NKG2D-bispecific enhances NK and CD8+ T cell antitumor immunity.

BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.

High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.

Cross-neutralization of influenza A viruses mediated by a single antibody loop.

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Effects of resistance training on force steadiness and common drive.

The purpose of this study was to investigate the influence of an 8-week resistance training program on force steadiness and common drive for the vastus lateralis muscle. Eight untrained men performed a resistance training program. Before the program and at the end of each week, the subjects performed a trapezoid isometric muscle action of the leg extensors, and bipolar surface electromyographic signals were recorded from the vastus lateralis. The signals were decomposed into action potential trains that were cross-correlated to measure common drive. Force steadiness was quantified as the standard deviation of force during the constant-force portion of the trapezoid muscle action. The training program did not consistently affect force steadiness or common drive for any of the subjects. Although future studies are needed with different muscles and training programs, changes in the overall motor control scheme are not likely with resistance training.

An examination of the time course of training-induced skeletal muscle hypertrophy.

Skeletal muscle hypertrophy is typically considered to be a slow process. However, this is partly because the time course for hypertrophy has not been thoroughly examined. The purpose of this study was to use weekly testing to determine a precise time course of skeletal muscle hypertrophy during a resistance training program. Twenty-five healthy, sedentary men performed 8 weeks of high-intensity resistance training. Whole muscle cross-sectional area (CSA) of the dominant thigh was assessed using a peripheral quantitative computed tomography scanner during each week of training (W1-W8). Isometric maximum voluntary contractions (MVC) were also measured each week. After only two training sessions (W1), the mean thigh muscle CSA increased by 5.0 cm(2) (3.46%; p < 0.05) from the pre-testing (P1) and continued to increase with each testing session. It is possible that muscular edema may have influenced the early CSA results. To adjust for this possibility, with edema assumedly at its highest at W1, the next significant increase from W1 was at W3. W4 was the first significant increase of MVC over P1. Therefore, significant skeletal muscle hypertrophy likely occurred around weeks 3-4. Overall, from the pre-testing to W8, there was an increase of 13.9 cm(2) (9.60%). These findings suggested that training-induced skeletal muscle hypertrophy may occur early in a training program.

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies.

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

Test-retest reliability of barbell velocity during the free-weight bench-press exercise.

The purpose of this study was to calculate test-retest reliability statistics for peak barbell velocity during the free-weight bench-press exercise for loads corresponding to 10-90% of the 1-repetition maximum (1RM). Twenty-one healthy, resistance-trained men (mean ± SD age = 23.5 ± 2.7 years; body mass = 90.5 ± 14.6 kg; 1RM bench press = 125.4 ± 18.4 kg) volunteered for this study. A minimum of 48 hours after a maximal strength testing and familiarization session, the subjects performed single repetitions of the free-weight bench-press exercise at each tenth percentile (10-90%) of the 1RM on 2 separate occasions. For each repetition, the subjects were instructed to press the barbell as rapidly as possible, and peak barbell velocity was measured with a Tendo Weightlifting Analyzer. The test-retest intraclass correlation coefficients (model 2,1) and corresponding standard errors of measurement (expressed as percentages of the mean barbell velocity values) were 0.717 (4.2%), 0.572 (5.0%), 0.805 (3.1%), 0.669 (4.7%), 0.790 (4.6%), 0.785 (4.8%), 0.811 (5.8%), 0.714 (10.3%), and 0.594 (12.6%) for the weights corresponding to 10-90% 1RM. There were no mean differences between the barbell velocity values from trials 1 and 2. These results indicated moderate to high test-retest reliability for barbell velocity from 10 to 70% 1RM but decreased consistency at 80 and 90% 1RM. When examining barbell velocity during the free-weight bench-press exercise, greater measurement error must be overcome at 80 and 90% 1RM to be confident that an observed change is meaningful.

Linearity and reliability of the mechanomyographic amplitude versus dynamic torque relationships for the superficial quadriceps femoris muscles.

The purpose of this investigation was to examine the linearity and reliability of the mechanomyographic (MMG) amplitude versus dynamic torque relationships for the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM) muscles. Nine healthy men and 11 healthy women performed submaximal to maximal, concentric, isokinetic muscle actions of the leg extensors at 30 degrees s(-1) on two occasions. Surface MMG signals were detected from the VL, RF, and VM of the dominant thigh during both trials. The ranges of the coefficients of determination for the MMG amplitude versus dynamic torque relationships were 0.01-0.94 for the VL, 0.01-0.84 for the RF, and 0.19-0.96 for the VM. The intraclass correlation coefficients for the linear MMG amplitude versus torque slope coefficients were 0.823 (VL), 0.792 (RF), and 0.927 (VM). These results indicate that, when analyzed for individual subjects, the MMG amplitude versus dynamic torque relationships demonstrated inconsistent linearity. When using MMG in the clinical setting, dynamic muscle actions of the superficial quadriceps femoris muscles do not appear to be appropriate for assessing changes in muscle function during strength training.

Relationships among peak power output, peak bar velocity, and mechanomyographic amplitude during the free-weight bench press exercise.

The purpose of this study was to examine the relationships among mechanomyographic (MMG) amplitude, power output, and bar velocity during the free-weight bench press exercise. Twenty-one resistance-trained men [one-repetition maximum (1-RM) bench press = 125.4+18.4 kg] performed bench press muscle actions as explosively as possible from 10% to 90% of the 1-RM while peak power output and peak bar velocity were assessed with a TENDO Weightlifting Analyzer. During each muscle action, surface MMG signals were detected from the right and left pectoralis major and triceps brachii, and the concentric portion of the range of motion was selected for analysis. Results indicated that power output increased from 10% to 50% 1-RM, followed by decreases from 50% to 90% 1-RM, but MMG amplitude for each of the muscles increased from 10 to 80% 1-RM. The results of this study indicate that during the free-weight bench press exercise, MMG amplitude was not related to power output, but was inversely related to bar velocity and directly related to the external load being lifted. In future research, coaches and sport scientists may be able to estimate force/torque production from individual muscles during multi-joint, dynamic constant external resistance muscle actions.

Linearity and reliability of the mechanomyographic amplitude versus concentric dynamic constant external resistance relationships for the bench press exercise.

The purpose of the present study was to examine the linearity and reliability of the mechanomyographic (MMG) amplitude versus concentric dynamic constant external resistance (DCER) relationships for the bench press exercise. Twenty-one resistance-trained men (mean +/- SD age = 23.5 +/- 2.7 yr; 1 repetition maximum [1RM] bench press = 125.4 +/- 18.4 kg) volunteered to perform submaximal bench press muscle actions as explosively as possible from 10% to 90% of the 1RM on 2 separate occasions. During each muscle action, surface MMG signals were detected from both the right and left pectoralis major and triceps brachii, and the concentric portion of the range of motion was selected for analysis. The coefficients of determination for the MMG amplitude versus concentric DCER relationships ranged from r2 = 0.010 to 0.980 for the right pectoralis major, r2 = 0.010 to 0.943 for the left pectoralis major, r2 = 0.010 to 0.920 for the right triceps brachii, and r2 = 0.020 to 0.915 for the left triceps brachii, thus indicating a wide range of linearity between subjects. The intraclass correlation coefficients (ICC) and corresponding standard error of measurements (SEM) for the linear slope coefficients for these relationships were 0.592 (39.3% of the mean value), 0.537 (41.9% of the mean value), 0.625 (42.0% of the mean value), and 0.460 (60.2% of the mean value) for the right pectoralis major, the left pectoralis major, the right triceps brachii, and the left triceps brachii, respectively. These data demonstrated that these relationships were neither linear nor reliable enough to be used for assessing issues such as the neural versus hypertrophic contributions to training-induced strength gains and the mechanisms underlying cross-education.

A comparison of techniques for estimating training-induced changes in muscle cross-sectional area.

The ability to accurately estimate changes in muscle cross-sectional area (CSA) could be a useful tool for strength and conditioning practitioners to assess the effectiveness of a resistance training program. The purpose of this study was twofold: (a) to compare the reliability of 2 separate anthropometric-based field estimations of thigh muscle CSA with that of a more accurate, sophisticated imaging technique (peripheral quantitative computed tomography [pQCT] scanner) and (b) to determine if the field methods would be sensitive enough to detect changes in CSA during a resistance training program. Twenty-five healthy, untrained men completed 8 weeks of resistance training. Cross-sectional area testing occurred twice before the start of training, for reliability and again every 2 weeks during the study. Testing consisted of a pQCT scan of the right thigh followed by circumference and skinfold measurements. Two separate equations (Moritani and deVries [M + D] and Housh multiple regression [HMR]) were used to estimate CSA from the anthropometric data. The M + D and HMR methods demonstrated intraclass correlations of 0.983 and 0.961, respectively, but both significantly underestimated thigh muscle CSA when compared to the pQCT. This error was consistent, however, and consequently, the field methods were able to demonstrate increases in muscle CSA with a pattern similar to those from the pQCT. Thus, these equations can be useful tools to evaluate an athlete's progress toward the goal of increasing muscle CSA. It is the authors' hope that the present study will increase awareness among practitioners of these useful field methods for estimating training-induced changes in muscle CSA.

An examination of mechanomyographic signal stationarity during concentric isokinetic, eccentric isokinetic and isometric muscle actions.

The purpose of the present study was to examine the stationarity of surface mechanomyographic (MMG) signals from the vastus lateralis, rectus femoris and vastus medialis during concentric isokinetic, eccentric isokinetic and isometric muscle actions of the leg extensors. Eleven healthy men (mean +/- SD age = 20.1 +/- 1.1 years) performed submaximal to maximal concentric isokinetic, eccentric isokinetic and isometric muscle actions of the leg extensors, and surface MMG signals were detected from the vastus lateralis, rectus femoris and vastus medialis. All surface MMG signals were tested for stationarity with a procedure that examined changes in the mean value, variance, mean frequency and standard deviation of the power spectrum over time. The results showed that the three muscle action types resulted in similar levels of signal nonstationarity. Thus, despite the fact that the isometric muscle actions were performed at a constant force level and with no change in muscle length, the level of signal nonstationarity was similar to that for the concentric and eccentric isokinetic muscle actions. Future studies need to be done to identify the mechanism(s) causing MMG signal nonstationarity during isometric muscle actions.

An examination of the linearity and reliability of the electromyographic amplitude versus dynamic constant external resistance relationships using monopolar and bipolar recording methods.

The purpose of this study was to examine the linearity and reliability of the electromyographic (EMG) amplitude versus dynamic constant external resistance (DCER) relationships for monopolar and bipolar recording techniques during concentric and eccentric muscle actions. Nineteen healthy men (mean ± SD age = 22.9 ± 2.5 years) performed a series of randomly ordered, submaximal to maximal, unilateral DCER muscle actions of the dominant forearm flexors on two occasions separated by at least 48 h. Specifically, the subjects lifted and lowered weights corresponding to 10-100% of the one repetition maximum (1-RM) in 10% increments. During each muscle action, monopolar and bipolar surface EMG signals were detected simultaneously from the biceps brachii. For the monopolar and bipolar methods, the coefficients of determination for the EMG amplitude versus DCER relationships ranged from 0.64-0.98 and 0.38-0.98 for the concentric muscle actions and 0.45-0.98 and 0.45-0.98 for the eccentric muscle actions, respectively. The intraclass correlation coefficients (ICC) and corresponding standard errors of measurement (SEM) for the linear slope coefficients for the EMG amplitude versus DCER relationships were 0.682 (18.4%) and 0.594 (21.8%) with the monopolar method and 0.810 (25.6%) and 0.774 (17.6%) with the bipolar method for the concentric and eccentric muscle actions, respectively. These findings indicated that monopolar and bipolar recording techniques may be used with a similar degree of linearity and reliability for the EMG amplitude versus concentric and eccentric DCER relationships.

Cross-correlation analysis of mechanomyographic signals detected in two axes.

The purpose of this study was to use laser displacement sensors to examine the cross-correlation of surface mechanomyographic (MMG) signals detected from the rectus femoris muscle in perpendicular and transverse axes during isometric muscle actions of the leg extensors. Ten healthy men (mean +/- SD age = 22.1 +/- 1.6 years) and ten healthy women (age = 24.4 +/- 2.8 years) volunteered to perform submaximal to maximal isometric muscle actions of the dominant leg extensors. During each muscle action, two separate MMG signals were detected from the rectus femoris with laser displacement sensors. One MMG sensor was oriented in an axis that was perpendicular (PERP) to the muscle surface, and the second sensor was oriented in an axis that was transverse (TRAN) to the muscle surface. For each subject and force level, the MMG signals from the PERP and TRAN sensors were cross-correlated. The results showed maximum cross-correlation coefficients that ranged from R(x)(,y) = 0.273 to 0.989, but all subjects demonstrated at least one coefficient greater than 0.89. These findings showed a high level of association between the MMG signals detected in the perpendicular and transverse axes. Thus, it may not be necessary to detect MMG signals in multiple axes.