Frequencies of red blood cell major blood group antigens and phenotypes in the Chinese Han population from Mainland China.

Alloantibodies directed to red blood cell (RBC) antigens play an important role in alloimmune-mediated haemolytic transfusion reactions and haemolytic disease of the foetus and newborn. The frequencies and phenotypes of RBC antigens are different in populations from different geographic areas and races. However, the data on major blood group antigens in the Chinese Han population from Mainland China are still very limited; thus, we aimed to investigate them in this study. A total of 1412 unrelated voluntary Chinese Han blood donors were randomly recruited. All donors were typed for blood group antigens: D, C, c, E, e, C(w) , Jk(a) , Jk(b) ,M, N, S, s, Le(a) , Le(b) , K, k. Kp(a) , Kp(b) , Fy(a) , Fy(b) , Lu(a) , Lu(b) , P1 and Di(a) using serological technology. Calculations of antigen and phenotype frequencies were expressed as percentages and for allele frequencies under the standard assumption of Hardy-Weinberg equilibrium. Amongst the Rh antigens, D was the most common (98.94%) followed by e (92.28%), C (88.81%), c (58.43%), E (50.78%) and C(w) (0.07%) with DCe/DCe (R1 R1 , 40.72%) being the most common phenotype. In the Kell blood group system, k was present in 100% of the donors and a rare phenotype, Kp (a+b+), was found in 0.28% of the donors. For the Kidd and Duffy blood group systems, Jk (a+b+) and Fy (a+b-) were the most common phenotypes (44.05% and 84.35%, respectively). In the MNS blood group system, M+N+S-s+ (45.54%) was the most common, whereas M+N-S-s- and M-N+S-s- were not found. The rare Lu (a-b-) and Lu (a+b+) phenotypes were identified in 0.43% and 1.13% of the donors, respectively. Le(a) and Le(b) were seen in 17.92% and 63.03% of donors, respectively. The frequency of Di(a) was 4.75%, which was higher than in the Chinese population in Taiwan region or the Caucasian and Black populations (P < 0.0001). This study systematically describes the frequencies of 24 blood group antigens in the Chinese Han population from Mainland China. The data can be helpful in creating a donor database for preparation of indigenous cell panels and providing antigen-negative blood to patients with multiple alloantibodies.

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi-dye nucleic acid amplification technology screening test.

BACKGROUND: The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. STUDY DESIGN AND METHODS: A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. RESULTS: Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads <12 IU/ml. CONCLUSION: The cobas(®) TaqScreen MPX test, version 2.0, has an advantage over the current Roche blood screening test, the cobas TaqScreen MPX test, for screening donations in countries with a high prevalence of occult HBV infections since the uncertainty associated with identifying samples with very low viremia is removed by the ability of the test to identify the viral target in samples that are reactive with the cobas TaqScreen MPX test, version 2.0.

Genetic characterization and genotyping of hepatitis B virus (HBV) isolates from donors with an occult HBV infection.

BACKGROUND AND OBJECTIVES: Screening of Thai blood donors has resulted in the detection of donors with an occult HBV infection (OBI), where HBsAg is undetectable, but hepatitis B virus (HBV) DNA is present in serum in low concentrations. This study was designed to determine whether the occurrence of OBI in donors was linked to the HBV genotype and possibly to mutations in the surface (S) and core (C) gene regions. MATERIALS AND METHODS: Mutations in the S and C gene regions in 48 Thai donors with OBI were mapped by sequencing. Genotyping was determined with the INNO-LiPA test and by phylogenetic analysis of sequences from the S and C genes. RESULTS: The majority of OBI samples were genotype C (81·3%) with 6·3% of samples being genotype B. In addition, two genotype I isolates were identified. Mutations in the S region (100%) were found especially in loop 1 of the major hydrophilic loop (MHL) at positions I110L, T114S, T126I and S113T, whereas mutations in the C region (65%) were within the basal core promoter region (position A1762T/G1764A) and precore region (position G1896A). CONCLUSION: The majority of OBI samples were HBV genotype C, although genotype I, which is newly emerging in Thailand, was also detected. The study demonstrated that OBI was probably not associated with a particular HBV genotype or with certain mutations in the S and C gene regions. However, mutations in the C gene region which could potentially impair viral replication and HBsAg production and potentially lead to OBI were identified.

Evaluation of two, commercial, multi-dye, nucleic acid amplification technology tests, for HBV/HCV/HIV-1/HIV-2 and B19V/HAV, for screening blood and plasma for further manufacture.

BACKGROUND: The cobas TaqScreen MPX Test, version 2.0, a multiplex, multi-dye nucleic acid amplification technology (NAT) test from Roche was evaluated by two European Blood Banks, the German Red Cross Blood Donor Service, Frankfurt, Germany and Centro de Hemoterapia y Hemodonación de Castilla y León, Valladolid, Spain. In addition, the cobas TaqScreen DPX Test was evaluated for the simultaneous detection and quantitation of parvovirus B19 and the detection of hepatitis A virus (HAV). STUDY DESIGN AND METHODS: The performances of the two tests were evaluated regarding the analytical sensitivity, the reproducibility of the tests using samples containing low concentrations of each virus and cross-contamination using samples containing high titres of virus. RESULTS: The analytical sensitivity of the MPX Test, version 2.0, obtained by the German Red Cross Blood Donor Service was 1·1, 3·9 and 43·3 IU/ml for HBV, HCV and HIV-1, respectively. The comparable analytical sensitivity at Centro de Hemoterapia y Hemodonación de Castilla y León was 3·5, 17·6 and 50·6 IU/ml for HBV, HCV and HIV-1, respectively. The analytical sensitivity of the DPX test determined by the German Red Cross Blood Donor Service was 0·6 and 3·8 IU/ml for HAV and B19. CONCLUSION: These multiplex and multi-dye blood screening assays represent a flexible NAT screening system for mini-pools between 6 and 96 samples per pool and fulfil all requirements of the European Pharmacopoeia for HCV and B19V testing of plasma for fractionation. The inclusion of a new multi-dye technology means discriminatory assays are no longer required for either test thus improving workflow, turn-around time and minimize the risk of obtaining a reactive result for which the virus cannot be identified.

Symptoms do not predict colorectal cancer in an FOB screened population.

BACKGROUND AND AIMS: The Scottish Bowel Screening Programme aims to detect cancer in asymptomatic individuals. We aimed to measure the prevalence of lower gastrointestinal symptoms in faecal occult blood (FOB) screen-positive patients, to correlate the symptoms with neoplasia and to compare the predictive value of FOB screening with urgent symptomatic referrals in Ayrshire and Arran. METHODS: Data were collected prospectively on FOB screen-positive patients undergoing colonoscopy. Patients completed a symptom questionnaire. Positive predictive values (PPVs) for detecting neoplasia were calculated and a chi-square test was performed to determine any influence of symptoms in diagnosing neoplasia. Symptomatic patients undergoing colonoscopy via a general practice fast-track system were compared. RESULTS: A total of 378 FOB screen-positive patients were included. In all, 198 (52%) had colorectal symptoms. Overall, 32 were diagnosed with colorectal cancer and 93 had polyps . FOB positivity and symptoms gave a PPV of 34% for neoplasia. FOB positivity without symptoms gave a PPV of 32% for neoplasia. Urgent referral of symptomatic patients had a lower PPV of 21% for neoplasia (p < 0.001). CONCLUSION: Half the FOB screen-positive patients had bowel symptoms. Symptoms in these patients had no correlation with an increased rate of neoplasia. The PPV for neoplasia is superior in symptomatic and asymptomatic screen-positive patients when compared to conventional urgent symptom-based referral.

Comparison of positive lymph node ratio with an inflammation-based prognostic score in colorectal cancer.

BACKGROUND: Two prognostic scoring systems have been proposed in colorectal cancer: the pathologically based positive lymph node ratio (pLNR) and the inflammation-based modified Glasgow Prognostic Score (mGPS). This study compared these two scores with the tumour node metastasis (TNM) staging system in terms of cancer survival. METHODS: Between 2003 and 2005, 206 patients, of mean(s.d.) age 69·9(10·6) (range 40-95) years, underwent curative resection for colorectal cancer in two centres. Age, sex, primary tumour site and whether radio/chemotherapy was given were recorded in addition to the three scores (TNM stage, pLNR and mGPS). Univariable and multivariable analyses of overall survival were performed. RESULTS: Age, rectal cancer, TNM stage, pLNR and mGPS were significant factors in univariable analysis. On multivariable analysis, N category and tumour stage (I-III) were removed from the model, leaving pLNR and mGPS as independent predictors of overall survival: hazard ratio 1·51 (95 per cent confidence interval 1·24 to 1·84; P < 0·001) and 1·56 (1·18 to 2·08; P = 0·020) respectively. C-statistic analysis, used to compare pLNR and mGPS directly, found only pLNR to be significant (P < 0·001) CONCLUSION: This study found pLNR to be the superior prognostic scoring system in determining long-term survival in patients undergoing resection for colorectal cancer.

Myofibroblastic inflammatory breast tumor and fibrosarcoma: a case report.

Myofibroblastic inflammatory tumors can occur in any human tissue. They are benign lesions and more common in young patients. We describe the case of a 41-year-old patient with a nodule measuring 2.5 x 1.5 cm in the superior lateral quadrant of the left breast. The pathological examination showed proliferation of the spindle cells in dense fascicles interspersed by colagen among frequent plasmocytes, lymphocytes and eosinophils, associated with scarce typical mitosis. In the same site ten years before the patient had undergone a nodulectomy and the diagnosis was a benign filloid tumor. The patient returned with a nodule in the surgical scar at follow-up and the incisional biopsy showed a malignant filloid tumor with a sarcomatous component. A mastectomy was performed.

Impact of national guidelines on family history breast cancer surveillance.

The breast cancer risk of women already under family history surveillance was accurately assessed according to national guidelines in an attempt to rationalize the service. Women attending two breast units in Glasgow between November 2003 and February 2005 were included. One thousand and five women under annual surveillance were assessed and had their relatives diagnoses verified. Four hundred and ninety-seven women were at significantly increased risk and eligible for follow-up. Five hundred and eight (50%) women attending were not eligible for family history surveillance, and 498 (98%) of these women accepted discharge. In conclusion, national guidelines have helped to more clearly define women who should undergo surveillance. This avoids unnecessary and potentially harmful routine investigations, and the service has been improved.

Does nosocomial COVID-19 result in increased 30-day mortality? A multi-centre observational study to identify risk factors for worse outcomes in patients with COVID-19.

This study aimed to determine whether nosocomial coronavirus disease 2019 (COVID-19) has a worse outcome compared with community-acquired COVID-19. This was a prospective cohort study of all hospitalized patients with confirmed COVID-19 in three acute hospitals on 9th April 2020. Patients were followed-up for at least 30 days. Nosocomial infection was defined as a positive swab after 7 days of admission. In total, one hundred and seventy-three patients were identified, and 19 (11.0%) had nosocomial infection. Thirty-two (18.5%) patients died within 30 days (all cause) of a positive swab test; there were no significant differences in 30-day all-cause mortality rates between the three groups (i.e. patients admitted with suspected COVID-19, patients with incidental COVID-19 and patients with nosocomial COVID-19): 21.1% vs 17.6% vs 21.6% (P=0.755). Nosocomial COVID-19 is not associated with increased mortality compared with community-acquired COVID-19.

Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes.

BACKGROUND: In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. METHODS: The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. RESULTS: Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. CONCLUSIONS: The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

Endoscopists' estimation of size should not determine surveillance of colonic polyps.

OBJECTIVE: Current British Society of Gastroenterology guidelines use adenomatous polyp size as one of the key factors in determining polyp follow-up. This study aimed to compare polyp size assessment by colonoscopists and pathologists before and after fixation to determine the optimal method for measurement. METHOD: Thirty-five colorectal polyps were found during pre-arranged colonoscopies in one centre. Polyp size was measured to the nearest 1 mm by three different methods: 1. by the endoscopist at colonoscopy; 2. by the pathologist fresh, following removal; 3. by the pathologist fixed, following fixation. The endoscopist and the pathologist were blinded to each other's measurements. RESULTS: Seventeen men, eighteen women with mean age of 66.2 years (SD: 9.4, range: 38.7-85.5) underwent polypectomy/s with all polyps removed intact. Polypectomies were performed by consultants (43%), nurse specialists (34%) and specialist registrars (23%). The median size (mm) of polyps measured were endoscopically, 6.5 (2-25 mm); fresh specimen 7.0 (4-28 mm) and fixed 7.0 (4-28 mm). Endoscopic measurements were significantly lower than that of fresh and fixed sizes (P < 0.001 and P = 0.003 respectively), with poor correlation [correlation of variance (CV): 21.0% and intraclass correlation coefficient (ICCC): 0.841 for endoscopic and fresh measurements; CV: 21.1% and ICCC: 0.838 for endoscopic and fixed measurements]. There was no statistical difference between fresh and fixed specimen measurements (P > 0.05; CV: 4.2%, ICCC: 0.974). In three patients, the endoscopic measurement was < 1 cm in polyps that were found to be >or= 1 cm on pathological measurement. CONCLUSIONS: Endoscopists consistently underestimated polyp size. Fixation had no effect on polyp size. Pathologists' measurement of polyp size on fixed specimens should determine the need for further colonoscopic follow-up.

Positive lymph node retrieval ratio optimises patient staging in colorectal cancer.

Alternative lymph node (LN) parameters have been proposed to improve staging in colorectal cancer. This study compared these alternative parameters with conventional TNM staging in predicting long-term survival in patients undergoing curative resection. A total of 295 consecutive patients (mean age 70 years; range 39-95; s.d. 10.4) underwent resection for colorectal cancer from 2001 to 2004. Age, sex, primary tumour site, TNM stage and chemotherapy/radiotherapy were recorded. Patients with colon and rectal cancers were analysed separately for LN parameters: LN total; adequate LN retrieval (> or =12) and inadequate (<12); total number of negative LN; total number of positive LN and the ratio of positive LN to total LN (pLNR). Univariate and multivariate survival analysis was performed. The median number of LN retrieved was 10 (1-57) with adequate LN retrieval in 147 cases (49.8%). For each T and N stage, inadequate LN retrieval did not adversely affect long-term survival (P>0.05). On multivariate analysis, only pLNR was an independent predictor of overall survival in both colon and rectal cancers (HR 11.65, 95% CI 5.00-27.15, P<0.001 and HR 13.40, 95% CI 3.64-49.10, P<0.001, respectively). Application of pLNR subdivided patients into four prognostic groups. Application of the pLNR improved patient stratification in colorectal cancer and should be considered in future staging systems.

Chronic antigen ingestion protects ovalbumin sensitized mice from severe manifestation of Leishmania major infection.

In the present work, the development of experimental leishmaniasis was examined in sensitized BALB/c mice that were chronically fed with antigen. After an oral challenge with egg white solution, the ovalbumin (Ova)-sensitized mice showed an increase in serum anti-Ova IgE and IgG1 antibodies. Lesions induced by Leishmania major infection were reduced by the ingestion of Ova in sensitized mice, as assessed by reduced footpad growth, lower parasite loads and improved pathological outcome compared to sham sensitized mice. Moreover, such findings were connected to a shift to a Th1 response involving higher IFN-gamma production and serum levels of IgG2a anti-Leishmania antigens. The data appear to corroborate the suggestion that chronic ingestion of an antigen by sensitized mice modulates the immunological system through a shift in cytokine release, exhibiting a healing response and resistance to L. major infection.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway.

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

Reshaping a human antibody to inhibit the interleukin 6-dependent tumor cell growth.

The mouse PM-1 monoclonal antibody binds to the human interleukin 6 receptor, inhibits IL-6 functions, and shows strong antitumor cell activity against multiple myeloma cells. In order to be effective as a therapeutic agent administered to human patients in repeated doses, reshaped human PM-1 antibodies consisting of human REI-based light chain and NEW-based heavy chain variable regions were designed and constructed with the assistance of a structural model of the mouse PM-1 variable regions. The best reshaped human PM-1 antibody is equivalent to mouse or chimeric PM-1 antibody in terms of antigen binding and growth inhibition against multiple myeloma cells. Only a few minor changes in the human framework regions were required to recreate the mouse PM-1 antigen-binding site within a human antibody. The reshaped human PM-1 antibody, therefore, could be efficacious in human multiple myeloma patients.

Natural history of drug-resistant clones of Streptococcus pneumoniae colonizing healthy children in Portugal.

A total of 3,539 Streptococcus pneumoniae (Pn) were recovered from 4,969 nasopharyngeal samples of children attending 13 day-care centers (DCCs) located in Lisbon, Portugal, during a surveillance study from January, 2001, through March, 2003, integrated in the European intervention project (EURIS, European Resistance Intervention Study). All Pn isolates were tested for anti-biotyping and drug-resistant pneumococci (DRPn) were further tested by serotyping and pulsed-field gel electrophoresis (PFGE). Overall carriage of Pn was very high (71.2%) and 39.9% of the isolates were resistant to antimicrobials (22.5% with decreased susceptibility to penicillin and 17.4% susceptible to penicillin and resistant to other antimicrobials). Serotypes 6B, 14, 23 F, 19F, and 19 A were prevalent among the 1,287 DRPn and 5.8% of the isolates were non-typeable. Eighty PFGE patterns were identified among 1,285 DRPn, and 93.1% of the DRPn belonged to 26 major clonal types that comprised: Pneumococcal Molecular Epidemiology Network (PMEN) clones (76.3%), Portuguese (PT)-DCC clones, previously detected in 1996-1999 (14.3%), and EURIS PT-DCC new clones, identified for the first time in the EURIS study, during 2001-2003 (9.4%). Comparing with previous Portuguese surveillance studies carried out since 1996, we observed that carriage increased from 47% to 71%, but no major changes were detected on the prevalence of pneumococcal serotypes. Moreover, although PMEN clones were predominant in all DCCs, in the present study the majority of them were gradually decreasing in time whereas several PT-DCC and new clones seemed to be increasing.

A single backmutation in the human kIV framework of a previously unsuccessfully humanized antibody restores the binding activity and increases the secretion in cos cells.

Humanization of rodent mAbs by CDR-grafting (also called "reshaping") is now a standard procedure for reducing immunogenicity and recruiting human effector functions. However, the design of the humanized mAb can sometimes prove circuitous. Attempts were made to humanize L-25, a mouse antibody against the human alpha-4 integrin subunit using the usual protocols. Despite reaching eight backmutations in the light chain, it was not possible to recover the binding activity to the level of the chimeric. In an effort to restore the binding activity, an analysis of the human kappa IV acceptor frameworks was undertaken. This analysis highlighted the Asp at position 9 in framework 1, which although a common amino acid in human kappa IV frameworks, was an unusual residue in mouse kappa frameworks. Backmutating this position to the mouse amino acid completely restored the binding of the humanized antibody and as a by-product also increased the secretion levels in cos cells. Mutating position 9 to the consensus residue for human kappa I also restored the binding and secretion levels although not to the same extent. The resulting humanized antibody had a light chain with only a single backmutation to the mouse sequence.

Humanization of a mouse anti-human interleukin-6 receptor antibody comparing two methods for selecting human framework regions.

Mouse monoclonal antibody AUK12-20 binds to human IL-6 receptor and inhibits IL-6 functions. It has been humanized by CDR-grafting for therapeutic use. In the design of reshaped human AUK12-20 VL region, the human framework regions (FRs) from the human Bence-Jones protein REI were used. The reshaped human AUK12-20 light chain, in combination with chimeric AUK12-20 heavy chain, bound to antigen as well as chimeric AUK12-20 antibody. In the design of reshaped human AUK12-20 VH region, two sets of the human FRs were chosen and compared. One set was from the consensus amino acid sequence for human VH regions subgroup (HSG)-I and the other set was from human antibody HAX, the most similar human VH region found in a database of human immunoglobulin sequences. The HSG-I-based and the HAX-based reshaped human AUK12-20 heavy chains in combination with the reshaped human AUK12-20 light chain, showed approximately 90 and 100% antigen-binding and competition-binding activities as compared to the chimeric or mouse AUK12-20 heavy chains. Most importantly, these humanized antibodies inhibited the IL-6-dependent tumor cell growth as well as the original mouse antibody suggesting that these humanized antibodies could be efficacious in human patients. Our results show that both approaches for the design of reshaped human antibodies can be used for successful humanization. The approach based on FRs from the most similar individual human antibody, however, seemed to be best for designing a reshaped human antibody that mimicked as closely as possible the original mouse antibody.