Jacqueline Sharon

Recombinant polyclonal antibodies for cancer therapy

Although monoclonal antibodies are increasingly used for cancer therapy, remissions are only temporary due to emergence of tumor cell escape variants that are no longer affected by the antibody. The emergence of escape variants could be minimized by multi‐targeting of tumor cells with polyclonal antibodies, which would also be more efficient than monoclonal antibodies at mediating effector functions for target destruction. A technology for generating recombinant polyclonal antibodies for cancer therapy has been developed based on the construction and selection of tumor‐reactive Fab phage display libraries. The selected Fabs are mass‐converted to full‐length polyclonal antibody libraries (PCALs) of any isotype and any species. Prototypic PCALs generated against human colorectal cancer cell lines showed that libraries of diverse recombinant antibodies, enriched for reactivity to the cancer cells compared to normal human cells, can be obtained. The success of recombinant polyclonal antibodies as cancer therapeutics will depend on the ability to generate, characterize, and mass‐produce PCALs with high ratios of cancer‐to‐normal reactivities that cross‐react with many cancers of the same type.

Recent efforts toward the development of peptide secondary structure mimetics at Molecumetics Ltd. for the discoveries of new drug candidates utilizing combinatorial chemistry with solid phase synthesis are described

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen escape variants, and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review her e our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics

Protective antibodies play an essential role in immunity to infection by neutralizing microbes or their toxins and recruiting microbicidal effector functions. Identification of the protective B‐cell epitopes, those parts of microbial antigens that contact the variable regions of the protective antibodies, can lead to development of antibody therapeutics, guide vaccine design, enable assessment of protective antibody responses in infected or vaccinated individuals, and uncover or localize pathogenic microbial functions that could be targeted by novel antimicrobials. Monoclonal antibodies are required to link in vivo or in vitro protective effects to specific epitopes and may be obtained from experimental animals or from humans, and their binding can be localized to specific regions of antigens by immunochemical assays. The epitopes are then identified with mapping methods such as X‐ray crystallography of antigen–antibody complexes, antibody inhibition of hydrogen–deuterium exchange in the antigen, antibody‐induced alteration of the nuclear magnetic resonance spectrum of the antigen, and experimentally validated computational docking of antigen–antibody complexes. The diversity in shape, size and structure of protective B‐cell epitopes, and the increasing importance of protective B‐cell epitope discovery to development of vaccines and antibody therapeutics are illustrated through examples from different microbe categories, with emphasis on epitopes targeted by broadly neutralizing antibodies to pathogens of high antigenic variation. Examples include the V‐shaped Ab52 glycan epitope in the O‐antigen of Francisella tularensis, the concave CR6261 peptidic epitope in the haemagglutinin stem of influenza virus H1N1, and the convex/concave PG16 glycopeptidic epitope in the gp120 V1/V2 loop of HIV type 1.

A bidirectional phage display vector for the selection and mass transfer of polyclonal antibody libraries

An approach to the creation of antigen-specific polyclonal libraries of intact antibodies is presented. A polyclonal library of Fab antibody fragments would be expressed using a phage display vector, and selected for reactivity with an antigen or group of antigens. For conversion into a sublibrary of intact polyclonal antibodies, the selected heavy (H) and light (L) chain variable (V) region gene combinations would be transferred in mass, as linked pairs, to a eukaryotic expression vector which provides immunoglobulin (Ig) constant (C) region genes. To enable this selection and transfer, a bidirectional phage display vector was generated, in which the V region gene pairs are linked head to head in opposite transcriptional orientations. The functionality of this vector was demonstrated by the selection, transfer and expression of linked V region gene pairs derived from an A/J mouse that had been immunized with p-azophenylarsonate (Ars)-coupled keyhole limpet hemocyanin (KLH). As expected, the expressed IgG2b anti-Ars antibodies with selected V region gene pairs were shown to have V region sequences and Ars-binding characteristics similar to those of anti-Ars hybridoma antibodies. The technology presented here has potential for many diagnostic and therapeutic applications. These include the generation of polyclonal antibody libraries against multiple epitopes on infectious agents or cancer cells, and of polyclonal libraries encoding chimeric molecules composed of antibody V regions and T cell receptor C regions.

Characterization of Monoclonal Antibodies to Terminal and Internal O-Antigen Epitopes of Francisella tularensis Lipopolysaccharide

The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia.

A typical preparation of Francisella tularensis O-antigen yields a mixture of three types of saccharides.

Tularemia is a severe infectious disease in humans caused by the Gram-negative bacterium Francisella tularensis (Ft). Because of its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen. Purification of LPS saccharides of defined length and composition is necessary to reveal the epitopes targeted by protective antibodies. In this study, we purified saccharides from LPS preparations from both the Ft subspecies holarctica live vaccine strain (LVS) and the virulent Ft subspecies tularensis SchuS4 strain using liquid chromatography. We then characterized the fractions using high-resolution mass spectrometry and tandem mass spectrometry. Three types of saccharides were observed in both the LVS and SchuS4 preparations: two consisting of OAg tetrasaccharide repeats attached to one of two core oligosaccharide variants and one consisting of tetrasaccharide repeats only (coreless). The coreless OAg oligosaccharides were shown to contain Qui4NFm (4,6-dideoxy-4-formamido-D-glucose) at the nonreducing end and QuiNAc (2-acetamido-2,6-dideoxy-O-D-glucose) at the reducing end. Purified homogeneous preparations of saccharides of each type will allow mapping of protective epitopes in Ft LPS.

Expression of a prototypic anti-colorectal cancer polyclonal antibody library in mammalian cells

We describe the production of a prototypic polyclonal antibody library (PCAL), a standardized mixture of full-length IgG polyclonal antibodies for which the genes are available. The PCAL was generated by mass transfer of heavy and light chain variable region gene pairs, selected for binding to human colorectal cancer cells, from a Fab phage display vector to a mammalian IgG expression vector. Following transfection of the IgG vector library into Sp2/0 myeloma cells, clones were characterized for IgG expression and binding to the colorectal cancer cells by ELISA, and for diversity by DNA fingerprinting, nucleotide sequencing, and immunoblot analysis. The results showed that 76-84% of the library clones produce IgG and of those 72-79% bind antigen. Furthermore, preliminary analysis showed clonal diversity at both the DNA and antigen-binding levels. When depleted of reactivity to normal tissue, polyclonal antibody libraries to cancer cells may be efficacious for cancer therapy.

Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia

Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non‐overlapping terminal and internal epitopes of the F. tularensis LPS O‐polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg‐binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg‐binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb‐treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B‐cell epitopes of F. tularensis OAg.

Polyclonal Fab phage display libraries with a high percentage of diverse clones to Cryptosporidium parvum glycoproteins

The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C. parvum-specific polyclonal antibody libraries, standardised, perpetual mixtures of polyclonal antibodies, for which the genes are available. A combinatorial Fab phage display library was generated from the antibody variable region gene repertoire of mice immunised with C. parvum surface and apical complex glycoproteins which are believed to be involved in mediating C. parvum attachment and invasion. The variable region genes used to construct this starting library were shown to be diverse by nucleotide sequencing. The library was subjected to one round of antigen selection on C. parvum glycoproteins or a C. parvum oocyst/sporozoite preparation. The two selected libraries showed specific reactivity to the glycoproteins as well as to the oocyst/sporozoite preparation, with 50-73% antigen-reactive members. Fingerprint analysis of individual clones from the two antigen-selected libraries showed high diversity, confirming the polyclonality of the selected libraries. Furthermore, immunoblot analysis on the oocyst/sporozoite and glycoprotein preparations with selected library phage showed reactivity to multiple bands, indicating diversity at the antigen level. These C. parvum-specific polyclonal Fab phage display libraries will be converted to libraries of polyclonal full-length antibodies by mass transfer of the selected heavy and light chain variable region gene pairs to a mammalian expression vector. Such polyclonal antibody libraries would be expected to mediate effector functions and provide optimal passive immunity against cryptosporidiosis.

The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide.

We have previously described two types of protective B‐cell epitopes in the O‐antigen (OAg) of the Gram‐negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti‐OAg monoclonal antibodies (mAbs), and a non‐overlapping epitope at the non‐reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non‐reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen‐binding bivalent avidity than internally binding anti‐OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen‐binding. The X‐ray crystal structure of N62 Fab showed that the antigen‐binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non‐reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions.