New approaches to engineering allogeneic stem cell grafts are starting to reduce the toll of recurrent hematological malignancies and graft-versus-host disease (GVHD). In vivo and ex vivo graft engineering can help maintain engraftment and infection protection while augmenting anti-tumor effects and reducing the reliance on systemic conditioning.

“Graft engineering is a growing field right now and quite exciting,” said Marie Bleakley, MD, PhD, MMsc, professor and director of cellular therapy and transplantation for pediatric leukemia and the Gerdin Family Endowed Chair for Leukemia Research, Fred Hutchinson Cancer Center.

Bleakley, one of the co-chairs of the 2025 Tandem Meetings Scientific Organizing Committee (SOC), moderated the Feb. 13 session Plenary: Graft Engineering to Reduce Relapse of Hematological Malignancies. The list of engineering approaches is long and growing, she noted. Early success has been reported using cytokine manipulation; natural killer (NK) cell activation; bi-specific engagers; genetically modified lymphocytes using CAR T cells, T cell receptor therapy (TCR T), and more; gene/baseline/prime editing of T cells and other components; and antibody-drug conjugates.

“Not all types, and certainly not all patients, benefit from transplantation,” said Simone Minnie, PhD, postdoctoral research fellow, Fred Hutchinson Cancer Center. “Why is that and what can we do about it?”

The answers to why lie in biology. Allogeneic stem cell transplantation changes immune surveillance in important ways. CD8+ T-cell exhaustion can appear, reducing tumor-killing capacity. The trajectory of T-cell exhaustion is not the same in all malignances or grafts, complicating the picture.

CD8 T-cell phenotype is driven largely by alloantigen, Minnie said. That means immunotherapy can be highly effective following autologous stem cell transplant but only drives GVHD after allogeneic grafting.

Calcineurin inhibitors broadly block T-cell exhaustion in all stages, at least in mouse models, but can also promote alloantigen activity. Post-transplantation cyclophosphamide (PTCy) enables tumor-driven CD8 T-cell exhaustion while decoy resistant interleukin-18 (DR-18) is a potent T-cell activator and enhances graft versus myeloma in some models. DR-18 can also augment CD19 CAR T-cell therapy.

Minor histocompatibility (minor H) antigens are the edge of translational research. Minor H antigens occur in most grafts and can create either rampant leukemia or GVHD depending on multiple factors, Blakely said.

Ubiquitous or epithelial expression favors GVHD, hematopoietic expression favors graft versus leukemia (GVL). A diverse minor H community favors GVHD, lower diversity favors GVL. The presence of numerous T cell clones favors GVHD, while fewer clones favor GVL.

It is not clinically practical to remove all T cells, Blakeley added, but it is possible to deplete specific T-cell types to improve graft activity and persistence. Stem cells and memory T cells are biologically important and key to infection protection, but naïve T cells (Tn) are specific for minor antigens and can be selectively depleted.

Mouse models supported the efficacy of Tn(CD45RA) depletion to reduce GVL. A proof-of-concept trial in 14 patients using a TCR T that was half CD8+ and half CD4+ T cells depleted of Tn cells after hematopoietic stem cell transplant (HCT) showed positive safety and efficacy. The phase 1 trial is scheduled to reopen in March.

Graft engineering is also moving into the CD19 world. The U.S. Food and Drug Administration (FDA) has approved five anti-CD19 CAR T-cell constructs, noted SOC Co-chair Melody Smith, MD, MS, assistant professor of blood and marrow transplantation and cellular therapy,  Stanford University. Three of the constructs are for B-cell acute lymphoblastic leukemia (B-ALL). All are effective, and half of patients relapse regardless of the construct used.

A bi-specific CD19/CD22 CAR T-cell therapy showed 100% response and 88% complete response, but patients still showed progression about five months after treatment. In vivo T-cell depletion with PTCy following allogeneic HCT is still needed to improve relapse rates and times.

Regulatory T cells (Tregs) suppress alloreactivity, which led to the Orca-T engineered allograft that combines infusion of hematopoietic stem and progenitor cells (HSPCs) and Tregs on day 0 followed by conventional T cells on day 2. Recipients showed low rates of GVHD.

The next step was to combine Orca-T and allogeneic CD19/CD22 CAR T cells for B-ALL. A phase 1 trial in 14 patients showed no acute or chronic GVHD. All of the patients were alive at the latest visit, including two at 24 months. Twelve of the 14 were negative for minimal residual disease at their last follow up visit. The median follow up was 304 days, including six patients with more than 12 months of follow-up.

There was no prolonged cytopenia, Smith reported, and donor CD19/CD22 CAR-T cells persisted beyond one year.

This and other sessions at the 2025 Tandem Meetings | Transplantation & Cellular Therapy Meetings of ASTCT® and CIBMTR® will be available for on-demand viewing for registered attendees following the live presentation.