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.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired “eXchange Unit between T domains” (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

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.

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.

The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen–antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti–mouse and anti–rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O⁶-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti–mouse and anti–rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays.

Antibody-drug conjugates (ADCs) consist of an antibody linked to a cytotoxic agent. To optimize the efficacy of ADCs, our study developed a pre-targeting drug delivery system based on the specific interaction between two synthetic coiled-coil proteins, Zip1 and Zip2. The targeting vehicle was composed of Zip2-fused single-chain variable fragments, while the toxic vehicle consisted of Zip1 conjugated to cytotoxic agent monomethyl auristatin E via SNAP-tag. The targeting activities of the pre-targeting reagents were evaluated using various techniques, including flow cytometry, fluorescence microscopy, cell viability and apoptosis assays, and ex vivo multiplex immunofluorescence. The pre-targeting complexes demonstrated specific binding and internalization in breast cancer cell lines, as assessed by flow cytometry and fluorescence microscopy. Furthermore, cell death was observed in antigen-expressing cell lines upon triggering apoptosis at nanomolar concentrations. Given the heterogeneous tumor environment and individual differences, separating the "targeting" and "killing" steps enables targeting molecules to bind to different antigens, followed by the application of the Zip1-drug complex without requiring antibody engineering. Our study highlights the potential of this pre-targeting system to efficiently deliver drugs, offering a promising strategy to address challenges faced by ADCs, such as poor tissue penetration, low accumulation, and "on-target, off-tumor" toxicity.