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Antibodies, Volume 7, Issue 3 (September 2018)

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Open AccessArticle Relative Contribution of Framework and CDR Regions in Antibody Variable Domains to Multimerisation of Fv- and scFv-Containing Bispecific Antibodies
Antibodies 2018, 7(3), 35; https://doi.org/10.3390/antib7030035
Received: 12 July 2018 / Revised: 29 August 2018 / Accepted: 30 August 2018 / Published: 31 August 2018
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Abstract
Bispecific antibodies represent an emerging class of antibody drugs that are commonly generated by fusion of Fv or scFv antigen binding domains to IgG or Fab scaffolds. Fv- or scFv-mediated multimerisation of bispecific antibodies via promiscuous vH-vL pairing can result in sub-optimal monomer
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Bispecific antibodies represent an emerging class of antibody drugs that are commonly generated by fusion of Fv or scFv antigen binding domains to IgG or Fab scaffolds. Fv- or scFv-mediated multimerisation of bispecific antibodies via promiscuous vH-vL pairing can result in sub-optimal monomer levels during expression, and hence, undesirable therapeutic protein yields. We investigate the contribution of disulphide stabilised Fv and scFv to Fab-Fv and Fab-scFv multimerisation. We show that monomer levels of isolated Fv/scFv cannot always be used to predict monomer levels of Fab-linked Fv/scFv, and that Fab-scFv monomer levels are greater than the equivalent Fab-Fv. Through grafting bispecifics with framework/CDR-‘swapped’ Fv and scFv, we show that monomer levels of disulphide stabilised Fab-Fv and Fab-scFv can be improved by Fv framework ‘swapping’. The Fab-Fv and Fab-scFv can be considered representative of the significant number of bispecific antibody formats containing appended Fv/scFv, as we also used Fv framework ‘swapping’ to increase the monomer level of an IgG-scFv bispecific antibody. This research may, therefore, be useful for maximising the monomeric yield of numerous pharmaceutically-relevant bispecific formats in pre-clinical development. Full article
(This article belongs to the Special Issue Bispecific Antibodies-Opportunities and Challenges)
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Open AccessCorrection Correction: Verkade, J.M.M.; et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates. Antibodies 2018, 7, 12
Antibodies 2018, 7(3), 34; https://doi.org/10.3390/antib7030034
Received: 17 August 2018 / Accepted: 29 August 2018 / Published: 30 August 2018
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Abstract
The conflict of interest section of the published paper [1] has been updated as follows[...] Full article
Open AccessAddendum Addendum: Hoffmann, C.; et al. Autoantibodies in Neuropsychiatric Disorders. Antibodies 2016, 5, 9
Antibodies 2018, 7(3), 33; https://doi.org/10.3390/antib7030033
Received: 21 August 2018 / Accepted: 23 August 2018 / Published: 27 August 2018
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Abstract
The conflict of interest section was missing in the published paper [1]. It has been updated as follows: [...] Full article
Open AccessCorrection Correction: Mehrling, T.; et al. Challenges in Optimising the Successful Construction of Antibody Drug Conjugates in Cancer Therapy. Antibodies 2018, 7, 11
Antibodies 2018, 7(3), 32; https://doi.org/10.3390/antib7030032
Received: 21 August 2018 / Accepted: 22 August 2018 / Published: 27 August 2018
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Abstract
The Conflict of Interest section of the published paper [1] has been updated as follows: [...] Full article
Open AccessCorrection Correction: Moeker, N.; et al. Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings. Antibodies 2017, 6, 15
Antibodies 2018, 7(3), 31; https://doi.org/10.3390/antib7030031
Received: 20 August 2018 / Accepted: 22 August 2018 / Published: 24 August 2018
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Abstract
The authors wish to make the following correction to the Conflict of Interests section in their published paper[...] Full article
Open AccessFeature PaperArticle Applicability of Traditional In Vitro Toxicity Tests for Assessing Adverse Effects of Monoclonal Antibodies: A Case Study of Rituximab and Trastuzumab
Antibodies 2018, 7(3), 30; https://doi.org/10.3390/antib7030030
Received: 30 July 2018 / Revised: 10 August 2018 / Accepted: 14 August 2018 / Published: 17 August 2018
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Abstract
Monoclonal antibody (mAb) therapeutics have a promising outlook within the pharmaceutical industry having made positive strides in both research and development as well as commercialisation, however this development has been hampered by manufacturing failures and attrition. This study explores the applicability of traditional
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Monoclonal antibody (mAb) therapeutics have a promising outlook within the pharmaceutical industry having made positive strides in both research and development as well as commercialisation, however this development has been hampered by manufacturing failures and attrition. This study explores the applicability of traditional in vitro toxicity tests for detecting any off-target adverse effect elicited by mAbs on specific organ systems using hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn), respectively. The mechanism of antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) via complement activation, and complement dependent cellular cytotoxicity (CDCC) were assessed. Major results: no apparent ADCC, CDCC, or CDC mediated decrease in cell viability was measured for HepG2 cells. For HDFn cells, though ADCC or CDCC mediated decreases in cell viability wasn’t detected, a CDC mediated decrease in cell viability was observed. Several considerations have been elucidated for development of in vitro assays better suited to detect off target toxicity of mAbs. Full article
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Open AccessFeature PaperArticle Tuning Relative Polypeptide Expression to Optimize Assembly, Yield and Downstream Processing of Bispecific Antibodies
Antibodies 2018, 7(3), 29; https://doi.org/10.3390/antib7030029
Received: 5 July 2018 / Revised: 3 August 2018 / Accepted: 6 August 2018 / Published: 10 August 2018
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Abstract
Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that
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Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that pair with a common heavy chain. Introduction of non-optimal codons into the sequence of a particular polypeptide is an effective strategy for down modulating its expression. Here we applied this strategy but restricted the modification of the codon content to the constant domain of one light chain. This approach facilitates parallel optimization of several bsAbs by using the same modified constant domains. Partial sequence de-optimization reduced expression of the targeted polypeptide. Stable cell pools could be isolated displaying increased bispecific antibody titers as well as changes in the abundance of undesired by-products that require elimination during downstream processing. Thus, modulating the relative expression of polypeptides can have a significant impact on bsAb titer and product related impurities; which are important factors for large scale manufacturing for clinical supply. Full article
(This article belongs to the Special Issue Bispecific Antibodies-Opportunities and Challenges)
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Open AccessReview Engineering IgG-Like Bispecific Antibodies—An Overview
Antibodies 2018, 7(3), 28; https://doi.org/10.3390/antib7030028
Received: 4 July 2018 / Revised: 24 July 2018 / Accepted: 24 July 2018 / Published: 1 August 2018
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Abstract
Monoclonal antibody therapeutics have proven to be successful treatment options for patients in various indications. Particularly in oncology, therapeutic concepts involving antibodies often rely on the so-called effector functions, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), which are
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Monoclonal antibody therapeutics have proven to be successful treatment options for patients in various indications. Particularly in oncology, therapeutic concepts involving antibodies often rely on the so-called effector functions, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), which are programed in the antibody Fc region. However, Fc-mediated effector mechanisms often seem to be insufficient in properly activating the immune system to act against tumor cells. Furthermore, long term treatments can lead to resistance against the applied drug, which is monospecific by nature. There is promise in using specific antibodies to overcome such issues due to their capability of recruiting and activating T-cells directly at the tumor site, for instance. During the last decade, two of these entities, which are referred to as Blinatumomab and Catumaxomab, have been approved to treat patients with acute lymphoblastic leukemia and malignant ascites. In addition, Emicizumab, which is a bispecific antibody targeting clotting factors IXa and X, was recently granted market approval by the FDA in 2017 for the treatment of hemophilia A. However, the generation of these next generation therapeutics is challenging and requires tremendous engineering efforts as two distinct paratopes need to be combined from two different heavy and light chains. This mini review summarizes technologies, which enable the generation of antibodies with dual specificities. Full article
(This article belongs to the Special Issue Bispecific Antibodies-Opportunities and Challenges)
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Open AccessFeature PaperArticle Design Principles for Bispecific IgGs, Opportunities and Pitfalls of Artificial Disulfide Bonds
Antibodies 2018, 7(3), 27; https://doi.org/10.3390/antib7030027
Received: 15 June 2018 / Revised: 16 July 2018 / Accepted: 24 July 2018 / Published: 28 July 2018
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Abstract
Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many
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Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many of them are small recombinant fragments, their utility could be limited. For some therapeutic applications, full-size IgGs may be the optimal format. Two challenges should be met to make bispecific IgGs; one is that each heavy chain will only pair with the heavy chain of the second specificity and that homodimerization be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and not with the light chain of the second specificity. The first solution to the first criterion (knobs into holes, KIH) was presented in 1996 by Paul Carter’s group from Genentech. Additional solutions were presented later on. However, until recently, out of >120 published bsAb formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested. We present a solution for the second challenge—correct pairing of heavy and light chains of bispecific IgGs; an engineered (artificial) disulfide bond between the antibodies’ variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL. We name antibodies produced according to this design “BIClonals”. Bispecific IgGs where the artificial disulfide bond is placed in the CH1-CL interface are also presented. Briefly, we found that an artificial disulfide bond between VH position 44 to VL position 100 provides for effective and correct H–L chain pairing while also preventing the formation of wrong H–L chain pairs. When the artificial disulfide bond links the CH1 with the CL domain, effective H–L chain pairing also occurs, but in some cases, wrong H–L pairing is not totally prevented. We conclude that H–L chain pairing seems to be driven by VH–VL interfacial interactions that differ between different antibodies, hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs, making it necessary to carefully evaluate the optimal solution for each new antibody. Full article
(This article belongs to the Special Issue Bispecific Antibodies-Opportunities and Challenges)
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Open AccessFeature PaperArticle BPSL1626: Reverse and Structural Vaccinology Reveal a Novel Candidate for Vaccine Design against Burkholderia pseudomallei
Antibodies 2018, 7(3), 26; https://doi.org/10.3390/antib7030026
Received: 31 May 2018 / Revised: 16 July 2018 / Accepted: 17 July 2018 / Published: 19 July 2018
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Abstract
Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel
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Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 Å resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules. Full article
(This article belongs to the collection Computational Antibody and Antigen Design)
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Open AccessArticle Molecular Recognition between Aβ-Specific Single-Domain Antibody and Aβ Misfolded Aggregates
Antibodies 2018, 7(3), 25; https://doi.org/10.3390/antib7030025
Received: 31 May 2018 / Revised: 6 July 2018 / Accepted: 9 July 2018 / Published: 13 July 2018
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Abstract
Aβ is the toxic amyloid polypeptide responsible for Alzheimer’s disease (AD). Prevention and elimination of the Aβ misfolded aggregates are the promising therapeutic strategies for the AD treatments. Gammabody, the Aβ-Specific Single-domain (VH) antibody, recognizes Aβ aggregates with high affinity and specificity and
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Aβ is the toxic amyloid polypeptide responsible for Alzheimer’s disease (AD). Prevention and elimination of the Aβ misfolded aggregates are the promising therapeutic strategies for the AD treatments. Gammabody, the Aβ-Specific Single-domain (VH) antibody, recognizes Aβ aggregates with high affinity and specificity and reduces their toxicities. Employing the molecular dynamics simulations, we studied diverse gammabody-Aβ recognition complexes to get insights into their structural and dynamic properties and gammabody-Aβ recognitions. Among many heterogeneous binding modes, we focused on two gammabody-Aβ recognition scenarios: recognition through Aβ β-sheet backbone and on sidechain surface. We found that the gammabody primarily uses the complementarity-determining region 3 (CDR3) loop with the grafted Aβ sequence to interact with the Aβ fibril, while CDR1/CDR2 loops have very little contact. The gammabody-Aβ complexes with backbone binding mode are more stable, explaining the gammabody’s specificity towards the C-terminal Aβ sequence. Full article
(This article belongs to the collection Computational Antibody and Antigen Design)
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Open AccessReview Engaging with Raman Spectroscopy to Investigate Antibody Aggregation
Antibodies 2018, 7(3), 24; https://doi.org/10.3390/antib7030024
Received: 18 June 2018 / Revised: 4 July 2018 / Accepted: 4 July 2018 / Published: 7 July 2018
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Abstract
In the last decade, a number of studies have successfully demonstrated Raman spectroscopy as an emerging analytical technique for monitoring antibody aggregation, especially in the context of drug development and formulation. Raman spectroscopy is a robust method for investigating protein conformational changes, even
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In the last decade, a number of studies have successfully demonstrated Raman spectroscopy as an emerging analytical technique for monitoring antibody aggregation, especially in the context of drug development and formulation. Raman spectroscopy is a robust method for investigating protein conformational changes, even in highly concentrated antibody solutions. It is non-destructive, reproducible and can probe samples in an aqueous environment. In this review, we focus on the application and challenges associated with using Raman spectroscopy as a tool to study antibody aggregates. Full article
(This article belongs to the Special Issue Aggregation of Antibodies)
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Open AccessFeature PaperArticle OptMAVEn-2.0: De novo Design of Variable Antibody Regions against Targeted Antigen Epitopes
Antibodies 2018, 7(3), 23; https://doi.org/10.3390/antib7030023
Received: 20 May 2018 / Revised: 28 June 2018 / Accepted: 29 June 2018 / Published: 30 June 2018
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Abstract
Monoclonal antibodies are becoming increasingly important therapeutic agents for the treatment of cancers, infectious diseases, and autoimmune disorders. However, laboratory-based methods of developing therapeutic monoclonal antibodies (e.g., immunized mice, hybridomas, and phage display) are time-consuming and are often unable to target a specific
[...] Read more.
Monoclonal antibodies are becoming increasingly important therapeutic agents for the treatment of cancers, infectious diseases, and autoimmune disorders. However, laboratory-based methods of developing therapeutic monoclonal antibodies (e.g., immunized mice, hybridomas, and phage display) are time-consuming and are often unable to target a specific antigen epitope or reach (sub)nanomolar levels of affinity. To this end, we developed Optimal Method for Antibody Variable region Engineering (OptMAVEn) for de novo design of humanized monoclonal antibody variable regions targeting a specific antigen epitope. In this work, we introduce OptMAVEn-2.0, which improves upon OptMAVEn by (1) reducing computational resource requirements without compromising design quality; (2) clustering the designs to better identify high-affinity antibodies; and (3) eliminating intra-antibody steric clashes using an updated set of clashing parts from the Modular Antibody Parts (MAPs) database. Benchmarking on a set of 10 antigens revealed that OptMAVEn-2.0 uses an average of 74% less CPU time and 84% less disk storage relative to OptMAVEn. Testing on 54 additional antigens revealed that computational resource requirements of OptMAVEn-2.0 scale only sub-linearly with respect to antigen size. OptMAVEn-2.0 was used to design and rank variable antibody fragments targeting five epitopes of Zika envelope protein and three of hen egg white lysozyme. Among the top five ranked designs for each epitope, recovery of native residue identities is typically 45–65%. MD simulations of two designs targeting Zika suggest that at least one would bind with high affinity. OptMAVEn-2.0 can be downloaded from our GitHub repository and webpage as (links in Summary and Discussion section). Full article
(This article belongs to the collection Computational Antibody and Antigen Design)
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Open AccessReview In Silico Methods in Antibody Design
Antibodies 2018, 7(3), 22; https://doi.org/10.3390/antib7030022
Received: 1 June 2018 / Revised: 28 June 2018 / Accepted: 28 June 2018 / Published: 29 June 2018
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Abstract
Antibody therapies with high efficiency and low toxicity are becoming one of the major approaches in antibody therapeutics. Based on high-throughput sequencing and increasing experimental structures of antibodies/antibody-antigen complexes, computational approaches can predict antibody/antigen structures, engineering the function of antibodies and design antibody-antigen
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Antibody therapies with high efficiency and low toxicity are becoming one of the major approaches in antibody therapeutics. Based on high-throughput sequencing and increasing experimental structures of antibodies/antibody-antigen complexes, computational approaches can predict antibody/antigen structures, engineering the function of antibodies and design antibody-antigen complexes with improved properties. This review summarizes recent progress in the field of in silico design of antibodies, including antibody structure modeling, antibody-antigen complex prediction, antibody stability evaluation, and allosteric effects in antibodies and functions. We listed the cases in which these methods have helped experimental studies to improve the affinities and physicochemical properties of antibodies. We emphasized how the molecular dynamics unveiled the allosteric effects during antibody-antigen recognition and antibody-effector recognition. Full article
(This article belongs to the collection Computational Antibody and Antigen Design)
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Open AccessArticle Thinking outside the Laboratory: Analyses of Antibody Structure and Dynamics within Different Solvent Environments in Molecular Dynamics (MD) Simulations
Antibodies 2018, 7(3), 21; https://doi.org/10.3390/antib7030021
Received: 2 May 2018 / Revised: 11 June 2018 / Accepted: 20 June 2018 / Published: 24 June 2018
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Abstract
Monoclonal antibodies (mAbs) have revolutionized the biomedical field, directly influencing therapeutics and diagnostics in the biopharmaceutical industry, while continuing advances in computational efficiency have enabled molecular dynamics (MD) simulations to provide atomistic insight into the structure and function of mAbs. Despite the success
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Monoclonal antibodies (mAbs) have revolutionized the biomedical field, directly influencing therapeutics and diagnostics in the biopharmaceutical industry, while continuing advances in computational efficiency have enabled molecular dynamics (MD) simulations to provide atomistic insight into the structure and function of mAbs. Despite the success of MD tools, further optimizations are still required to enhance the computational efficiency of complex mAb simulations. This issue can be tackled by changing the way the solvent system is modelled to reduce the number of atoms to be tracked but must be done without compromising the accuracy of the simulations. In this work, the structure of the IgG2a antibody was analyzed in three solvent systems: explicit water and ions, implicit water and ions, and implicit water and explicit ions. Root-mean-square distance (RMSD), root-mean-square fluctuations (RMSF), and interchain angles were used to quantify structural changes. The explicit system provides the most atomistic detail but is ~6 times slower in its exploration of configurational space and required ~4 times more computational time on our supercomputer than the implicit simulations. Overall, the behavior of the implicit and explicit simulations is quantifiably similar, with the inclusion of explicit ions in the implicit simulation stabilizing the antibody to reproduce well the statistical fluctuations of the fully explicit system. Therefore, this approach holds promise to maximize the use of computational resources to explore antibody behavior. Full article
(This article belongs to the collection Computational Antibody and Antigen Design)
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