Chimeric antigen receptors (CARs) combine antigen-recognizing variable regions (scFVs) from monoclonal antibodies (mAb) with intracellular T-cell signaling domains. First generation receptors utilized the CD3ζ chain of the T cell receptor (TCR) complex, while second and third generation receptors incorporate either one or two costimulatory singals, respectively, typically derived from B7 family molecules (CD28, ICOS) and/or the TNF superfamily (4–1BB, OX40, CD27). CARs are typically inserted into primary T cells via retroviral-mediated gene transfer. Image Credit: Discovery Medicine.

Clinical T Cell Modulation with CARs Holds Promise for Specific Cancer Targets

February 16th, 2016

Recent research by a variety of teams including those associated with Dr. Stanley Riddell and Fred Hutch have been working to identify novel approaches to the treatment of a specific array of cancers including treatment-resistant cases of B-cell malignancies such as acute lymphoblastic leukemia, Non-Hodgkin lymphoma and chronic lymphocytic leukemia.

“T-cells are a living drug, and in particular they have the potential to persist in our body for our whole lives.”

Chiara Bonini,

The treatments involve the collection and genetic modification of specific T-cells either from a patient’s own body or an immunologically appropriate donor. Adoptive T-cell transfer is an experimental immune-system modulation process in which specific subtypes of immune system cells undergo molecular modifications that incline them to sustained assault of specific target cancers. The process involves equipping the T-cells with CARs (chimeric antigen receptors).

“This is a living therapy. When you put it in [human bodies] the cells will undergo expansion in vivo.”

— Stanley Riddell

In an as yet unpublished trial, 27 of 29 patients with acute lymphoblastic leukemia achieved total remission. The remaining 2 perished during or shortly after treatment. This is an extrodinarily high remission rate, however, for reasons not yet clearly articulated, there are catastrophic side effects still associated with the process. Present challenges include the radical reduction of toxic side-effects and the necessity of rapidly expanding our library of cancer-expressed target molecules and T-cell receptor pairings that will allow us to more effectively target cancers of a variety of kinds.

Absent from public discussion are the possible dangers of introducing such cells into human biomes in terms of unforeseen repercussions for offspring or those who such patients may relate with. It is supposed, perhaps that any effects of the treatment ‘live and die within a single patient’, yet our advancing understandings of epigenetics and other possible non-germ-cell transports of heritability may require we revisit such concerns in the future.

The material supplied for extended reference below was excerpted from the 2016 AAAS Program Guide.

T cell immunity has evolved to recognize and respond to health threats and provide a lifelong memory that prevents recurrent disease. However, with chronic diseases, T cells often become inactive. Recent advances have brought the idea of fighting chronic infections, and even cancers, by restoring protective T cell responses much closer to reality for mainstream clinical practice. This session focuses mainly on a promising approach known as adoptive T cell therapy, in which a patient receives “killer” immune cells that target a disease agent. However, several obstacles to widespread clinical use must be addressed: identifying or generating T cells that will be most effective for each individual case, whether from the patient or from a suitable donor; avoiding or countering potential side-effects; and finding ways to shorten the path from bench to bedside. This symposium reports on progress on all three fronts. This discussion covers the current status of clinical trials; the importance of distinct T cell subsets with stem cell-like characteristics for achieving durable responses; gene therapy for providing effective antigen-receptors and minimizing side-effects; and innovations in clinical cell processing and purification that could, among other things, open the way for faster regulatory approvals.

Stanley Riddell, Fred Hutchinson Cancer Research Center, Seattle, WA

Engineering T cells for safe and effective cancer immunotherapy

Stanley Riddell, Fred Hutchinson Cancer Research Center

Recent advances in understanding the mechanisms by which cancers evade immune recognition have led to new immunotherapies that have the potential to transform the management of many human cancers. T lymphocytes are critical to adaptive immunity to pathogens and tumors because of their longevity and ability to clonally expand, and are increasingly being manipulated for the treatment of human diseases, including cancer. Advances in gene transfer methods using viral and non-viral delivery have made it possible to introduce genes into T cells that encode natural T cell or synthetic receptors that bind to molecules on the surface of cancer cells, and redirect the specificity of the T cell to mediate tumor cell destruction. Several groups, including our own have shown that the adoptive transfer of T cells that are genetically engineered to express a synthetic chimeric antigen receptor (CAR) that targets the B cell lineage molecule CD19 exert potent antitumor activity in patients with chemotherapy refractory B-cell malignances including acute lymphoblastic leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma. T cells exist in different subsets with cell intrinsic properties that affect their ability to persist and proliferate after adoptive transfer. By selecting T cells with optimal attributes for genetic modification and adoptive therapy, eradication of massive tumor burdens can be accomplished with remarkably low doses of engineered T cells, and with predictable T cell expansion kinetics and toxicity. A remaining challenge is to identify target molecules that are expressed on common human cancers and to design receptors that safely target them so that adoptive immunotherapy with genetically redirected T cells can be broadly applied.

Chiara Bonini, San Raffaele Scientific Institute, Milan, Italy

Tumor-specific T lymphocytes are rare cells, able to recognize cancer cells through their T cell receptors (TCR), and kill them. To exploit tumor-specific T cells for cancer treatment, and overcome their paucity, it is today possible to transfer in patients’ T cells genes encoding for rare tumor-specific TCR. However, such genetically modified T cells express four different TCR chains, that might mispair, leading to unpredictable toxicity and to an overall dilution of the tumor specific TCR on cell surface, thus limiting the efficacy of therapeutic cellular product.

To overcome these issues, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to alfa and beta chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes by lentiviral vectors (Provasi et al, Nature Medicine 2102). The TCR gene editing technology, proved safer and more effective than conventional TCR gene transfer in vitro and in animal studies. While successful, complete editing requires multiple manipulation steps and four transduction procedures. To reduce the duration and complexity of the procedure, we recently developed and tested the ‘single TCR editing’ (SE) approach, that allows to redirect T cell specificity in clinically suitable conditions.

We validated SE using an HLA-A2 restricted TCR specific for NY-ESO-1157–165, a cancer testis antigen expressed by several tumor types, including multiple myeloma. Conventional TCR gene transfer (TR) and SE cells were compared in terms of efficacy and safety in vitro and in vivo. SE rapidly produced high numbers of tumor specific T cells, with an early differentiation phenotype, including memory stem T cells, associated to long term survival in adoptive immunotherapy clinical trials (Oliveira et al., STM 2015). SE proved highly efficient, and similar to TR T cells, in killing multiple myeloma in vitro and in immunodeficient NSG mice. However, while TR lymphocytes proved highly alloreactive, SE cells showed a favorable safety profile. Indeed, in contrast to the TR cells, SE cells mediated tumor rejection without inducing xenogeneic graft versus host disease in NSG mice, thus promoting a significantly higher survival rate. The detrimental alloreactive effect mediated by TR cells led to a xenogeneic GvHD rate comparable to that of unmodified allogenic T cells.

Overall, the single TCR gene editing procedure provides a rapid and efficient method for generating primary T cells that highly express a tumor specific TCR and are devoid of their endogenous TCR repertoire, and thus represents a protean platform for the effective and safe adoptive transfer of allogeneic and autologous T lymphocytes redirected towards any desired tumor associated antigen.

Dirk Busch, TUM, Munich, Germany

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. The definition of optimal targets and antigen receptors as well as the differentiation status of transferred T cells are emerging as crucial parameters for generating cell products with predictable efficacy and safety profiles. Our laboratory has demonstrated that defined subsets within the memory CD8+ T cell compartment fulfill all key characteristics of adult tissue stem cells and are essential for robust and long-term maintained responses upon adoptive transfer. In experimental animal models we could show that even the transfer of a single memory stem T cell can be enough to reconstitute protective immunity, demonstrating the therapeutic power of distinct T cell subsets. In order to enrich defined T cell populations for clinical applications, we have developed clinical multi-parameter enrichment technologies, and transferred these to GMP-conform cell separation units. A first proof-of-concept clinical trial (treatment of CMV infection in patients upon allogeneic hematopoetic stem cell transplantation with donor-derived antigen-specific T cells) has just been finished and confirmed that therapeutic adoptive transfer of low numbers of highly purified antigen-specific T cells (without any further in vitro culture) is clinically safe and confers specific immunity to infection.

Infusing a T cell product containing memory stem cells can be highly effective therapeutically, but might also bear some risk of toxicity. Therefore, safeguards that allow selective depletion of transferred cells in the case of un-tolerable side effects may be needed. In this context, we explored the capacity of a truncated version of Epidermal Growth Factor Receptor (EGFRt) co-expressed with adoptively transferred T cells. EGFRt is functionally inert, as it cannot bind EGFR-ligands and lacks signaling components, and is non-immunogenic. However, it still binds to Cetuximab, an EGFR-specific antibody already used for clinical applications. We can show in a pre-clinical animal models that EGFRt-expressing engineered T cells can be effectively depleted via Cetuximab treatment in vivo. For example, B cell aplasia (lack of B lymphocytes), which is a common long-lasting side effect of CD19 CAR-T cell treatment, can be reverted by antibody-mediated in vivo depletion.