The Effect of Vaccination against COVID-19 in Cancer Patients: Final Results of the COICA Trial.

Background: The COICA study is an ambispective, observational trial that was conceived to assess the clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cancer patients. A recently published, population-based, case-control study reported a reduced vaccine efficacy at 3-6 months in cancer patients compared to individuals without cancer. Objectives: The aim of the study was to describe coronavirus disease 19 (COVID-19) outcomes in cancer patients and analyze differences in SARS-CoV-2 outcomes between vaccinated and unvaccinated patients. Methods: Descriptive statistics and frequency counts were used to summarize characteristics of the study population. χ2 test and the log-rank test were used to compare outcomes between vaccinated and unvaccinated patients. Results: A total of 141 cancer patients (80 males, 61 females) were recruited at two participating Institutions from March 2020 until April 2022 and observed from the time of positive SARS-CoV-2 test to the time of negativization or death. Approximately 35% of patients had been vaccinated at the time of infection with 2 (16 patients) or 3 (33 patients) vaccine doses. Vaccinated patients consistently and significantly showed improved COVID-19 outcomes compared to unvaccinated patients, with CT-diagnosed pneumonia, hospitalization, O2 therapy, and death reported in 0% versus 48.6%, 2.0% versus 15.2%, 0% versus 14.1%, and 0% versus 7.6%, respectively, of assessable patients (p < 0.05). Vaccinated versus unvaccinated patients showed a significantly shorter time to negativization, with a median (95% confidence interval) time of 12 (10-14) versus 20 (17-23) days, respectively (p < 0.001). Conclusions: Vaccination consistently improved all COVID-19 outcomes. No death was recorded among vaccinated patients. Additional research is especially warranted to establish optimal timing and patient selection for administration of the fourth vaccination dose.

The Impact of Routine Molecular Screening for SARS-CoV-2 in Patients Receiving Anticancer Therapy: An Interim Analysis of the Observational COICA Study.

Introduction: Cancer aggravates COVID-19 prognosis. Nosocomial transmission of SARS-CoV-2 is particularly frequent in cancer patients, who need to attend hospitals regularly. Since March 2020, all cancer patients having access to the Oncology Unit at the "Andrea Tortora" Hospital (Pagani, Salerno - referred to as "the Hospital") as inpatients or outpatients receiving intravenous therapy have been screened for SARS-CoV-2 using RT-PCR nasal swab. The ongoing COICA (COVID-19 infection in cancer patients) study is an ambispective, multicenter, observational study designed to assess the prognosis of SARS-CoV-2 infection in cancer patients. The aim of the study presented here was to explore potential differences in COVID-19-related outcomes among screening-detected versus nonscreening-detected SARS-CoV-2-infected patients. Methods: The COICA study enrolled cancer patients who had received any anticancer systemic therapy within 3 months since the day they tested positive for SARS-CoV-2 on RT-PCR. The target accrual is 128 patients, and the study was approved by the competent Ethics Committee. Only the subgroup of patients enrolled at the Hospital was considered in this unplanned interim analysis. Logistic regression analysis was used to evaluate the association of screening-based versus nonscreening-based diagnosis. Results: Since March 15, 2020, until August 15, 2021, a total of 931 outpatients and 230 inpatients were repeatedly screened for SARS-CoV-2 using RT-PCR nasal swab at the Hospital. Among these, 71 asymptomatic patients were positive on routine screening and 5 patients were positive for SARS-CoV-2 outside the institutional screening. Seven patients died because of COVID-19. At univariate analysis, nonscreening- versus screening-detected SARS-CoV-2 infection was associated with significantly higher odds of O2 therapy (OR = 16.2; 95% CI = 2.2-117.1; p = 0.006), hospital admission (OR = 31.5; 95% CI = 3.1-317.8; p = 0.003), admission to ICU (OR = 23.0; 95% CI = 2.4-223.8; p = 0.007), and death (OR = 8.8; 95% CI = 1.2-65.5; p = 0.034). Conclusion: Routine screening with RT-PCR may represent a feasible and effective strategy in reducing viral circulation and possibly COVID-19 mortality in patients with active cancer having repeated access to hospital facilities.

COVID 19 therapies and anti-cancer drugs: A systematic review of recent literature.

BACKGROUND: It is reasonable to think that cancer patients undergoing chemotherapy, targeted therapy or immunotherapy could have a more aggressive course if positive for Coronavirus disease CoV-2 (COVID- 19). METHODS: We conducted a literature review on https://www.ncbi.nlm.nih.gov/pubmed/, https://scholar.google.com, www.arxiv.org, www.biorxiv.org, of all articles published using the keywords COVID-19 therapy or treatment and cancer until May 2, 2020. A total of 205 articles were identified and 53 were included in this review. RESULTS: We describe the ongoing COVID-19 therapies that should be known by oncologists and highlight the potential interactions with antineoplastic drugs, commonly used in clinical practice. The main drug interactions were found with tocilizumab, ruxolitinib and colchicine. CONCLUSIONS: The literature provides an inconclusive picture on potential preferred treatments for COVID-19 and their interactions with antineoplastic agents. Future clinical trials are needed to better understand the interactions between different drugs in the context of COVID-19 pandemic.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab.

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study.

Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca2+-activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm2 (resting tension), 146.8 ± 13.8 mN/mm2 (maximal active tension, P0), 5.4 ± 0.22 s-1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s-1 (linear relaxation rate), and 10.8 ± 1.3 s-1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca2+-sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.

The homozygous K280N troponin T mutation alters cross-bridge kinetics and energetics in human HCM.

Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (k ACT) and the rate constant of isometric relaxation (slow k REL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces k ACT, slow k REL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases k ACT, slow k REL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein.

Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G4S)2] of a fused heavy chain containing in total 2 (G4S)2-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins.

R4496C RyR2 mutation impairs atrial and ventricular contractility.

Ryanodine receptor (RyR2) is the major Ca(2+) channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca(2+) leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca(2+)]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca(2+)]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca(2+) transient was normal, despite the reduced SR Ca(2+) content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca(2+) transients because they are incapable of properly increasing the amount of Ca(2+) stored in the SR because of a larger SR Ca(2+) leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca(2+) channel current (ICa-L) and SR Ca(2+) content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca(2+) release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca(2+) content, but cannot compensate for the inability to increase SR Ca(2+) content during inotropic challenge.

Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding ß-myosin heavy chain (ß-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 µmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.

HDAC6 contributes to pathological responses of heart and skeletal muscle to chronic angiotensin-II signaling.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab.

Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcγR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab')2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B.

Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy.

Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

Mutations in MYH7 reduce the force generating capacity of sarcomeres in human familial hypertrophic cardiomyopathy.

AIMS: Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling. METHODS AND RESULTS: Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick (MYBPC3, MYH7) and thin (TPM1, TNNI3, TNNT2) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m²) and HCMsmn (26 ± 3 kN/m²) compared with donor (36 ± 2 kN/m²). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 µm²). Low Fmax in MYBPC3mut, TNNI3mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7mut, TPM1mut, and TNNT2mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7mut, TPM1mut, and TNNT2mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m²). In addition, force was lower in MYH7mut cardiomyocytes compared with MYBPC3mut, HCMsmn, and donor at submaximal [Ca²âº]. CONCLUSION: Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca²âº]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.

Determination of antibody glycosylation by mass spectrometry.

Immunoglobulin (Ig) G is formed by two antigen-binding moieties termed Fabs and a conserved Fc -portion, which interacts with components of the immune systems. Within the Fc, N-linked carbohydrates are attached to each conserved asparagine residue at position 297 within the CH2 domain. These oligosaccharide moieties introduce a higher degree of heterogeneity within the molecule, by influencing stability of the antibody and its mediated effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). The carbohydrate moieties can vary strongly depending on the production host and can be manipulated by different fermentation conditions, thereby influencing the function of the antibody. Therefore it is necessary to carefully monitor changes in the carbohydrate composition during cell line development and production processes. This chapter describes two different mass spectrometry based methods used for analyses of the carbohydrate moieties attached to the Fc-part of human IgG1. In the first approach, the glycans are released from the antibody by endoglycosidase (Peptide N Glycosidase F) digestion and monitored by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS), whereas in the second method the carbohydrate structures, still attached to an enzymatically produced Fc-fragment, are analyzed by electrospray ionization mass spectrometry.

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition.

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation.

IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 "stalk" domain, exCD23>sCD23>derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.