LB-ARHGDIB-1R As Novel Minor Histocompatibility Antigen For Therapeutic Application

Patients with hematological malignancies can be successfully treated with allogeneic hematopoietic stem cell transplantation (alloSCT). Beneficial Graft-versus-Leukemia (GvL) reactivity, however, is often accompanied with undesired Graft-versus-Host Disease (GvHD). In HLA-matched alloSCT, donor T-cells can mediate GvL reactivity by recognition of minor histocompatibility antigens (MiHA) on malignant cells, but also GvHD when MiHA are recognized on non-hematopoietic tissues. To decrease the incidence and severity of GvHD, T-cells can be (partially) depleted from the graft and re-administered later as donor lymphocyte infusion (DLI). MiHA are polymorphic peptides that are presented in HLA-molecules and can be recognized as non-self by donor-derived T-cells. Only a minority of MiHA have therapeutic relevance based on hematopoietic-restricted expression and only 25% and 40% of recipients transplanted with sibling and unrelated donors, respectively, are eligible for T-cell therapies in which one of the known hematopoietic-restricted MiHA is targeted. Therefore, to increase the efficacy and limit the toxicity of T-cell therapy, more therapeutic MiHA are needed. Recently, we identified a MiHA encoded by ARHGDIB, a gene that has been described to be highly expressed on hematopoietic cells, and we here studied the therapeutic relevance of LB-ARHGDIB-1R in detail.

First, we confirmed hematopoietic-restricted expression of ARHGDIB by microarray gene expression analysis and demonstrated >10-fold overexpression in the majority of malignant and healthy hematopoietic versus non-hematopoietic cells. In line with its hematopoietic-restricted gene expression profile, LB-ARHGDIB-1R in the context of HLA-B* 07:02 was specifically recognized on different hematological malignancies, but not on non-hematopoietic fibroblasts and keratinocytes cultured in the absence or presence of IFN-γ, which was added to mimick the pro-inflammatory cytokine milieu of the early post-transplantation period. Next, we investigated the cytolytic capacity of LB-ARHGDIB-1R specific T-cells, and demonstrated specific lysis of patient, but not donor, EBV-B cells and specific lysis of an ALL sample in a 10 hrs ⁵¹Cr-release assay. Specific lysis of additional ALL and AML samples could be measured after 48 hrs of co-incubation in a flowcytometry-based cytotoxicity assay.

To determine the in vivo immunogenicity of LB-ARHGDIB-1R, 11 MiHA-disparate HLA-B* 07:02 positive patient-donor pairs were screened for specific CD8⁺ T-cells by dual color tetramer analysis. All patients received partial T-cell depleted alloSCT and sampling was done at different time points after alloSCT (and DLI). In 4 out of 11 patients, LB-ARHGDIB-1R-specific T-cells (>0.01%) could be detected directly ex vivo and in 4 additional patients after 7 days of in vitro peptide stimulation, indicating that LB-ARHGDIB-1R is highly immunogenic. High frequencies of LB-ARHGDIB-1R specific T-cells were measured ex vivo in a patient whose hematological relapse was successfully treated with DLI, without development of GvHD, further supporting the therapeutic relevance of LB-ARHGDIB-1R.

In summary, we confirmed hematopoietic-restricted expression of LB-ARHGDIB-1R and demonstrated T-cell mediated lysis of primary leukemic cells in long-term co-incubation assays. Furthermore, we showed that LB-ARHGDIB-1R is highly immunogenic and that specific T-cells could be detected in a patient who responded to DLI in the absence of GvHD. Altogether, our data support the clinical relevance of LB-ARHGDIB-1R as therapeutic MiHA with the potential to shift the delicate balance between GvL and GvHD in favor of a desired anti-tumor effect.