In vitro affinity maturation of HuCAL antibodies: complementarity determining region exchange and RapMAT technology

Immunotherapy ◽  
2009 ◽  
Vol 1 (4) ◽  
pp. 571-583
Author(s):  
Josef Prassler ◽  
Stefan Steidl ◽  
Stefanie Urlinger
Keyword(s):  
Monoclonal Antibodies ◽  
Affinity Maturation ◽  
Optimization Techniques ◽  
Human Diseases ◽  
Therapeutic Antibodies ◽  
Naturally Occurring ◽  
Human Immunoglobulins ◽  
Antibody Libraries ◽  
Complementarity Determining Region

Monoclonal antibodies gain ever-increasing importance in the treatment of human diseases across a broad range of indications. Diverse technologies currently exist, which are used to generate recombinant therapeutic antibodies that are basically indistinguishable from naturally occurring human immunoglobulins. We describe how human combinatorial antibody libraries are used together with unique optimization techniques to produce such therapeutically relevant proteins, for instance in the areas of oncology and inflammation.

scholarly journals Prophylactic Administration of a Complementarity-Determining Region Derived from a Neutralizing Monoclonal Antibody Is Effective against Respiratory Syncytial Virus Infection in BALB/c Mice

1998 ◽  
Vol 72 (1) ◽  
pp. 807-810 ◽  
Author(s):  
C. Bourgeois ◽  
J. B. Bour ◽  
L. S. Aho ◽  
P. Pothier
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
Lung Infection ◽  
Neutralizing Monoclonal Antibody ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Complementarity Determining Region ◽  
Single Peptide

ABSTRACT Immunotherapy with antibodies against respiratory syncytial virus (RSV) is a treatment option given the absence of any vaccine or other available satisfactory treatment. We selected one of our monoclonal antibodies, RS-348, that is highly neutralizing. We showed that a single peptide (PEP3H) derived from complementarity-determining region 3 (CDR3) of its heavy chain was capable of neutralizing the virusin vitro. When intranasally administered 24 h before challenge, this peptide protected BALB/c mice against RSV lung infection. These results indicate that a single CDR can be effective against RSV infection.

Human Anti-Mouse Antibodies

2000 ◽  
Vol 124 (6) ◽  
pp. 921-923 ◽  
Author(s):  
George G. Klee
Keyword(s):  
Monoclonal Antibodies ◽  
In Vivo Imaging ◽  
Laboratory Measurements ◽  
Imaging Studies ◽  
Short Article ◽  
Human Immunoglobulins ◽  
Monoclonal Mouse

Abstract Human anti-mouse antibodies (HAMA) are human immunoglobulins with specificity for mouse immunoglobulins. This topic currently is of interest because of the increased use of monoclonal mouse antibodies as diagnostic reagents both for in vitro laboratory measurements and for in vivo imaging studies. Monoclonal mouse antibodies also are being used therapeutically. This short article reviews the production of HAMA in patients receiving monoclonal antibodies and illustrates the potential ways that HAMA can interfere with immunoassay measurements. Methods for measuring and neutralizing HAMA also are discussed.

scholarly journals Potent germline-like monoclonal antibodies: Rapid identification of promising candidates for antibody-based antiviral therapy

2021 ◽  
Author(s):  
Xiaoyi Zhu ◽  
Fei Yu ◽  
Yanling Wu ◽  
Tianlei Ying
Keyword(s):  
Monoclonal Antibodies ◽  
Rational Design ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Viral Infections ◽  
Somatic Hypermutation ◽  
Affinity Maturation ◽  
Human Mabs

Abstract Recent years, fully human monoclonal antibodies (mAbs) are making up an increasing share of the pharmaceutical market. However, to improve affinity and efficacy of antibodies, many somatic hypermutation could be introduced during affinity maturation, which cause several issues including safety and efficacy and limit their application in clinic. Here, we propose a special class of human mAbs with limited level of somatic mutations, referred to as germline-like mAbs. Remarkably, germline-like mAbs could have high affinity and potent neutralizing activity in vitro and in various animal models, despite lacking of extensive affinity maturation. Furthermore, the germline nature of these mAbs implies that they exhibit lower immunogenicity and can be elicited relatively fast in vivo compared with highly somatically mutated antibodies. In this review, we summarize germline-like mAbs with strong therapeutic and protection activity against various viruses that caused large-scale outbreaks in the last decade, including influenza virus H7N9, Zika virus (ZIKV), Dengue virus (DENV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also illustrate underlying molecular mechanisms of these germline-like antibodies against viral infections from the structural and genetic perspective, thus providing insight into further development as therapeutic agents for treatment of infectious diseases and implication for rational design of effective vaccines.

scholarly journals Efficient Construction and Effective Screening of Synthetic Domain Antibody Libraries

2019 ◽  
Vol 2 (1) ◽  
pp. 17 ◽  
Author(s):  
Arghavan Solemani Zadeh ◽  
Alissa Grässer ◽  
Heiko Dinter ◽  
Maximilian Hermes ◽  
Katharina Schindowski
Keyword(s):  
Phage Display ◽  
Selection Process ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Effective Screening ◽  
Naturally Occurring ◽  
Domain Antibody ◽  
Efficient Construction ◽  
Antibody Libraries ◽  
Complementarity Determining Region

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.

scholarly journals Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies

2021 ◽  
Author(s):  
Denice T.Y. Chan ◽  
Maria A.T. Groves
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Sequence Space ◽  
Therapeutic Potential ◽  
Dna Recombination ◽  
Affinity Maturation ◽  
Therapeutic Antibodies ◽  
Effective Strategies ◽  
Key Features

Affinity maturation is a key technique in protein engineering which is used to improve affinity and binding interactions in vitro, a process often required to fulfil the therapeutic potential of antibodies. There are many available display technologies and maturation methods developed over the years, which have been instrumental in the production of therapeutic antibodies. However, due to the inherent limitations in display capacity of these technologies, accommodation of expansive and complex library builds is still a challenge. In this article, we discuss our recent efforts in the affinity maturation of a difficult antibody lineage using an unbiased approach, which sought to explore a larger sequence space through the application of DNA recombination and shuffling techniques across the entire antibody region and selections using ribosome display. We also highlight the key features of several display technologies and diversification methods, and discuss the strategies devised by different groups in response to different challenges. Particular attention is drawn to examples which are aimed at the expansion of sequence, structural or experimental diversity through different means and approaches. Here, we provide our perspectives on these methodologies and the considerations involved in the design of effective strategies for the directed evolution of antibodies.

Structural insight into a matured humanized monoclonal antibody HuA21 against HER2-overexpressing cancer cells

2019 ◽  
Vol 75 (6) ◽  
pp. 554-563 ◽  
Author(s):  
Zhenyi Wang ◽  
Liansheng Cheng ◽  
Gongrui Guo ◽  
Baoyun Cheng ◽  
Siyi Hu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cancer Cells ◽  
Growth Factor Receptor ◽  
Affinity Maturation ◽  
Chimeric Antibody ◽  
Humanized Monoclonal Antibody ◽  
Egfr Family ◽  
Insight Into

HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.

scholarly journals In vitro evaluation of therapeutic antibodies against a SARS-CoV-2 Omicron B.1.1.529 isolate

2022 ◽  
Author(s):  
Franck Touret ◽  
Cecile Baronti ◽  
Hawa Sophia Bouzidi ◽  
Xavier de Lamballerie
Keyword(s):  
Monoclonal Antibodies ◽  
Significant Loss ◽  
The Other ◽  
Clinical Strain ◽  
Significant Activity ◽  
Therapeutic Antibodies ◽  
Combination Therapies ◽  
Fold Reduction ◽  
Rapid Spread

The emergence and rapid spread of the Omicron variant of SARS-CoV-2, which has more than 30 substitutions in the spike glycoprotein, compromises the efficacy of currently available vaccines and therapeutic antibodies. Using a clinical strain of the Omicron variant, we analyzed the neutralizing power of eight currently used monoclonal antibodies compared to the ancestral B.1 BavPat1 D614G strain. We observed that six of these antibodies have lost their ability to neutralize the Omicron variant. Of the antibodies still having neutralizing activity, Sotrovimab/Vir-7831 shows the smallest reduction in activity, with a factor change of 3.1. Cilgavimab/AZD1061 alone shows a reduction in efficacy of 15.8, resulting in a significant loss of activity for the Evusheld cocktail (42.6 fold reduction) in which the other antibody, Tixagevimab, does not retain significant activity against Omicron. Our results suggest that the clinical efficacy of the initially proposed doses should be rapidly evaluated and the possible need to modify doses or propose combination therapies should be considered.

562 SO-C101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A596-A596
Author(s):  
Zuzana Antosova ◽  
Nada Podzimkova ◽  
Marketa Jiratova ◽  
Eva Nedvedova ◽  
Guy de Martynoff ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nk Cells ◽  
Nk Cell ◽  
Therapeutic Antibodies ◽  
Cell Cytotoxicity ◽  
Sequential Administration ◽  
Dependent Cell ◽  
Anti Tumor Activity

BackgroundSO-C101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SO-C101 specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment.MethodsHuman NK cell proliferation, the expression of NK cell receptors and ADCC activity of human PBMC after stimulation with SO-C101 in vitro in combination with monoclonal antibodies were detected by flow cytometry. The anti-tumor efficacy of SO-C101 in combination with Daratumumab was assessed in a multiple myeloma SCID xenograft mouse model in vivo.ResultsIn this study, we show that SO-C101 induced proliferation and expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+. Furthermore, SO-C101 induced expression of the cytotoxic receptors NKp30 and NKG2D whereas no upregulation of the inhibitory receptors CD158a, CD158b and NKG2A was detected. Both NK cell subsets activated by SO-C101 exhibited cytotoxicity towards cancer cells in vitro. Using human PBMCs, we show that SO-C101 potentiated killing of tumor cells induced by several clinically approved therapeutic monoclonal antibodies such as Cetuximab, Daratumumab and Obinutuzumab in vitro. SO-C101 and Daratumumab monotherapy treatment inhibited tumor growth in vivo, however, their combination showed the strongest anti-tumor efficacy. Specifically, sequential administration of Daratumumab, followed by SO-C101 promoted complete tumor regression, compared to partial anti-tumor responses induced by the respective monotherapies.ConclusionsSO-C101 augments the anti-tumor activity of therapeutic antibodies by increasing NK cells mediated antibody-dependent cell cytotoxicity. These results support the evaluation of SO-C101 in combination with monoclonal therapeutic antibodies in clinical studies.Ethics ApprovalThe anti-tumor efficacy studies in mice were approved by the internal ethics board of the respective contract research organization (CRO).

Minimal contribution of cell-bound antibodies to the immunoscintigraphy of inflamed joints with 99mTc-anti-CD4 monoclonal antibodies

2002 ◽  
Vol 41 (03) ◽  
pp. 129-134 ◽  
Author(s):  
A. Wolski ◽  
E. Palombo-Kinne ◽  
F. Wolf ◽  
F. Emmrich ◽  
W. Becker ◽  
...  
Keyword(s):  
T Cells ◽  
Monoclonal Antibodies ◽  
Synovial Membrane ◽  
In Vitro Release ◽  
Peritoneal Macrophages ◽  
Lymphoid Organs ◽  
Whole Body ◽  
Free Form ◽  
Ankle Joints

Summary Aim: The cellular joint infiltrate in rheumatoid arthritis patients is rich in CD4-positive T-helper lymphocytes and macrophages, rendering anti-CD4 monoclonal antibodies (mAbs) suitable for specific immunoscintigraphy of human/ experimental arthritis. Following intravenous injection, however, mAbs are present both in the free form and bound to CD4-positive, circulating monocytes and T-cells. Thus, the present study aimed at analyzing the relative contribution of the free and the cell-bound component to the imaging of inflamed joints in experimental adjuvant arthritis (AA). Methods: AA rat peritoneal macrophages or lymph node T-cells were incubated in vitro with saturating amounts of 99mTc-anti-CD4 mAb (W3/25) and injected i.v. into rats with AA. Results: In vitro release of 99mTc-anti-CD4 mAb from the cells was limited (on average 1.57%/h for macrophages and 0.84%/h for T-cells). Following i.v. injection, whole body/joint scans and tissue measurements showed only negligible accumulation of radioactivity in inflamed ankle joints (tissue: 0.22 and 0.34% of the injected activity, respectively), whereas the radioactivity was concentrated in liver (tissue: 79% and 71%, respectively), kidney, and urinary bladder. Unlike macrophages, however, anti-CD4 mAb-coated T-cells significantly accumulated in lymphoid organs, the inflamed synovial membrane of the ankle joints, as well as in elbow and knee joints. Conclusion: While the overall contribution of cell-bound mAbs to the imaging of arthritic joints with anti-CD4 mAbs is minimal, differential accumulation of macrophages and T-cells in lymphoid organs and the inflamed synovial membrane indicates preferential migration patterns of these 2 cell populations in arthritic rats. Although only validated for 99mTc-anti-CD4 mAbs, extrapolation of the results to other anticellular mAbs with similar affinity for their antigen may be possible.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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