Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Igh ^p.Abbreviations used in this paper Igh immunoglobulin heavy chain - SDS sodium dodecyl sulfate     

Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells

Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG₁, IgG₂ and IgG₄ thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG_2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

A defucosylated anti-PD-L1 monoclonal antibody 13-mG2a-f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established an anti-PD-L1 monoclonal antibody (mAb), L₁Mab-13 (IgG₁, kappa), by immunizing mice with PD-L1-overexpressing CHO-K1 cells. L₁Mab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines in flow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues via immunohistochemical analysis. In this study, we investigated whether L₁Mab-13 reacts with oral cancer cell lines and exerts antitumor activities. Because L₁Mab-13 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we first converted the subclass of L₁Mab-13 from IgG₁ into IgG_2a (13-mG_2a), and further produced a defucosylated version (13-mG_2a-f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 13-mG_2a-f was confirmed using fucose-binding lectins, such as Aleuria aurantia and Pholiota squarrosa lectins. The dissociation constants (K_D) for 13-mG_2a-f in SAS and HSC-2 oral cancer cells were determined via flow cytometry to be 2.8 × 10^?9 M and 4.8 × 10^?9 M, respectively, indicating that 13-mG_2a-f possesses extremely high binding affinity. In vitro analysis demonstrated that 13-mG_2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 13-mG_2a-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Taken together, these data demonstrate that treatment with 13-mG_2a-f may represent a useful therapy for patients with PD-L1-expressing oral cancers.     

Hormone activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressivein vitro effects ofimmunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11–20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10⁻¹¹−10⁻⁷ M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strainSalmonella typhimurium 415. By using a¹²⁵I-labeled “addressing” fragment of ACTH {[¹²⁵I]ACTH (13–24)}, we showed that MT-4 cells express specific receptors for ACTH (K _d 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [¹²⁵I]ACTH-(13–24) to these receptors withK _i1 of 0.38 andK _i2 of 0.34 nM, respectively. Specific receptors for ACTH (K _d 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to compete with labeled ACTH-(13–24) for binding to these receptors (K _i=1.8 nM), and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

Schistosoma mansoni: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

Detection of immunoglobulin heavy chain IgG3 polymorphism in wild mice with xenogeneic monoclonal antibodies

We have generated four xenogeneic rat antimouse IgG₃ monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG₃ molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG₃ isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG₃ molecules). An IgG₃ variant was also isolated from hybridomas derived from Mus spretus.Abbreviations Igh-C immunoglobulin heavy chain constant region - PVC polyvinyl chloride - RIA radioimmune assay - ELISA enzyme linked immunosorbent assay     

Experimental trichinosis-II. Quantitative and qualitative variations of serum in CBA mouse and Wistar rat (author's transl)]

Variations of serum globuline and immunoglobulins were measured by electrophoresis on cellulose acétate and radial immunodiffusion during the course of infection in inbred CBA mice and Wistar rats.In the two animal species, electrophoresis showed a significant decrease of serum albumin attending an increase of γ globulins. This phenomenon was earlier and greater in mice than in rats. This difference might be explained by suggesting that Trichinella is better adapted to rats than to mice.Beginning in the second week of infection, all classes of immunoglobulins were increased in the mouse with IgG_2a and IgA showing the greatest increase. The different classes of immunoglobulins continued to increase during the 4th month of infection but the rate of increase diminished after the second month except for IgA which increased between the 60th and 120th day.     

Serological changes in adjuvant-treated mice,their specificity and relevance to tumor immunity

Summary The effects of a variety of adjuvant protocols on immunoglobulin levels in normal and tumor bearing CBA mice have been investigated together with their ability to elicit immunoglobulin which bind to tumor cells in vitro and inhibit the growth of a transplanted syngeneic MC-induced fibrosarcoma.A marked increase in serum levels of certain immunoglobulins (especially IgG _2a , IgG _2b and IgM) and immunoglobulin interacting with tumor cells in vitro was noted in normal and tumor bearing mice following the administration of C. parvum (strain No. CN6134 and 10387), P. freudenreichii (strain No. 10470) and B. pertussis while a modest increase in some of these accompanied BCG injection. The Freund's complete and incomplete adjuvant protocols adopted had little effect on any of these parameters. The C. parvum protocols alone inhibited tumor growth.The immunoglobulins evoked by C. parvum strain No. 6134 which bound to tumor cells in vitro were extremely heterogeneous, activity being detected in all Ig classes and subclasses. This organism also evoked immunoglobulins which interacted with syngeneic embryonic fibroblasts, and adult syngeneic kidney and spleen cells.Tumor cells which had been preincubated in sera rich in immunoglobulins binding to tumor cells in vitro (i.e. from C. parvum-treated mice) did not exhibit reduced growth following i.v. or s.c. transplantation to syngeneic recipients.     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

Crystallization of intact monoclonal antibodies

Attempts were made to crystallize four monoclonal antibodies, one IgG_2ak and three IgG_1k. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG_2ak antibody specific for canine lymphoma cells,^1,2 crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG_1k antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 Å, b = 193 Å, c = 74 Å, and β = 110°. These crystals have an entire IgG_1k molecule as the asymmetric unit and they diffract to at least 3.2 Å resolution. © 1995 Wiley-Liss, Inc.     

An allele (Pk-1 b ) from wild-caught mice that affects the activity and kinetics of erythrocyte and liver pyruvate kinase

A true breeding strain was made from a wild-caught mouse with low erythrocyte pyruvate kinase (E.C. 2.7.1.40) activity. This variation showed additive inheritance and segregated as an allele at a single locus (Pk-1 ^b). Mice homozygous for the reduced blood pyruvate kinase activity cosegregated for reduced liver activity. In both these tissues the variant enzyme had a lowered heat stability and reduced K _m values for ADP. An increased stimulation by FDP was also detected in the liver pyruvate kinase. No difference in the isoelectric point of the variant enzyme in either erythrocyte or liver was observed when compared with the enzyme from C57BL mice (Pk-1 ^a/Pk-1 ^a). It is concluded that Pk-1 is the structural gene for the erythrocyte and the major liver pyruvate kinase. No other tissue pyruvate kinase showed altered characteristics.This work was supported by a Medical Research Council grant.     

Anti-allergic effects of His-Ala-Gln tripeptide in vitro and in vivo

We examined the inhibitory effects of HAQ (His-Ala-Gln) peptide on type-1 allergy in vitro and in vivo. HAQ peptide inhibited β-hexosaminidase release and intracellular Ca²⁺ levels of rat basophilic leukemia RBL-2H3 cells. Oral administration of a HAQ peptide-added diet (1 mg/mouse/administration) to C3H/HeJ mice for 14 days led to significant suppression of allergic symptoms, but did not reduce allergen-specific IgE or IgG₁.     

Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine s chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1^b (IgG_2a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1^a, Igh-1^d, Igh-1^e and Igh-1^j allotypes. The genetic studies of (BALB/c x C57BL/6)F₁ and backcross F₂ animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.Abbreviations used in this paper PCA passive cutaneous anaphylaxis - RID radioimmunodiffusion - i.p. intraperitoneally - EA egg albumin - Igh-C immunoglobulin heavy chain constant region locus - DNP 2,4-dinitrophenyl - PBS phosphate-buffered saline - NMS normal mouse serum - KLH keyhole limpet hemocyanin Visiting investigator supported by the Scientific and Humanistic Development Council from the Central University of Venezuela, currently at the following address: Consejo de Desarrollo Cientifico y Universidad Central de Venezuela, Av. Principal Urb. La Floresta Ota., Silenia Caracas, Venezuela.     

Variable Region Identical Immunoglobulins Differing in Isotype Express Different Paratopes

The finding that the antibody (Ab) constant (C) region can influence fine specificity suggests that isotype switching contributes to the generation of Ab diversity and idiotype restriction. Despite the centrality of this observation for diverse immunological effects such as vaccine responses, isotype-restricted antibody responses, and the origin of primary and secondary responses, the molecular mechanism(s) responsible for this phenomenon are not understood. In this study, we have taken a novel approach to the problem by probing the paratope with ¹⁵N label peptide mimetics followed by NMR spectroscopy and fluorescence emission spectroscopy. Specifically, we have explored the hypothesis that the C region imposes conformational constraints on the variable (V) region to affect paratope structure in a V region identical IgG₁, IgG_2a, IgG_2b, and IgG₃ mAbs. The results reveal isotype-related differences in fluorescence emission spectroscopy and temperature-related differences in binding and cleavage of a peptide mimetic. We conclude that the C region can modify the V region structure to alter the Ab paratope, thus providing an explanation for how isotype can affect Ab specificity.     

Somatic cell hybridization of mouse myeloma cells

Somatic cell hybrids between different mouse myeloma cell lines have been readily isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antigen expression. Three hybrid cell lines are described here in detail: an IgG_2b, ? X IgG_2a, ?; an IgG₁, ? X IgG_2b, ?; and an IgG₁, ? X IgM, λ. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.     

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.     

Relationships between IgE/IgG4 Epitopes,Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

Background

Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies.The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown.The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG₄ binding regions.

Methodology/Principal Findings

The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg²⁺, but not Ca²⁺, binding was determined by band shift using SDS-PAGE. IgE and IgG₄ epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients.The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG₄. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.

Conclusions/Significance

This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG₄ binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.     

L445P mutation on heavy chain stabilizes IgG4 under acidic conditions

ABSTRACT

IgG₄, a common type of therapeutic antibody, is less stable during manufacturing processes compared with IgG₁. Aggregation and fragmentation are the two main challenges. Here, we report instability of the heavy chain (HC) C-terminal region under acidic conditions, which leads to cleavage and aggregation. Leu⁴⁴⁵, at the C-terminal region of the HC in IgG_4, plays a critical role in its acid-induced fragmentation and subsequent aggregation. We found that mutating HC C-terminal Leu⁴⁴⁵ to Pro (the corresponding residue in IgG₁) in IgG₄_CDR-X significantly reduces fragmentation and aggregation, while mutating Pro⁴⁴⁵ to Leu in IgG₁_CDR-X promotes fragmentation and aggregation. HC C-terminal Gly⁴⁴⁶ cleavage was observed in low pH citrate buffer and resulted in further fragmentation and aggregation, whereas, glycine buffer can completely inhibit the cleavage and aggregation. It is proposed that cleavages occur through acid-induced hydrolysis under acidic conditions and glycine stabilizes IgG₄ via two main mechanisms: 1) product feedback inhibition of the hydrolysis reaction, and 2) stabilization of protein conformation by direct interaction with the peptide backbone and charged side chains. Experiments using IgG₄ molecules IgG₄_CDR-Y and IgG₄_CDR-Z with the same CH domains as IgG₄_CDR-X, but different complementarity-determining regions (CDRs), indicate that the stability of the HC C-terminal region is also closely related to the sequence of the CDRs. The stability of IgG₄_CDR-X is significantly improved when binding to its target. Both observations suggest that there are potential interactions between Fab and CH2-CH3 domains, which could be the key factor affecting the stability of IgG antibodies.