High-affinity Human Antibodies Research Articles

The Immunocytokine L19–IL2 Eradicates Cancer When Used in Combination with CTLA-4 Blockade or with L19-TNF

Systemic high-dose IL2 promotes long-term survival in a subset of metastatic melanoma patients, but this treatment is accompanied by severe toxicities. The immunocytokine L19-IL2, in which IL2 is fused to the human L19 antibody capable of selective accumulation on tumor neovasculature, has recently shown encouraging clinical activity in patients with metastatic melanoma. In this study, we have investigated the therapeutic performance of L19-IL2, administered systemically in combination with a murine anti-CTLA-4 antibody or with a second clinical-stage immunocytokine (L19-TNF) in two syngeneic immunocompetent mouse models of cancer. We observed complete tumor eradications when L19-IL2 was used in combination with CTLA-4 blockade. Interestingly, mice cured from F9 tumors developed new lesions when rechallenged with tumor cells after therapy, whereas mice cured from CT26 tumors were resistant to tumor rechallenge. Similarly, L19-IL2 induced complete remissions when administered in a single intratumoral injection in combination with L19-TNF, whereas the two components did not lead to cures when administered as single agents. These findings provide a rationale for combination trials in melanoma, as the individual therapeutic agents have been extensively studied in clinical trials, and the antigen recognized by the L19 antibody has an identical sequence in mouse and man.

Abstract 2848: Preclinical activity of OM-RCA-01, a humanized anti-FGFR1 antibody, in renal cell carcinoma (RCC)

Abstract Background: Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for treatment of metastatic RCC. Early we reported that anti-FGFR1 monoclonal antibody inhibited ligand-induced phosphorylation of FGFR1, activation of MAP kinase and Akt downstream signaling pathways in FGFR1 expressing endothelial (bovine adrenal cortex capillary endothelial cells) and human RCC (Caki-1) lines (Tsimafeyeu et al. AACR/JCA joint conference 2010). Today we investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody with high affinity (Kd of 1.59 nM), in RCC. Methods: To assess the effect of anti-FGFR1 antibody on FGF-mediated signaling, the human renal carcinoma Caki-1 FGFR1-expressing cells were incubated (0.5% FBS) and were dosed with OM-RCA-01 at 100, 10, and 1 mcg/ml. Control wells were left untreated. Three hours after dosing, basic FGF was added at a concentration of 50 ng/ml. Additional control wells were treated with OM-RCA-01 without FGF-stimulation. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Charles River female NCr nu/nu mice (6-12 weeks of age) were set up with 1 mm3 Caki-1 tumor fragments sc in flank. Tumor sizes were measured in a blind fashion twice a week with a vernier caliper. Mice with established tumors (an average size of 80 - 120 mg) were randomly divided into vehicle, non-specific IgG or OM-RCA-01 groups per 10 animals in group. Animals were treated with antibody using different doses (1 or 10 mg/kg). Endpoint was significant differences in tumor growth delay. Results: In vitro study showed that basic FGF significantly increased proliferation of the human FGFR1-expressing renal carcinoma cells (P=0.011). No effect of basic FGF on cell line proliferation was observed when the cells were incubated with OM-RCA-01 antibody at any concentrations up to 100 mcg/kg in comparison with FGF-untreated control (P=0.855). In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors (P=0.006) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses (P=0.917). Conclusions: High-affinity humanized anti-FGFR1 antibody OM-RCA-01 suppressed ligand-induced proliferation of human RCC cells in vitro. OM-RCA-01 has significant early anti-tumor efficacy in Caki-1 xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2848. doi:1538-7445.AM2012-2848

Expression of high-affinity human antibody fragments in bacteria

Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

Mammalian cell–based discovery of high-affinity human antibodies from IgG libraries

Mammalian cell–based discovery of high-affinity human antibodies from IgG libraries

Polyreactive antibodies in adaptive immune responses to viruses.

B cells express immunoglobulins on their surface where they serve as antigen receptors. When secreted as antibodies, the same molecules are key elements of the humoral immune response against pathogens such as viruses. Although most antibodies are restricted to binding a specific antigen, some are polyreactive and have the ability to bind to several different ligands, usually with low affinity. Highly polyreactive antibodies are removed from the repertoire during B-cell development by physiologic tolerance mechanisms including deletion and receptor editing. However, a low level of antibody polyreactivity is tolerated and can confer additional binding properties to pathogen-specific antibodies. For example, high-affinity human antibodies to HIV are frequently polyreactive. Here we review the evidence suggesting that in the case of some pathogens like HIV, polyreactivity may confer a selective advantage to pathogen-specific antibodies.

Rise and Fall of an Anti-MUC1 Specific Antibody

BackgroundSo far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.ResultsA human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10−10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.ConclusionsAntibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these “best in class” binding parameters, the therapeutic success of this antibody was prevented by the target biology.

Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform

Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10 9 members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2 nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer.

K11-Linked Polyubiquitination in Cell Cycle Control Revealed by a K11 Linkage-Specific Antibody

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.

Antibody-mediated neutralization of Sema4D reduces tumor angiogenesis and growth in vivo.

e13154 Background: It has been demonstrated that semaphorin 4D (Sema4D; CD100) is a potent pro-angiogenic molecule. Sema4D binding to its receptor plexin-B1 (Plex-B1) on endothelial cells transactivates c-Met and promotes formation of new blood vessels and tumor growth in vivo. Sema4D is overexpressed in a wide array of tumor types, and is also produced by inflammatory cells present in the tumor microenvironment. It has recently been demonstrated that in an environment lacking Sema4D, the ability of mouse breast cancer cells to originate tumor masses and metastases is severely impaired, and that Sema4D produced by tumor-associated macrophages is required for tumor angiogenesis and growth. In addition to its effects on endothelial cells, Sema4D binding to Plex-B1 on tumor cells results in c-Met transactivation and migration of tumor cells. It has been further reported that overexpression of Plex-B1 and Met in breast and ovarian cancers is a negative prognostic factor. Tumors coexpressing Plex-B1 and Met were characterized by worse grading and a higher incidence of lymph node metastases. Collectively, these results suggest that expression of Sema4D, either by tumor cells or by tumor associated macrophages, may represent a widely used strategy by tumors to induce angiogenesis and that expression of the Sema4D receptor in tumors may further induce tumor invasion, thereby enhancing their growth, survival, and metastatic potential. Antibody neutralization of Sema4D (CD100) thus may represent a new therapeutic strategy for cancer treatment. Methods: We selected a high affinity human IgG4 antibody that is specific for both mouse and human Sema4D, and utilized several in vitro functional assays to demonstrate that this antibody blocks Sema4D – Plex-B1 interactions. Results: Using both transplanted and orthotopic tumor models we demonstrated that antibody mediated Sema4D neutralization in vivo inhibits tumor growth and tumor angiogenesis. Conclusions: This human antibody is currently in IND-directed toxicology testing and we anticipate the initiation of human clinical studies in 2H 2010. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Vaccinex, Inc. Vaccinex, Inc.

Abstract LB-36: Antibody mediated neutralization of sema4D reduces tumor angiogenesis and growth In Vivo.

Abstract It has been demonstrated that semaphorin 4D (SEMA4D; CD100) is a potent pro-angiogenic molecule. SEMA4D binding to its receptor plexin-B1 (PLXNB1) on endothelial cells transactivates c-MET and promotes formation of new blood vessels and tumor growth in vivo. SEMA4D is over-expressed in a wide array of tumor types, and is also produced by inflammatory cells present in the tumor microenvironment. It has recently been demonstrated that in an environment lacking SEMA4D, the ability of mouse breast cancer cells to originate tumor masses and metastases is severely impaired, and that SEMA4D produced by tumor associated macrophages is required for tumor angiogenesis and growth. In addition to its effects on endothelial cells, SEMA4D binding to PLXNB1 on tumor cells results in MET transactivation and migration of tumor cells. It has been further reported that overexpression of PLXNB1 and MET in breast and ovarian cancers is a negative prognostic factor. Tumors co-expressing PLXNB1 and MET were characterized as having a higher grade and an increased frequency of metastases. Collectively, these results suggest that expression of SEMA4D, either by tumor cells or by tumor associated macrophages, may represent a mechanism of tumor neovascularization, and that expression of the SEMA4D receptor in tumors may further induce tumor invasion, thereby enhancing their growth, survival, and metastatic potential. Antibody neutralization of SEMA4D thus may represent a new therapeutic strategy for cancer treatment. We selected a high affinity human IgG4 antibody that is specific for both mouse and human SEMA4D, and utilized several in vitro functional assays to demonstrate that this antibody blocks SEMA4D - PLXNB 1 interactions. Using both transplanted and orthotopic tumor models we demonstrated that antibody mediated SEMA4D neutralization in vivo inhibits tumor growth and tumor angiogenesis. This human antibody is currently in IND-enabling toxicology testing and we anticipate the initiation of human clinical trials in 2H 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-36.

Isolation and characterization of selective and potent human Fab inhibitors directed to the active-site region of the two-component NS2B–NS3 proteinase of West Nile virus

There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes.

Isolation of high affinity human scFv antibodies against mouse and human tumor endothelial marker 1 (TEM1) using yeast-display scFv library derived from an autoimmune patient. (42.4)

Abstract TEM1 is predominantly expressed on neovasculature, thus ideally suited for targeted imaging and therapy for cancer. Yeast has emerged as an alternative technology for display and selection of recombinant antibody fragments (scFv). Because autoimmune disorders may produce self-reactive specificities, an autoimmune repertoire derived from a patient with thrombotic thrombocytopenic purpura (TTP) was screened to isolate anti-TEM1 scFv. A yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage. The library (10^9 members) was flow sorted with decreasing antigen concentration until a scFv pool detecting 2 nM of rhTEM1/GST was obtained. Secreted scFv were expressed and 470 scFv were screened by ELISA for binding to rhTEM1/GST. Nearly 50% of the scFv gave colorimetric signals higher than 3 standard deviations above background. No cross-reactivity with GST control protein was detected. Sequencing of 29 scFv identified 8 unique antibodies. Two of these clones exhibited Kd's in the 100 pM range and bound to both mouse and human TEM1. Such cross-reactive anti-mouse/human TEM will facilitate proof of concept for the development of novel anti-angiogenic targeted therapies. Our data suggest that the use of large scFv libraries derived from autoimmune patient repertoires may provide a rich source of high affinity antibody reagents to therapeutically relevant molecules. This work was supported by the Claneil Foundation

409 POSTER A Phase I clinical trial of the oral PPAR gamma agonist, CS-7017 in patients with advanced malignancies

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

MAbs from mammalian cells

Cytos has modified its cloning technology for mammalian cell display of antibodies to isolate repertoires of high-affinity human antibodies faster—and possibly with greater diversity—than phage display. But the platform may be limited to antibody targets such as viruses while excluding cancer and self-antigens.

Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

The Human Combinatorial Antibody Library HuCAL GOLD Combines Diversification of All Six CDRs According to the Natural Immune System with a Novel Display Method for Efficient Selection of High-Affinity Antibodies

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.

Modification and identification of a vector for making a large phage antibody library

The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies. scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated. The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression. The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

Production of High-Affinity Human Monoclonal Antibody Fab Fragments to the 19-Kilodalton C-Terminal Merozoite Surface Protein 1 ofPlasmodium falciparum

A combinatorial immunoglobulin gene library was constructed from peripheral blood lymphocytes of eight patients infected with Plasmodium falciparum and was screened for the production of human monoclonal antibody Fab fragments to the C-terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1(19)). Three Fab clones recognized recombinant MSP-1(19) under nonreducing conditions. Indirect immunofluorescence microscopy demonstrated that three Fab clones stained the surfaces of late trophozoites/schizonts and merozoites of the FCR3 and 3D7 strains, suggesting the Fabs' reactivities to a conserved epitope. Sequence analysis of the heavy-chain genes revealed that the closest germ line V segments were VH1-8 and VH7-81, with 91% to 98% homology. The closest germ line D segment was D3-10, and the closest germ line J segment was JH4 or JH5, with 90% to 97% homology. In the light-chain genes, the closest germ line V segment was A27 for the Jkappa2, Jkappa4, and Jkappa5 segments. The dissociation constants of these Fab fragments for recombinant MSP-1(19) ranged from 1.09 x 10(-9) to 2.66 x 10(-9) M. The binding of the three Fab fragments to MSP-1(19) was competitively inhibited by the anti-MSP-1(19) mouse monoclonal antibody 12.8, which inhibits erythrocyte invasion by merozoites. However, the human Fab fragment with the highest affinity did not inhibit in vitro growth of P. falciparum. This is the first report of gene analysis and bacterial expression of human monoclonal antibodies to P. falciparum MSP-1(19). The combinatorial immunoglobulin gene library derived from malaria patients provides a potential tool for producing high-affinity human antibodies specific for P. falciparum.

A high affinity human antibody antagonist of P-selectin mediated rolling

We have characterized the IgG form of a previously isolated and engineered single-chain Fv (scFv), named RR2r3s4-1, that binds to human PSGL-1. This fully human IgG was determined to have a K d of 1.8 ± 0.7 nM by fluorescence quenching titration. It better inhibits P-selectin–PSGL-1 interactions than a commercially available murine monoclonal antibody KPL1 and better inhibits neutrophil rolling than KPL1. Thus, RR2r3s4-1 is the most effective antibody at inhibiting P-selectin–PSGL-1 interactions known. Specificity analysis reveals that RR2r3s4-1 does not cross react with murine PSGL-1 and thus requires more than tyrosine sulfate for binding to human PSGL-1. This evidence demonstrates the therapeutic potential of this antibody as a potent anti-inflammatory therapeutic.