Steven M. Dunn

Directed evolution of human T-cell receptors with picomolar affinities by phage display

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide–human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to ∼1–100 μM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax11–19 peptide–HLA-A*0201 complex and the NY-ESO-1157–165 tumor-associated peptide antigen–HLA-A*0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.

Control of HIV-1 immune escape by CD8 T-cells expressing enhanced T-cell receptor

HIVs considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A*02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (KD < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.

Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157–165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3α and CDR2β amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1+/HLA-A*02+ tumor cell lines by TCR gene-modified CD4+ T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3α chain was identified that enhanced Ag-specific reactivity in gene-modified CD4+ and CD8+ T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27–35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4+ T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

High-Affinity TCRs Generated by Phage Display Provide CD4+ T Cells with the Ability to Recognize and Kill Tumor Cell Lines

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 μM to 26 pM). CD8+ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (KD value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, KD 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with KD values of 450 nM and 4 μM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8+ T cells, introduction of these TCR dramatically increased the reactivity of CD4+ T cells to tumor cell lines.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity.

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell‐surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide‐independent cross‐reactivity of native TCRs is generally avoided through cell‐mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line‐encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high‐affinity TCRs with therapeutic and diagnostic potential.

An Intrathecal Fentanyl Dose-Response Study in Lower Extremity Revascularization Procedures

Background:Intrathecal opioids routinely are administered to surgical patients to provide prolonged postoperative analgesia. This study evaluated the dose-response effects of intrathecal fentanyl in an elderly patient population undergoing lower extremity revascularization procedures. Methods:Surgical anesthesia was induced using a continuous spinal technique. No patient received perioperative antiemetics or opioids. Postoperatively, after complete regression of spinal anesthesia, patients received via the spinal catheter either 0, 5,10, 20,40, or 50 µg fentanyl. Blood pressure, heart rate, respiratory rate, oxyhemoglobin saturation, and visual analog pain scores were recorded approximately every minute for 15 min after study drug administration. After this Initial observation period, blood pressure, heart rate, respiratory rate, oxyhemoglobin saturation were recorded every 15 min for the next 2 h, then every 30 mln thereafter. A verbal analog pain score, with 0 equivalent to no pain and 10 the worst pain imaginable, was obtained with each set of vital signs. The study was concluded when the verbal analog pain score was ≥3, the patient was medicated, and the time was recorded. Any side effects, such as respiratory depression, nausea, vomiting, and pruritus, were recorded. Results:Minimal analgesia was derived from the 0-, 5-, and 10-µg doses. Beginning at 20 µg, patients experienced the onset of satisfactory analgesia (visual analog pain scores < 3) within 4 mln. All patients in the 40- and 50-µg groups had excellent analgesia (visual analog pain scores < 1) within 10 min. No patient experienced respiratory depression (respiratory rate < 9 breaths/min), hypoxemia (oxyhemoglobin saturation < 90%), or any hemodynamic alterations (20% change greater or less than baseline heart rate or blood pressure). In the 50-µg group, five of ten patients complained of pruritus. Conclusions:Results indicate that 40 µg intrathecal fentanyl provides satisfactory analgesia for approximately 5 h in an elderly patient population, with a low incidence of side effects.

Comparison of epidural fentanyl versus epidural sufentanil for analgesia in ambulatory patients in early labor

Epidural sufentanil, after a lidocaine and epinephrine test dose, provides adequate analgesia and allows for ambulation during early labor. Epidural fentanyl has not been evaluated in this setting. The current study was designed to determine whether there is an analgesic difference between epidural fentanyl and epidural sufentanil in laboring patients. Forty-six laboring nulliparous women, at <5-cm cervical dilation, who requested epidural analgesia were enrolled. After a 3-mL test dose of lidocaine with epinephrine, patients were randomized to receive either sufentanil 20 &mgr;g or fentanyl 100 &mgr;g. After administration of the analgesic, pain scores and side effects were recorded for each patient at 5, 10, 15, 20, and 30 min and every 30 min thereafter, by an observer blinded to the technique used. There were no demographic differences between the two groups. Pain relief was rapid for all patients. The mean durations of analgesia were similar between the sufentanil group (138 ± 50 min) and the fentanyl group (124 ± 42 min). Side effects were similar between the two groups. In early laboring patients, epidural fentanyl 100 &mgr;g, after a lidocaine and epinephrine test dose, provides analgesia comparable to that of sufentanil 20 &mgr;g. Implications In early laboring patients, epidural fentanyl 100 &mgr;g, after a lidocaine and epinephrine test dose, provides analgesia comparable to that of sufentanil 20 &mgr;g.

T cell receptor engagement of peptide-major histocompatibility complex class I does not modify CD8 binding

Activation of cytotoxic T cells is initiated by engagement of the T-cell receptor (TCR) with peptide-major histocompatibility class I complexes (pMHCI). The CD8 co-receptor also binds to pMHCI, but at a distinct site, and allows the potential for tripartite TCR/pMHCI/CD8 interactions, which can increase T cell antigen sensitivity. There has been a substantial interest in the effect of the pMHCI/CD8 interaction upon TCR/pMHCI engagement, and several conflicting studies have examined this event, using the soluble extracellular domains of CD8 and the TCR, by surface plasmon resonance. However, the evidence to date suggests that the TCR engages cognate pMHCI before CD8 recruitment, so the question of whether TCR engagement alters CD8 binding is likely to be more relevant to the biological order of T cell antigen encounter. Here, we have examined the binding of CD8 to several variants of the HLA A2-restricted telomerase(540-548) antigen (ILAKFLHWL) and the HLA A2-restricted NY-ESO-1(157-165) antigen (SLLMWITQC) that bind to their cognate TCRs with distinct affinities and kinetics. These interactions represent a range of agonists that exhibit different CD8 dependency for activation of their respective T cells. By using engineered affinity enhanced TCRs to these ligands, which have extended off-rates of approximately 1h compared to seconds for the wildtype TCRs, we have examined pMHCI/CD8 binding before and during TCR-engagement. Here we show that the binding of the extracellular domain of the TCR to pMHCI does not transmit structural changes to the pMHCI-CD8 binding site that would alter the subsequent pMHCI/CD8 interaction.

The use of fentanyl added to morphine-lidocaine-epinephrine spinal solution in patients undergoing cesarean section

Because of its slow onset of action, intrathecal morphine may not be the optimal drug for intraoperative analgesia during short cases, such as cesarean sections. It is not known whether adding fentanyl to a morphine-lidocaine spinal solution would provide any benefits to patients undergoing cesarean sections. Sixty-two women scheduled for elective cesarean section received intrathecal 5% lidocaine with dextrose (50-70 mg), epinephrine 200 micrograms, preservative-free morphine 0.2 mg, and either 10 micrograms of fentanyl (study group) or preservative-free normal saline (placebo group) in a 0.2-mL volume. Patients were asked to rate their severity of pain on a visual analog scale (VAS) intraoperatively as the uterus was exteriorized and again when the dermatomal level had receded to L1. Intravenous fentanyl was administered if the patient experienced intraoperative discomfort. The VAS scores were 0.8 +/- 1.5 and 2.3 +/- 1.6 (mean +/- SD) in the placebo group at the time of uterine extrusion and in the post-anesthesia care unit (PACU). The corresponding scores in the fentanyl group were 0.4 +/- 1.1 and 2.7 +/- 2.2. There was a significant difference between the two groups in the VAS scores intraoperatively (P < 0.014) but not in the PACU (P not significant). There was also a significant difference (P < 0.015) in the need for supplementation with intravenous (i.v.) fentanyl. Six patients in the placebo group received i.v. fentanyl as compared with none of the patients in the fentanyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrasonography and Pediatric Caudals

Ultrasound is an important tool for performing pediatric regional blocks, including caudal blocks. We present a case in which the availability of ultrasound allowed us to proceed with a successful caudal block which we otherwise might have abandoned in an infant with difficult anatomy.