# Patent

## Title
Products comprising inactivated yeasts or moulds and active VHH-type antibodies

## Abstract

The use of VHHs in the preparation of products to provide stability of antibody specificity under destabilizing conditions whereby normally lower eukaryotes or traditional antibodies are killed or inactivated.

## Background
RELATED APPLICATIONS
The present application is a U.S. National Phase Application of International Application PCT/EP99/01678, filed Mar. 12, 1999, which claims the benefit of European Application No. 98104479.5, filed Mar. 12, 1998 and European Application No. 99200439.0, filed Feb. 16, 1999, all of which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention is in the field of applied biotechnology and relates in particular to an economic way of introducing antibodies into a variety of products, especially consumer product such as food products and personal care products and animal feed.
BACKGROUND OF THE INVENTION
To fulfil many unmet demands of the society, there is a clear need to provide new products from which consumers may benefit, in particular in the field of food products including animal feed and personal care products such as laundry detergents, deodorants etc. One of the problems with the presently used consumer products is that if they contain functional compounds, these compounds are often not very specific.
For instance, for killing microorganisms in food products the functional compound may be a salt or an acid. If the concentration of either of these compounds is sufficiently high, they are able to kill microorganisms. However, often the level of salt renders the taste of the product less attractive to the consumer. Moreover, high levels of salt are not recommended by health authorities. The same holds for products that contain considerable amounts of acids or preservatives.
Non-food consumer products such as personal care products e.g. laundry detergent products may contain compounds that are able to bleach stains, but as they do not specifically recognise stains, considerable amounts of these bleach generating compounds (e.g. percarbonate/TAED) are necessary.
Another example is that some hair care products contain compounds that kill the microorganisms involved in dandruff. Also these compounds are not specific and in addition to the causative microorganisms of dandruff they also kill other microorganisms on the skin which are beneficial to the consumer.
Still another example of an unmet demand of the society is the overproduction of manure due to cattle farming. Part of this problem is that the conversion of animal feed into nutrients is not optimal due to the presence of negative compounds like phytic acid.
The above given examples of unmet consumer needs and society needs can be extended, but it will already be clear that consumer products and animal feed with compounds which specifically meet the demand of the consumer or society will be of great importance.
The main characteristic of processes in living cells or living species is that they are often highly specific. Enzymes only recognise specific substrates and the specificity of antibodies is unsurpassed. Therefore in recent years the industries involved in the manufacturing of (chemical and biotechnological) consumer products or animal feed are increasingly applying biological molecules in order to make their products more specific.
A successful example of this development is the introduction of proteases, lipases, cellulases and amylases in laundry products. These enzymes have a certain specificity and because the production of these enzymes has been improved considerably by recombinant DNA techniques, these microbial enzymes can be produced by microorganisms at costs affordable for consumer products. The introduction of enzymes in detergents has indeed met demands of our society. Over the last decades the energy usage for cleaning of laundry has been reduced with about 50% and the amount of chemicals with about 40%.
In the area of animal feed enzymes can play an important role to reduce the environmental pollution, as has been nicely demonstrated by the application of the enzyme phytase in animal feed. Again this development was possible due to the enormous improvement of the production of phytase by rDNA techniques.
However, often a higher specificity than just recognising a protein or fat is highly desirable to fulfil the unmet demands and therefore the consumer goods and animal feed industry is looking for ways to get specificities in the same order as antibodies have.
In nature microorganisms do not produce antibodies. Although it is well known that by using rDNA technology microorgansims can produce almost any protein, irrespective of its origin, the yield of homologous proteins is much higher than for heterologous proteins. Moreover, expensive purification processes are needed to recover the heterologous protein from its producing cell, since it is not allowed to bring living rDNA organisms into the environment. These two factors, the low production yield and the fact that rDNA organisms have to be separated from the products that they produce have slowed down the introduction of heterologous proteins in consumer products other than in laundry products and some food products. In animal feed only the addition of phytase is a real success up to now.
The present invention relates in particular to introducing certain categories of antibodies into a variety of products, especially food products, personal care products and animal feed.
Hamers-Casterman et al., Nature (1993) 363:446-448 disclose isolated immunoglobulins from the serum of Camelidae comprising two heavy polypeptide chains sufficient for the formation of a complete antigen binding site, which immunoglobulins further being devoid of light polypeptide chains.
WO 94/25591 discloses the production of antibodies or functionalised fragments thereof derived from heavy chain immunoglobulins of Camelidae, using transformed lower eukaryote host organisms.
Spinelli et al., Nature structural biology (1996) 3:752-757 disclose the crystal structure of a llama heavy chain variable domain.
In many processes involving recombinant DNA techniques and culturing or fermenting transformed microorganisms, it is necessary at some stage, for example at the conclusion of the fermentation, to kill the active cells in order to prevent any viable recombinant organisms from being released into the environment.
A conventional way of killing cells is using heat. U.S. Pat. No. 4,601,986 is an example of the use of heat to kill the cells and stop the growth of microorganism cultures. Other conventional ways of killing cells are by lysing the cells, for example by applying high osmotic pressures or by adding enzymes which break down the cell walls or membranes. These techniques are exemplified in U.S. Pat. Nos. 4,299,858, 3,816,260, 3,890,198, and 3,917,510.
In many systems host microorganisms, for example lower eukaryote cells, are difficult to kill. Conventional methods, such as heat, are too severe and may destroy or alter the desired product before the cells are killed. This applies also for the immobilized systems of binding proteins on lower eukaryotes disclosed in WO 94/18330, when the binding protein is an antibody or a functional fragment thereof.
It is often desirable to manufacture, store or use products under conditions which would normally be detrimental to the stability of biological systems such as microorganisms or antibodies. For example products may desirably be heated, e.g. pasteurised during manufacture or use (e.g. high temperature washing) or its composition and/or pH may be such that the biological systems do not survive storage over pro-longed periods.
Similarly, there is still a need for stable functional systems possibly involving microorganisms, where the microorganisms are inactivated or killed and/or their growth is surpressed while the functional activity of the system is substantially maintained.
In this light there is a need to use components in products, especially consumer products, whereby on the one hand said components have a acceptable or even good specificity and on the other hand are stable under conditions of manufacture and/or storage and/or use.
BRIEF DESCRIPTION OF TERMS
As used herein, the term “antibody” refers to an immunoglobulin which may be derived from natural sources or may be synthetically produced, in whole or as antibody fragment.
An “antibody fragment” is a portion of a whole antibody which retains the ability to exhibit antigen binding activity. Functionalized antibody fragments are also embraced in this term.
The term “functionalized antibody fragment” is used for indicating an antibody or fragment thereof to which one or more functional groups, including enzymes and other binding polypeptides, are attached resulting in fusion products of such antibody fragment with another biofunctional molecule.
The term “traditional antibody” is used for an antibody which normally consists of two heavy and two light chains or fragments thereof.
The term “VHH” refers to the single heavy chain variable domain antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains; synthetic and naive VHH can be construed accordingly.
SUMMARY OF THE INVENTION
In one aspect the present invention relates to a method of preparing, storing or using a product comprising active antibodies, wherein said method involves the application of conditions whereby normally lower eukaryotes or traditional antibodies are killed or inactivated and whereby the active antibodies are VHHs.
In another aspect the invention relates to the use of VHHs in the preparation of products to provide stability of antibody specificity under conditions whereby normally lower eukaryotes or traditional antibodies are killed or inactivated.
These and other aspects of the invention will be described in further detail in the description which follows.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a typical dilution curve for the antibodies used, from which the sub-saturation point was determined.
FIG. 2 shows the antigen binding after temperature treatment of hCG specific mouse MAbs (A) and llama VHHs (B) or RR-6 specific mouse MAbs (C) and llama VHHs (D). Antibodies were incubated for 2 hrs at different temperatures, cooled down to room temperature and subsequently ELISA was performed.
FIG. 3 shows the antigen binding in presence of ammonium thiocyanate at sub saturation point of hCG specific mouse MAbs (A) and llama VHHs (B) or RR-6 specific mouse MAbs (C) and llama VHHs (D).
FIG. 4 represents a restriction map of plasmid pUR 4482
FIG. 5 represents a restriction map of plasmid pUR 4588.
FIG. 6 represents a restriction map of plasmid pUR 4676
FIG. 7 represents a restriction map of plasmid pUR 4659
FIG. 8 shows the influence of different levels of OMO (without protease) on the stability of anti-RR120 VHHs in 25 mM NaCO3 and pH 10.3, whereby the amount of binding in 25 mM NaCO3 at pH 10.3 is set at 100% immunoreactivity.
FIG. 9 shows the influence of different levels of LAS on the stability of anti-RR120 VHHs in 25 mM NaCO3 and pH 10.3, whereby the amount of binding in 25 mM NaCO3 at pH 10.3 is set at 100% immunoreactivity.
FIG. 10 shows the amino acid sequence of anti-RR120 VHHs A38 (SEQ ID NO: 23) and A44 (SEQ ID NO: 24) (obtained by screening in PBS), A306 (SEQ ID NO: 25), A307 (SEQ ID NO: 26) and A308 (SEQ ID NO: 27) (obtained by screening in LAS) and A309 (obtained by screening in Dutch Omo without protease; SEQ ID NO: 28).



DETAILED DESCRIPTION OF THE INVENTION
Traditional antibodies, consisting of two heavy and two light chains are rather stable when they circulate in the blood and lymph systems to protect the host against invaders. This stability is caused by several S—S bridges between the heavy chains and the strong interaction between the heavy and the light chain. However, production of complete antibodies in microorganisms is very difficult and economically not yet feasible for introduction into consumer products or animal feed.
Recently, it has been shown that antibody fragments of traditional antibodies can be produced by certain microorganisms, but in general the yield of these fragments is rather low. The stability of fragments of traditional antibodies is rather low and the physics of protein stability is still not sufficiently developed to predict protein stability from its amino acid sequence.
The present invention is based on the surprising finding, after extensive research and experimentation, that on average VHHs are more stable against destabilising physical and/or chemical conditions such as one or more of: high temperature, ultra high pressure, pulse electric field, radiation, presence of anti-microbial, presence of (mixed) surfactants, presence of ethanol, pH altering materials and presence of chaotropic agents as compared to eukaryotic cells or as compared to traditional antibodies. It is even more surprising that certain variable domains of Camelidae antibodies from llamas appear to be very stable under pasteurisation conditions.
Therefore in a first aspect the inventions relates to a method of preparing (including the identification of antibodies), storing or using a product comprising active antibodies, wherein said method involves the application of destabilising conditions whereby normally lower eukaryotes or traditional antibodies are killed or inactivated and wherein the active antibodies are VHHs.
In another aspect the invention relates to the use of VHHs in the preparation of products to provide stability of antibody specificity under destabilising conditions whereby normally lower eukaryotes or traditional antibodies are killed or inactivated.
The invention is further based on the finding that physical techniques or chemical treatment, or a combination of physical techniques and chemical treatment or a combination of special types of mild techniques which are destabilising conditions in that they are able to kill or inactivate lower eukaryotes e.g. Saccharomyces Cerevisiae. Typically the lower eukaryotes would be reduced by a factor of at least 106, or which are able to kill or inactivate traditional antibodies, surprisingly can be used whereby the VHHs retain their immunoreactivity to a large extent, typically >60%, and preferably from about 70% to 100% or even 90% to 100%.
Preferably the destabilising conditions are such that normally lower eukaryote cells are inactivated with a factor of at least 106. Typically the conditions are chosen such that under these conditions lower eukaryotes would be reduced by a factor of at least 106, because this killing efficiency is often acceptable for the health authorities as killing efficiency for pathogenic bacteria in food products. A preferred reduction is with a factor of 107 to 109. Particularly the conditions are chosen such that the activity of Saccharomyces cerevisiae would be reduced by a factor of at least 106.
In a preferred embodiment the destabilising conditions are such that normally the immunoreactivity of traditional antibodies is reduced to less than 70%, more preferred less than 10%, e.g. less than 1% or even less than 0.01%. Typical examples of traditional antibodies are mouse monoclonal antibodies. Especially preferably the destabilising conditions are such that the immunoreactivity of mouse monoclonal antibodies agains hCG as described in Gani M, Coley J and Porter P Hybridoma 6: (6) 637-643, December 1987 (hereafter referred to as HCGMABS) is reduced to less than 70%, more preferred less than 10%, e.g. less than 1% or even less than 0.01%.
The VHHs for use in accordance to the invention are stable under the conditions whereby the lower eukaryotic cells or traditional antibodies are normally killed or substantially inactivated. In particular they preferably recognise their binding target e.g. harmful microorganisms or toxins with a binding constant of >104, preferably >106 or >10′, most preferably >108 and/or retain their immunoreactivity for at least 70%, more preferred from 80-100%, most preferred from 90 to 100% after application of the destabilising conditions for eukaryotic cells and/or traditional antibodies.
The destabilising conditions preferably comprise physical treatments and/or chemical conditions which are applied during manufacture, storage or use of the product.
Suitable examples of physical treatment as destabilising conditions are selected from one or more of:
(a) a high temperature treatment, for example at more than 70° C., for example 80°, 90° or 100° C., especially 70-100° C. The duration of the heat treatment is not very critical but will normally be between 30 seconds and 2 hours, e.g. 1-60 minutes. Preferably the duration is such that on the one hand destabilising conditions are present for lower eukaryotes or traditional antibodies, but on the other hand the VHHs are not substantially deactivated. Typical examples of such conditions are for example pasteurisation of the product during manufacture or use of the product under high temperature conditions e.g. high temperature (e.g. >70° C. washing).
(b) ultra high pressure treatment, for example at 250 Mpa or more, say 250-500 Mpa. Again the duration of the high pressure treatment is not very critical, but will normally be between 1 second and 2 hours, e.g. 1-60 minutes.
(c) irradiation treatment, for example at least 10 kGray gamma radiation, for example 10-50 kGray gamma radiation.
(d) pulse electric field treatment. Suitable application conditions of pulse electric field are well-known in the art.
Optionally physical treatment destabilising conditions can be used in combination with one or more chemical destabilising conditions. If this is done, generally it is possible to use relatively mild physical conditions in combination with relatively mild chemical conditions. In particular it was found that combinations of mild physical treatments and antimicrobial compounds, such as acids or antimicrobial peptides were even more effective to kill the lower eukaryotes, while maintaining the functionality of the VHH. Using these combinations, killing effects of >107 could be achieved, while the functionality of the antibody remained substantially unaffected.
In another embodiment of the invention the destabilising conditions involve chemical treatment e.g. selected from one or more of:
(a) the presence of an effective amount of antimicrobial agent, for example sorbic acid, benzoic acid, nisin, MB21, or another bacteriocin. Typically the effective amount of antimicrobial agents are chosen such that they are capable to contribute to the destabilising conditions. The exact effective level is usually dependent on the type of agent and the whether or not other destabilising treatments are applied but may normally be from 1 ppm to 5 wt %, e.g. 0.01 to 2 wt %.
(b) the presence of an effective amount of a cell wall degrading enzyme, for example β-(1,3)-glucanase, β-(1,6)-glucanase, chitinase and/or a redox enzyme. Typically the effective amount of such enzymes are chosen such that they are capable to contribute to the destabilising conditions. The exact effective level is usually dependant on the type of enzyme and whether or not other destabilising treatments are applied but may normally be from 1 ppm to 1 wt %, e.g. 0.0001 to 0.1 wt %.
(c) the presence of solvent materials e.g. alcohol at for example at a level of at least 10 wt %. These high levels of alcohol are often destabilising especially for lower eukaryotes such as yeasts. Typical destabilising levels for solvent materials may vary in a wide range dependant on the type of solvent and the other destabilising conditions which are applied. Normally levels of 3-96 wt % would be used, e.g. 10 to 35 wt % of the product. A convenient solvent material is ethanol.
(d) the presence of surfactant materials, especially mixtures of two or more surfactants e.g. selected from the group of anionic, nonionic or zwitterionic detergency materials for example at a level of at least 0.2 g/l. Typical examples of suitable surfactant materials are well-known in the art e.g in the formulation of household detergent or personal washing systems such as shampoo. The level of the surfactant materials may vary e.g. between 5 and 50 wt % of the product, e.g. 10-30 wt % or much lower levels such as 0.01 to 20 g/l in the washing solution or the screening medium. Advantageously, mixtures of anionic and nonionic surfactants are used in a weight ratio of from 10:1 to 1:10.
e) the presence of chaotropic materials e.g. ammonium thiocyanate (ATC), urea and/or ATC elution;
(f) the presence of materials e.g. acidifying or buffering materials which render the pH less than 5 or more than 9, examples of these are NaCO3, acetic acid, citric acid etc.
As described above the chemical treatment may advantageously be used in combination with one or more physical treatments.
In accordance to an aspect of the invention the destabilising conditions are applied during one or more of preparation, storage or use of the product. For the purpose of this invention the term “preparation of the product” refers to all steps carried out in the preparation of the product in the presence of VHH such as on the one hand “selection” of the VHH, and on the other hand actual “manufacture” of the product e.g. mixing of ingredients with VHHs, pasteurization, drying, packaging etc.
Surprisingly it has been found that VHHs are often more stable than traditional antibodies. One of the problems in the manufacture of products especially the stage of identification of suitable antibodies for use in products e.g. food, personal care or animal feed products is that the selection is usually done in a model system. The good stability of VHHs now renders it possible to carry out the identification of the effective VHHs under destabilisation conditions. For example the VHHs can be screened in a medium comparable to the product in which the VHH is to be used. For example antibodies for use in personal care products such cleaning formulations e.g. laundry detergents or shampoos can be screened in the product. This is an easy and effective way to identify VHHs which on the one hand have adequate binding affinity towards the target molecule, but which are on the other hand stable under product conditions.
Although applicants have found that VHHs are on average more stable than traditional antibodies or lower eukaryotes, it may still be desirable to apply a selection step to VHHs whereby the VHHs which are most stable under the destabilising conditions are selected. Such a selection step may be carried out by any suitable method, however as described above a preferred method involves the screening of VHHs under product conditions e.g. in the presence of a system which is equal or similar to the product in which the VHH is to be used. Therefore in a preferred embodiment of the invention both the selection of suitable VHHs and at least one further step selected from manufacture, use or storage takes place under destabilising conditions.
Therefore the invention also relates to a method to prepare a product comprising VHHs, comprising the steps of: selection of suitable VHHs by screening under conditions which are equal or similar to the conditions of the product; and incorporating the selected VHHs in the product. Preferably such product is manufactured, used or stored under destabilising conditions for lower eukaryotes or traditional antibodies as described above. The invention also encompasses a product obtainable by such a method.
In another embodiment of the invention, the manufacture of the product involves the combination of ingredients and application of destabilising conditions. For example it is often desirable in the production of a food, personal care or animal feed product to heat the product, e.g. to ensure pasteurisation to ensure keepability. If traditional antibodies are present during such a heating step, they are generally inactivated and hence they can only be added after the pasteurization step, which is clearly less desired. Surprisingly it has been found that VHHs are often more stable against the use of destabilising conditions applied in the manufacturing process of products.
In another embodiment of the invention the storage of the product involves the application of destabilising conditions. For example the chemical composition of the product may contain ingredients which would normally destabilise traditional antibodies and/or eukaryotes. Surprisingly it has been found that VHHs have an enhanced stability wrt said destabilising conditions which renders it possible to store the product for pro-longed periods e.g 1 week to 1 year without an unacceptable reduction of immunoreactivity.
In another embodiment the use of the product involves the application of destabilising conditions. For example the use of a food product may include the heating thereof to temperatures which would normally destabilise lower eukaryotes and/or traditional antibodies. Alternatively personal care products e.g. laundry detergents may be used under high temperature conditions. Surprisingly it has been found that VHHs can be used and retain their stability for the antibody activity when destabilising conditions are applied during use.
Preferred products in which VHHs can be applied include, for example, consumer products. A preferred group of products are food products such as ice-cream, oil based products such as margarines, oils, mayonnaise, dressings, soups, sauces, soft drinks, for example tea based drinks, meals etc. Another preferred group of products are personal care product such as cleaning products like soap bars, washing liquids, gels, laundry detergents e.g. in powder, paste, tablet or liquid form, and deodorants, creams, lotions, shampoo etc. A third group of products are animal feed products in dry or wet form.
Typical amounts of VHHs in the product are from 0.001 μM to 1 M, under the destabilising conditions e.g. in the preparation, storage or use the amounts can typically be from 0.1 to 100 μM, e.g. 2 to 20 μM.
The invention will be illustrated by means of the following examples which also show the use of Mouse monoclonal antibodies.
Mouse monoclonal antibodies (MAbs) or fragments thereof have many potential applications in addition to for example cancer therapy and diagnostic kits. However, for most applications large quantities are needed whereas costs have to be reduced. For MAbs or fragments thereof these conditions often cannot be met. The Camelidae heavy-chain antibodies offer a solution to this problem. Probably because of their simple one chain structure and their solubility (Spinelli et al., 1996), VHHs, especially the variable part of llama heavy chain can be secreted relatively pure and in high amounts by the yeast S. cerevisiae. For this reason VHHs, especially llama VHHs are suitable for large scale applications.
As will be shown in the experimental part, VHHs, especially llama VHHs are far more heat stable compared to mouse MAbs. Most VHHa were still able to bind after two hours at temperatures as high as 90° C. The thermal denaturation at lower temperatures of mouse MAbs can be explained because of heavy and light chain separation. After cooling down, these fragments will not associate, or only randomly, resulting substantially in non-functional antibodies.
In general, VHHs and mouse MAbs are comparable in antigen binding in the presence of ATC (ammonium thiocyanate). HCG specific antibodies could bind at higher ATC concentrations than RR-6 specific antibodies. This is probably due to the nature of the antigen and or antibody-antigen interaction.
Regarding the specificity, VHHs have about the same potential as mouse MAbs in recognition of antigen. Within the small subset of anti-hCG, llama VHHs, some were found specific to separate alpha-subunit, beta-subunit and intact hCG. The anti-RR6 llama VHHs did not crossreact with other azodyes, resembling RR-6 in structure. The small subset tested seems to be very specific to RR-6.
Most of the VHHs have apparent affinities (KD) range of 10−7-10−9 M, which is of high affinity. Some mouse MAbs used were found to have higher affinity (109-1011 M−1) which can be contributed to avidity because mouse MAbs are bivalent.
In summary, when comparing the biochemical properties of VHHs, especially llama VHHs and mouse MAbs for stability, specificity and affinity, it appears that VHHs have physical chemical properties which make them excellent candidates for use both in existing and novel applications. These applications are in a variety of fields and which are immediately evident to the man skilled in the art. As most food products have to be pasteurized, the addition of VHHs create really new options. For example, VHHs that bind and neutralize redox enzymes thereby preventing colour changes of food products, or VHHs that recognize proteins or polysaccharide of food products thereby providing or improving the structure to the product, or VHHs that bind off flavours (scavenging) or bind flavours (controlled release).
The invention is further illustrated by the following experimental work which however is not intended to limit the invention in any respect.
Example I
Materials and Methods
To investigate if VHHs such as llama antibodies can be used in a range of applications, a number of physical chemical and functional characteristics was investigated, thereby concentrating on three characteristics: stability, specificity and affinity. Llama VHHs and “classical” bivalent mouse monoclonal IgG antibodies (“mouse Mabs”), specific for either the human pregnancy hormone or the azodye Reactive Red-6 (“RR-6”), were compared with respect to heat stability and antigen binding in chaotropic environment (ammonium thiocyanate (ATC) elution). Furthermore, llama VHHs were tested for their antigen specificity and affinity. Both llama and mouse antibodies consist of two subsets: one specific for the protein antigen Chorionic Gonadotropin (hCG) and one specific for the hapten azodye RR-6.
The results show that llama especially regarding functional binding at high temperature (about 90° C.) llama VHHs are extraordinarily stable, compared to mouse MAbs.
Example I.1
Purification of Llama VHHs and Mouse MAbs
Llama VHHs HI-6, H-13, H-14, HI-15, HI-113 (anti-hCG) and R2, R4, R7, R8, R9, R10 (anti RR-6), described in EMBL data library accession numbers: AJ236095 (HI-6), AJ236096 (H-13), AJ236094 (H-14), AJ236097 (HI-15), AJ236098 (HI-113), AJ236100 (R2), AJ236102 (R4), AJ236105 (R7), AJ236106 (R8), AJ236107 (R9), and AJ236108 (R10), were secreted by S. cerevisiae as a fusion protein comprising a VHH with a C-terminal myc-tag and a (His)6-tag. For stability studies, yeast culture supernatants containing llama VHHs were ultrafiltrated using membranes with cut off limits of 50 kD and 5 kD, respectively, (Filtron, USA) and dialysed against phosphate buffered saline (PBS, 145 mM NaCl, 7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 0.01% sodium azide).
The final VHH preparation had a purity between 80-90% as judged by Coomassie Brilliant Blue (R-250, Sigma, Zwijndrecht, the Netherlands) staining of sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE).
Mouse MAbs were obtained from hybridoma culture supernatant and purified using protein A and subsequent dialysis against PBS. The hCG specific mouse MAbs used was mAB 3299 (anti-Thyroid Stimulating Hormone). The RR-6 specific mouse MAbs used was URL 08-04.
Example I.2
Sub-Saturation Point
Using ELISA, dilution curves were made for all antibodies. From these dilution curves subsaturation point was determined (FIG. 1). The sub-saturation point was defined as the highest concentration on the linear part of the curve. Using this sub-saturation point any effect on antigen binding can be observed as a decrease in signal.
Example I.3
Temperature
Llama VHHs and mouse MAbs were incubated at various temperatures (4° C., 60° C., 70° C., 80° C. and 90° C.) for two hours. Subsequently, antibodies were put 30 minutes at 25° C. and stored at 4° C. Samples were diluted to sub saturation point and ELISA was performed (FIG. 2). It can be concluded that the VHH fragments are more stable than the MAbs.
Example I.4
Binding in the Presence of Ammonium Thiocyanate
Double ATC (0-8M) concentrations were mixed 1:1 with double sub saturation concentrations of antibodies and were incubated for 10 min at room temperature. Standard ELISA was performed using antibody-ATC mixtures. See FIG. 3.
From FIGS. 3B and 3D it can be concluded that the llama VHH fragments HI-15 and R7 are able to bind at higher concentrations ammonium thiocyanate than any of the RR-6 or hCG specific mouse MAbs tested. The binding of HI-15 was reduced only 20% in 4M ammonium thiocyanate, whereas none of the other antibodies tested were able to bind at this concentration. Llama VHH fragment R7 was completely inhibited in RR-6 binding at 4M ammonium thiocyanate, whereas the best anti RR-6 Mabs (URL 08-01 and URL 08-13) were already completely inhibited at 2M ammonium thiocyanate (FIG. 3C).
Based on these findings applicants are of the opinion that it is well within the ability of the skilled person to find VHHs which are stable under conditions which are normally destabilising for traditional antibodies such as Mouse Mabs.
Example I.5
Specificity of Llama VHHs
ELISA was performed on separate alpha-, beta-subunit, or native hCG. The results are shown in Table 1.







TABLE 1







Specificity llama VHHs anti-hCG












Llama VHH
Alpha
Beta
Intact







HI-6
−
+
+



H-13
+
−
+



H-14
+
−
+



HI-15
+
−
+



HI-113
−
−
+










Within this small subset, llama VHHs were found specific to separate alpha, beta, or native hCG. This indicates that llama VHHs have the same potential as mouse MAbs in recognition of antigen.
Example I.6
Affinity
Apparent affinities of a selection of llama VHHs were determined by kinetic measurements using the IAsys Biosensor (Affinity Sensors, Cambridge, UK). The results are given in Table 2 below.







TABLE 2







Affinity values llama VHHs and mouse MAbs












Llama VHH
Kd (nM)
Mouse Mab
Kd (nM)
















H-14
300-400
URL 08-04
1



R2
22
Fab URL 08-04
8.4



R7
45
Mab 3299
3



R8
20





R9
83





R10
58










Example II.1
Induction of Humoral Immune Responses in Llama
A male llama was immunized with K88ac fimbriae, also known as F4 fimbriae, which were purified from E. coli strain 1087 according to Van Zijderveld et al. (1990). Immunizations were performed both subcutaneously and intramuscularly using 1 ml 50 mg/l K88ac per immunization site. The first two immunizations were performed with a three week interval and using a water in oil emulsion (4:5 (v/v) antigen in water:specol) as described by Bokhout et al. (1981 and 1986). The third and fourth immunization were done without adjuvant, five and nine weeks after the first immunization. The immune response was followed by antigen specific ELISA's.
Polystyrene microtiter ELISA plates (Greiner HC plates) were activated overnight at 4° C. with 100 μl/well of 5 mg/l K88ac antigen in 0.05 M sodium carbonate buffer at pH 9.5. After each incubation plates were washed four times with 0.05% (v/v) Tween 20 in demi water in order to remove unbound proteins. The wells were then successively incubated 1 hr at ambient temperature with 100 Fl antibody solution in blocking buffer (PBS containing 2% (w/v) BSA; 0.05% (v/v) Tween 20; 1% (v/v) culture supernatant of a S. cerevisiae strain, SU 50 (Giuseppin, et al., 1993) grown on YPD; 1% (v/v) of a cleared lysate of E. coli JM109 cells). The antigen sensitized plates were then successively incubated with (1) serially diluted llama serum samples, (2) 2000-fold diluted polyclonal rabbit anti llama serum (obtained via immunizing rabbits with llama immunoglobulines which were purified via ProtA and ProtG columns), (3) 2000-fold diluted swine-anti-rabbit immunoglobulins conjugated with horse radish peroxidase. The bound peroxidase activity was determined using the substrate 3,3′,5,5′-tetramethylbenzidine.
Example II.2
Cloning, Expressing and Screening of Llama VHH Fragments
II.2.1 Isolation of Gene Fragments Encoding Llama VHH Domains.
From an immunized llama in accordance to Example II a blood sample of about 200 ml was taken and an enriched lymphocyte population was obtained via Ficoll (Pharmacia) discontinuous gradient centrifugation. From these cells, total RNA was isolated by acid guanidium thiocyanate extraction (e.g. via the method described by Chomczynnski and Sacchi (1987). After first strand cDNA synthesis (e.g. with the Amersham first strand cDNA kit), DNA fragments encoding VHH fragments and part of the long or short hinge region where amplified by PCR using specific primers:











(see SEQ. ID. NO: 1)




PstI



VH-2B
5′-AGGTSMARCTGCAGSAGTCWGG-3′






S=C and G, M=A and C, R=A and G, W=A and T,










(see SEQ. ID. NO: 2)



HindIII


Lam-07
5′-AACAGTTAAGCTTCCGCTTGCGGCCGCGGAGCTGGGGTC


 



TTCGCTGTGGTGCG-3′ (short hinge)















(see SEQ. ID. NO: 3)



HindIII


Lam-08
5′-AACAGTTAAGCTTCCGCTTGCGGCCGCTGGTTGTGGTTT


 



TGGTGTCTTGGGTT-3′ (long hinge)






Upon digestion of the PCR fragments with PstI (coinciding with codon 4 and 5 of the VHH domain, encoding the amino acids L-Q) and BstEII (located at the 3′-end of the VHH gene fragments, outside and upstream of the 08 primer coinciding with the amino acid sequence Q-V-T), the DNA fragments with a length between 300 and 450 bp (encoding the VHH domain, but lacking the first three and the last three codons) were purified via gel electrophoresis and isolation from the agarose gel.
II.2.2 Construction of Saccharomyces cerevisiae Expression Plasmids Encoding Llama VHH Domains
Plasmids pUR4547 (CBS 100012) and pUR4548 (CBS 100013) are Saccharomyces cerevisiae episomal expression plasmids, derived from pSY1 (Harmsen et al., 1993). Both plasmids contain the GAL7 promoter and PGK terminator sequences as well as the invertase (SUC2) signal sequence. In both plasmids the DNA sequence encoding the SUC2 signal sequence is followed by the first 5 codons (encoding Q-V-Q-L-Q; see SEQ. ID. NO: 4) of the VHH domain (including the BstII site), a stuffer sequence, the last six codons (encoding Q-V-T-V-S-S; see SEQ. ID. NO: 5) of the VHH domain. In pUR4547, this is followed by two stop codons, an AflII and HindIII site. In pUR4548, the Q-V-T-V-S-S (SEQ. ID. No: 5) sequence is followed by eleven codons encoding the myc-tag, two stop codons, an AflII and HindIII site.
Plasmids pUR4547 and pUR4548 were deposited under the Budapest Treaty at the Centraal Bureau voor Schimmelcultures, Baarn (The Netherlands) on 18 Aug. 1997 with deposition numbers CBS 100012 and CBS 100013, respectively. In accordance with Rule 28(4) EPC, or a similar arrangement from a state not being a contracting state of the EPC, it is hereby requested that a sample of such deposit, when requested, will be submitted to an expert only.
Upon digesting pUR4548 with PstI and BstEII, the about 6.4 kb vector fragment was isolated and ligated with the PstI-BstEII fragments of between 300 and 450 bp obtained as described above. After transformation of S. cerevisiae via electroporation, transformants were selected from minimal medium agar plates (comprising 0.7% yeast nitrogen base, 2% glucose and 2% agar, supplemented with the essential amino acids and bases).
II.2.3 Screening for Antigen Specific VHH Domains
For the production of llama VHH fragments with myc-tail, individual transformants were grown overnight in selective minimal medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with the essential amino acids and bases) and subsequently diluted ten times in YPGal medium (comprising 1% yeast extract, 2% bacto pepton and 5% galactose). After 24 and 48 hours of growth, the culture supernatant of the colonies was analysed by ELISA for the presence of VHH fragments which specifically bind to the E. coli K88 antigen, in essential the same way as described in Example 1. In this case, however, the presence of specifically bound VHH fragments was detected by incubation with monoclonal anti-myc antibodies, followed by incubation with polyclonal rabbit-anti-mouse conjugate with alkaline phosphatase.
In this way a number of VHH fragments have been obtained, which specifically recognise the E. coli K88 antigen.
Two examples of such fragments are given below:









(see SEQ. ID. NO: 6)



K607



QVQLQESGGG LVQPGGSLRL SCAASGSIFS ASAMTWYRQA



 



PGKSREYVAR IFFSGGTNYA DSVKGRFTIS RDNAKNTMYL



 



QMNDLKREDT AVYYCNLLSY WGQGTQVTVS S















(see SEQ. ID. NO: 7)



K609



QVQLQESGGG LVQPGGSLRL SCAASGGTFS WYAMGWFRQA



 



PGKEREFVAT VSRGGGSTYY ADSVKGRFTI SRDNAKNTVY



 



LQMNSLKPED TAAYYCAAGR GSPSDTGRPD EYDYWGQGTQ



 



VTVSS






Example II.3
Construction of “Self-Cloning” Yeast Expression Cassette, Encoding a Chimeric Protein Anchored to the Cell Wall of Yeast, Comprising a Binding Domain and a Cell Wall Anchor
In order to display a binding domain at the yeast cell wall, a genetic fusion of the gene encoding the binding domain and a gene encoding the cell wall anchor has to be constructed, either with or with out a linker sequence, essentially as described in WO 94/25591. As an example, the construction of a “self cloned” yeast, displaying a llama VHH fragment fused via the “long hinge” region to the a-agglutinin cell wall anchor will be described below. Obviously this can be done in much the same way for different binding domains, different linkers/hinges and different cell wall anchors.
II.3.1 Isolation of the Llama “Heavy Chain” Long Hinge Sequence
For the isolation of the hinge regions of the llama “heavy chain” antibodies, the cDNA which was obtained as described in Example 1, was amplified by PCR using the primers VH-2B and Lam-03. The nucleotide sequence of the latter primer was based on a consensus sequence based on the CH2 domains of different species. The PCR reaction resulted in three DNA fragments of about 450 (a), 550 (b) and 850 (c) bp.









(see SEQ. ID. NO: 8)



       BamHI



Lam-03  GGTATGGATCCACRTCCACCACCACRCAYGTGACCT






Upon digesting these fragments with PstI and BamHI and size fractionation, they were ligated into pEMBL (Dente, 1983) and subjected to nucleotide sequence analysis. The clones obtained from the DNA fragment with an average length of  ̃450 bp (a) were found to comprise a short hinge region, having the following sequence:








(see SEQ ID NOs: 9 and 29)



          VHH    ><                 short hinge               >



GGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCAGCTCCAAGTGTCCCAAATGCCCA


  V  T  V  S  S  A  H  H  S  E  D  P  S  S  K  C  P  K  C  P


                <-      LAM 07 WB         <-1






The clones obtained from the DNA fragment with an average length of  ̃550 bp (b) was found to comprise a long hinge region, having the following sequence:








(see SEQ ID NOS: 10 and 30)



BstEII   VHH    ><                                       long hinge



GGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAACCACAACCACAACCACAACCACAACCCAAT


  V  T  V  S  S  E  P  K  T  P  K  P  Q  P  Q  P  Q  P  Q  P  Q  P  N


 


                <-        LAM 08 WB       <-1


                                >


CCTACAAYAGAATCCAAGTGTCCCAAGTGTCCA


 P  T  T  E  S  K  C  P  K  C  P






For the construction of pUR4588 (FIG. 5), a NheI site was introduced in the 3′-end of the long hinge region via PCR using the primers:







(see SEQ. ID. NO: 11)


             NheI


BOLI 18 CGCGGCTAGCCTTGGATTCTGTTGTAGGATTGGGTTG













(see SEQ. ID. NO: 12)


        BstEII


LH CCCAGGTCACCGTCTCCTCAGAACCCAAG






via which the following sequence:








(see SEQ ID NOs: 13 and 31)


CCTACAACAGAATCCAAGTGTCCCAAGTGTCCA


 P  T  T  E  S  K  C  P  K  C  P






became








(see SEQ ID NOs: 14 and 32)


                   NheI


CCTACAACAGAATCCAAGGCTAGC


 P  T  T  E  S  K  A  S






Upon digestion with BstEII and NheI, an about 90 bp fragment was obtained encoding the last 4 amino acids of the VHH domain and the long hinge region except for the last 5 amino acids.
II.3.2 Adaptation of the α-Agglutinin Gene
Plasmid pUR4482 (FIG. 4) is a yeast episomal expression plasmid for expression of a fusion protein with the invertase signal sequence, the CHv09 variable region, the Myc-tail and the Camel “X-P-X-P” Hinge region (see Hamers-Casterman et al., 1993), and the α-agglutinin cell wall anchor region.
The PstI-site at position 1665 in the α-agglutinin gene was removed via a three step PCR using primers PAF01 to PAF04. For easy cloning and sequencing in pEMBL8, EcoRI and HindIII sites were introduced at the 3′- and 5′-end of the gene fragment encoding α-agglutinin, respectively. PCR-A using primers PAF01 and PAF02 on a pUR4482 template resulted in an about 707 bp fragment with the EcoRI restriction site at the 3′-end. PCR-B using primers PAF03 and PAF04 on a pUR4482 template resulted in an about 427 bp fragment with the HindIII site at the 5′-end. A third PCR was performed with the fragments obtained from reaction A and B, together with the primers PAF01 and PAF04. After digesting the obtained PCR product ( ̃1135 bp), which comprises the modified α-agglutinin gene without the PstI restriction site, with EcoRI and HindIII, the resulting  ̃1130 bp fragment was ligated into the vector fragment of pEMBL8 which was digested with the same enzymes resulting in pRL03.







PCR-A


(see SEQ. ID. NO: 15)


PAF01 GGAATTCGTCTCCTCAGAACAAAAAC


 


(see SEQ. ID. NO: 16)


PAF02 GCTGCTGCAAAAGGAATTTA













PCR-B


(see SEQ. ID. NO: 17)


PAF03 AAATTCCTTTTGCAGCAGC


 


(see SEQ. ID. NO: 18)


PAF04 GGGAAGCTTCGACAAAAGCAGAAAAATGA






In essentially the same way as for the removal of the PstI site, the SapI site in the α-agglutinin coding sequence at position 1618 was removed from plasmid pRL03. PAF01 and BOLI-20 were used in a PCR reaction with pRL03 as template to generate an approximately 660 bp fragment and primers BOLI19 and PAF04 were used to make an approximately 504 bp fragment which were linked by splicing by overlap extension using primers PAF01 and PAF04 (PCR-C).







PCR-A


PAF01   see above


 


(see SEQ. ID. NO: 19)


BOLI-20 TTACAAAAGTGGGTTCTTCAGATGGAA













PCR-B


(see SEQ. ID. NO: 20)


BOLI-19 TTCCATCTGAAGAACCCACTTTTGTAA


 


PAF04   see above






PCR-C

Product of PCR-A and PCR-B and PAF01 and PAF04.

Again an about 1130 bp EcoRI-HindIII fragment was obtained and cloned in pEMBL8.
Finally, the SacI site at position 2041 can be removed and a HindIII site can be introduced downstream of the stop codon by replacing the sequence:







(see SEQ ID NOs: 33 and 34)


SacI                             HindIII


gagctCGGTTCGATCATTTTTCTGCTTTTGTCGAagctt


 


cTCGAGCCAAGCTAGTAAAAAGACGAAAACAGCTTCGAa


E  L  G  S  I  I  F  L  L  L  S






with a synthetic linker having the sequence:









(see SEQ. ID. NOs: 21 and 35)



(SacI)                                                         HindIII



gagctGGGTTCGATCATTTTTCTGCTTTTGTCGTACCTGCTATTCTAAGATCTGATTAAACGCGTGAagctt


 


cTCGACCCAAGCTAGTAAAAAGACGAAAACAGCATGGACGATAAGATTCTAGACTAATTTGCGCACTTCGAa


E  L  G  S  I  I  F  L  L  L  S  Y  L  L  F  *






In this way a plasmid was obtained containing an about 990 bp NheI-HindIII fragment encoding the C-terminal 320 amino acids of the α-agglutinin in which the PstI, SapI and SacI sites were removed via silent mutations in order to facilitate further construction work.
II.3.3 Construction of pUR4588
The following fragments were subsequently joined:
i) the about 100 bp SacI-PstI fragment of pUR4548: comprising a part of the Gal7 promoter, the SUC2 nucleotide sequence and the first 4 codons of the VHH fragment,
ii) the about 350 bp PstI-BstEII fragment, which was obtained as described in Example 2: encoding a truncated VHH fragment, missing both the first 4 (QVQL; see SEQ. ID. NO: 4) and the last 5 (VTVSS; see SEQ. ID. NO: 5) amino acids of the VHH fragment,
iii) the about 90 bp BstEII-NheI fragment obtained as described in Example 3.1: encoding the last 5 amino acids of the VHH fragment and the long hinge region, and
iv) the about 1 kb NheI-HindIII fragment obtained as described in 3.2: the adapted α-agglutinin gene.
Finally, the thus obtained about 1.5 kb SacI-HindIII fragment was ligated into the about 7.5 kb vector fragment of pUR2822 which was digested with the same enzymes, resulting in pUR4588 (FIG. 7).
Plasmid pUR2822 was deposited under the Budapest Treaty at the Centraal Bureau voor Schimmelcultures, Baarn (The Netherlands) on 29 Sep. 1998 with deposition number CBS 101292. In accordance with Rule 28(4) EPC, or a similar arrangement from a state not being a contracting state of the EPC, it is hereby requested that a sample of such deposit, when requested, will be submitted to an expert only.
II.3.4 Production of Yeast Strains “Functionally” Expressing the Chimeric Protein
Plasmid pUR4588 (FIG. 5) was digested with SapI, after which an about 6.7 kb fragment was purified, via gel electrophoresis and isolated from the agarose gel. This fragment was introduced into yeast strains via electroporation. Transformants containing (multicopies of) the DNA fragment integrated into the rDNA locus of their genome were selected as described in Example 2. Induction of the production and display of the binding domain was also done as described in Example 2. In order to determine the functionality of the binding domains displayed at the yeast cell wall, the following assay was performed:
The yeast cells were diluted in PBS containing 0.05% (v/v) Tween 20 (PEST) to an A660=1 and 10 μl of this suspension was mixed with 1 μg K88ac antigen in 100 μl PBST in an Eppendorf tube and incubated for 1 hr at ambient temperature. Unbound antigen was removed from the yeast cells by three washes in 1 ml PBST. Cells were collected after each wash by centrifugation and aspiration of the supernatant. The yeast cells were subsequently resuspended in 100 μl of PBST containing a conjugate of monoclonal antibody AD11, which is specific for K88, and horse radish peroxidase (AD11-HRPO; Van Zijderveld et al., 1990). After 1 hr incubation at ambient temperature the cells were washed again three times with PBST. The amount of AD11-HRPO bound to the yeast cells was taken as a measure of the amount of functional K88-binding domains displayed at the yeast cell wall. This value was determined by performing a peroxidase assay on serial twofold dilutions of these cells, using 3,3′,5,5′-tetramethylbenzidine as a substrate, and measuring the A450. The recovered functionality (RF) of the displayed antibody fragments after a physical treatment to inactivate the S. cerevisae cells is defined as the A450 before treatment (is F0) divided by the A450 after treatment (is Ft).
Example III
Heat Inactivation of VHHs
III.1 Production of Biomass Suspension
Saccharomyces cerevisiae strain SU50 containing the genomically integrated expression plasmid pUR4588 (FIG. 5) was used. The strain was grown in a 10 l scale fermentor using a fed batch fermentation set up (Mendoza-Vega et al., 1994). Such a fermentation consists of two steps: the first step (the batch phase) is performed in a Yeast extract and Peptone (YP) medium containing glucose. In the second step (the feed phase) YP medium is added containing glucose and galactose, in order to induce the promoter for the production of the VHH-agglutinine fusion. In this way a culture was obtained with a cell density of about 109-1010 per ml, comparable to large scale fermentations.
Cell density or viable counts (N) were determined by making decimal dilutions of a cell culture in sterile pepton physiological salt (PFS; 0.85% NaCl, 0.1% Bactopepton (Difco), pH 7). Samples of these dilutions were plated on YPD agar and incubated for 5 days at 30° C. after which the number of colonies were counted. The reduction of viable counts (RVC) after treatment to inactivate the S. cerevisiae cells is defined as the logarithm of the viable counts after treatment (is Nt) divided by the number of viable counts before treatment (is N0); Log(Nt/N0).
III.2 Heat Treatment
Determination of the killing efficiency of heat treatment on high cell density cultures was performed using one of two different approaches, as desired.
III.2.1 Using Glass Capillaries
Sterile glass capillaries (Fisher Scientific Den Bosch, l=100 mm, d=2 mm) were filled with 0.1 ml of the yeast culture medium and sealed. After incubating the filled, sealed capillaries, they were placed for a set time in a water bath at the desired temperature (experiments were performed at quadruplets). After this the recovered functionality (RF) and the reduction in viable counts (RVC) for the different samples was determined as described above.
III.2.2 Using Aluminium Vials
Sterile aluminium cryo vials (Omnilabo, volume 3 ml) were filled with 0.5 ml of the yeast culture for each temperature/time combination (experiments performed in duplo). The vials were placed in a water bath at the desired temperature and time. After this the recovered functionality (RF) and the reduction in viable counts (RVC) for the different samples was determined as described above.
III.3 Viable Counts and GI-ELISA
From the yeast culture the viable counts (N0) and the GI-ELISA signal (F0) was determined before heat treatment. The same was done for the samples obtained as described above (Nt and Ft, respectively). The results are presented in Table 3 below.












TABLE 3







Temp (° C.)
time (sec)
RVC (=log (Nt/N0))
RF (=Ft/F0)









60
300 
 −4
100%



62
60
>−6
100%



70
30
>−7
>85%



74
15
>−7
>85%










Example IV
Inactivation of S. cerevisiae SU50 (pUR 4588) with Ultra High Pressure
From literature it is known that at pressures over 300 MPa proteins FF ATP-ase can be inactivated (Wouters et al, 1998).
S. cerevisiae SU50 (pUR4588), displaying anti-E. coli K88 VHH fragments, was grown at 10 liter scale as described above. Samples of 1.5 ml were taken and transferred to bags for Ultra High Pressure treatment in a “Food Lab Multi Vessel” (Stansted Fluid Power, UK). Samples were incubated at three different pressures 250, 300 and 325 Mpa for different periods of time. The survival of the yeast cells and the functionality of the displayed VHHs was determined as described above. The results are presented in Table 4 below.












TABLE 4








time





Pressure (Mpa)
(min)
RVC (=log (Nt/N0))
RF (=Ft/F0)









250
60
−9
100%



300
 3
−6
>70%




20
−9
>70%



325
 1
−6
 70%




 3
−9
 70%




20
−9
 70%










It was further found that yeast cells that do not have the capacity to synthesize trehalose are much more sensitive for UHP (Fernandes et al., 1997). Therefore, a preferred embodiment of the inactivation of yeast with UHP is, instead of using a wild type yeast strain, using a variant strain defective in the synthesis of trehalose, e.g. a strain in which the TPS1 gene has been inactivated e.g. by partial or complete deletion. In this way the UHP treatment could be reduced from 300 MPa to 200 MPa with the same reduction in viable counts.
Example V
Killing of Yeast Cells by Irradiation
An alternative for inactivation of yeast cells is gamma irradiation. S. cerevisiae SU50 (pUR4588), was grown at 10 liter scale as described above. After fermentation the biomass slurry was transferred to polypropylene drums and exposed to 10 kGray or 25 kGray of gamma irradiation (GAMMASTAR, Ede). The survival of the yeast cells and the functionality of the displayed VHHs was determined as described above. For both cases, the RVC was better than −10 (no viable counts left after irradiation), whereas the RF of the VHHs was 100%. The anti-K88 activity was not affected by the irradiation.
A number of genes involved in DNA repair are known (e.g rad9, rad30, rad52, sir2, sir3, sir4). Yeast strains carrying mutations in these genes, are known to be more sensitive towards radiation (Friedberg et al., 1991; Tsukamoto et al., 1997). In a preferred embodiment of this invention, a yeast strain with one or more mutations in one or more of these genes is used.
Example VI
Combined Effect of Heat Inactivation and Antimicrobials on Saccharomyces cerevisiae 
S. cerevisiae SU50 (pUR4588) cells were cultivated and harvested as described above and subjected to heat treatment as described above in the presence of antimicrobials like ethanol, potassium sorbate and antimicrobial peptides like nisin and MB21. According to the methods described in the previous examples the inactivation efficiency and the recovery of the functionality of the VHH antibodies have been determined. The results are given in Table 5 below.







TABLE 5







Inactivation of S. cerevisiae and VHH as function of the


temperature and the concentration of antimicrobials











Temp

Time
RVC
RF


(° C.)
Antimicrobials
(min)
(=log Nt/No)
(=Ft/Fo)





50
3% ethanol
60 
 0
100%


50
0.2% K-sorbate
60 
 0
100%


60
—
5
−4
100%


60
3% ethanol
5
−7
 90%


60
0.2% K-sorbate
5
−8
 95%









It has been reported that yeast cells that do not have the full capacity of synthesising multi drug resistant proteins are much more sensitive to weak acids, e.g. sorbic acid (PDR12; Piper et al., 1998). Therefore, a preferred embodiment of inactivation of yeast by a combination of heat and acid is to use, instead of the wild type yeast strain, a strain in which the PMR gene is inactivated, e.g. via partial or complete deletion. In this way either the temperature or the amount of acid or both can be reduced considerably.
Example VII
Animal Feeding Trials with Yeast Displaying Anti E. coli K88 VHHs on their Surface
In this experiment two groups of piglets were used. A mild E. coli K88 infection was induced in all animals by an oral dose of 106 E. coli K88 cells. For group I (exp. group) the feed was supplemented with 6 ml of a concentrated suspension (1010-1011 cells per ml) of SU50 (pUR4588), and for group II (control) with the same amount of wild type SU50 yeast. The yeast cells were inactivated with gamma irradiation according to the procedure as described in Example 6.
Number of affected piglets due to dosing of >106 E. coli K88/g in the meal.
See Table 6 below.









TABLE 6





Day
Control
Exp group







0
 0
 0


1
22
17


2
50
19









Conclusion: protective effect clearly visible after 2 days.
Example VIII
Dye Transfer Prevention Using HCV Antibodies
Cotton swatches dyed with RR6 were prepared by soaking pieces of cotton (5*5 cm) in a solution of RR6 for 30 min (0.1 M borate buffer, pH 8.5, 0.15 M NaCl). After drying, these swatches were washed in the presence of various concentrations of a VHH(R7 anti-RR6 as disclosed in example 1). For the monitoring of dye transfer, a white cotton swatch (dye pick-up swatch) was added. As wash solution, a carbonate buffer (pH 9) was used, to which in a number of experiments surfactants were added. These were alkyl benzene sulfonate (LAS, 600 mg/l), and a LAS/Synperonic A7 mixture (290 mg/l LAS, 660 mg/l SA7).
After the wash the swatches were line dried and the reflectance spectra were measured using a Minolta spectrometer. The data thereby obtained were transferred to the CIELAB L*a*b* colour space parameters. In this colour space, L* indicates lightness and a* and b* are the chromaticity coordinates. The colour differences between the swatches prior to washing and after the wash, were expressed as ΔE indicating the distance in the colour space between before and after washing.
The results, given as the ΔL, Δa, Δb, and ΔE of the dye pick-up swatches after the experiment, with respect to white cotton are given in the table below.













TABLE 7







Conditions applied
ΔL
Δa
Δb
ΔE









Buffer, pH 7 containing VHH







against RR6 at concentration:







  0 μM
2.9
−5.7
2.2
6.8



1.25 μM 
1.9
−4.1
1.7
4.9



2.5 μM
2.0
−4.3
1.9
5.2



5.0 μM
0.6
−1.4
0.8
1.8



10.0 μM 
0.6
−1.1
0.6
1.4



Buffer, pH 9 containing VHH







against RR6 at concentration:







  0 μM
3.3
−5.4
2.1
6.7



1.25 μM 
2.1
−4.8
2.0
5.7



2.5 μM
1.6
−3.5
1.5
4.2



5.0 μM
0.7
−1.7
0.8
2.1



10.0 μM 
0.1
−0.5
0.4
0.7



LAS solution, pH 9, containing







VHH against RR6 at concentration:







  0 μM
2.0
−4.3
1.9
5.1



10.0 μM 
1.1
−2.6
1.2
3.1



LAS/SA7 solution, pH 9, containing







VHH against RR6 at concentration:







  0 μM
2.0
−4.2
1.8
5.0



10.0 μM 
0.1
−0.2
0.1
0.3










These results illustrate that anti-RR6 antibodies are able to prevent dye transfer, especially at levels of above 2.5 μM during use and that these VHHs remain stable in the presence of surfactants especially mixed surfactants and can hence continue to be effective during the use of laundry detergents.
Example IX
Induction of a Humoral Immune Response in Llama
A male llama was immunised with an oil emulsion (1:9 V/V, antigen in PBS:Specol (Bokhout et al.) subcutaneously and intramuscularly. Per site about 0.75-1.5 ml emulsion was injected containing about 100 μg of a conjugate consisting of the azo-dye RR120 (Cibacron Red 4G-E, Ciba Geigy) which was coupled to BSA via its reactive triazine group.
Immunisations were performed according to the following time schedule: the second immunisation was performed three weeks after the first injection, and the third immunisation two weeks after the second one. The immune response against RR120 was followed by titration of serum samples in ELISA by using Nunc Covalink plates, which where coated with the azo-dye-RR120 (100 μl/well of 2.5 mM RR120 in coating buffer (0.1 M di-sodium tetraborate decahydrate, 0.15 M NaCl pH 8.5), incubation overnight at 50° C.)
After incubation with serum, the bound llama antibodies were detected with poly-clonal rabbit-anti-llama antiserum (obtained via immunising rabbits with llama immunoglobulines purified via ProtA and ProtG columns; ID-DLO) and, as a second step, swine-anti-rabbit immunoglobulines (Dako) conjugated with alkaline phosphatase were used after which the alkaline phosphatase enzyme-activity was determined upon incubation with p-nitro-phenyl phosphate and the optical density was measured at 405 nm.
Example IX.2
Cloning, Selection and Screening of Llama VHH Fragments
IX.2.1 Isolation of Gene Fragments Encoding Llama VHH Domains
From an immunised llama a blood sample of about 200 ml was taken and an enriched lymphocyte population was obtained via centrifugation on a Ficoll (Pharmacia) discontinuous gradient. From these cells, total RNA was isolated by acid guanidium thiocyanate extraction (e.g. via the method described by Chomczynnski and Sacchi, 1987). After first strand cDNA synthesis using MMLV-RT (Gibco-BRL) and random oligonucleotide primers (Pharmacia), or by using the Amersham first strand cDNA kit, DNA fragments encoding VHH fragments and part of the long or short hinge region were amplified by PCR using specific primers of SEQ ID 1, SEQ ID 2 and SEQ ID 3 as described above.
IX.2.2 Construction and Screening of S. cerevisiae Libraries which Secrete VHH Domains in the Culture Medium
The DNA-fragments generated by PCR were digested with PstI (coinciding with codon 4 and 5 of the VHH domain, encoding the amino acids L-Q) and BstEII (naturally occurring in FR4 region of the majority of the VHH-genes and coinciding with the amino acid sequence Q-V-T). The digested PCR-products, with a length between 300 and 400 bp (encoding the complete VHH domain, but lacking the first and the last three codons), were separated by electrophoresis on agarosegel and, after purification from the gel-slices with the Qiaex-II extraction kit, cloned in the Saccharomyces cerevisiae episomal expression plasmids pUR4547 or pUR4548
RR120 specific VHH fragments were isolated via screening of culture supernatants from individual clones in ELISA using either

    
    
        rabbit anti-llama VHH poly-clonal antibody followed by incubation with goat anti-rabbit poly-clonal antibodies conjugated to horse radish peroxidase (Bio-rad) (for VHH fragments cloned in pUR4547); or
        mouse anti-myc mono-clonal antibody 9E10 followed by incubation with poly-clonal goat-anti-mouse conjugate with horse radish peroxidase (for VHH genes cloned in pUR4548) for detection.
    
    


For the production of llama VHH fragments in S. cerevisiae, individual transformants were grown overnight in selective minimal medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with the essential amino acids and bases) and subsequently diluted ten times in YPGal medium (comprising 1% yeast extract, 2% bacto pepton and 5% galactose). After 24 and 48 hours of growth, the culture supernatant of the colonies was analysed by ELISA for the presence of antigen specific VHH fragments.
IX.2.3 Construction and Screening of S. cerevisiae Libraries which Display VHH Domains at the Cell Surface
The PCR-products obtained as described in IX.2.1 can be digested with PstI and BstEII and cloned in the yeast display vector pUR4659 (FIG. 7) as gene-fragments encoding the VHH-domain including the hinge region. In this way VHH antibody domains were displayed on yeast cell surface by means of N-terminal fusions onto the cell wall protein α-agglutinin (see patent WO 94/01567). Such a yeast display library with 1×106 clones was constructed in the episomal yeast expression vector pUR4659. Transformed yeast cells were pooled and grown for about 17 hours on selective minimal medium until the OD660 was 2. Subsequently, these cells were inocculated into YPGal for expression, secretion and display of the VHH antibody fragments on the cell surface and grown for appr. 17 hours. Secreted VHH-fragments were removed by washing the cells with PBS, thereby avoiding a disturbing competition for binding to antigen between yeast bound and free VHH domains.
For the isolation of RR120 binding VHH fragments, the VHH displaying S. cerevisiae cells were incubated for two hours with in vitro biotinylated BSA-RR120 in PBS followed by an incubation with FITC-labelled streptavidin. Fluorescence of individual yeast cells was analysed by means of a FACS (Fluorescence Activated Cell Sorter) and the cells with give above average fluorescent signals were selected and inocculated in minimal medium for a second round selection. In this second round, cells with the highest fluorescent signals were selected and propagated as single colonies for analysis or grown for further rounds of selections.
Individual S. cerevisiae clones were grown on YPgal for 48 hours in wells of microtiter plates. During the second part of this incubation, fusion protein consisting of a VHH fragment and α-agglutinin is secreted in the growth-medium. Culture supernatants containing these chimeric molecules were tested in ELISA for binding to RR120 using the myc-TAG for detection as described above. For further analysis of selected clones, VHH-encoding DNA fragments from these yeast display vectors were recloned in the episomal yeast secretion vector pUR4548 and analysed as described in paragraph 2.2.
IX. 2.4 Construction and Screening of a Phage Library Displaying VHH Domains
FIG. 6 gives a schematic representation of the phagemid vector pUR4676 which was assembled from:

    
    
        the about 2.85 kbp NcoI/ScaI fragment of pFab5c(Ørum et al., 1993). The BstEII site present in the lacI gene was removed via a silent mutation using PCR mutagenesis.
        the about 2.7 kbp NcoI/ScaI fragment of pHEN1 (Hoogeboom et al., 1991).
    
    


The DNA fragment between the PstI site and the first codons of geneIII were replaced with the following sequence (SEQ ID No. 22):








PstI                      BstEII          NotI          HindIII



       <-  MPE 28 WB (22)  -><-    MPE 42 WB (26)    -><- MPE 44 WB (25)


  ctgcaGGAGTCATAATGAGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCAAGCGGAAGCTTAGAACAAA


 


  gACGTCCTCAGTATTACTCCCTGGGTCCAGTGGCAGAGGAGTCGCCGGCGTTCGCCTTCGAATCTTGTTT


   <-      MPE 27 WB (36)            -><-     MPE 41 WB (26)   -><-


  [->            cassette HC-V          <-]                       [->


 


                                                                  (HindIII)


        -><-     MPE 32 WB (30)       -><-      MPE 46 WB (27)   ->


  AACTCATCTCAGAAGAGGATCTGAATGGGGCCGCACATCACCATCATCACCATGGGGCCGCATAGagcttAACTGTTGAA


 


  TTGAGTAGAGTCTTCTCCTAGACTTACCCCGGCGTGTAGTGGTAGTAGTGGTACCCCGGCGTATCTCGAATTGACAACTT


  MPE 43 (25)     -><-        MPE 31 WB (30)    -><-  MPE 45 WB (21) ->


      myc tail          <-]         [->His (6) tail<-]        amber     [-> G III






resulting in pUR4676.

Upon digesting this vector with PstI and HindIII and subsequent ligation in the presence of the PCR fragments produced as described in 2.1 and digested with PstI/HindIII, the VHH phagemid libraries were obtained.
IX.2.2.1. Selection of the VHH Library.
From the phagemid libraries, VHH displaying phage particals were produced via superinfection with phage M13-K07 (Promega) essentially as described by (Marks et al., 1991). Selection of the VHH phage library was performed essentially as described by (Marks et al., 1991) via three rounds of panning using RR120 coated immunotubes. The immunotubes (Nunc; Maxisorp, 75×12 mm) were coated with 4 ml 2.5 mM RR120 in coating buffer (0.1 M di-sodium tetraborate decahydrate and 0.15 M NaCl pH 8.5) overnight at 50° C. After blocking with Marvel (2% in PBS) and washing with PBS, about 10E12 phages (preincubated in 4 ml 2% Marvel in PBS for 15 minutes) were added to the tubes. Upon incubation for 1 hour (on turntable) the tubes were washed 15 times with PBST (PBS with 0.1% tween) and 2 times with PBS. Bound phages were subsequently eluted by adding 2 ml 100 mM triethylamine.
The eluted phages were added to 1 M Tris pH 7.4 for neutralisation before adding E. coli cells for infection/phage rescue. This process was repeated three times.
Subsequently, single E. coli colonies were transferred to microtiter plates and grown overnight in 150 microliter 2× TY medium containing ampicillin (100 microgram per ml) and IPTG (0.8 mM). The culture supernatant was subsequently tested in an antigen specific ELISA.
In this way a large number of RR120 binding VHH domains could be isolated.
For further analysis of selected clones, VHH-encoding DNA fragments from these phage display vectors can be recloned in the episomal yeast secretion vector pUR4548 and analysed as described in paragraph IX.2.2.
IX.3 Selection of RR120 Recognising VHH Domains Via Yeast Display in Consumer Products under Application Conditions
To enrich for VHH fragments stable in product formulations the screening as described in 2.3 was repeated using application conditions.
In this example we have dissolved the yeast cells in commercially available detergent formulations resp 6 g/l Dutch Omo® (without protease, hereafter referred to as Omo) followed by the addition of the biotinylated BSA-RR120. After a one hour incubation under application conditions, unbound RR120 and detergent were discarded by centifugation. Subsequently, the cells were incubated with FITC-labelled streptavidin and analysed as described above.
IX.4 Selection of RR120 Recognising VHH Domains Via Phage Display in Consumer Products Under Application Conditions
IX.4.1 Initial selection
Selection of phages binding to the RR120 antigen under application conditions was essentially performed as described in example 2.4, but using the following conditions during the selection of the phages in the immunotubes:
1. 0.6 g/l OMO-MA in 25 mM sodiumcarbonate pH 9.0 with 2% marvel (0.6+M(9))
2. 3 g/l OMO-MA in 25 mM sodiumcarbonate pH 9.0 with 2% marvel (3+M(9))
3. 6 g/l OMO-MA in 25 mM sodiumcarbonate pH 9.0 with 2% marvel (6+M(9))
4. 6 g/l OMO-MA in 25 mM sodiumcarbonate pH 9.0 (6 (9))
5. 0.6 g/l LAS in 25 mM sodiumcarbonate pH 9.0 with 2% marvel (0.6L+M(9))
6. 0.6 g/l LAS in 25 mM sodiumcarbonate pH 10.3 with 2% marvel (0.6L+M(10.3))
7. 3 g/l OMO-MA in water with 2% marvel (3+M(10.3))
The results after three rounds of panning are presented in the following table 8
















Conditions
round 1
Round 2
round 3









1 0.6 + M(9)
1 104
4 103
—



2 3 + M(9)
1 104
4 103
—



3 6 + M(9)
5 103
8 103
—



4 6 (9)
12
—
—



5 0.6L + M(9)
1 105
2 106
8 104



6 0.6L + M(10.3)
1 105
1 106
8 104



7 3 + M(10.3)
3 105
7 105
4 104



8 PBS + M
1 105
5 105
8 104










As a comparison the results of the panning in PBS (see I.1) and 2% marvel are included.
IX.4.2 Screening of Free VHH Domains Produced by Selected Clones Under Application Conditions
The fragments indicated in FIG. 10, and Table 9 were selected for further investigation







TABLE 9







Selection conditions










α-RR120 VHH
Selection







A38, A44
PBS pH 7.4



A306
0.6 g l−1 LAS pH 9 (25 mM NaCO3-buffer)



A307, A308
0.6 g l−1 LAS pH 10.3 (25 mM NaCO3-buffer)



A309
  3 g l−1 OMO pH 10.3 (water)










After purification via ion exchange chromatography, the fragments were subjected to an ELISA assay according to the following approach:
An ELISA was performed using serial dilutions (in PBS) of the selected α-RR120 antibody fragments resulting in a set of standard dilution curves. The OD 450 nm signal was plotted against the log antibody concentration. The concentration at halve the maximal OD (450) is defined as the midpoint concentration and set as 100% immunoreactivity for the standard curve.
Subsequently this experiment was repeated using serial dilutions made in product formulations at different concentrations. Again the immunnoreactivity was determined (VHH concentration resulting in half the max OD450 achieved in that particular product concentration).
The relative immunoreactivity of the dilution curves in different product concentrations was then expressed as a percentage of the 100% immunoreactivity as determined from the standard curve and represented in FIGS. 8 and 9.




  
    %
    ⁢
    
      
    
    ⁢
    immunoreactivity
  
  =
  
    
      
        mid
        ⁢
        
          -
        
        ⁢
        point
        ⁢
        
          
        
        ⁢
        standard
        ⁢
        
          
        
        ⁢
        conditions
      
      
        mid
        ⁢
        
          -
        
        ⁢
        point
        ⁢
        
          
        
        ⁢
        application
        ⁢
        
          
        
        ⁢
        conditions
      
    
    ×
    100
  




IX. 4.3 Sequences of VHH Domains which Bind to RR120 Under Application Conditions

FIG. 10 represents the sequences of VHH domains which bind to RR120 under the conditions as described above.
LITERATURE REFERRED TO IN THE EXAMPLES


    Bokhout, B. A., Van Gaalen, C., and Van Der Heijden, Ph. J., (1981), A selected water-in-oil emulsion: composition and usefulness as an immunological adjuvant. Vet. Immunol. Immunopath., 2:491-500
    Bokhout, B. A., Bianchi, A. T. J., Van Der Heijden, Ph. J., Scholten, J. W. and Stok, W., (1986), The influence of a water-in-oil emulsion on humoral immunity. Comp. Immun. Microbiol. Infect. Dis., 9:161-168.
    Chomczynnski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Analytical Biochem. 162:156-159.
    Dente, L., Cesareni, G., Cortese, R. (1983) pEMBL—A new family of single stranded plasmids Nucleic Acids Research 11:1645-1655.
    Faber, K. N., Haima, P., Harder, W., Veenhuis, M. and Geert, A. B., (1994) Highly efficient electrotransformation of the yeast Hansenula polymorpha. Current Genetics, 25: 305-310
    Fernandes, P. M. B., Panek, A. D., and Kurtenbach, E. (1997) Effect of hydrostatic pressure on a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene. FEMS Microbiolology letters 152:17-21.
    Friedberg, E. C., Siede, W. and Cooper, A. J. (1991) Cellular responses to DNA damage in yeast pp 147-192 in: The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae. Eds. Broach, J. R, Pringel, J. R and Jones, E. W. Cold Spring Harbor Laboratory Press.
    Giuseppin, M. L. F., Almkerk, J. W., Heistek, J. C., Verrips, C. T., (1993) Comparative Study on the Production of Guar Alpha-Galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in Continuous Cultures. Applied and Environmental Microbiology 59:52-59.
    Giuseppin, M. L. F., Lopes, M. T. S., Planta, R. J., Verbakel, J. M. A., Verrips, C. T. (1991) Process for preparing a protein by a yeast transformed by multicopy integration of an expression vector. PCT application WO 91/00920 (UNILEVER)
    Harmsen, M. M., Langedijk, A. C., van Tuinen, E., Geerse, R. H., RauP, H. A., Maat, J., (1993) Effect of pmr1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba-galactosidase by S. cerevisiae. Gene 125 115-123
    Hodgkins, M., Mead, D., Ballance, D. J., Goodey, A. and Sudbery, P., (1993) Expression of the Glucose Oxidase Gene from Aspergillus niger in Hansenula Polymorpha and its use as a reporter gene to isolate regulatory mutations. Yeast, 9:625-635
    Klis, F. M., Schreuder, M. P., Toschka, H. Y. and Verrips, C. T., (1994) Process for immobilising enzymes to the cell wall of a microbial cell by producing a fusion protein. PCT application WO 94/01567 (UNILEVER)
    Marks J. D., Hoogenboom H. R., Bonnert T. P., et al. (1991) By-passing immunization—Human-antibodies from V-gene libraries displayed on phage: Journal of Molecular Biology 222: (3) 581-597 Dec. 5 1991
    Mendoza-Vega, O., Sabatie, J, & Brown, S. W. (1994) Industrial-production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cerevisiae. FEMS Microbiology Reviews 15:369-410.
    Piper, P., Mahe, Y., Thompson, S., Pandjaitan, R., Holyoak, C., Egner, R., Muhlbauer M., Coote P. and Kuchler, K. (1998) The pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. EMBO J. 17:4257-4265.
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## Claims

1. A method of producing a composition containing active VHH antibodies, the method comprising:
(a) generating a binding domain display library comprising nucleic acid molecules encoding VHH antibodies, wherein binding domains of the VHH antibodies capable of binding antigens are presented on the surface of the library;
(b) selecting for binding domain display library members wherein the VHH antibodies presented on the surface of the library retain binding activity at pH value of less than 5 or more than 9;
(c) expressing VHH antibodies selected for in step (b) using nucleic acid encoded by binding domain display library members; and
(d) adding the VHH antibodies expressed in (c) into the composition.

2. The method of claim 1, wherein prior to performing step (b) the method further comprises:
treating the VHH antibodies of step (a) with a chaotropic material, and
selecting for binding domain display library members wherein the VHH antibodies presented on the surface of the library retain binding activity in the presence of said chaotropic material.

3. The method of claim 2, wherein the chaotropic material is ammonium thiocyanate.

4. The method of claim 2, wherein the chaotropic material is urea.

5. The method of claim 1, whereby the VHH antibodies retain at least 70% of their immunoreactivity.

6. The method of claim 1, wherein the composition is a food composition.

7. The method of claim 1, wherein the composition is a personal care composition.

8. The method of claim 1, wherein the binding domain display library is selected from the group consisting of a yeast cell surface display library or a phage display library.