The final objectives of the proposed project were to provide a system by which human antibodies could be easily derived (using the phage antibody system) from human B cells which were immunised with different antigens in vitro. Furthermore, by the use of immunogens derived from proteins expressed by bacteria (either as fusion proteins or on the bacterial cell surface), it was hoped that the derivation of human mAbs against the protein products of cloned genes would be facilitated.
The collaborative part of this project could not be completed as planned due to the difficulty of obtaining reproducible in vitro immunisation, an unsolved scientific problem still being tackled by many groups worldwide. However, the individual groups continued to develop their respective technologies, and as a result the realisation of the concept of selecting a (human) antibody against the product of a cloned gene is far closer than it was when the project started.
A number of conditions for the growth and in vitro immunisation of human splenic and tonsil B cells have been tested by the UL group. In common with many other groups working in this field, the conditions required for perfect and reproducible in vitro immunisation have not been found. However, growing splenic B cells on OKT3 coated macroplates in culture for 6 days and immunising with Tetanus toxoid - DNP (TT-DNP) resulted in good B cell survival and occasional specific antibody responses against TT-DNP. Single positive antibody secreting cells could be recognised by a sensitive ELISAspot assay adapted for this purpose. The problem in developing effective in vitro immunisation may be related to the maximum number of cells which can be immunised in vitro (no more than 1E7 at a time, not all of which will be responsive), a number far lower than the number of different specificities in either a mouse or a large phage antibody (phAb) library (1E10). In parallel with these experiments, the immunisation of human cells transferred to scid-mice has been attempted. Secondary immune responses against antigens (TT) to which donors had been previously exposed could be obtained, as could primary responses against antigens coupled to TT (e.g. canine albumin-TT). However primary responses against antigens (ovalbumin, TT or canine albumin-TT), which donors had not previously encountered, could not be obtained.
In the absence of in vitro immunised cells from which phage antibody (phAb) libraries could be made, the group at CAT continued to work on the development of methodologies to create large phage(mid) antibody libraries. By comparison with control scFvs, the main bottlenecks in the creation of such libraries were shown to involve the de novo cloning of the V regions, the assembly of the scFv and efficient restriction digestion of the final PCR product. These problems were overcome by optimising the primers used for amplification, cloning heavy and light chains separately and combining them in a two fragment PCR assembly using long DNA overhangs which allowed efficient restriction prior to cloning. The application of these methods led to the creation of a library with a diversity of 6E9, from which antibodies with affinities as high as 300pM could be obtained. In addition to optimising conventional cloning procedures, in vivo recombination methods were also studied, but found to be far less effective than the conventional scFv phagemid library.
The use of bacterial 'living columns' to isolate phAbs against the protein products of cloned genes has been developed by the SIRS group under this grant. This experimental technique involves the display of an epitope or protein on the surface of bacteria and using this as a column to select from phAb libraries. The feasibility of the approach was demonstrated by the expression of an experimental epitope (p21ras) on the surface of E. coli (within the context of lamB) which was shown to be capable of purifying an anti-ras phAb from a background of irrelevent phAbs as well as binding phAbs from a naive library. Subsequently, the expression of NGF both on the surface of bacteria, and as a soluble protein, was attempted using a number of different protein display systems (lamB, Lpp/OmpA, pelB periplasmic). This was to have served as a model system (the structure of this neurotrophin (NT) is known and a panel of Abs which recognise it are available) for the display of other NTs and the derivation of antibodies against them. Unfortunately, although NGF was expressed in the bacteria, it was not displayed on the bacterial cell surface, probably due to the complex nature of the protein (cysteine knot structure), and neither could soluble NGF be obtained. As a result it was decided that a new target should be studied. Loops from the presenillin 1 gene (involved in Alzheimer's disease) were cloned into surface expression vectors and used to select phage antibodies from three published libraries. phAb clones whose specificity is being investigated have been obtained from one of these.
A natural extension of the use of the living column is the possibility of developing selection by infection. Following other work performed in the laboratory which show that by manipulation of fd g3p (by adding the IKe g3p binding domain), fd host range can be augmented to infect bacteria bearing N pili as well as those bearing F pili, we have attempted to identify the protein recognised by IKe g3p on the N pilus in order to use it as a display vector. This was not successful. We have also attempted to select phAbs from both naive libraries and libraries made from mice immunised with N pili. However, in no case was positive selection obtained. These experiments will be repeated with more representative libraries.
Funding SchemeCSC - Cost-sharing contracts
SG8 6EJ Royston