Targeting cancer with antibody-drug conjugates: Promises and challenges.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of biotherapeutics that utilize antibodies to selectively deliver cytotoxic drugs to the tumor site. As of May 2021, the U.S. Food and Drug Administration (FDA) has approved ten ADCs, namely Adcetris®, Kadcyla®, Besponsa®, Mylotarg®, Polivy®, Padcev®, Enhertu®, Trodelvy®, Blenrep®, and Zynlonta™ as monotherapy or combinational therapy for breast cancer, urothelial cancer, myeloma, acute leukemia, and lymphoma. In addition, over 80 investigational ADCs are currently being evaluated in approximately 150 active clinical trials. Despite the growing interest in ADCs, challenges remain to expand their therapeutic index (with greater efficacy and less toxicity). Recent advances in the manufacturing technology for the antibody, payload, and linker combined with new bioconjugation platforms and state-of-the-art analytical techniques are helping to shape the future development of ADCs. This review highlights the current status of marketed ADCs and those under clinical investigation with a focus on translational strategies to improve product quality, safety, and efficacy.

Immunohistochemical Detection of p40 Expression in Lung Cancer Clinical Samples.

Immunohistochemistry is the technique by which antigens in tissues are detected by means of antigen-antibody reaction. The p40 antibody is directed against the ΔN domain of the ΔNp63 isoform of p63 and is a highly specific marker for the squamous cell carcinoma subtype of non-small cell lung carcinomas (NSCLC). As such, immunohistochemical detection of this antigen in NSCLC biopsies is extremely valuable to assess tumor histological subtype. Herein we describe a manual procedure for performing p40 immunohistochemistry on formalin-fixed paraffin-embedded tissue sections by the indirect polymer-based two-step technique using hydrogen peroxide and 3-3'diaminobenzidine detection system.

Immunoblot Validation of Phospho-Specific Antibodies Using Lung Cancer Cell Lines.

The cancer phenotype is usually characterized by deregulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both preclinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines. The protocol consists in the dephosphorylation of the Rb-containing protein lysates by treating them with bovine intestinal phosphatase, followed by assessment of the dephosphorylation by immunoblot.

Targeting a neoantigen derived from a common TP53 mutation.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.

Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches.

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

UK NEQAS ICC & ISH Ki-67 Data Reveal Differences in Performance of Primary Antibody Clones.

We examined data from 374 laboratories staining for Ki-67 as part of external quality assessment over 8 runs between 2013 and 2017 (total data sets=2601). One of 5 primary antibodies was used for 94.8% of submissions, with MIB-1 (Agilent Dako) comprising 58.8% of the total. Examining assessment score as a continuous variable showed the 30-9 (Ventana) and K2 (Leica Biosystems) clones were associated with the highest mean scores (17.0; 95% confidence interval, 16.8-17.2 and 16.3; 95% confidence interval, 15.9-16.6, respectively). Stain quality was not significantly different between them. Both were associated with significantly better staining compared with MIB-1 (Agilent Dako), MM1 (Leica Biosystems), and SP6 from various suppliers (P<0.05). Similarly, categorical assessment of "Good" versus "Not good" staining quality showed that the 30-9 and K2 clones were both significantly associated with "Good" staining (both P<0.001). Other methodological parameters were examined for significant primary antibody-specific effects; none were seen for 30-9, K2, or SP6. The MM1 clone was more likely to be associated with good quality staining when it was used with Leica Biosystems sourced antigen retrieval, detection, and platform, all statistically significant at P<0.01. MIB-1 was more likely to be associated with good quality staining results when it was used with Agilent Dako antigen retrieval, detection, and staining platforms (P<0.0001), and less likely at the same significance level when used with Leica Biosystems reagents and equipment. The data presented here show the importance of not just primary antibody choice but also matching that choice to other methodological factors.

Evaluation of an antibody-PNA conjugate as a clearing agent for antibody-based PNA-mediated radionuclide pretargeting.

Radionuclide molecular imaging of cancer-specific targets is a promising method to identify patients for targeted antibody therapy. Radiolabeled full-length antibodies however suffer from slow clearance, resulting in high background radiation. To overcome this problem, a pretargeting system based on complementary peptide nucleic acid (PNA) probes has been investigated. The pretargeting relies on sequential injections of primary, PNA-tagged antibody and secondary, radiolabeled PNA probe, which are separated in time, to allow for clearance of non-bound primary agent. We now suggest to include a clearing agent (CA), designed for removal of primary tumor-targeting agent from the blood. The CA is based on the antibody cetuximab, which was conjugated to PNA and lactosaminated by reductive amination to improve hepatic clearance. The CA was evaluated in combination with PNA-labelled trastuzumab, T-ZHP1, for radionuclide HER2 pretargeting. Biodistribution studies in normal mice demonstrated that the CA cleared ca. 7 times more rapidly from blood than unmodified cetuximab. Injection of the CA 6 h post injection of the radiolabeled primary agent [131I]I-T-ZHP1 gave a moderate reduction of the radioactivity concentration in the blood after 1 h from 8.5 ± 1.8 to 6.0 ± 0.4%ID/g. These proof-of-principle results could guide future development of a more efficient CA.

89Zr-Labeled AR20.5: A MUC1-Targeting ImmunoPET Probe.

High expression levels of the tumor-associated antigen MUC1 have been correlated with tumor aggressiveness, poor response to therapy, and poor survival in several tumor types, including breast, pancreatic, and epithelial ovarian cancer. Herein, we report the synthesis, characterization, and in vivo evaluation of a novel radioimmunoconjugate for the immuno-positron emission tomography (immunoPET) imaging of MUC1 expression based on the AR20.5 antibody. To this end, we modified AR20.5 with the chelator desferrioxamine (DFO) and labeled it with the positron-emitting radiometal zirconium-89 (t1/2 ~3.3 d) to produce [89Zr]Zr-DFO-AR20.5. In subsequent in vivo experiments in athymic nude mice bearing subcutaneous MUC1-expressing ovarian cancer xenografts, [89Zr]Zr-DFO-AR20.5 clearly delineated tumor tissue, producing a tumoral activity concentration of 19.1 ± 6.4 percent injected dose per gram (%ID/g) at 120 h post-injection and a tumor-to-muscle activity concentration ratio of 42.4 ± 10.6 at the same time point. Additional PET imaging experiments in mice bearing orthotopic MUC1-expressing ovarian cancer xenografts likewise demonstrated that [89Zr]Zr-DFO-AR20.5 enables the visualization of tumor tissue-including metastatic lesions-with promising tumor-to-background contrast.

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA.

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.

Humanized Anti-Tumor-Associated Glycoprotein-72 for Submillimeter Near-Infrared Detection of Colon Cancer in Metastatic Mouse Models.

BACKGROUND: Tumor-associated glycoprotein (TAG)-72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72-specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models. METHODS: Anti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49-IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy. RESULTS: Subcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 µg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light. CONCLUSIONS: Anti-huCC49-IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

Utility of Claudin-4 versus BerEP4 and B72.3 in pleural fluids with metastatic lung adenocarcinoma.

INTRODUCTION: Lung adenocarcinoma (LADC) is the most common occult primary in patients presenting with a malignant pleural effusion. Distinguishing metastatic LADC from reactive mesothelial cells (RMC) and malignant mesothelioma (MM) based on morphology alone has been a persistent diagnostic challenge in cytopathology. Claudin-4, a major functional constituent of tight junctions, has been shown to help distinguish LADC from RMC and MM in surgical specimens. Our goal was to further validate and assess the utility of Claudin-4 in comparison to BerEP4 and B72.3 in malignant effusions with a focus on metastatic LADC. MATERIALS AND METHODS: We evaluated 58 pleural effusions (40 LADC, 10 RCM, and 8 MM). Immunohistochemistry was performed using Claudin-4, Ber-EP4, and B72.3 on cell blocks. Staining patterns, quantity of tumor cells, and intensity of staining (weak, moderate, or strong) were assessed. RESULTS: All cases of LADC were positive for Claudin-4 with an overall sensitivity of 100% (40 of 40) and specificity of 100% (18 of 18). In addition, Claudin-4 showed the highest quantity and quality of staining with 3+ staining intensity in 73% (29 of 40) of cases, compared with 35% (14 of 40) of cases using BerEP4 and 52% (21 of 40) of cases using B72.3. The sensitivity and specificity for BerEP4 were 90% and 78%, respectively. The sensitivity and specificity for B72.3 were 87.5% and 100%, respectively. CONCLUSIONS: In this study, Claudin-4 performed superiorly compared to BerEP4 and B72.3 in distinguishing lung adenocarcinoma from RMC or MM in pleural effusions. Our results show Claudin-4 is a useful marker for distinguishing RMC and MM from lung adenocarcinoma, with high sensitivity (100%) and specificity (100%), compatible with studies shown in the literature.

Antibody-modified magnetic nanoparticles as specific high-efficient cell-separation agents.

Separation of tumor cells is a promising approach that helps not only in early detection of cancer but also as an efficient tool that holds great importance in prohibiting cancer cell mutation, drug resistance to treatments, and in granting successful adjuvant therapies. As one of the highly efficient processes for the separation of single cells, tumor cells, and specific proteins from fresh whole blood, a magnetic iron oxide nanoparticle (IONP)-based immunomagnetic separation technique has been developed in this article. The synthesized IONPs were modified with antibodies (Abs) against human epithelial growth factor receptor 2 (HER2), which is overexpressed and/or amplified in about 15% of breast cancer patients with several types of human cancer cells. The prepared Ab-conjugated IONPs (Ab-IONPs) attach HER2-positive cancer cells exclusively and can serve as specific high-efficient single-cell separation agents. The results showed that the magnetic IONPs have been successfully attached to the Abs via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide linkers. Maximum targeting efficiency of the Ab-IONP complex, which was 94.5 ± 0.8% for BT474 and 70.6 ± 0.4% for mixture of cells (BT474 and MCF7), was achieved with a minimum amount of Abs, to provide an economically efficient single-cell detection device.

Lipid-based phagocytosis nanoenhancer for macrophage immunotherapy.

Tumor associated macrophages (TAMs) play an important role in initiating the immunosuppressive environment that negatively impacts the immunotherapy efficacy and has long been linked with cancer progression. On the other hand, activated macrophages display immense phagocytic potential and can be used as an effector cell for cancer therapy. But, activating TAMs to effectively phagocytose cancer cells is challenging. Cancer cells upregulate CD47, a "don't eat me" receptor that ligates with SIRPα present on macrophages to downregulate the phagocytosis. Since phagocytosis is a physical phenomenon based on engulfment of aberrant cells, we hypothesized that the phagocytic function of macrophages can be enhanced by blocking both CD47 and SIRPα in tandem and at the same time, engaging both macrophages and cancer cells can favor increased macrophage-cancer cellular interactions. Here, we demonstrate that a simple approach of anti-CD47 and anti-SIRPα antibodies conjugated lipid-based phagocytosis nanoenhancer (LPN) can perform both of these functions. The LPNs were stable in both physiological and biologically relevant conditions, bound to both macrophages and cancer cells and significantly enhanced phagocytosis of cancer cells as compared to combination of free antibodies. LPN treatment showed significant tumor growth inhibition and increased survival in B16F10 melanoma tumor bearing mice with no systemic toxicity. Mechanistic analysis of efficacy revealed an increase in intra-tumoral infiltration of effector T cells and NK cells. Cytokine analysis revealed increased secretion of intracellular iNOS, a hallmark of activated macrophages. This study shows that LPN can simultaneously block both CD47 and SIRPα and can effectively engage macrophage and cancer cell in close proximity. Combining these facets provide a simple approach to enhance phagocytosis and improve anti-cancer macrophage immunotherapy.

Validation of the QR1 Antibody for the Evaluation of PD-L1 Expression in Non-Small Cell Lung Adenocarcinomas.

The evaluation of Programmed cell Death Ligand 1 (PD-L1) expression in the tumor cells with immunohistochemistry is a mandatory diagnostic step in the treatment of lung cancer. It is important to utilize validated antibodies that can reliably detect PD-L1 positive cells. Different antibodies have already been studied. In this present study, we compared a new clone (QR1, Quartett) with reference clones to determine if it can be used in place of previously identified reference clones. We built a tissue micro array (TMA) from 110 lung adenocarcinomas and compared it using immunohistodetection of four different clones: QR1, 22c3, Sp263, and E1L3N. We analyzed the correlation between the sample duplicates for each clone and then a correlation and the concordance between the clones were calculated. A total of 101 patients were exploitable; the duplicates for each clone had a strong correlation. The correlation was the strongest (r=0.82) between QR1 and 22c3 and less strong with the other clones. Totals of 78%, 79%, and 97% of the QR1 cases were concordant with 22c3 for the thresholds of <1%, 1% to 49%, and ≥50%, respectively. The sensitivities and specificities of QR1, compared with 22c3, were >75% and 81%, respectively. PD-L1 expression, analyzed in lung adenocarcinomas with QR1, is highly correlated and concordant with the main reference clone used in most laboratories (22c3). It can be used to replace the latter in clinical routine.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody.

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5-2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

STAT6 Expression in Solitary Fibrous Tumor and Histologic Mimics: a Single Institution Experience.

STAT6 stain has proved to be a good surrogate marker for the genetic alteration (NAB2-STAT6 gene fusion) in solitary fibrous tumor (SFT). This study aims to validate the use of STAT6 rabbit monoclonal antibody in differentiating SFT from its histologic mimics. Forty-five cases of SFT and 110 cases from 9 other spindle cell tumors were collected for STAT6 immunostaining. Positive nuclear STAT6 staining was present in all 45 SFT cases (100% sensitivity). No nuclear staining was identified in other spindle cell neoplasms (0/13, dedifferentiated liposarcoma; 0/17, synovial sarcoma; 0/16, malignant peripheral nerve sheath tumors; 0/25, undifferentiated pleomorphic sarcoma; 0/10, dermatofibrosarcoma protuberans; 0/9, low-grade fibromyxoid sarcoma; 0/6, angiofibroma; 0/5, deep fibromatosis; 0/9, gastrointestinal tumor). The STAT6 staining in SFT was usually diffuse (5+ in 19 cases; 4+ in 17 cases) and strong (40 cases). Monoclonal STAT6 stain is highly sensitive and specific for SFTs and particularly useful in the diagnosis of difficult SFT cases.

A Comparison of E6H4 and G175-405 p16-specific Monoclonal Antibodies in Oropharyngeal Squamous Cell Carcinoma.

The purpose of this investigation is to directly compare G175-405 and E6H4 p16-specific antibodies as immunomarkers of HPV-driven oropharyngeal carcinoma. The investigators designed a retrospective analysis using specimens from an archived tissue bank with known in situ hybridization and polymerase chain reaction status for HPV DNA. Fifty randomly selected oropharyngeal specimens were evaluated with both the G175-405 and E6H4 p16-specific monoclonal antibodies. Two pathologists, blinded to the HPV-specific testing status, evaluated p16 positivity for both antibody clones. Interrater agreement was determined using a Cohen κ coefficient. Sensitivity and specificity values were calculated using a standard 2×2 contingency table, then compared using McNemar test. Interrater agreement for interpretation of p16 expression was 92% (κ=0.84) for the G175-405 clone and 100% for the E6H4 clone (κ=1.0). The G175-405 stain had a sensitivity of 0.917 and specificity of 0.846. The E6H4 stain had a sensitivity of 1.000 and specificity of 0.769. Using McNemar test, there were no significant differences found for sensitivity (P=0.480) or specificity (P=0.480) values. The results of this study suggest that though both G175-405 and E6H4 antibody stains are statistically comparable immunomarkers for HPV-driven oropharyngeal carcinoma, the E6H4 clone offers improved interobserver reliability.

A two-dimensional immunomagnetic nano-net for the efficient isolation of circulating tumor cells in whole blood.

An immunomagnetic "nano-net" was designed and synthesized for specifically capturing rare cells of interest from mixtures. The nano-net, Ab@Lipo-MNP-GO, consists of conjugated antibody molecules on a lipid coated magnetic nanoparticle-graphene oxide sheet complex. The magnetism, chemical composition, and the morphology of the construct and its precursors were characterized by SQUID, FTIR, TGA, DLS and SEM, to confirm the feasibility of the synthetic steps and the resulting properties suitable for solution phase immuno-recognition for cell capture. When applied to capturing circulating tumor cells (CTCs) in oral, colon and lung cancer clinical patients' blood samples, the nano-net construct exhibited far superior ability whereas conventional immunomagnetic beads in some cases were unable to capture any CTCs, even by increasing the bead concentration. Confocal images showed that the nano-net wrapped around the CTCs while the immunomagnetic beads attached them with point contacts. A stable, patch-like multivalent matrix nano-net was demonstrated to tackle the shortcomings of single point contact of immunomagnetic beads to the target cell. This strategy is universal for any cell separation in complex fluids.

P-Glycoprotein Antibody Decorated Porous Hydrogel Particles for Capture and Release of Drug-Resistant Tumor Cells.

Multidrug resistance is one of the leading causes of chemotherapy failure in cancer patients. Early detection and capture of drug-resistant tumor cells can facilitate the monitoring of the therapy process and improve the prognosis of patients. In this study, novel P-glycoprotein (P-gp) antibody modified porous hydrogel particles are proposed for drug-resistant tumor cells capture. The hydrogel particles employ a highly biocompatible hydrogel, methacrylate gelatin (GelMA), as the carrier and replicate from the silica colloidal crystal beads. By the modification of P-gp antibody probes on their surfaces, the hydrogel particles are endowed with the ability to capture drug-resistant tumor cells, which overexpress specific components of P-gp on their membranes. Additionally, the acquired ordered porous nanostructure of the particles can provide not only more surface area for antibody immobilization but also a nanopatterned platform for highly efficient target cell capture. The above advantages make the porous hydrogel particles ideal for efficient capture and detection of the drug-resistant tumor cells, which can be expected to facilitate the point-of-care pharmacotherapy and promisingly improve the patient outcomes.