Assessment of CYP-Mediated Drug Interactions for Enasidenib Based on a Cocktail Study in Patients with Relapse or Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome.

As a selective and potent inhibitor targeting the isocitrate dehydrogenase-2 (IDH2) mutant protein, enasidenib obtained approval from the US Food and Drug Administration (FDA) in 2017 for adult patients with acute myeloid leukemia (AML) with an IDH2 mutation. In vitro investigations demonstrated that enasidenib affects various drug metabolic enzymes and transporters. This current investigation aimed to assess enasidenib on the pharmacokinetics (PKs) of CYP substrates, including dextromethorphan (CYP2D6 probe drug), flurbiprofen (CYP2C9 probe drug), midazolam (CYP3A4 probe drug), omeprazole (CYP2C19 probe drug), and pioglitazone (CYP2C8 probe drug), in patients with AML or myelodysplastic syndrome. Results showed that following the co-administration of enasidenib (100 mg, once daily) for 28 days, the PK parameters AUC(0-∞) and Cmax of dextromethorphan increased by 1.37 (90% confidence interval (CI): 0.96, 1.96) and 1.24 (90% CI: 0.94, 1.65)-fold, respectively, compared to dextromethorphan alone. For flurbiprofen, these parameters increased by 1.14 (90%CI: 1.01, 1.29) and 0.97 (90% CI 0.86, 1.08)-fold, respectively, when compared to flurbiprofen alone. Conversely, midazolam exhibited decreases to 0.57 (90% CI 0.34, 0.97) and 0.77 (90% CI 0.39, 1.53)-fold, respectively, in comparison to midazolam alone. The parameters for omeprazole increased by 1.86 (90% CI: 1.33, 2.60) and 1.47 (0.93, 2.31)-fold, respectively, compared to omeprazole alone, while those for pioglitazone decreased to 0.80 (90% CI: 0.62, 1.03) and 0.87 (90% CI: 0.65, 1.16)-fold, respectively, in comparison to pioglitazone alone. These findings provide valuable insights into dose recommendations concerning drugs acting as substrates of CYP2D6, CYP2C9, CYP3A4, CYP2C19, and CYP2C8 when administered concurrently with enasidenib.

Relative bioavailability of fedratinib through various alternative oral administration methods in healthy adults.

Fedratinib is an oral Janus kinase 2-selective inhibitor for the treatment of adult patients with intermediate-2 or high-risk myelofibrosis; however, some patients have difficulty with oral dosing. This randomized, phase 1, open-label, 2-part crossover study evaluated the relative bioavailability, safety, tolerability, taste, and palatability of fedratinib resulting from various alternative oral administration methods in healthy adults. Participants could receive fedratinib 400 mg orally as intact capsules along with a nutritional supplement; as contents of capsules dispersed in a nutritional supplement, delivered via nasogastric tube; or as a divided dose of 200 mg orally twice daily as intact capsules with a nutritional supplement. Fifty-eight participants received treatment. Total exposure to fedratinib was similar after oral administration of intact capsules or when dispersed in a nutritional supplement (area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration geometric mean ratio [AUC0-t GMR] [90% CI], 1.007 [0.929-1.092]). Total exposure to fedratinib was slightly reduced following nasogastric administration (AUC0-t GMR 0.850 [0.802-0.901]) and as a divided dose (AUC0-t GMR 0.836 [0.789-0.886]). No new safety signals were identified for fedratinib, and most participants found the taste and palatability acceptable when dispersed in a nutritional supplement. Overall, results suggest no clinically meaningful differences in total exposure to fedratinib between the tested oral administration methods. These findings may facilitate administration of fedratinib to patients who are intolerant of swallowing the capsule dosage form. (ClinicalTrials.gov: NCT05051553).

Effect of fluconazole on the pharmacokinetics of a single dose of fedratinib in healthy adults.

PURPOSE: Fedratinib is an orally administered Janus kinase (JAK) 2-selective inhibitor for the treatment of adult patients with intermediate-2 or high-risk primary or secondary myelofibrosis. In vitro, fedratinib is predominantly metabolized by cytochrome P450 (CYP) 3A4 and to a lesser extent by CYP2C19. Coadministration of fedratinib with CYP3A4 inhibitors is predicted to increase systemic exposure to fedratinib. This study evaluated the effect of multiple doses of the dual CYP3A4 and CYP2C19 inhibitor, fluconazole, on the pharmacokinetics of a single dose of fedratinib. METHODS: In this non-randomized, fixed-sequence, open-label study, healthy adult participants first received a single oral dose of fedratinib 100 mg on day 1. Participants then received fluconazole 400 mg on day 10 and fluconazole 200 mg once daily on days 11-23, with a single oral dose of fedratinib 100 mg on day 18. Pharmacokinetic parameters were calculated for fedratinib administered with and without fluconazole. RESULTS: A total of 16 participants completed the study and were included in the pharmacokinetic population. Coadministration of fedratinib with fluconazole increased maximum observed plasma concentration (Cmax) and area under the plasma concentration-time curve from time 0 to the last quantifiable concentration (AUC0-t) of fedratinib by 21% and 56%, respectively, compared with fedratinib alone. Single oral doses of fedratinib 100 mg administered with or without fluconazole were well tolerated. CONCLUSIONS: Systemic exposure after a single oral dose of fedratinib was increased by up to 56% when fedratinib was coadministered with fluconazole compared with fedratinib alone. TRIAL REGISTRY: CLINICALTRIALS.GOV: NCT04702464.

Impact of fedratinib on the pharmacokinetics of transporter probe substrates using a cocktail approach.

INTRODUCTION: Fedratinib, an oral, selective Janus kinase 2 inhibitor, has been shown to inhibit P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 2, and multidrug and toxin extrusion (MATE) 1 and MATE2-K in vitro. The objective of this study was to evaluate the influence of fedratinib on the pharmacokinetics (PK) of digoxin (P-gp substrate), rosuvastatin (OATP1B1/1B3 and BCRP substrate), and metformin (OCT2 and MATE1/2-K substrate). METHODS: In this nonrandomized, fixed-sequence, open-label study, 24 healthy adult participants received single oral doses of digoxin 0.25 mg, rosuvastatin 10 mg, and metformin 1000 mg administered as a drug cocktail (day 1, period 1). After a 6-day washout, participants received oral fedratinib 600 mg 1 h before the cocktail on day 7 (period 2). An oral glucose tolerance test (OGTT) was performed to determine possible influences of fedratinib on the antihyperglycemic effect of metformin. RESULTS: Plasma exposure to the three probe drugs was generally comparable in the presence or absence of fedratinib. Reduced metformin renal clearance by 36% and slightly higher plasma glucose levels after OGTT were observed in the presence of fedratinib. Single oral doses of the cocktail ± fedratinib were generally well tolerated. CONCLUSIONS: These results suggest that fedratinib has minimal impact on the exposure of P-gp, BCRP, OATP1B1/1B3, OCT2, and MATE1/2-K substrates. Since renal clearance of metformin was decreased in the presence of fedratinib, caution should be exercised in using coadministered drugs that are renally excreted via OCT2 and MATEs. TRIAL REGISTRATION: Clinicaltrials.gov NCT04231435 on January 18, 2020.

Effects of strong and moderate CYP3A4 inducers on the pharmacokinetics of fedratinib in healthy adult participants.

PURPOSE: Fedratinib is an oral and selective Janus kinase 2 inhibitor that is indicated for treatment of adults with intermediate-2 or high-risk primary or secondary myelofibrosis. Fedratinib is metabolized by cytochrome P450s (CYPs), primarily CYP3A4. The objective of this study was to determine the effects of the strong CYP3A4 inducer rifampin and moderate CYP3A4 inducer efavirenz on the pharmacokinetics of single doses of fedratinib. METHODS: This Phase 1, open-label, two-part study (Part 1 for rifampin and Part 2 for efavirenz) was conducted in healthy adult men and women. A single dose of fedratinib (500 mg) was administered on Day 1. Participants received rifampin 600 mg daily or efavirenz 600 mg daily on Days 9-18. On Day 17, a single dose of fedratinib (500 mg) was coadministered with rifampin or efavirenz. Plasma fedratinib concentrations were measured using validated liquid chromatography-tandem mass spectrometry. RESULTS: Maximum observed plasma fedratinib concentrations were lowered by approximately 70% and 30% during coadministration with rifampin or efavirenz, respectively, compared with fedratinib alone. Geometric means of fedratinib area under the plasma concentration-time curve from 0 to infinity were decreased by 81% (90% confidence interval [CI], 77-83%) and 47% (90% CI, 40-53%) during coadministration with rifampin or efavirenz, respectively. Fedratinib was generally well tolerated when administered alone or in combination with rifampin or efavirenz. CONCLUSION: Significant reductions in fedratinib exposure were observed in the presence of strong or moderate CYP3A4 inducers. These results suggest that agents that are strong or moderate inducers of CYP3A4 should be avoided when coadministered with fedratinib. TRIAL REGISTRATION NUMBER: NCT03983239 (Registration date: June 12, 2019).

Drug-Drug Interaction Study to Assess the Effect of Cytochrome P450 Inhibition and Induction on the Pharmacokinetics of the Novel Cereblon Modulator Avadomide (CC-122) in Healthy Adult Subjects.

Avadomide (CC-122) is a novel immunomodulatory drug that binds to cereblon, a member of the Cullin 4-RING E3 ubiquitin ligase complex. Avadomide has multiple pharmacologic activities including potent immune modulation, antiangiogenic, antitumor, and antiproliferative activity and is being evaluated as an oncology treatment for hematologic malignancies and advanced solid tumors. In vitro study has indicated that cytochrome P450 (CYP) 3A and CYP1A2 appear to be the major enzymes involved in the oxidative metabolism of avadomide. The effects of CYP3A inhibition/induction and CYP1A2 inhibition on the pharmacokinetics of avadomide in healthy adult subjects were assessed in 3 parts of an open-label, nonrandomized, 2-period, single-sequence crossover study. Following a single oral dose of 3 mg, avadomide exposure when coadministered with the CYP1A2 inhibitor fluvoxamine was 154.81% and 107.59% of that when administered alone, for area under the plasma concentration-time curve from time 0 to infinity (AUC0-inf ) and maximum observed plasma concentration (Cmax ), respectively. Avadomide exposures, when coadministered with the CYP3A inhibitor itraconazole, were 100.0% and 93.64% of that when administered alone, for AUC0-inf and Cmax , respectively. Avadomide exposures when coadministered with the CYP3A inducer rifampin were 62.83% and 88.17% of that when administered alone, for AUC0-inf and Cmax , respectively. Avadomide was well tolerated when administered as a single oral dose of 3 mg alone or coadministered with fluvoxamine, itraconazole, or rifampin. These results should serve as the basis for avadomide dose recommendations when it is coadministered with strong CYP3A and CYP1A2 inhibitors and with rifampin.

Exposure-response analysis to assess the concentration-QTc relationship of CC-122.

CC-122 hydrochloride is a novel pleiotropic pathway modifier compound that binds cereblon, a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex. CC-122 has multiple activities including modulation of immune cells, antiproliferative activity of multiple myeloma and lymphoma cells, and antiangiogenic activity. CC-122 is being developed as an oncology treatment for hematologic malignancies and advanced solid tumors. Cardiovascular and vital sign assessments of CC-122 have been conducted in hERG assays in vitro and in a 28-day good laboratory practice monkey study with negative signals. To assess the potential concentration-QTc relationship in humans and to ascertain or exclude a small QT effect by CC-122, a plasma concentration exposure- and ΔQTcF-response model of CC-122 was developed. Intensive CC-122 concentration and paired triplicate electrocardiogram data from a single ascending dose study were included in the analysis. The parameters included in the final linear exposure-response model are intercept, slope, and treatment effect. The slope estimate of 0.0201 with 90% CI of (0.009, 0.035) indicates a weak relationship between ΔQTcF and CC-122 concentration. The upper bounds of the 90% CI of the model-predicted ΔΔQTcF effect at C max from the 4 mg clinical dose and the supratherapeutic dose of 15 mg (1.18 ms and 8.76 ms, respectively) are <10 ms threshold, suggesting that the risk of CC-122 QT prolongation effect at the relevant therapeutic dose range from 1 mg to 4 mg is low.

Biomarkers in Pharmaceutical Research.

BACKGROUND: Biomarkers are important tools in drug development and are used throughout pharmaceutical research. CONTENT: This review focuses on molecular biomarkers in drug development. It contains sections on how biomarkers are used to assess target engagement, pharmacodynamics, safety, and proof-of-concept. It also covers the use of biomarkers as surrogate end points and patient selection/companion diagnostics and provides insights into clinical biomarker discovery and biomarker development/validation with regulatory implications. To survey biomarkers used in drug development--acknowledging that many pharmaceutical development biomarkers are not published--we performed a focused PubMed search employing "biomarker" and the names of the largest pharmaceutical companies as keywords and filtering on clinical trials and publications in the last 10 years. This yielded almost 500 entries, the majority of which included disease-related (approximately 60%) or prognostic/predictive (approximately 20%) biomarkers. A notable portion (approximately 8%) included HER2 (human epidermal growth factor receptor 2) testing, highlighting the utility of biomarkers for patient selection. The remaining publications included target engagement, safety, and drug metabolism biomarkers. Oncology, cardiovascular disease, and osteoporosis were the areas with the most citations, followed by diabetes and Alzheimer disease. SUMMARY: Judicious biomarker use can improve pharmaceutical development efficiency by helping to select patients most appropriate for treatment using a given mechanism, optimize dose selection, and provide earlier confidence in accelerating or discontinuing compounds in clinical development. Optimal application of biomarker technology requires understanding of candidate drug pharmacology, detailed modeling of biomarker readouts relative to pharmacokinetics, rigorous validation and qualification of biomarker assays, and creative application of these elements to drug development problems.

High-dimensional analysis of the aging immune system: verification of age-associated differences in immune signaling responses in healthy donors.

BACKGROUND: Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors. METHODS: In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)]. RESULTS: Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets. CONCLUSIONS: These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.

A dual function of NKG2D ligands in NK-cell activation.

NK-cell killing requires both the expression of activating receptor ligands and low MHC class I expression by target cells. Here we demonstrate that the expression of any of the murine ligands for the NK-cell activating receptor NKG2D results in a concomitant reduction in MHC class I expression. We show this both in tumor cell lines and in vivo. NK-cell lysis is enhanced by the decrease in MHC class I expression, suggesting the change is biologically relevant. These results demonstrate that NKG2D ligand expression on target cells not only allows for activating receptor recognition, but also actively reduces expression of the inhibitory ligand, MHC class I, leading to enhanced recognition and killing by NK cells.

Cutting edge: FcR-like 5 on innate B cells is targeted by a poxvirus MHC class I-like immunoevasin.

Under selective pressure from host immunity, viruses have retained genes encoding immunoevasins, molecules interfering with host viral recognition and clearance. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. We previously described an orthopoxvirus MHC class I-like protein (OMCP) that binds with high affinity to the activating receptor NKG2D on NK and T cell subsets, implicating NKG2D in antiorthopoxvirus immunity. In this study, we report that OMCP also binds in an NKG2D-independent manner to B cells and monocytes/macrophages. We identify murine FcR-like 5 (FCRL5), an orphan immunoregulatory protein highly expressed by innate B lymphocytes, as a specific receptor for OMCP. The three N-terminal Ig domains of FCRL5 are required for OMCP binding. The targeting of FCRL5 by an orthopoxvirus immunoevasin strongly implicates it in contributing to host defense against zoonotic orthopoxviruses.

Murine trophoblast cells induce NK cell interferon-gamma production through KLRK1.

Murine models suggest that natural killer (NK) cells are important for normal implantation site development, in part, through the production of interferon gamma (IFNG). As KLRK1 (NKG2D) is expressed on human and murine uterine NK (uNK) cells, we examined the role of KLRK1 in the interaction between murine trophoblasts and NK cells. Flow cytometric analysis revealed that both murine trophoblast stem (TS) cells and differentiated trophoblast giant cells expressed the KLRK1 ligand retinoic acid early transcript 1, or RAET1. Coculture of activated NK cells with either TS cells or giant cells led to the production of IFNG, as measured by ELISA. In addition, coculture with TS cells led to the downregulation of KLRK1. Both responses were inhibited by soluble KLRK1 ligand, but not by irrelevant protein. Further studies demonstrated the presence of KLRK1 ligand on uterine cells derived from either virgin or pregnant mice, although uterine RAET1 protein expression was upregulated in vitro by progesterone, but not estradiol. We suggest that the interaction of KLRK1 and RAET1 may be involved in IFNG production by uNK cells, and thus, this receptor-ligand pair may contribute to successful murine implantation site development.

Immunodeficient mouse strains display marked variability in growth of human melanoma lung metastases.

PURPOSE: Immunodeficient mice serve as critical hosts for transplantation of xenogeneic cells for in vivo analysis of various biological processes. Because investigators typically select one or two immunodeficient mouse strains as recipients, no comprehensive study has been published documenting differences in human tumor engraftment. Taking advantage of the increased metastatic potential of RhoC-expressing human (A375) melanoma cells, we evaluate four immunodeficient mouse strains: severe combined immunodeficiency (scid), nonobese diabetic (NOD)-scid, NOD-scid beta2m(null), and NOD-scid IL2Rgamma(null) as xenograft tumor recipients. EXPERIMENTAL DESIGN: Bioluminescence, magnetic resonance imaging, and histopathology were used to monitor serial tumor growth. Natural killer (NK) cell function was examined in each mouse strain using standard (51)Chromium release assays. RESULTS: Melanoma metastases growth is delayed and variable in scid and NOD-scid mice. In contrast, NOD-scid beta2m(null) and NOD-scid IL2Rgamma(null) mice show rapid tumor engraftment, although tumor growth is variable in NOD-scid beta2m(null) mice. NK cells were detected in all strains except NOD-scid IL2Rgamma(null), and in vitro activated scid, NOD-scid, and NOD-scid beta2m(null) NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands MHC class I chain-related A and B molecules renders melanoma susceptible to murine NK cell-mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D. CONCLUSIONS: Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction used by NK cells in immunodeficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rgamma(null) animals makes this strain an excellent recipient of melanoma and potentially other human malignancies.

IFN-dependent down-regulation of the NKG2D ligand H60 on tumors.

In this study, we show that IFN-gamma or IFN-alpha reduce expression of H60 on 3'-methylcholanthrene (MCA) sarcomas from 129/Sv mice. As determined by flow cytometry using either NKG2D tetramers or NKG2D ligand-specific mAb, H60 was identified as the NKG2D ligand most frequently expressed on these sarcomas, and its expression was selectively down-regulated by either IFN-gamma or IFN-alpha in a manner that was dose- and time-dependent and reversible. Down-regulation occurred at the transcript level and was STAT1-dependent. It also had functional consequences. IFN-gamma-treated MCA sarcomas with high levels of H60 were resistant to killing by IL-2-activated NK cells. Resistance was not solely dependent on enhanced MHC class I expression but rather also required H60 down-regulation. IFN-gamma-treated tumor cells also displayed diminished capacity to down-regulate NKG2D on freshly isolated NK cells. Transplanted tumor cells reisolated from immunocompetent mice displayed reduced H60 expression and increased MHC class I expression compared with tumor cells that were either left unmanipulated or reisolated from mice treated with neutralizing IFN-gamma-specific mAb. This report thus represents the first demonstration that certain cytokines and specifically the IFNs regulate expression of specific NKG2D ligands on murine tumors. This process most likely helps to specify the type of immune effector cell populations that participate in host-protective antitumor responses.

Recognition of infected cells by natural killer cells.

Under the influence of cytokines associated with innate immunity, natural killer (NK) cells rapidly become activated and migrate to sites of infection. Upon contact with infected parenchyma they proliferate, release cytokines and/or kill cells harboring pathogens. Multiple stimulatory and inhibitory receptors can provide the integrated signals that trigger this contact-mediated NK-cell function. Recent work has begun to define the ligands for these receptors in the context of infection by certain well-studied viruses. These results, in addition to future work involving other pathogens, will provide an understanding of the molecules present on parasitized cells that mark them as targets of innate immunity.

Cutting edge: murine UL16-binding protein-like transcript 1: a newly described transcript encoding a high-affinity ligand for murine NKG2D.

Murine NKG2D is known to recognize H60 and five RAE1 variants. The human homologue recognizes both inducible MHC class I chain-related gene and constitutive (UL16-binding protein (ULBP)) ligands. Widely expressed, the latter are thought to mark transformed or infected cells for destruction by NK cells in the context of down-regulated cell surface class I (i.e., the "missing self"-response). Unlike MIC and ULBP however, mRNA for the murine ligands appears only in very limited contexts in the mature animal. In this study, we describe a NKG2D ligand termed "murine ULBP-like transcript 1 (MULT1) whose mRNA appears to be widely expressed in adult parenchyma. This molecule possesses MHC class I-like alpha1 and alpha2 domains as well as a large cytoplasmic domain. Recombinant MULT1 binds NKG2D with relatively high affinity (K(D) approximately 6 nM) and low k(off) (approximately 0.006s(-1)). Expression of MULT1 by normally resistant RMA cells results in their susceptibility to lysis by C57BL/6 splenocytes.

Costimulation of multiple NK cell activation receptors by NKG2D.

The activation of NK cells is mediated through specific interactions between activation receptors and their respective ligands. Little is known, however, about whether costimulation, which has been well characterized for T cell activation, occurs in NK cells. To study the function of NKG2D, a potential NK costimulatory receptor, we have generated two novel hamster mAbs that recognize mouse NKG2D. FACS analyses demonstrate that mouse NKG2D is expressed on all C57BL/6 IL-2-activated NK (lymphokine-activated killer (LAK)) cells, all splenic and liver NK cells, and approximately 50% of splenic NKT cells. Consistent with limited polymorphism of NKG2D, its sequence is highly conserved, and the anti-NKG2D mAbs react with NK cells from a large number of different mouse strains. In chromium release assays, we show that stimulation of NK cells with anti-NKG2D mAb can redirect lysis. Also, enhanced lysis of transfected tumor targets expressing NKG2D ligand could be inhibited by addition of anti-NKG2D mAb. Interestingly, stimulation of LAK cells via NKG2D alone does not lead to cytokine release. However, stimulation of LAK via both an NK activation receptor (e.g., CD16, NK1.1, or Ly-49D) and NKG2D leads to augmentation of cytokine release compared with stimulation through the activation receptor alone. These results demonstrate that NKG2D has the ability to costimulate multiple NK activation receptors.

Ligands for murine NKG2D display heterogeneous binding behavior.

NKG2D transmits stimulatory signals to natural killer cells and other hematopoietic cells, leading to enhanced proliferation, cytokine secretion and target killing. Murine and human NKG2D each recognize five known class I-related molecules with distinct primary structures. Here, we used surface plasmon resonance to examine the binding of murine NKG2D to its cognate ligands: RAE-1B6 (a newly described C57BL/6J variant of RAE-1), RAE-1 delta (common to BALB and C57BL6/J), and H60 (expressed in BALB, but not C57BL/6J). While RAE-1B6 and H60 display relatively high affinities for NKG2D with K(D) in the 20-30 nM range and k(off )in the 0.03s(-1) to 0.06s(-1) range (t(1/2) approximately 10-20s); the RAE-1 delta variant binds with a lower affinity: K(D) of approximately 750 nM. Furthermore, RAE-1 delta displays biphasic kinetics with dominant k(off) of approximately 0.2s(-1) (t(1/2) approximately 3s), partially explaining the lower affinity. Thus, H60 and RAE-1B6 bind NKG2D with almost identical kinetics while sharing only 20% amino acid sequence identity; whereas other RAE-1 molecules demonstrate faster dissociation and lower affinities than RAE-1B6 despite sharing 90% sequence identity. C57BL/6J mice, although not expressing the H60 gene product, retain a high-affinity ligand for NKG2D in the form of RAE-1B6.