EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies.

Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains - two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cε2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).

Interplay between kinase domain autophosphorylation and F-actin binding domain in regulating imatinib sensitivity and nuclear import of BCR-ABL.

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML.

Polymorphism in ARE-I region of prostate-specific antigen gene associated with low serum testosterone level and high-grade prostate cancer.

OBJECTIVES: To examine the impact of polymorphism in the androgen-responsive element I region of the prostate-specific antigen (PSA) gene on the serum testosterone level and Gleason score in patients with newly diagnosed, untreated prostate cancer (PCa). High-grade PCa is associated with a low serum testosterone level, and the testosterone level has been negatively correlated with the expression of PSA. METHODS: Endocrine factors (including testosterone, follicle-stimulating hormone, and luteotropic hormone), PSA level, prostate volume, and Gleason score were measured in 134 patients with untreated, biopsy-verified PCa. PSA polymorphism was determined by polymerase chain reaction-based methods using DNA from peripheral blood samples. RESULTS: Patients with the PSA G/G genotype had lower serum testosterone levels (3.5 +/- 1.2 ng/mL) than those with the A/A genotype (4.3 +/- 1.6 ng/mL) or the A/G genotype (4.4 +/- 1.5 ng/mL). The PSA level in the A/A and A/G genotype groups were significantly lower than that in the G/G genotype group (18.2 +/- 55.0 ng/mL versus 20.5 +/- 27.6 ng/mL, P = 0.013). In a multiple logistic regression model, the odds ratio for the G/G polymorphism was significantly increased for Gleason score (odds ratio 2.4, 95% confidence interval 1.6 to 10.4; P = 0.02) and serum testosterone level (odds ratio 0.44, 95% confidence interval 0.36 to 0.94; P = 0.01) relative to genotypes A/A and A/G. CONCLUSIONS: Our results showed that the PSA G/G genotype is associated with a greater Gleason score and serum PSA level but lower serum testosterone level and could be considered a risk factor for a poor outcome of PCa.

Associations of serum testosterone with microvessel density, androgen receptor density and androgen receptor gene polymorphism in prostate cancer.

PURPOSE: We investigate potential associations of serum testosterone with microvessel density, androgen receptor expression and AR gene polymorphism in men with untreated prostate cancer. MATERIAL AND METHODS: Serum luteinizing hormone, follicle-stimulating hormone, estradiol and testosterone were determined in men with newly diagnosed prostate cancer. The number of tumor vessels per 0.46 mm. and androgen receptor density (as the percent positive nuclei) were quantified immunohistochemically on prostate cancer areas of prostate biopsy specimens. Polymorphisms within the AR gene (number of CAG repeats) were determined by polymerase chain reaction and restriction fragment length polymorphism analysis using DNA from peripheral blood. RESULTS: The 39 men entered into this study were grouped into 16 with low (3 ng./ml. or less, group 1) and 23 with normal (greater than 3 ng./ml., group 2) serum testosterone. Mean prostate specific antigen +/- SD was significantly lower in group 1 than in group 2 (18.8 +/- 11.1 versus 27.2 +/- 12.2 ng./ml., p = 0.03). Mean Gleason score (7.4 +/- 1.3 versus 6.0 +/- 1.2, p = 0.01), androgen receptor density (96.6% +/- 2.8% versus 84.8% +/- 7.2%, p = 0.03) and tumor vessel density (63.0 +/- 30.8/0.46 versus 39.0 +/- 22.9/0.46 mm.2, p = 0.007) were significantly higher in group 1 than in group 2. The number of CAG repeats within the AR gene did not correlation with serum androgen. CONCLUSIONS: Low serum testosterone in men with newly diagnosed prostate cancer is associated with higher tumor microvessel and androgen receptor density as well as with higher Gleason score, suggesting enhanced malignant potential.

Polymorphic CAG repeats in the androgen receptor gene, prostate-specific antigen polymorphism and prostate cancer risk.

As the development of prostate cancer is androgen-dependent, it has been hypothesized that variation in transcriptional activity by the androgen receptor (AR) related to polymorphic CAG repeats in exon 1, influences prostate cancer risk. The AR regulates gene transcription by binding to androgen-response elements (AREs) in target genes, such as the prostate-specific antigen (PSA). In the ARE-I sequence of the PSA gene an adenine to guanine polymorphism is described. It has been hypothesized that the AR binds the two PSA alleles (A and G) with differing affinities and may, thereby, differentially influence prostate cancer risk. To examine the role of the polymorphisms in the AR and PSA genes in prostate cancer susceptibility, we conducted a case-control study of Austrian Caucasians with 190 newly diagnosed prostate cancer patients and 190 age-matched control men with benign prostatic hyperplasia (BPH). The polymorphisms were determined by polymerase chain reaction (PCR)-based methods using DNA from peripheral white blood cells. Logistic regressions were performed to calculate odds ratios (OR) and confidence limits (CL) and to control for possible confounders. Our data provide no evidence for an association between prostate cancer and CAG repeat length. However, we found a significant influence of the ARE-I PSA polymorphism on prostate cancer risk, when calculating the combination of the A/G and G/G genotypes relative to subjects with the A/A genotype (OR = 0.63; 95% CL 0.39-0.99; P = 0.048), suggesting that the G allele has a protective effect. In a case analysis according to Gleason score, the PSA G/G genotype was significantly more frequent in patients with Gleason score >7 (35.1%) than in patients with Gleason score <7 (21.5%), providing evidence that the PSA G/G genotype is associated with more advanced disease at time of diagnosis. However, the ambivalent role of the PSA during prostate carcinogenesis needs further investigation.

Association of polymorphisms within androgen receptor, 5alpha-reductase, and PSA genes with prostate volume, clinical parameters, and endocrine status in elderly men.

BACKGROUND: The aim of this study was to assess the impact of polymorphisms of three genes within the androgen pathway on prostate volume, clinical parameters, and endocrine status. METHODS: Elderly men with lower urinary tract symptoms underwent clinical and endocrine work-up. In parallel, polymorphisms within the 5alpha-reductase gene (SRD5A2 V89L and A49T), the androgen receptor gene (AR; number of CAG repeats), and the prostate specific antigen (PSA) gene (A --> G substitution at position-158) were determined by polymerase chain reaction and restriction-length polymorphism analysis by using DNA from peripheral blood. RESULTS: A total of 190 men (66.5 +/- 9.2 yr) were analyzed. The number of CAG repeats within the AR and the PSA polymorphism revealed no associations to clinical and endocrine parameters. Individuals carrying the mutated SRD5A2 A49T allele (5.3% of the total population) had larger prostates (54.1 vs. 39.3 ml), higher PSA levels (12.2 vs. 4.3 ng/ml), and a 35% reduction in prostatic stroma/epithelial cell ratio. Men with the mutated SRD5A2 V89L gene had lower testosterone levels. CONCLUSIONS: In contrast to prostate cancer, polymorphisms within AR and PSA genes do not seem to be of importance for benign prostatic hyperplasia. Polymorphisms within the 5alpha-reductase gene are interesting biomarkers for the development of benign prostatic hyperplasia and benign prostatic enlargement.