JP2005526498A - Method and apparatus for whole cell panning - Google Patents

Abstract

New methods and devices for whole cell panning employ a homogeneous population of cells separated by a membrane that is permeable to phage / phagemid particles. The apparatus separates a first chamber that may contain a first population of cells of a first type; a second chamber that may contain a second population of cells of a second type; and the two chambers A membrane comprising a membrane that is permeable to phage particles and phagemid particles while preventing mixing of the first population and the second population of cells.

Description

(background)
(Technical field)
The present disclosure relates to methods and apparatus for whole cell panning using phage / phagemid libraries for homogeneous cell culture.

(Background of related technology)
In the phage or phagemid particle display technique, a pre-assembled antibody or antibody fragment library is attached to the phage coat protein gene of the phage / phagemid. The antibody fragment is then expressed and displayed on the surface of the phage / phagemid particle. Antibodies with affinity for the target antigen are captured, propagated in bacteria, and further enriched through successive rounds of selection. This technique is referred to as panning, as reviewed in Page Display of Proteins and Peptides: A Laboratory Manual (Siegel, 2001). In most cases, the panning method uses a purified antigen immobilized on a solid support. Purification of the target antigen is often not possible either because the antigen is unknown, as with putative stem cell markers or tumor-specific antigens, or because the antigen cannot be produced in sufficiently large quantities. Also, purification of some membrane targets (ie, 7 transmembrane receptors) can cause improper folding in solution. Furthermore, purification can affect the native binding properties of the antigen. For these reasons, it is desirable to pan for antibody-carrying phage / phagemid particles that bind to their native antigen, such as on the intact cell surface.

  Phage / phagemid non-specific binding tends to be a problem in all types of panning. This is due to a variety of causes including the use of complex antigens with “sticky surfaces”. In panning techniques based on whole cells, this problem is amplified by the presence of unwanted non-specific antigens or ligands present on the cell surface. These antigens can take the form of endogenous proteins, carbohydrates, and lipids. Problems associated with non-specific binding in cell-based panning techniques can be partially avoided by employing negative selection, where the phage / phagemid library is pre-expressed with cells lacking the antigen of interest. Incubate. Unbound phage / phagemid is then used as a starting library to pan the target cells. Negative selection schemes are rather inadequate due to the low affinity of non-specific binding interactions. Furthermore, the weak specific interaction between the phage / phagemid and the target antigen is masked by negative selection that rapidly depletes the phage / phagemid library and reduces the chance of selecting new antibody-antigen interactions. obtain.

  Recently, methods have been developed that employ simultaneous positive and negative selection. These methods employ a heterogeneous mixture of antigen positive cells and an excess of antigen negative adsorbent cells. This antigen-negative cell serves as a sink for non-specific phage / phagemid, while the target cell captures any phage / phagemid that exhibits specific binding properties. The fidelity of specific binding interactions can be controlled by adjusting the ratio of antigen positive cells to antigen negative cells. Methods that employ these heterogeneous mixtures must also employ a specific scheme to separate the two cell types after incubation with the phage / phagemid. There are many ways to do this separation. For example, one method employs flow cytometry and fluorochrome labeled antibodies that recognize orthogonal antibodies on target cells (see US Pat. No. 6,265,150BI). This method predicts the presence of marker antigens on target cells that distinguish target cells from adsorbent cells, and specific fluorescent dye-labeled antibodies, and the demand is always easily achieved, especially in tumor cell targets Not a translation.

  Another commonly used method is to label target cells with magnetic beads and then mix with adsorbent cells (see US Pat. No. 5,876,925). Bacterial phage encoding Ig fragments are panned against a mixture of antigen positive biotinylated cell targets pre-coated with streptavidin-conjugated magnetic microbeads and excess unlabeled antigen negative cells. After phage incubation, the cells are separated by use of a magnetic affinity column. One disadvantage of this technique is that Ig fragment panning is done on non-native cell surfaces displaying biotin / streptavidin / magnetic bead complexes.

  In yet another method, target cells with bound phage can be aggregated from a heterogeneous mixture using immunoprecipitation (see US Pat. No. 5,985,543).

  Whole cell panning using the above method has several drawbacks. After panning with the phage library, the target cell population must be separated from non-specific adsorbent cells. This requires that target cells in a heterogeneous sample have markers on the cell surface that distinguish them from adsorbent cells, and that secondary antibodies that recognize these markers are available. Second, the action of adding components to detect markers on the cell surface of the target cell can disrupt the native environment of the antibody on the cell surface, on tumor cells with attached magnetic bead complexes. This results in the selection of antibodies that bind the displayed antigen. Finally, antibody-antigen interactions can be disrupted due to the process of separation (eg, shear forces).

  A more general method for whole cell panning that retains the advantages of a heterogeneous mixture of target / adsorbent cells but eliminates the need for cell separation to obtain the desired phage / phagemid may be advantageous.

(wrap up)
New methods and apparatus for whole cell panning employ a homogeneous population of cells separated by a membrane permeable to phage / phagemid particles. One population of cells (eg, target cells) consists of target cells that are maintained on the first side of the membrane and display possible antigens of interest. A second population of cells is maintained on the second side of the membrane and consists of non-specific adsorbent cells. In this protocol, there is no point to mix the two cell populations.

  A cell culture device maintains the separation of two or more cell lines in two or more different chambers by one or more removable barriers. This isolated chamber allows separate growth of non-specific cells and target antigen positive cells. A semi-permeable membrane can be sandwiched between two solid barriers. If different cell lines require different growth media, they can be placed in different chambers and grow in situ with the solid pieces, preventing mixing of any material between the chambers. After growth is complete, the solid barrier is removed. When the barrier between chambers is removed, both cell populations contact a semipermeable membrane that allows passive lateral diffusion between adjacent chambers. This membrane has a pore size that prevents cells from passing through it but allows smaller particles (eg, phage or phagemid) to pass through. A cell type specific library of phage / phagemid particles is then added. This membrane allows lateral diffusion of phage / phagemid particles carrying antibody fragments between chambers of the device while maintaining cell line separation. The entire device can be loosely covered with a lid that allows air circulation.

  In another embodiment, each culture chamber has openings on both the top and sides. The side openings accommodate solid or semi-permeable inserts. During cell growth, the individual chambers are provided with solid side inserts that allow isolated cell growth. This solid side insert can be removed and the two chambers can be mounted on either side of the semi-permeable membrane insert. The phage / phagemid library can then be added to one or both of the cell population. This semi-permeable membrane allows passive diffusion of phage / phagemid particles between chambers while keeping the adsorber and target cell types separated. The entire device can be loosely covered with a lid to allow air circulation.

Detailed Description of Preferred Embodiments
As can be seen in FIG. 1, the new apparatus 10 for whole cell panning includes a container 11 that defines a first chamber 12 and a second chamber 22. The container 11 can be made from any inert material that can support a solution of cells. Suitable materials for construction include, but are not limited to, glass and polymers such as polycarbonate or polystyrene. Preferably, the construction material is transparent. Chamber 12 holds a first suspension 13 containing a population of cells (eg, target cells). Chamber 22 holds a second suspension 23 containing a population of cells (eg, adsorbent cells). A loosely fitting cover 16 prevents contamination of the suspensions 13,23. One side of the chamber 12 is defined by a solid removable insert 14. One side of the chamber 22 is defined by a solid removable insert 24. The solid removable inserts 14, 24 create a water tight seal so that mixing of the contents of the chambers 12, 22 is prevented. There is a semi-permeable membrane 15 sandwiched between solid removable inserts 14,24. This semipermeable membrane is designed to have openings that are too small for cells to move through the membrane. However, the pores of membrane 15 must be large enough to allow the passage of the display library through this membrane. In the case of a phage or phagemid library, the membrane pore size is in the range of 0.2-7 microns, preferably about 5 microns. The membrane can be made from any inert material that can be provided with an appropriately sized pore.

  Upon removal of the solid removable inserts 14, 24, the first suspension and the second suspension 13 and 23 are brought into contact with the membrane 15, respectively. Since this membrane pore is too small to allow cell passage, the two populations of cells remain distinct. A display library is added to one or both of the containers 12,22. Individual members of the library pass freely through this membrane 15. Certain members of the display library bind preferentially to target cells in the first suspension 13. Any binding that may occur after a predetermined time has already occurred, and the first suspension 13 can be removed and the binding members of the library can be removed from the target cells and recovered. The second suspension 23 and unbound members of the library can be discarded.

  An alternative embodiment of a whole cell panning device is shown in FIG. 3, where chambers 112 and 122 are defined by separate containers 111, 121, each having a cover 116, 126. The chamber 112 holds a first suspension 113 that contains a population of cells (eg, target cells). Chamber 122 holds a second suspension 123 containing a population of cells (eg, adsorbent cells). Upper portions of the containers 111 and 121 have screw portions 117 and 127, respectively. Covers 116, 126 can be tightly screwed to avoid leakage during handling or transport, but can remain loose to allow air circulation during cell culture. When proper cell growth occurs and the cell suspension 113, 123 is to be used for panning, the covers 116, 126 are removed and the display library is placed in one or both of the suspensions 113, 123. Added and a common cover 130 is applied to the containers 111, 121. The common cover 130 has a first threaded portion 135 that can be tightly screwed into the threaded portion 117 of the container 111 and a second threaded portion 137 that can be tightly screwed into the threaded portion 127 of the container 121. The membrane 115 defines the central portion of the common cover 130. As shown in FIG. 4, once the assembled device is placed on its side, both the suspensions 113 and 123 come into contact with the membrane 115. As in the previous embodiment, the pores of the membrane are too small to allow cell passage, so the two populations of cells remain distinct. However, individual members of the library pass freely through this membrane 115. Certain members of this display library bind preferentially to target cells in the first suspension 113. Any binding that may occur after a predetermined time has already occurred and the first suspension 113 can be removed, and the binding members of the library can be removed from the target cells and recovered. The second suspension 123 and unbound members of the library can be discarded.

  In yet another embodiment (shown in FIG. 5), chambers 212 and 222 are defined by separate containers 211, 221 in the form of rectangular test tubes. Each container 211, 221 has a watertight side cover 218, 228 that snap-fits onto a side flange 219, 229, respectively, to cover the top cover 216, 226 and membranes 215, 225. Chamber 212 holds a first suspension 213 that contains a population of cells (eg, target cells). Chamber 222 holds a second suspension 223 containing a population of cells (eg, adsorbent cells). In this embodiment, the suspension 213 is in direct contact with the membrane 215 and the suspension 223 is in direct contact with the membrane 225. When appropriate cell growth occurs and the cell suspension 213, 223 is to be used for panning, the display library is added to one or both of the suspensions 213, 223. The side covers 218, 228 are removed and a connection sleeve 230 is applied to the containers 211, 221 instead of the side covers 218, 228. This connecting sleeve 230 connects the two containers 211, 221 so that fluids and library members (not cells) can pass from one chamber 212 to the other chamber 222. As in the previous embodiment, the pores of these membranes 215 and 225 are too small to allow cell passage, so the two populations of cells remain distinct. However, individual members of the library are free to pass through these membranes 215,225. Certain members of this display library bind preferentially to target cells in the first suspension 213. After a predetermined time, any binding that may occur has already occurred and the first suspension 213 can be removed and the binding members of the library can be removed from the target cells and recovered. The second suspension 223 and unbound members of the library can be discarded.

  In a variation of the embodiment shown in FIG. 5, it is possible to employ only a single membrane on the connecting sleeve 230 instead of the membrane on each container. It may also be advantageous to employ agitation (eg, with a magnetic stirrer) in one or both of the chambers of any of the above embodiments. Further, in any of the previously described embodiments, pumping can be advantageously employed to assist in moving the library members across the membrane. Pumping can also be useful if the washing step is to be performed while the cell suspension is still in place in the chamber of the device. Suitable types of pumps include, but are not limited to, peristaltic pumps. It is also contemplated that two syringe-like containers may be employed, where one or more syringe plungers are used manually to provide a pumping action.

  The whole cell panning method described herein retains the existing advantages while eliminating major drawbacks. Since the target cells and adsorbent cells are always maintained as separate and homogeneous populations in different chambers of the device, no cell separation step is required and therefore no cell marker is required. In addition, cell lines can be grown on different media using solid separators. These solid separators can be removed, stripping the semipermeable membrane. A phage / phagemid library is then added. The phage / phagemid particles are free to move through the membrane to both chambers while different cell types are maintained in isolation.

  While the above description includes many specific details of the method according to the present invention, these specific details should not be construed as limitations on the scope of the invention, but are merely exemplary of preferred embodiments thereof. is there. Those skilled in the art will envision many other possible variations that are within the scope and spirit of the invention as defined by the claims appended hereto. For example, instead of a threaded cover, the container may be covered with what is commonly known to those skilled in the art as a “snap cap”. These “snap caps” have a first position that is loose on the container and a second snap-down position that forms a water tight seal. As another example, the watertight side cover in the embodiment shown in FIG. 5 can be replaced with a peel-off cover that provides a single use device for cell panning. Accordingly, the preceding description is to be considered as illustrative and not restrictive.

FIG. 1 is a cross-sectional view of a whole cell panning device according to the present disclosure having a solid barrier in place. FIG. 2 is a cross-sectional view of the whole cell panning apparatus of FIG. 1 with the solid barrier removed. FIG. 3 is a cross-sectional view of two individual chambers useful in assembling a whole cell panning device according to the present disclosure. 4 is a cross-sectional view of the two individual chambers of FIG. 3 when assembled to provide a whole cell panning device according to the present disclosure. FIG. 5 is a cross-sectional view of an alternative embodiment of two individual chambers useful in assembling a whole cell panning device according to the present disclosure with a solid barrier in place.

Claims (17)

A first chamber that may contain a first population of cells of a first type;
A second chamber that may contain a second population of cells of a second type; and a membrane that separates the two chambers to prevent mixing of the first population of cells with the second population And a membrane that is permeable to phage particles and phagemid particles. The apparatus of claim 1, further comprising at least one removable solid insert adjacent to the membrane. The apparatus of claim 1, further comprising a first removable solid insert and a second solid insert sandwiching the membrane. The apparatus of claim 1, further comprising a lid. A first container that may contain a suspension of a first population of cells and has an open end;
A second container comprising a second population of cells and having an open end; and a coupling adapted to receive the open end of both the first container and the second container Coupling having a membrane disposed therein and maintaining the first population of cells from the second population of cells while allowing passage of phage particles and phagemid particles therethrough A device comprising: The apparatus of claim 5, wherein each of the first container and the second container has a threaded portion adjacent to the open end. The apparatus of claim 6, wherein the coupling includes a first threaded portion and a second threaded portion that receive threaded portions of the first container and the second container. 6. The method of claim 5, further comprising: a first lid that can seal the open end of the first container in a watertight manner; and a second lid that can seal the open end of the second container in a watertight manner. The device described. The apparatus of claim 8, wherein the first lid and the second lid are screw caps. The apparatus of claim 6, wherein the coupling receives the first container and the second container in a snap fit relationship. A first container that may comprise a first suspension of a first population of cells in a medium and has an open end;
A semi-permeable membrane covering the open end of the first container and impermeable to the passage of cells;
A second container that may contain a second suspension of the second population of cells in the medium and has an open end;
A semi-permeable membrane that covers the open end of the second container and is impermeable to the passage of cells; and both the first container and the second container A device comprising a coupling adapted to receive an open end in a manner to allow contact of the medium of the first suspension and the second suspension. The apparatus of claim 11, wherein each of the first container and the second container has a threaded portion adjacent to the open end. The apparatus of claim 12, wherein the coupling includes a first threaded portion and a second threaded portion for receiving threaded portions of the first container and the second container. 12. The apparatus of claim 11, further comprising a first lid that can seal the open end of the first container in a watertight manner and a second lid that can seal the open end of the second container in a watertight manner. The device described. The apparatus of claim 14, wherein the first lid and the second lid are screw caps. The apparatus of claim 11, wherein the coupling receives the first container and the second container in a snap fit relationship. A method of screening an antibody library for antibodies that bind to an antigen comprising:
Providing a first suspension comprising a population of antigen positive cells;
Providing a second suspension comprising a population of antigen negative cells;
Adding a display library comprising a plurality of members to at least one of the first suspension or the second suspension;
Separation of the population of antigen positive cells from the population of antigen negative cells allows passage of members of the display library into both the first suspension and the second suspension. While maintaining with a semi-permeable membrane; and recovering cells from the antigen positive population and identifying members of the display library bound thereto.

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