Symposium Abstracts

Toward Advance in GVHD Therapy

Bruce Blazar, M.D.
University of Minnesota

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is hindered by GVHD. Regulatory T cells (Tregs) can reduce GVHD in mice and patients but may be variably suppressive in vivo even at high doses. To enhance suppressor function, murine Treg were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated hCD19 transgenic, hemizygous, allogeneic recipients. Compared to recipients receiving controlled transduced Tregs, those receiving hCAR19 Tregs had a significant decrease in acute GVHD. GVHD amelioration was accomplished without abrogating anti-tumor effects, as murine hCD19 and luciferase transduced B-cell lymphoma cells (hCD19+TBL-12luc) were both cleared by allogeneic hCAR19 Tregs. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. In vivo, perforin-/- CAR19 Tregs were inferior to wildtype Tregs in mediating GVL in lethally irradiated recipients of semi-alloHSCT bone marrow, Tconventional cells and hCD19+TBL12 cells. Further, cyclophosphamide treated hCD19 hemizygous mice given hCAR19 CTLs without allo-HSCT experienced rapid lethality due to cytokine release syndrome, whereas hCAR19 Tregs avoided this complication and suppressed hCAR19 CTL side-effects. CAR19 Tregs are a novel and effective strategy to suppress GVHD without GVL loss.

Broadening Applicability of Antigen-Specific T Cells Post-BMT

Catherine Bollard, M.D., M.B.Ch.B.
Children's National
George Washington University

Cell therapy performs an important role to prevent and treat relapse as well as virus disease after transplant.  Outside of T-cell engineering using chimeric antigen receptors and artificial T-cell receptors, T-cell therapies in the BMT setting have utilized ex vivo expanded antigen-specific T-cells targeting viral antigens and non-viral tumor-associated antigens. The application of antigen-specific T cell therapeutics post BMT is evidenced by donor lymphocyte infusion strategies, selective depletion and ex vivo expansion of antigen-specific T cells. In this presentation we will discuss how antigen-specific T-cell therapy targeting virus and/or tumor associated antigens may contribute to enhanced overall survival in the BMT setting and how such approaches have broadening applicability in the field. Specifically, bench to bedside studies that have utilized multi-antigen specific T cell therapeutics post BMT including in the multi-center setting will be discussed and reviewed.

T-Cell Genome Editing for Cancer Immunotherapy

Chiara Bonini, M.D.
University Vita-Salute San Raffaele
IRCCS Ospedale San Raffaele

Adoptive T cell therapy (ACT) is a therapeutic modality largely indebted to the field of hematopoietic stem cell transplantation. In this setting, allogeneic donor T cells infused with the graft promote clinical responses against hematological malignancies. At its dawn, ACT solely relied on tumor-specific T cells isolated from the tumor masses and expanded in vitro, and feasibility was limited. The development of genetic engineering technologies and, more recently, of genome editing tools dramatically changed the landscape of ACT, rapidly making this treatment accessible to an unprecedent number of patients and tumor types. By inserting a chimeric antigen receptor (CAR), or an exogeneous tumor reactive T cell receptor (TCR) into patient’s T cells, the specificity could be precisely redirected toward selected tumor antigens. This new opportunity shifted the research focus and raised up novel questions: the main issue was no more how to harvest a sufficient number of tumor-specific T cells from each single patient, but how to isolate design and combine tools to proficiently generate and expand the most fit engineered T cells for each target disease. The selected tools and protocols should ideally allow T cells to infiltrate the tumor mass, to recognize relevant tumor antigens, to survive and resist the immunosuppressive signals present in the tumor microenvironment and to persist as memory cells, to patrol the organism for recurrence. Challenges and opportunities towards the generation of optimal T cell therapy products will be discussed.

Advanced TCR Engineering for Repertoire Analysis and Adoptive T Cell Therapy

Dirk Busch, Ph.D.
Technical University of Munich (TUM) School of Medicine

Effective antigen-specific T cell immune responses are usually characterized by the initial recruitment of polyclonal populations from the naïve compartment, consisting of a repertoire of different T cell receptors (TCRs) with varying affinities/avidities for cognate pMHC ligands. The underlying mechanisms of recruitment and maintenance of polyclonality, as well as its relevance for the quality of immunity during acute and chronic diseases, as well for immune memory are not well understood. This is mainly due to technical hurdles in studying functionally ‘polyclonality’ in preclinical experimental models as well as directly in humans.

By implementing and developing advanced technologies (like single cell TCR sequencing/identification, traceable TCR retrogenic T cells, Crispr-Cas9-mediated orthotopic TCR replacement, structural and functional TCR avidity measurement, high-throughput TCR reporter cell screening) we are now in a position to analyze T cell polyclonality in hitherto unmatched detail. First findings during infections and anti-tumor responses demonstrate that T cells with high-avidity antigen receptors are rare within the naïve repertoire and initially a much larger fraction of low-avidity T cells gets recruited until high-avidity T cells dominate the acute response. During chronic antigen-exposure, evolutionary changes occur and lower-avidity T cells can become the prevalent subset.

We believe that a better understanding of T cell polyclonality during normal physiological immune responses in health and disease will not only help to improve our understanding of immunity and immune pathology, this knowledge will also be relevant for the engineering of most effective T cell products (including CAR-T cells) for adoptive T cell therapy.

Improving Adoptive T Cell Therapies With Synthetic Immunology

Justin Eyquem, Ph.D.
University of California, San Francisco

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Non-toxic delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knock-in efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knock-ins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knock-out screen and identified QA2 as the primary receptor for Ark313. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR/Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables pre-clinical modeling of Trac targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens new avenues in experimental T cell immunology.

Translating Innate Natural Killer Cell Memory as Cancer Immunotherapy

Todd A. Fehniger, M.D., Ph.D.
Washington University

Natural kill (NK) cells are innate lymphoid cells that protect from infection, mediate anti-cancer immune responses, and contribute to the graft versus leukemia effect in hematopoietic cell transplantation. They decide to respond to a target cell based on integration of input from activating, inhibitory, and cytokine receptors. Distinct from adaptive immunologic memory, NK cells exhibit innate memory-like responses following brief, combined activation with interleukins 12, 15 and 18. The biology of memory-like NK cells includes improved multifunctional responses, better recognition of cancer target cells, and prolonged in vivo persistence. Mechanistically, this biology results from a distinct transcriptional and epigenetic state.  Memory-like NK cells were translated to the clinic within several clinical trials, demonstrating an excellent safety profile, and reporting complete remissions in patients with acute myeloid leukemia. Allogeneic adoptive transfer of a single dose of memory-like NK cells results in persistent macro chimerism for up to 3 weeks, providing an “off the shelf” cellular therapy amendable to multiple dosing. In contrast, when memory-like NK cells are administered as a single dose in an immune compatible setting, they functionally persist for > 3 months. Pre-clinical studies have demonstrated that memory-like NK cells also exhibit superior ADCC and responses to solid tumors, compared to conventional NK cells. CAR-engineered memory-like NK cells also exhibit enhanced responses to NK-resistant cancers. Thus, memory-like NK cell differentiation provides platform for further innovation to improve multiple aspects of NK cellular therapy.

The Development and Applications of Adoptive T Cell Therapy

Carl June, M.D.
University of Pennsylvania

Advances in the understanding of basic immunology have ushered in two major approaches for cancer therapy over the past 10 years. The first is checkpoint therapy to augment the function of the natural immune system. The second uses the emerging discipline of synthetic biology and the tools of molecular biology and genome engineering to create new forms of engineered cells with enhanced functionalities.  The emergence of synthetic biology approaches for cellular engineering provides a broadly expanded set of tools for programming immune cells for enhanced function. Barriers to therapy of solid tumors will be discussed.

From Bench to Bedside to the FDA: T Cell Costimulation Blockade for the Prevention of Acute GVHD

Leslie Kean, M.D., Ph.D.
Dana-Farber Cancer Institute

The Kean laboratory has studied immune tolerance-induction and the impact of T cell costimulation blockade on immune tolerance for over a decade. This work has included preclinical large animal studies and clinical trials. Our preclinical work underscored the potential efficacy of CD28:CD80/86 blockade with CTLA4-Ig in controlling acute GVHD (AGVHD). On the strength of this preclinical data, and also based on work performed by the Storb and Blazar laboratories in canine and murine models, we launched a series of clinical trials designed to determine whether the abatacept formulation of CTLA4-Ig could prevent AGVHD. Based on these trials and on Real World Evidence from the CIBMTR, abatacept was recently FDA-approved for AGVHD prevention. The Phase 2 trial of abatacept for AGVHD prevention (‘ABA2’) demonstrated a significant decrease in Grade 2-4 AGVHD after both 8/8 and 7/8 unrelated-donor hematopoietic stem cell transplantation in patients prophylaxed with CNI/MTX + abatacept versus those receiving CNI/MTX alone. The randomized double-blind, placebo-controlled design of the 8/8 arm has enabled a rigorous determination of the immunologic drivers of the success of abatacept in preventing AGVHD. Combined flow cytometric and transcriptomic analysis have provided compelling evidence that T cell allo-proliferation was a harbinger of Gr 2-4 AGVHD onset in placebo patients, and that abatacept was able to control this proliferative escape. Unlike the proliferation-dominated breakthrough AGVHD profile in placebo patients, RNA-Seq revealed a unique AGVHD signature in ABA patients. Thus, patients who developed Gr 2-4 AGVHD despite abatacept exhibited a GSEA gene expression profile in both CD4+ and CD8+ T cells enriched for  naïve T cell gene sets, prominently including IL6 signaling pathway transcripts. These data underscore the biologic and clinical impact of CD28:CD80/86 costimulation blockade on AGVHD, and point us toward future studies to enhance immune tolerance after transplantation.

Hematopoietic Stem Cell Gene Therapy

Donald Kohn, M.D.
UCLA Broad Stem Cell Research Center

Treatment of inherited blood cell diseases using autologous transplantation of gene-modified stem cells (gene therapy) has been advancing over the past 3 decades.  Adenosine deaminase Severe Combined Immune Deficiency (ADA SCID) was the first disorder approached by gene therapy.  In studies done in collaboration with investigators at University College London/Great Ormond Street Hospital (UCL/GOSH), we treated 50 ADA SCID patients with the EFS-ADA lentiviral vector (LV) and busulfan reduced intensity conditioning (RIC)h busulfan. 48/50 (96%) achieved sustained engraftment of ADA gene-corrected stem cells with immune reconstitution.  Similar high frequencies of immune reconstitution have been achieved in a trial using a LV and RIC for X-linked SCID (XSCID) in a trial performed at UCL/GOSH, Boston Children’s Hospital and UCLA with all 11 patients achieving sustained immune reconstitution. We have also performed clinical trials of gene therapy for two disorders of neutrophil dysfunction, X-linked Chronic Granulomatous Disease (XCGD) and Leukocyte Adhesion Deficiency I (LAD I).  Both trials used the same LV backbone with a chimeric myeloid enhancer/promoter driving expression of the relevant cDNA (CYBB and ITGB2, respectively) and cytoablative busulfan conditioning (target AUC 65-75 mg/L*hr.).  While the 5 adult patients with XCGD achieved sustained engraftment of gene-corrected HSC with >10% oxidase (DHR)+ neutrophils and absence of subsequent opportunistic infections, all 4 pediatric patients suffered significant decline in gene-marked cells in peripheral blood cells and BM CD34, 3-6 months after gene therapy, stabilizing at ~0.5% DHR+ neutrophils.  The basis for the decline in gene-marked cells in the pediatric patients is unknown.  In contrast, all 9 of the LAD I patients, treated between 0.5-9 years of age, have shown stable persistence of gene marked blood cells and ~20-70% CD18-expressing neutrophils.  We are currently investigating the use of adenine base editing in HSC to correct a founder mutation  in the CD3D gene that causes SCID in a Mennonite population; base editing in HSC is highly efficient and restores the ability of the corrected HSPC to produce mature T lymphocytes in vitro in an Artificial Thymic Organoid system.  These studies demonstrate the potential to apply HSC gene therapy for the treatment of blood cell diseases.  

Programming Cell Therapies with CRISPR

Alexander Marson, M.D., Ph.D.
University of California, San Francisco

The goal of the Marson lab is to understand genetic circuits that control human immune cell function in health and disease. We have begun to identify autoimmunity risk variants that disrupt immune cell circuits, and how pathogenic circuits may be targeted with novel therapeutics. Our lab has developed new tools for CRISPR genome engineering in primary human T cells. We are now pursuing a comprehensive strategy to test how coding and non-coding genetic variation controls functional programs in the immune system. Genome engineered human T cells hold great potential for the next generation of cell-based therapies for cancer, autoimmunity, and infectious diseases.

Advanced Genetic Engineering of Hematopoiesis To Treat Inherited Disease and Cancer

Luigi Naldini, M.D., Ph.D.
San Raffaele Telethon Institute for Gene Therapy,
University Vita-Salute San Raffaele 
IRCCS Ospedale San Raffaele

Hematopoietic stem cells (HSC) gene therapy (GT) by lentiviral vectors is providing substantial benefit to patients affected by primary immunodeficiencies, hemoglobinopathies and storage disorders. Long-term follow up shows stable hematopoietic reconstitution by high numbers of corrected HSC without signs of clonal expansion or exhaustion, providing a reassuring molecular picture underlying the long-lasting clinical benefit.

Precise engineering by gene editing may further improve the safety of HSC GT by achieving in situ gene correction or targeted transgene integration. We reported the first targeted gene editing of human HSC followed by studies highlighting barriers limiting its efficacy and novel strategies overcoming them. Homology-driven repair, however, remains limiting. We now report that the choice of template can increase efficiency and safety of the procedure. Moreover, the emergence of base and prime editors that minimize or bypass the requirement for DNA DSB allows editing single/few mutant nucleotides with limited activation of DNA damage response.

Another long-sought goal of HSC GT is to make space for the infused cells without relying on genotoxic conditioning, which entails acute and chronic serious adverse effects. We report that HSC mobilization opens a window of opportunity for engraftment of donor cells, which can effectively outcompete those in the circulation for engraftment in the depleted niches. Competitive advantage results from the rescue in culture of a detrimental impact of mobilizing agents on HSC and can be further enhanced by transient over-expression of engraftment effectors. These findings were obtained in mouse models of diseases to prove their therapeutic potential and in human hematochimeric mice to validate them for human HSC.

Overall, our work should advance HSC GT by a combination of transformative approaches leveraging on precision genetic engineering while alleviating the morbidity of the procedure, broadening application to several diseases and patients worldwide. 

Generation of Engineered Tregs and B Cells for Novel Therapeutic Applications

David Rawlings, M.D.
Seattle Children's Hospital
University of Washington

We are using homology-directed repair (HDR)- based gene editing to generate novel cell therapies including engineered T regulatory cells (EngTreg) and drug-secreting plasma B cells (PCs). While Treg therapy may provide a transformative approach to restore tolerance, key technical hurdles that limit application include: Treg rarity, lack of specificity, and in vivo competition. Introduction of a constitutive promoter into FOXP3 converts CD4 T cells into EngTreg with robust suppressive activity. Expanding on this concept, we utilize dual-HDR editing to simultaneously drive FOXP3 and introduce a tissue-Ag specific TCR and a signaling complex that mimics IL-2. Islet-antigen specific EngTreg, generated via this approach, exert bystander suppression of polyclonal T1D Teff and block disease in animals; supporting use of this platform across tissue specific immune diseases. Similarly, we utilize gene editing in primary B cells and in vitro differentiation to generate long-lived PCs capable of sustained engraftment and exogenous protein production in vivo, supporting future use of this approach for a broad range of therapies.

Tissue Metabolic Adaptations in Regulation of Immunotoxicities

Pavan Reddy, M.D. 
Baylor College of Medicine

Gastrointestinal microbial dysbiosis regulates immune mediated diseases, in part through the regulation of host metabolism. However, the mechanism for dysbiosis and whether it is a cause, a consequence, a regulator or a marker is unclear. The immune target cell intrinsic mechanisms that regulate their sensitivity to immune and inflammatory effectors also remains poorly understood. Recent insights suggest that in T cell mediated intestinal diseases such as GVHD and IBD, the intestinal target cells (IECs) metabolism is altered and that impacts the threshold of their sensitivity to T cell mediated cytotoxicity. The IECs demonstrate reduction in the function of mitochondrial complex II that precludes appropriate utilization of oxygen. The impact of the IEC metabolic defect on the microbiome will be presented. Strategies that regulate the impact of metabolic reprogramming and promote a milieu that facilitates healthy microbiome will be discussed. 

Local Maintenance of GVHD Within Target Tissues

Warren Shlomchik, M.D. 
University of Pittsburgh Cancer Institute

In allogeneic hematopoietic stem cell transplantation, donor ab T cells attack recipient tissues, causing graft-vs-host disease (GVHD), a major cause of morbidity and mortality. A central question in the field has been how GVHD is sustained despite T cell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of new alloreactive effectors from blood into affected tissues. Here we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1203 alloreactive T cell clones we fit a mathematical model predicting that within each tissue a small number of progenitor T cells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that the narrow targeting of subpopulations of tissue-resident alloreactive T cells—preventing their development, their early trafficking to GVHD target tissues or inhibiting their function once established—could be effective with less global immunosuppression than occurs with current approaches.

Allogeneic Hematopoietic Cell Transplantation: From Lab to Clinic and Back

Rainer Storb, M.D.
Fred Hutchinson Cancer Center

After 60 years of investigations into the use of allogeneic hematopoietic cell transplantation, this procedure has progressed from one that was thought to be plagued with insurmountable complications to a standard treatment for many serious hematological diseases. How have these complications been overcome and how was the therapy expanded to include patients who were too old or medically infirm to tolerate conventional transplantation approaches?

The Role of the Intestinal Microbiome in Cancer Immunotherapy

Marcel R. M. van den Brink, M.D.
Memorial Sloan Kettering Cancer Center

Intestinal microbiomes and their mammalian hosts have co-evolved, resulting in mutualistic interactions that affect health and disease. A role for the gut microbiota in the development of graft-versus-host disease (GVHD) after Allogeneic Hematopoietic Cell Transplantation (allo-HCT) was first described in the 1970s by van Bekkum, who demonstrated that mice kept under germ-free (GF) conditions exhibited less mortality from acute GVHD than conventionally housed animals. In recent years several studies have demonstrated that the intestinal microbiome can modulate cancer immunotherapy, especially check point inhibition. We began our investigation into the role of the intestinal microbiome in alloin 2009 using next-generation sequencing. We have demonstrated a relationship between microbiota composition and clinical outcomes, including acute GVHD, chronic GVHD, infection, engraftment, relapse, and immune reconstitution. We will briefly highlight some of the main findings of a few of these studies.

In a study of 8,767 fecal samples obtained from 1,362 patients from four centers we found that patterns of microbiota disruption, characterized by loss of α-diversity during allo-HCT were similar across transplantation centers and geographic locations. Higher diversity of intestinal microbiota both before transplantation and at the time of neutrophil engraftment was associated with a lower risk of death in independent cohorts. After allo-HCT, almost all patients incur a period of microbial domination by certain bacteria, most frequently Enterococcus, which is associated with higher risk of acute GVHD-related mortality and shortened overall survival (OS). We found similar expansion of Enterococcus in GVHD mouse models and demonstrated in gnotobiotic models that Enterococcus administration aggravates acute GVHD. We observed a) enterocyte damage resulting in less expression of lactase and the anti-microbial protein Reg3g/b (which controls Enterococcus) and b) dysbiosis characterized by contraction of salutary commensal flora, all contributing to alloreactivity. Enterococcus growth is dependent on the disaccharide lactose, and we demonstrated that withholding dietary lactose attenuates Enterococcus outgrowth and reduces the severity of GVHD in mice. Finally, allo-HCT patients with a lactase-deficiency genotype showed compromised clearance of post-antibiotic Enterococcus domination.  

We also analyzed 1,161 fecal samples collected from 534 autologous HCT recipients at two transplantation centers and found that, similar to allo-HCT, high fecal intestinal diversity in the peri-engraftment period was associated with longer progression-free and overall survival.

We expanded our analyses of microbiome to outcomes after therapy with chimeric antigen receptor (CAR) T cells. We found in a retrospective cohort of 228 patients from two centers that exposure to broad-spectrum antibiotics within the 4 weeks prior to anti-CD19 CAR T cell infusion was associated with shorter overall survival and increased risk of neurotoxicity. Importantly, we also identified species within the class Clostridia that were associated with day-100 complete response.

In our recent unpublished studies in mouse and man we have found relationships between diet, drugs (also non-antibiotic drugs) and bile acids and the composition of the intestinal microbiome and clinically relevant outcomes after allo-HCT.

In conclusion, the concordance of microbiota disruption patterns and their associations with clinical outcomes suggests that approaches to manipulate the intestinal microbiota could improve cancer immunotherapy, such as allo-HCT and CART cell therapy.

Updates on Personalized Cancer Vaccines

Catherine Wu, M.D.
Dana-Farber Cancer Institute

Multiple lines of evidence have convincingly demonstrated tumor neoantigens as an important class of immunogenic tumor antigens, and have motivated the development of personalized cancer neoantigen targeting approaches. Neoantigens arise from amino acid changes encoded by somatic mutations in the tumor cell and have the potential to bind to and be presented by personal HLA molecules.  As a field, we have now successfully moved beyond the first wave proof-of-concept studies that have demonstrated the safety, feasibility and high immunogenicity of these personalized vaccines.  An imperative now is to address the challenges of discovering and optimizing the selection of antigens to target, the delivery approach, and extending this promising approach to a broader array of cancer settings.