Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.Monoclonal antibodies (mAbs) are a rapidly growing class of therapeutics that combine high binding affinities and specificities with long in vivo half-lives. A large number of mAbs have been approved for therapeutic use or are in development (1–6). Early therapeutic mAbs were derived from mouse sources, retained mouse sequences, and were thus immunogenic when used in human patients, limiting the ability to dose repeatedly. The use of humanized and/or fully human antibodies avoided immunogenicity problems and allowed long-term treatment of chronic diseases. Thus, a variety of systems were developed to create humanized or fully human therapeutic antibodies (7–18). One popular approach involves the in vitro isolation of antigen-specific antibodies using display strategies involving human antibody fragments expressed on the surface of phage, bacteria, or yeast (12). Although these synthetic approaches can be quite powerful and can rapidly generate leads, they potentially result in increased immunogenicity in vivo, and the initial display-derived antibody fragments can subsequently require extensive post hoc protein reengineering efforts when reformatted into conventional antibody formats to overcome issues such as insolubility, aggregation, and proteolysis (1, 12, 19). Natural selection of antibodies in vivo within mammalian systems tends to optimize desirable biochemical and pharmacokinetic properties, avoiding the need for extensive post hoc reengineering. Thus, as first proposed based on the finding that human Ig genes efficiently rearrange when introduced into mouse pre-B cells (20), another popular approach for generating human therapeutic mAbs was developed using transgenic mice genetically engineered to produce fully human antibodies (15–18). These so-called HumAb mice were engineered using a “KO-plus-transgenic” strategy in which the endogenous murine Ig genes were disrupted to eliminate the endogenous mouse immune response, whereas transgenic introduction of human Ig loci at different random genetic loci drove production of fully human antibodies.Although HumAb mice generated using this KO-plus-transgenic approach represented a transforming advance in the field, they suffered, however, from partial immune deficiencies compared with WT mice, limiting their ability to produce robust Ab responses to some antigens (21–23). The immune deficiencies of these first-generation HumAb mice may be due to the manner in which they were genetically engineered. First, the genomic context of the randomly inserted human transgenes may contribute to their inefficient functionality, as they may lack extended locus control regions such as the 3′ enhancers (24) and regulatory region (25) of the Ig heavy (IgH) locus, which have been shown to play critical roles in Ig expression and class switching or even alter the 3D location of the Ig genes within B-cell nuclei (26, 27) and thus perturb function in unanticipated ways. In addition, the immune deficiencies of the first-generation HumAb mice may be partly explained by their use of human constant regions. Immunoglobulins interact with other components of the B-cell receptor (BCR) signaling complex via their constant regions, and such interactions are required for appropriate signaling required for antigen-independent B-cell development in bone marrow, as well as antigen-dependent natural selection processes in the periphery (28–36). Others previously noted that interactions between constant regions and BCR coreceptors do not operate efficiently across species (29). Thus, we reasoned that the immune deficiencies in first-generation HumAb mice may in part be due to inefficient interspecies protein-protein interactions between human constant regions and the mouse coreceptors. Finally, the secreted human immunoglobulins produced in the HumAb mice may also interact inefficiently with various mouse Fc receptors, further adversely affecting the humoral immune response (37, 38).As described in the companion article (39), we attempted to exploit the advantages and overcome the limitations of the first-generation HumAb approaches by precisely replacing the entire mouse germ-line variable region gene repertoire with the equivalent human germ-line variable sequences in situ, while maintaining all mouse constant regions and all known gene expression control elements within the natural mouse genomic location. We reasoned that the introduced human Ig variable gene segments would rearrange normally, be linked to mouse constant regions, and furthermore be expressed from the endogenous mouse Ig loci at physiologically appropriate levels. Because these “reverse chimeric” antibody molecules would bear human antigen-binding variable domains fused to mouse constant domains, we presumed they would interact in a species appropriate way with mouse BCR coreceptors and mouse Fc receptors, resulting in a fully functional immune system. We refer to these humanized Ig variable domain mice as VelocImmune mice because they were generated using VelociGene technology (40).In this paper, we show that VelocImmune mice have a humoral immune system indistinguishable from that of WT mice, with normal cell populations at all stages of B-cell development and normal lymphoid organ structures. Sequences of antibodies derived from the humanized Ig loci exhibit normal variable segment rearrangement, somatic hypermutation, and class switching. Immunizations of VelocImmune mice generate robust humoral responses from which a large diversity of monoclonal antibodies can be isolated. Thus, VelocImmune mice are a unique platform for the efficient production of fully human antibodies, several of which have already entered clinical development.     

从外周血单个核细胞扩增多样性人免疫球蛋白基因的研究

目的 建立成熟的体外扩增多样性人免疫球蛋白基因的实验方法。方法 设计多对具有简并性特点的人IgG、IgM扩增引物，从人外周血单个核细胞(PBMCs)中提取RNA并反转录为cDNA，以其为模板聚合酶链反应(PCR)扩增人免疫球蛋白κ轻链基因和重链Fd段基因，对扩增产物进行凝胶电泳和DNA指纹分析鉴定。结果 利用不同引物进行的PCR反应均成功扩增出相应的免疫球蛋白基因，经鉴定所获得的基因产物具有良好的多样性，在天然抗角蛋白自身抗体研究和抗体库的构建中得到初步应用。结论 合理设计引物能够从PBMCs中成功扩增出多样性的人免疫球蛋白基因，为人抗体和相关免疫分子以及自身免疫性疾病的研究提供了方便条件．     

Viral load change and sequential evolution of entire hepatitis C virus genome in Irish recipients of single source-contaminated anti-D immunoglobulin*

In hepatitis C virus (HCV) infection, serum viral load is important in the prediction of therapeutic efficacy. However, factors that affect the viral load remain poorly understood. To identify viral genomic elements responsible for the viral load, we investigated samples from a population of Irish women who were iatrogenically infected from a single HCV source by administration of HCV 1b-contaminated anti-D immune globulin between 1977 and 1978 (Kenny-Walsh, N Engl J Med 1999; 340: 1228). About 15 patients were divided into two groups, viral load increasing group (11 patients) and decreasing group (4 patients). Pairs of sera were collected from each patient at interval between 1.1 and 5.8 years. Full-length sequences of HCV genome were determined, and analyzed for changes in each patient. Sliding window analysis showed that the decreasing group had significantly higher mutation rates in a short segment of NS5B region that may affect the activity of RNA-dependent RNA polymerase. By comparing each coding regions, significantly higher mutation numbers were accumulated in NS5A region in the increasing group than the decreasing group (0.92 vs 0.16 nucleotides/site/year, P = 0.021). The mutation in certain positions of the HCV genome may be determinant factors of the viral load in a relatively homogeneous patient population.     

Assignment of genes for immunoglobulin kappa and heavy chains to chromosomes 6 and 12 in mouse.

Using somatic cell hybrids from fusions of lymphocytes of two different mouse stocks with the myeloma cell line X63-Ag8, we have assigned genes for the immunoglobulin heavy and kappa-type light chains to chromosomes 12 and 6, respectively. The two mouse stocks exhibit karyotypes consisting of nine pairs of metacentric chromosomes as a result of centric fusions of acrocentric chromosomes in different combinations. In the hybrid cells these metacentric chromosomes can be distinguished from the acrocentric chromosomes of myeloma origin, permitting correlation of Ig chain expression with mitotic loss of individual metacentric chromosomes.     

Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes

Background

Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive.

Design and Methods

To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters.

Results

Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets.

Conclusions

Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.     

Dual rearrangement of immunoglobulin and T-cell receptor genes in blast crisis of CML

Dual rearrangement of immunoglobulin and T-cell antigen receptor (beta, delta) genes was demonstrated in a case of Philadelphia chromosome-positive chronic myeloid leukemia (CML) in blast crisis. The blast cells, showing L2 morphology and high activity of TdT, expressed pre-B cell (CD19+, Ia+) and myeloid (CD13+, CD34+) surface antigens but lacket T-cell antigens (CD2-, CD7-). Cytogenetic studies on bone marrow and peripheral blood revealed the Phl chromosome in all metaphases analyzed, majority of which also had the additional chromosome changes, +8, +10, +21. Furthermore, molecular analysis of the breakpoint cluster region (bcr) on chromosome 22 showed a rearrangement, confirming the CML origin of the blast cells.     

抗幽门螺杆菌重组VacA卵黄抗体保护剂的探讨

目的比较硫糖铝和多种糖类对抗幽门螺杆菌重组VacA卵黄抗体保护作用,为制备能耐受胃酸和蛋白酶的IgY制剂奠定基础。方法诱导DH5-αvacA-pQE30大量表达VacA蛋白,制备纯化的VacA IgY。在不同酸性的IgY液中加入同浓度各种保护剂,在相同酸性的IgY液中加入不同浓度的各种保护剂,然后加入不同浓度硫糖铝和正常胃内浓度或高浓度的胃蛋白酶,以ELISA法测定试验组IgY的剩余抗体活性（AR）。结果在pH2.0和3.0条件下,各保护剂均能提高IgY抗体活性,且硫糖铝对IgY抗体活性的保护作用优于糖类,在此条件下50%和40%硫糖铝均可完全保持IgY抗体活性。在pH2.0-3.0及正常或高胃蛋白酶浓度下,30%以上硫糖铝均可有效保护IgY的活性。结论低pH下硫糖铝对IgY抗体活性的保护作用优于糖类,且30%以上硫糖铝可增强VacA IgY对胃蛋白酶的耐受能力,是较理想的IgY保护剂。     

Icteric acute hepatitis E with no response of immunoglobulin M class anti‐hepatitis E virus antibody

A 68‐year‐old Japanese man developed icteric acute hepatitis during periodic care after undergoing gastrectomy due to early gastric cancer. The routine serological markers for hepatitis A, B and C viruses were all negative. Although the liver enzymes spontaneously recovered without any specific therapy, cholestasis was relatively prolonged and successfully treated with prednisolone. Determination of serum hepatitis E virus (HEV) RNA revealed the transient infection of HEV, and both immunoglobulin (Ig)A and IgG class anti‐HEV antibodies were detected after the disease onset, whereas those were negative when measured 3 weeks prior to the onset. In addition, the titer of serum IgA class antibody was associated with the clinical signs of hepatitis. In contrast, no IgM class antibody was detected throughout the course. This case suggests that screening only with IgM class antibody is not sufficient to detect acute HEV infection.     

High levels of immunoglobulin A anti-tissue transglutaminase antibodies at diagnosis are a predictive factor for celiac hepatitis

Introduction: Celiac hepatitis is characterized by the presence of liver injury in patients with celiac disease that resolves after gluten-free diet.

Aim: To evaluate predictive factors of celiac hepatitis at celiac disease diagnosis.

Methods: Retrospective study including 46 adult patients with the diagnosis of celiac disease.

Results: Eighty-seven percent were women, with a mean age of 33?±?11 years, 87% had a Marsh 3 and 46% (n?=?21) had celiac hepatitis. These patients had a median Immunoglobulin A anti-tissue transglutaminase antibody (TTG-IgA) level of 208.0?U/ml (p25–p75: 89–1316?U/ml), a mean aspartate aminotransferase of 42?±?24?U/L, alanine aminotransferase 50?±?28?U/L, alkaline phosphatase 111?±?64?U/L, at the time of diagnosis. Median TTG-IgA one year after diagnosis was 9U/ml (p25–p75: 4.5–30.5?U/ml) and 33% of the patients had normal values. At diagnosis, patients without celiac hepatitis had a median TTG-IgA of 77U/ml (p25–p75: 24–288?U/ml), mean aspartate aminotransferase of 23?±?4?U/L, alanine aminotransferase 20?±?6?U/L, alkaline phosphatase 69?±?17?U/L. Median of TTG-IgA one year after diagnosis was 6?U/ml (p25–p75: 3–19?U/ml) and 48% had normal values. The celiac hepatitis group patients had higher values of TTG-IgA (p?=?0.007) at diagnosis. There was a statistically significant positive correlation between TTG-IgA and alanine aminotransferase (r?=?0.324, p?=?0.028) at diagnosis. The odds of having celiac hepatitis was almost 5-fold higher in patients with a TTG-IgA level higher than 310?U/ml (OR?=?4.8, 95%CI?=?1.213–18.781, p?=?0.025).

Conclusions: Higher TTG-IgA levels are a predictive factor for celiac hepatitis in adult patients with celiac disease at diagnosis.     

Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia

We have previously shown that injection of anti-glycoprotein (GP) IIb induces murine immune thrombocytopenia (ITP) and that intravenous immunoglobulin (IVIg) ameliorates ITP. We hypothesise that murine ITP may be associated with platelet apoptosis, which is upregulated by anti-GPIIb and downregulated by IVIg. The current study demonstrated that anti-GPIIb injection induced three critical apoptosis manifestations in platelets: (i) mitochondrial inner transmembrane potential (delta psi m) depolarisation; (ii) caspase-3 activation; and (iii) phosphatidylserine (PS) exposure. IVIg administration inhibited caspase-3 activation and PS exposure, but not delta psi m-depolarisation, in anti-GPIIb-treated platelets, demonstrating that IVIg ameliorates thrombocytopenia concomitantly with inhibiting late, but not early mechanisms of platelet apoptosis.     

Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes

Summary. The variable region genes used to encode the immunoglobulin expressed by tumour cells of a patient with follicular lymphoma have been identified and sequenced. Initially, a lymph node biopsy was analysed and revealed usage of V_H and V_κ genes which had numerous substitutions as compared with the closest germ line genes. The pattern of mutations in V_H was consistent with a role for positive selection by antigen. In addition, there was evidence in both V_H and V_κ sequences for clonal heterogeneity.
After 5 years, which included treatment with chemotherapy, the patient relapsed with tumour cells present in the blood. Analysis of the V-genes used by the emerging tumour revealed a single homogeneous sequence for both V_H and V_L, which, in each case, matched closely one of the sequences in the original lymph node biopsy.
These results indicate that selection. possibly mediated by antigen, can operate on a cell destined to give rise to lymphoma, and that intraclonal variation can occur after the neoplastic event. However, in this case, late relapse in the blood is dominated by a single clone.     

Pharmacokinetics and safety of Intragam 10 NF, the next generation 10% liquid intravenous immunoglobulin, in patients with primary antibody deficiencies

Background and aims: Intragam® 10 NF is the next generation 10% intravenous immunoglobulin with three pathogen reduction steps and a noncarbohydrate stabiliser. This open label, cross‐over study in patients with primary immunodeficiency was designed to evaluate whether Intragam 10 NF differed in its pharmacokinetics (PK) compared with Intragam P and to assess Intragam 10 NF safety and tolerability. Methods: Nineteen primary immunodeficiency patients were administered one cycle of their existing Intragam P dose (0.2–0.8 g/kg 3–4 weekly), followed by seven cycles of Intragam 10 NF administered at the same dosing schedule as Intragam P. The primary objective was to compare serum immunoglobulin G (IgG) trough levels. Secondary endpoints were PK variables, safety and tolerability. Results: There was no significant within‐patient difference in the average trough immunoglobulin G concentration between Intragam P and Intragam 10 NF (8.76 g/L, 8.55 g/L respectively) (geometrical mean ratio 1.034; 95% confidence interval 0.996–1.073; P = 0.079). Mean PK parameters for both products were similar, with all 95% confidence interval encompassing 1.0 except for time to maximum concentration. Time to maximum concentration occurred earlier with Intragam 10 NF compared with Intragam P, with a shorter infusion time (mean 1.75 h vs 2.52 h respectively; P < 0.05). Headache was the most frequent treatment‐related event following both products. There were no study withdrawals, deaths, or notable changes in laboratory values or vital signs. Conclusion: Intragam 10 NF was well tolerated and exhibited similar PK to Intragam P, with the advantage of a 45 min shorter infusion time.     

Non-lymphoid blast crisis of CML with rearrangement of immunoglobulin and T-cell receptor delta genes

We report a patient with chronic myeloid leukaemia (Philadelphia-positive with M-BCR rearrangement) in transformation whose blast cells had myelomonocytic morphology, absent terminal deoxynucleotidyl transferase expression and non-lymphoid cell surface markers (CD10-, CD19-, CD33+, CD14+, CD11+). Leukaemia cell DNA showed rearrangement of both immunoglobulin heavy chain and T-cell receptor delta genes. Such rearrangements may be a feature of a small proportion of patients with non-lymphoid transformation of CML as they are in a minority of cases of de novo acute non-lymphoblastic leukaemia.     

Expression and rearrangement of homologous immunoglobulin VH genes in two mouse strains.

A family of murine anti-p-azophenylarsonate (Ars) antibodies share a variable (V) region serologically defined marker, the 36-60 idiotype (Id36-60). Most mouse strains possess five genes highly homologous to the gene encoding the heavy (H) chain V region of antibodies bearing Id36-60 (VH36-60); however, only one of these genes is ever utilized by hybridomas whose antibodies bind Ars and bear Id36-60. The relevant VH genes were cloned from A/J and BALB/c mouse DNA libraries. Their DNA sequences were found to differ at only two positions. Southern blot analysis, protein sequence determination, and nucleic acid sequence determination indicate that the above hybridomas utilize the same joining (JH3), diversity (D), and VH gene segments regardless of BALB/c or A/J strain origin. Despite this virtual identity, BALB/c and A/J mouse strains express quite different serum levels of Id36-60-bearing antibodies when immunized with Ars. The basis of this regulatory process is discussed.     

Renal transplantation in HLA sensitized patients: Traversing the immunological barrier

Successful renal transplantation across HLA barrier in sensitized individuals has been on the rise during the past decade, primarily due to improved desensitization regimes. The aim of this study was to share outcome of desensitization in renal transplant recipients with donor‐specific anti‐HLA antibodies (DSA). This was a retrospective analysis of all HLA immunized individuals who were prospective renal transplant recipients. All such patients underwent preconditioning as per the institutional desensitization protocol. Complement‐dependent cytoxicity‐based crossmatch (CDC‐XM), luminex‐based crossmatch (LM‐XM) and flowcytometry‐based crossmatch (FC‐XM) were done in all cases. If any of these tests turned out positive, single antigen bead assay (SAB) was performed. Desensitization for DSA was performed in 55 patients and all patients were followed‐up for 1 year to assess graft function and patient outcome. CDC‐XM being a less sensitive assay, could not detect incompatibility in 29 (52.73%) cases. After desensitization, even though SAB and LM‐XM results revealed an MFI within acceptable range, FC‐XM being an extremely sensitive assay, continued to give a positive result in eight (14.55%) cases. The mean ± SD number of pretransplant TPE were 3.44 ± 0.98 (2‐11). Out of 55, there were 10 patients who were lost to follow up. Patient and graft survival of 45 patients at 1 year was found to be 100%. Preconditioning for renal transplants in the form of immunosuppression with TPE is an extremely useful auxiliary for transplantation in HLA sensitized renal transplant recipients.     

Escherichia coli extract-catalyzed recombination in switch regions of mouse immunoglobulin genes.

We have shown that Escherichia coli extracts catalyze recombination between mouse immunoglobulin mu and alpha genes inserted separately in lambda phage vectors carrying different genetic markers. Most of the recombination sites in the inserts are located in the switch regions of the heavy chain genes, as previously found in the expressed genes of myeloma cells. The recombination took place at relatively high frequency (10(-4)). The recombinational system in E. coli or lambda phage seems to prefer short nucleotide sequences similar to those used in the class switch recombination.     

Humoral immune failure defined by immunoglobulin class and immunoglobulin G subclass deficiency is associated with shorter treatment‐free and overall survival in Chronic Lymphocytic Leukaemia

Immune dysfunction attributed to hypogammaglobulinaemia is common in chronic lymphocytic leukaemia (CLL) and infection is a major contributor to morbidity and mortality. A higher incidence of multiple immunoglobulin and immunoglobulin G (IgG) subclass deficiency was associated with more advanced disease (P < 0·001 and P < 0·001, respectively) in a cohort of 147 CLL patients. Multiple immunoglobulin and IgG subclass deficiency were significantly associated with shorter treatment‐free survival (TFS) (P < 0·001 and P = 0·006, respectively). The association between disease stage and immune dysfunction demonstrated by these data suggest aspects of immune deficiency correlate with disease severity and may be associated with shorter TFS in CLL.     

Intrahepatic synthesis of immunoglobulin in liver disease

ABSTRACT— Patterns of intrahepatic immunoglobulin production were investigated by an in vitro biosynthetic labelling technique which measured the rate of Ig production in liver biopsy fragments. This technique depends on the incorporation of ³H-leucine into proteins synthesized by cells in the biopsy fragment and subsequently released into the culture medium, and precipitation of Ig with monospecific antisera. Intrahepatic Ig production was expressed as counts of radioactivity precipitated/g of liver tissue/24 h. Mean values were high in various inflammatory diseases of the liver, including alcoholic hepatitis (AH) (17 cases), IgG, 87.8, IgA, 105.6 and IgM, 14.7, chronic active hepatitis (CAH) (19 cases), IgG, 86.0, IgA, 56:1 and IgM, 12.6, and acute viral hepatitis (3 cases), IgG, 116.0, IgA, 61.0 and IgM, 32.0, but low in histologically normal livers (6 cases), IgG, 4.5, IgA, 4.8 and IgM, 4.7, alcoholic fatty liver (11 cases), IgG, 9.4, IgA, 11.4 and IgM, 7.1, and miscellaneous non-inflammatory conditions (10 cases), IgG, 8.7, IgA, 11.1 and IgM, 5.0. Photomicrographs were used to measure the density of plasma cells, expressed as cells/mm² of liver biopsy tissue: mean counts were for AH 5.1, CAH 16.2 and normal liver 0.0. Intrahepatic Ig production in vitro did not correlate with the density of plasma cells in biopsy samples from cases of AH or CAH, nor with serum Ig levels.