Monoclonal antibody (MoAb) for cancer treatment has been greatly progressed during this decade. Six kinds of MoAbs are now in clinical use in Japan. Here we review on the development of MoAbs such as rituximab, bevacizumab and cetuximab. The characterization and the nomenclature of MoAb are also described. Panitumumab, a new MoAb targeting epithelial growth factor receptor(EGFR), and the resistance of colorectal cancer with KRAS mutation for anti-EGFR MoAbs are on recent topics. The concept of bevacizumab beyond progression (BBP) and the unfavorable results of bevacizumab in elder nonsmall cell lung carcinoma patients are so suggestive considering the strategy of the individualized cancer therapy. Finally, "drug lag" for the development of anti-cancer agents between Eastern and Western has to be discussed.

Antibody therapy in cancer

Conventional methods for extraction of DNA are expensive, time-consuming and tedious. To overcome these limitations, a paper-based DNA extraction device was developed that incorporates sponge-based buffer storage, a paper-based valve and channels of different length to autonomously direct the reagent and sample to the Fusion 5 disk for DNA capturing. With this device, DNA can be extracted within 2 min from only 30 μL samples of whole blood, serum, breast cancer cell, saliva, sputum and bacterial suspension. The device can also extract Hepatitis B Virus DNA from clinical blood samples and after quantification shows a detection limit as low as 104 copies⋅mL−1. This highlights its potential use in future diagnostics. The performance of the device was similar to that of the commercial QIAamp DNA micro kits and the FTA card. In our perception, this simple, inexpensive, portable and disposable device holds great promise in terms of POC testing in resource-poor settings.

Paper-based device with on-chip reagent storage for rapid extraction of DNA from biological samples

In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.

Advances in paper-based sample pretreatment for point-of-care testing.

As a rapidly developing field, paper-based microfluidic devices (μPADs) are expected to have profound implications, especially for in vitro diagnosis. In particular, integrated μPADs, with the merits of capillary-driven force, cost effectiveness, mass production and ease of use, are emerging as ideal tools for personal diagnosis; sample collection/pretreatment, signal transduction and results output can be simplified into one μPAD with minimal need for complex operation. In this review, we emphasize the latest developments of integrated μPADs and highlight the accomplishments and challenges of each component, including sample collection/pretreatment, signal transduction and amplification, and results output. Moreover, we give an overview of the growing trend of novel integrated μPADs and analyze existing challenges as well as potential directions for integrated μPADs for point-of-care testing (POCT).

Integrated paper-based microfluidic devices for point-of-care testing

Allosteric drugs and mutations: chances, challenges, and necessity.

Current therapeutic antibodies such as Trastuzumab, are typically of the blood circulatory IgG1 class (Cκ/ CHγ1). Due to the binding to Her2 also present on normal cell surfaces, side effects such as cardiac failure can sometimes be associated with such targeted therapy. Using antibody isotype swapping, it may be possible to reduce systemic circulation through increased tissue localization, thereby minimising unwanted side effects. However, the effects of such modifications have yet to be fully characterized, particularly with regards to their biophysical properties in antigen binding. To do this, we produced all light and heavy chain human isotypes/subtypes recombinant versions of Trastuzumab and Pertuzumab, and studied them with respect to recombinant production and Her2 binding. Our findings show that while the light chain constant region changes have no major effects on production or Her2 binding, some heavy chain isotypes, in particularly, IgM and IgD isotypes, can modulate antigen binding. This study thus provides the groundwork for such isotype modifications to be performed in the future to yield therapeutics of higher efficacy and efficiency.

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The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding.

Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development.

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Effect of VH-VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding.

Background Variable heavy chain (VH) family frameworks (FWRs) have been reported to affect antibody receptor and superantigen binding; however, such effects in IgE remain largely unknown. Given that VH family biases have been previously reported in IgE of certain allergies, there is a need to investigate this phenomenon for biotechnological and therapeutic purposes. Objective We sought to investigate the effects of VH families on IgE interaction with FceRIα, anti-IgE omalizumab, antigen, and superantigen protein A (spA) by using the pertuzumab and trastuzumab IgE models. Methods Pertuzumab VH1–VH7 family variants of IgE with the same complementarity-determining regions were investigated with regard to their binding interactions to FceRIα, Her2, omalizumab, and spA. Notable FceRIα-IgE observations were cross-checked against appropriate trastuzumab IgE VH variants. Computational structural modeling and simulations were also performed for insight into the mechanism of interactions with various VH FWRs. Results The pertuzumab VH5 IgE variant, but not the trastuzumab VH5 IgE, was found to interact with FceRIα significantly longer than the respective VH family variants within each model antibody. No significant differences in interaction were found between IgE and omalizumab for the pertuzumab VH variants. Although trastuzumab VH3 interacted with spA, none of our pertuzumab VH variants, including VH3, associated with spA. Conclusion We found unexpected varying allosteric communications caused by the VH family FWRs to the FceRIα-, Her2-, and spA-binding regions of pertuzumab IgE, with implications for use of IgE/anti-IgE therapeutics to treat allergy and IgE therapeutics in allergo-oncology.

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

Antibody research has traditionally focused on heavy chains, often neglecting the important complementary role of light chains in antibody formation and secretion. In the light chain, the complementarity-determining region 3 (VL-CDR3) is specifically implicated in disease states. By modulating VL-CDR3 exposure on the scaffold through deletions in the framework region 3 (VL-FWR3), we further investigated the effects on secretion in recombinant production and antigen binding kinetics. Our random deletions of two residues in the VL-FWR3 of a Trastuzumab model showed that the single deletions could impact recombinant production without significant effect on Her2 binding. When both the selected residues were deleted, antibody secretion was additively decreased, and so was Her2 binding kinetics. Interestingly, we also found allosteric effects on the Protein L binding site at VL-FWR1 elicited by these deletions in VL- FWR3. Together, these findings demonstrate the importance of light chain FWR3 in antigen binding, recombinant production, and antibody purification using Protein L.

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The role of Antibody Vκ Framework 3 region towards Antigen binding: Effects on recombinant production and Protein L binding

The reductionist approach is prevalent in biomedical science. However, increasing evidence now shows that biological systems cannot be simply considered as the sum of its parts. With experimental, technological, and computational advances, we can now do more than view parts in isolation, thus we propose that an increasing holistic view (where a protein is investigated as much as a whole as possible) is now timely. To further advocate this, we review and discuss several studies and applications involving allostery, where distant protein regions can cross-talk to influence functionality. Therefore, we believe that an increasing big picture approach holds great promise, particularly in the areas of antibody engineering and drug discovery in rational drug design.

Jun-Jie Poh

Papers

The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding.

Effect of VH-VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding.

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

The role of Antibody Vκ Framework 3 region towards Antigen binding: Effects on recombinant production and Protein L binding

Perspective: The promises of a holistic view of proteins-impact on antibody engineering and drug discovery.