A Model of Minor Histocompatibility Antigens in Allogeneic Hematopoietic Cell Transplantation

Minor histocompatibility antigens (mHAg) composed of peptides presented by HLA molecules can cause immune responses involved in graft-versus-host disease (GVHD) and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (HCT). The current study was designed to identify individual graft-versus-host genomic mismatches associated with altered risks of acute or chronic GVHD or relapse after HCT between HLA-genotypically identical siblings. Our results demonstrate that in allogeneic HCT between a pair of HLA-identical siblings, a mHAg manifests as a set of peptides originating from annotated proteins and non-annotated open reading frames, which i) are encoded by a group of highly associated recipient genomic mismatches, ii) bind to HLA allotypes in the recipient, and iii) evoke a donor immune response. Attribution of the immune response and consequent clinical outcomes to individual peptide components within this set will likely differ from patient to patient according to their HLA types.

Consequence of Histoincompatibility beyond GvH-Reaction in Cytomegalovirus Disease Associated with Allogeneic Hematopoietic Cell Transplantation: Change of Paradigm

Hematopoietic cell (HC) transplantation (HCT) is the last resort to cure hematopoietic malignancies that are refractory to standard therapies. Hematoablative treatment aims at wiping out tumor cells as completely as possible to avoid leukemia/lymphoma relapse. This treatment inevitably co-depletes cells of hematopoietic cell lineages, including differentiated cells that constitute the immune system. HCT reconstitutes hematopoiesis and thus, eventually, also antiviral effector cells. In cases of an unrelated donor, that is, in allogeneic HCT, HLA-matching is performed to minimize the risk of graft-versus-host reaction and disease (GvHR/D), but a mismatch in minor histocompatibility antigens (minor HAg) is unavoidable. The transient immunodeficiency in the period between hematoablative treatment and reconstitution by HCT gives latent cytomegalovirus (CMV) the chance to reactivate from latently infected donor HC or from latently infected organs of the recipient, or from both. Clinical experience shows that HLA and/or minor-HAg mismatches increase the risk of complications from CMV. Recent results challenge the widespread, though never proven, view of a mechanistic link between GvHR/D and CMV. Instead, new evidence suggests that histoincompatibility promotes CMV disease by inducing non-cognate transplantation tolerance that inhibits an efficient reconstitution of high-avidity CD8+ T cells capable of recognizing and resolving cytopathogenic tissue infection.

Possible NK cell-mediated immune responses against iPSC-derived cells in allogeneic transplantation settings

In the regenerative medicine field, allogenic transplantation of regenerated tissues has been promoted because autologous transplantation setting is costly and time-consuming to prepare and therefore unsuitable for emergent treatment. To avoid a T cell-mediated immune rejection in the allogenic transplantation setting, induced pluripotent stem cells (iPSCs) derived from different HLA haplotype-homozygous (HLA-homo) donors have been prepared to be used as source of regenerated tissues. However, there still remain immunological issues, even when HLA-homo iPSCs are used. One issue is the immune response against minor histocompatibility antigens expressed on the regenerated tissues, and the other is the immune rejection mediated by NK cells. In this article, we introduce our research on NK cell reactivity against the regenerated tissues in the HLA homo-to-hetero transplantation setting. We further introduce several approaches taken by other groups that address the NK-mediated immune rejection issue.

Population Distribution of GvL and GvH Minor Histocompatibility Antigens

Background: Donor-derived T cells that target minor histocompatibility antigens (mHAs) in allogeneic hematopoietic cell transplant (HCT) mediate graft versus leukemia (GvL) and graft versus host (GvH) effects. Prediction of mHAs that drive GvL has garnered interest for targeted immunotherapy, but there have been few large-scale studies of population prevalence of predicted mHAs. Prioritization of mHAs that are shared among patients would allow for treatment of more individuals with mHA-targeting therapies. We report here population metrics of predicted mHAs in a dataset of over 3000 patients treated with HCT and reported to CIBMTR from 2000-2011. Our goal is to identify the most common mHAs within leukemia and Myelodysplastic Syndrome (MDS) patient populations to target with T cell immunotherapies or graft engineering techniques. Methods: Data is derived from two cohorts of donor recipient HCT pairs (DRPs) treated for Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL), and MDS from the CIBMTR and previously analyzed in DISCOVeRY-BMT. Cohort 1 included 2609 10/10 HLA-matched DRPs treated from 2000-2008, and Cohort 2 included 572 10/10 HLA-matched DRPs treated from 2009-2011 plus 351 8/8 HLA-matched DRPs treated from 2000-2011 (Hahn et al. 2015, Biol Blood Marrow Transplant). Cohorts were combined for analyses. Approximately 20,000 missense SNPs were extracted from Illumina HumanOmni Express genotyping data. Computational mHA prediction was performed according to prior work from our lab (Lansford et al. 2018, Blood Adv.). Minor mismatches were predicted based on coding SNPs present in the recipient but not donor. mHAs were defined as mismatches that would lead to variant peptides predicted to bind at least 1 recipient HLA molecule and be expressed in leukemia cells (GvL mHA) and/or acute GvHD target organs (GvH mHA). GvL mHAs were categorized as "GvL,No_GvH" or "GvL" based on transcripts per million (TPM) corresponding to GvH organs, with "GvL" indicating between 5-50 TPM and "GvL,No_GvH" indicating <5 TPM. GvH mHAs were categorized similarly with respect to expression in leukemia cells. "GvL,GvH" indicated mHAs expressed highly in both leukemia and in GvHD target organs. Results: Patient demographics and number of total predicted mHAs from each ethnic group are shown in Table 1. Number of predicted mHAs per patient varied widely both within and between HLA types (Figure 1). Despite underrepresentation of some ethnic groups in our dataset, we identified thousands of potential mHAs in each group (Figure 2A). GvL mHA and GvH mHA proportions were similar across recipient ethnicities (Figure 2A-B). GvL mHA made up approximately half of predicted mHAs for each ethnic group (Figure 2B). Total numbers of mHAs per HCT recipient were significantly different between recipient ethnic groups within each cohort (Figure 2C). Although proportions of GvL vs GvH mHAs were stable across HLA alleles, there were substantial differences in number of predicted mHA by allele (Figure 3). Despite limited representation of some HLA types in our dataset, we were able to identify GvL mHAs for potential therapeutic targeting corresponding to 56 HLA alleles. We generated ranked lists of the most common shared mHA for each HLA allele, using an implementation of the standard greedy algorithm solution to the maximum set coverage problem. With this method, we identified the fewest number of mHA peptides needed to cover desired percentages of the recipient population with at least one mHA. For example, for HLA A*02:01, HLA*B07:02, and HLA*C07:01, engineering T cells to target the top nine to twelve peptides would allow for treatment of 80% of the patient population in our cohorts (Figure 4). These represent common HLA alleles in Caucasian, African American, Hispanic, and Asian populations, indicating that this technique can identify targets that could be therapeutically beneficial for a greater diversity of patients than standard treatments. mHA pools can also be filtered on peptide expression or HLA binding to ensure that the targeted peptides are highly expressed and presented. Conclusions: Despite differences in predicted number of mHA by ethnicity and HLA alleles, shared GvL mHA exist across common HLA. To the extent that these are targetable by adoptive cellular therapy, we can expand equal access to mHA targeted immunotherapies, improving upon traditional models where only the most prevalent HLA types are covered. Disclosures Armistead: Cell Microsystems: Patents & Royalties: Patent application U.S. 16/347,104 "Automated collection of a specified number of cells"; GeneCentric: Consultancy. Vincent:GeneCentric Therapeutics: Consultancy.

156 Discovery of TSC-100: A natural HA-1-specific TCR to treat leukemia following hematopoietic stem cell transplant therapy

BackgroundApproximately 30–40% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options.1 2 Rare patients that naturally develop an HA-1-specific graft-versus-leukemia T cell response, however, show substantially lower relapse rates.3 4 HA-1 (VLHDDLLEA, genotype RS_1801284 A/G or A/A) is an HLA-A*02:01-and hematopoietically restricted minor histocompatibility antigen, making it an ideal candidate for TCR immunotherapy for liquid tumors.5MethodsWe developed a high-throughput TCR discovery platform that enables rapid cloning of antigen-specific TCRs from healthy donors. We then used this platform to screen 178.3 million naïve CD8+ T cells from six unique HA-1- (VLRDDLLEA, genotype RS_1801284 G/G) donors, identifying 329 HA-1-specific TCRs. We tested each TCR for expression and the ability to kill HA-1+ target cells, using a previously published, clinical-stage HA-1-specific TCR as a benchmark for these studies.6 In parallel, we tested TCR constant region modifications to promote expression and proper pairing of exogenous TCR alpha and beta chains and designed a lentiviral vector to co-deliver CD8 coreceptors as well as a CD34 enrichment tag to enable purification of engineered T cells. The top 11 candidates were cloned into our optimized backbone and evaluated for cytotoxicity, cytokine production, and T cell proliferation using a panel of HLA-A*02:01+ HA-1+ cell lines. Finally, the top two TCRs were evaluated for allo-reactivity and off-target cross-reactivity using our proprietary genome-wide T-Scan platform.ResultsThe TCR discovery and evaluation platform described here identified 329 HA-1-specific TCRs from a total of 178.3 million naïve T cells, and TSC-100 as the most active TCR. Defined mutations in the constant region of TSC-100 enhanced its surface expression while decreasing expression of endogenous TCRs, and co-introduction of CD8 enabled efficient engagement and function of engineered CD4 cells. Overall, TSC-100 exhibited comparable activity to a clinical-stage benchmark TCR when challenged with cell lines expressing moderate to high levels of HA-1, and superior activity when incubated with cell lines expressing low levels of both HA-1 and MHC-I.6 In addition, TSC-100 exhibited no detectable allo-reactivity to 108 different HLA types tested, and minimal off-target effects when challenged with a genome-wide library expressing peptides derived from human proteins.ConclusionsTSC-100 exhibits comparable or superior activity to a clinical-stage therapeutic TCR, with minimal allo-reactivity or off-target effects. Based on these results, TSC-100 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2021.Ethics ApprovalAll clinical samples used in the study were collected by STEMCELL Technologies, StemExpress and HemaCare using their IRB approved protocols.ReferencesPavletic SZ, Kumar S, Mohty M, et al. NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from The Committee on the Epidemiology and Natural History of Relapse following Allogeneic Cell Transplantation. Biol Blood Marrow Transplant. 2010;16(7):871–890.Miller JS, Warren EH, van den Brink MR, et al. NCI First International Workshop on The Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on the Biology Underlying Recurrence of Malignant Disease following Allogeneic HSCT: Graft-versus-Tumor/Leukemia Reaction. Biol Blood Marrow Transplant 2010;16(5):565–586.Marijt WAE, Heemskerk MHM, Kloosterboer FM, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. PNAS 2003;100:2742–2747.Spierings E, Kim Y, Hendriks M, et al. Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-Matched related and unrelated hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 1244–1253.Bleakley M, Riddell SR. Exploiting T cells specific for human minor histocompatibility antigens for therapy of leukemia. Immunol Cell Biol 2011;89(3):396–407.Dossa RG, Cunningham T, Sommermeyer D, et al. Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse. Blood 2018;131(1):108–120.