CAR T Cells: A Look Under the Hood and Down the Road

Mark J. Adler, MD

Disclosures

September 16, 2014

Editor's Note: Cancer immunotherapy is among the fastest-growing areas of oncology research, but it leaves many oncologists scratching their heads. The deluge of promising data in immunotherapy means it is worth the effort to catch up on the science. To put things in perspective, Medscape asked Mark J. Adler, MD, Co-founder and Director of the San Diego Cancer Research Institute, and former Director of the San Diego Cancer Center, to provide an overview of cancer immunotherapy, and to take one of the most promising approaches—CAR T cells—for a test drive.

The success of early childhood immunizations proves that the immune system can provide both specific and long-lasting defense against many foreign foes, including polio, smallpox, and diphtheria, but how effective is it against cancer? The very existence and ubiquity of cancer is testament to the failure or insufficiency of the immune system against domestic enemiesour own rogue and malignant cells. Although recent advances in cancer immunotherapy have garnered the status of 2013 "breakthrough of the year" by Science,[1] we should recall numerous headlines of the past that prematurely trumpeted interferon and the "magic bullet" antibodies as conquering heroes. Will CAR T cells bring us some victorious battles in our war on cancer? Some historical context will permit us to see how far we are on the road to cancer immunotherapy and how much hard driving remains ahead.

From a Mishmash of Dead Bacteria

The long history of immunologic approaches to cancer is strewn with disappointing outcomes and only occasional success. In the 1890s, William Coley, then at New York Hospital, used his eponymous "vaccine"a mishmash of dead bacteriato elicit profound systemic immune and inflammatory reactions in cancer patients. Although there were anecdotal remissions, such generalized immune and inflammatory eruptions were not reliable therapeutic tools. Within the past quarter century, we have developed an increasingly differentiated view of the specific players, and their specialized roles, that are involved in the immune response. We have separated antibody-based immune reactions from T-cell-based immunity and identified concerted immune operations involving various cell types, antibodies, and chemical stimulants (or cytokines). Recipes of separate and combined immune players, in varying doses and schedules, have been thrown into battle against malignancy.

For over 30 years, interferon, a cytokine whose physiologic role includes antiviral function, has brought modest improvement in the treatment of solid tumors such as renal cancer and melanoma, and more recently in Kaposi sarcoma, a tumor that emerged through the weakened immunity of many AIDS patients. Another cytokine, interleukin-2 (IL-2), was found capable of eliciting a far more profound immunologic response by T cells. With high-dose intravenous IL-2 administration, high-quality durable remissions have been achieved in melanoma[2] and renal cancer.[3] Unfortunately, such victories were associated with furious immune reactions that took a severe and sometimes lethal toll.

Antibodies, or so-called "magic bullets" aimed at specific tumor targets, took immune therapy to a new level when rituximab (added to the standard cytotoxic regimen) increased complete response rates and overall survival in some of the most aggressive lymphomas of B-cell origin.[4] A large number of antibodies to solid tumors followed, including trastuzumab, which targets the human epidermal growth factor receptor 2 (HER2) antigen found in about one third of breast cancers; and cetuximab, which targets the epidermal growth factor receptor (EGFR) found in a subset of colon cancers and head and neck cancers.

In a novel twist, rather than taking direct aim at tumors, antibodies recently have been fashioned to attack and interfere with apparatus that regulates, and sometimes inhibits, T-cell immunity against cancer. Ipilimumab antibody, for example, targets cytotoxic T-lymphocyte antigen 4 (CTLA-4), a surface protein receptor on a T-cell population that enhances immune tolerance to tumor cells and thereby inhibits tumor destruction. Impairing these "immune brakes" and freeing T cells for anti-tumor function have yielded breakthrough response rates in melanoma.[5] Although dramatic responses have generally been ephemeral, some cases have been long-lasting, giving a tantalizing glimpse of the possibility of a "resetting" of the immune system.[6] Newer antibodies directed against so-called checkpoint inhibitors, such as programmed death 1 receptor (PD-1) and its ligand, PD-L1, have been shown to successfully neutralize mediators of cell-cycle arrest on inhibitory T cells. Antibody to PD-1 achieved objective responses in up to one fourth of patients with non-small cell lung cancer, renal cancer, and melanoma, with two thirds of these remissions lasting at least a year, as reported by Suzanne Topalian and colleagues.[7] In an update of that report, 31% of melanoma patients who achieved an objective remission with antibody to PD-1 had a median survival of two years.[8] A combination of immune blockade inhibitors, using both anti-CTLA and anti-PD-1 in patients with advanced melanoma, led to at least 80% disease reduction in the 50% of patients who responded to the maximal dose.[9]

Progress with PD-1 and PD-L1 drugs, mediated through cellular immunity, led to renewed interest in T cells themselves and in strategies to directly engineer their enhanced function. Earlier direct use of T cells was accomplished by harvesting and growing lymphocytes found proximate to cancer cells, or so-called tumor-infiltrating lymphocytes (TILs). TILs used in conjunction with IL-2 treatment brought occasional complete responses in melanoma, as well as some objective responses in patients treated previously with IL-2,[10] but harvesting TILs often required extensive surgical intervention, and then only a narrow range of tumors were candidates for treatment.

Subsequent advance came with the design of recombinant T-cell receptors (TCRs) fashioned to allow T cells to recognize and react to specific surface peptide targets. However, because such peptides needed to be presented by antigen-presenting cells (APCs) within the major histocompatibility complex (MHC), the clinical value of recombinant TCRs was again limited to the few tumors that could present antigens in this context. Additional problems included suboptimal receptor-to-target avidity, cross-reactivity to endogenous nontumor antigens, and the potential for severe unintended autoimmune reactions.[11] Overall, the results of therapies using direct antitumor antibodies, cytokines, TILs, and TCRs have been less impressive than those of strategies intended to release cellular immune inhibition and thereby induce tumor killing.

CARs: The Nuts and Bolts

The advent of so-called chimeric antigen receptor (CAR) T cells (sometimes referred to as CARs) represents a further refinement in cellular immune engineering, and one that shows highly promising early results. Of greatest interest is the potential of this technology to free cellular immune approaches from some important physiologic limitations.[11]

The use of this therapy starts with harvesting of T cells from a patient's blood, transducing these cells ex vivo by retro- or lentiviral transfer of CAR, amplifying the population of CAR-transduced cells, and then infusing them back into the patient. The sophistication and technical virtuosity involved with CAR T-cell production represent a tour de force of modern molecular biology. Instead of the mythological chimera, with its lion head joined to a snake tail, these creations consist of antibody components at the membrane surface to confer specific binding properties, connected to intracellular tails capable of inducing proliferation of CAR T cells, secretion of cytokines, and destruction (or lysis) of tumor cells. Thus, in a sense, these synthetic creations combine the specificity of B cells with the destructive capacity of T cells. More specifically, the engineered CAR prototype utilizes a single-stranded fusion protein that combines antibody specificity with T-cell activation functions. The extracellular component of this fusion molecule consists of the variable domain of antibody heavy chain linked to the variable domain of light chain. Attached to this extracellular receptor is an intracellular signaling moiety, typically derived from sequences of the TCR/CD3 complex. Activation of this intracellular portion, or "tail," is set in motion when the external receptor binds to antigen; such activation, in turn, induces cytokine secretion (including IL-2), tumor lysis, and proliferation of CAR T cells. Use of antibody binding rather than typical TCR binding components confers several advantages to CAR T cells compared with their natural counterparts, foremost of which is the MHC independence of the process. Not only does this open up a far wider range of peptide targets than either natural or previously engineered TCRs, but CARs can also bind with high affinity to other molecular classes, including carbohydrates and inorganic chemicals. This permits surface markers such as CA19-9—the carbohydrate antigen most associated with pancreatic cancer—to be targeted in an MHC-independent fashion.[12]

Second- and third-generation CAR constructs incorporate costimulatory elements that further surmount physiologic constraints upon activation and proliferation. A critical refinement includes the addition of the costimulatory molecule CD28, which is usually presented by APCs; CD28 induces proliferation of the stimulated cell rather than cell death and resultant anergy. Second-generation CAR receptors artificially fuse this intracellular CD28 stimulatory domain into one molecule with the previously described antigen-binding and T-cell activating domains of the CAR construct. The resulting receptor therefore enhances APC-ligand independence and, by induction of proliferation, multiplies the number of these therapeutic agents.

CARs and the Drive to the Clinic

Most clinical trials involving CAR T cells have been in hematologic malignancies, a natural starting ground given that circulating tumor cells are easily accessible and express several well-characterized surface tumor antigens. Even though we are still at the starting line, CARs have achieved complete and lasting remissions in refractory CD19-positive B-cell malignancies, including chronic lymphocytic leukemia, acute lymphocytic leukemia, and non-Hodgkin lymphoma. A recent review[11] noted that only 14 clinical trials on hematologic malignancies have been published, and all but one of these targeted B-cell malignancies with CARs aimed at CD19 or CD20 antigens. CAR T-cell use in solid tumors is far less developed.

Carl June and colleagues, in reviewing their own and other early investigations into CARs, made a number of important observations about CARs in B-cell malignancies.[13] Of note, acute lymphocytic leukemia, the most aggressive of these malignancies, demonstrated the best overall response rate at around 80%, regardless of variation in protocols, trial design, institution, or method of producing CARs. Evidence showed that engraftment or growth and persistence of CAR T cells after reinfusion was necessary for successful responses; a sustained presence of CAR T cells over several months was probably required when this therapy was used to replace allogeneic transplantation. Toxicities were generally associated with immune activation, including cytokine release syndrome and macrophage activation. Future studies will need to focus on the extent to which some ill effects are also therapeutic, in hopes of avoiding symptomatic remedies that interfere with critical elements of immune activation.[13]

Caution: Roadwork Ahead

Before we become prematurely exuberant over CAR T cells, it is worthwhile to review some of the roadblocks hindering the broader adoption of this therapeutic approach. Among the most general challenges is a problem common to all anticancer therapy—tumor heterogeneity, particularly the variance in surface expression of tumor antigens. Then there is the tendency of tumor cells to "evolve." Anticancer therapy can be stymied by persistence of dormant, nondifferentiated, and non-antigen-expressing progenitors (stem cells or otherwise) waiting to emerge when competitors are cleared. In a striking case of two complete responses in childhood acute lymphocytic leukemia that were achieved using anti-CD19 CAR constructs, one of the patients relapsed with blast cells that were CD19-negative, no longer expressing the target.[14] Other challenges to CAR T-cell therapy include the toxicity of cytokine release syndrome, cross-reactivity, and autoimmune consequences, as well as the formidable logistics and expense of this complex, patient-specific therapy.

Use of CARs to treat solid tumors presents daunting challenges not present in hematologic malignancies. The size alone of solid tumor masses, typically millions to billions of times larger than separate single, floating cells, has long been a barrier to immune strategies. T-cell immunity, often metaphorically described as "soldier" cells engaged in cell-to-cell combat, would, in the case of CARs and solid tumors, be the equivalent of sending foot soldiers to destroy a massive fortress. Within such a mass, moreover, local factors would include impaired vascular access and a different milieu within areas of central tumor necrosis. Even if CARs could find their way inside these masses, differences in such metabolic parameters as local pH and O2 content could vastly alter antigen affinity. Altered biochemical mechanisms ranging from cytokine release and action to cellular and protein interactions could neutralize CAR effectiveness. Assuming that these towering obstacles could be overcome, the CAR methodology might find a useful adjunctive role of "cleaning up" residual disease after surgical or chemical debulking.

The lack of constancy, homogeneity, and uniformity of cells and targets has been the bane of oncologic approaches in solid tumors. We have seen dramatic responses, frequently complete remissions—even in that most lethal beast, small cell lung cancer; unfortunately, even after complete remissions, chemoresistant cells pop up, multiply, and rapidly overpower the patient. We see hormone-resistant tumors emerge after early success with hormonal therapy for breast and prostate cancers. Even with hematologic malignancies, which appear to uniformly express CD19, we have seen relapse following the emergence of CD19-negative leukemic cell populations after CAR T-cell therapy.[13,14] Cancer is a dynamic and feisty opponent.

CARs and TRUCKs

The recognition of cancer cell variants that do not express the desired target has led to an exciting strategy utilizing so called TRUCKs (T cells Redirected for Universal Cytokine Killing). To construct TRUCKs, CAR T cells are engineered with the additional capacity to induce IL-12 production; activation of CARs by specific antigen recognition causes release of IL-12 which, in turn, attracts immune cells, including natural killer cells and macrophages, to attack tumor cells. Heterogeneous tumor cells in the vicinity, which lacked the antigen and were therefore invisible to CAR recognition, can now be destroyed.[11] Preclinical data are encouraging and clinical trials using TRUCKs have commenced.

Toxicities observed with CAR T cells might be considered relatively modest compared with classic cytotoxic agents and sometimes with targeted biological small drugs such as the tyrosine kinases. However, the toxicities associated with immune storms or cytokine release syndromes are not trivial and indeed may be life-threatening. One of the perennial challenges in trying to ameliorate side effects and symptoms of all immune-based therapy remains: identification and separation of mechanisms responsible for unnecessary toxicity from those that are crucial for the intended therapeutic effect.

Beyond the expected risks of exaggerated, generalized immune reactions, severe damage has been attributable in some instances to attack upon the right antigen in the wrong place. In one of the few solid tumor studies using CAR T cells, CARs targeting HER2/neu were used to treat a patient with antigen-expressing metastatic colon cancer.[15] Rapidly following infusion of the CAR T cells, the patient experienced progressive and severe respiratory distress and died within 5 days despite intensive medical care. Evaluation of circulating cytokines suggested a cytokine storm, which researchers speculated to be triggered by the presence of antigen on pulmonary epithelial cells. This effect was analogous in some respects to the well-recognized cardiac toxicity of trastuzumab when its use against breast cancer leads to inadvertent binding to HER2 antigen on cardiac muscle.

The Road Ahead

Overall, CAR T cells represent advancement toward therapy with greater specificity and efficacy and less toxicity. The processes required to generate CARs remain logistically complex, individualized, confined to a narrow tumor spectrum, and expensive. However, efforts are under way to eliminate the need for patient-specific T cells, with the ultimate goal of developing universal, off-the-shelf T-cell products.[16]

Just as major advances against solid tumors (including testicular and ovarian cancers) came with the advent of combination chemotherapy, so too may we anticipate combined immunologic approaches. Already, different tyrosine kinase inhibitors are being combined, and preclinical work has shown evidence of increased therapeutic effect when CAR T cells are given in conjunction with antibody to PD-1.[17]

The current surge of novel immune-based therapies, particularly the engineering feats of CARs and TRUCKs, have raised hopes for cancer immunotherapies, but researchers and clinicians have many miles to go on the road ahead.

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