Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination.

Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis.

Eukaryotic DNA Polymerases in Homologous Recombination.

Homologous recombination (HR) is a central process to ensure genomic stability in somatic cells and during meiosis. HR-associated DNA synthesis determines in large part the fidelity of the process. A number of recent studies have demonstrated that DNA synthesis during HR is conservative, less processive, and more mutagenic than replicative DNA synthesis. In this review, we describe mechanistic features of DNA synthesis during different types of HR-mediated DNA repair, including synthesis-dependent strand annealing, break-induced replication, and meiotic recombination. We highlight recent findings from diverse eukaryotic organisms, including humans, that suggest both replicative and translesion DNA polymerases are involved in HR-associated DNA synthesis. Our focus is to integrate the emerging literature about DNA polymerase involvement during HR with the unique aspects of these repair mechanisms, including mutagenesis and template switching.

Expression of human BRCA2 in Saccharomyces cerevisiae complements the loss of RAD52 in double-strand break repair.

BRCA2 is a tumor-suppressor gene that is normally expressed in the breast and ovarian tissue of mammals. The BRCA2 protein mediates the repair of double-strand breaks (DSBs) using homologous recombination, which is a conserved pathway in eukaryotes. Women who express missense mutations in the BRCA2 gene are predisposed to an elevated lifetime risk for both breast cancer and ovarian cancer. In the present study, the efficiency of human BRCA2 (hBRCA2) in DSB repair was investigated in the budding yeast Saccharomyces cerevisiae. While budding yeast does not possess a true BRCA2 homolog, they have a potential functional homolog known as Rad52, which is an essential repair protein involved in mediating homologous recombination using the same mechanism as BRCA2 in humans. Therefore, to examine the functional overlap between Rad52 in yeast and hBRCA2, we expressed the wild-type hBRCA2 gene in budding yeast with or without Rad52 and monitored ionizing radiation resistance and DSB repair efficiency. We found that the expression of hBRCA2 in rad52 mutants increases both radiation resistance and DSB repair frequency compared to cells not expressing BRCA2. Specifically, BRCA2 improved the protection against ionizing radiation by at least 1.93-fold and the repair frequency by 6.1-fold. In addition, our results show that homology length influences repair efficiency in rad52 mutant cells, which impacts BRCA2 mediated repair of DSBs. This study provides evidence that S. cerevisiae could be used to monitor BRCA2 function, which can help in understanding the genetic consequences of BRCA2 variants and how they may contribute to cancer progression.

Saccharomyces cerevisiae Mus81-Mms4 prevents accelerated senescence in telomerase-deficient cells.

Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae.

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

Mpox gastrointestinal manifestations: a systematic review.

INTRODUCTION: Mpox is a viral infection caused by the monkeypox virus, a member of the Poxviridae family and Orthopoxvirus genus. Other well-known viruses of the Orthopoxvirus genus include the variola virus (smallpox), cowpox virus and vaccinia virus. Although there is a plethora of research regarding the dermatological and influenza-like symptoms of mpox, particularly following the 2022 mpox outbreak, more research is needed on the gastrointestinal (GI) effects. OBJECTIVES: This systematic review is to outline the GI manifestations of the monkeypox virus. METHODS: The authors conducted this systematic review using guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. A search was conducted through the PubMed, EMBASE and MEDLINE databases from January 1958 to June 2023. The authors selected English language papers that discussed the GI symptoms in mpox patients. A manual search was also conducted in the reference sections of these publications for other relevant papers. RESULTS: 33 papers involving 830 patients were selected for this review. The GI manifestations in mpox patients are proctitis, vomiting, diarrhoea, rectal pain, nausea, tenesmus, rectal bleeding and abdominal pain. Although various papers explored transmission routes, one paper established a direct connection between anal-receptive sex transmission route and the development of a GI complication (proctitis). Another study reported that the mode of transmission could potentially impact the occurrence of GI symptoms and severity of the disease. The reviewed papers did not discover a relation between the severity of dermatological and influenza-like symptoms and the GI manifestations mentioned. CONCLUSION: This systematic review confirms that GI manifestations are observed in mpox patients. GI symptoms of mpox are crucial for gastroenterologists and other healthcare professionals to recognise in order to address patient discomfort and further understand the pathophysiology of the virus.

Rad51 determines pathway usage in post-replication repair.

Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro . A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 Å resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.

Ocular Manifestations and Potential Treatments of Alport Syndrome: A Systematic Review.

Objectives: Alport syndrome (AS) is a severe, rare hereditary disorder that can lead to end-stage renal disease, auditory degeneration, and ocular abnormalities. Despite extensive research on AS in relation to auditory and renal disorders, more research is needed on the ocular presentations of AS. This systematic review aims to summarize the common ocular abnormalities in patients with AS and to explore the potential treatment options for these irregularities. Methods: The PubMed, MEDLINE, and EMBASE databases were systematically searched from January 1977 to April 2022. Only papers that were published in the English language and explored the ocular abnormalities in AS patients were selected. We manually searched reference lists of included papers for additional studies. Results: A total of 23 articles involving 195 patients were included in this review. The common ocular manifestations in AS patients are lenticonus, macular holes, fleck retinopathy, and thinning of the macula. Although published literature has described the use of cataract surgeries and vitrectomies as standard surgical techniques to alleviate ocular abnormalities in non-AS patients, it must be noted that surgical techniques have not been evaluated in a large research study as a solution for AS abnormalities. Another prospective treatment for AS is gene therapy through the reversion of causative COL4 variants to wild type or exon-skipping therapy for X-linked AS with COL4A5 truncating mutations. Gene therapy, however, remains unable to treat alterations that occur in the fetal and early development phase of the disease. Conclusions: The review found no definitive conclusions regarding the efficacy and safety of surgical techniques and gene therapy in AS patients. Recognition of ocular abnormalities through an ophthalmic examination with an optical coherence tomography (OCT) and slit-lamp examination is critical to the medical field, as ophthalmologists can aid nephrologists and other physicians in diagnosing AS. Early diagnosis and care can minimize the risk of detrimental ocular outcomes, such as blindness and retinal detachment.

Mating type influences chromosome loss and replicative senescence in telomerase-deficient budding yeast by Dnl4-dependent telomere fusion.

As we age, the majority of our cells gradually lose the capacity to divide because of replicative senescence that results from the inability to replicate the ends of chromosomes. The timing of senescence is dependent on the length of telomeric DNA, which elicits a checkpoint signal when critically short. Critically short telomeres also become vulnerable to deleterious rearrangements, end-degradation and telomere-telomere fusions. Here we report a novel role of non-homologous end-joining (NHEJ), a pathway of double-strand break repair in influencing both the kinetics of replicative senescence and the rate of chromosome loss in telomerase-deficient Saccharomyces cerevisiae. In telomerase-deficient cells, the absence of NHEJ delays replicative senescence, decreases loss of viability during senescence, and suppresses senescence-associated chromosome loss and telomere-telomere fusion. Differences in mating-type gene expression in haploid and diploid cells affect NHEJ function, resulting in distinct kinetics of replicative senescence. These results suggest that the differences in the kinetics of replicative senescence in haploid and diploid telomerase-deficient yeast are determined by changes in NHEJ-dependent telomere fusion, perhaps through the initiation of the breakage-fusion-bridge cycle.

Cooperation between non-essential DNA polymerases contributes to genome stability in Saccharomyces cerevisiae.

DNA polymerases influence genome stability through their involvement in DNA replication, response to DNA damage, and DNA repair processes. Saccharomyces cerevisiae possess four non-essential DNA polymerases, Pol λ, Pol η, Pol ζ, and Rev1, which have varying roles in genome stability. In order to assess the contribution of the non-essential DNA polymerases in genome stability, we analyzed the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant in microhomology mediated repair, due to recent studies linking some of these DNA polymerases to this repair pathway. Our results suggest that the length and quality of microhomology influence both the overall efficiency of repair and the involvement of DNA polymerases. Furthermore, the non-essential DNA polymerases demonstrate overlapping and redundant functions when repairing double-strand breaks using short microhomologies containing mismatches. Then, we examined genome-wide mutation accumulation in the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant compared to wild type cells. We found a significant decrease in the overall rate of mutation accumulation in the quadruple mutant cells compared to wildtype, but an increase in frameshift mutations and a shift towards transversion base-substitution with a preference for G:C to T:A or C:G. Thus, the non-essential DNA polymerases have an impact on the nature of the mutational spectrum. The sequence and functional homology shared between human and S. cerevisiae non-essential DNA polymerases suggest these DNA polymerases may have a similar role in human cells.

Telomere dysfunction drives increased mutation by error-prone polymerases Rev1 and zeta in Saccharomyces cerevisiae.

Using a model system, we have shown that replicative senescence is accompanied by a 16-fold increase in base substitution and frameshift mutations near a chromosome end. The increase was dependent on error-prone polymerases required for the mutagenic response to DNA lesions that block the replication fork.

Lymphocyte activation antigen CD70 expressed by renal cell carcinoma is a potential therapeutic target for anti-CD70 antibody-drug conjugates.

Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.

Antibody targeting of B-cell maturation antigen on malignant plasma cells.

B-cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. BCMA binds to two ligands that promote tumor cell survival, a proliferation inducing ligand (APRIL) and B-cell activating factor. To selectively target BCMA for plasma cell malignancies, we developed antibodies with ligand blocking activity that could promote cytotoxicity of multiple myeloma (MM) cell lines as naked antibodies or as antibody-drug conjugates. We show that SG1, an inhibitory BCMA antibody, blocks APRIL-dependent activation of nuclear factor-kappaB in a dose-dependent manner in vitro. Cytotoxicity of SG1 was assessed as a naked antibody after chimerization with and without Fc mutations that enhance FcgammaRIIIA binding. The Fc mutations increased the antibody-dependent cell-mediated cytotoxicity potency of BCMA antibodies against MM lines by approximately 100-fold with a > or = 2-fold increase in maximal lysis. As an alternative therapeutic strategy, anti-BCMA antibodies were endowed with direct cytotoxic activity by conjugation to the cytotoxic drug, monomethyl auristatin F. The most potent BCMA antibody-drug conjugate displayed IC(50) values of < or = 130 pmol/L for three different MM lines. Hence, BCMA antibodies show cytotoxic activity both as naked IgG and as drug conjugates and warrant further evaluation as therapeutic candidates for plasma cell malignancies.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

Development of potent monoclonal antibody auristatin conjugates for cancer therapy.

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.

Efficient elimination of B-lineage lymphomas by anti-CD20-auristatin conjugates.

The anti-CD20 antibody rituximab is useful in the treatment of certain B-cell malignancies, most notably non-Hodgkin's lymphoma. Its efficacy has been increased when used in combination with chemotherapy, yet anti-CD20 monoclonal antibodies (mAbs) directly conjugated with drugs such as doxorubicin (Dox) have failed to deliver drug or to demonstrate antitumor activity. We have produced anti-CD20 antibody-drug conjugates that possess potent antitumor activity by using the anti-mitotic agent, monomethyl auristatin E (MMAE), linked via the lysosomally cleavable dipeptide, valine-citrulline (vc). Two anti-CD20 conjugates, rituximab-vcMMAE and 1F5-vcMMAE, were selectively cytotoxic against CD20(+) B-lymphoma cell lines, with IC(50) values ranging from 50 ng/mL to 1 microg/mL. Unlike rituximab, which showed diffuse surface localization, rituximab-vcMMAE capped and was internalized within 4 hours after binding to CD20(+) B cells. Internalization of rituximab-vcMMAE was followed by rapid G(2)-M phase arrest and onset of apoptosis. Anti-CD20 antibody-drug conjugates prepared with Dox were internalized and localized as with rituximab-vcMMAE, yet these were not effective for drug delivery (IC(50) > 50 microg/mL). Consistent with in vitro activity, rituximab-vcMMAE showed antitumor efficacy in xenograft models of CD20-positive lymphoma at doses where rituximab or rituximab-Dox conjugates were ineffective. These data indicate that anti-CD20-based antibody-drug conjugates are effective antitumor agents when prepared with a stable, enzyme-cleavable peptide linkage to highly potent cytotoxic agents such as MMAE.

Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate.

PURPOSE: An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 monoclonal antibody (mAb) cAC10, with eight drug moieties per mAb, was previously shown to have potent cytotoxic activity against CD30(+) malignant cells. To determine the effect of drug loading on antibody-drug conjugate therapeutic potential, we assessed cAC10 antibody-drug conjugates containing different drug-mAb ratios in vitro and in vivo. EXPERIMENTAL DESIGN: Coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10 created an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatography to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2, E4, and E8, respectively). Antibody-drug conjugate potency was tested in vitro against CD30(+) lines followed by in vivo xenograft models. The maximum-tolerated dose and pharmacokinetic profiles of the antibody-drug conjugates were investigated in mice. RESULTS: Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC(50) values E8

Comparison of immunoscintigraphy, efficacy, and toxicity of conventional and pretargeted radioimmunotherapy in CD20-expressing human lymphoma xenografts.

UNLABELLED: Pretargeted radioimmunotherapy (RIT) using streptavidin (sAv)-conjugated antibodies before radiolabeled-biotin is a promising approach to improve absorbed dose ratios and achieve high durable remission rates with diminished systemic toxicity. This study compared the immunoscintigraphy, toxicity, and therapeutic efficacy of pretargeted RIT with conventional RIT using an anti-CD20 antibody. METHODS: Athymic mice bearing Ramos human Burkitt's lymphoma xenografts were injected intraperitoneally with a 1F5-sAv conjugate followed 24 h later by a galactosylated, biotinylated clearing agent (CA) and, finally, 3 h later by (111)In- or (90)Y-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin. The comparison groups consisted of mice injected with conventional, directly labeled (111)In- or (90)Y-1F5. RESULTS: Rapid tumor uptake of radioactivity within 2 h was observed with the pretargeting approach, resulting in high-contrast tumor images at 24 h with minimal blood-pool radioactivity. Although conventional radiolabeled antibodies produced clear tumor images at 24 h, a large amount of radioactivity was present in the blood pool. The tumor-to-blood ratio was 3.5:1 with pretargeting compared with 0.4:1 with conventional (111)In-1F5. Pretargeted RIT with 29.6 MBq (800 micro Ci) (90)Y-DOTA-biotin cured 100% of mice with tolerable toxicity, whereas conventional RIT with (90)Y-1F5 at a dose of 14.8 MBq (400 micro Ci) produced no cures, induced profound pancytopenia, and was lethal to all mice. CONCLUSION: These results suggest that anti-CD20 pretargeted RIT may be superior to conventional radiolabeled antibodies in terms of radioimmunoscintigraphy, toxicity, and therapeutic efficacy for treatment of B-cell lymphomas.

Telomerase deficiency affects the formation of chromosomal translocations by homologous recombination in Saccharomyces cerevisiae.

Telomerase is a ribonucleoprotein complex required for the replication and protection of telomeric DNA in eukaryotes. Cells lacking telomerase undergo a progressive loss of telomeric DNA that results in loss of viability and a concomitant increase in genome instability. We have used budding yeast to investigate the relationship between telomerase deficiency and the generation of chromosomal translocations, a common characteristic of cancer cells. Telomerase deficiency increased the rate of formation of spontaneous translocations by homologous recombination involving telomere proximal sequences during crisis. However, telomerase deficiency also decreased the frequency of translocation formation following multiple HO-endonuclease catalyzed DNA double-strand breaks at telomere proximal or distal sequences before, during and after crisis. This decrease correlated with a sequestration of the central homologous recombination factor, Rad52, to telomeres determined by chromatin immuno-precipitation. This suggests that telomerase deficiency results in the sequestration of Rad52 to telomeres, limiting the capacity of the cell to repair double-strand breaks throughout the genome. Increased spontaneous translocation formation in telomerase-deficient yeast cells undergoing crisis is consistent with the increased incidence of cancer in elderly humans, as the majority of our cells lack telomerase. Decreased translocation formation by recombinational repair of double-strand breaks in telomerase-deficient yeast suggests that the reemergence of telomerase expression observed in many human tumors may further stimulate genome rearrangement. Thus, telomerase may exert a substantial effect on global genome stability, which may bear significantly on the appearance and progression of cancer in humans.

Contribution of linker stability to the activities of anticancer immunoconjugates.

The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.