Emily Streaker

Cross-Reactive HIV-1-Neutralizing Human Monoclonal Antibodies Identified from a Patient with 2F5-Like Antibodies

ABSTRACT The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140JR-FL. Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW664–666 core of the 2F5 epitope and two additional upstream residues (L660,663). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5—they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.

A novel human monoclonal antibody that binds with high affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity

Mesothelin is a potential new target for cancer immunotherapy because it is present at relatively low levels only in mesothelial cells of pleura, peritoneum, and pericardium of healthy people, but is significantly elevated in a number of tumors, including mesothelioma, ovarian, pancreatic, and lung cancers. However, all currently available antibodies against mesothelin are either murine or chimeric, which could limit their use because of increased likelihood of immunogenicity compared with fully human antibodies. Here, we report the identification and characterization of a novel fully human monoclonal antibody, m912, which was isolated from a human Fab library by panning against recombinant mesothelin. This antibody in scFv, Fab, and IgG1 formats bound specifically and with high affinity (equilibrium dissociation constant in the nmol/L range) to cell surface-associated human mesothelin and to recombinant mesothelin. It specifically lysed cancer cells engineered to express mesothelin in the presence of peripheral blood mononuclear cells isolated from healthy donors most likely by antibody-dependent cellular cytotoxicity. M912 is the first reported fully human monoclonal antibody to mesothelin, which has potential for cancer treatment and diagnosis. [Mol Cancer Ther 2009;8(5):1113–8]

A folate receptor beta-specific human monoclonal antibody recognizes activated macrophage of rheumatoid patients and mediates antibody-dependent cell-mediated cytotoxicity

IntroductionFolate receptor beta (FRβ) is only detectable in placenta and limited to some hematopoietic cells of myeloid lineage in healthy people. Studies have indicated that FRβ is over-expressed in activated macrophages in autoimmune diseases and some cancer cells. In this study we aimed to develop an FRβ-specific human monoclonal antibody (mAb) that could be used as a therapeutic agent to treat rheumatoid arthritis and other autoimmune diseases, as well as FRβ positive cancers.MethodsFunctional recombinant FRβ protein was produced in insect cells and used as antigen to isolate a mAb, m909, from a human naïve Fab phage display library. Binding of Fab and IgG1 m909 to FRβ was measured by ELISA, surface plasmon resonance, immune fluorescence staining, and flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with FRβ positive CHO cells as target cells and isolated peripheral blood monocytes as effector cells in an in vitro assay.ResultsFab m909 bound with relatively high affinity (equilibrium dissociation constant 57 nM) to FRβ. The IgG1 m909 showed much higher (femtomolar) avidity as measured by ELISA, and it bound to FRβ positive cells in a dose-dependent manner, but not to parental FRβ negative cells. m909 did not compete with folate for the binding to FRβ on cells. m909 was not only able to select FRβ positive, activated macrophages from synovial fluid cells of arthritis patients as efficiently as folate, but also able to mediate ADCC in FRβ positive cells.ConclusionsUnlike folate-drug conjugates, m909 selectively binds to FRβ, does not recognize FRα, and has at least one effector function. m909 alone has potential to eliminate FRβ positive cells. Because m909 does not compete with folate for receptor binding, it can be used with folate-drug conjugates in a combination therapy. m909 can also be a valuable research reagent.

454 antibody sequencing - error characterization and correction

Background454 sequencing is currently the method of choice for sequencing of antibody repertoires and libraries containing large numbers (106 to 1012) of different molecules with similar frameworks and variable regions which poses significant challenges for identifying sequencing errors. Identification and correction of sequencing errors in such mixtures is especially important for the exploration of complex maturation pathways and identification of putative germline predecessors of highly somatically mutated antibodies. To quantify and correct errors incorporated in 454 antibody sequencing, we sequenced six antibodies at different known concentrations twice over and compared them with the corresponding known sequences as determined by standard Sanger sequencing.ResultsWe found that 454 antibody sequencing could lead to approximately 20% incorrect reads due to insertions that were mostly found at shorter homopolymer regions of 2-3 nucleotide length, and less so by insertions, deletions and other variants at random sites. Correction of errors might reduce this population of erroneous reads down to 5-10%. However, there are a certain number of errors accounting for 4-8% of the total reads that could not be corrected unless several repeated sequencing is performed, although this may not be possible for large diverse libraries and repertoires including complete sets of antibodies (antibodyomes).ConclusionsThe experimental test procedure carried out for assessing 454 antibody sequencing errors reveals high (up to 20%) incorrect reads; the errors can be reduced down to 5-10% but not less which suggests the use of caution to avoid false discovery of antibody variants and diversity.

Origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing

Our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as HIV-1, SARS coronavirus (SARS CoV), and Hendra and Nipah viruses (henipaviruses). Although broadly neutralizing antibodies (bnAbs) against the HIV-1 were observed in patients, elicitation of such bnAbs remains a major challenge when compared to other viral targets. We previously hypothesized that HIV-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. To further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human IgM antibodies which had been used for selecting antibodies against SARS CoV receptor-binding domain (RBD), and soluble G proteins (sG) of henipaviruses. We found that the human IgM repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity, and a lower extent of somatic mutation. In this study, we identified antibody maturation intermediates that are related to bnAbs against the HIV-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. Further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnAbs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics.

Characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: Implications for mechanisms of immune evasion and design of vaccine immunogens

Abstract We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens.

Characterization of human IgG repertoires in an acute HIV-1 infection

All known broadly neutralizing antibodies (bnAbs) are highly somatically mutated and therefore significantly differ from their germline predecessors. Thus although the mature bnAbs bind to conserved epitopes of the HIV-1 envelope glycoprotein (Env) with high affinity their germline predecessors do not or weakly bind Envs failing to initiate an effective immune response. The identification of less somatically mutated bnAbs and/or antibody maturation intermediates that are clonally related to bnAbs may be useful to circumvent the major problem of initiating immune responses leading to elicitation of bnAbs. Here, we describe the identification of IgG antibodies from an acutely HIV-1-infected patient using a combination of phage display and high-throughput sequencing. We found two antibodies with only a single point mutation in the V region of their heavy chain variable domains compared to their putative germline predecessors which bound with high affinity to several Envs. They targeted the Env gp41 and did not neutralize HIV-1. Using high-throughput sequencing, we identified several highly abundant CDR3s, germline-like as well as somatically mutated V genes in the VH/VL repertoires of the patient which may provide antibody intermediates corresponding to known bnAbs as templates for design of novel HIV-1 vaccine immunogens.

N-terminal truncation of an isolated human IgG1 CH2 domain significantly increases its stability and aggregation resistance.

Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions, and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that the removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5 °C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity, and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak binding of CH2 to shFcRn as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins.

Putative rhesus macaque germline predecessors of human broadly HIV-neutralizing antibodies: differences from the human counterparts and implications for HIV-1 vaccine development.

Broadly neutralizing antibodies (bnAbs) are likely to be a key component of protective immunity conferred by an effective HIV-1 vaccine. We and others have reported that putative human germline predecessors of known human bnAbs lack measurable binding to HIV-1 envelope glycoproteins (Env), which could be a new challenge for eliciting human bnAbs. Rhesus macaques have been used as nonhuman primate models for testing vaccine candidates, but little is known about their germline Abs. Here we show the similarities and differences between putative rhesus macaque and human germline predecessors and possible intermediate antibodies of one of the best characterized bnAbs, b12. Similar to the human counterpart, a putative rhesus macaque b12 germline antibody lacks measurable binding to HIV-1 Envs, suggesting that initiation of somatic maturation of rhesus macaque germline b12 predecessor may also be a challenge. However, differences in sequence characteristics and binding properties between macaque and human b12 germline and intermediate antibodies suggest that the two germline predecessors may undergo different maturation pathways in rhesus macaques and in humans. These results indicate that immunogens that could initiate the immune responses and drive somatic mutations leading to elicitation of b12 or b12-like bnAbs in rhesus macaques and in humans are likely to be different. This has important implications for HIV-1 vaccine development.