HLA-restricted Antigen Research Articles

Association between the HLA-DRB1*03:01-DQB1*02:01 haplotype and PF4/heparin antibodies

Key Points HLA-DRB1*03:01-DQB1*02:01 haplotype patients develop higher anti–platelet factor 4/heparin (PF4/H) levels following IV heparin exposure. HLA-restricted antigen presentation may play a role in the formation of anti-PF4/H antibodies.

CAR-T with License to Kill Solid Tumors in Search of a Winning Strategy.

Artificial receptors designed for adoptive immune therapies need to absolve dual functions: antigen recognition and abilities to trigger the lytic machinery of reprogrammed effector T lymphocytes. In this way, CAR-T cells deliver their cytotoxic hit to cancer cells expressing targeted tumor antigens, bypassing the limitation of HLA-restricted antigen recognition. Expanding technologies have proposed a wide repertoire of soluble and cellular “immunological weapons” to kill tumor cells; they include monoclonal antibodies recognizing tumor associated antigens on tumor cells and immune cell checkpoint inhibition receptors expressed on tumor specific T cells. Moreover, a wide range of formidable chimeric antigen receptors diversely conceived to sustain quality, strength and duration of signals delivered by engineered T cells have been designed to specifically target tumor cells while minimize off-target toxicities. The latter immunological weapons have shown distinct efficacy and outstanding palmarès in curing leukemia, but limited and durable effects for solid tumors. General experience with checkpoint inhibitors and CAR-T cell immunotherapy has identified a series of variables, weaknesses and strengths, influencing the clinical outcome of the oncologic illness. These aspects will be shortly outlined with the intent of identifying the still “missing strategy” to combat epithelial cancers.

Antigen Presentation by Individually Transferred HLA Class I Genes in HLA-A, HLA-B, HLA-C Null Human Cell Line Generated Using the Multiplex CRISPR-Cas9 System.

Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients, graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference, zinc finger nuclease, and the CRISPR/Cas9 system. To improve HLA gene editing, we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore, artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.

Carfilzomib alters the HLA-presented peptidome of myeloma cells and impairs presentation of peptides with aromatic C-termini.

Recent studies suggest that multiple myeloma is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell immunotherapy. As standard of care in myeloma includes proteasome inhibitor therapy, it is of great importance to characterize the effects of this treatment on HLA-restricted antigen presentation and implement only robustly presented targets for immunotherapeutic intervention. Here, we present a study that longitudinally and semi-quantitatively maps the effects of the proteasome inhibitor carfilzomib on HLA-restricted antigen presentation. The relative presentation levels of 4780 different HLA ligands were quantified in an in vitro model employing carfilzomib treatment of MM.1S and U266 myeloma cells, which revealed significant modulation of a substantial fraction of the HLA-presented peptidome. Strikingly, we detected selective down-modulation of HLA ligands with aromatic C-terminal anchor amino acids. This particularly manifested as a marked reduction in the presentation of HLA ligands through the HLA allotypes A*23:01 and A*24:02 on MM.1S cells. These findings implicate that carfilzomib mediates a direct, peptide motif-specific inhibitory effect on HLA ligand processing and presentation. As a substantial proportion of HLA allotypes present peptides with aromatic C-termini, our results may have broad implications for the implementation of antigen-specific treatment approaches in patients undergoing carfilzomib treatment.

A Pre-Clinical Model Of Hematopoietic Stem Cell Based Immunotherapy For Cancer Utilizing The NY-ESO-1 T-Cell Receptor and sr39TK PET Reporter / Suicide Gene

Immunotherapy using peripheral blood T-cells engineered with T-cell receptor (TCR) or chimeric antigen receptor genes is a promising approach for the treatment of malignant diseases, and has demonstrated clinical efficacy capable of curing late stage cancer patients. Unfortunately the complete response rate remains low, and the majority of patients respond transiently and then relapse. The massive ex vivo expansion of autologous cells required to generate a therapeutic bolus may exhaust the replicative capacity of the infused cell product. An approach using cells with greater regenerative capacity is an attractive solution to this problem. We hypothesize that gene transfer of an NY-ESO-1 cancer/testes antigen specific TCR into human hematopoietic stem cells (HSCs) is capable of generating a continuous supply of effector T-cells capable of killing cancer in vivo.To evaluate this approach, we utilize a humanized mouse model where peripheral blood stem cells (PBSC) enriched for the stem and progenitor marker CD34+ are transduced with a lentivirus encoding codon optimized NY-ESO-1 TCR and HSV-sr39TK transgenes, then adoptively transferred to preconditioned (100 cGy TBI) NSG-HLA-A2.1 neonatal mice. Development of NY-ESO-1 TCR bearing effector T-cells was detected in the peripheral blood of mice as early as 2 months post-transplant, and persisted for at least 5 months post-transplant. Ex vivo assay of T-cells developed from engineered HSCs showed robust release of interferon-γ when cocultured with NY-ESO-1 antigen and HLA-A2.1 matched tumor cells but not when HLA was mismatched, indicating HLA restricted antigen recognition. Furthermore cytotoxicity assays showed that engineered T-cells were capable of specifically killing tumor cells when antigen and HLA matched.The inclusion of the PET imaging/suicide gene HSV-sr39TK allows both the non-invasive tracking of progeny derived from gene modified HSCs, and their ablation in the event of on-target / off-cancer reactivity or hematopoietic dysplasia due to insertional mutagenesis of gene modified cells. [18F]-FHBG PET imaging of TCR/TK engineered humanized mice allowed the detection of gene modified cells in the marrow compartments of mice, namely the long bones of the legs and arms. Importantly, strong thymus signal was also observed indicating robust thymic population/thymopoiesis of gene modified cells. No specific signal was detected in mock transduced HSC transplanted humanized mice, with identical biodistribution of signal as observed in non-transplanted NSG-A2.1 mice.To examine the suicide gene function of the HSV-sr39TK cassette, we treated engineered humanized mice with vehicle or ganciclovir, the prodrug for suicide gene function of HSV-sr39TK. Ganciclovir treatment resulted in ablation of hematopoietic compartment specific PET signal, while vehicle did not. Mice were subsequently euthanized, and the presence of gene modified cells in organ compartments was examined by digital qPCR for the lentiviral psi element. We detected an order of magnitude decrease in the amount of gene modified cells in ganciclovir treated animals compared with controls.Our studies demonstrate the feasibility of using TCR engineered HSCs for immunotherapy, as functional, HLA-restricted NY-ESO-1 T-cells developed in vivo from transplanted HSCs. In vivo imaging of gene modified cells supports visual resolution of gene modified HSC progeny at a level not previously described, and validation of the suicide gene function of HSV-sr39TK is of particular importance to gene therapy studies. Further work is focused on examining the immunological memory phenotype of TCR modified cells that develop from TCR modified HSCs, the effect of in vivo activation of these cells by dendritic cell pulse, and the ability of NY-ESO-1 humanized mice to mount a persistent anti-tumor response. Disclosures:No relevant conflicts of interest to declare.

Dual Transgenesis of T Cells with a Novel CD44v6-Specific Chimeric Antigen Receptor and a Suicide Gene for Safe and Effective Targeting of Chemoresistance in Hematopoietic Tumors

Abstract 3125 Background:We have previously demonstrated that the genetic induction of a conditional suicidal phenotype in donor T cells allows for an operational dissociation of the GVL effect from GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, leukemia often escapes the immunological pressure of alloreactive donor T cells by losing “passenger” mismatched HLAs. Conversely, redirecting T cells against a non HLA-restricted antigen critically involved in the neoplastic phenotype may circumvent tumor escape due to the emergence of antigen-loss variants. The isoform variant 6 of CD44 is expressed by different epithelial and hematological cancers, and is possibly involved in tumor-cell survival and proliferation. Clinical experience with chemo-conjugated CD44v6-specific mAbs in epithelial tumors showed substantial efficacy, which was however limited by skin toxicity due to background expression of CD44v6 on keratinocytes Aim:By analogy with our experience in HSCT, we reasoned that CD44v6 targeting with suicide gene-modified T cells would provide a major therapeutic effect against hematological tumors, while granting a safety switch in case of toxicity. To this aim, we designed a novel CD44v6-specific chimeric antigen receptor (CAR) and developed a strategy for its co-expression with a suicide gene Results:CD44v6 expression by FACS was observed at high levels in 6/17 (37%) cases of acute myeloid leukemia (AML), but did not associate with enhanced leukemia initiation after infusion into NSG mice (83% vs 88%). In all cases, however, AML cells isolated from the bone marrow (BM) of engrafting mice were brightly positive for CD44v6, suggesting in vivo regulation by microenvironmental factors. In vitro, co-culturing primary AML cells with human BM-derived mesenchymal stromal cells (MSCs) caused a selective up-regulation of CD44v6 (P<0.01). The phenomenon was causally linked to MSC-induced acquisition of resistance to the chemotherapeutic agents daunorubicin and ara-c, as demonstrated by lentiviral vector (LV)-assisted short-hairpin (sh) RNA interference. At difference with AML, CD44v6 was constitutively expressed in 9/11 (81%) cases of multiple myeloma (MM). Knocking-down CD44v6 by shRNA interference significantly increased baseline sensitivity of MM cells to bortezomib. After validating CD44v6 as a common target of chemoresistant AML and MM cells, we generated a 2G CAR by cloning the scFv of a humanized CD44v6-specific mAb in a CD28/TCR zeta chain backbone and expressed it along with the Herpes simplex virus thymidine kinase (HSV-tk) suicide gene by means of a LV carrying a bi-directional promoter. After LV transduction, primary T cells concomitantly acquired CD44v6-specific in vitro cytotoxicty against autologous AML and MM cells, and a selective sensitivity to the suicide gene-activating prodrug ganciclovir. CD44v6-specific recognition associated with T-cell proliferation, IL-2 and IFN-gamma production, and complete clearance of AML cells in a BM-niche model with MSCs at very low E :T ratios (1:5–1:10). Interestingly, in the same model CD34+CD38- healthy cells were not eliminated by CD44v6-redirected T cells, consistently with stable lack of CD44v6 surface expression on healthy HSC. Once infused into NSG mice, CD44v6-redirected T cells had a major antitumor effect against previously engrafted CD44v6-positive AML and MM cell lines (THP1, P<0.001 and MM1.S, P<0.05 vs control CAR, respectively) and against autologous primary AML cells (P<0.005). Since the performance of the suicide gene is felt to be critical for controlling possible toxicities of CAR-redirected T cells, we also evaluated and inducible form of caspase 9 (icasp9) as a possible alternative to HSV-tk. Casp9 activation by its prodrug (the AP1903 dimerizer) permitted efficient CD44v6-redirected T-cell elimination even in the absence of cell proliferation and with a much faster kinetics than HSV-tk (>90% elimination: 18 hrs vs 112 hrs average, P<0.005) Conclusions:We demonstrated that LV-mediated dual transgenesis of primary human T cells with a novel CD44v6-specific CAR and a suicide gene is feasible, results into a powerful antitumor effect against chemoresistant AML and MM cells, and enables effective T-cell ablation in case of toxicity. The premise that suicide gene-modified CAR-redirected T cells can widen the therapeutic index of CD44v6 targeting awaits clinical confirmation Disclosures:Bordignon:Molmed SpA: Employment. Bonini:MolMed: Consultancy.

Increased effector–target cell conjugate formation due to HLA restricted specific antigen recognition

The T cell receptor (TCR) orchestrates T cell mediated-cytotoxicity through a complex interaction that results in an antigen-specific effector-target cell conjugate formation. While it is well recognized that specific TCR/antigen interactions generate the immunological synapse, their direct contribution to the effector-target cell conjugate has not been conclusively demonstrated. Moreover, since human cytotoxic T lymphocyte (CTL) clones are also susceptible to antigen-independent adhesion to target cells, it remains unclear whether effector-target cell conjugate formation can serve as an indicator of specific antigen recognition by the TCR. To address this question, a well-characterized epitope-specific CTL clone recognizing the melanoma-associated antigen epitope gp100:209-217 in association with HLA-A*0201 was tested against melanoma cell lines lacking or expressing the HLA-A*0201 allele and/or gp100. In this model, TCR/HLA/antigen interactions cooperated with accessory/adhesion molecules to facilitate effector-target cell conjugate formation. HLA-restricted antigen recognition played a dominant role resulting in up to 2-fold increases in conjugate frequency, and a 50% increase of CTL binding to tumor cells over background. The increased number of CTL contained in conjugates correlated with the number of IFN-gamma producing CTL. These results warrant further investigation to evaluate conjugate assays as a potential tool to detect and isolate viable and functionally active CTL. Since conjugate formation analysis does not require knowledge of the target antigen, this assay could potentially be used for enrichment of CTL directed against novel antigens.

Human Leukocyte Antigen Class I-Restricted Activation of CD8 + T Cells Provides the Immunogenetic Basis of a Systemic Drug Hypersensitivity

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

Role of IL-12B Promoter Polymorphism in Adamantiades–Behcet's Disease Susceptibility: An Involvement of Th1 Immunoreactivity against Streptococcus Sanguinis Antigen

Adamantiades-Behcet's disease (ABD) is a chronic inflammatory multisystem disorder. Although the precise etiology is unclear, high prevalence of human leukocyte antigen (HLA)-B51 predisposition and predominantly involved T-helper type 1 cells (Th1)-type proinflammatory cytokines and extrinsic Streptococcal infection suggest a substantial association with an immunogenetic basis and strengthens the hypothesis that IL-12, a potent inducer of Th-1 immune reaction, is a putative candidate in its pathogenesis. These clinicopathological findings led us to examine interleukin 12 p40 (IL-12B) promoter polymorphism, for which the 4-base pair (bp) heterozygous insertion has been shown to affect the gene transcription and subsequent protein production. We analyzed IL-12B promoter genotypes in 194 Japanese subjects (92 with ABD and 102 normal controls) by PCR-based restriction enzyme digestion. The frequency of the insertion heterozygosity was significantly higher in patients than in controls (49/92, 53.3% vs 39/102, 38.2%, respectively). Comparing these with HLA haplotype data, this trend was more significant in HLA-B51-negative patients (29/42, 69.0% vs 20/50, 40.0%; P = 0.005). As assessed by semiquantitative reverse transcription-PCR and ELISA, stimulation with Streptococcal antigens specifically increased expression of IL-12 p40 mRNA and protein, in conjunction with IL-12 p70 induction, in peripheral blood mononuclear cells from heterozygous patients. Our results provide evidence for anti-bacterial host response toward Th1-immunity mediated by IL-12 in patients with ABD, and the possible insight into the genetic susceptibility that is independent of HLA background.

Individualized Synthetic Peptide Vaccines with GM-CSF in Locally Advanced Melanoma Patients

We report on 10 patients with resected American Joint Committee on Cancer (AJCC)stage IIA-IIIC melanoma receiving individualized adjuvant peptide vaccinations derived from the melanosomal antigens MelanA/MART1, gp100 and tyrosinase, according to patient tumor associated HLA restricted antigen expression, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Except for 1 patient, all patients had received systemic pretreatment with immunotherapy (n = 8), chemoimmunotherapy (n = 1), chemotherapy (n = 1), or cefalectin therapy (n = 1). Upon prior therapy, 7 of 10 patients had progressed with subcutaneous/cutaneous (n = 2), lymph node (n = 3), or subcutaneous/cutaneous and lymph node (n = 2)metastases, which were subsequently resected prior to vaccination. After a mean of 6.5 vaccination cycles, progression-free survival was 6 months (median, range 2-10). Five patients were relapse-free for 1+ up to 21+ months, 3 patients developed a solitary cutaneous metastasis, and 2 patients developed multiple metastases during vaccination. Overall, vaccine treatment was well tolerated, with no severe side-effects. Eight of 10 patients developed local delayed type hypersensitivity (DTH)reactions to synthetic peptides after the first or second injection. In 2 patients, transient fever, nausea, diarrhea, and muscle pain of National Cancer Institute (NCI)Grade I occurred. In summary, individualized synthetic peptide vaccination, combined with GM-CSF, was feasible and warrants further clinical investigation.

Prospects for immunotherapy of malignant disease.

The majority of T cell-recognized tumour antigens in humans are encoded by genes that are also present in normal tissues. Low levels of gene expression in normal cells can lead to the inactivation of high-avidity T cells by immunological tolerance mechanisms. As a consequence, low-avidity T cell responses in patients are often inadequate in providing tumour protection. Recently, several technologies have been developed to overcome tolerance, allowing the isolation of high-affinity, HLA-restricted receptors specific for tumour-associated peptide epitopes. Furthermore, transfer of HLA-restricted antigen receptors provides an opportunity to empower patient T cells with new tumour-reactive specificities that cannot be retrieved from the autologous T cell repertoire.

Design of cassette vectors permitting cloning of all types of human TCR variable α and β regions

T cell clones are an irreplaceable asset for the study of immune responses relevant to human pathologies. Such cells, however, cannot always be maintained in long-term culture. In order to reconstitute functional human T cell receptors (TCRs) into stable and fast growing hybridoma T cells, we developed a general approach based on a versatile cassette system, which allows cloning of all types of human T cell receptor variable α and β region genes fused to murine constant regions. These chimeric constructs are easily excised and transferred into expression vectors that can be used to transfect a human CD4-expressing murine T cell hybridoma recipient. The resulting transfectants are highly stable both in terms of T cell receptor-CD3 expression and IL-2 response to the specific antigenic stimulus. Using these cassette vectors, we reconstituted the original HLA-restricted antigen specificity for two human T cell clones, one recognizing an immunodominant epitope of HIV-1 gp120, and the other recognizing an immunodominant epitope of HIV-1 reverse transcriptase. We found that the reconstituted hybridomas maintain the ability of the original T cell clones to recognize the appropriate epitope in the context of the relevant MHC either as a synthetic peptide or after processing. Their unlimited growth capacity makes them particularly suited for in vitro studies.

Molecular characterization of a subtype of DQw1 recognized by hapten-specific T cells.

Previous studies of HLA-restricted antigen recognition by cloned T cells have frequently demonstrated reactivity that did not correlate precisely with the expression of serologically defined HLA specificities. To further explore such discrepancies, we utilized monoclonal antibody (MoAb) blocking, partial NH2-terminal amino acid sequencing, and Southern blot hybridization techniques to analyze the fine specificity of four autologous trinitrophenyl-specific T cell lines restricted to DR2-linked epitopes. MoAb blocking studies demonstrated that two of these lines recognized determinants on DR molecules while the other two recognized determinants on the same molecule that expresses the DQw1 determinant. However, these latter two lines appeared to recognize a DQw1-related determinant found primarily in association with DR2, but not the other DQw1-associated DR alleles, DR1 and DRw6. To ascertain whether these lines were defining a functional split of DQw1, we performed partial NH2-terminal amino acid sequencing of the molecules precipitated with a DQw1-specific MoAb (Genox 3.53) from different stimulator lines. The results showed that these T cell lines recognized a subtype of DQw1 that is in linkage disequilibrium with DR2. Moreover, we identified characteristic restriction fragment length polymorphisms with a DQ beta-specific cDNA that correlated with stimulatory capacity for the DQw1-restricted lines. These results demonstrate that: DQ molecules may provide restriction determinants that are incorrectly assigned to DR molecules on stimulator panel analyses; cloned antigen-specific T cell lines recognize polymorphic regions of class II molecules not distinguished by either conventional typing antisera or xenogeneic MoAb; and the DQw1 epitope(s) is located on a heterogeneous group of DQ molecules that differ from each other in the primary sequence of their beta chains.