Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.     

VL-VH expression by monoclonal antibodies recognizing avian lysozyme

Seven BALB/c hybridoma antibodies directed against the protein antigen, hen egg-white lysozyme c (HEL), were characterized on the basis of their ability to bind lysozymes from 10 species of birds, and their ability to bind HEL competitively. The hybridomas were separable into three complementation groups based upon competitive interactions. The fine specificities of all antibodies were distinct, but two, HyHEL-8 and HyHEL-10, had very similar and overlapping reactivity patterns. To test the hypothesis that VL-VH pairing correlates with binding specificity, the N-terminal amino acid sequences were determined to identify the VL and VH isotopes (subgroups) of the anti-HEL antibodies. HyHEL-8 and -10 shared the VK23 light chain isotype and nearly identical heavy chains in Kabat subgroup I, whereas the heavy and light chain isotypes of all other antibodies differed from HyHEL-8 and -10 and from each other. The heavy and light chain isotypes expressed by HyHEL-8 and -10 are also expressed by XRPC-25, a DNP-binding myeloma protein that does not bind lysozyme. These results are discussed with respect to the contributions of various genetic sources of structural diversity to antibody functional diversity.     

The limited diversity of the mouse gamma-chains anti-GAT repertoire does not seem to be noticeably amplified upon class switch

NH2-terminal sections of H and L chains isolated from five monoclonal anti-GAT antibodies derived from BALB/c mice have been sequenced upon to residue 43. Four among these five antibodies, sharing similar public idiotypic determinants, possess extremely conserved sequences, both for the H, which is apparented to the VH II type, and the L chains, which belong to the V kappa I subgroup. VH sequences are identical up to residue 43 and contain the common sequences (residues 1 to 32) defined for the H chains derived from the DBA/2 IgM anti-GAT monoclonal antibodies. Light chains are also remarkably conserved, a rather unusual situation for kappa-chains. The fifth antibody that expresses only part of the public idiotypic determinants contains very distinctive H and L chains. Its heavy chains are close to the VH I subgroup, whereas its kappa-chains permit definition of a new V kappa subgroup. The repertoire appears to be highly conserved between BALB/c and DBA/2 mice, and does not seem larger in IgG than in IgM antibodies. This latter observation does not speak in favor of a switch-linked amplification of diversity.     

Different VL and VH germ-line genes are used to produce similar combining sites with specificity for alpha(1----6)dextrans

Monoclonal antibodies to alpha(1----6)dextrans produced in mice immunized with the T-independent antigens alpha(1----6)dextran or the stearylisomaltosyl oligosaccharides have been characterized immunochemically. To correlate the immunochemical properties of these monoclonal antibodies with their primary structure, we have sequenced the variable (V) regions of the light (L) and heavy (H) chains. Three V kappa germ-line genes belonging to two major gene families were used; differential J usages also contribute to diversity. Five different VH germ-line genes belonging to three major VH families were used. The VH genes were further modified by junctional diversity and differential J usage and possibly by somatic mutations. The effects of these modifications on the fine specificities of anti-alpha(1----6)dextrans are discussed. Thus far, six different combinations of VLJL-VH(D)JH chains that form groove-type combining sites specific for alpha(1----6)dextran have been found. We conclude that entirely different VL and VH can form combining sites specific for the internal linear sequence of alpha(1----6)dextran.     

Structural evidence for a polymorphic or allelic form of the heavy chain variable region.

The heavy (H) chains of anti-phosphocholine (PC) antibodies from C57BL/6J and CBA/J were sequenced through the N-terminal 36 residues and compared with previously published sequences of A/J anti-PC antibody and BALB/c PC-binding myeloma proteins T15, M603, and M511. Each of these antibody preparations contained molecules having light (L) chains and idiotypic determinants of T15, M511, and M603 indicating the presence of at least three different anti-PC antibodies in each pool. The structures of the C57BL/6J and CBA/J H chains each revealed a single sequence from positions 1 to 36 (which includes the first complementarity determining region (CDR), and they were identical. The first CDR was identical to that previously found for BALB/c and A/J indicating that this portion of these antibody molecules is highly conserved throughout inbred mice and is probably critical to PC-binding. A surprising finding was that both C57NL/6 and CBA sequence differed from the BALB/c and A/J sequences at two positions, residue 14 and 16. Since each of these strains differs at the allotype locus, the data indicates that the evolution of allotypy in mice occurred after variable region diversity for the particular genes.     

Matching nucleotide sequences of human antibodies with other known sequences

From an evolutionary point of view, the complementarity-determining regions of antibodies are distinct from other proteins including the framework regions of antibodies. A search for identical nucleotide sequences of eighty-four 15 consecutive bp in the complementary-determining regions of human antibody heavy chains with other known sequences yielded four matches: two sequential 15-bp matches, or one 16-bp match, with the coding region of a sea-urchin testis histone H2b-2, one 15-bp match with the promotor region of a cauliflower mosaic virus inclusion body protein, and a 15-bp match with an intron between exons 1 and 2 of human factor IX. As a control, an identical search of eighty-four 15 consecutive bp in the framework regions of human antibody heavy chains yielded no matches with other sequences except those from other antibody framework regions. Since the currently available nucleotide sequence database used in the search consisted of about 1 x 10(7) bp, finding such matches in the complementarity-determining regions might not be random.     

Neutralizing Capacity of Monoclonal Antibodies That Recognize Peptide Sequences Underlying the Carbohydrates on gp41 of Simian Immunodeficiency Virus

Extensive glycosylation of the envelope spikes of human and simian immunodeficiency virus (HIV and SIV) is an important factor for the resistance of these viruses to neutralization by antibodies. SIVmac239 gp41 has three closely spaced sites for N-linked carbohydrate attachment. Rhesus macaques experimentally infected with mutant versions of SIVmac239 lacking two or three of these carbohydrate sites developed strong serum reactivity against mutated peptide sequences at the site of these glycosylations, as well as high titers of neutralizing activity to the mutant viruses (E. Yuste et al., J. Virol. 82:12472–12486, 2008). However, whether antibodies that recognize these underlying peptides have neutralizing activity has not been directly demonstrated. Here we describe the isolation and characterization of three gp41-specific monoclonal antibodies (4G8, 6G8, and 7D6) from one of these mutant-infected monkeys. All three antibodies reacted with mutant gp41 from viral particles and also with peptides corresponding to mutated sequences. Slight differences in peptide specificities were observed among the three antibodies. Sequence analysis revealed that the heavy chains of all three antibodies were derived from the same germ line heavy-chain segment (IGHV4-59*01), but they all had very different sequences in complementarity-determining region 3. The light chains of all three antibodies were very closely related to one another. All three antibodies had neutralizing activity to mutant viruses deficient in gp41 carbohydrate attachment, but they did not neutralize the parental SIVmac239. These results demonstrate unambiguously that antibodies with specificity for peptide sequences underlying gp41 carbohydrates can effectively neutralize SIV when these carbohydrates are absent. However, the presence of these gp41 carbohydrates effectively shields the virus from antibodies that would otherwise neutralize viral infectivity.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

Immunoglobulin idiotype and anti-anti-idiotype utilize the same variable region genes irrespective of antigen specificity

Idiotypic determinants characterizing certain antibody specificities have been proven valuable structural and genetic markers in studies of antibody diversity and regulation. The heritable predominant idiotype associated with the response to p-azophenylarsonate in A/J mice consists of a set of highly homologous (greater than 95%) heavy and light chain variable region amino acid sequences probably arising by somatic mutation from one or a few V region genes. We examined a peculiar set of monoclonal antibodies that have been defined as CRI by serologic analysis, but that have no affinity for the hapten Ars. These antibodies were elicited by immunization with anti-CRI rather than by the conventional immunization with antigen. The amino acid sequences of the amino terminal half of the V regions of these anti-(anti-CRI) antibodies are indistinguishable from those of conventional Ars-binding CRI antibodies. Thus, Ars-binding CRI and Ars-nonbinding anti-(anti-CRI) are derived from similar or identical VH and VL genes.     

Structural studies on induced antibodies with defined idiotypic specificities. I. The heavy chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

Amino acid sequence analysis has been performed on three groups of heavy (H) chains of A/J mice. H chains derived from unimmunized animals were compared to anti-p-azophenylarsonate (anti-Ar) antibodies which were further subdivided into those possessing and those depleted of a cross-reacting idiotype (CRI). It was found that anti-Ar antibodies bearing the CRI are homogeneous through the first hypervariable region of the H chain. The same sequence was obtained for pooled antibody isolated from the ascites fluid of 18 A/J mice or from a single mouse. The H chains appear to belong to a minor V-H subgroup. In the first 30 positions Anti-Ar antibodies depleted of the CRI had the same sequence as those containing the CRI (with small amounts of heterogeneity at some positions), but contained a mixture of sequences in the first hypervariable region of the H chain. These studies indicate that antibodies with similar specificity and with identical framework sequences, but which differ in their hypervariable regions, contain different idiotypic determinants, and support the concept that the idiotypic determinants reside primarily within hypervariable regions.     

N端测序作为单克隆抗体常规放行分析方法的探讨

旨在探讨N端测序作为单克隆抗体常规放行分析方法的适用性。应用Edman降解法、质量肽图法对两个针对不同靶点的单抗进行N端测序,用肽图法寻找二者的特征性鉴别肽段,用离子色谱、毛细管区带电泳和成像毛细管等点聚焦电泳进行异质性分析。Edman降解法显示两个单抗轻链和重链的15个氨基酸分别完全一致,质量肽图法显示二者轻重链的T1肽段分别完全一致,而肽图法和3种异质性分析方法则可对两个抗体进行有效鉴别。由于人源化或人源单抗序列框架数量较为有限,两个单抗的N末端序列完全相同,运用Edman降解法进行N端测序是否能作为单抗的常规放行分析方法值得进一步商榷,同时上述多种方法可运用于单抗的鉴别分析,并可对其异质性进行控制,较N端测序分析更具有客观性。     

Structural studies on induced antibodies with defined idiotypic specificities. II. The light chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

N-terminal amino acid sequence analyses have been performed on three preparations of light chains of A/J mice. Light chains derived from the IgG of unimmunized animals were compared to light chains of anti-p-azo-phenylarsonate (anti-Ar) antibodies possessing a cross-reacting idiotype (CRI); the latter were derived from the ascites fluid of a single A/J mouse, or from the pooled ascites fluids of 18 A/J mice. The heavy chains of these same two antibody preparations had previously been shown to comprise a single, homogeneous sequence to position 40. With few exceptions, the first 26 positions of light chains derived from unimmunized animals were extremely heterogeneous; the heterogeneity is comparable to that observed in a composite of sequence data on light chains of BALB/c myeloma proteins. Although the light chains obtained from anti-Ar antibodies possessing the CRI (whether from the pool of 18 A/J mice or from a single mouse) were more restricted in their sequence, at several positions as many as four alternative amino acids were detected. These studies indicate that an antibody population with defined idiotypic specificity, and very possibly identical heavy chain sequences, may contain at least four distinct light chains. The feasibility of structural studies on antibodies induced in individual mice is further demonstrated.     

Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.     

Light chain diversity of murine anti-streptococcal antibodies: IgCH-linked effects on L chain expression.

L chains derived from anti-group A streptococcal carbohydrate antibodies raised in A/J, BALB/cJ, C57BL/6J, CB-20, BAB-14, and CAL-20 mice were examined by isoelectric focusing. Multiple strain-associated differences in the degree and frequency of expression of particular L chain spectrotypes were observed. Analysis of L chain-focusing patterns in allotype-congenic mice revealed that IgCH-linked genes can have profound effects on the L chain phenotypes expressed by strains with identical L chain genotypes. Lastly, the overall spectrotypic diversity of L chains from anti-GAC antibodies appears to be less extensive than the diversity of the antibodies from which these L chains derive, documented by similar techniques. These results are interpreted in light of the significance of combinatorial diversity in generating antibody heterogeneity.     

LC-MS characterization and purity assessment of a prototype bispecific antibody

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MS^E peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.     

Looking for therapeutic antibodies in next-generation sequencing repositories

ABSTRACT

Recently it has become possible to query the great diversity of natural antibody repertoires using next-generation sequencing (NGS). These methods are capable of producing millions of sequences in a single experiment. Here we compare clinical-stage therapeutic antibodies to the ~1b sequences from 60 independent sequencing studies in the Observed Antibody Space database, which includes antibody sequences from NGS analysis of immunoglobulin gene repertoires. Of 242 post-Phase 1 antibodies, we found 16 with sequence identity matches of 95% or better for both heavy and light chains. There are also 54 perfect matches to therapeutic CDR-H3 regions in the NGS outputs, suggesting a nontrivial amount of convergence between naturally observed sequences and those developed artificially. This has potential implications for both the legal protection of commercial antibodies and the discovery of antibody therapeutics.     

Molecular basis of an isogeneic anti-idiotypic response.

The nucleotide sequences of the variable region genes expressed in the heavy and light chains of six isogeneic anti-idiotope antibodies recognizing idiotopes on two closely related antibodies with specificity for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were determined. In two independently derived anti-idiotope cell lines the same or strongly homologous V kappa, VH and D region genes had originally been rearranged. The two lines express long and partly homologous N sequences (presumed to be not of germ line origin) at the border of D, resulting in CDR3s of unusual length. An unusually long CDR3, partly encoded by N sequences, is also present in the heavy chain of a third anti-idiotope antibody. The VH regions of the three remaining anti-idiotope antibodies originate from a single VH gene which belongs to the same VH group as the VH genes expressed in the other anti-idiotopes. Two of these antibodies, expressing similar V, D and J elements, had been isolated from the same mouse and appear to have diverged from the same B cell precursor by at least two rounds of somatic mutation. Somatic point mutations have occurred in most, if not all anti-idiotope V region sequences. In two instances somatic mutations in J increase the structural homology between anti-idiotopes. The anti-idiotypic response in this system is thus genetically restricted and may depend upon the selection of non-germ line sequences, suggesting an explanation for the low frequency at which anti-idiotope antibodies are expressed in this system.     

Phage displayed antibodies to heat stable alkaline phosphatase: framework region as a determinant of specificity

Human antibodies against specific targets of tumor cells are the most desirable molecules for possible immunotherapy. They could be developed by using the combinatorial antibody library displayed on a phage. We selected four human antibody fragments (scFv) binding to the oncoplacental antigen Heat Stable Alkaline Phosphatase (HSAP, the placental isozyme of alkaline phosphatase) from a synthetic human antibody library. Characterization of these scFvs showed they bound HSAP with moderate affinity but did not have isozyme specificity, as determined by binding to cell lines exhibiting differential expression of isozymes of alkaline phosphatase. The V(H) sequences of two of these scFvs were similar and although both bound to HSAP only one was cross-reactive with albumin. The sequences revealed a difference in the framework region (FR1) of these antibodies, indicating a role for this region in the determination of specificity. This is also significant considering that the heavy chains generated the diversity of the synthetic library used in this study, and only a single light chain showing binding to BSA was used for the entire library.     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.