Monique Cosman Balhorn

A selective high affinity ligand (SHAL) designed to bind to an over-expressed human antigen on non-Hodgkin's lymphoma also binds to canine B-cell lymphomas

Therapies using antibodies directed against cell surface proteins have improved survival for human patients with non-Hodgkins lymphoma (NHL). It is possible that similar immuno-therapeutic approaches may also benefit canine NHL patients. Unfortunately, variability between human and canine epitopes often limits the usefulness of such therapies in pet dogs. The Lym-1 antibody recognizes a unique epitope on HLA-DR10 that is expressed on the majority of human B-cell malignancies. The Lym-1 antibody has now been observed to bind to dog lymphocytes and B-cell NHL. Sequence comparisons and computer modeling of a human and three canine DRB1 proteins identified several orthologs of human HLA-DR10 expressed by dog lymphocytes. Immuno-staining confirmed the presence of proteins containing the Lym-1 epitope on dog lymphocytes and B-cell NHL. In addition, a selective high affinity ligand (SHAL) SH-7139 designed to bind within the Lym-1 epitope of HLA-DR10 was also observed to bind to canine B-cell NHL tissue. This SHAL, which is selectively cytotoxic to cells expressing HLA-DR10 and has been shown to cure mice bearing human B-cell lymphoma xenografts, may prove useful in treating B-cell malignancies in pet dogs.

New monoclonal antibodies specific for mammalian protamines P1 and P2

ABSTRACT The expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline

Abstract 2991: Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10

SH7139, the first in a series of selective high affinity ligand (SHAL) therapeutics designed to treat non-Hodgkin9s 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 Burkitt9s 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-Hodgkin9s 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.

Abstract 1305: Metabolism studies of SH7139, a small molecule drug targeting B-cell malignancies overexpressing HLA-DR10, confirm its prodrug function

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, Burkitt9s (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.

Abstract 5498: Multiple mechanisms of action may contribute to the lymphoma cell-killing activity of SH7139

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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 its 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 SH7139s 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

Abstract 4494: Physical and metabolic stability of SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10

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-Hodgkin9s 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 Burkitt9s 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 drug9s 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-Hodgkin9s 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

Abstract 2703: Effect of route and dosing regimen on efficacy of SH7139 in mouse Burkitt's lymphoma xenografts

SH7139, our most advanced small molecule therapeutic for non-Hodgkin9s 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 Burkitt9s 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 Burkitt9s 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

Abstract 5460: Intracellular uptake and metabolism of SH7139: Is it a targeted prodrug for B-cell lymphomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 Burkitts 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 SHALs 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 2453: Optimizing a modular synthetic approach for producing SH7139, a new drug candidate for treating B-cell lymphomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Abstract 4419: Pilot toxicology study of the B-cell lymphoma drug candidate SH7139 demonstrates lack of toxicity in beagles.

A group of small molecule drug candidates for non-Hodgkin9s 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 naive 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, HE 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