Potent synergistic anti-tumor activity of a novel humanized anti-HER2 antibody hersintuzumab in combination with trastuzumab in xenograft models.

Immunotherapy of HER2-overexpressing cancers by FDA approved monoclonal antibodies (mAbs) such as trastuzumab and pertuzumab has shown promising results. We have recently produced a novel humanized anti-HER2 mAb, hersintuzumab, which did not sterically inhibit binding of trastuzumab and pertuzumab to HER2, thus recognizing a distinct epitope on subdomain I + II of HER2. In this study, we assessed the in vitro and in vivo anti-tumor activity of this mAb individually and in combination with trastuzumab. Different HER2-overexpressing human cancer cell lines, including SKOV3, NCI-N87 HCC1954 and BT-474 were cultured and binding reactivity of Hersintuzumab to these cell lines was analyzed by flow cytometry. In addition, the inhibitory effect of different concentrations of hersintuzumab, trastuzumab and their combination on tumor cells growth was assessed by XTT assay. For Assessment of tumor growth inhibition in xenograft model, Balb/c athymic nude mice were subcutaneously injected with NCI-N87 and SKOV3 tumor cells and then treated intravenously with these mAbs. Our results showed that hersintuzumab could bind to all HER2-overexpressing cell lines similar to trastuzumab. In vitro experiments showed that both hersintuzumab and trastuzumab individually and in combination inhibited growth of all cell lines with the exception of HCC-1954.Inhibitory effect of the combination of mAbs was significantly higher than that of each mAb alone. Similar results were obtained in the gastric (NCI-N87) and ovarian (SKOV-3) tumor xenograft models. Hersintuzumab in combination with trastuzumab induces synergic anti-tumor effects on HER2-overexpressing cells in vitro and in vivo and is potentially a therapeutic tool for treatment of HER2-overexpressing cancers.

Monoclonal antibody directed to the PilQ -PilA DSL region in Pseudomonas aeruginosa improves survival of infected mice with antibiotic combination.

The infections caused by Pseudomonas aeruginosa are related to high mortality and morbidity in critically ill patients because of multidrug resistance. Thus, we performed the efficacy of the monoclonal antibody (mAb) against PilQ -PilA DSL region (QA) in combination with antibiotics in a model of P. aeruginosa infection. In the present study, three clinically applicable antibiotics (levofloxacin, ceftazidime and gentamicin) and the anti-QA mAb were utilized for treatment of P. aeruginosa sepsis in mice. Reliably, in comparison with other treatment groups (antibody or antibiotic administration), the combination of antibiotic and anti-QA mAb essentially enhanced the survival of mice infected with P. aeruginosa PAO1. This synergistic effect was due to improved bactericidal effect, which prevented bacterial dissemination to different organs. Consequently, the antibiotic and anti-QA mAb combination gives a new effective strategy for the treatment of P. aeruginosa sepsis, particularly when large numbers of exceptionally virulent strains are present.

Translation, cross-cultural adaptation, reliability and validity of the Persian version of the ACL-QOL questionnaire in patients with anterior cruciate ligament reconstruction.

BACKGROUND: Quality of life (QOL) measures can be used to make sound clinical decisions after reconstruction of the anterior cruciate ligament (ACL). The purpose of the present study was to translate and cross-culturally adapt the Anterior Cruciate Ligament Quality of Life (ACL-QOL) questionnaire into the Persian language and to evaluate its psychometric properties. METHODS: The process of translation and cross-cultural adaptation followed the World Health Organization method. One hundred and forty-five patients with ACL reconstruction (ACL-R) filled out the Persian versions of ACL-QOL and the SF-36 Questionnaire. The measurement properties of internal consistency, agreement, criterion validity, floor and ceiling effects were measured. 40 out of 145 patients with ACL-R completed the Persian version of the ACL-QOL questionnaire twice for the test-retest reliability. RESULTS: The questionnaire had high internal consistency (Cronbach's α = 0.96). The intraclass correlation was excellent for reliability and agreement in five domains and overall score (ICC 0.87, 0.74, 0.90, 0.85, 0.81 and 0.89; p < 0.001). The standard error of measurement and the minimum detectable change were found to be 3.28 points and 9.9 points, respectively. There was a strong correlation between each item and the total score of the Persian version of ACL-QOL questionnaire. The questionnaire showed strong and moderate criterion validity (r = 0.61, r = 0.37) with SF-36 physical component score and mental component score, respectively. No ceiling and floor effects were observed. CONCLUSIONS: Persian version of the ACL-QOL questionnaire has acceptable reliability and validity and can be used in assessing Iranians quality of life after ACL reconstruction.

Antibody response to HER2 extracellular domain and subdomains in mouse following DNA immunization.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-20 % of breast cancer patients and is an appropriate target for immunotherapy in these patients. Monoclonal antibodies (mAbs) specific to HER2 are currently applied to treat breast cancer patients with HER2 overexpression. Active immunization with HER2 DNA or protein has been considered as a suitable alternative. The aim of this study is to evaluate anti-HER2 antibody response in serum of mice immunized with DNA constructs containing full extracellular domain (fECD) or subdomains of human HER2. Four extracellular subdomains and also fECD of HER2 were cloned into pCMV6-Neo vector. Different groups of Balb/C mice were immunized with HER2 DNA constructs and boosted with HER2 recombinant protein. The anti-HER2 antibody was subsequently determined by ELISA, flow cytometry, and immunohistochemistry. Anti-HER2 antibody was detected only in serum of mice immunized with fECD DNA. None of HER2 extracellular subdomains induced appreciable levels of anti-HER2 antibody. However, boosting with fECD or extracellular subdomain III (DIII) recombinant protein resulted in enhanced anti-HER2 fECD as well as anti-HER2 subdomain antibody responses. In this regard, almost all (99 %) of HER2-overexpressing BT474 cells could be detected by serum antibody from mice immunized with HER2 subdomain DNA and boosted with recombinant HER2 protein by flow cytometry. Similarly, serum of mice immunized with DIII DNA construct and boosted with recombinant DIII protein could also recognize these cells, but to a lesser extent (50 %). Our findings suggest that combination of HER2 DNA and protein immunization could effectively induce anti-HER2 antibody response in Balb/C mice.

The Correlation between Intersegmental Coordination Variability and Frontal Plane Hip Kinematics during Running in Persons with Patellofemoral Pain.

Background: Despite the existing evidence indicating altered hip kinematics as well as the studies showing altered movement coordination variability in persons with patellofemoral pain (PFP), there is no study investigating the correlation between hip joint kinematic and movement coordination variability in persons with patellofemoral pain (PFP). Objective: This study aims to evaluate the correlation between peak hip adduction and variability of thigh frontal-shank transverse coordination during running in persons with PFP. Material and Methods: In this cross-sectional correlational study, kinematic data were collected from 34 females (17 with and 17 without PFP) aged 18-35 years during treadmill running at preferred and fixed speeds, each for 30 s. The continuous relative phase method was used to calculate the coordination of thigh frontal-shank transverse. To calculate the deviation phase as the variability of intersegmental coordination, the standard deviation of the ensemble continuous relative phase curve points was averaged. The parameters of interest were peak hip adduction and coordination variability of thigh frontal-shank transverse. The Pearson Correlation Coefficient (r) was used to calculate the correlation between the variables. Results: The Pearson correlation coefficient showed a significant negative correlation between the peak hip adduction angle and variability of thigh frontal- shank transverse during running at both fixed (r=-0.553, P<0.05) and preferred (r=-0.660, P<0.01) speeds in persons with PFP while the control group showed a small nonsignificant correlation (r<0.29, P>0.05). Conclusion: The results indicated that greater adduction of the hip joint in persons with PFP during running is contributed to lesser variability of thigh frontal-shank transverse.

Establishment of a murine model of breast cancer expressing human epidermal growth factor receptor 2 (4T1-HER2).

AIM: Although people with HER2-positive breast cancer benefit from approved HER2-targeted therapy, acquiring resistance to the therapies occurs. Animal models can play a part in gaining a deep understanding of such a process and addressing questions concerning developing and improving immunotherapy approaches. MATERIALS AND METHODS: To develop such a model, we transfected murine 4T1 cells with the pCMV6-Neo-HER2 construct and evaluated HER2 expression and its effects on the established cell line behavior in vitro and in vivo. RESULTS: Data illustrated that human HER2 protein was expressed on isolated 4T1-HER2 clones in vitro and in vivo. Except for proliferation over 48 hours, such expression did not change 4T1-HER2 characteristics compared to 4T1 in vitro. Notwithstanding the reduction in proliferation, the rate of tumorigenicity was 90% in challenged mice and Herceptin therapy significantly decreased tumors' growth and metastasis compared to the control group. CONCLUSION: We describe a murine model for HER2-positive breast cancer not only helping shed light on the mechanisms by which the tumor evades antitumor immunity but also playing a key role in making breast cancer more sensitive to novel immunotherapy modalities.

Comparing position sense and isokinetic strength of the muscles of elbow joint between aikidokas and non-athlete people.

BACKGROUND: Aikido is a martial art comprises of locking techniques and falls. During the locking techniques, the elbow joint is forced into extended position. Moreover, the elbow hits the ground during the falling techniques. These might compromise joint position sense (JPS). The objectives of this study were to compare JPS and strength of the muscles of elbow joint between Aikidokas and a non-athlete group and to evaluate the correlation between JPS and muscle strength among Aikidokas. METHODS: All male Jiyushinkai style Aikidokas and a healthy matched non-athlete group participated in this cross-sectional study. Passive JPS at a speed of 4°/s and the isokinetic strength of elbow flexors and extensors were assessed. RESULTS: Evaluating the isokinetic parameters revealed no significantly difference between the groups in either flexion or extension at speeds of 60 (P-value range: 0.2-0.99) and 120 °/s (P-value range: 0.05-0.96). Also, the groups had no significant difference regarding different types of reconstruction error including constant error (P-value range: 0.38-0.91), variable error (P-value range: 0.09-0.87), and total variability (P-value range: 0.30-0.80). Moreover, very weak to weak correlation was observed between isokinetic parameters and passive JPS (r-value range: 0.01-0.39). CONCLUSIONS: JPS was not impaired in Aikidokas in spite of the repetitive stress applied to the elbow joint during the performance of Aikido techniques. The lack of significant difference in isokinetic between Aikidokas and healthy non-athletes, and the absence of an acceptable correlation between IPS and muscle strength in Aikidokas, might be attributed to the soft nature of Aikido.

A Novel Fc-Engineered Anti-HER2 Bispecific Antibody With Enhanced Antitumor Activity.

Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.

Comparison of the knee joint reaction force between individuals with and without acute anterior cruciate ligament rupture during walking.

BACKGROUND: Anterior cruciate ligament plays a significant role in knee joint stability. It is claimed that the incidence of knee osteoarthritis increases in individuals with anterior cruciate ligament (ACL) rupture. The aim of this study was to evaluate the knee joints reaction force in ACL rupture group compared to normal subjects. METHOD: Fifteen patients with acute ACL rupture and 15 healthy subjects participated in this study. The ground reaction force (GRF) and kinematic data were collected at a sampling rate of 120 Hz during level-ground walking. Spatiotemporal parameters, joint angles, muscle forces and moments, and joint reaction force (JRF) of lower extremity were analyzed by OpenSIM software. RESULTS: The hip, knee and ankle joints reaction force at loading response and push-off intervals of the stance phase during walking was significantly higher in individuals with ACL rupture compared to healthy controls (p value < 0.05). Walking velocity (p value < 0.001), knee (p value = 0.065) and ankle (p value = 0.001) range of motion in the sagittal plane were significantly lower in the patients with ACL rupture compared to healthy subjects. The mean value of vertical GRF in the mid-stance, the peak of the hip adduction moment in loading response and push-off phases, the hip abductor, knee flexor and vastus intermedius part of quadriceps muscle forces were significantly higher compared to healthy subjects (p < 0.05) while vastus medialis and vastus lateralis produced significantly lower force (p < 0.001). CONCLUSIONS: Based on results of this study, lower limb JRF was higher in those with ACL rupture compared to healthy subjects may be due to the compensatory mechanisms used by this group of subjects. An increase in knee JRF in patients with ACL rupture may be the reason for the high incidence of knee OA.

Trunk, pelvis, and knee kinematics during running in females with and without patellofemoral pain.

BACKGROUND: Females are two times more likely to develop patellofemoral pain (PFP) than males. Abnormal trunk and pelvis kinematics are thought to contribute to the pathomechanics of this condition. However, there is a scarcity of evidence investigating proximal segments kinematics in females with PFP. RESEARCH QUESTION: The purpose of this study was to investigate whether females with PFP demonstrate altered trunk, pelvis, and knee joint kinematics compared with healthy controls during running. METHODS: Thirty-four females (17 PFP, 17 controls) underwent a 3-dimensional motion analysis during treadmill running at preferred and fixed speeds, each trial for 30 s. Variables of interest included magnitudes of peak angles for trunk (forward flexion, ipsilateral trunk lean), pelvis (anterior tilt, contralateral drop), knee (flexion, valgus, internal rotation), range of motion (RoM) of trunk and pelvis in sagittal and frontal planes and RoM of knee joint in the three cardinal planes of motion. Kinematic data were compared between groups using mixed model repeated measure analysis of variance with the trial as the repeated measure. RESULTS: The PFP group displayed significantly less pelvis frontal plane RoM, greater knee frontal plane RoM, and less knee sagittal plane RoM during running compared with controls, irrespective of running trial. No differences were found in peak kinematic variables between PFP and healthy groups. SIGNIFICANCE: These results may suggest a rigid stabilization strategy at the pelvis, which the body has adapted to prevent further frontal plane knee malalignment. Less knee sagittal plane RoM may be indicative of another protective strategy in the PFP group to avoid patellofemoral joint reaction force. Clinical assessments and rehabilitative treatments may benefit from considering a global program with focus on pelvis kinematics in addition to the knee joint in females with PFP.

Effects of Kinesiotape vs Soft and Semirigid Ankle Orthoses on Balance in Patients With Chronic Ankle Instability: A Randomized Controlled Trial.

BACKGROUND: Chronic ankle instability (CAI) is a frequent complication of ankle sprain that may be associated with long-term consequences. Although taping and bracing are common interventions that are widely used by clinicians and athletic trainers for patients with CAI, no studies have compared the effects of kinesiotaping and bracing on balance performance in these patients. The present study aimed to compare the effects of ankle kinesiotaping, a soft ankle orthosis, and a semirigid ankle orthosis on balance performance in patients with CAI. METHODS: Sixty patients with CAI were randomly assigned to 4 groups that received kinesiotaping, a soft orthosis, a semirigid orthosis, or no treatment (control group). Dynamic and static balance were measured with the modified Star Excursion Balance Test, single leg hop test, and single leg stance test before and after a 4-week intervention period. RESULTS: Significant between-group differences were seen in all evaluated outcomes (P ≤ .003). The lowest reach distances in all directions in the modified Star Excursion Balance Test were found in the control group, and these patients also had a significantly shorter measured distance in the single leg hop test, and more errors in the single leg stance test compared with the 3 intervention groups. No significant differences were found among the 3 intervention groups. CONCLUSION: Use of kinesiotaping and a soft or a semirigid ankle brace for 4 weeks were all beneficial in improving static and dynamic balance in individuals with CAI. None of the interventions was superior to the other 2. LEVEL OF EVIDENCE: Level I, randomized controlled trial.

Can fibular reposition taping improve balance performance in individuals with chronic ankle instability? A randomized controlled trial.

BACKGROUND: Lateral ankle sprain results in positional faults in the fibula which are thought to limit accessory motion in the ankle, leading to hypomobility and negatively influencing sensorimotor function and postural control. Although it has been shown that fibular reposition taping (FRT) is effective in the prevention of recurrent lateral ankle sprain, its ability to produce significant changes in balance measures in patients with chronic ankle instability is inconclusive. OBJECTIVE: This study aimed to determine whether a FRT intervention affects balance performance in patients with chronic ankle instability. DESIGN: Randomized controlled trial. METHODS: Sixty individuals with chronic ankle instability were randomly allocated to three groups: FRT, sham taping, or no intervention (control group). Kinesiotape was applied and then re-applied on 3 occasions per week for 2 weeks. Static and dynamic balance were measured with three functional tests before and 1 day after the last session of intervention with the tape removed: single-leg stance test, single-leg hop test for distance, and modified Star Excursion Balance Test (mSEBT). RESULTS: The results of ANCOVA showed that there were no significant differences between the three groups except for mSEBT reach distance in the posterolateral direction, which was significantly greater in the FRT group than the control group (p = 0.03). CONCLUSION: Applying FRT for 2 weeks did not significantly affect static or dynamic balance measures in individuals with chronic ankle instability, hence its clinical efficacy to influence balance remains uncertain in this population. CLINICAL TRIAL REGISTRATION NUMBER: IRCT20171122037576N2.

A Novel Anti-HER2 Bispecific Antibody With Potent Tumor Inhibitory Effects In Vitro and In Vivo.

Overexpression of HER2 has been reported in many types of cancer, making it a perfect candidate for targeted immunotherapy. The combination of two FDA approved monoclonal antibodies (mAbs), trastuzumab and pertuzumab, has more robust anti-tumor activity in patients with HER2-overexpressing breast cancer. We recently produced a new humanized anti-HER2 mAb, hersintuzumab, which recognizes a different epitope than trastuzumab and pertuzumab on HER2. This mAb, in combination with trastuzumab, exhibits more potent anti-tumor activity than each parental mAb alone. Here we have developed a novel bispecific anti-HER2 antibody (BsAb) designated as trasintuzumab, composed of trastuzumab and hersintuzumab, using dual variable domain immunoglobulin (DVD-Ig) technology. Both variable domains of trasintuzumab are fully functional and have similar affinities to the parental mAbs and are also able to bind to natural HER2 on the surface of several HER2-expressing cell lines. Trasintuzumab was found to inhibit the growth of different types of tumor cell lines through suppression of the AKT and ERK signaling pathways as efficiently as the combination of the parental mAbs. It also induced tumor regression as potently as the combination of the two mAbs in nude mice bearing ovarian and gastric cancer xenografts. Our data suggest that trasintuzumab may be a promising BsAb therapeutic candidate for the treatment of HER2-overexpressing cancers.

Production and Characterization of Monoclonal Antibodies against Human Prostate Specific Antigen.

BACKGROUND: Prostate Specific Antigen (PSA) is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies (mAbs) against PSA have been presented. METHODS: Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer (PCa), Benign Prostatic Hyperplasia (BPH) and brain cancer tissues by Immunohistochemistry (IHC). RESULTS: Five anti-PSA mAbs (clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/К) and clones (2C8-E9, 2G3-E2, IgG2a/К) were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot. CONCLUSION: These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.

A novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells.

BACKGROUND: Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem cells (HSCs) and the small- vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a marker for diagnosis and classification of leukemia. Anti CD34 antibodies are used for isolation and purification of HSCs from bone marrow, peripheral blood and cord blood. OBJECTIVE: To characterize a newly produced monoclonal antibody against a human CD34 peptide. METHODS: Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. RESULTS: ELISA experiment revealed that the antibody recognized CD34 peptide. Western blot analysis on TF1 cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody. CONCLUSIONS: Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.