International Journal of Molecular Sciences

Research

16 pages, 2156 KiB

Open AccessArticle

¹⁸F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography

by Katie S. Ardipradja, Christian W. Wichmann, Kevin Hickson, Angela Rigopoulos, Karen M. Alt, Hannah A. Pearce, Xiaowei Wang, Graeme O’Keefe, Andrew M. Scott, Karlheinz Peter, Christoph E. Hagemeyer and Uwe Ackermann

Int. J. Mol. Sci. 2022, 23(13), 6886; https://doi.org/10.3390/ijms23136886 - 21 Jun 2022

Cited by 3 | Viewed by 1833

Abstract

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on [...] Read more.

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[¹⁸F]fluorobenzamido)ethyl]maleimide ([¹⁸F]FBEM). ScFv_anti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[¹⁸F]fluorobenzamido)ethyl]maleimide ([¹⁸F]FBEM). Radiolabelled scFv was injected into mice with FeCl₃-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer ¹⁸F-scFv_anti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFv_anti-LIBS against activated platelets using site-specific bio conjugation. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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13 pages, 4480 KiB

Open AccessArticle

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment

by Chaoyu Zhang, Wenjie Sheng, Marwah Al-Rawe, T. M. Mohiuddin, Marcus Niebert, Felix Zeppernick, Ivo Meihold-Heerlein and Ahmad Fawzi Hussain

Int. J. Mol. Sci. 2022, 23(11), 6122; https://doi.org/10.3390/ijms23116122 - 30 May 2022

Cited by 7 | Viewed by 3450

Abstract

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new [...] Read more.

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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14 pages, 2590 KiB

Open AccessArticle

Antibody-Drug Conjugate Targeting c-Kit for the Treatment of Small Cell Lung Cancer

by Kwang-Hyeok Kim, Jin-Ock Kim, Jeong-Yang Park, Min-Duk Seo and Sang Gyu Park

Int. J. Mol. Sci. 2022, 23(4), 2264; https://doi.org/10.3390/ijms23042264 - 18 Feb 2022

Cited by 12 | Viewed by 3788

Abstract

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15–25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. [...] Read more.

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15–25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of K_D = 5.5 × 10⁻⁹ M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC₅₀ ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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13 pages, 2237 KiB

Open AccessArticle

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases

by Xiaojie Chu, Zehua Sun, Du-San Baek, Wei Li, John W. Mellors, Steven D. Shapiro and Dimiter S. Dimitrov

Int. J. Mol. Sci. 2021, 22(20), 11136; https://doi.org/10.3390/ijms222011136 - 15 Oct 2021

Cited by 11 | Viewed by 2411

Abstract

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we [...] Read more.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and V_H 1D1.43 from two large phage-displayed human Fab and V_H libraries. After fusion with human IgG1 Fc, both of them (V_H-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with V_H-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both V_H-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. V_H-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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Review

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18 pages, 2827 KiB

Open AccessReview

Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy

by Wenjie Sheng, Chaoyu Zhang, T. M. Mohiuddin, Marwah Al-Rawe, Felix Zeppernick, Franco H. Falcone, Ivo Meinhold-Heerlein and Ahmad Fawzi Hussain

Int. J. Mol. Sci. 2023, 24(4), 3086; https://doi.org/10.3390/ijms24043086 - 4 Feb 2023

Cited by 18 | Viewed by 8702

Abstract

Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect [...] Read more.

Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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18 pages, 998 KiB

Open AccessReview

Near Infrared Photoimmunotherapy: A Review of Recent Progress and Their Target Molecules for Cancer Therapy

by T. M. Mohiuddin, Chaoyu Zhang, Wenjie Sheng, Marwah Al-Rawe, Felix Zeppernick, Ivo Meinhold-Heerlein and Ahmad Fawzi Hussain

Int. J. Mol. Sci. 2023, 24(3), 2655; https://doi.org/10.3390/ijms24032655 - 31 Jan 2023

Cited by 16 | Viewed by 3055

Abstract

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed molecular targeted cancer treatment, which selectively kills cancer cells or immune-regulatory cells and induces therapeutic host immune responses by administrating a cancer targeting moiety conjugated with IRdye700. The local exposure to near-infrared (NIR) light causes [...] Read more.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed molecular targeted cancer treatment, which selectively kills cancer cells or immune-regulatory cells and induces therapeutic host immune responses by administrating a cancer targeting moiety conjugated with IRdye700. The local exposure to near-infrared (NIR) light causes a photo-induced ligand release reaction, which causes damage to the target cell, resulting in immunogenic cell death (ICD) with little or no side effect to the surrounding normal cells. Moreover, NIR-PIT can generate an immune response in distant metastases and inhibit further cancer attack by combing cancer cells targeting NIR-PIT and immune regulatory cells targeting NIR-PIT or other cancer treatment modalities. Several recent improvements in NIR-PIT have been explored such as catheter-driven NIR light delivery, real-time monitoring of cancer, and the development of new target molecule, leading to NIR-PIT being considered as a promising cancer therapy. In this review, we discuss the progress of NIR-PIT, their mechanism and design strategies for cancer treatment. Furthermore, the overall possible targeting molecules for NIR-PIT with their application for cancer treatment are briefly summarised. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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35 pages, 1775 KiB

Open AccessReview

Bispecific Antibody-Based Immune-Cell Engagers and Their Emerging Therapeutic Targets in Cancer Immunotherapy

by Ha Gyeong Shin, Ha Rim Yang, Aerin Yoon and Sukmook Lee

Int. J. Mol. Sci. 2022, 23(10), 5686; https://doi.org/10.3390/ijms23105686 - 19 May 2022

Cited by 10 | Viewed by 9148

Abstract

Cancer is the second leading cause of death worldwide after cardiovascular diseases. Harnessing the power of immune cells is a promising strategy to improve the antitumor effect of cancer immunotherapy. Recent progress in recombinant DNA technology and antibody engineering has ushered in a [...] Read more.

Cancer is the second leading cause of death worldwide after cardiovascular diseases. Harnessing the power of immune cells is a promising strategy to improve the antitumor effect of cancer immunotherapy. Recent progress in recombinant DNA technology and antibody engineering has ushered in a new era of bispecific antibody (bsAb)-based immune-cell engagers (ICEs), including T- and natural-killer-cell engagers. Since the first approval of blinatumomab by the United States Food and Drug Administration (US FDA), various bsAb-based ICEs have been developed for the effective treatment of patients with cancer. Simultaneously, several potential therapeutic targets of bsAb-based ICEs have been identified in various cancers. Therefore, this review focused on not only highlighting the action mechanism, design and structure, and status of bsAb-based ICEs in clinical development and their approval by the US FDA for human malignancy treatment, but also on summarizing the currently known and emerging therapeutic targets in cancer. This review provides insights into practical considerations for developing next-generation ICEs. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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27 pages, 7623 KiB

Open AccessReview

Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence

by Ricardo Piña, Alma I. Santos-Díaz, Erika Orta-Salazar, Azucena Ruth Aguilar-Vazquez, Carola A. Mantellero, Isabel Acosta-Galeana, Argel Estrada-Mondragon, Mara Prior-Gonzalez, Jadir Isai Martinez-Cruz and Abraham Rosas-Arellano

Int. J. Mol. Sci. 2022, 23(3), 1426; https://doi.org/10.3390/ijms23031426 - 26 Jan 2022

Cited by 28 | Viewed by 23330

Abstract

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is [...] Read more.

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay. Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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Other

Jump to: Research, Review

2 pages, 186 KiB

Open AccessReply

Reply to Sheval, E.V. Comment on “Piña et al. Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence. Int. J. Mol. Sci. 2022, 23, 1426”

by Ricardo Piña, Isabel Acosta-Galeana and Abraham Rosas-Arellano

Int. J. Mol. Sci. 2022, 23(8), 4375; https://doi.org/10.3390/ijms23084375 - 15 Apr 2022

Cited by 1 | Viewed by 1115

Abstract

We have carefully read the interesting explanatory comment by Eugene V. Sheval [...] Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

2 pages, 185 KiB

Open AccessComment

Comment on Piña et al. Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence. Int. J. Mol. Sci. 2022, 23, 1426

by Eugene V. Sheval

Int. J. Mol. Sci. 2022, 23(8), 4372; https://doi.org/10.3390/ijms23084372 - 15 Apr 2022

Cited by 1 | Viewed by 1347

Abstract

With great interest, I have read the article “Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence” written by Piña et al. [...] Full article

(This article belongs to the Special Issue Recent Advances of Site-Specific Antibody Drug Conjugations for Clinical Application)

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