Low Cell Surface Expression Research Articles

P011 Advances in HLA clinical relevant exon novelty detection from over 21,000 donor recruitment samples

Aim The assignment of exon novel alleles is clinically relevant in hematopoietic stem cell transplantation (HSCT), in particular for donor selection. Here we investigate the exon novelty in over 21,000 National Marrow Donor Program (NMDP) registry donors by using next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (HLA-A, -B, -C) from promoter to 3’ UTR. Class II genes (-DQB1, -DRB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2 × 250 bp paired-end sequencing on the Illumina MiSeq. Exon novelty was assigned using GenDx NGSengine v2.4.0 and Omixon Twin v1.0.7 software to ensure high confidence in allele calling. Exon novelties were confirmed by Sanger sequencing and 3D protein structure was predicted using Phyre2. Results Using this automated workflow, over 21,000 NMDP registry donors were successfully typed using high-resolution by NGS. NGS-HLA typing identified 94 exon novelties (0.044%) at five loci (HLA-A = 33, -B = 16, -C = 18, -DQB1 = 12, and -DRB1 = 15). We discerned 29 synonymous and 65 non-synonymous mutations. Among them 17 novel alleles (HLA-A = 3, -B = 5, -C = 2, -DQB1 = 4, and -DRB1 = 3) were detected within key exons involved in antigen recognition (exon 2 and 3 for Class I, and exon 2 for Class II). Interestingly, there are three frameshift novelties: A∗33:01:01NEW (1598InsC), A∗66:03NEW (1598delC), and B∗38:01:01NEW (9 bp del 1705-1713). Based on the 3D protein structure, these three proteins are truncated on α 3. Future experiments will determine if these proteins lose function or have lower cell surface expression. Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. High resolution HLA typing provides more accurate matching of the HLA-A, -B, -C, -DQB1, and -DRB1 alleles to improve patient survival. This work is the first step to define permissive mismatches in the context of HSCT, and it emphasize the need to improve our knowledge on donor selection.

Adoptively transferred Vγ9Vδ2 T cells show potent antitumor effects in a preclinical B cell lymphomagenesis model.

A central issue for adoptive cellular immunotherapy is overcoming immunosuppressive signals to achieve tumor clearance. While γδ T cells are known to be potent cytolytic effectors that can kill a variety of cancers, it is not clear whether they are inhibited by suppressive ligands expressed in tumor microenvironments. Here, we have used a powerful preclinical model where EBV infection drives the de novo generation of human B cell lymphomas in vivo, and autologous T lymphocytes are held in check by PD-1/CTLA-4-mediated inhibition. We show that a single dose of adoptively transferred Vδ2+ T cells has potent antitumor effects, even in the absence of checkpoint blockade or activating compounds. Vδ2+ T cell immunotherapy given within the first 5 days of EBV infection almost completely prevented the outgrowth of tumors. Vδ2+ T cell immunotherapy given more than 3 weeks after infection (after neoplastic transformation is evident) resulted in a dramatic reduction in tumor burden. The immunotherapeutic Vδ2+ T cells maintained low cell surface expression of PD-1 in vivo, and their recruitment to tumors was followed by a decrease in B cells expressing PD-L1 and PD-L2 inhibitory ligands. These results suggest that adoptively transferred PD-1lo Vδ2+ T cells circumvent the tumor checkpoint environment in vivo.

CD33 Splicing Polymorphism Is a Strong Predictor of Therapeutic Efficacy of Gemtuzumab Ozogamicin in De Novo AML: Report from COG-AAML0531 Study

▪Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging as therapeutic for AML. We have previously discovered polymorphisms in CD33 coding region that might be associated with outcome in patients treated with GO. One particular coding polymorphism, CD33-SNP rs12459419-C>T (Ala14Val), is located within the splice enhancer region of exon-2, leading to expression of an alternate splice isoform lacking exon-2. This alternate splice isoform (D2-CD33), would encode a protein product lacking the IgV domain, which is the binding site for GO and most if not all CD33 antibodies used for diagnostic immunophenotyping. We therefore hypothesized that the SNP rs12459419 genotype would be associated with differential expression of the D2-CD33 transcript levels and differential cell surface CD33 expression leading to genotype determined differential response to GO.We evaluated the genotype frequency and functional significance of rs12459419, its association with CD33 cell-surface expression on leukemic blasts, and clinical response in 816 children and young adults with AML randomized to GO vs. No-GO in the COG trial AAML0531.The CD33 SNP rs12459419 genotype frequency was-CC=51%, CT=39% and TT=10% in patients, similar to the observed frequency in the general population. Correlation of SNP allele frequency with CD33 transcript levels and surface CD33 expression (as determined by p67.6 antibody) demonstrated that the T-allele was significantly associated with higher levels of D2-CD33 transcript (P=4.7e-11, Figure 1) and with lower diagnostic leukemic cell CD33 surface intensity (P=1.93e-29).Clinical outcome based on the SNP genotype demonstrated that patients with CC-genotype had significantly lower RR of 26%±7% in the GO arm whereas those in the No-GO arm had a RR of 49%±9% (HR=0.468, P<0.001). The corresponding DFS for CC genotype patients in the GO and No-GO arms was 65%±7% and 46%±9%, respectively (HR=0.597, P=0.004; Figure 1). In contrast, in those with heterozygous CT or homozygous TT genotype, GO exposure provided no clinical benefit in RR (CT: 38%±9% vs. 37±10%, P=0.975; TT: 46%±20% vs. 46%±20%, P=0.798) nor DFS (CT: 56%±9% vs. 60%±10% GO vs. No-GO, P=0.821; TT: 51%±20% vs. 54%±18%, GO vs. No-GO, P=0.972, Figure 1).We further evaluated the impact of the CD33 genotype on the efficacy of GO in different risk groups as well as in high vs. low CD33 expression cohorts. Patients in the low-risk (LR) group with the CC genotype treated with GO had a RR of 10%±8% vs. 37%±13% (P< 0.001) from remission. Standard-risk patients with CC genotype had a RR of 41±12% vs. 59±12% (P=0.056) and high-risk patients had a RR of 36%±27% vs. 70%±32% (P=0.073) for the GO and No-GO arms. In contrast there was no benefit of addition of GO in patients with the CT or TT genotypes within each risk group.Since CD33 expression has been recently associated with GO efficacy, we evaluated the association of rs12459419 genotypes in patients with low (N=153) and high CD33 (N=436) cell surface expression quartile-1 and quartiles-2-4, respectively. Significant improvement in RR was observed in patients with the CC genotype in the GO arm over those in the No-GO arm (P=0.001) within quartiles 2-4, but not with the CT/TT genotype (P=0.112). Within lower CD33 expression (quartile-1) cohort, a similar trend towards improvement in RR compared with those in the No-GO arm (P=0.055) was observed although the CC genotype was less frequent.Multivariate cox regression analysis that included genotype, risk status, and CD33 expression demonstrated that CD33 CC genotype was an independent predictor of response to GO (HR= 0.45, P<0.001 for RR and HR=0.57, P=0.003 for DFS).The rs12459419 genotype mediates expression of the GO binding site and informs on which patients should receive GO. The knowledge of CD33 genotype and prediction of response to GO provides opportunities to use patient genotypes for selecting CD33-targeted therapies. Given only half of the patients are expected to have a response to GO, we propose that all prior GO containing studies be re-evaluated for response based on patient CD33 genotype. The efficacy of GO in patients with an appropriate antibody binding domain also raises the possibility of developing next-generation CD33 immunoconjugates with epitopes targeted to regions not affected by alternative splicing and SNPs. [Display omitted] DisclosuresLoken:Hematologics: Employment, Equity Ownership.

Abstract A037: IMCgp100 ImmTAC: A new immunotherapeutic reagent for the treatment of malignant melanoma

Abstract For years cancer therapies were mainly concentrated around surgery, chemotherapy and radiotherapy. Recently, a new domain has emerged. Immunotherapies aim to exploit, enhance and optimise the patient's immune system to be more potent at eradicating tumours. T-cell immunotherapies, however, often encounter challenges with low cell surface expression of tumour-associated-antigens (TAAs), down-regulation of class I MHC molecules and low T-cell avidity for TAAs, all contributing to tumour escape. To address these challenges, Immunocore Ltd. has developed ImmTAC (Immune Mobilising mTCR Against Cancer) technology that aims to recruit and redirect circulating T-cells to target and kill cancer cells with high specificity. ImmTACs are composed of an affinity enhanced monoclonal T-cell receptor (mTCR) combined with an anti-CD3 specific antibody fragment (CD3-scFv). The mTCR targets cancer cells that express TAAs displayed on class I MHC molecules, with high affinity. The CD3-scFv effector domain then drives the recruitment and activation of T-cells. ImmTACs enable an immune synapse to form, leading to the destruction of cancer cells by T-cells. TAA candidates are selected according to their level of gene expression by measuring their mRNA levels by RT-PCR in healthy and cancer cells, in order to reduce off-target toxicity, as well as by measuring their cell surface expression on class I MHCs by mass spectrometry, to verify target presentation. We present here our current clinical ImmTAC candidate, IMCgp100, which targets the melanoma-associated gp100 antigen presented on HLA-A2. IMCgp100 has been extensively studied in vitro and is undergoing a phase IIa clinical trial in advanced melanoma patients in the UK and US. The maximum tolerated dose has been established and a dose expansion phase is currently underway. T-cell mobilisation, relevant cytokine release as well as tumour shrinkage have been monitored. The very encouraging results observed so far in our clinical trials show ImmTAC technology to be a promising efficient immunotherapy for cancer. Citation Format: Johanne Pentier, Mary Connolly, Martina Canestraro, Ruth Ryan, Luise Weigand, Namir Hassan, Annelise Vuidepot, Bent Jakobsen. IMCgp100 ImmTAC: A new immunotherapeutic reagent for the treatment of malignant melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A037.

Allele Specific Expression of MICA Variants in Human Fibroblasts Suggests a Pathogenic Mechanism.

The major histocompatibility complex class I chain-related gene A (MICA) is involved in immune responses of both nature killer (NK) cells and subsets of T cells with its receptor NKG2D. MICA is highly polymorphic in sequence which leads to MICA protein variants with distinct features. Specific polymorphisms of MICA have been associated with inflammatory diseases, including ankylosing spondylitis (AS), ulcerative colitis (UC) and Behçet’s disease. Studies herein characterize expression features of three MICA variants including MICA*008, a common variant in general population, and *MICA*007 and *019, which are associated with susceptibility to inflammatory diseases. MICA*019 was highly expressed on the surface of fibroblasts whereas expression of MICA*007 was the lowest in the culture supernatant. MICA*008 had low cell surface expression but was the only MICA allele in which exosomal material was detected. Surface or membrane-bound MICA activates NKG2D-mediated cytotoxicity, whereas soluble and exosomal MICAs down-regulate NKG2D. Therefore, comparisons of these three MICA variants in fibroblasts provides insight into understanding how MICA associated immune responses could be regulated to influence levels of inflammation.

TREM2 Protein Expression Changes Correlate with Alzheimer's Disease Neurodegenerative Pathologies in Post-Mortem Temporal Cortices.

Triggering receptor expressed by myeloid cells 2 (TREM2), a member of the immunoglobulin superfamily, has anti‐inflammatory phagocytic function in myeloid cells. Several studies have shown that TREM 2 gene variant rs75932628‐T increased the risks for Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. It has been suggested that the risks could be resulted from the loss of TREM2 function caused by the mutation. Indeed, new evidence showed that several mutations in the immunoglobulin‐like V‐region led to low cell surface expression of TREM2 and reduced phagocytic function. Because of the emerging importance in understanding TREM2 expression and functions in human neurodegenerative diseases, we conducted biochemical and morphological studies of TREM2 expression in human post‐mortem temporal cortical samples from AD and normal cases. Increased expression of TREM2 protein was found to significantly correlate with increases of phosphorylated‐tau and active caspase 3, a marker of apoptosis, and also loss of the presynaptic protein SNAP25. Strong intensities of TREM2 immunoreactivity were observed in the microglia associated with amyloid plaques and in neuritic pathology‐enriched areas. Based on the findings that TREM2 expression correlated with neurodegenerative markers, further investigation on whether there is abnormality of TREM2 functions in AD brains with nonmutated TREM2 is needed.

Functional analysis of the distal region of the third intracellular loop of PROKR2

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270–272) resulted in undetectable cell surface expression, whereas RKR (264–266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.

A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44high/EGFRlow, CD44high/EGFRhigh and CD44low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44high/EGFRlow population showed a spindle-shaped EMT-like morphology, while the CD44low population was dominated by cobblestone-shaped cells. The CD44high/EGFRlow population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44high/EGFRlow cells in comparison to CD44low cells. Moreover, CD44high/EGFRlow cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44high/EGFRhigh and CD44low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.

Sensitivity of T-cell Acute Lymphoblastic Leukemia to TRAIL Induced Apoptosis

Objective To observe the sensitivity of T-cell acute lymphoblastic leukemia(T-ALL) to tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-mediated apoptosis and to discuss the possible mechanism.Methods 3 T-ALL cell lines and a primary cell were used and the expression of TRAIL receptors on the cell surface as well as the viability and apoptosis after incubation with TRAIL were measured.Results Most of the cell lines and the primary leukemia cell were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors.Conclusion Low expression of cellular surface death receptors might be one of the mechanisms for resistance to TRAIL-mediated cellular immunity.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Heterogeneity in the processing of CLCN5 mutants related to Dent disease

Mutations in the electrogenic Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC-5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss of function in Dent disease.

Contribution of Thomsen-Friedenreich antigens to bladder cancer malignancy: Characterization of cell line models

A particular type of glycans has been associated with cancer, the O-linked glycosidic Thomsen-Friedenreich (TF) antigens -T and Tn, and the sialylated forms sT and sTn. The expression of TF antigens in bladder cancer (BC) has also been reported, although the correlation with prognosis is controversial. Furthermore, the molecular basis underlying their expression and the role played by the enzymes sialyltransferases (STs), responsible for the expression of sialylated forms, are unknown [1]. For a better understanding, we chose to study the cell line models: MCR and HT1376, exhibiting a low cell surface expression of the sTn and sT respectively, that were transduced with “ST6GalNac.I” or “ST3Gal.I” STs cDNA. The resulted transduced cells MCRST6GalNac1 and HT1376ST3Gal1 where purified by immune-magnetic sorting for the sTn, and selected clones for the expression of sT. Our preliminary results show differences concerning to cell adhesion ability and immunogenicity. Namely, dendritic cells (DCs) and MCRST6GalNac1 in co-cultures, results in an increased expression of TGF-b and IL-10, and decreased of TNF-a and IL-6. After adhesion, MCR negative control (MCRNC) induces higher maturation on DCs (higher expression of HLA-DR, CD80, CD83 and CD86). Concerning to phagocytosis, it was seen that HT1376ST3Gal1 are apparently more resistant to phagocytosis by macrophages, in respect to negative control (HT1376NC), and MCRST6GalNac1 less resistant to phagocytosis by DCs, in respect to MCRNC. Author details CEDOC, Departamento de Imunologia, Faculdade de Ciencias Medicas, Universidade Nova de Lisboa, Lisboa, Portugal. Departimento di Patologia Sperimentale, Universita di Bologna, Bologna, Italia.

Resistance of T-cell acute lymphoblastic leukemia to tumor necrosis factor−related apoptosis-inducing ligand-mediated apoptosis

Cytotoxic ligands are involved in tumor immunity and graft-vs.-leukemia effect after allogeneic stem cell transplantation for leukemia. To clarify the susceptibility of T-cell acute lymphoblastic leukemia (T-ALL) to tumor immunity, sensitivity to recombinant human soluble Fas ligand (rhsFasL) and tumor necrosis factor-related apoptosis-inducing ligand (rhsTRAIL) was determined. Sensitivity to rhsFasL and rhsTRAIL and cell surface expression of their receptors were tested in T-ALL cell lines (n = 7) and patients' samples (n = 17) and compared with those in B-precursor ALL cell lines (n = 30). Expression of components of the death-inducing signaling complex and the TRAIL receptor genes (DR4/DR5), and the methylation status and promoter activity of the DR4/DR5 gene were tested in T-ALL cell lines. T-ALL cell lines showed higher level of Fas expression and higher sensitivity to rhsFasL than did B-precursor ALL cell lines. Despite comparable expression of components of death-inducing signaling complex, cell lines and patients' samples of T-ALL showed TRAIL-resistance associated with low cell surface expression of DR4/DR5. Gene expression of DR4/DR5 in T-ALL cell lines was significantly lower than that in B-precursor ALL cell lines, and the methylation status of the gene promoter in T-ALL cell lines was associated with the gene expression level at least for DR4. The demethylating agent, 5-aza 2'deoxycytidine, upregulated the gene expression of DR4/DR5, but was insufficient for their surface expression due to low basal promoter activity. In contrast to higher sensitivity to FasL, T-ALL showed resistance to TRAIL, which might be responsible for resistance to TRAIL-mediated cellular immunity.

Insertion of an NPVY sequence into the cytosolic domain of the erythropoietin receptor selectively affects erythropoietin-mediated signalling and function

EPO (erythropoietin), the major hormone regulating erythropoiesis, functions via activation of its cell-surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell-surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anaemia. In a recent study [Nahari, Barzilay, Hirschberg and Neumann (2008) Biochem. J. 410, 409-416] we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell-surface expression. In the present study we demonstrate that this NPVY EPO-R insert has a selective effect on EPO-mediated downstream signalling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by increased phosphorylation of the NPVY-EPO-R (on Tyr479), Akt, JAK2 (Janus kinase 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2), but not STAT5 (signal transducer and activator of transcription 5), as compared with cells expressing wild-type EPO-R. This enhanced signalling is reflected in augmented proliferation at low EPO levels (0.05 units/ml) and protection against etoposide-induced apoptosis. Increased cell-surface levels of NPVY-EPO-R are most probably not sufficient to mediate these effects as the A234E-EPO-R mutant that is expressed at high cell-surface levels does not confer an augmented response to EPO. Taken together, we demonstrate that insertion of an NPVY sequence into the cytosolic domain of the EPO-R confers not only improved maturation, but also selectively affects EPO-mediated signalling resulting in an improved responsiveness to EPO reflected in cell proliferation and protection against apoptosis.

Optimization of the HA-1-Specific T Cell Receptor for Gene Therapy of Hematological Malignancies.

Abstract 4093Poster Board III-1028Relapsed hematological malignancies after HLA-matched allogeneic stem cell transplantation (allo-SCT) are treated by donor lymphocyte infusion (DLI), inducing long-lasting complete remissions. However, treatment of relapsed hematological malignancies after allo-SCT with DLI is associated with induction of graft versus host disease (GvHD). It has been demonstrated that T cells recognizing minor histocompatibility antigens (mHags) selectively expressed on hematopoietic cells mediate anti-leukemic reactivity after allo-SCT without causing GvHD. mHags are derived from genetically polymorphic proteins that can be differentially expressed between donor and recipient. The mHag HA-1 is presented in the context of HLA-A2. The HA-1 tissue distribution is restricted to hematopoietic cells and carcinomas, making it an attractive target antigen to treat hematological malignancies relapsing after allo-SCT when the patient is HA-1+ and the donor is HA-1-. Therefore, adoptive transfer of HA-1-TCR gene modified T cells might be an attractive strategy to separate GvHD from graft versus leukemia effect (GvL). For optimal anti-leukemic reactivity, high expression of introduced TCRs and persistence of the gene modified T cells is important. Based on the previously reported low HA-1-TCR expression on HA-1-TCR modified T cells, optimization of the strategy is required. Several strategies to improve expression of the introduced TCR have been described. Protein expression of the TCR chains can be enhanced by codon optimization. In addition, preferential pairing facilitated by introduction of an extra disulfide bond in the constant regions of the TCR chains can increase the cell surface expression of the transferred TCR. Another strategy based on the fact that TCRs differ in their capacity to compete for cell surface expression, is to select recipient T cells with weak competitor phenotypes. In this study, we investigated the cause of low HA-1-TCR expression after gene transfer, and used the different strategies to increase HA-1-TCR expresssion. To study whether low HA-1-TCR expression was due to inefficiency of the TCRα and β chains to pair, TCR-deficient jurkat cells were transduced with the individual TCRα and TCRβ chains in combination with 17 different TCRα chains and TCRβ chains. Results indicated that low HA-1-TCR expression was not due to inefficient pairing of the HA-1-TCR chains, but caused by low HA-1-TCRβ chain expression on the cell surface. To investigate whether low cell surface expression of the HA-1-BV6S4 chain was due to intrinsic properties, the CDR1 and CDR3 region of the HA-1-TCR BV6S4 chain were exchanged with the CDR1 and CDR3 region of the HA-2-TCRβ (BV6S2) chain. We demonstrated that exchange of the HA-1-TCRβ CDR1 region with the HA-2-TCRβ CDR1 region resulted in improved TCR-expression, however, the HA-1-specificity was completely abolished, indicating that the HA-1-TCRβ CDR1 region is crucial for HA-1-specificity. Furthermore, since there is exclusive TCRBV6S4 chain usage of HA-1-specific T cells, we were unable to select for another HA-1-TCR for clinical use, and were pressed to optimize the HA-1-TCRβ chain. Both codon optimization of the HA-1-TCR chains aiming at improving protein expression and inclusion of cysteine residues in the HA-1-TCR chains aiming at inducing preferential pairing resulted in a significant increase in HA-1-TCR expression. Combining the two strategies increased the HA-1-TCR expression even more, resulting in 70% and 35% of tetramer positive HA-1-TCR transferred weak competitor and strong competitor T cells, respectively. In addition, the HA-1-TCR engineered T cells were able to efficiently recognize target cells that endogenously process and present HA-1, independent of whether the recipient T cells were strong or weak competitor T cells. These results illustrate that engineering of the HA-1-TCR by codon optimization and introduction of an extra cysteine bond resulted in high numbers of high-avidity HA-1-TCR in any T cell of choice irrespective of the properties of the endogenous TCR. Disclosures:No relevant conflicts of interest to declare.

Engineering of a parainfluenza virus type 5 fusion protein (PIV-5 F): Development of an autonomous and hyperfusogenic protein by a combinational mutagenesis approach

The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope with the target cell membrane. For the paramyxovirus parainfluenza virus type 5 (PIV-5), this fusion involves an attachment protein (HN) and a class I viral fusion protein (F). We investigated the effect of 20 different combinations of 12 amino-acid substitutions within functional domains of the PIV-5 F glycoprotein, by performing cell surface expression measurements, quantitative fusion and syncytia assays. We found that combinations of mutations conferring an autonomous phenotype with mutations leading to an increased fusion activity were compatible and generated functional PIV-5 F proteins. The addition of mutations in the heptad-repeat domains led to both autonomous and hyperfusogenic phenotypes, despite the low cell surface expression of the corresponding mutants. Such engineering approach may prove useful not only for deciphering the fundamental mechanism behind viral-mediated membrane fusion but also in the development of potential therapeutic applications.

Disruption of Cdk5-Associated Phosphorylation of Residue Threonine-161 of the δ-Opioid Receptor: Impaired Receptor Function and Attenuated Morphine Antinociceptive Tolerance

Morphine is the most commonly used and most effective analgesic in the clinic. However, its use is limited by the tolerance. Evidence indicates that the delta-opioid receptor (DOR) is essential for morphine antinociceptive tolerance; however, their underlying mechanisms are poorly understood. Here, we show that cyclin-dependent kinase 5 (Cdk5), activated in morphine antinociceptive tolerance, directly phosphorylates DOR at Thr-161 in DRG neurons. Cdk5 was found to phosphorylate Thr-161 in the second loop of DOR, but not the corresponding residue in the mu-opioid receptor (MOR). Phosphorylation at Thr-161 is required for normal cell surface expression of DOR, and the formation of DOR-MOR heterodimers. Our studies indicated that inhibition of Cdk5 activity or overexpression of a DOR mutant lacking the Cdk5 phosphorylation site displayed relatively low cell surface expression and relatively low abilities to form heterodimers of DOR and MOR; intrathecal delivery of a construct expressing the T161A mutant of DOR attenuated morphine antinociceptive tolerance in rats, suggesting that Thr-161 phosphorylation of DOR contributed to Cdk5-mediated morphine antinociceptive tolerance. Furthermore, an engineered Tat fusion-interfering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L), reduced the cell surface expression of DOR, disrupted the formation of DOR-MOR heterodimers, and significantly attenuated the development of morphine antinociceptive tolerance after intrathecal injection. The present study indicates that Cdk5-mediated phosphorylation of DOR at Thr-161 plays a crucial role in the development of morphine tolerance and suggests the possibility of targeting DOR phosphorylation at Thr-161 to attenuate morphine antinociceptive tolerance during pain management.

A Novel Trafficking Signal within the HLA-C Cytoplasmic Tail Allows Regulated Expression upon Differentiation of Macrophages

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.

Development of Novel 68Ga- and 18F-Labeled GnRH-I Analogues with High GnRHR-Targeting Efficiency

A large majority of tumors of the reproductive system express the gonadotropin releasing hormone receptor (GnRHR). Blockade and activation of this receptor with various antagonistic and agonistic analogues of native GnRH-I (pGlu(1)-His(2)-Trp(3)-Ser(4)-Tyr(5)-Gly (6)-Leu(7)-Arg(8)-Pro(9)-Gly(10)-NH2), respectively, has shown efficient suppression of tumor growth. In this study, the GnRH-receptor system has been evaluated with respect to its suitability as a target for in vivo peptide receptor targeting using radiolabeled GnRH-analogues, and in parallel, new (18)F- and (68Ga)-labeled GnRH analogues have been developed. In vitro radioligand binding assays performed with various GnRHR-expressing human cell lines using [(125)I]Triptorelin (D-Trp(6)-GnRH-I) as the standard radioligand revealed a very low level of GnRH receptor expression on the cell surface. Generally, total cellular activity was very low (approximately 3% of the applied activity), and only a small fraction (max. 40%) of cell-associated activity could be attributed to receptor-specific radioligand binding/internalization. However, substitution of fetal calf serum by NU serum in the culture medium led to increased and stable GnRHR-expression, especially in the ovarian cancer cell line EFO-27, thus allowing for a stable experimental setup for the evaluation of the new radiolabeled GnRH-I analogues. The new radiolabeled GnRH-I analogues developed in this study were all based on the D-Lys(6)-GnRH-I-scaffold. For (68)Ga-labeling, the latter was coupled with DOTA at D-Lys(6). To allow (18)F-labeling via chemoselective oxime formation, D-Lys(6)-GnRH-I was also conjugated with Ahx (aminohexanoic acid) or beta-Ala, which in turn was coupled with Boc-aminooxyacetic acid. (18)F-labeling via oxime formation with 4-[(18)F]fluorobenzaldehyde was performed using the Boc-protected precursors. Receptor affinities of [(68)Ga]DOTA-GnRH-I, D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I, and D-Lys(6)-betaAla([(18)F]FBOA)-GnRH-I (FBOA = fluorobenzyloxime acetyl) were determined using GnRHR-membrane preparations, and internalization efficiency of the new radioligands was determined in EFO-27 cells. Both quantities were highest for D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I (IC 50 = 0.50 +/- 0.08 nM vs 0.13 +/- 0.08 nM for Triptorelin; internalization: 86 +/- 16% of the internal reference [(125)I]Triptorelin), already substantially reduced in the case of the -betaAla([(18)F]FBOA)-derivative (IC 50 = 0.86 +/- 0.13 nM; internalization: 42 +/- 3% of [(125)I]Triptorelin), while the [(68)Ga]DOTA-analogue showed almost complete loss of binding affinity and ligand internalization (IC50 = 13.3 +/- 1.0 nM; internalization: 2.6 +/- 1.0% of [(125)I]Triptorelin). Generally, the lipophilic residue [(18)F]FBOA is much better tolerated as a modification of the D-Lys(6)-side chain, with receptor affinity of the respective analogues strongly depending upon spacer length between the D-Lys(6)-side chain and the [(18)F]FBOA-moiety. In summary, D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I shows the highest potential for efficient GnRHR-targeting in vivo of the compounds investigated. Unfortunately, however, the very low cell surface expression of GnRH-receptors and thus very low radioligand uptake by GnRHR-positive tumor cells found in vitro was also confirmed by a preliminary biodistribution study in OVCAR-3 xenografted nude mice using the standard GnRHR radioligand [(125)I]Triptorelin. Tumor uptake was lower than blood activity concentration at 1 h p.i. (0.49 +/- 0.05 vs 0.96 +/- 0.13 for tumor and blood, respectively). These data seriously challenge the suitability of the GnRHR-system as a suitable target for in vivo peptide receptor imaging using radiolabeled GnRH-I derivatives, despite the availability of high-affinity radiolabeled receptor-ligands such as D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I.

Intrinsic Differences in the Response of the Human Lutropin Receptor Versus the Human Follitropin Receptor to Activating Mutations

In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.