Core Spotlight: Cell Manipulation

December, 2017

In this DF/HCC News Spotlight Edition, we present the Connell & O’Reilly Families’ DF/HCC Cell Manipulation Core Facility (CMCF), which produces safe and effective cellular components for subjects enrolled in novel therapeutic clinical research protocols and standard of care cellular therapies. In this context, the Core supports translational research and works closely with members in the Cancer Immunology Program and multiple Clinical Programs spanning Bone Marrow Transplantation, Lymphoma, Myeloma, and Leukemia as well as multiple solid malignancy services. DF/HCC users of core services work in diverse areas, including hematopoietic stem cell transplantation, cancer vaccines, adoptive cellular therapies for cancer, and even non-oncologic regenerative medicine.  There are no equivalent academic facilities in New England that can provide the unique services that the CMCF contributes in support of clinical research. 

Recent Updates: 

Currently, the CMCF releases over 1200 standard and research products a year, under more than 40 clinical trials which are operated under more than 15 FDA INDs.  Recent initiatives have been the introduction of genetically-modified therapies including Chimeric Antigen Receptor (CAR) T cells and tumor vaccines, use of other Immune Effector Cells such as regulatory T cells to induce immune tolerance, and forays into mesenchymal stem cell and induced pluripotent stem cell production to support organ healing and regeneration.

Most excitingly, this rapid growth in cell therapy demand is expected to continue and will be enabled by the opening of DFCI’s new CMCF processing facility on the 11th and 12th floors of the Smith Building expected in May, 2018.  This advance will triple current manufacturing space, centralize other cell processing infrastructure, and bring the lab into closer proximity to gene-therapy researchers. 

Key Services: 

The cellular components produced by the Core currently fall into three major categories:

Cellular Product Diseases Targeted
Routine HPC Products or Selected Subsets Lymphoma, Leukemia, Myeloma
Genetically-modified Stem Cells Congenital Immune Deficiencies
CAR T Cells Lymphoma, Leukemia, Myeloma
Engineered TCR Cells Sarcoma
Tumor-Based Vaccines Leukemia, Colorectal and Ovarian Cancer
Dendritic Cell Vaccines Prostate Cancer, Glioblastoma
Dendritic, Natural Killer and Cytotoxic T Cells Leukemia, EBV-related Cancers, CMV
Mesenchymal Stromal Cells Neurodegenerative Disease, Kidney or Respiratory Failure
Limbal Stem Cells Corneal Blindness
Induced Pluripotent Stem Cells Parkinson's Disease, Diabetes Mellitus

Project Highlights

DF/HCC PROTOCOL 12-217 (BB-IND 7248)

Title: A Randomized Placebo-controlled Phase II Trial of Irradiated, Adenovirus Vector-transfected GM-CSF-secreting Autologous Leukemia Cell Vaccination (GVAX) Versus Placebo Vaccination in Patients with Advanced MDS/AML after Allogeneic Hematopoietic Stem Cell Transplantation
PI: Vincent Ho, Stem Cell Transplantation.

The early post-transplant period after allogeneic hematopoietic stem cell transplantation (HSCT) is characterized by severe lymphopenia and intense homeostatic proliferation of donor T, B and NK cells. Dr Ho and colleagues hypothesized that administration of autologous tumor vaccines during this period would elicit strong leukemia-specific responses from the donor immune cells. 

Two previous Phase I non-randomized studies were conducted at DFCI to examin the safety of autologous AML/GVAX in patients with refractory MDS, AML or advanced CML when administered early after non-myeloablative allogeneic HSCT. These trials confirmed the safety and immunogenicity of this approach with no increase in acute or chronic GvHD and durable complete responses were seen in high-risk subjects, suggesting potentiation of a graft-versus-leukemia effect. In the current study, autologous cellular vaccines are generated in the CMCF by adenoviral vector-mediated GM-CSF gene transfer into myeloblasts harvested prior to HSCT. After engraftment, patients begin a series of 6 vaccinations with lethally irradiated ad-GM-CSF infected autologous leukemia cells (Figure, upper right) or placebo in a blinded fashion. The CMCF has successfully manufactured autologous AML vaccines or placebo for all subjects enrolled to date. Planned accrual is 152 patients and the study is ongoing. Similar products have been manufactured for trials involving patients with colorectal cancer, chronic lymphocytic leukemia, and follicular lymphoma.

Publication: Ho V, Vanneman M, Kim H et al., Proc Natl Acad Sci USA 2009

DF/HCC PROTOCOL 12-306 (BB-IND 15368)

Title: A Phase I Trial of a Dendritic Cell Activating Scaffold Incorporating Autologous Melanoma Cell Lysate (WDVAX) in Metastatic Melanoma Patients.
PI: F.S. Hodi, Melanoma

Cancer immunotherapy is progressing at a rapid pace, particularly in melanoma where trials of CTLA-4 and PD-1/PDL-1 monoclonal antibody blockade have demonstrated clinical benefit. In this setting, tumor regression is mediated by endogenous T cells in the tumor microenvironment that are specific for antigens expressed by the patient’s tumor.
Immunogenic biodegradable scaffold

Immunogenic biodegradable scaffold

To optimize the activation of endogenous T cells in the tumor microenvironment, investigators in the Cancer Immunology Program collaborated with investigators at the Wyss Institute for Biologically Inspired Engineering at Harvard to create a novel immunogenic biodegradable scaffold that effectively induced T-cell tumor immunity and regression of well-established melanoma in murine model systems. This novel approach is now being explored in a “first-in-human” clinical trial of WDVAX technology in patients with advanced melanoma.

This vaccine is compounded at the CMCF of a subcutaneously implanted porous, poly-lactideco-glycolide (PLG) biodegradable polymer scaffold loaded with autologous melanoma necrotic cell lysate, human GM-CSF and CpG oligodeoxynucleotide, a TLR9 agonist (Picture). The Phase I clinical trial has enrolled 3 cohorts of patients with 5 participants in each cohort and is currently enrolling intothe expansion cohort.

Each patient is scheduled to receive 4 vaccine implants with decrease of intervals between scaffold implantation from 4 weeks in dose level 1 to 2 weeks in dose level 3. The CMCF has successfully manufactured vaccine scaffolds for 22 patients and impressive local immune responses have been observed in patients who have completed all administrations. 

Publication:  Oral Abstract Presentation - Daley, Negre, Kim et al., International Society for Cellular Therapy Regional Meeting, Memphis, TN, 2016.

DF/HCC PROTOCOL 13-281 (BB-IND 15640)

Title: A Phase I Trial of Regulatory T-cells plus Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-versus-Host Disease
PI: J. Koreth, Stem Cell Transplantation 

Allogeneic hematopoietic stem cell transplantation (HSCT) has curative potential in advanced/aggressive hematologic malignancies, but chronic graft-vs-host disease (cGVHD) arising from inappropriate allo- and auto-immune activation remains a major source of morbidity and mortality in HSCT survivors. Regulatory T-cells (Treg) are naturally evolving CD4+CD25+FOXP3+ lymphocytes capable of controlling adaptive and innate immune responses and dominantly suppressing autoreactivity. Previous DF/HCC clinical trials have demonstrated that daily administration of low-dose interleukin-2 (IL-2) to patients with active chronic GVHD results in the selective expansion of CD4 Treg in vivo with objective clinical responses in over 50% of patients. To determine whether combining adoptive infusion of donor Treg and daily low-dose IL-2 would result in enhanced in vivo expansion of transferred Treg and improved clinical outcome, J. Korethand colleagues just completed a Phase I clinical trial to evaluate the safety and efficacy of combining these 2 approaches. In this trial, CD4 Treg are purified in the CMCF from apheresis products obtained from the original stem cell donor (Figure, Left panel). Treg purification is accomplished by depletion of CD8+ and CD19+ cells followed by positive selection of CD25+ cells (Figure, Middle panel) using monoclonal antibody conjugated magnetic beads and the Miltenyi CliniMacs device (Figure, Right panel). Following a single Treg infusion at one of 3 escalating dose level, patients begin daily subcutaneous injections of low-dose IL-2 for 8 weeks. The Core developed standard operating procedures, carried out validations of the manufacturing process, and assisted in the IND submission. The CMCF successfully provided purified CD4 Treg for all 25 patients in the study. There have been no unanticipated toxicities and clinical efficacy is encouraging. TCR sequencing of the Treg-enriched products and subsequent post-infusion blood samples is being used to track persistence and potential expansion of infused donor Treg. 

Publication: Oral Abstract Presentation - Nikiforow, Kim, Jones et al., American Society of Hematology Annual Meeting, Atlanta, GA, 2017.




DF/HCC PROTOCOL 14-386 (BB-IND 16042)

Title: A Phase 1 Study of Chimeric Antigen Receptor (CAR)-Modified T Cells Targeting NKG2D Ligands in Patients with Acute Myeloid Leukemia/Advanced Myelodysplastic Syndrome and Multiple Myeloma
PI: S Nikiforow, Stem Cell Transplantation

Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers (Figure, Panel B versus native immunologic synapse, Panel A). Based on successful murine tumor models employing T cells expressing NKG2D CARs by Charles Sentman and colleague at Dartmouth School of Medicine, this first-in-human study investigates a novel CAR fusing full-length human NKG2D with the CD3ç signaling domain (Figure, Right panel). In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but are absent or poorly expressed on healthy tissues. To determine safety of these NKG2D CAR-transduced T cells in patients with Acute Myeloid Leukemia/Advanced Myelodysplastic Syndrome and Multiple Myeloma, escalating doses from 1x106 to 1x107 cells were infused without prior lymphodepleting chemotherapy to patients with relapsed and/or refractory disease. The CMCF transduced autologous T cells stimulated via antiCD3 antibodies and interleukin 2 with a retroviral vector containing the NKG2D CAR construct described above and then expanded them to sufficient numbers over an 8-day culture period. Extensive product characterization in the CMCF Quality Control Lab and sponsor’s facility was followed by infusion of fresh cells.  Products were successfully manufactured for all 12 eligible patients enrolled and no dose-limiting toxicities were seen. This NKG2D CAR manufacturing approach was found to be feasible and well-tolerated. This study launched a larger Phase 1 multicenter trial involving both patients with hematologic malignancies and solid tumors and results are being submitted for publication.

Poster Abstract Presentation - Nikiforow, Werner, Murad et al., American Society of Hematology Annual Meeting, San Diego, CA, 2016.

For More Information:

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