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.

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.

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.

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.

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.

Target-Specific Magnetic Resonance Imaging of Human Prostate Adenocarcinoma Using NaDyF4-NaGdF4 Core-Shell Nanoparticles.

We illustrate the development of NaDyF4-NaGdF4 core-shell nanoparticles (NPs) for targeting prostate cancer cells using a preclinical 9.4 T magnetic resonance imaging (MRI) of live animals. The NPs composed of paramagnetic Dy3+ and Gd3+ (T2- and T1-contrast agents, respectively) demonstrate proton relaxivities of r1 = 20.2 mM-1 s-1 and r2 = 32.3 mM-1 s-1 at clinical 3 T and r1 = 9.4 mM-1 s-1 and r2 = 144.7 mM-1 s-1 at preclinical 9.4 T. The corresponding relaxivity values per NP are r1 = 19.4 × 105 mMNP-1 s-1 and r2 = 33.0 × 105 mMNP-1 s-1 at 3 T and r1 = 9.0 × 105 mMNP-1 s-1 and r2 = 147.0 × 105 mMNP-1 s-1 at 9.4 T. In vivo active targeting of human prostate tumors grown in nude mice revealed docking of anti-prostate-specific membrane antigen (PSMA) antibody-tagged NPs at tumor sites post-24 h of their intravenous injection. On the other hand, in vivo passive targeting showed preferential accumulation of NPs at tumor sites only within 2 h of their injection, ascribed to the enhanced permeation and retention effect of the tumor. A biodistribution study employing the harvested organs of mice, post-24 h injection of NPs, quantified active targeting as nearly twice as efficient as passive targeting. These outcomes provide potential opportunities for noninvasive diagnosis using NaDyF4-NaGdF4 core-shell NPs for target-specific MRI.

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.

Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs. METHODS: We used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs. RESULTS: Human VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression. CONCLUSIONS: Fully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

The PSMA-targeting Half-life Extended BiTE Therapy AMG 160 has Potent Antitumor Activity in Preclinical Models of Metastatic Castration-resistant Prostate Cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. EXPERIMENTAL DESIGN: We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. RESULTS: AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6-42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68-labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. CONCLUSIONS: The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675.

Fc-Binding Antibody-Recruiting Molecules Targeting Prostate-Specific Membrane Antigen: Defucosylation of Antibody for Efficacy Improvement*.

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity and the need for intravenous delivery. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc-binding antibody-recruiting molecules (Fc-ARMs). Fc-ARMs can theoretically recruit most endogenous antibodies, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) to eliminate cancer cells. Herein, we describe new Fc-ARMs that target prostate cancer (Fc-ARM-Ps). Fc-ARM-Ps recruited antibodies to cancer cells expressing prostate-specific membrane antigen but did so with lower efficiency compared with Fc-ARMs targeting the folate receptor. Upon recruitment by Fc-ARM-P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N-glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc-ARM strategy.

Pasotuxizumab, a BiTE® immune therapy for castration-resistant prostate cancer: Phase I, dose-escalation study findings.

Aim: We report results of a first-in-human study of pasotuxizumab, a PSMA bispecific T-cell engager (BiTE®) immune therapy mediating T-cell killing of tumor cells in patients with advanced castration-resistant prostate cancer. Patients & methods: We assessed once-daily subcutaneous (SC) pasotuxizumab. All SC patients developed antidrug antibodies; therefore, continuous intravenous (cIV) infusion was assessed. Results: A total of 47 patients received pasotuxizumab (SC: n = 31, 0.5-172 µg/d; cIV: n = 16, 5-80 µg/d). The SC maximum tolerated dose was 172.0 µg/d. A sponsor change stopped the cIV cohort early; maximum tolerated dose was not determined. PSA responders occurred (>50% PSA decline: SC, n = 9; cIV, n = 3), including two long-term responders. Conclusion: Data support pasotuxizumab safety in advanced castration-resistant prostate cancer and represent evidence of BiTE monotherapy efficacy in solid tumors. Clinical trial registration: NCT01723475 (ClinicalTrials.gov).

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization.

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen.

The clinical translation of theranostic 177Lu-radiopharmaceuticals based on inhibitors of the prostate-specific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a potential PSMA-immunotheranostic agent by conjugation of a single-chain variable fragment of the IgGD2B antibody (scFvD2B) to DOTA, to obtain a 177Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated 177Lu-scFvD2B was obtained in high yield and stability. In vitro, 177Lu-scFvD2B disclosed a higher binding and internalization in LNCaP (PSMA-positive) compared to PC3 (negative control) human PCa cells. In vivo studies in healthy nude mice revealed that 177Lu-scFvD2B present a favorable biokinetic profile, characterized by a rapid clearance from non-target tissues and minimal liver accumulation, but a slow wash-out from kidneys. Micro-SPECT/CT imaging of mice bearing pulmonary microtumors evidenced a slow uptake by LNCaP tumors, which steadily rose up to a maximum value of 3.6 SUV at 192 h. This high and prolonged tumor uptake suggests that 177Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy.

A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing.

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity.

Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer.

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA+ cells and that accumulate specifically in PSMA+ tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA+ cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

Metal-Labeled Aptamers as Novel Nanoprobes for Imaging Mass Cytometry Analysis.

Imaging mass cytometry (IMC) is an emerging imaging technology that exploits the multiplexed analysis capabilities of the CyTOF mass cytometer to make spatially resolved measurements for tissue sections. In a comprehensive view of tissue composition and marker distribution, recent developments of IMC require highly sensitive, multiplexed assays. Approaching the sensitivity of the IMC technique, we designed a novel type of biocompatible metal-labeled aptamer nanoprobe (MAP), named 167Er-A10-3.2. The small molecular probe was synthesized by conjugating 167Er-polymeric pentetic acid (167Er-DTPA) with an RNA aptamer A10-3.2. For demonstration, 167Er-A10-3.2 was applied for observing protein spatial distribution on prostatic epithelium cell of paraffin embedded Prostatic adenocarcinoma (PaC) tissue sections by IMC technology. The 167Er-A10-3.2 capitalizes on the ability of the aptamer to specifically bind target cancer cells as well as the small size of 167Er-A10-3.2 can accommodate multiple aptamer binding antigen labeled at high density. The detection signal of 167Er-A10-3.2 probe was 3-fold higher than that of PSMA antibody probe for a targeted cell under lower temperature epitope retrieval (37 °C) of PaC tissue. Furthermore, we successfully demonstrated the simultaneously staining ability of aptamer probes in IMC analysis. The successful imaging acquisition using aptamers probes in IMC technology may offer opportunity for the diagnosis of malignancies in the future.

Chimeric cytokine receptor enhancing PSMA-CAR-T cell-mediated prostate cancer regression.

Objective: Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated an unprecedented therapeutic efficacy in hematological malignancies; however, its effectiveness in solid tumors remains elusive. In order to enable CAR-T cells more effective to solid tumors, a inverted chimeric cytokine receptor (ICR) was designed, which is consists of the TGF-ß extracellular domain, IL-7 receptor intracellular domain, and co-expression on CAR-T cells.Materials and Methods: We selected prostate specific membrane antigen (PSMA) as a target for CAR-T cells, constructed corresponding effector cells, and verified the anti-tumor activity of this enhanced PSMA-CAR-T cell by a series of repeated target cell stimulation experiments in vitro and the anti-tumor capabilities by using mice xenograft model in vivo.Results: In vitro experiments showed that co-expression of ICR could significantly enhance sustained anti-tumor capabilities of PSMA-CAR-T cells. Moreover, in vivo experiments also confirmed that the enhanced PSMA-CAR-T cells exhibited significant superior anti-tumor capabilities and could prolong the survival time in the xenograft and PDX models of prostate cancer.Conclusions: PSMA-CAR-T cells co-expressing ICR can be envisaged as a new therapeutic strategy for prostate cancer and support the translation of this enhanced approach in the clinical setting.