treated the first patient in the United States to undergo a radioimmunotherapy procedure for non-Hodgkin lymphoma.His decades-long career paralleled the development of nuclear medicine, from early scintigraphy studies to sophisticated combined molecular imaging and therapy.

Abstract Selective high affinity ligands (SHALs) are novel targeting agents composed of lysine/mini-polyethylene glycol scaffolds linking two or three ‘recognition’ ligands together. The tridentate SHAL SH7139 is selectively cytotoxic at sub-nanomolar concentrations to lymphoma cells over-expressing HLA-DR10. Electron microscopy examinations of Burkitt's lymphoma xenografts from mice treated with SH7139 suggest one mechanism for cell killing may involve cell signaling-induced apoptosis analogous to that observed with Lym-1 antibody. An alternative mechanism is that intracellular hydrolysis of the SHAL's amide bonds following its uptake and metabolism by lymphoma cells could lead to the release of the three recognition ligands and the subsequent inhibition (or promotion) of cellular activities that trigger apoptosis or induce cell death and necrosis. Each of the three recognition ligands (Dv, Ct, and Cb) is independently, but non-selectively, toxic to both Raji (HLA-DR10 positive) and Jurkat (HLA-DR10 negative) cells. The Ct ligand, 3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid, is a structural analog of aryloxyphenoxypropionate herbicides that inhibit acetyl-CoA carboxylase (ACC), a key enzyme in fatty acid biosynthesis that is upregulated in many cancers to meet their need for rapid growth and proliferation. Our studies show that although Ct could be accommodated into the herbicide binding pocket of ACC, this ligand was not found to inhibit human and rat ACC1 or ACC2. However, metabolic cleavage of the single amide bond present in Ct would lead to the production of two smaller metabolites, an aniline derivative and malonate, both of which are known to be biologically active and potentially toxic compounds. The Dv ligand, dabsyl-L-valine, belongs to a family of biphenylsulfonamides, many of which have been shown to inhibit matrix metalloproteinases such as MMP9 and MMP14 that are upregulated in tumor cells. The Cb ligand, 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzene carboxylic acid, is a structural analog of a large number of inhibitors that have been shown to block the assembly of the contractile ring and the initiation of cytokinesis by inhibiting guanosine nucleoside binding to MgcRacGAP, a protein that participates in the activation of the myosin motor and the initiation of cleavage furrow ingression. A combination of SH7139 metabolism studies, computer modeling, and enzyme inhibition assays have been performed to determine if Ct, Dv and Cb and their metabolites are produced and whether they contribute to lymphoma cell killing. Citation Format: Monique C. Balhorn, Gary Mirick, Dong Cheng, Zhengping Ma, Edmond Y. Lau, Saphon Hok, Gerald L. DeNardo, Rod Balhorn. Intracellular uptake and metabolism of SH7139: Is it a targeted prodrug for B-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2014-5460

Abstract SH7139, our most advanced small molecule therapeutic for non-Hodgkin's lymphoma, was designed to bind to a unique site on HLA-DR10, a cell surface receptor found on B-cell lymphocytes and over-expressed on B-cell derived malignancies. Studies conducted with lymphoma and other cell lines have shown that SH7139 binds selectively and is highly cytotoxic only to tumor cells expressing HLA-DR10. A previous study also showed SH7139 to be highly effective in treating human Burkitt's lymphoma (Raji) in a mouse xenograft model. We have used this same mouse model to compare the efficacy of SH7139 delivered by different routes and using different treatment regimens. The results show that the greatest survival times were achieved with mice given SH7139 by i.p. injection on days 0, 7 and 14. Doses delivered i.p. on days 0, 2 and 4 and orally on days 0,7,14,21,28 and 35 were slightly less effective, and a single dose delivered i.p. on day 0 and three doses delivered orally on days 0, 7, and 14 were the least effective. The concentration of SH7139 that achieved these effects in vivo, which was determined by measuring xenograft uptake of 111-In-labeled SH7139, was found to be in the sub-nanomolar range. In addition to identifying the best route and dosing regimen for future studies and confirming the drug works in vivo at concentrations similar to those found to be effective in cells cultured in vitro, these studies also show the drug can be effective when administered orally. Citation Format: Rodney Balhorn, Gary Mirick, Gerald L. DeNardo, Laurel Beckett, Judy Li, Saphon Hok, Monique Balhorn. Effect of route and dosing regimen on efficacy of SH7139 in mouse Burkitt's lymphoma xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2014-2703

There is a need for effective "broad spectrum" therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of (188)Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi (188)Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of (188)Re-6D2 was escalated to 54 mCi. SPECT/CT revealed (188)Re-6D2 uptake in melanoma metastases. The mean effective half-life of (188)Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that (188)Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

Hadassah Medical Center, Hebrew University, Kiryat Hadassah, 91120 Jerusalem, Israel 2 Chaim Sheba Medical Center, Tel Hashomer, 52621 Ramat Gan, Israel 3 Sackler Faculty of Medicine, Tel Aviv Medical Center, 69978 Tel Aviv, Israel 4 Rambam Health Care Campus, 31096 Haifa, Israel 5 Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 6 Pain erapeutics, Inc., Austin, TX 78731, USA Davis Medical Center, University of California, Sacramento, CA 95817, USA

Although the term has been coined recently, the concepts underlying theranosis have been applied in patient care for more than one-half century. However, advanced technologies are used now. Theranosis describes processes used to tailor therapy for a patient. It is the use of diagnostic tests to identify those patients better-suited for a drug (or drugs) or to determine how well a drug is working. 131I-iodide for imaging and for therapy of hyperthyroidism and thyroid cancer is an excellent example of personalized theranosis and has withstood challenge for more than 50 years. Radioimmunotherapy for non-Hodgkin lymphoma is a more recent example of theranosis. Either of 2 anti-CD20 monoclonal antibodies, one labeled with indium for imaging or 90Y for radiotherapy or a second labeled with 131I for both imaging and radiotherapy, is used for salvage and first-line therapy of multifocal non-Hodgkin lymphoma. The efficacy of these drugs is greater than that of alternative therapies. To mimic the molecular specificity and cell selectivity of a monoclonal antibody, smaller molecules that also bind to proteins upregulated by malignant cells can be used to transport cytotoxic agents to the malignant cells. Smaller carrier molecules like peptides, aptamers, affibodies, and selective, high-affinity ligands facilitate intensification of therapy because of their size. Personalized genomics, proteomics, and molecular imaging are among technologies currently used for theranosis. Molecular emission tomographic imaging with radiolabeled drugs has been used to examine the pharmacology of anticancer therapies and their effectiveness. Increased glycolysis, a molecular phenotype of many malignancies, can be imaged using 18F-fluoro-2-deoxyglucose (FDG). Tomographic imaging using FDG allows stratification of patients into those responding and likely to respond to the therapy and those better treated in another manner. Prediction of therapeutic response avoids useless therapy so that FDG imaging is included in official response evaluation criteria. Although a fixed approach to therapy may be more practical, an individualized approach is more likely to ensure that each patient receives an effective drug and drug dose that has acceptable and definable tissue effects. Drugs that work in one individual may be ineffective or cause adverse events in others.

Cancer Biotherapy & RadiopharmaceuticalsVol. 25, No. 5 EditorialPretherapy Prediction of Nephrotoxicity After Peptide Radionuclide Receptor Therapy (PRRT)Gerald L. DeNardo and Daniel J. MaceyGerald L. DeNardoProfessor Emeritus, Internal Medicine, Radiology and Pathology University of California at Davis, Sacramento, California.Search for more papers by this author and Daniel J. MaceyConsultant medical physicist, Birmingham, AL.Search for more papers by this authorPublished Online:15 Oct 2010https://doi.org/10.1089/cbr.2010.0875AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 25Issue 5Oct 2010 InformationCopyright 2010, Mary Ann Liebert, Inc.To cite this article:Gerald L. DeNardo and Daniel J. Macey.Pretherapy Prediction of Nephrotoxicity After Peptide Radionuclide Receptor Therapy (PRRT).Cancer Biotherapy and Radiopharmaceuticals.Oct 2010.507-509.http://doi.org/10.1089/cbr.2010.0875Published in Volume: 25 Issue 5: October 15, 2010Online Ahead of Print:September 19, 2010PDF download

Although most patients with locoregional cancer are cured by surgery, radiotherapy, chemotherapy, and combinations thereof, those with distant metastases are not despite systemic chemotherapy. These patients respond to local radiotherapy but generally need systemic therapy. Non-Hodgkin's lymphoma (NHL) provides a paradigm for the role of molecular targeted radiotherapy (MTRT) because these patients have multifocal disease in most cases. Although patients with NHL achieve remissions after multiple cycles of chemotherapy, less than one half of those with aggressive NHL are cured and almost none of those with low grade NHL. Furthermore, NHL, like other cancers, becomes chemoresistant, yet remains responsive to radiotherapy. MTRT, radiation targeted by molecules, is a good strategy for the treatment of multifocal and radiosensitive cancers. Radioimmunotherapy (RIT) is an MTRT approach using MAbs, or parts thereof, to target the radionuclide that delivers radiation. Two anti-CD20 monoclonal antibodies (MAbs), one labeled with 111In for imaging or 90Y for therapy and a second labeled with 131I for imaging and therapy, have proven effective and safe for MTRT for NHL patients. The importance of the radiation is demonstrated in the data from the randomized pivotal trial of 90Y-ibritumomab; response rates were distinctly better in the 90Y-ibritumomab arm than in the rituximab arm. Furthermore, the efficacy of 131I-tositumomab was greater than that of the same MAb alone in another pivotal trial. Although hematologic toxicity is dose limiting for MTRT, febrile neutropenia is uncommon. MTRT is also not associated with mucositis, hair loss, or persistent nausea or vomiting, unlike chemotherapy. Randomized trials of MTRT in different strategies have not been conducted, but there is evidence of better outcomes, particularly for strategies that provide dose intensification, such as pretargeted MTRT, multiple dosing ("fractionation"), and MTRT with stem cell transplantation (SCT). Pretargeted RIT separates delivery of the targeting molecule from radionuclide delivery, provides dose escalation, and is more effective than direct one-step RIT, although more complicated to implement. Improved drugs and strategies for MTRT have documented potential for better patient outcomes. Smaller radionuclide carriers, such as those used for pretargeted MTRT, should be incorporated into the management of patients with NHL and other cancers soon after the patients have proven incurable. Expected improvements using better drugs, strategies, and combinations with other drugs seem likely to make MTRT integral in the management of many patients with cancer and likely to lead to cures of NHL.

Like rituximab, monoclonal antibodies reactive with human leukocyte antigen have potent antilymphoma activity. However, size limits their vascular and tissue penetration. To mimic monoclonal antibody binding, nanomolecules have been synthesized, shown specific for the beta subunit of HLA-DR10, and selective for cells expressing this protein. Selective high affinity ligands (SHALs) containing the 3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid (Ct) ligand residualized and had antilymphoma activity against expressing cells. Herein, we show the extraordinary potency in mice with human lymphoma xenografts of a tridentate SHAL containing this ligand. After titrating antilymphoma activity in cell culture, a randomized preclinical study of a tridentate SHAL containing the Ct ligand was conducted in mice with established and aggressive human lymphoma xenografts. Mice having HLA-DR10 expressing Raji B- or Jurkat's T-lymphoma xenografts were randomly assigned to receive either treatment with SHAL at a dose of 100 ng i.p. weekly for 3 consecutive weeks, or to be untreated. Primary end-points were cure, overall response rates and survival. Toxicity was also evaluated in these mice, and a USFDA general safety study was conducted in healthy Balb/c mice. In Raji cell culture, the threshold and IC50 concentrations for cytotoxic activity were 0.7 and 2.5 nmol (pm/ml media), respectively. When compared to treated Jurkat's xenografts or untreated xenografts, Raji xenografts treated with the SHAL showed an 85% reduction in hazard of death (P=0.014; 95% confidence interval 32-95% reduction). There was no evidence for toxicity even after i.p. doses 2000 times greater than the treatment dose associated with cure of a majority of the mice with Raji xenografts. When compared with control groups, treatment selectively improved response rates and survival in mice with HLA-DR10 expressing human lymphoma xenografts at doses not associated with adverse events and readily achievable in patients.

BACKGROUND:A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma.RESULTS:An analog of the SHAL (DvLPBaPPP)2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus.CONCLUSION:The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.

The promise of hyperthermia has yet to be realized, but the fundamental idea and the effects of heat on (cancer) cells are well known. Cell death from exposure to heat is a function of both the intensity of the heat and the length of the exposure. Cells die by necrosis and by apoptosis. Sublethal heat doses sensitize cancer cells to radiation and drugs. Because of advances in chemistry and physics, harnessing the power of heat to kill cancer cells seems achievable now! Using novel systems embodied in the combination of molecular-targeted nanoparticles and hysteretic heating of the nanoparticles with "focused" alternating magnetic frequencies (AMFs), heat delivery can be better controlled. Importantly, hyperthermia does not damage, and may actually enhance, the immune system. Trials in patients are needed to settle the clinical role of new thermal treatment.

UNLABELLEDNanoparticle thermotherapy (NPTT) uses monoclonal antibody-linked iron oxide magnetic nanoparticles (bioprobes) for the tumor-specific thermotherapy of cancer by hysteretic heating of the magnetic component of the probes through an externally applied alternating magnetic field (AMF). The present study investigated the effect of NPTT on a human prostate cancer cell line, DU145. The concept of total heat dose (THD) as a measure for NPTT was validated on a cellular level and THD was correlated to cell death in vitro. The study, furthermore, explored the potential enhancement of the NPTT effect through added external beam radiation therapy (EBRT), because both forms of treatment have a different, and potentially complementary, mechanism of causing cell death.METHODSUsing carbodiimide, (111)In-DOTA-ChL6 was conjugated to dextran iron oxide 20-nm particles with polyethylene glycol COOH groups on the surface and purified as (111)In-bioprobes. NPTT and EBRT were applied alone and combined to cells labeled with the bioprobes. Cell response was monitored by measuring lactate dehydrogenase (LDH), a product of cytolysis, in the medium. This distinct focus on the response to NPTT was possible, since we found in previous studies that the LDH assay was relatively insensitive to the response of cells (without bioprobes) to EBRT in the dose levels given here.RESULTSNPTT showed a significantly increased cell death at a total calculated heat dose of 14.51 and 29.02 J/g cells (50% and 100% AMF duty, 350 Oe, 136 kHz, 12 cycles, 20 minutes total), compared with AMF exposure in the absence of bioprobes. Adding EBRT to NPTT did not increase cell death, as measured by LDH. However, EBRT given to cells labeled with bioprobes caused significant cell death at radiation doses of 10 Gy and higher.CONCLUSIONSIn human prostate cancer cell cultures, NPTT applied as a single modality caused cell death that correlated with THD estimation; complete cell death occurred at 14.51 J/g cells. Consequently, enhancement of the NPTT effect through the addition of EBRT could not be addressed. Interestingly, EBRT induced cell death on bioprobe-labeled cells at EBRT levels that did not show cell death in the absence of bioprobes; this phenomenon is worth investigating further.

BACKGROUND:Noninvasive, focused hyperthermia can be achieved by using an externally applied alternating magnetic field (AMF) if effective concentrations of nanoparticles can be delivered to the target cancer cells. Targeting agents, for example, monoclonal antibodies or peptides, linked to magnetic iron oxide nanoparticles (NP), represent a promising strategy to target cancer cells and hyperthermia.METHODS:We have developed a new radioconjugate NP ((111)In-DOTA-di-scFv-NP), using recombinantly generated antibody fragments, di-scFv-c, for the imaging and therapy of anti-MUC-1-expressing cancers, because aberrant MUC-1 is abundantly expressed on the majority of human epithelial cancers. Anti-MUC-1 di-scFv-c (50 kDa) were engineered, generated, and selected to link maleimide functionalized nanoparticles (NP-M). DOTA chelate was conjugated with di-scFv-c for radionuclide chelation to trace the radioimmunonanoparticles (RINPs) in vivo.RESULTS:Heat-inducing NP-M were prepared with maleimide density >15 per particle for site-specific thiolation. The specific activity of the RINP was 4-5 microCi (111)In/mg with >10 molecules of di-scFv per NP. We characterized the RINP by polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, size-exclusion chromatography, and tumor-cell binding. RINP had a >90% di-scFv conjugated to NP and an immunoreactivity >80% relative to unmodified di-scFv-c on HBT 3477 and DU145 tumor cells. Pharmacokinetics and whole-body autoradiography studies demonstrated that a 5% injected dose was targeted in tumor after 24 hours.CONCLUSIONS:Further development of this new preparation of RINP may provide uniquely high tumor-targeting NP for AMF-driven tumor hyperthermia with less spleen and kidney accumulation.

1388 Objectives: Effective methods of treatment of currently deadly metastatic melanoma are urgently needed. Previously we established the feasibility of targeting melanin in experimental human melanoma with 188-Rhenium (188Re)-labeled 6D2 mAb resulting in tumor regression. We carried out additional pre-clinical development of 188Re-6D2 for use in Phase I clinical trial in patients with metastatic melanoma. Methods: The developmental work included chemistry, pharmacokinetics, efficacy, and acute hematologic toxicity studies in an experimental human melanoma. Results: Tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP) was tested as potential agent for generation of –SH groups on 6D2 mAb for subsequent radiolabeling with 188Re. TCEP turned out to be both effective in generating a sufficient number of -SH groups on 6D2 mAb to ensure high radiolabeling yields and facile in preserving mAbs structural integrity. 188Re-6D2 cleared from the blood with the half-life of approximately 5 hrs and from the body – with the half-life of 10 hr. Therapy of A2058 melanoma tumor-bearing mice showed that while the lowest dose of 0.15 mCi per mouse had no effect on the tumor progression relative to untreated mice - doses of 0.5, 1.0 and 1.5 mCi significantly (P Conclusions: 188Re-6D2 was immunoreactive and stable over time, cleared fast from the blood and major organs and was therapeutic in experimental human melanoma. Research Support: Pain Therapeutics, Inc. and AECOM Cancer Center.