Over the last decade, the development of multiple strategies to allow the safe transfer from the donor to the patient of high numbers of partially HLA-incompatible T cells has dramatically reduced the toxicities of haploidentical hematopoietic cell transplantation (haplo-HCT), but this was not accompanied by a similar positive impact on the incidence of post-transplantation relapse. In the present review, we will elaborate on how the unique interplay between HLA-mismatched immune system and malignancy that characterizes haplo-HCT may impact relapse biology, shaping the selection of disease variants that are resistant to the "graft-vs.-leukemia" effect. In particular, we will present current knowledge on genomic loss of HLA, a relapse modality first described in haplo-HCT and accounting for a significant proportion of relapses in this setting, and discuss other more recently identified mechanisms of post-transplantation immune evasion and relapse, including the transcriptional downregulation of HLA class II molecules and the enforcement of inhibitory checkpoints between T cells and leukemia. Ultimately, we will review the available treatment options for patients who relapse after haplo-HCT and discuss on how a deeper insight into relapse immunobiology might inform the rational and personalized selection of therapies to improve the largely unsatisfactory clinical outcome of relapsing patients.

Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).

Background Genomic loss of mismatched HLAs ("HLA loss") represents a frequent modality by which acute myeloid leukemia (AML) evades immune recognition from donor T cells after partially HLA-incompatible allogeneic hematopoietic cell transplantation (allo-HCT). One important consequence of this post-transplantation relapse mechanism is that infusions of lymphocytes from the original donor become ineffectual, prompting the search for alternative therapeutic options. Here, to circumvent the loss of physiological T cell receptor-HLA interactions in these patients, we tested the ability of an anti-CD3/CD33 bispecific antibody (BsAb) to re-target donor T cells towards HLA loss relapses. Methods For short-term in vitro experiments, T cells were co-cultured with the MOLM-13 AML cell line or with primary patient blasts for 96 hours in presence or absence of an anti-CD3/CD33 BsAb. As readouts, we measured T cell activation (as surface expression of CD25 and CD69) and the absolute counts and relative proportion of effectors and targets. For long-term in vitro experiments, we established mixed lymphocyte cultures (MLCs) of T cells purified from two patients after haploidentical HCT and primary AML blasts obtained from the same patients at the time of diagnosis. After sequential stimulations, the co-cultures were tested against targets of interest, with or without addition of the BsAb. Functional readouts were T cell degranulation (measured as CD107a expression), antigen-specific activation (as CD137/41-BB expression) and target-specific cytotoxicity (measured by time-lapse live cell imaging over a 48 hour time span). For in vivo experiments, human leukemic cells were infused intravenously into non-irradiated NSG mice, followed by intraperitoneal infusion of T cells and daily administration of the BiTE compound. Results First, we retrospectively analyzed immunophenotypic data of 36 AML patients who experienced HLA loss relapses at our Institution, documenting robust expression of CD33 on the surface of the relapsed leukemia in 35 of them (97%; Figure 1A). By short-term co-culture experiments we titrated the BsAb concentration to be used for subsequent in vitro assays to 100 ng/ml, and the most informative effector:target ratio to 1:3. Then, we established MLCs by stimulating T cells collected from two patients after partially HLA-incompatible allo-HCT with AML blasts collected from the same patients at the time of diagnosis. In both cases, donor-derived T cells robustly responded against the patient blasts both in term of degranulation (Figure 1B) and of antigen-specific activation (Figure 1C). As expected, when we tested the same T cells against the patient leukemia at time of HLA loss relapse, we detected no T cell-mediated responses. Noticeably, when the BsAb was added, in both cases we detected a strong response not only against the diagnosis but also against the HLA loss variants, indicating that T cells were effectively re-targeted towards leukemic cells. Similar results were obtained also by live cell imaging, measuring target cell apoptosis over 48 hours of recording: also in this assay, in fact, donor T cells recognized and killed leukemia at diagnosis (45% of detection area positive for apoptosis dye) and failed to recognize its HLA loss relapse counterpart (32% of area positive for apoptosis dye). Addition of the BsAb to the co-cultures had a minor effect on recognition of the original disease (45% of area positive for apoptosis dye) but drove dramatic cell death of HLA loss blasts (80% of area positive for apoptosis dye), demonstrating that the BsAb induced not only T cell activation but also and most importantly target cell killing (Figure 1D). Finally, we modeled the BsAb activity in vivo, showing that, whereas the sole infusion of human T cells is not able to prevent the outgrowth of leukemia in the bone marrow of NSG mice, addition of the bispecific antibody leads to effective disease clearance (Figure 1E). Conclusions Our results demonstrate that anti-CD3/CD33 BsAbs can effectively redirect donor T cells against HLA loss leukemia variants, resulting in their rapid and effective killing. Taken together, these promising findings strongly support translation of this approach to ad hoc designed early-phase clinical trials, to provide a rational therapy for this increasingly recognized but still treatment-orphan modality of post-transplantation relapse. Figure 1 Disclosures Subklewe: Janssen: Consultancy; Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Roche: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding. Vago:Moderna Therapeutics: Research Funding; GenDx: Research Funding.