Photoimmunotherapy of Prostate Cancer With Antibody and Fab Fragments Targeting the Prostate Specific Membrane Antigen.

BACKGROUND/AIM: The standard treatment for localized prostate cancer involves surgical removal of the prostate with curative intent. However, when tumor cells persist in the operation site, there is high risk of local recurrence and tumor spread, leading to stressful follow-up treatments, impaired quality of life, and reduced overall survival. This study examined photoimmunotherapy (PIT) as a new treatment option for prostate cancer cells. MATERIALS AND METHODS: We generated conjugates consisting of either a humanized antibody or Fab fragments thereof targeting the prostate specific membrane antigen (PSMA), along with our silicon phthalocyanine photosensitizer dye WB692-CB1. PSMA-expressing prostate cancer cells were incubated with the antibody dye or Fab dye conjugates and cell binding was measured using flow cytometry. Cells were irradiated with varying doses of red light for dye activation, and cytotoxicity was determined by erythrosin B staining and subsequent analysis using a Neubauer counting chamber. RESULTS: Specific cytotoxicity was induced with the antibody dye conjugate in the prostate cancer cells in a light dose-dependent manner. Treatment of the cells with the Fab dye conjugate resulted in lower cytotoxicity, which could be attributed to a reduced binding affinity and a reduced dye uptake of the Fab fragment. CONCLUSION: Our new antibody dye and Fab dye conjugates offer potential for future intraoperative PIT in patients with localized prostate cancer, with the aim to ensure complete removal of tumor cells from the surgical area, to avoid local recurrence, and to improve clinical outcome.

Milk Fat Globule Proteins Are Relevant Bovine Milk Allergens in Patients with α­Gal Syndrome.

Alpha-gal syndrome (AGS) is a mammalian meat allergy associated with tick bites and specific IgE to the oligosaccharide galactose-α-1,3-galactose (α-gal). Recent studies have shown that 10-20% of AGS patients also react to the dairy proteins. Considering the already described role of the meat lipid fraction in AGS manifestations, the aim of this work has been to investigate whether the milk fat globule proteins (MFGPs) could be involved in AGS. The MFGPs are extracted and their recognition by the IgE of AGS patients is proved through immunoblotting experiments. The identification of the immunoreactive proteins by LC-HRMS analysis allows to demonstrate for the first time that butyrophillin, lactadherin, and xanthine oxidase (XO) are α-gal glycosylated. The role of xanthine oxidase seems to be prevalent since it is highly recognized by both the anti-α-gal antibody and AGS patient sera. The results obtained in this study provide novel insights in the characterization of α-Gal carrying glycoproteins in bovine milk, supporting the possibility that milk, especially in its whole form, may give reactions in AGS patients. Although additional factors are probably associated with the clinical manifestations, the avoidance of milk and milk products should be considered in individuals with AGS showing symptoms related to milk consumption.

Plasmodium yoelii surface-related antigen (PySRA) modulates the host pro-inflammatory responses via binding to CD68 on macrophage membrane.

Malaria, one of the major infectious diseases in the world, is caused by the Plasmodium parasite. Plasmodium antigens could modulate the inflammatory response by binding to macrophage membrane receptors. As an export protein on the infected erythrocyte membrane, Plasmodium surface-related antigen (SRA) participates in the erythrocyte invasion and regulates the immune response of the host. This study found that the F2 segment of P. yoelii SRA activated downstream MAPK and NF-κB signaling pathways by binding to CD68 on the surface of the macrophage membrane and regulating the inflammatory response. The anti-PySRA-F2 antibody can protect mice against P. yoelii, and the pro-inflammatory responses such as IL-1ß, TNF-α, and IL-6 after infection with P. yoelii are attenuated. These findings will be helpful for understanding the involvement of the pathogenic mechanism of malaria with the exported protein SRA.

Lutetium-177 Labelled Anti-PSMA Monoclonal Antibody (Lu-TLX591) Therapy for Metastatic Prostate Cancer: Treatment Toxicity and Outcomes.

INTRODUCTION: Whilst prostate cancer is the fourth most common cancer globally, effective therapies for patients with advanced disease are lacking. In recent years, interest in using theranostic agents to treat castrate-resistant prostate cancer (CRPC) and metastatic prostate cancer has emerged. Lu-TLX591 monoclonal antibody is a potential agent of significance; however, to date, reports on its toxicity and efficacy have been limited to small clinical trials in heavily pretreated patients. This retrospective study describes the real-world toxicity and efficacy profile of Lu-TLX591. METHODS: Eighteen patients received Lu-TLX591 at two private oncology centres in Australia. Patients were eligible if they had CRPC or metastatic prostate cancer and prostate-specific membrane antigen (PSMA)-avid disease confirmed by PSMA-positron emission tomography (PET). Patients received two cycles of Lu-TLX591 monoclonal antibody (177 Lu-DOTA-rosopatamab) each dosed from 1.01-2.85 GBq, 14 days apart. Patient side effects, blood test results and radiology reports were recorded on the patient's electronic medical record (eMR). RESULTS: Prominent side effects included fatigue (55.6%), anorexia (16.7%), nausea (11.1%), and transfusion reactions (11.1%). All-grade haematological toxicities included lymphopenia (61.1%), anaemia (22.2%), leukopenia (27.8%), neutropenia (27.8%), and thrombocytopenia (27.8%). Grade 4 toxicity included lymphopenia (6.7%) and thrombocytopenia (6.7%). Patients' prostate-specific antigen (PSA) responses were as follows; ≥ 30% PSA decline (27.8%), ≥ 50% PSA decline (11.4%) and any PSA decline (38.9%). Follow-up radiology revealed 54.5% stable disease, 45.4% disease progression and 9.1% disease regression. CONCLUSION: Lu-TLX591 was safely administered at acceptable toxicity and its efficacy reflects previous clinical trials. Larger studies are required and are underway (NCT04786847; NCT05146973; NCT04876651) to determine Lu-TLX591 effectiveness amongst different prostate cancer populations and compare its efficacy against peptide-based radiopharmaceutical agents.

Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

A combined biomarker panel shows improved sensitivity and specificity for detection of ovarian cancer.

BACKGROUND: Combined biomarkers can improve the sensitivity and specificity of ovarian cancer (OC) diagnosis and effectively predict patient prognosis. This study explored the diagnostic and prognostic values of serum CCL18 and CXCL1 antigens combined with C1D, FXR1, ZNF573, and TM4SF1 autoantibodies in OC. METHODS: CCL18 and CXCL1 monoclonal antibodies and C1D, FXR1, ZNF573, and TM4SF1 antigens were coated with microspheres. Logistic regression was used to construct a serum antigen-antibody combined detection model; receiver-operating characteristic curve (ROC) was used to evaluate the diagnostic efficacy of the model; and the Kaplan-Meier method and Cox regression models were used for survival analysis to evaluate the prognosis of OC. Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) projects and online survival analysis tools were used to evaluate prognostic genes for OC. The CIBERSORT immune score was used to explore the factors influencing prognosis and their relationship with tumor-infiltrating immune cells. RESULTS: The levels of each index in the blood samples of patients with OC were higher than those of the other groups. The combined detection model has higher specificity and sensitivity in the diagnosis of OC, and its diagnostic efficiency is better than that of CA125 alone and diagnosing other malignant tumors. CCL18 and TM4SF1 may be factors affecting the prognosis of OC, and CCL18 may be related to immune-infiltrating cells. CONCLUSIONS: The serum antigen-antibody combined detection model established in this study has high sensitivity and specificity for the diagnosis of OC.

Pilot study of the diagnostic utility of 89 Zr-df-IAB2M and 68 Ga-PSMA-11 PET imaging and multiparametric MRI in localized prostate cancer.

BACKGROUND: Accurate diagnosis of localized prostate cancer (PCa) is limited by inadequacy of multiparametric (mp) MRI to fully identify and differentiate localized malignant tissue from benign pathologies. Prostate-specific membrane antigen (PSMA) represents an excellent target for molecular imaging. IAB2M, an 85-kD minibody derived from a de-immunized monoclonal antibody directed at the extracellular domain of human PSMA (huJ591), and PSMA-11, a small molecule ligand have been previously tested as probes for visualization of recurrent/metastatic PCa with PET/CT. This pilot, non-randomized trial studied their diagnostic utility in patients (pts) with localized PCa. METHODS: Pts planned for radical prostatectomy (RP) were enrolled and underwent mpMRI and PET/CT imaging with 89 Zr-df-IAB2M and/or 68 Ga-PSMA-PET/CT. Image results were read by a radiologist blinded to clinical information and pathology results, mapped and compared to corresponding histopathology findings from all lesions, both clinically significant and nonsignificant. The detection rates of all three imaging modalities were measured and correlated. RESULTS: 20 pts with median age of 64.5 (46-79) years and PSA level of 7.5 (1.6-36.56) ng/ml were enrolled. 19 pts underwent RP and were imaged pre-operatively with 89 Zr-Df-IAB2M PET/CT and mpMRI. Nine of those were imaged using 68 Ga-PSMA-11 as well. Out of 48 intraprostatic lesions verified on surgical pathology, IAB2M PET/CT was able to detect 36 (75%). A similar proportion of pathologically confirmed, clinically significant lesions (22/29, 76%) was detected. IAB2M PET/CT was also able to identify 14/19 (74%) extraprostatic lesions. The performance of mpMRI was inferior, with 24/48 detectable lesions (50%) and 18/29 clinically significant intraprostatic lesions (62%). Compared to the current standard (mpMRI), IAB2M PET/CT had a sensitivity of 88%, specificity 38%, positive predictive value 58%, and accuracy 63%. In 9 pts who underwent Ga-PSMA-11 as well, the latter yielded a detection rate of 70% (14/20), which was also seen in clinically significant lesions (10/14, 71%). Ga-PSMA-11 PET/CT also detected 4/6 (67%) extraprostatic lesions. CONCLUSIONS: In this pilot study, the performance of 89 Zr-df-IAB2M was superior to mpMRI and similar to 68 Ga-PSMA-11 PET/CT. The higher detection rate of PSMA-PET supports its use as a diagnostic tool with consequent management change implications in men with localized PCa.

Virus-Like Particles Containing the E2 Core Domain of Hepatitis C Virus Generate Broadly Neutralizing Antibodies in Guinea Pigs.

A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct-acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384 to 661) that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bNAbs) than the parental form (receptor-binding domain [RBD]). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S, RBD-S, Δ123A7-S, and RBDA7-S; in the last two, 7 cysteines were replaced with alanines. While all four E2-S variant VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bNMAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (NAbs), with three of those animals generating bNAbs against 7 genotypes. Immune serum generated by animals with NAbs mapped to major neutralization epitopes located at residues 412 to 420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease, including cirrhosis and cancer. Broadly neutralizing antibodies that recognize the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here, we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus-like particles that display epitopes recognized by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.

Pretherapeutic estimated glomerular filtration rate predicts development of chronic kidney disease in patients receiving PSMA-targeted radioligand therapy.

BACKGROUND: Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) may be associated with renal toxicity. We aimed to identify predictive parameters for the development of chronic kidney disease (CKD) in patients with metastatic castration resistant prostate cancer (mCRPC) undergoing RLT. METHODS: In 46 mCRPC patients scheduled for Lu-177-PSMA-RLT, pretherapeutic estimated glomerular filtration rate (eGFR [ml/min/1.73 m2 ]), Tc-99m-mercaptoacetyltriglycine (Tc-99m-MAG3) clearance and baseline Ga-68-PSMA-ligand positron emission tomography (PET)-derived renal cortical uptake and PSMA-tumor volume (TV) were determined. We tested the predictive capability of these parameters and clinical risk factors for the occurrence of CKD (defined as CTCAE vers. 5.0 grade 2 or higher) during follow-up. RESULTS: After 4 ± 3 cycles of RLT average eGFR declined from 76 ± 17 to 72 ± 20 ml/min/1.73 m2 (p = 0.003). Increased estimated renal radiation dose (eRRD) was significantly associated with renal functional decline (p = 0.008). During follow-up, 16/46 (30.4%) developed CKD grade 2 (no grade 3 or higher). In receiver operating characteristic (ROC) analysis, pretherapeutic eGFR was highly accurate in identifying the occurrence of CKD vs no CKD with an area under the curve (AUC) of 0.945 (p < 0.001; best threshold, 77 ml/min/1.73 m2 ), followed by Tc-99m-MAG3-derived tubular extraction rate (TER; AUC, 0.831, p < 0.001; best threshold, 200 ml/min/1.73 m2 ). Renal PET signal (p = 0.751) and PSMA-TV (p = 0.942), however, were not predictive. Kaplan-Meier analyses revealed adverse renal outcome for patients with lower eGFR (p = 0.001) and lower scintigraphy-derived TER (p = 0.009), with pretherapeutic eGFR emerging as the sole predictive parameter in multivariate analysis (p = 0.007). CONCLUSION: Serious adverse renal events are not a frequent phenomenon after PSMA-targeted RLT. However, in patients developing moderate CKD after RLT, pretherapeutic eGFR is an independent predictor for renal impairment during follow-up.

A brief history of OspA vaccines including their impact on diagnostic testing for Lyme disease.

The only United States Food and Drug Administration approved vaccine preparation to prevent Lyme disease consisted of a single recombinant outer surface protein A (OspA), which was marketed for use from late 1998 until early 2002, with no vaccine currently available for humans for nearly 20 years. OspA vaccines generate an antibody-mediated, transmission blocking immunity, that prevents Borrelia burgdorferi from being transmitted during a tick bite. Although this OspA vaccine was safe and effective, it likely would have required booster doses to maintain immunity, and vaccination regularly caused false positive results on first-tier serologic testing for Lyme disease, when a whole cell-based enzyme immunoassay was used. Clinical trials are in progress to test a new multivalent OspA vaccine designed to prevent Lyme disease in both the United States and Europe.

Identification of Antibodies against Neutrophil Surface Antigens in Two Iranian Patients with Autoimmune Neutropenia.

Autoimmune neutropenia is a type of immune-mediated neutropenia, caused by antibody-induced neutrophil destruction. Here we report two cases (3-year-old boy and 9-year-old girl) with suspected autoimmune neutropenia. The presence of neutrophil antibodies in sera of these patients was investigated using standard neutrophil antibody screening tests such as granulocyte immunofluorescence test (GIFT), granulocyte agglutination test (GAT), and lymphocyte immunofluorescence test (LIFT). A positive reactivity with two panel cells was found in GIFT. No reactivities with panel cells were observed in GAT and LIFT. To the best of our knowledge, this is the first report for detecting the neutrophil reactive antibodies using genotyped neutrophils in patients with autoimmune neutropenia in Iran. The final diagnosis of our patients was primary autoimmune neutropenia for the boy and autoimmune neutropenia associated with familial Mediterranean fever for the girl.

A Novel Insight into the Role of PLA2R and THSD7A in Membranous Nephropathy.

Membranous nephropathy (MN) is an organ-restricted autoimmune disease mainly caused by circulating autoantibodies against podocyte antigens, including the M-type phospholipase A2 receptor (PLA2R) and thrombospondin domain-containing 7A (THSD7A). Antibodies against PLA2R are present in 70%-80% and against THSD7A in 2% of adult patients, which provides a paradigm shift in molecular diagnosis and management monitoring. Both antigens share some similar characteristics: they are expressed by podocytes and have wide tissue distributions; they are bound by autoantibodies only under nonreducing conditions, and the subtype of most autoantibodies is IgG4. However, the factors triggering autoantibody production as well as the association among air pollution, malignancy, and the pathogenesis of MN remain unclear. In this review, we discuss the similarity between the pathological mechanisms triggered by disparate antigens and their associated diseases. Furthermore, we demonstrated the possibility that PM2.5, malignancy, and gene expression specifically induce exposure of these antigens through conformational changes, molecular mimicry, or increased expression eliciting autoimmune responses. Thus, this review provides novel insights into the pathological mechanism of MN.

Identification of a novel variant erythrocyte surface antigen-1 (VESA1) in Babesia orientalis.

Babesia orientalis, belonging to the phylum Apicomplexa, is mainly accountable for water buffalo babesiosis, which adversely affected the livestock industry in China. Variant erythrocyte surface antigen-1 (VESA1), an antigen that helps infected erythrocytes to escape from host immune responses, was first reported in Babesia bovis. Various VESA1 proteins have also been characterized in other Babesia species. Nevertheless, there is no research on the identification and characterization of VESA1 proteins in Babesia orientalis. In this study, the BoVESA1 gene was amplified from both gDNA and cDNA. The results revealed that it is an intronless gene with a full length of 753 bp, encoding a protein of 250 amino acids with a predicted molecular weight of 28 kDa. The coding sequence (CDS) was cloned into the pGEX-6p-1 vector using a homologous recombination kit and expressed as a glutathione-S-transferase (GST)-fusion protein with a molecular weight of 53 kDa. The tertiary structure of BoVESA1 was predicted using the I-TASSER software. The recombinant protein was subjected to western blotting; the immunogenicity of recombinant BoVESA1 (rBoVESA1) was identified by incubating it with B. orientalis-positive serum. The native BoVESA1 was identified using the lysates of B. orientalis-infected water buffalo erythrocytes incubated with the anti-rBoVESA1 mouse serum. The results showed a band of ~ 28 kDa, which is similar to the predicted size. Immunofluorescence assay (IFA) using anti-rBoVESA1 serum probed indicated a strong signal in the infected RBCs, while the negative control showed no signal. In conclusion, the VESA1 protein was first identified in B. orientalis. This study facilitated further investigation of B. orientalis, and the results indicated that BoVESA1 may serve as a potential candidate antigen for diagnosis and detection of B. orientalis infection.

Characterization of Circulating Extracellular Vesicle Surface Antigens in Patients With Primary Aldosteronism.

[Figure: see text].

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Comparative analysis of antibody responses to outer surface protein (Osp)A and OspC in dogs vaccinated with Lyme disease vaccines.

Lyme disease (LD), the most common tick-borne disease of canines and humans in N. America, is caused by the spirochete Borreliella burgdorferi. Subunit and bacterin vaccines are available for the prevention of LD in dogs. LD bacterin vaccines, which are comprised of cell lysates of two strains of B. burgdorferi, contain over 1000 different proteins and cellular constituents. In contrast, subunit vaccines are defined in composition and consist of either outer surface protein (Osp)A or OspA and an OspC chimeritope. In this study, we comparatively assessed antibody responses to OspA and OspC induced by vaccination with all canine bacterin and subunit LD vaccines that are commercially available in North America. Dogs were administered a two-dose series of the vaccine to which they were assigned (3 weeks apart): Subunit-AC, Subunit-A, Bacterin-1, and Bacterin-2. Antibody titers to OspA and OspC were determined by ELISA and the ability of each vaccine to elicit antibodies that recognize diverse OspC proteins (referred to as OspC types) assessed by immunoblot. While all of the vaccines elicited similar OspA antibody responses, only Subunit-AC triggered a robust and broadly cross-reactive antibody response to divergent OspC proteins. The data presented within provide new information regarding vaccination-induced antibody responses to key tick and mammalian phase antigens by both subunit and bacterin LD canine vaccine formulations.

Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release.

BACKGROUND: Therapeutic options currently available for metastatic castration-resistant prostate cancer (mCRPC) do not extend median overall survival >6 months. Therefore, the development of novel and effective therapies for mCRPC represents an urgent medical need. T cell engagers (TCEs) have emerged as a promising approach for the treatment of mCRPC due to their targeted mechanism of action. However, challenges remain in the clinic due to the limited efficacy of TCEs observed thus far in solid tumors as well as the toxicities associated with cytokine release syndrome (CRS) due to the usage of high-affinity anti-CD3 moieties such as OKT3. METHODS: Using genetically engineered transgenic rats (UniRat and OmniFlic) that express fully human IgG antibodies together with an NGS-based antibody discovery pipeline, we developed TNB-585, an anti-CD3xPSMA TCE for the treatment of mCRPC. TNB-585 pairs a tumor-targeting anti-PSMA arm together with a unique, low-affinity anti-CD3 arm in bispecific format. We tested TNB-585 in T cell-redirected cytotoxicity assays against PSMA+ tumor cells in both two-dimensional (2D) cultures and three-dimensional (3D) spheroids as well as against patient-derived prostate tumor cells. Cytokines were measured in culture supernatants to assess the ability of TNB-585 to induce tumor killing with low cytokine release. TNB-585-mediated T cell activation, proliferation, and cytotoxic granule formation were measured to investigate the mechanism of action. Additionally, TNB-585 efficacy was evaluated in vivo against C4-2 tumor-bearing NCG mice. RESULTS: In vitro, TNB-585 induced activation and proliferation of human T cells resulting in the killing of PSMA+ prostate tumor cells in both 2D cultures and 3D spheroids with minimal cytokine release and reduced regulatory T cell activation compared with a positive control antibody that contains the same anti-PSMA arm but a higher affinity anti-CD3 arm (comparable with OKT3). In addition, TNB-585 demonstrated potent efficacy against patient-derived prostate tumors ex vivo and induced immune cell infiltration and dose-dependent tumor regression in vivo. CONCLUSIONS: Our data suggest that TNB-585, with its low-affinity anti-CD3, may be efficacious while inducing a lower incidence and severity of CRS in patients with prostate cancer compared with TCEs that incorporate high-affinity anti-CD3 domains.

Fixing and Binding Antibodies to Suspension Cells in Preparation for Staining.

Staining of suspension cells after fixation, described here, is normally used only to detect cell-surface antigens. Once cells are fixed and permeabilized, the antibodies are added. The antibodies can be labeled directly or they can be detected by using a labeled secondary reagent that will bind specifically to the primary antibody. Detection reagents for cell staining can be labeled with fluorochromes, enzymes, gold, or iodine. Because the cells are in suspension, the washing steps are tedious, and care should be taken not to centrifuge for long durations or at high speeds.

Prostate Cancer Targeted X-Ray Fluorescence Imaging via Gold Nanoparticles Functionalized With Prostate-Specific Membrane Antigen (PSMA).

PURPOSE: The gold nanoparticle (GNP) as a promising theranostic probe has been increasingly studied. The tumor-targeting efficiency of GNPs is crucial to increase the therapeutic ratio. In this study, we developed PSMA-targeted GNPs to enhance GNP uptake in prostate cancer and developed an x-ray fluorescence imaging system to noninvasively monitor and assess GNP delivery. METHODS AND MATERIALS: For targeted therapy of prostate cancer, anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PEGylated GNPs through 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) chemistry. In vivo imaging was implemented using an in-house-developed dual-modality computed tomography (CT) and x-ray fluorescence CT (XFCT) system on mice bearing subcutaneous LNCaP prostate tumors. After intravenous administration of GNPs (15 mg/mL, 200 µL), the x-ray fluorescence signals from the tumor were collected at various time points (5 minutes to approximately 30 hours) for GNP pharmacokinetics analysis. At 24 hours after administration, x-ray fluorescence projection (XRFproj) and XFCT imaging were conducted to evaluate the prostate tumor uptake of active- and passive-targeting GNPs. Inductively coupled plasma mass spectrometry analysis was adopted as a benchmark to verify the quantification accuracy of XRFproj/XFCT imaging. RESULTS: Fluorescence microscopic imaging confirmed the enhanced (approximately 4 times) targeting efficiency of PSMA-targeted GNPs in vitro. The pharmacokinetics analysis showed enhanced tumor uptake/retention of PSMA-targeted GNPs and revealed that the peak tumor accumulation appeared at approximately 24 hours after intravenous administration. Both XRFproj and XFCT imaging presented their accuracy in quantifying GNPs within tumors noninvasively. Moreover, XFCT imaging verified its unique capabilities to simultaneously determine the heterogeneous spatial distribution and the concentration of GNPs within tumors in vivo. CONCLUSIONS: In conjunction with PSMA-targeted GNPs, XRFproj/XFCT would be a highly sensitive tool for targeted imaging of prostate cancer, benefiting the elucidation of mechanisms of GNP-assisted prostate-cancer therapy.