Net charge of antibody complementarity-determining regions is a key predictor of specificity

Abstract Specificity is one of the most important and complex properties that is central to both natural antibody function and therapeutic antibody efficacy. However, it has proven extremely challenging to define robust guidelines for predicting antibody specificity. Here we evaluated the physicochemical determinants of antibody specificity for multiple panels of antibodies, including >100 clinical-stage antibodies. Surprisingly, we find that the theoretical net charge of the complementarity-determining regions (CDRs) is a strong predictor of antibody specificity. Antibodies with positively charged CDRs have a much higher risk of low specificity than antibodies with negatively charged CDRs. Moreover, the charge of the entire set of six CDRs is a much better predictor of antibody specificity than the charge of individual CDRs, variable domains (VH or VL) or the entire variable fragment (Fv). The best indicators of antibody specificity in terms of CDR amino acid composition are reduced levels of arginine and lysine and increased levels of aspartic and glutamic acid. Interestingly, clinical-stage antibodies with negatively charged CDRs also have a lower risk for poor biophysical properties in general, including a reduced risk for high levels of self-association. These findings provide powerful guidelines for predicting antibody specificity and for identifying safe and potent antibody therapeutics.

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A Two-Step Approach for the Design and Generation of Nanobodies

International Journal of Molecular Sciences ◽

10.3390/ijms19113444 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3444 ◽

Cited By ~ 7

Author(s):

Hanna Wagner ◽

Sarah Wehrle ◽

Etienne Weiss ◽

Marco Cavallari ◽

Wilfried Weber

Keyword(s):

Heavy Chain ◽

Animal Experiments ◽

Affinity Maturation ◽

Phage Library ◽

Second Step ◽

Selection Procedures ◽

Variable Domains ◽

Phage Libraries ◽

Complementarity Determining Regions ◽

Conventional Antibody

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

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Abdominal Computed Tomography Predicts Progression in Patients With Rai Stage 0 Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2006.08.4194 ◽

2007 ◽

Vol 25 (12) ◽

pp. 1576-1580 ◽

Cited By ~ 40

Author(s):

Ana Muntañola ◽

Francesc Bosch ◽

Pedro Arguis ◽

Eduardo Arellano-Rodrigo ◽

Carmen Ayuso ◽

...

Keyword(s):

Computed Tomography ◽

Chronic Lymphocytic Leukemia ◽

Early Stage ◽

Strong Predictor ◽

Clinical Stage ◽

Prognostic Significance ◽

Clinical Information ◽

Lymphocytic Leukemia ◽

Abdominal Ct ◽

Work Up

Purpose Whether computed tomography (CT) should be routinely included in the diagnostic work-up in patients with chronic lymphocytic leukemia (CLL) has not yet been determined. The aim of this study was to analyze the prognostic significance of abdominal CT in patients with CLL in Rai clinical stage 0. Patients and Methods Abdominal CT was performed at diagnosis in 140 patients consecutively diagnosed with CLL in Rai stage 0 disease. Results An abnormal abdominal CT was found in 38 patients (27%). Abnormal CT correlated with increased bone marrow infiltration (P = .024), high lymphocyte count (P = .001), increased ZAP-70 expression (P = .003), and short lymphocyte doubling time (LDT; P = .007). Patients with abnormal CT progressed more frequently and had a shorter time to progression than those with normal CT (median, 3.5 years v not reached, respectively; P < .001) and required earlier treatment intervention. In a multivariate analysis, only high ZAP-70 expression (relative risk = 3.60) and an abnormal abdominal CT (RR = 2.71) correlated with disease progression. Conclusion In this series, an abnormal abdominal CT was a strong predictor of progression in patients with early-stage CLL. The inclusion of CT scans in the initial work-up of patients with early clinical stage on clinical grounds can, therefore, provide relevant clinical information.

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Effects of net charge and the number of positively charged residues on the biological activity of amphipathic α-helical cationic antimicrobial peptides

Advances in Experimental Medicine and Biology - Peptides for Youth ◽

10.1007/978-0-387-73657-0_246 ◽

2009 ◽

pp. 561-562 ◽

Cited By ~ 19

Author(s):

Ziqing Jiang ◽

Adriana I. Vasil ◽

John Hale ◽

Robert E. W. Hancock ◽

Michael L. Vasil ◽

...

Keyword(s):

Biological Activity ◽

Antimicrobial Peptides ◽

Net Charge ◽

Cationic Antimicrobial Peptides ◽

Charged Residues ◽

Positively Charged

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Reproductive organ involvement in female patients undergoing radical cystectomy.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.6_suppl.274 ◽

2013 ◽

Vol 31 (6_suppl) ◽

pp. 274-274

Author(s):

Hooman Djaladat ◽

Eila C. Skinner ◽

Gus Miranda ◽

Jie Cai ◽

Siamak Daneshmand

Keyword(s):

Bladder Cancer ◽

Radical Cystectomy ◽

Strong Predictor ◽

Clinical Stage ◽

Reproductive Organs ◽

Reproductive Organ ◽

Organ Involvement ◽

Palpable Mass ◽

Clinical Criteria ◽

Female Patients

274 Background: Anterior pelvic exenteration is traditionally the treatment of choice for women with invasive bladder carcinoma. Female reproductive organ involvement is reported to be low, but there is not enough evidence to abandon this part during radical surgery. We evaluated the pathological involvement of reproductive organs (RO) in female patients undergoing radical cystectomy for invasive bladder urothelial carcinoma. Methods: 2,098 patients with bladder cancer underwent cystectomy between 1971 and 2008 at USC, of whom 458 were female. 411 (90%) had urothelial and 47 had non-urothelial bladder cancer. The cohort of female cystectomy patients with pathologic RO involvement is reviewed and their RFS and OS are discussed. Results: In the TCC group, 20 patients (4.8%) had RO involvement by urothelial cancer (UC-RO); 10 (2.5%) had vaginal, 2 (0.5%) cervical and one (0.25%) only uterus involvement with the rest (7) having a combination. In non-UC group, only two (4%) had RO involvement. In the UC-RO cohort, median age was 71 yo (38-89). Only one patient (5%) underwent orthotopic diversion. 19 cases (95%) were high grade TCC. Clinical stage T4a was diagnosed in 25% of cases preoperatively. Associated CIS, multifocality, LVI and histologic type of cancer had no significant correlation with RO involvement. Patients with a palpable mass or hydronephrosis were 5 times more likely to have RO involvement (P<0.001). RO involvement was associated with higher chance of positive lymph nodes (60%) (P=0.001). Three (15%) and seven (35%) patients underwent neo-adjuvant and adjuvant chemotherapy respectively. Urethral pathology was positive in 1 patient. 5 (25%) developed local recurrence and 9 (45%) developed distant metastasis at a median follow up of 12.2 yrs (0.1 to 35.5 yrs). Two-year RFS and five-year OS in UC-RO group were both 10%. Conclusions: The risk of RO involvement in female patients undergoing cystectomy for bladder cancer is about 5%, with the vagina being the most commonly involved organ. A palpable mass and/or hydronephrosis are a strong predictor of RO involvement, although other clinical criteria are not predictive.

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Dramatic Activation of an Antibody by a Single Amino Acid Change in Framework

10.21203/rs.3.rs-862975/v1 ◽

2021 ◽

Author(s):

Wei-Ching Liang ◽

Jianping Yin ◽

Patrick Lupardus ◽

Jianhuan Zhang ◽

Kelly M. Loyet ◽

...

Keyword(s):

Heavy Chain ◽

Amino Acid Change ◽

Single Amino Acid ◽

Inhibitory Antibody ◽

Chain Molecules ◽

High Affinity ◽

Single Amino Acid Change ◽

Rabbit Monoclonal Antibody ◽

Complementarity Determining Regions ◽

Antibody Function

Abstract Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFβ2 and TGFβ31. Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFβ2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFβ2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFβ2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.

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Icosahedral viruses defined by their positively charged domains: a signature for viral identity and capsid assembly strategy

10.1101/600981 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rodrigo D. Requião ◽

Rodolfo L. Carneiro ◽

Mariana Hoyer Moreira ◽

Marcelo Ribeiro-Alves ◽

Silvana Rossetto ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Level ◽

Protein Domain ◽

Capsid Proteins ◽

Capsid Assembly ◽

Genome Packaging ◽

Structure Comparison ◽

Net Charge ◽

Icosahedral Viruses ◽

Positively Charged

AbstractCapsid proteins often present a positively charged arginine-rich region at the N and/or C-termini that for some icosahedral viruses has a fundamental role in genome packaging and particle stability. These sequences show little to no conservation at the amino-acid level and are structurally dynamic so that they cannot be easily detected by common sequence or structure comparison. As a result, the occurrence and distribution of positively charged protein domain across the viral and the overall protein universe are unknown. We developed a methodology based on the net charge calculation of discrete segments of the protein sequence that allows us to identify proteins containing amino-acid stretches with an extremely high net charge. We observed that among all organisms, icosahedral viruses are especially enriched in extremely positively charged segments (Q ≥ +17), with a distinctive bias towards arginine instead of lysine. We used viral particle structural data to calculate the total electrostatic charge derived from the most positively charged protein segment of capsid proteins and correlated these values with genome charge arising from the phosphates of each nucleotide. We obtained a positive correlation (r = 0.91, p-value < 0001) for a group of 17 viral families, corresponding to 40% of all families with icosahedral structures described so far. These data indicated that unrelated viruses with diverse genome types adopt a common underlying mechanism for capsid assembly and genome stabilization based on R-arms. Outliers from a linear fit pointed to families with alternative strategies of capsid assembly and genome packaging.Significance StatementViruses can be characterized by the existence of a capsid, an intricate proteinaceous container that encases the viral genome. Therefore, capsid assembly and function are essential to viral replication. Here we specify virus families with diverse capsid structure and sequence, for each capsid packing capacity depends on a distinctive structural feature: a highly positively charged segment of amino acids residues, preferentially made of arginine. We also show that proteins with the same characteristics are rarely found in cellular proteins. Therefore, we identified a conserved viral functional element that can be used to infer capsid assembly mechanisms and inspire the design of protein nanoparticles and broad-spectrum antiviral treatments.

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Discovering Selected Antibodies From Deep-Sequenced Phage-Display Antibody Library Using ATTILA

Bioinformatics and Biology Insights ◽

10.1177/1177932220915240 ◽

2020 ◽

Vol 14 ◽

pp. 117793222091524 ◽

Cited By ~ 1

Author(s):

Andréa Queiroz Maranhão ◽

Heidi Muniz Silva ◽

Waldeyr Mendes Cordeiro da Silva ◽

Renato Kaylan Alves França ◽

Thais Canassa De Leo ◽

...

Keyword(s):

Phage Display ◽

Fold Change ◽

Robust Solution ◽

Performance Accuracy ◽

Variable Domains ◽

Ngs Data Analysis ◽

Complementarity Determining Regions ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Generation Sequencing

Phage display is a powerful technique to select high-affinity antibodies for different purposes, including biopharmaceuticals. Next-generation sequencing (NGS) presented itself as a robust solution, making it possible to assess billions of sequences of the variable domains from selected sublibraries. Handling this process, a central difficulty is to find the selected clones. Here, we present the AutomaTed Tool For Immunoglobulin Analysis (ATTILA), a new tool to analyze and find the enriched variable domains throughout a biopanning experiment. The ATTILA is a workflow that combines publicly available tools and in-house programs and scripts to find the fold-change frequency of deeply sequenced amplicons generated from selected VH and VL domains. We analyzed the same human Fab library NGS data using ATTILA in 5 different experiments, as well as on 2 biopanning experiments regarding performance, accuracy, and output. These analyses proved to be suitable to assess library variability and to list the more enriched variable domains, as ATTILA provides a report with the amino acid sequence of each identified domain, along with its complementarity-determining regions (CDRs), germline classification, and fold change. Finally, the methods employed here demonstrated a suitable manner to combine amplicon generation and NGS data analysis to discover new monoclonal antibodies (mAbs).

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Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding

10.1101/2020.02.12.946350 ◽

2020 ◽

Cited By ~ 1

Author(s):

Simon P. Kelow ◽

Jared Adolf-Bryfogle ◽

Roland L. Dunbrack

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Data Bank ◽

Antigen Binding ◽

Sequence Variability ◽

Antigen Binding Site ◽

Sequencing Studies ◽

Variable Domains ◽

Complementarity Determining Regions ◽

Hiv 1

AbstractAntibody variable domains contain “complementarity determining regions” (CDRs), the loops that form the antigen binding site. CDRs1-3 are recognized as the canonical CDRs. However, a fourth loop sits adjacent to CDR1 and CDR2 and joins the D and E strands on the antibody v-type fold. This “DE loop” is usually treated as a framework region, even though mutations in the loop affect the conformation of the CDRs and residues in the DE loop occasionally contact antigen. We analyzed the length, structure, and sequence features of all DE loops in the Protein Data Bank, as well as millions of sequences from HIV-1 infected and naïve patients. We refer to the DE loop as H4 and L4 in the heavy and light chain respectively. Clustering the backbone conformations of the most common length of L4 (6 residues) reveals four conformations: two κ-only clusters, one λ-only cluster, and one mixed κ/λ cluster. The vast majority of H4 loops are length-8 and exist primarily in one conformation; a secondary conformation represents a small fraction of H4-8 structures. H4 sequence variability exceeds that of the antibody framework in naïve human high-throughput sequences, and both L4 and H4 sequence variability from λ and heavy germline sequences exceed that of germline framework regions. Finally, we identified dozens of structures in the PDB with insertions in the DE loop, all related to broadly neutralizing HIV-1 antibodies, as well as antibody sequences from high-throughput sequencing studies of HIV-infected individuals, illuminating a possible role in humoral immunity to HIV-1.

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Fc Receptor Variants and Disease: A Crucial Factor to Consider in the Antibody Therapeutics in Clinic

International Journal of Molecular Sciences ◽

10.3390/ijms22179489 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9489

Author(s):

Jin Kim ◽

Ji Young Lee ◽

Han Gil Kim ◽

Min Woo Kwak ◽

Tae Hyun Kang

Keyword(s):

Signal Transduction ◽

Endothelial Cells ◽

Individual Differences ◽

Genetic Variants ◽

Promoter Region ◽

Control Point ◽

Protective Function ◽

Effector Functions ◽

Antibody Therapeutics ◽

Antibody Function

The fragment crystallizable (Fc) domain of antibodies is responsible for their protective function and long-lasting serum half-life via Fc-mediated effector function, transcytosis, and recycling through its interaction with Fc receptors (FcRs) expressed on various immune leukocytes, epithelial, and endothelial cells. Therefore, the Fc–FcRs interaction is a control point of both endogenous and therapeutic antibody function. There are a number of reported genetic variants of FcRs, which include polymorphisms in (i) extracellular domain of FcRs, which change their affinities to Fc domain of antibodies; (ii) both cytoplasmic and intracellular domain, which alters the extent of signal transduction; and (iii) the promoter region of the FcRs gene, which affects the expression level of FcRs, thus being associated with the pathogenesis of disease indications. In this review, we firstly describe the correlation between the genetic variants of FcRs and immunological disorders by individual differences in the extent of FcRs-mediated regulations. Secondly, we discuss the influence of the genetic variants of FcRs on the susceptibility to infectious diseases or cancer in the perspective of FcRs-induced effector functions. Overall, we concluded that the genetic variants of FcRs are one of the key elements in the design of antibody therapeutics due to their variety of clinical outcomes among individuals.

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