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Commercially approved conventional antibody-drug conjugates (ADCs) are produced as heterogeneous mixtures containing a stochastic distribution of payloads decorating the antibody molecules resulting in decreased efficacy and thus lowering their therapeutic index. Control of the DAR and conjugation site in the development of next-generation ADCs is believed to assist in increasing the therapeutic index of these targeted biologics leading to overall enhanced clinical efficacy and reduced toxicity. A chemical site-specific conjugation technology termed AJICAP® allows ADC developers to control both the location and quantity of the payload conjugation to an antibody. Furthermore, this simplified ADC composition enables a streamlined chemical analysis. Here we report the chromatographic separation of site-specific ADCs produced by AJICAP® technology using an analytical affinity chromatography HPLC column containing a recombinant FcγIIIa receptor-ligand immobilized on a non-porous polymer resin (NPR). These HPLC analyses provided visually clear chromatogram results reflecting the heterogeneity of each ADC. The affinity strength was also measured by biolayer interferometry (BLI) and predicted by molecular structure analysis. The results indicate that AJICAP® technology is a promising solution to link hydrophobic payloads to antibodies without compromising antibody receptor function. This study also shows that FcγIIIa-NPR column can be used to characterize site-specific conjugated ADCs compared to ADCs synthesized using conventional methods.

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The naturally extracellular hemoglobin (erythrocruorin) of the Canadian nightcrawler, Lumbricus terrestris (LtEc), is a unique oxygen transport protein that may be an effective substitute for donated human blood. Indeed, this ultra-high molecular weight (~3.6 MDa) hemoglobin has already been shown to avoid the side effects associated with previous hemoglobin-based oxygen carriers and its high thermal stability (Tm = 56°C) and resistance to heme oxidation (kox = 0.04 hr-1 × 103 at 20°C) allow it to be stored for long periods of time without refrigeration. However, before it can be tested in human clinical trials, an effective and scalable purification process for LtEc must be developed. We have previously purified LtEc for animal studies with tangential flow filtration (TFF), which allows rapid and scalable purification of LtEc based on its relatively large size, but that type of size-based purification may not be able to specifically remove some impurities and high MW (>500 kDa) contaminants like endotoxin (MW = ~1-4 MDa). Anion exchange (AEX) and immobilized metal affinity chromatography (IMAC) are two purification methods that have been previously used to purify mammalian hemoglobins, but they have not yet been used to purify large invertebrate hemoglobins like LtEc. Therefore, the goal of this study was to determine if AEX and IMAC resins could successfully purify LtEc from crude earthworm homogenate, while also preserving its macromolecular structure and function. Both processes were able to produce purified LtEc with low levels of endotoxin, but IMAC purification induced significantly higher levels of heme oxidation and subunit dissociation than AEX. In addition, the IMAC process required an additional desalting step to enable LtEc binding. In contrast, AEX produced highly pure LtEc that was not dissociated. LtEc purified by AEX also exhibits similar oxygen binding characteristics (P50 = 27.33 ± 1.82 mm Hg, n = 1.58 ± 0.17) to TFF-purified LtEc (P50 = 28.84 ± 0.40 mm Hg, n = 1.93 ± 0.02). Therefore, AEX appears to be the optimal method for LtEc purification.

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In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one-quarter of cancers in women aged 40-49 years, and one-fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5-year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a Tc-99m-chelator peptide on the N-terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc-99m-chelator PNA-peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu-64-chelator PNA-peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4-24 h after tail-vein administration of MYC or CCND1 Tc-99m-chelator PNA-peptides, but not after administration of mismatch controls. Similarly, human Panc-1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail-vein administration of a KRAS Tc-99m-chelator PNA-peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail-vein adminstration of a KRAS Cu-64-chelator PNA-peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might in the future provide additional genetic characterization of pre-invasive and invasive breast cancers.

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Transposon Tn7 inserts itself into the attTn7 target DNA sequence at the 3' end of the Escherichia coli glmS gene with high specificity and efficiency. This site in the E. coli genome displays amino acid conservation and nucleotide similarity with orthologous sequences in Archaebacteria and eukaryotes. On the basis of the high degree of nucleotide similarity, particularly with eukaryotes, we examined the interactions of a set of 20-bp duplex DNA sequences with the Tn7 protein TnsD. The protein was overexpressed in the IPTG-inducible vector pET14b-TnsD in E. coli BL21(DE3)-RIL-Codon-Plus, and purified by nickel chelation and ion exchange chromatography. Changes in the conformation of DNA duplexes upon interaction with TnsD were monitored by circular dichroism (CD) spectroscopy. TnsD binding to and dissociation from immobilized DNA duplexes were monitored by total internal reflectance (TIR). CD and TIR results were analyzed to calculate k(on), k(off), and K(D) values. The 20-bp DNA duplex corresponding to attTn7 at the 3' end of E. coli glmS displayed strong affinity for TnsD protein, with K(D) approximately 20 nM. Eukaryotic orthologs of attTn7 from yeast and mammalian GFPT1 displayed lower affinity, with K(D) approximately 500 nM. Mutant DNA sequences with a single central mismatch did not display any detectable interaction with TnsD. The physical studies validate our biological observation of Tn7 transposition into a plasmid containing the mammalian attTn7 ortholog sequence [Cleaver, S. H., and Wickstrom, E. (2000) Gene 254, 37-44], and suggest that 1-2 amino acid substitutions in TnsD might be sufficient to permit binding to mammalian orthologs that is as strong as wild-type TnsD binding to attTn7.

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