherapy before CAR T cell infusion. Anti-CD19 CAR T cells. The CY dose 2 (up to 3 g/m ) is the lowest amount of conditioning treatment among the published trials evaluating CD19targeted CAR T cells for NHL.

ACCEPTED MANUSCRIPT

Kalos et al. [2]

2011

3

CLL

Per discretion of treating physician

Lentivirus

CD19

1.46 x 10 7 1.6 x 10

Savoldo et al. [17]

2011

6

NHL

None

Retrovirus

CD19

2 x 10 /m 8 2 2 x 10 /m

Kochenderfer et al. [18]

2010

1

Lymphoma

FLU/CY

Retrovirus

CD19

1-3 x 10

Jensen et al. [19]

2010

2

DLBCL

FLU

Retrovirus

CD19

2

FL

5

9

FLU or CY

Electroporation

CD20

M AN U TE D

8

2

0

100

0

First use of anti-CD19 CAR

100

0

0

0

29

14

Retroviral FMC63 anti-CD19 scFvCD3 with thymidine kinase suicide gene Anti-CD20 CAR T cells. CAR T cells persisted 5-9 weeks

2

1 x 10 /m 9 2 3.3 x 10 /m

SC

NHL

EP

7

AC C

2008

0

2

2 x 10 /m 8

Till et al. [20]

0

RI PT

7

Patients were given a single course of chemotherapy during the week before infusion Retroviral FMC63 anti-CD19 scFvCD3f and anti-CD19 scFv – CD28CD3. CAR persistance for 6 weeks.

100

ACCEPTED MANUSCRIPT Center

Comments

Clinicaltrials.gov ID

Clinicaltrials.gov Status

Retrovirus

CD19

MSKCC

Dose-escalation

NCT00466531

Recruiting

With ipilimumab

NCT00586391

Active, not recruiting

Dose escalation

NCT00608270

Active, not recruiting

After AlloHCT, viral co-specificity

NCT00840853

Active, not recruiting

With IL-2

NCT00924326

Active, not recruiting

NCT00968760

Active, not recruiting

NCT01044069

Recruiting

Upfront therapy

NCT01416974

Active, not recruiting

MSKCC

After AlloHSCT, viral co-specificity

NCT01430390

Recruiting

MDACC

After AlloHSCT

NCT01497184

Active, not recruiting

NCT01593696

Active, not recruiting

Retrovirus

CD19

RI PT

B-cell malignancies

Target

BCM

Retrovirus

CD19

NCI

B-cell lymphoma

Retrovirus

CD19

MDACC

B-ALL

Retrovirus

CD19

MSKCC

CLL

Retrovirus

CD19

MSKCC

B-ALL

Retrovirus

CD19

B-cell malignancies

NA

CD19

B-cell malignancies

NA

CD19

CD19+ ALL

NA

With or without IL-2

EP

TE D

B-cell malignancies

SC

CLL

Gene Transfer

M AN U

Disease

AC C

NCI

CD19

Seattle Children’s

EGFR+ construct (may allow deletion)

NCT01683279

Active, not recruiting

CD19

ACCUP

2 dose level comparison

NCT01747486

Recruiting

After autologous SCT

NCT01840566

Active, not recruiting

NCT01860937

Recruiting

CLL/SLL

Lentivirus

Aggressive B-NHL, relapsed/refractory

Retrovirus

CD19

MSKCC

B-ALL

Retrovirus

CD19

MSKCC

ACCEPTED MANUSCRIPT B-cell malignancies

NA

CD19

Beijing

NCT01864889

Recruiting

B-cell malignancies

Lentivirus

CD19

FHCRC

NCT01865617

Recruiting

B-cell NHL

Lentivirus

CD19

Penn

NCT02030834

Recruiting

B-ALL

Lentivirus

CD19

Seattle Children’s

NCT02028455

Recruiting

B-ALL

Lentivirus

CD19

ACCUP

NCT02030847

Recruiting

B-cell malignancies

Retrovirus

CD19

BCM

NCT02050347

Recruiting

NA

CD19

Beijing

NCT02081937

Recruiting

B-cell malignancies

Retrovirus

CD19

Sweden

NCT02132624

Recruiting

B-cell NHL

Retrovirus

CD19

Japan

NCT02134262

Recruiting

B-cell malignancies

NA

CD19

China

NCT02846584

Recruiting

B-cell malignancies

NA

CD19

China

NCT02782351

Recruiting

B-cell malignancies

NA

CD19

NCI

NCT02659943

Recruiting

B-cell lymphoma

NA

CD19

China

NCT02547948

Recruiting

B-cell lymphoma

NA

CD19

Peking University

NCT02247609

Recruiting

B-cell malignancies

NA

CD20

China

NCT02710149

Recruiting

B-cell malignancies

NA

CD22

China

NCT02794961

Recruiting

CD30+ Lymphoma (CARCD30)

EBV CTLs

NCT01192464

Active, not recruiting

HL / NHL (CART CD30)

EBV CTLs

B-cell malignancy or myeloma CLL / SLL

RI PT

M AN U

SC

After AlloHSCT

TE D

Sequential CAR-T Bridging HSCT

EP

AC C

MCL

EGFR+ construct (may allow deletion)

EBV CTLs Expressing CD30 Chimeric Receptors

CD30

BCM

CD30

UNCLCCC

NCT01316146

Active, not recruiting

Retrovirus

Kappa light chain

BCM

NCT00881920

Recruiting

NA

ROR1

MDACC / UCSD

NCT02194374

Active, not

ACCEPTED MANUSCRIPT recruiting

UCART19 -Universal donor CAR-T cells

TE D

M AN U

SC

RI PT

CD19

EP

Lentivirus

AC C

ALL

UCL, Great Ormond Hospital UK

NCT02808442

Recruiting

ACCEPTED MANUSCRIPT

REFERENCES TABLE 1

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

RI PT

SC

5.

M AN U

4.

TE D

3.

EP

2.

Brudno JN, Somerville RPT, Shi V, et al. Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of BCell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-Versus-Host Disease. Journal of Clinical Oncology 2016;34:1112-1121. Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73. Porter DL, Hwang WT, Frey NV, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med 2015;7:303ra139. Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015;385:517-528. Kochenderfer JN, Dudley ME, Kassim SH, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 2015;33:540-549. Chapuis AG, Ragnarsson GB, Nguyen HN, et al. Transferred WT1-Reactive CD8+ T Cells Can Mediate Antileukemic Activity and Persist in Post-Transplant Patients. Science Translational Medicine 2013;5:174ra127-174ra127. Davila ML, Riviere I, Wang X, et al. Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia. Science Translational Medicine 2014;6:224ra225-224ra225. Brentjens RJ, Davila ML, Riviere I, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 2013;5:177ra138. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014;371:15071517. Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368:15091518. Cruz CR, Micklethwaite KP, Savoldo B, et al. Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study. Blood 2013;122:2965-2973. Kochenderfer JN, Dudley ME, Carpenter RO, et al. Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation. Blood 2013;122:4129-4139. Ritchie DS, Neeson PJ, Khot A, et al. Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther 2013;21:2122-2129. Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 2012;119:2709-2720. Till BG, Jensen MC, Wang J, et al. CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood 2012;119:3940-3950. Brentjens RJ, Riviere I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 2011. Savoldo B, Ramos CA, Liu E, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 2011;121:1822-1826. Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 2010;116:4099-4102. Jensen MC, Popplewell L, Cooper LJ, et al. Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans. Biol Blood Marrow Transplant 2010;16:1245-1256.

AC C

1.

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Till BG, Jensen MC, Wang J, et al. Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells. Blood 2008;112:2261-2271.

AC C

20.