Genomic attributes of prostate cancer across primary and metastatic noncastrate and castrate resistant disease states: a next generation sequencing study of 183 patients.

Primary prostatic adenocarcinoma (pPC) undergoes genomic evolution secondary to therapy-related selection pressures as it transitions to metastatic noncastrate (mNC-PC) and castrate resistant (mCR-PC) disease. Next generation sequencing results were evaluated for pPC (n = 97), locally advanced disease (involving urinary bladder/rectum, n = 12), mNC-PC (n = 21), and mCR-PC (n = 54). We identified enrichment of TP53 alterations in high-grade pPC, TP53/RB1 alterations in HGNE disease, and AR alterations in metastatic and castrate resistant disease. Actionable alterations (MSI-H phenotype and HRR genes) were identified in approximately a fifth of all cases. These results help elucidate the landscape of genomic alterations across the clinical spectrum of prostate cancer.

Preliminary Analysis of a Phase II Trial of Stereotactic Body Radiation Therapy for Prostate Cancer With High-Risk Features After Radical Prostatectomy.

Purpose: There are limited data regarding using stereotactic body radiation therapy (SBRT) in the postprostatectomy setting. Here, we present a preliminary analysis of a prospective phase II trial that aimed to evaluate the safety and efficacy of postprostatectomy SBRT for adjuvant or early salvage therapy. Materials and Methods: Between May 2018 and May 2020, 41 patients fulfilled inclusion criteria and were stratified into 3 groups: group I (adjuvant), prostate-specific antigen (PSA) < 0.2 ng/mL with high-risk features including positive surgical margins, seminal vesicle invasion, or extracapsular extension; group II (salvage), with PSA ≥ 0.2 ng/mL but < 2 ng/mL; or group III (oligometastatic), with PSA ≥ 0.2 ng/mL but < 2 ng/mL and up to 3 sites of nodal or bone metastases. Androgen deprivation therapy was not offered to group I. Androgen deprivation therapy was offered for 6 months for group II and 18 months for group III patients. SBRT dose to the prostate bed was 30 to 32 Gy in 5 fractions. Baseline-adjusted physician reported toxicities (Common Terminology Criteria for Adverse Events), patient reported quality-of-life (Expanded Prostate Index Composite, Patient-Reported Outcome Measurement Information System), and American Urologic Association scores were evaluated for all patients. Results: The median follow-up was 23 months (range, 10-37). SBRT was adjuvant in 8 (20%) patients, salvage in 28 (68%), and salvage with the presence of oligometastases in 5 (12%) patients. Urinary, bowel, and sexual quality of life domains remained high after SBRT. Patients tolerated SBRT with no grade 3 or higher (3+) gastrointestinal or genitourinary toxicities. The baseline adjusted acute and late toxicity grade 2 genitourinary (urinary incontinence) rate was 2.4% (1/41) and 12.2% (5/41). At 2 years, clinical disease control was 95%, and biochemical control was 73%. Among the 2 clinical failures, 1 was a regional node and the other a bone metastasis. Oligometastatic sites were salvaged successfully with SBRT. There were no in-target failures. Conclusions: Postprostatectomy SBRT was very well tolerated in this prospective cohort, with no significant effect on quality of life metrics postirradiation, while providing excellent clinical disease control.

A new drug delivery method of bispecific ligand-directed toxins, which reduces toxicity and promotes efficacy in a model of orthotopic pancreatic cancer.

OBJECTIVE: Biologicals targeting epidermal growth factor (EGF) and interleukin 13 receptors not only react with overexpressed markers on cancer cells but also react with receptors on normal cells. Because we developed novel bispecific ligand-directed toxins synthesized by cloning EGF and interleukin 13 on the same molecule with toxin, our objective was to determine whether we could block normal receptors while still targeting receptors overexpressed on cancer cells, thereby decreasing toxicity while maintaining efficacy. METHODS: A method, toxicity blocking (ToxBloc), was developed in which a bolus intraperitoneal dose of recombinant EGF13 (without toxin) was given to mice approximately 15 to 20 minutes before DTEGF13. Experiments were then performed to determine whether the maximal tolerated dose (MTD) was reduced and whether we were still able to eliminate progression of aggressive human, metastatic, pancreatic cancer induced by orthotopic injection (OT) in nude mice. RESULTS: ToxBloc permitted us to safely exceed the DTEGF13 maximal tolerated dose by 15-fold. This approach permitted repetitive high dosing with the bispecific ligand-directed toxin resulting in tumor regression (P < 0.01). Tumor effects were documented using a tumor imaging model in which OT tumor growth was monitored noninvasively in real time. ToxBloc was selective because other bispecific peptides did not block. CONCLUSIONS: ToxBloc represents a new method of drug delivery and a potential solution to the problem of toxicity.

A novel reduced immunogenicity bispecific targeted toxin simultaneously recognizing human epidermal growth factor and interleukin-4 receptors in a mouse model of metastatic breast carcinoma.

PURPOSE: To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE(38)KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo. EXPERIMENTAL DESIGN: A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE(38)). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231. RESULTS: EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by approximately 90% with no apparent loss in in vitro or in vivo activity. CONCLUSIONS: Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials.

Anti-glioblastoma effect of a recombinant bispecific cytotoxin cotargeting human IL-13 and EGF receptors in a mouse xenograft model.

To improve activity of a recombinant IL-13 cytotoxin (CT) comprised of IL-13 spliced to truncated diphtheria toxin (DT(390)), epidermal growth factor (EGF) was added to the same single chain protein. This new recombinant bispecific CT, called DTEGF13, enhanced the killing potency against the human glioblastoma lines, U87MG (0.015 nM) and U118MG (0.02 nM). A similar enhancement was observed against the lung carcinoma cell line, Calu-3 (0.0018 nM). Enhanced activity could not be explained by an increased number of cytokines available for binding since a combination of monospecific DTEGF and DTIL13 did not cause the same enhanced activity. Enhanced activity was dependent on the presence of both cytokines on the same single chain molecule and killing was receptor specific since target receptor negative leukemia cells were unaffected by the highly selective DTEGF13 and cytotoxicity could be blocked with anti-EGFR and anti-IL-13 antibodies. In a xenograft flank tumor model, intratumoral injection of DTEGF13, but not monospecific DTEGF or DTIL13, significantly inhibited the growth of established U87 tumors in nude mice (P < 0.04). In this model, the human EGF and IL-13 components of DTEGF13 are reactive with mouse EGFR and IL-13R, respectively. These studies show that a new co-targeting agent that simultaneously recognizes EGFR and IL-13R is more effective than its monospecific counterparts and that DTEGF13 has therapeutic advantages for glioblastoma.

A bispecific recombinant cytotoxin (DTEGF13) targeting human interleukin-13 and epidermal growth factor receptors in a mouse xenograft model of prostate cancer.

PURPOSE: Overexpressed cytokine receptors are considered valid targets for new biologicals targeting prostate cancer. However, current reagents are limited in efficacy. Our goal was to determine the advantages of simultaneously targeting two established targets, epidermal growth factor receptor and interleukin-13 (IL-13) receptor, with a new bispecific cytotoxin in which both EGF and IL-13 cytokines were cloned onto the same single-chain molecule with truncated diphtheria toxin (DT(390)). EXPERIMENTAL DESIGN: In vitro experiments measured the potency of bispecific DTEGF13 and compared its activity to its monospecific counterparts, DTEGF and DTIL13. We determined whether the presence of both cytokine ligands on the same molecule was responsible for its superior activity. In vivo, DTEGF13 was given i.t. to athymic nude mice with established PC-3 human prostate cancer tumor xenografts on their flanks. RESULTS: In vitro, DTEGF13 was more potent than the monospecific cytotoxins against human prostate cancer lines. Enhanced activity was related to the presence of both cytokines on the same single-chain molecule and was not attributed to enhanced binding capacity. Killing was receptor specific. Cytotoxicity could be blocked with anti-EGF and anti-IL-13 antibodies. In vivo, DTEGF13, but not monospecific DTEGF or DTIL13, significantly inhibited the growth of established PC-3 tumors in nude mice (P < 0.0001). CONCLUSIONS: These data show for the first time that simultaneous targeting of cytokine receptors with two ligands on the same molecule has pronounced anticancer advantages. In an animal model in which human DTEGF13 is cross-reactive with mouse, DTEGF13 was highly effective in checking aggressive prostate tumor progression and was reasonably tolerated.

Increasing anticarcinoma activity of an anti-erbB2 recombinant immunotoxin by the addition of an anti-EpCAM sFv.

PURPOSE: erbB2, the product of the Her2-neu gene, is a well-established therapeutic target for antibody-based biologicals, but anti-erbB2 antibody-toxin fusion proteins are limited in their activity. The goal of this study was to determine if genetically adding an sFv targeting epithelial cell adhesion molecule (EpCAM) to an anti-Her2 sFv immunotoxin would result in enhanced antitumor activity. EXPERIMENTAL DESIGN: In vitro studies were done in which the new bispecific immunotoxin DTEpCAM23 was compared with monospecific immunotoxins (DTEpCAM and DT23) to quantitate immunotoxin activity. Mixtures of monospecific immunotoxins were tested to determine if they were as effective as the bispecific immunotoxin. Binding and internalization studies were also done. In vivo, bispecific immunotoxins were given i.t. to athymic nude mice bearing HT-29 human colon cancer flank tumors and i.p. to mice with i.p. tumors. RESULTS: DTEpCAM23 bispecific immunotoxins showed far greater activity than monospecific immunotoxin (sometimes over 2,000-fold) against most tumor lines. Bispecific immunotoxin was superior and selective in its activity against different carcinoma cell lines. Bispecific immunotoxin had greater activity than monospecific immunotoxin indicating an advantage of having both sFv on the same single-chain molecule. Binding and internalization studies did not explain the differences between bispecific immunotoxin and monospecific immunotoxin activity. Orientation of the sFvs on the molecule had a significant effect on in vitro and in vivo properties. The bispecific immunotoxins were more effective than the monospecific immunotoxin in the flank tumor mouse model. CONCLUSIONS: The synthesis of bispecific immunotoxin created a new biological agent with superior in vitro and in vivo activity (over monospecific immunotoxin), more broad reactivity, more efficacy against tumors in vivo, and diminished toxic effects in mice.