Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands

We have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display we selected several CTLA-4-based variants capable of binding to human alphavbeta3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities.     

Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides.

We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide. Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced. These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage. Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3. CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4. The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. Proteins 1999;36:217-227.     

Molecular determinants of inverse agonist activity of biologicals targeting CTLA-4

Ligation of CD28 or CTLA-4 with some biologicals can activate T cells due to an unexpected superagonist or inverse agonist activity, respectively. The risk of such an outcome limits the therapeutic development of these reagents. Thus, identifying the molecular determinants of superagonist/inverse agonist properties for biologicals targeting costimulatory/inhibitory receptors has not only fundamental value but also important therapeutic implications. In this study, we show that ligation of CTLA-4 with either soluble B7.1 Ig (but not B7.2 Ig) or with a recombinant bispecific in-tandem single chain Fv known as 24:26 induces TCR-independent, T cell activation. Such an inverse agonist activity requires CD28 expression and high CTLA-4 expression and is not seen when CTLA-4 is ligated by membrane-bound B7.1 or B7.2. At the molecular level, the inverse agonist activity of B7.1 Ig or 24:26 correlates with their ability to induce the formation of unique dimer-based, CTLA-4 oligomers on the T cell surface and involves CTLA-4 signaling through its cytoplasmic domain. Our results provide a potential mechanism to explain and to predict inverse agonist activity for CTLA-4 ligands.     

Overexpression of the Ctla-4 isoform lacking exons 2 and 3 causes autoimmunity

CTLA-4 is a potent inhibitor of T cell activation, primarily upon binding to its costimulatory ligands (B7.1 and B7.2) expressed on APCs. However, variants of CTLA-4 can also function independently of B7 molecules. 1/4CTLA-4 is a highly conserved isoform encoded by exons 1 and 4 of the Ctla4 gene that lacks the ligand-binding and the transmembrane domains, and as yet, its function is not known. To investigate the function of 1/4CTLA-4, we generated transgenic (Tg) mice overexpressing this variant. Cytokine production by 1/4CTLA-4 Tg T cells was elevated compared with wild type T cells. The frequency of CD44(high) memory T cells in 1/4CTLA-4 Tg mice was increased, and as the mice aged, the frequency further increased. 1/4CTLA-4 Tg mice >1 y old had increased expression of T cell activation markers and developed spontaneous autoimmunity, including elevated production of autoantibodies. In contrast with young 1/4CTLA-4 Tg mice, aged 1/4CTLA-4 Tg mice had elevated frequencies of Foxp3(+) regulatory T cells, but the regulatory T cells from these mice were not able to inhibit colitis development. Collectively, these data suggest that the function of the 1/4CTLA-4 isoform is distinct from that of CTLA-4 in that it enhances T cell activation and promotes autoimmunity rather than inhibiting immune responses.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

Fine specificity of ligand-binding domain 1 in the polymeric Ig receptor: importance of the CDR2-containing region for IgM interaction

The human polymeric Ig receptor (pIgR), also called transmembrane secretory component, is expressed basolaterally on exocrine epithelia, and mediates specific external transport of dimeric IgA and pentameric IgM. The extracellular part of pIgR consists of five Ig-like domains (D1-D5), and a highly conserved D1 region appears to mediate the initial noncovalent ligand interaction. While the human pIgR binds both dimeric IgA and pentameric IgM with high affinity, the rabbit counterpart has virtually no binding capacity for pentameric IgM. This remarkable disparity constitutes evidence that the binding site of the two ligands differs with regard to essential receptor contact elements. Therefore, we expressed human/rabbit chimeric pIgRs in Madin-Darby canine kidney cells and found that human pIgR D1 is crucial for the interaction with pentameric IgM when placed in the context of a full-length receptor regardless of its backbone species. D1 contains three complementarity-determining region-like loops (CDR1-3), and to further map human D1 regions involved in pentameric IgM binding, we transfected Madin-Darby canine kidney cells with human/rabbit chimeric receptors in which the regions containing the CDR-like loops had been interchanged. Our results showed that the region containing the CDR2-like loop is the most essential for pentameric IgM binding. The region containing the CDR1-like loop also contributed substantially to this interaction, whereas only little contribution was provided by the region containing the CDR3-like loop, although it appeared to be necessary for maximal pentameric IgM binding.     

A DNA vaccine against cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) prevents tumor growth

Co-stimulatory signaling pathway triggered by the binding of B7.1/B7.2 (CD80/86) of antigen-presenting cells (APCs) to CD28 of T cells is required for optimal T-cell activation. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding with a greater affinity. Ipilimumab, a monoclonal antibody against CTLA-4, has shown positive efficacy in a pivotal clinical trial for the treatment of metastatic melanoma and was approved by FDA. However, the cost of monoclonal antibody-based therapeutics might limit the number of patients treated. To develop a novel therapeutics specifically targeting CTLA-4, we constructed a DNA vaccine by cloning the sequence of CTLA-4 fused with a transmembrane domain sequence of placental alkaline phosphatase (PLAP) into a mammalian expression plasmid, pVAC-1. Immunization with the resulting construct, pVAC-1-hCTLA-4, elicited antibody specific to human CTLA-4 with cross reactivity to murine CTLA-4, which was sufficient for inhibiting B16F10 tumor growth in c57BL/6 mice in the absence of measurable toxicity. Coupling liposome with pVAC-1-mCTLA-4 could break tolerance to self-antigen in BALB/c mice and induce potent immunity against murine CTLA-4, and suppress growth of subcutaneous renal cell carcinoma (Renca).     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Hierarchical regulation of CTLA-4 dimer-based lattice formation and its biological relevance for T cell inactivation

CTLA-4 is an activation-induced, homodimeric inhibitory receptor in T cells. Recent crystallographic reports have suggested that it may form lattice-like arrays on the cell surface upon binding B7.1/B7.2 (CD80, CD86) molecules. To test the biological relevance of these CTLA-4-B7 lattices, we introduced a C122A point mutation in human CTLA-4, because this residue was shown to be essential for dimerization in solution. Surprisingly, we found that up to 35% of C122A CTLA-4 dimerized in human T lymphocytes. Moreover, C122A CTLA-4 partitioned within lipid rafts, colocalized with the TCR in the immunological synapse, and inhibited T cell activation. C122-independent dimerization of CTLA-4 involved N-glycosylation, because further mutation of the N78 and N110 glycosylation sites abrogated dimerization. Despite being monomeric, the N78A/N110A/C122A triple mutant CTLA-4 localized in the immunological synapse and inhibited T cell activation. Such functionality correlated with B7-induced dimerization of these mutant molecules. Based on these data, we propose a model of hierarchical regulation of CTLA-4 oligomerization by which B7 binding ultimately determines the formation of dimer-dependent CTLA-4 lattices that may be necessary for triggering B7-dependent T cell inactivation.     

B7-independent inhibition of T cells by CTLA-4

CTLA-4 is an inhibitory molecule that regulates T cell expansion and differentiation. CTLA-4 binding to B7-1/B7-2 is believed to be crucial for its inhibitory signal both by competing for CD28 binding to the same ligands and aggregating CTLA-4 to deliver negative signals. In this study, we demonstrate that B7 binding is not essential for CTLA-4 activity. CTLA-4 knockout T cells are hyperresponsive compared with wild-type T cells in B7-free settings. Expression of a B7-nonbinding CTLA-4 mutant inhibited T cell proliferation, cytokine production, and TCR-mediated ERK activation in otherwise CTLA-4-deficient T cells. Finally, transgenic expression of the ligand-nonbinding CTLA-4 mutant delayed the lethal lymphoproliferation observed in CTLA-4-deficient mice. These results suggest that ligand binding is not essential for the CTLA-4 function and supports an essential role for CTLA-4 signaling during T cell activation.     

Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1

The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.     

A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.     

Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites

By examining a large database of amino acid sequences of antibodies of various specificities, we have found that many antibodies of distinctly different specificities assemble identical VL domains with different VH domains. In contrast, rarely is the same VH domain found in sets of antibodies of different specificities. We identified additional sets of antibodies of different specificities and identical sequences covering amino acid residues VH 1 to 94 and VL 1 to 95. In addition, there were segments of additional antibodies for which complete sequences were not available, but identities were seen in VL CDR1, VL CDR2, and VL CDR3 up to the VL-JL junction. The finding that there are many identical VL 1 to 95 segments with different VH 1 to 94 sequences, and vice versa, raises important questions as to the role of VH in influencing the conformation of VL and, conversely, the role of VL in influencing the conformation of VH. Evidence is also cited indicating that a single amino acid change may seriously disrupt site structure and in some instances abolish binding. Our findings suggest that it will be important in the future to investigate further conformational effects on antibody structure by using X-ray crystallography, nuclear magnetic resonance spectroscopy, or other methods, to obtain a better understanding of the functions and topography of antibody-combining sites.     

CTLA-4 is not required for induction of CD8(+) T cell anergy in vivo.

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.     

Small molecule ligands define a binding site on the immune regulatory protein B7.1.

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. These compounds inhibit the binding of B7.1 to both CD28 and CTLA4. Both classes of compounds appear to bind the same site, a relatively small portion of the GFCC'C" face of the N-terminal V-set domain of human B7.1, not present in the homologous B7.2 or even mouse B7.1. This site may represent a rare hot spot for small molecule antagonist design of inhibitors of cell-cell interactions, whose ligands may yield leads for the development of novel immunomodulatory medicines.     

Crystallographic Analysis of Antigen–Antibody Complexes: End-on Insertion of Ligands in Antibodies—CDR3 Loops as Arbiters

As the dominant constituents of the active sites, complementarity-determining regions (CDRs) and particularly the CDR3 loops strongly influence the size and shape of this interdomain space. Six sets of CDR3 loops were extracted from our collection of crystal structures and examined for their modes of association. The CDR3 loops of the NC6.8 Fab face each other across a small crevice that is expanded further by end-on insertion of its high-affinity ligand (a trisubstituted guanidine sweet-tasting compound). This wedging event triggers a series of extensive local and transmitted conformational changes. In the 4-4-20 Fab, the CDR3 loops provide scaffolding for the high-affinity binding of fluorescein and shield the ligand from bulk solvent in the interdomain space above and below the very compact binding slot. Constituents of the CDR2 and CDR3 loops of the BV04-01 Fab interact to form “false floors” over potential cavity-type sites and thereby eliminate end-on insertion. Instead, fragments of single-stranded DNA are bound with low affinity in a groove whose course is altered on complex formation by global movements of V_Hrelative to V_Land by local shifts of HCDR3. Trafficking of even small peptide ligands between the V domains of the Pot Fv is prevented by the collapse of the large HCDR3 segment into the residual interdomain space. This protein is better suited to polyreactive binding of a variety of large protein antigens on its external surfaces. By comparison, the space available between the CDR3 loops of the Mcg light-chain dimer is very large. It has proved to be accessible for end-on insertion of peptides and other ligands ranging over seven orders of magnitude in affinity. Recently, an insect neuropeptide hormone, with pGlu as its penetrating agent, has been found to pierce the entire V dimer interface from the entrance of the traditional active site to the solvent pool between the V and C domains. In an Mcg × Hud heterodimer, the Hud CDR3 plays a role similar to that of the Pot H chain and blocks access to the interdomain space by close interactions with the CDR3 of Mcg.     

Insertion of host-derived costimulatory molecules CD80 (B7.1) and CD86 (B7.2) into human immunodeficiency virus type 1 affects the virus life cycle

Human immunodeficiency virus type 1 (HIV-1) carries virus-encoded and host-derived proteins. Recent advances in the functional characterization of host molecules inserted into mature virus particles have revealed that HIV-1 biology is influenced by the acquisition of host cell membrane components. The CD28/B7 receptor/ligand system is considered one of the fundamental elements of the normal immune response. Two major cell types that harbor HIV-1 in vivo, i.e., monocytes/macrophages and CD4+ T cells, express the costimulatory molecules CD80 (B7.1) and CD86 (B7.2). We investigated whether CD80 and CD86 are efficiently acquired by HIV-1, and if so, whether these host-encoded molecules can contribute to the virus life cycle. Here we provide the first evidence that the insertion of CD80 and CD86 into HIV-1 increases virus infectivity by facilitating the attachment and entry process due to interactions with their two natural ligands, CD28 and CTLA-4. Moreover, we demonstrate that NF-kappaB is induced by CD80- and CD86-bearing virions when they are combined with the engagement of the T-cell receptor/CD3 complex, an event that is inhibited upon surface expression of CTLA-4. Finally, both CD80 and CD86 were found to be efficiently incorporated into R5- and X4-tropic field strains of HIV-1 expanded in cytokine-treated macrophages. Thus, besides direct interactions between the virus envelope glycoproteins and cell surface constituents, such as CD4 and some specific chemokine coreceptors, HIV-1 may attach to target cells via interactions between cell-derived molecules incorporated into virions and their natural ligands. These findings support the theory that HIV-1-associated host proteins alter virus-host dynamics.     

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies

Phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties since conventional hybridoma technologies have yielded mostly low-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties in immobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for protein antigens cannot be readily applied. We adapted phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterization of phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. We first constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) using Man3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences were isolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residues at nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Furthermore, first round characterization of scFvs expressed from respective phages indicated good affinity and specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness of this strategy in constructing human scFv against various carbohydrate antigens. Further studies on the purification and characterization of these scFvs are presented in an accompanying paper in this issue.