Pharmacokinetic characterization in xenografted mice of a series of first-generation mimics for HLA-DR antibody, Lym-1, as carrier molecules to image and treat lymphoma.

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 (<2 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-4-amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-N,N',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 (111)In 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/microg (105 microCi/microg) for the (111)In-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.     

Antioxidant potential of a novel tetrapeptide derivative in isoproterenol-induced myocardial necrosis in rats

A novel tetrapeptide derivative Boc-Lys(Boc)-Arg-Asp-Ser(tbu)-OtBu (PEP1261) has been tested in vivo in isoproterenol (ISO) hydrochloride (HCl)-induced myocardial necrosis in rats. ISO x HCl induces myocardial necrosis in rats which is accompanied by the distinct increase in heart weight, marked electrocardiographic changes, increase in the levels of serum marker enzymes and lipid peroxides and decrease in the levels of antioxidants. PEP1261 (5 mg/kg body weight i.p.) pre- and post-treatment effectively decreases serum marker enzyme levels, while the electrocardiographic changes get restored towards normalcy. PEP1261 also inhibits the action of the free radicals toxicity by increasing the levels of antioxidants and histological studies confirm the above findings. This study shows that PEP1261 could serve as an excellent cardioprotective agent possessing membrane-stabilizing action.     

High-dose radioimmunotherapy combined with fixed, low-dose paclitaxel in metastatic prostate and breast cancer by using a MUC-1 monoclonal antibody, m170, linked to indium-111/yttrium-90 via a cathepsin cleavable linker with cyclosporine to prevent human anti-mouse antibody.

PURPOSE: Although radioimmunotherapy alone is effective in lymphoma, its application to solid tumors will likely require a combined modality approach. In these phase I studies, paclitaxel was combined with radioimmunotherapy in patients with metastatic hormone-refractory prostate cancer or advanced breast cancer. EXPERIMENTAL DESIGN: Patients were imaged with indium-111 (111In)-1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid-peptide-m170. One week later, yttrium-90 (90Y)-m170 was infused (12 mCi/m2 for prostate cancer and 22 mCi/m2 for breast cancer). Initial cohorts received radioimmunotherapy alone. Subsequent cohorts received radioimmunotherapy followed 48 hours later by paclitaxel (75 mg/m2). Cyclosporine was given to prevent development of human anti-mouse antibody. RESULTS: Bone and soft tissue metastases were targeted by 111In-m170 in 15 of the 16 patients imaged. Three prostate cancer patients treated with radioimmunotherapy alone had no grade 3 or 4 toxicity. With radioimmunotherapy and paclitaxel, two of three prostate cancer patients developed transient grade 4 neutropenia. Four breast cancer patients treated with radioimmunotherapy alone had grade 3 or 4 myelosuppression. With radioimmunotherapy and paclitaxel, both breast cancer patients developed grade 4 neutropenia. Three breast cancer patients required infusion of previously harvested peripheral blood stem cells because of neutropenic fever or bleeding. One patient in this trial developed human anti-mouse antibody in contrast to 12 of 17 patients in a prior trial using m170-radioimmunotherapy without cyclosporine. CONCLUSIONS: 111In/90Y-m170 targets prostate and breast cancer and can be combined with paclitaxel with toxicity limited to marrow suppression at the dose levels above. The maximum tolerated dose of radioimmunotherapy and fixed-dose paclitaxel with peripheral blood stem cell support has not been reached. Cyclosporine is effective in preventing human anti-mouse antibody, suggesting the feasibility of multidose, "fractionated" therapy that could enhance clinical response.     

Antioxidative activity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid.

The (-)-(1S, 3S) isomer of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid was synthesised by Pictet-Spengler condensation of tryptophan with acetaldehyde. It was evaluated for its antioxidative activity in the linoleic acid autooxidation system by the ferric thiocynate method. Butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), and alpha-tocopherol were used as the reference standards. The compound showed moderate antioxidative activity and also synergistic effect with the reference standards. The synergistic effect was in the increasing order of BHT, alpha-tocopherol and BHA. The synergistic effect was higher at higher concentrations studied.     

Thermal dosimetry predictive of efficacy of 111In-ChL6 nanoparticle AMF--induced thermoablative therapy for human breast cancer in mice.

Antibody (mAb)-linked iron oxide nanoparticles (bioprobes) provide the opportunity to develop tumor specific thermal therapy (Rx) for metastatic cancer when inductively heated by an externally applied alternating magnetic field (AMF). To evaluate the potential of this Rx, in vivo tumor targeting, efficacy, and predictive radionuclide-based heat dosimetry were studied using (111)In-ChL6 bioprobes (ChL6 is chimeric L6) in a human breast cancer xenograft model. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 (DOTA is dodecanetetraacetic acid) was conjugated to polyethylene glycol-iron oxide-impregnated dextran 20-nm particles and purified as (111)In-bioprobes. (111)In doses of 740-1,110 kBq (20-30 muCi) (2.2 mg of bioprobes) were injected intravenously into mice bearing HBT3477 human breast cancer xenografts. Pharmacokinetic (PK) data were obtained at 1, 2, 3, and 5 d. AMF was delivered 72 h after bioprobe injection at amplitudes of 1,410 (113 kA/m), 1,300 (104 kA/m), and 700 (56 kA/m) oersteds (Oe) at 30%, 60%, and 90% "on" time (duty), respectively, and at 1,050 Oe (84 kA/m) at 50% and 70% duty over the 20-min treatment. Treated and control mice were monitored for 90 d. Tumor total heat dose (THD) from activated tumor bioprobes was calculated for each Rx group using (111)In-bioprobe tumor concentration and premeasured particle heat response to AMF amplitudes. Tumor growth delay was analyzed by Wilcoxon rank sum comparison of time to double, triple, and quintuple tumor volume in each group, and all groups were compared with the controls. RESULTS: Mean tumor concentration of (111)In-bioprobes at 48 h was 14 +/- 2 percentage injected dose per gram; this concentration 24 h before AMF treatment was used to calculate THD. No particle-related toxicity was observed. Toxicity was observed at the highest AMF amplitude-duty combination of 1,300 Oe and 60% over 20 min; 6 of 10 mice died acutely. Tumor growth delay occurred in all of the other groups, correlated with heat dose and, except for the lowest heat dose group, was statistically significant when compared with the untreated group. Electron microscopy showed (111)In-bioprobes on tumor cells and cell death by necrosis at 24 and 48 h after AMF. CONCLUSION: mAb-guided bioprobes (iron oxide nanoparticles) effectively targeted human breast cancer xenografts in mice. THD, calculated using empirically observed (111)In-bioprobe tumor concentration and in vitro nanoparticle heat induction by AMF, correlated with tumor growth delay.     

Characteristics of dimeric (bis) bidentate selective high affinity ligands as HLA-DR10 beta antibody mimics targeting non-Hodgkin's lymphoma

Despite their large size, antibodies have proven to be suitable radioisotope carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. To mimic antibody (Ab) targeting behavior while decreasing size by 50-100x, a combination of computational and experimental methods were used to generate molecules that bind to unique sites within the HLA-DR epitopic region of Lym-1, an Ab shown effective in patients. Lym-1 Ab mimics (synthetic high afinity ligands; SHALs) were generated and studied in vitro, using live cell binding assays, and/or pharmacokinetic studies over 24 h in xenografted mice given 1 or 20 microg SHAL doses i.v. Multimilligram amounts of each of the dimeric (bis) SHALs were synthesized at high purity, and labeled with indium-111 at high specific activity and purity. These SHALs were selective for HLA-DR and HLA-DR expressing malignant cells and had functional affinities that ranged from 10(-9) M (nanomolar) to 10(-10) M. Blood clearances ranged from 3.6 to 9.5 h and body clearances ranged from 15.2 to 43.0 h for the 6 bis DOTA-SHALs studied in a mouse model for non-Hodgkin's lymphoma (NHL). While localization was shown in Raji NHL xenografts, biodistribution was influenced by 'sinks' for individual ligands of the SHALs. Highly pure, dimeric mimics for HLA-DR Ab were synthesized, biotinylated and radiolabeled, and showed selectivity in vitro. Pharmacokinetic behavior in mice was influenced by the ligands and by the linker length of the dimeric SHALs. Nanomolar or better functional affinity was observed when a suitably long linker was used to connect the two bidentate SHALs. The concept and methodology are of interest because applicable for targeting most proteins; the SHAL synthetic platform is highly efficient and adaptive.     

Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1–directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti–PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm³) and large tumors (150 mm³), whereas the activity of anti–PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1–positive and PD-L1–negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.Lymphocyte activity is regulated by a complex series of stimulatory, costimulatory, and inhibitory cues. Although the central regulator of T-lymphocyte function is the T-cell receptor (TCR), the balance between positive and negative signaling inputs deeply shapes the response that lymphocytes mount upon exposure to reactive peptide/MHC complexes (1). As a cause and consequence of their development, cancer cells accumulate somatic mutations that distinguish them from noncancerous cells. These tumor “neo-antigens” can be recognized by the TCRs of cytotoxic T lymphocytes and in many cases prompt an endogenous antitumor response by the immune system (2). Thus, immunogenic tumors must exploit, or at least indirectly benefit from, immunosuppressive pathways to escape immune destruction (3).Expression of the inhibitory programmed cell death ligand-1 (PD-L1) in the tumor environment is a key exemplar of this phenomenon. PD-L1 normally serves to prevent autoimmunity by engaging its receptor, programmed cell death protein-1 (PD-1), on activated T cells (4). Upon binding to either of its two ligands, PD-L1 and PD-L2, PD-1 initiates an inhibitory signaling cascade through its intracellular signaling domains, including an immunoreceptor tyrosine-based inhibitory motif and immunoreceptor tyrosine-based switch motif (5). The result is activation of SHP phosphatases that oppose TCR signaling. Although beneficial in preventing excessive or harmful inflammation under normal conditions, in the context of cancer, tumor and stromal PD-L1 expression presents a barrier to immune function by contributing to the exhaustion of the antitumor lymphocytes that might otherwise clear the malignancy (6). Consequently, the PD-1:PD-L1 pathway has emerged as a critical target for cancer immunotherapy, and monoclonal antibodies that block either side of this inhibitory interaction have demonstrated impressive activity across a broad set of cancer subtypes, even at advanced and metastatic stages of disease (7–11).Despite their proven utility, antibodies have specific drawbacks as therapeutics, which may be especially pertinent when targeting the PD-1:PD-L1 signaling pathway. For example, PD-1–expressing effector T cells are found infiltrated within solid tissue of PD-L1–expressing tumors (6). This is problematic for antibodies, which are impeded from entering tumors due to their large size (12). It follows that antibodies may therefore fail to completely antagonize PD-1:PD-L1 signaling at the intended therapeutic site within tumors, leading to suboptimal efficacy. An additional limitation of antibodies is their ability to activate cytotoxic immune responses through natural killer cells and macrophages (e.g., ADCC/ADCP) (13). Although this Fc-mediated effect is in fact required for the efficacy of some immunotherapeutic antibodies (14), it may in part be counterproductive in the case of this receptor–ligand pair. Both PD-1 and PD-L1 are expressed on the surface of antitumor cytotoxic T cells (15, 16), and as such, antibodies targeting PD-1 and PD-L1 may paradoxically result in the undesirable depletion of the very lymphocytes they are intended to activate. Consistent with this hypothesis, treatment with anti–PD-1 antibodies of anti–PD-1 antibodies has been reported to correlate with lower circulating T-cell numbers in patients (17).To date, most studies of PD-1 or PD-L1 blockade have used monoclonal antibodies. In principle, a soluble fragment of the PD-1 ectodomain could be administered as a competitive antagonist of PD-L1. At 14 kDa in size, this agent would be approximately an order of magnitude smaller than a monoclonal antibody (150 kDa) and also lack an antibody Fc moiety. We thus sought to determine whether such an alternative agent could exhibit improved antitumor responses by avoiding antibody-intrinsic limitations.     

The Remarkable Stability of Chimeric,Sialic Acid-derived α/δ-Peptides in Human Blood Plasma

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-α-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric α/δ-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en α/δ-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with ¹¹¹In, incubated in human blood plasma at 37 °C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural α-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.     

Acute liver injury upregulates microRNA‐491–5p in mice,and its overexpression sensitizes Hep G2 cells for tumour necrosis factor‐α‐induced apoptosis

Background: MicroRNAs (miRNAs) have emerged as novel genetic regulators of cell functions such as proliferation, apoptosis and cancer. Aims: The aim of this study was to evaluate the role of a specific miRNA in modulating hepatic cell functions. Methods: C57Bl/6 mice were administered anti‐fas receptor antibodies to induce liver cell apoptosis. miRNAs were purified from the liver tissue and evaluated using an miRNA microarray. The role of miRNA‐491_5p, which was overexpressed in the model, in modulating hepatic cell functions was evaluated. miRNA‐491_5p was overexpressed in Hep G2 cells, followed by the addition of tumour necrosis factor (TNF)‐α, and induction of apoptosis as well as genes involved in apoptosis pathways were evaluated. The effect of miRNA‐491_5p target genes on apoptosis was also analysed by inhibiting their expression by siRNA‐induced gene silencing. Results: Upregulation of miRNA‐491_5p was found in a high‐dose anti‐fas receptor antibody group. Overexpression of microRNA‐491_5p sensitized Hep G2 cells for TNF‐α‐induced apoptosis, and also caused an inhibition of α‐fetoprotein, (AFP), heat shock protein‐90 (hsp‐90) and nuclear factor‐κB (NF‐κB). Overexpression of miRNA‐491_5p or inhibition of AFP and hsp‐90 resulted in an increased apoptosis in TNF‐α‐treated Hep G2 cells. Conclusions: One of the miRNAs that is associated with the acute liver injury mouse model, miRNA‐491_5p, sensitizes Hep G2 cells for TNF‐α‐induced apoptosis, at least in part, by inhibiting AFP, hsp‐90 and NF‐κB.