Chimeric antigen receptor technology: a breakthrough in immuno-oncology

Daniel BATY,MD, PhD
Department of Pediatrics
Yong Loo Lin School of Medicine
National University of Singapore

Chimeric antigen receptor technology: a breakthrough in immuno-oncology

by D. Campana, Singapore

Chimeric antigen receptors (CARs) can redirect the specificity of immune cells. An antibody-derived single-chain fragment variable (scFv) provides specific capacity to bind a surface antigen expressed by cancer cells. The scFv is linked, via hinge and transmembrane protein segments, to signaling domains that trigger T-cell activation when the CAR binds to its cognate antigen. Contemporary CARs typically contain a primary signaling molecule such as CD3ζ, and one or two costimulatory molecules, such as CD28 and/or4-1BB (CD137).Costimulation sustains T-cell proliferation and suppresses activation-induced cell death. CAR-engineered T cells can exert powerful cytotoxicity against cancer cells in vitro and in animal models. Results of recent clinical studies have demonstrated the remarkable potential of this technology, with durable remissions achieved in patients with refractory B-cell leukemia and lymphoma targeted with anti-CD19 CARs. Whether such responses will also be seen in other malignancies is still unknown. Infusion of CAR-engineered T cells can have serious side effects, such as cytokine release and tumor lysis syndromes, and “on-target off-tumor” activity caused by expression of tumor antigens in normal cells. These issues must be addressed and better ways for large-scale production of CAR T cells must be developed. Ultimately, the generation of highly optimized “living drugs” that can be administered on demand with predictable activity should lead to the incorporation of CAR T cells into mainstream cancer treatment

Medicographia. 2015;37:280-286 (see French abstract on page 286)

The perception that the immune system could be a tool to fight cancer is not new. More than 100 years ago William Coley observed that sarcoma could regress in some patients after infections, prompting administration of bacterial toxins to treat cancer; around the same time, Paul Ehrlich reasoned that the immune system could control tumor cell growth.1 During the last 2-3 decades, the anticancer potential power of immune cells became clear to hematologists performing hematopoietic stem cell transplants for patients with leukemia. The strong association between severity of T cell–mediated graft-vs-host disease (GvHD) and probability of remaining in remission posttransplant led to the practice of infusing T lymphocytes from the stem cell donor posttransplant in efforts to suppress impending relapse.2 Nevertheless, the risk of potentially lethal GvHD from donor lymphocyte infusions is high. Separating the graft-vs-leukemia effect from the GvHD effect has been the main preoccupation of transplant hematologists for the last 4 decades.

If one could redirect autologous T cells towards tumor cells, then it might be possible to have graft vs leukemia without GvHD. This was made possible by the advent of chimeric antigen receptors (CARs).

Your CAR is here

A CAR is an artificial multimolecular receptor. Its specificity derives from a single-chain fragment variable (scFv) of an antibody, ie, a fusion protein containing the portions of an antibody specifically binding to a target antigen connected with a short peptide. The scFv is linked to a transmembrane domain that ensures expression on the cell membrane; a hinge region is generally placed between the scFv and the transmembrane domain to give flexibility to the CAR and facilitate antigen binding and signal transduction. The power house of the receptor is in the intracellular portion and consists of a signaling domain, typically a T-cell receptor (TCR)–associated signaling molecule, such as cluster of differentiation 3ζ(CD3ζ). Thus, scFv ligation to its unique antigen triggers signal transduction, similar to the one triggered by the TCRs normally expressed on T lymphocytes when they encounter a viral peptide. This results in T-cell activation, proliferation, and cytotoxicity (Figure 1). Therefore, by expressing a CAR in T lymphocytes, one can instantaneously generate a massive cohort of immune cells reacting against a tumor.

The concept of CAR (also referred to as “chimeric T-cell receptor,” “chimeric antibody/T-cell receptor,” or “T-body”) was first reported 25 years ago by Zelig Eshhar and his colleagues working at the Weizmann Institute of Science in Israel.3 In their initial studies, a prototype CAR was made by splicing the antigen- binding regions of an antibody against 2,4,6-trinitrophenyl hapten to a TCR. The construct was transfected into a mouse T-cell hybridoma that could kill cells expressing hapten trinitrophenyl.4 While the constructs were developed with the intention of studying T-cell activation mechanisms rather than developing a novel cancer treatment,3 the pioneering article already states that “This approach can be exploited, for example, to direct cytotoxic T lymphocytes to kill tumor or virally infected cells.”4 Later, the group refined the original prototype by linking scFv to CD3ζ(the subunit of the TCR/CD3 complex that transduces signals) or FcεRIγ chains, containing signaling cassettes known as immunoreceptor tyrosine-based activation motifs (ITAMs).5 Together with investigators at the National Cancer Institute in Bethesda, they published a study that tested the CAR concept in a more realistic experimental scenario.6 This new CAR was made with a scFv that reacted against a folate receptor overexpressed by ovarian cancer cells and was expressed in CD8+ tumor-infiltrating lymphocytes (TILs). Expanding the range of targetable antigens, a CAR reacting with the tumor-associated antigen human epidermal growth factor receptor 2 (HER2/neu) was reported soon after.7

The portfolio of antigens targetable by CARs has progressively increased. Those expressed by solid tumors included tumor-associated glycoprotein 72 (TAG-72),8 and epithelial glycoprotein 2 (EGP-2), both overexpressed in multiple carcinomas,9 as well as GD-2,10 expressed in neuroblastoma, sarcomas, and melanoma. CARs directed against antigens expressed in lymphomas and leukemias were also reported, including CD30, expressed in Hodgkin and anaplastic large cell lymphoma,11 CD20, expressed in B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL),12 and CD33, expressed in acute myeloid leukemia (AML).

Figure 1
Figure 1. Basic mechanisms of chimeric antigen receptor (CAR)
activity against cancer cells.

CARs expressed in T lymphocytes trigger signal transduction upon ligation of
their specifically recognized antigen on the surface of cancer cells. The signaling
cascade results in T-cell activation and proliferation. In cytotoxic T cells, it
also triggers exocytosis of lytic granules and killing of target cells.
Abbreviation: scFv, single-chain fragment variable.

New generations of CARs

During immune responses, TCR activation alone cannot provide a sufficiently robust stimulus to T lymphocytes; without participation of other “costimulatory” receptors, T-cell activation is short-lived, ultimately leading to T-cell unresponsiveness and/or apoptosis.13,14 One of the most extensively studied costimulatory molecules in T lymphocytes is CD28, which interacts with its ligands B7-1 (CD80) and B7-2 (CD86) expressed by antigen-presenting cells.14 Another costimulatory molecule is 4-1BB (CD137), which also increases lymphocyte activation and supports proliferation (reviewed in reference 15). Some studies suggested that 4-1BB stimulation could elicit more effective antitumor responses than those provoked by CD28, and that it preferentially expanded memory T cells.15

In general, costimulatory ligands are poorly expressed by tumor cells.16,17 Therefore, stimulation via a CAR containing only CD3ζ as a stimulatory module might result in unpredictable activation, depending on the degree of expression of costimulatory ligands in target cells. Elegant experiments performed by Brentjens et al18 illustrated this concept well. They found that in immunodeficient mice engrafted with tumor cells, the antitumor activity of T lymphocytes expressing an anti-CD19 CAR was considerably higher if the target cells were induced to overexpress the CD28 ligand, CD80.

A solution to the problem of variable costimulatory capacity by tumor cells is to integrate the costimulatory signal directly into the CAR.19-22 CARs bearing costimulatory signaling domain are designated as “second generation” to distinguish them from the “first-generation” CARs that could only deliver a primary, ITAM-derived, signal (Figure 2). Second-generation CARs provoke a much more reliable and robust T-cell stimulation than their predecessors. Finney et al19 found that CD28-containing CARs triggered higher interleukin 2 (IL-2) production as compared with constructs containing only CD3ζ without CD28; placing CD28 proximal to the cell membrane led to more efficient CAR expression. Maher et al22 reported that T lymphocytes expressing a CAR directed against prostate-specific membrane antigen were considerably more effective at triggering cytokine secretion, and proliferation, and tumor cell killing if CD28 was added to CD3ζ.

Because 4-1BB also plays an important costimulatory role in T cells, we constructed anti-CD19 CAR signaling via CD3ζ and added a 4-1BB signaling domain.17 The receptor contained hinge and transmembrane domains derived from human CD8α; all components were joined in a unique chimeric sequence. The expression of this CAR achieved in peripheral blood lymphocytes by retroviral transduction was high (median, 64%) and the addition of 4-1BB did not affect levels of expression.17 We found that this CAR could also be expressed by electroporation of the corresponding messenger RNA.23,24 When cocultured with CD19+ leukemic cells for 24 hours, T cells expressing the anti-CD19-BB-ζCAR produced about 5 times more IL-2 than T cells expressing an equivalent receptor lacking 4-1BB.17 T lymphocytes expressing the 4- 1BB CAR expanded for 3 weeks or more when cultured in the presence of CD19+ target cells without exogenous IL-2, while T cells transduced with the CAR lacking 4-1BB did not grow and survived for less than 2 weeks.17 The addition of 4-1BB also increased tumor cell killing, and the difference with receptors lacking 4-1BB was particularly evident at a low effector: target ratio in cultures extended to 5 days instead of the standard 4-hour assays.17 Research from other laboratories also demonstrated that 4-1BB contributes significantly to CAR function.25-27

Figure 2
Figure 2.: Modular composition of chimeric antigen receptors (CARs).

First-generation CAR contain one signaling molecule, typically CD3ζ or FcεRIγ. Second-generation
CARs also have a costimulatory molecule, eg, CD28 or 4-1BB (CD137). Third-generation
CARs with more than one costimulatory molecule have been described.
Abbreviation: scFv, single-chain fragment variable.

What is the best CAR?

At present, it is not clear what CAR configuration is optimal, although it seems unquestionable that “second-generation” constructs are more potent than “first-generation” constructs.

We found that 4-1BB induced higher production of IL-2, while CD28 induced higher production of interferon γ (IFNγ).15,17 Increased production of IFNγ by CD28 was also reported by others. 25,28 We could not detect differences in cytotoxicity mediated by the two CARs, but observed that the 4-1BB CAR induced better expansion of T cells in the presence of low concentrations of IL-2.15,17

Higher proliferation with the 4-1BB CAR was confirmed by Milone et al26 in immunodeficient mice. So-called “third-generation” CARs contain more than one costimulatory domain; it is not clear whether this will produce a proportionally more potent receptor. Some studies indicated that adding both 4-1BB and CD28 to a CAR increased cytokine production, proliferation, and cytotoxicity as compared with a single costimulus.15 Others, however, could not detect clear differences between single- and dual-costimulation CARs.15 Data on CARs with other costimulatory molecules (OX40, CD27, etc) are not extensive.

Figure 3
Figure 3. Preparation of chimeric antigen receptor
(CAR)–engineered T lymphocytes for clinical use.

Peripheral blood is obtained from the patient, eg, by leukapheresis.
In a facility operating under Current Good Manufacturing
Practice (CGMP) guidelines, T lymphocytes are activated
with anti-CD3 +/- anti-CD28 antibodies, and interleukin 2 (IL-2).
During T-cell proliferation, viral supernatant is added and viral
transduction procedures are performed. After approximately
10 days from the beginning of the culture, CAR-engineered
T cells are reinfused in the patients. Patients often receive lymphodepleting
chemotherapy during ex vivo cell processing to
facilitate the engraftment of the infused cells.

When building a CAR, cloning its various components is only the beginning; putting them together to maximize CAR expression and function requires much attention. As initially described for CD28,19 our anti-CD19-BB-ζ receptor requires the 4-1BB to be placed proximal to the transmembrane domain; we also modified the hinge and transmembrane portion derived from CD8α to allow optimal delivery of 4-1BB signaling.15,17 Simply inserting 4-1BB into any CAR might not produce the same results without attention to these details. The same probably applies to CD28 and to “third-generation” CARs. To this end, Kochenderfer et al29 showed that combining CD28 and 4-1BB actually decreased IL-2 production as compared with CD28 alone, but the transmembrane domains of the compared CARs were different (CD8 vs CD28). Along the same lines, Haso et al30 reported that CARs with single 4-1BB or CD28 domains were better than those containing both, but again, transmembrane domains were different as were levels of expression for different CARs. Another important variable that can influence results of CAR comparisons is the type of test used to assess function. For example, Finney et al28 did not observe any advantage when they added 4-1BB to their CAR, but T cells were tested only in short-term assays. Indeed, we found that the superior proliferation and cytotoxicity of 4-1BB CARs became obvious only when the experiments were extended for days and performed at low effector:target ratios.15,17 Finally, the type of antigen targeted may also play a role: in addition to scFv affinity,30,31 the length of hinge domain can also be a critical factor, as it has been shown for CARs targeting ROR131 and MUC1.32

CAR-engineered T lymphocytes in the clinic

The typical process for preparing CAR-engineered T cells consists of collecting blood from the patient via leukapheresis, followed by T-cell activation and expansion for about 10 days using anti-CD3 or anti-CD3 plus anti-CD28 stimulation (either with soluble antibodies or antibodies bound to a solid phase) and IL-2. During the culture, cells are exposed to retroviral or lentiviral supernatant containing the CAR construct in a viral vector. In future trials, additional genes might be included in the constructs, such as those encoding proteins that can facilitate the elimination of engineered cells to limit adverse effects, or allow the infusion of allogeneic cells. At the completion of the culture, cells are washed, concentrated, and infused. The cell product might be cryopreserved before infusion to allow for sterility and potency testing to take place. Before infusion, the patient may receive lymphodepleting chemotherapy (eg, fludarabine and cyclophosphamide) to facilitate the expansion of the infused T cells (Figure 3).33 The cell preparation typically takes place in facilities that work under Current Good Manufacturing Practice (CGMP) regulations enforced by the US Food and Drug Administration (FDA), or similar regulations imposed by equivalent authorities in other countries.

In the first reported study using CAR-engineered T cells, 14 patients with metastatic ovarian cancer received T cells expressing a first-generation CAR against an &lapha;-folate receptor.34 The infused cells were initially present in large numbers, but then declined and became nearly undetectable even by polymerase chain reaction (PCR) after 1 month; no antitumor effect was observed.34 In another early study, T cells expressing a firstgeneration CAR against carboxy anhydrase IX were administered to 12 patients with metastatic renal cell carcinoma.35 Levels of infused T cells in blood peaked at around day 6 and were detectable after about 1 month by PCR. Liver toxicity (possibly due to “on-target off-tumor” activity) was observed, but no antitumor activity.35 Because both of these studies used a first-generation CAR, lack of vigorous T-cell expansion and persistence might have contributed to the lack of antitumor activity, although it is possible that other factors may have played a role. Neither study used lymphodepletion prior to T-cell infusion, and T-cell activation ex vivo was performed without costimulation.

Among other early studies with first-generation constructs, an anti-GD2 CAR was used to treat 11 patients with neuroblastoma and active disease at the time of infusion, producing remission in three of these patients36,37; CAR T cells persisted for up to 3.6 years.37 No responses were seen in four patients with B-cell NHL who received T cells expressing anti-CD19 or anti-CD20 CARs; short persistence of the infused cells was noted, with evidence of immune rejection, which might have been related to the incorporation of selection and suicide genes in the construct.38

Much more encouraging results were obtained in patients with B-cell malignancies using T cells engineered with second- generation CARs. In 2010, Kochenderfer et al reported the case of a patient with follicular lymphoma and progressive disease treated with T cells expressing an anti-CD19 CAR signaling via CD3ζ and CD28.39 T cells were infused after lymphodepleting chemotherapy and a major disease regression with remission lasting 32 weeks was observed. There was also concomitant B-cell aplasia and hypogammaglobulinemia.39 In a recent update including 15 patients with NHL or CLL, complete remission was achieved in 8 of the 15 patients and a partial remission in 4, with 3 remissions in patients with diffuse large B-cell lymphoma continuing beyond 9-22 months.40 This group also reported results of infusing CARmodified T cells in patients with B-cell malignancies postallogeneic stem cell transplant, where the T cells were from the stem cell donor.41 While disease was resistant to infusion of unmodified donor lymphocytes, 3 of the 10 patients had disease regression following anti-CD19 CAR infusion. Interestingly, none of the 10 patients developed GvHD.40 In a recent report, this CAR was used to modify autologous T cells in 20 children and young adults with acute lymphocytic leukemia (ALL); 14 achieved complete remission, 12 with minimal residual disease negativity (Table I).42

Table I
Table I. Response to anti-CD19 chimeric antigen receptor (CAR)
in patients with acute lymphocytic leukemia (ALL).

*Minimal residual disease (MRD) studies were not performed in 2 of the 27 patients
who achieved complete remission.
Based on data from references 42, 45, and 47.

In 2011, using an anti-CD19 CAR costimulating via 4-1BB, Porter et al reported major responses in all 3 patients with treated CLL, with durable complete remission in 2 patients.43,44 Infused T cells expanded and persisted for at least 6 months. As expected, there was B-cell aplasia and hypogammaglobulinemia. Grupp et al used the same anti-CD19 to treat patients with relapsed or refractory ALL. In their most recent report, a complete remission was achieved in 27 of 30 patients, with a 6-month event-free survival rate of 67% and an overall survival rate of 78% (Table I).45 Three patients relapsed with CD19-negative leukemia.45

Anti-CD19 CARs costimulating via CD28 were used in other studies. Brentjens et al reported responses in 3 of 4 patients with chemotherapy-refractory CLL.46 In a study from this group using the same CAR to treat 16 patients with relapsed or refractory ALL, a complete response was achieved in 14 patients, with molecular remission in 12, allowing patients to receive allogeneic hematopoietic stem cell transplants.47 Cruz et al48 expressed the anti-CD19 CAR in virus-specific, donorderived T cells and infused them in 6 patients with relapsed CLL or ALL after hematopoietic stem cell transplant. There was no GvHD and cells persisted for several weeks in blood. Responses were seen in 2 of 6 patients.

A CD20 CAR, this time a “third-generation” construct costimulating via both CD28 and 4-1BB, was used to treat 4 patients with relapsed indolent B-cell and mantle cell NHL. Response is difficult to evaluate as 2 patients remained progression-free for 12 and 24 months, but did not have evaluable disease before infusion. The third patient had a partial remission with relapse 12 months later. Modified T cells were detected by PCR at tumor sites and up to 1 year in peripheral blood.49 CARs can also be expressed in natural killer cells,24,50 and clinical studies with such cells are ongoing.

Areas for improvement

Ehrlich’s concept of “magic bullets” that specifically bind to certain cells and eliminate them while sparing others was formulated at the dawn of the 19th century and inspired legions of oncologists to identify such agents. This seemed to be embodied by monoclonal antibodies, but clinical results, albeit exciting, have rarely met Ehrlich’s criteria of “therapia sterilisans magna,” which in oncology requires complete eradication of tumor. The excitement around CAR-engineered T cells is that they hold promise to achieve such a goal. The clinical data summarized here demonstrated dramatic and durable responses in patients with leukemia and lymphoma resistant to contemporary intensive chemotherapy and stem cell transplant, and therefore are extremely encouraging.

Although effective, CAR technology needs improvement. Successful studies with CARs have been performed in patients with lymphoid malignancies and it is not clear whether a similar success can be achieved in other types of cancer. To this end, there is a relative paucity of good targets and much research is needed. An elegant approach that may be helpful in this respect is that of using dual CARs that are either active or inhibited depending on the simultaneous expression of two antigens.51 Infusion of CAR-engineered T cells can have serious side effects, such as cytokine release and tumor lysis syndromes.47 Developing ways to better control T-cell activation would be useful. Also, it has been shown that ALL treated with anti-CD19 CARs can relapse as a CD19-negative ALL.45,52 Targeting multiple antigens simultaneously might help preventing such relapses. We developed a new chimeric receptor, CD16V-BB-ζ, whose specificity is directed by immunotherapeutic antibodies, instead of scFv53; hence, one can target multiple antigens with one single receptor. In B-cell malignancies, for example, the CD16V-BB-ζ receptor would allow T-cell therapy simultaneous targeting CD19, CD20, and CD22 with existing humanized antibodies. Moreover, T-cell activation could be, in principle, stopped if required, by withdrawal of antibody administration. With the exception of studies performed in the transplant setting using donor T cells, CAR-engineered T cells have so far been used in the autologous setting. This makes it difficult to evaluate responses and side effects across patients, as essentially every product is one-of-a-kind. Moreover, production of engineered T cells from individual patients presents considerable logistical problems. A major step forward in this area would be the development of an allogeneic product devoid of GvHD capacity. This off-the-self “living drug” would represent a major advance in this area.

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Keywords: acute lymphocytic leukemia; chimeric antigen receptor; chronic lymphocytic leukemia; costimulatory molecule; non-Hodgkin lymphoma; T lymphocyte