Imaging granzyme biochemistry in CAR T cell immunotherapy with restricted interaction peptides and PET

Purpose: Recent clinical successes in the use of chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer therapy. However, only 20% to 30% of patients achieve long term survival benefits, and distinguishing responders from non-responders early remains a challenge with conventional imaging techniques. Thus, there is an urgent need to develop new biomarkers to distinguish responsive and resistant patients, both to improve standard of care and to assess the antitumor activity of experimental immunotherapies. This study aims to determine the efficacy of a novel granzyme activated imaging probe to image treatment response to CAR T cell therapy in a mouse model.Methods: Immunodeficient mice were obtained and inoculated subcutaneously with Raji tumors. CD8+ T cells were obtained from donor blood and transduced to express anti-CD19 receptors. CAR T cells were then expanded in vitro with IL-2. After tumors were palpable, mice were treated with empty or anti-CD19 CAR T cells intravenously. Mice were then injected with 64Cu-GRIP B at 24 hours post CAR T cell administration. PET/CT studies were performed on a dedicated Inveon small animal scanner. Post mortem radiotracer uptake was quantified as %injected dose/g (%ID/g) values for tumor and normal tissue. Tumors from one representative animal per group was prepared for autoradiography. Results: ROI analysis of static PET/CT images indicated that 64Cu-GRIP B uptake in treated tumors rose from 0.5 to 2 hours post injection. Tumoral uptake of the probe was higher in anti-CD19 CAR T versus vehicle treated arm at 2 hours post injection. The mean tumoral standard uptake values for anti-CD19 CAR T and vehicle arms were 1.5%ID/cc and 0.4%ID/cc, respectively. Biodistribution data demonstrated similar uptake of the 64Cu-GRIP B probe between treatment arms as mean %ID/g for both arms was roughly 0.75. Digital autoradiography suggested substantially higher and localized uptake of the probe in the treatment arm compared to the vehicle arm in several mice. Conclusions: Using 64Cu-GRIP B, a peptide-based chemosensor whose biodistribution was engineered to be controlled by the proteolytic activity of secreted GZMB allows for imaging granzyme B mobilization by cytotoxic T cells in CAR T cell therapy. Further studies with larger samples sizes and standardized batches of CAR T cells will provide more conclusive results

Imaging granzyme biochemistry in CAR T cell immunotherapy with restricted interaction peptides and PET

Purpose: Recent clinical successes in the use of chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer therapy. However, only 20% to 30% of patients achieve long term survival benefits, and distinguishing responders from non-responders early remains a challenge with conventional imaging techniques. Thus, there is an urgent need to develop new biomarkers to distinguish responsive and resistant patients, both to improve standard of care and to assess the antitumor activity of experimental immunotherapies. This study aims to determine the efficacy of a novel granzyme activated imaging probe to image treatment response to CAR T cell therapy in a mouse model.Methods: Immunodeficient mice were obtained and inoculated subcutaneously with Raji tumors. CD8+ T cells were obtained from donor blood and transduced to express anti-CD19 receptors. CAR T cells were then expanded in vitro with IL-2. After tumors were palpable, mice were treated with empty or anti-CD19 CAR T cells intravenously. Mice were then injected with 64Cu-GRIP B at 24 hours post CAR T cell administration. PET/CT studies were performed on a dedicated Inveon small animal scanner. Post mortem radiotracer uptake was quantified as %injected dose/g (%ID/g) values for tumor and normal tissue. Tumors from one representative animal per group was prepared for autoradiography. Results: ROI analysis of static PET/CT images indicated that 64Cu-GRIP B uptake in treated tumors rose from 0.5 to 2 hours post injection. Tumoral uptake of the probe was higher in anti-CD19 CAR T versus vehicle treated arm at 2 hours post injection. The mean tumoral standard uptake values for anti-CD19 CAR T and vehicle arms were 1.5%ID/cc and 0.4%ID/cc, respectively. Biodistribution data demonstrated similar uptake of the 64Cu-GRIP B probe between treatment arms as mean %ID/g for both arms was roughly 0.75. Digital autoradiography suggested substantially higher and localized uptake of the probe in the treatment arm compared to the vehicle arm in several mice. Conclusions: Using 64Cu-GRIP B, a peptide-based chemosensor whose biodistribution was engineered to be controlled by the proteolytic activity of secreted GZMB allows for imaging granzyme B mobilization by cytotoxic T cells in CAR T cell therapy. Further studies with larger samples sizes and standardized batches of CAR T cells will provide more conclusive results

Elevated labile iron in castration-resistant prostate cancer is targetable with ferrous iron-activatable antiandrogen therapy.

Clinical responses to second generation androgen signaling inhibitors (e.g., enzalutamide) in metastatic castration-resistant prostate cancer (mCRPC) are variable and transient, and are associated with dose limiting toxicities, including rare but severe CNS effects. We hypothesized that changes to iron metabolism coincident with more advanced disease might be leveraged for tumor-selective delivery of antiandrogen therapy. Using the recently described chemical probes SiRhoNox and 18F-TRX in mCRPC models, we found elevated Fe2+ to be a common feature of mCRPC in vitro and in vivo. We next synthesized ferrous-iron activatable drug conjugates of second and third-generation antiandrogens and found these conjugates possessed comparable or enhanced antiproliferative activity across mCRPC cell line models. Mouse pharmacokinetic studies showed that these prototype antiandrogen conjugates are stable in vivo and limited exposure to conjugate or free antiandrogen in the brain. Our results reveal elevated Fe2+ to be a feature of mCRPC that might be leveraged to improve the tolerability and efficacy of antiandrogen therapy

Theranostic Targeting of CUB Domain-Containing Protein 1 (CDCP1) in Multiple Subtypes of Bladder Cancer.

PURPOSE: Despite recent approvals for checkpoint inhibitors and antibody-drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain-containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies. EXPERIMENTAL DESIGN: CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376). RESULTS: CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively. CONCLUSIONS: These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy

Imaging the Granzyme Mediated Host Immune Response to Viral and Bacterial Pathogens In Vivo Using Positron Emission Tomography

Understanding how the host immune system engages complex pathogens is essential to developing therapeutic strategies to overcome their virulence. While granzymes are well understood to trigger apoptosis in infected host cells or bacteria, less is known about how the immune system mobilizes individual granzyme species in vivo to combat diverse pathogens. Toward the goal of studying individual granzyme function directly in vivo, we previously developed a new class of radiopharmaceuticals termed "restricted interaction peptides (RIPs)" that detect biochemically active endoproteases using positron emission tomography (PET). In this study, we showed that secreted granzyme B proteolysis in response to diverse viral and bacterial pathogens could be imaged with [64Cu]Cu-GRIP B, a RIP that specifically targets granzyme B. Wild-type or germline granzyme B knockout mice were instilled intranasally with the A/PR/8/34 H1N1 influenza A strain to generate pneumonia, and granzyme B production within the lungs was measured using [64Cu]Cu-GRIP B PET/CT. Murine myositis models of acute bacterial (E. coli, P. aeruginosa, K. pneumoniae, and L. monocytogenes) infection were also developed and imaged using [64Cu]Cu-GRIP B. In all cases, the mice were studied in vivo using mPET/CT and ex vivo via tissue-harvesting, gamma counting, and immunohistochemistry. [64Cu]Cu-GRIP B uptake was significantly higher in the lungs of wild-type mice that received A/PR/8/34 H1N1 influenza A strain compared to mice that received sham or granzyme B knockout mice that received either treatment. In wild-type mice, [64Cu]Cu-GRIP B uptake was significantly higher in the infected triceps muscle versus normal muscle and the contralateral triceps inoculated with heat killed bacteria. In granzyme B knockout mice, [64Cu]Cu-GRIP B uptake above the background was not observed in the infected triceps muscle. Interestingly, live L. monocytogenes did not induce detectable granzyme B on PET, despite prior in vitro data, suggesting a role for granzyme B in suppressing their pathogenicity. In summary, these data show that the granzyme response elicited by diverse human pathogens can be imaged using PET. These results and data generated via additional RIPs specific for other granzyme proteases will allow for a deeper mechanistic study analysis of their complex in vivo biology.</p

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [18F] FDG Yield Potent Sensors of Living Bacteria In Vivo

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with β-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (β-1,3), and cellobiose (β-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications

Chemoenzymatic syntheses of fluorine-18-labeled disaccharides from [18F] FDG yield potent sensors of living bacteria in vivo

Chemoenzymatic techniqueshave been applied extensively to pharmaceuticaldevelopment, most effectively when routine synthetic methods fail.The regioselective and stereoselective construction of structurallycomplex glycans is an elegant application of this approach that isseldom applied to positron emission tomography (PET) tracers. We soughta method to dimerize 2-deoxy-[F-18]-fluoro-d-glucose([F-18]FDG), the most common tracer used in clinical imaging,to form [F-18]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation.When [F-18]FDG was reacted with & beta;-d-glucose-1-phosphatein the presence of maltose phosphorylase, the & alpha;-1,4- and & alpha;-1,3-linkedproducts 2-deoxy-[F-18]-fluoro-maltose ([F-18]FDM)and 2-deoxy-2-[F-18]-fluoro-sakebiose ([F-18]FSK)were obtained. This method was further extended with the use of trehalose(& alpha;,& alpha;-1,1), laminaribiose (& beta;-1,3), and cellobiose(& beta;-1,4) phosphorylases to synthesize 2-deoxy-2-[F-18]fluoro-trehalose ([F-18]FDT), 2-deoxy-2-[F-18]fluoro-laminaribiose ([F-18]FDL), and 2-deoxy-2-[F-18]fluoro-cellobiose ([F-18]FDC). We subsequentlytested [F-18]FDM and [F-18]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [F-18]FDM and [F-18]FSK were stable inhuman serum with high accumulation in preclinical infection models.The synthetic ease and high sensitivity of [F-18]FDM and[F-18]FSK to S. aureus including methicillin-resistant(MRSA) strains strongly justify clinical translation of these tracersto infected patients. Furthermore, this work suggests that chemoenzymaticradiosyntheses of complex [F-18]FDG-derived oligomers willafford a wide array of PET radiotracers for infectious and oncologicapplications

CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease.

PurposeWith the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets.Experimental designCDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post-enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice.ResultsCDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4-2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4-2B xenografts.ConclusionsThe data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC