Search

SH7139, the first of a series of selective high affinity ligand (SHAL) oncology drug candidates designed to target and bind to the HLA-DR proteins overexpressed by B-cell lymphomas, has demonstrated exceptional efficacy in the treatment of Burkitt lymphoma xenografts in mice and a safety profile that may prove to be unprecedented for an oncology drug. The aim of this study was to determine how frequently the HLA-DRs targeted by SH7139 are expressed by different subtypes of non-Hodgkin's lymphoma and by other solid cancers that have been reported to express HLA-DR. Binding studies conducted with SH7129, a biotinylated analog of SH7139, reveal that more than half of the biopsy sections obtained from patients with different types of non-Hodgkin's lymphoma express the HLA-DRs targeted by SH7139. Similar analyses of tumor biopsy tissue obtained from patients diagnosed with eighteen other solid cancers show the majority of these tumors also express the HLA-DRs targeted by SH7139. Cervical, ovarian, colorectal and prostate cancers expressed the most HLA-DR. Only a few esophageal and head and neck tumors bound the diagnostic. Within an individual's tumor, cell to cell differences in HLA-DR target expression varied by only 2 to 3-fold while the expression levels in tumors obtained from different patients varied as much as 10 to 100-fold. The high frequency with which SH7129 was observed to bind to these cancers suggests that many patients diagnosed with B-cell lymphomas, myelomas, and other non-hematological cancers should be considered potential candidates for new therapies such as SH7139 that target HLA-DR-expressing tumors.

Non-Hodgkin lymphoma is one of the most common types of cancer. Relapsed and refractory diseases are still common and remain significant challenges as the majority of these patients eventually succumb to the disease. Herein, we report a translatable concurrent chemo-immuno-radiotherapy (CIRT) strategy that utilizes fully synthetic antibody mimic Selective High-Affinity Ligand (SHAL)-functionalized doxorubicin-encapsulated nanoparticles (Dox NPs) for the treatment of human leukocyte antigen-D related (HLA-DR) antigen-overexpressed tumors. We demonstrated that our tailor-made antibody mimic-functionalized NPs bound selectively to different HLA-DR-overexpressed human lymphoma cells, cross-linked the cell surface HLA-DR, and triggered the internalization of NPs. In addition to the direct cytotoxic effect by Dox, the internalized NPs then released the encapsulated Dox and upregulated the HLA-DR expression of the surviving cells, which further augmented immunogenic cell death (ICD). The released Dox not only promotes ICD but also sensitizes the cancer cells to irradiation by inducing cell cycle arrest and preventing the repair of DNA damage. In vivo biodistribution and toxicity studies confirm that the targeted NPs enhanced tumor uptake and reduced systemic toxicities of Dox. Our comprehensive in vivo anticancer efficacy studies using lymphoma xenograft tumor models show that the antibody-mimic functional NPs effectively inhibit tumor growth and sensitize the cancer cells for concurrent CIRT treatment without incurring significant side effects. With an appropriate treatment schedule, the SHAL-functionalized Dox NPs enhanced the cell killing efficiency of radiotherapy by more than 100% and eradicated more than 80% of the lymphoma tumors., Antibody mimic Selective High-Affinity Ligand-functionalized doxorubicin-encapsulated nanoparticles have been engineered for concurrent chemo-immuno-radiotherapy of hematological cancer.

It is shown that the averaged chemical shift (ACS) of a particular nucleus in the protein backbone empirically correlates well to its secondary structure content (SSC). Chemical shift values of more than 200 proteins obtained from the Biological Magnetic Resonance Bank are used to calculate ACS values, and the SSC is estimated from the corresponding three-dimensional coordinates obtained from the Protein Data Bank. ACS values of (1)H(alpha) show the highest correlation to helical and sheet structure content (correlation coefficient of 0.80 and 0.75, respectively); (1)H(N) exhibits less reliability (0.65 for both sheet and helix), whereas such correlations are poor for the heteronuclei. SSC estimated using this correlation shows a good agreement with the conventional chemical shift index-based approach for a set of proteins that only have chemical shift information but no NMR or x-ray determined three-dimensional structure. These results suggest that even chemical shifts averaged over the entire protein retain significant information about the secondary structure. Thus, the correlation between ACS and SSC can be used to estimate secondary structure content and to monitor large-scale secondary structural changes in protein, as in folding studies.

SH7139, the first of a new class of cancer therapeutics developed for treating non-Hodgkin’s lymphoma, is unusual in that both targeting and pro-drug functionalities have been incorporated into the same small molecule. Functioning similar to an antibody-drug conjugate, SH7139 targets a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope within the β-subunit are reported to be over-expressed in approximately 85% of B-cell lymphomas. Upon binding to HLA-DR molecules located on the tumor cell’s surface, SH7139 is transported into the cytoplasm where it is concentrated and subsequently metabolized. A series of metabolic products derived from the SHAL’s recognition elements (three small molecules that are linked together to create SH7139 and to provide targeting selectivity) are generated as the drug is broken down, each of which inhibits one or more activities required for tumor cell survival. Preclinical studies with SH739 have demonstrated remarkable efficacy in treating B-cell lymphoma xenografts in mice, providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.41 μg/kg. Biopsy tissue binding studies conducted with SH7129, a biotinylated form of SH7139, and streptavidin-horse radish peroxidase detection have shown the drug binds to a significant fraction of tumors obtained from patients diagnosed with multiple myeloma and each of the B-cell lymphoma subtypes tested to date (DLBC, Burkitt’s, Mantle Cell, Follicular, MALT, and CLL). SH7129 was also observed to bind to tumor biopsies obtained from of a number of patients diagnosed with peripheral T-cell and nodular sclerosis Hodgkin’s lymphomas, a result consistent with observations reported by others that HLA-DRs are expressed in a subset of these lymphomas. HLA-DR expression has also been reported to occur in or be linked to a number of other types of cancer, including melanoma, cervical, ovarian, pancreatic and lung cancers. SH7129 staining of tumor microarrays have shown biopsy cores from a subset of patients diagnosed with each of these cancers also bind SH7139. While in vivo efficacy has only been tested in Burkitt’s (Raji), Mantle cell (Granta-519), and T-cell (Jurkat, a cell line control lacking HLA-DR and showing no efficacy) lymphoma xenografts, these tissue binding results suggest that in addition to the majority of the NHL subtypes, nodular sclerosis Hodgkin’s lymphoma, multiple myelomas, as well as a subset of melanomas, ovarian, cervical, pancreatic, and lung cancers may also respond to SH7139 therapy. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Monique Cosman Balhorn, Rod Balhorn. Therapeutic applications of the selective high affinity ligand SH7139 may extend beyond NHL to many other types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2017-1171

Selective High Affinity Ligands (SHALs) are small molecules that can be designed to bind selectively and with high affinity to any protein target by linking together several ligands that recognize and bind to different sites on the protein’s surface. SH7139, our first SHAL therapeutic for treating advanced non-Hodgkin’s lymphoma, has shown remarkable efficacy in B-cell lymphoma xenograft models by providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.4 μg/kg. Functioning similar to an antibody-drug conjugate and a pro-drug, SH7139 targets and binds to a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope are expressed in approximately 85% of B-cell lymphomas. Upon binding to its HLA-DR target, SH7139 is carried into the cytoplasm where it is concentrated and subsequently metabolized, releasing toxic metabolites (derived from each of the linked ligands) that inhibit a series of cellular activities required for tumor cell function. In preparation for an IND application submission and advancing SH7139 into clinical trials, acute dose range finding (i.v. bolus) and multiple dose (i.v. bolus on days 1, 8, and 15) toxicology and safety studies have been conducted with SH7139 in rats and beagle dogs. In the rat studies, doses up to 30 mg/kg (NOAEL = 30 mg/kg, 11,854 fold higher than the efficacy dose), were well tolerated with no findings associated with the drug. Beagle dogs were found to be the most sensitive species (NOAEL = 0.3mg/kg, 395 fold higher than the efficacy dose), with doses at 1 and 10mg/kg producing a reversible allergic-type reaction. A maximum tolerated dose (MTD) was reached in rats (100 mg/kg). No attempt was made to identify the MDT in dogs. In vitro assays conducted to evaluate potential cardiac safety showed SH7139 had no effect on potassium (hERG) and calcium (hCaV1.2) ion channel function up to the highest concentration tested (25 μM). A detectable effect above background was only observed with the sodium channel hNaV1.5 at 25 μM, a plasma concentration that would only be achieved in patients administered a dose ~2,000 fold higher than the efficacy dose. SH7139 was found to be negative in inducing gene mutation in five tester strains of Salmonella and E. coli (AMES assay). In a CHO-K1 micronucleus assay, no genotoxicity was observed in the presence of S9 over the entire concentration range tested. In the absence of S9, a positive response (3.3 fold) just above the triggering threshold (3 fold) of the assay was observed at the highest SH7139 concentration (500 μM). Based on these results and others derived from mouse xenograft efficacy studies, a maximum recommended Phase I trial starting dose has been tentatively set at 0.5 μg/kg with a safety factor of 324. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Rod Balhorn, Monique Cosman Balhorn. Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2017-4079

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N -acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP- N -methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn , and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342-347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N2 position of guanine.

An improved nuclear magnetic resonance (NMR) experimental scheme to measure self-diffusion coefficients of biomolecules in 95% H2O using pulsed-magnetic-field gradients is presented. The radiation damping of water, which deleteriously effects the quality of the spectra in high-field NMR, is advantageously used in reducing the water signal. The enhancements of the new sequence, designated BPP-SED, a novel combination of BPP-LED (bipolar-gradient-pulse pair longitudinal eddy current delay) and SED (selective echo dephasing) is demonstrated by measuring self-diffusion coefficients of dilute protein samples (0.5 mM) in limited volumes (250 μl).

Structure–dynamics interrelationships are important in understanding protein function. We have explored the empirical relationship between rotational correlation times (τc and the solvent accessible surface areas (SASA) of 75 proteins with known structures. The theoretical correlation between SASA and τc through the equation SASA = Krτc(2/3) is also considered. SASA was determined from the structure, τccalc was determined from diffusion tensor calculations, and τcexpt was determined from NMR backbone13 C or 15N relaxation rate measurements. The theoretical and experimental values of τc correlate with SASA with regression analyses values of Kr as 1696 and 1896 m2s-(2/3), respectively, and with corresponding correlation coefficients of 0.92 and 0.70.

Site-specifically modified oligonucleotides were derived from the reactions of stereoisomeric polycyclic aromatic diol epoxide metabolite model compounds with oligonucleotides of defined base composition and sequence. The NMR solution structures of ten different adducts studied so far are briefly described, and it is shown that stereochemical factors and the nature of the oligonucleotide context of the complementary strands, exert a powerful influence on the conformational features of these adducts.

The direct synthesis and characterization of site-specific adducts derived from the binding of (+)-1R,2S-dihydroxy-3S,4R-epoxide-1,2,3,4-tetrahydro-5-methylchrysene and the (-)-1S,2R,3R,4S-enantiomer [(+)- and (-)-5-MeCDE, respectively], to the N 2 -guanine residues in the oligonucleotide d(CCATCGCTACC) are described. The spectroscopic characteristics of the 5-MeCDE-modified oligonucleotides are discussed, and it is shown that their CD characteristics can be used to distinguish between the trans-addition products of the binding of the (+)- and (-)-enantiomers of 5-MeCDE (C4 position). The 11-met duplexes with the normal complementary strands are destabilized by the site-specific, covalently bound 5-MeCDE residues : the melting points, T m , are 5-10° lower than in the case of the unmodified duplex. Stereoselective exonuclease enzyme digestion patterns of the single-stranded (+)- and (-)-trans-5-MeCDE-modified oligonucleotides (Mao et al., 1993, Biochemistry, 32, 11785-11793) were used to probe the orientations of the covalently bound 5-MeCDE residues relative to the modified guanine and the 5'-3' strand polarity ; the aromatic residues are positioned either on the 5'-side [(+)-5-MeCDE], or the 3'-side [(-)-5-MeCDE adduct] of the modified guanine residues. The electrophoretic mobilities of the (+)-5-MeCDE-modified 11-mer duplexes in native polyacrylamide gels are slower than those of unmodified and modified duplexes containing the stereoisomeric (-)-5-MeCDE-N 2 -dG lesions. This indicates that the lesions derived from the tumorigenic (+)-5-MeCDE induce greater degrees of bending or local flexibility than the non-tumor-igenic (-)-5- MeCDE enantiomer. These differences in the orientational and structural characteristics are similar to those observed with analogous DNA adducts derived from the tumorigenic (+)-7R-8S-dihydroxy-9S-10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the non-tumorigenic 7S,8R,9R,10S-enantiomer, respectively. The adducts derived from BPDE and 5-MeCDE enantiomers thus display similar characteristics that depend primarily on the PAH diol epoxide enantiomer stereochemistry. This direct synthesis approach can be used to generate milligram quantities of site-specific 5-MeCDE-modified oligonucleotides that are suitable for NMR studies (Cosman et al., 1995, Biochemistry, 34, 6247-6260).

SH7139 is a small molecule drug developed to target B-cell malignancies overexpressing HLA-DR10. It contains a DOTA metal chelating ring and three recognition ligands (Ct, Cb, and Dv) that are linked together using a scaffold composed of lysines and mini-polyethylene glycols. The amide bonds within the scaffold have D-configurations to make them resistant to hydrolysis by native proteolytic enzymes. However, two of the ligands contain bonds and chemical groups that can be cleaved or modified by metabolic enzymes to produce known cytotoxic compounds. LCMS was used to identify the metabolites of SH7139 produced in cryopreserved hepatocytes, Burkitt's (Raji) lymphoma cells and the liver microsomes of several mammalian species. Following uptake of SH7139 by human, beagle and rat hepatocytes, which occurs slowly, the cleavage of an amide bond within the Ct ligand produced 2-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyaniline (M8) and a large fragment containing the remainder of SH7139 (M2). Phase II metabolism led to rapid glucuronidation of M8 to facilitate its excretion. This result may explain the lack of SH7139 liver toxicity in animals studied to date. Raji lymphoma cells produce the same two Phase I metabolites, but glucuronidation of M8 is not observed. Thus, M8 is free to inhibit multiple activities required for tumor cell survival, including the processing of misfolded proteins, lipogenesis and sumoylation. Experiments performed with microsomes isolated from Raji cells and human, Cynomologus monkey, beagle, rat and mouse livers identified eight additional metabolites. Removal of a single methyl group from intact SH7139 produced M7. Hydrolysis of the azo group within the SH7139 ligand Dv in both M2 and M7 led to the production of small and large fragments in each case: dimethylphenylenediamine (M9) and M1 and methylphenylenediamine (M10) and M5, respectively. M9 and the phenylenediamine that is usually produced by the further metabolism of M9 and M10 have been shown by others to inhibit ATP production in mitochondria. A final set of demethylation, amide cleavage, oxidation, reduction and glucuronidation reactions involving M7 and M2 produced the remaining metabolite M6. While metabolic processing of the Cb ligand did not produce a biologically reactive metabolite, other experiments have found that the Cb ligand in intact SH7139 inhibits the hydrolysis of GTP by MgcRacGAP-Rac1 and the completion of cytokinesis. Collectively, these results confirm that SH7139 functions similar to both an antibody drug conjugate (ADC) and a pro-drug. In contrast to an ADC, however, the same SH7139 ligand that participates in the targeting function is also involved, either directly or following metabolic processing, in tumor cell killing. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Monique Cosman Balhorn, Rod Balhorn. Metabolism studies of SH7139, a small molecule drug targeting B-cell malignancies overexpressing HLA-DR10, confirm its prodrug function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1305.

SH7139, the first in a series of selective high affinity ligand (SHAL) therapeutics designed to treat non-Hodgkin's lymphoma, has been shown to be selectively cytotoxic to lymphoma cells over-expressing HLA-DR10. Recent efforts to elucidate the mechanisms of action of SH7139 show that the small molecule drug functions similar to both an antibody drug conjugate and a pro-drug. SH7139 is comprised of three small molecule recognition elements that, when linked together, collectively target the drug to HLA-DR10. Following its binding to HLA-DR10, SH7139 is shuttled into the interior of the lymphoma cell where the subsequent metabolism of these recognition elements releases a series of metabolites that inhibit multiple activities required for tumor cell growth and replication. Studies performed using the Burkitt's lymphoma cell line Raji have shown that SH7139 is metabolized by Raji cells, and the metabolic cleavage of two of the recognition elements (Ct and Dv) produce cytotoxic compounds that contribute to tumor cell killing. While the third recognition element, Cb, is not cleaved off the SHAL scaffold or hydrolyzed to release a cytotoxic metabolite, its structural similarity to known inhibitors of the GTPase activating protein (GAP) MgcRacGAP suggested that it might be active in this pathway. MgcRacGAP functions as a switch that stimulates by many orders of magnitude the activity of the Rac1 GTPase, which is required for cleavage furrow formation, ingression, and the completion of cytokinesis. Experiments conducted with the MgcRacGAP-Rac1 complex have shown that the intact SH7139 molecule (IC50 = 10.6±1.6μM) as well as SH7139 fragments containing the Cb recognition element is effective in inhibiting the GTPase activity of the MgcRacGAP:Rac1 complex. These results confirm that one mechanism action of SH7139 is the inhibition of the Rac1-dependent effector pathways that control the rounding of cells undergoing mitosis, confine Rho activation to the equator of the cell for proper cleavage furrow formation and other processes involved in the completion of cytokinesis. This research was supported by the National Cancer Institute Phase II SBIR award R44CA159843 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Citation Format: Rodney Balhorn, Arjan J. van Adrichem, Saphon Hok, Monique C. Balhorn. Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2991.

Purpose: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non–Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. Experimental Design: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the β subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. Results: These SHALs bind with nanomolar to picomolar Kd's only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non–Hodgkin's lymphomas mimicking the selectivity of Lym-1. Conclusions: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab. Methods: A lysine-polyethylene glycol (PEG) backbone was used to synthetically link 2 of the following ligands: deoxycholate, 5-leuenkephalin, triiodothyronine, thyronine, dabsyl-L-valine, and N-benzoyl-L-arginyl-4amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-NN',N '',N'''-tetraacetic acid (DOTA) chelate attached to the linker. These SHALs have been assessed for their selectivity in binding to HLA-DR10-expressing cells and for their pharmacokinetics and tissue biodistribution in mice. Biotinylated versions of these SHALs discriminated cell lines positive for HLA-DR10 expression with near-nanomolar affinity. The DOTA versions of 4 SHALs were labeled with In-111 for pharmacokinetic studies in mice with HLA-DR10-expressing malignant Raji xenografts. Results: The bidentate, biotinylated, and DOTA-SHALs were synthesized in high-purity, multimilligram amounts. Mean radiochemical and product yields and purities were 90%, 75%, and 90% at mean specific activities of 3.9 MBq/mu g (105 mu Ci/mu g) for the In-111-labeled SHALs. As expected, rapid blood clearance and tumor targeting were observed. The pharmacokinetics of the SHALs was influenced by the component ligands. Biliary clearance, kidney localization, and serum receptor binding contributed to less favorable tumor targeting. Conclusion: A series of SHALs was readily synthesized in multimilligram amounts and showed the expected selective binding in vitro. Better selection of the SHAL components should provide second-generation SHALs with improved properties to fulfill the substantial potential of these novel molecular carriers for targeting.

The tridentate selective high affinity ligand (SHAL) SH7139 is selectively cytotoxic at sub-nanomolar concentrations to lymphoma cells over-expressing HLA-DR10. Recent studies suggest SH7139 is unusual in that multiple mechanisms may contribute to its killing of targeted tumor cells. Electron microscopy data suggest one mechanism involves the triggering of a cell-signaling event that leads to the induction of apoptosis when SH7139 binds to a unique site on HLA-DR10 (the same region/epitope recognized by the Lym-1 antibody). A second mechanism appears to involve the internalization of SH7139 by tumor cells and the subsequent release of it's three individual recognition elements/ligands by metabolic cleavage of the amide bonds that link them to a scaffold (a unique feature of SHALs). Analogous to the process of toxin delivery by antibody-drug conjugates, once these ligands are released inside the cell, they can each inhibit a different key pathway required for tumor cell survival. Additional metabolic conversions of these ligands by the tumor cells to toxic metabolites may also contribute to cell killing through the inhibition of a number of other critical cellular functions. The third proposed mechanism is based on the fact that the target receptor HLA-DR10 participates in the activation of T-cells. The binding of SH7139 to the antigen binding pocket of HLA-DR10 may trigger an immune response that targets the cancer cells with bound drug. All three proposed mechanisms are currently under study. If multiple diverse mechanisms of action contribute to SH7139's cytotoxicity, it will be extremely difficult for lymphoma cells to develop a resistance to the drug. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843-02 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Citation Format: Monique Cosman Balhorn, Saphon Hok, Rod Balhorn. Multiple mechanisms of action may contribute to the lymphoma cell-killing activity of SH7139. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5498. doi:10.1158/1538-7445.AM2015-5498

Oncology drugs must remain sufficiently stable during storage, following formulation, after introduction into the bloodstream and during their entry into cells to ensure accurate dosing. After uptake by cells, the biologically active form of the drug also needs to remain stable long enough to achieve an optimal anti-tumor response. SH7139, our most advanced small molecule therapeutic for non-Hodgkin's lymphoma, was designed to target and bind to a unique site on HLA-DR10, a cell surface receptor found on B-cell lymphocytes and over-expressed on many B-cell derived malignancies. Previous 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. SH7139 has also been shown to be highly effective in treating human Burkitt's lymphoma (Raji) in a mouse xenograft model. All of the studies performed to date suggest SH7139 meets the necessary stability requirements. No degradation of the drug has been observed during storage at -20°C over a period of 30 months. SH7139 is also stable when dissolved in water or saline over a wide range of pH (pH 2 to 9). Instability is only observed at a highly alkaline pH. At pH 12, a slow auto-catalytic cleavage reaction releases a fragment of one of the recognition elements. The amide bonds linking the three recognition elements to the lysine and miniPEG scaffold of SH7139 remain stable in plasma. Hepatocytes metabolize SH7139 at a moderate rate and produce two primary metabolites. The same metabolites are produced by both canine and human hepatocytes, and the smaller product, 2-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyaniline, is rapidly glucuronidated to facilitate its excretion. Both the physical and metabolic stability of SH7139 are consistent with the drug's observed anti-lymphoma activity. This research was supported by the National Cancer Institute Phase II SBIR award R44CA159843-02 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Citation Format: Rod Balhorn, Saphon Hok, Monique Cosman Balhorn. Physical and metabolic stability of SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4494. doi:10.1158/1538-7445.AM2015-4494

Many carcinogens have been shown to cause tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and is usually attributed to differences in organ metabolism. For heterocyclic amines, potent carcinogens that are formed in well-done meat, the ability to either bind to the estrogen receptor and activate or inhibit an estrogenic response will have a major impact on carcinogenicity. Here we describe our work with the human estrogen receptor alpha (hERa) and the mutagenic/carcinogenic heterocyclic amines PhIP, MeIQx, IFP, and the hydroxylated metabolite of PhIP, N2-hydroxy-PhIP. We found that PhIP, in contrast to the other heterocyclic amines, increased cell-proliferation in MCF-7 human breast cancer cells and activated the hERa receptor. We show mechanistic data supporting this activation both computationally by homology modeling and docking, and by NMR confirmation that PhIP binds with the ligand binding domain (LBD). This binding competes with estradiol (E2) in the native E2 binding cavity of the receptor. We also find that other heterocyclic amines and N2-hydroxy-PhIP inhibit ER activation presumably by binding into another cavity on the LBD. Moreover, molecular dynamics simulations of inhibitory heterocyclic amines reveal a disruption of the surface of the receptor protein involved withmore » protein-protein signaling. We therefore propose that the mechanism for the tissue specific carcinogenicity seen in the rat breast tumors and the presumptive human breast cancer associated with the consumption of well-done meat maybe mediated by this receptor activation.« less

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying bi-molecular interactions at the atomic scale. Our NMR lab is involved in the identification of small molecules, or ligands that bind to target protein receptors, such as tetanus (TeNT) and botulinum (BoNT) neurotoxins, anthrax proteins and HLA-DR10 receptors on non-Hodgkin's lymphoma cancer cells. Once low affinity binders are identified, they can be linked together to produce multidentate synthetic high affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors or biochips for the detection of pathogen proteins in environmental samples or body fluids. Here, we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy (trNOESY) that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known ''marker'' ligand, doxorubicin. Using this assay, we can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.

Aberrant association of autoantibodies with myelin oligodendrocyte glycoprotein (MOG), an integral membrane protein of the central nervous system (CNS) myelin, has been implicated in the pathogenesis of multiple sclerosis (MS). Sensitization of nonhuman primates (Callithrix jacchus marmosets) against the nonglycosylated, recombinant N-terminal domain of rat MOG (residues 1-125) reproduces an MS-like disease in which MOG-specific autoantibodies directly mediate demyelination. To assess the interrelationship between MOG structure and the induction of autoimmune CNS diseases and to enable structure-based rational design of therapeutics, a homology model of human MOG(2-120) was constructed based on consensus residues found in immunoglobulin superfamily variable-type proteins having known structures. Possible sites for posttranslational modifications and dimerization have also been identified and analyzed. The B cell and T cell epitopes have been identified in rat MOG-immunized marmosets, and these sequences are observed to map primarily onto accessible regions in the model, which may explain their ability to generate potent antibody responses.

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

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

The scale-up of synthetic methods for producing small molecule therapeutics can often prove to be problematic and expensive. Synthetic mechanisms can be inefficient and purification methods used to select specific isomers or remove reactants or other contaminants can often be difficult to scale. The modular approach we developed for creating small molecule targeting agents called SHALs (Selective High Affinity Ligands) as therapeutics and diagnostics for B-cell lymphoma circumvents many of these problems by using off the shelf components as “recognition elements” that collectively contribute to the molecules high affinity and selectivity and miniPEG and common amino acids to build up the scaffold using solid phase chemistry. Here we present the results of our efforts to scale up the synthesis of the first of these promising new drug candidates, SH7139, to provide the compounds needed for pre-IND animal testing and a pet dog lymphoma clinical trial. Working with AmbioPharm Inc,, a CRO with extensive expertise in the GMP synthesis of peptide pharmaceuticals, we recently completed small pilot production scale syntheses of SH7139 and several closely related analogs. The simple solid phase peptide-based chemistry used to synthesize the compounds coupled with preparative HPLC adapted well to scale-up and resulted in the production of highly purified compounds identical in structure (as determined by mass spectrometry and nuclear magnetic resonance spectroscopy) to the bench scale syntheses we performed in house. However, one unexpected and notable difference between the scaled-up synthetic products and their bench-scale synthesis counterparts was their solubility. SH7139 and two of its analogs, which are essentially insoluble in water and PBS when synthesized in house, were observed to be highly soluble (>100mg/ml) when synthesized using the scale-up protocol. While the reason for this is still under investigation, microscopic analyses of the dried product suggest the solubility difference may relate to the lyophilization step used to produce the dried product. The majority of the insoluble SH7139 we produce in bench-scale syntheses is amorphous in form, while the soluble SH7139 produced by AmbioPharm appears to be in a semi-crystalline or glass-like state. This research was supported, in part, by NIH/NCI SBIR grant 1R43CA159843-01 to Rod Balhorn. Citation Format: Monique Balhorn, Saphon Hok, Rod Balhorn. Optimizing a modular synthetic approach for producing SH7139, a new drug candidate for treating B-cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2453. doi:10.1158/1538-7445.AM2013-2453

A group of small molecule drug candidates for non-Hodgkin's lymphoma, called Selective High Affinity Ligands (SHALs), have been developed to target the cell surface antigen HLA-DR10 that is known to be over expressed on many B-cell derived malignancies. These compounds are created by chemically linking together two or three small molecule “recognition elements” that have been identified to bind to neighboring unique sites on the surface of the target protein. One of these compounds, SH7139, has been shown previously to be effective in treating human B cell lymphoma in a mouse xenograft model at extremely low doses (5μg/kg). Earlier experiments conducted in mice have also shown the compound to exhibit no signs of toxicity at 2000 times (10mg/kg) this dose. In an effort to obtain preliminary toxicology and safety data on SH7139 and assess the reversibility or persistence of any potential toxic effects prior to its being evaluated in a pet dog B-cell lymphoma trial for treating canine B-cell lymphomas, a pilot 28-day toxicology study was performed in three naïve Beagles. SH7139 was administered intravenously in three doses injected on days 0, 7 and 14. The dose tested, 50μg/kg, was ten times the dose required to cure 69% of mice carrying Raji xenografts. During the in-life period, animals were observed daily for mortality, moribundity, and food consumption, detailed clinical examinations were conducted on the days of dose, 24 hrs post-dose and weekly thereafter, and individual body weights and temperatures were recorded on the days of dosing and selected days after dosing. Clinical pathology samples (urinalysis, hematology, coagulation, clinical chemistry) were collected prior to dose administration on the days of dose and during the recovery phase (Days 21 and 28). On Day 29, all animals were euthanized and a complete necropsy was conducted with tissue collection and preservation. The key tissues were embedded in paraffin, H&E stained and examined microscopically by a board-certified pathologist. The results of this study show the treatment with SH7139 is well tolerated by healthy, naïve dogs. No treatment-related effects on body weight, body temperature, food consumption or clinical pathology parameters were observed. In addition, the lack of significant changes in hematology, clinical chemistry, urinalysis or coagulation parameters also indicate SH7139 exhibited no apparent systemic toxicity or side effects. Citation Format: Rod Balhorn, Saphon Hok, Monique Balhorn. Pilot toxicology study of the B-cell lymphoma drug candidate SH7139 demonstrates lack of toxicity in beagles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4419. doi:10.1158/1538-7445.AM2013-4419

Selective High Affinity Ligands (SHALs) are a new class of small molecule targeting agents that can be designed to bind selectively and with high affinity to unique sites located on almost any protein. A series of SHAL-based therapeutics have been developed to target the cell surface antigen HLA-DR10 that is over-expressed on many B-cell derived malignancies. SHALs are created by first identifying subsets of potential ligands, or “recognition elements,” that bind to two or three neighboring unique sites on the surface of the target protein. Bidentate or tridentate SHALs are produced by linking two or three of these recognition ligands together using carbodiimide chemistry and a miniPEG and lysine scaffold. SHAL synthesis is rapid, uses relatively inexpensive, commercially available components, and is very versatile. Multiple forms of the SHAL can be created by combining different recognition elements (ligands) or by attaching a variety of tags (biotin, chelating groups, dyes, inhibitors) to the end of the linker. Using this approach, SH7139 has been developed as a promising therapeutic for treating non-Hodgkin's lymphoma. Previous studies have shown SH7139 to be effective in treating human B-cell lymphoma in a mouse xenograft model at extremely low doses (3.3μg/kg); 70% of the tumors disappeared within 30 days after treatment and did not reoccur during the remainder of the life of the animal. Here we report that a biotinylated derivative of SH7139 can be used as companion diagnostic to prescreen needle aspirates or biopsies from B-cell lymphomas and determine whether or not the tumor is likely to respond to SH7139 therapy. The staining protocol involves detecting the bound biotinylated SHAL using commercially available alkaline phosphatase reagents, and it works equally well with both fixed and unfixed biopsy sections. Biotinylated SH7139 has been used to identify potentially responsive B-cell lymphomas in both canine and human patients. The development of SHAL-based diagnostics in parallel with new SHAL-based therapeutics will help advance individualized therapies and minimize the systemic trauma and valuable time lost by patients given treatments that prove to be ineffective. Our results also suggest this diagnostic may also prove useful in determining whether malignancies over-expressing HLA-DR10 have metastasized and individual lymphoma cells have migrated out of the tumor into surrounding normal tissue. This research was supported, in part, by NIH/NCI SBIR grant 1R43CA159843-01 to Rod Balhorn. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4591. doi:1538-7445.AM2012-4591

We have synthesized, separated, and purified approximately 10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-anti-benzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N2-dG adduct positioned opposite dC and flanked by G.C pairs in the d[C1-C2-A3-T4-C5-(BP)G6-C7-T8-A9-C10-C11].d[12- G13-T14-A15-G16-C17-G18-A19-T20-G 21-G22] duplex +ADdesignated (BP)G.C 11-mer+BD. We have determined the solution structure centered about the BP covalent adduct site in the (BP)G.C 11-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugar-phosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8].d[A15-G16-C17-G18-A19] segment centered about the adduct site with Watson-Crick alignment for both the (BP)G6.C17 pair and flanking G.C pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of approximately 45 degrees with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other steroisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomer-dependent spectrum of mutational and carcinogenic activities.