WO2001077361A1

WO2001077361A1 - Human immunoglobulin-producing gnotobiotics

Info

Publication number: WO2001077361A1
Application number: PCT/US2001/011698
Authority: WO
Inventors: Emer Leahy; Mark A. Hofer
Original assignee: American Biogenetic Sciences, Inc.
Priority date: 2000-04-10
Filing date: 2001-04-10
Publication date: 2001-10-18
Also published as: AU2001253339A1

Abstract

The present invention pertains to a colony of genetically transformed, antigen-free mice of both sexes which are produced by rearing genetically transformed mice in an essentially antigen-free environment. After several generations, this ultimately allows for the production of antigen-free mice whose immune system is highly sensitive to weak immunogens. Prior to entering the antigen-free environment, the mice, via a germline configuration, undergo molecular modification by having their endogenous immune system functionally deleted and effectively replaced with a substantially complete human immunoglobulin locus. The resultant antigen-free, genetically modified mice are capable of synthesizing high affinity human immunoglobulins to weak immunogens.

Description

HUMAN IMMUNOGLOBULLN-PRODUCING GNOTOBIOTICS

BACKGROUND

Generally, when a protein is perceived by a host's immune system as being foreign, then the healthy host's humoral and cellular immune defense system is triggered. This triggering of the specific immune system is dependent upon whether this protein is recognized by the recipient's immune system as being foreign.

The humoral immune system is mediated by B-cells. Immature B-cells display surface immunoglobulins (slg) which have the ability to interact with a specific epitopic sequence of a complex protein (antigen). Once there is affinity binding between the slg and epitope of the antigen, the immature B-cell undergoes differentiation forming mature B-cells which elaborate soluble immunoglobulins (Ig) that are specific for the original epitopic determinant on, or part of, the antigen. This response displayed by the humoral system receives assistance from the cellular immune system. A parallel process occurs in the cellular arm of the specific immune system. This T- cell response is of significant import in that cytokines and other soluble message elements are produced and released by certain T-cells which then can interact with immature B-cells of the humoral system in order to facilitate the transition from immature to mature B-cells. T-cells also play other vital roles in the immune response. Specific interaction between a foreign immunogen and T-cell stimulates the T-cell resulting in the secretion of cytokines which then assist B-cells in their process of differentiation. Additionally, there are other elaborated cytokines produced by stimulated T-cells which affect cellular components of the non-specific immune system which can ultimately lead to an inflammatory and/or anaphylactic response. Returning to the humoral system, this system consists of essentially two types of mature B-cells. Memory B-cells serve an anamnestic role in the immune system by having the ability to respond to the same immunogen that triggered a primary response of the immune system. Plasma cells are mature B-cells which elaborate soluble antibodies specific for an epitopic site. These soluble antibodies interact with epitopic sites on the foreign protein, thus facilitating its demise.

Due to the specificity of antibodies, these proteins can be used in many different ways other than defending against foreign invaders. For example, antibodies can be used in the diagnosis of certain pathologies which have characteristic surface, or elaborated, antigens. Antibodies can be used in a therapeutic setting, whereby the antibody can be introduced to treat an individual suffering from a foreign invader, such as a pathological bacterial species. Antibodies can be used in pure research. For instance, in the area of immunohistochemistry, labeled antibodies specific for a target protein on a specific cell can be used to isolate and visualize that cell, or cells, using the aid of a microscope.

There are a plethora of uses of antibodies, especially monoclonal antibodies. Monoclonal antibodies contain homogenous antibodies specific for only one eptiopic site whereas in contrast, polyclonal sera contains a heterogenous mixture of antibodies generally directed to the same antigen, however, individual antibodies may be directed to different epitopic sites.

Generally, for the uses described above, monoclonal antibodies are most desirable. Typically, monoclonal antibodies are produced using a murine system. As is described in greater detail below, a mouse is challenged with a foreign protein with the desire of eliciting an immune response from the recipient mouse's immune system. Sera obtained from this mouse, after a suitable period of time, will contain polyclonal antibodies directed against the foreign protein. The spleen of the mouse is isolated with the goal of isolating individual colonies of plasma cells that produce a homogenous antibody, i.e., monoclonal antibodies. The desired plasma cells can then be fused and transformed into perpetual antibody producing plasma cells. In practice, it is difficult if not impossible to produce plasma cells which elaborate high affinity monoclonal antibodies which will react against a weak immunogen. Without being bound by theory, it is believed that due to the antigen saturated environment in which the mouse is reared, the mouse will have a lower sensitivity towards weak immunogens. Therefore, the probability of such a mouse producing high affinity monoclonal antibodies against weak immunogens is very low. Yet, there exists a need to produce and obtain monoclonal antibodies having a high affinity for low immunogens.

As mentioned above, antibodies find utility in numerous therapeutic and diagnostic regimes. For example, antibodies can be used to treat a pathological process whose etiology is a virus which uses one of a host cell's surface receptors to enter the cell, thereby commandeering that cell. Antibodies directed against such a receptor can be used to block entry of the virus into the host cell. It is highly desirable to produce and obtain monoclonal antibodies for such a purpose. However, as previously mentioned, monoclonal antibodies are typically of murine, porcine, rabbit, or primate origin. Such monoclonal antibodies may be effective in interacting with the designed target, however, such antibodies will be considered a foreign protein if used in a different species. Consider for example, a human patient being treated using a murine antibody. The host, or human in this case, will consider the murine antibody as foreign resulting in the triggering of the host's immune system which can not only inactivate the antibody thereby reducing or eliminating its therapeutic effectiveness but may also result in other adverse side effects, all of which are undesirable.

There exists a current need for a new method to efficiently produce high affinity human immunolglobulins against molecules with low immunogenicity.

SUMMARY

The present invention pertains to a new method for producing human antibodies to molecules of low immunogenicity using an antigen-free, human immunoglobulin-based non- human mammal. That is, a non-human mammal reared in essentially an antigen-free environment having a near complete human immunoglobulin locus as part of its genome.

The invention also pertains to methods for producing an antigen-free genetically transformed non-human mammal comprising a near complete human immunoglobulin locus. While a preferable non-human mammal is the mouse, it should be understood that reference to the mouse in this text is merely for illustrative convenience and that the principles apply equally as well for other non-human mammals and that such other mammals are also included within the principles of the instant invention.

In one embodiment, a colony of antigen-free mice of both sexes are produced by rearing them in an essentially antigen-free environment. These antigen-free mice can then propagate progeny in this antigen-free environment. This ultimately allows for the production of progeny whose immune system is highly sensitive to weak immunogens. Such a highly sensitive immune system is able to produce, for example, high affinity antibodies to weak immunogens. Such weak immunogens if presented to a non-antigen-free mouse typically result in the production of a low affinity antibody, if there is a response at all.

In this embodiment, a near complete human immunoglobulin locus is ideally introduced into the genome of the mice which enter the antigen- free environment or alternately in antigen-free mice which have been raised in the antigen-free environment. This genetically transformed mouse preferably has a near complete human immunoglobulin loci, including both a heavy chain locus and a human kappa light chain locus. This genetically transformed antigen-free mouse has the ability to synthesize and elaborate a high affinity human immunoglobulin when challenged with a weak immunogenic molecule.

In another embodiment, mice having essentially complete human immunoglobulin loci are introduced into an essentially antigen-free environment. After two to five generations, the progeny of such mice not only have the capacity to synthesize and elaborate human immunoglobulins thereby obviating the need to "humanize" murine antibodies, but they also have the ability to produce high affinity human immunoglobulins when challenged with a weak immunogen.

DETAILED DESCRIPTION

A colony of antigen-free genetically transformed mice comprising human immunoglobulin loci have the ability to synthesize and elaborate moderate to high affinity human immunoglobulins when challenged with a weak immunogen. Both polyclonal and monoclonal human antisera can be obtained from such mice. This invention also pertains to methods for producing such antigen-free genetically transformed mice. By producing human antibodies rather than murine antibodies, the need to humanize the murine antibodies (e.g., substitute sections of human antibodies for the mouse antibody by molecular biology techniques such as those described by Winter et al, EP 023400A) is completely obviated. In one embodiment, mice, via a germline reconfiguration, undergo molecular modification by having their endogenous immune system functionally deleted or deactivated, and augmented or replaced with a complete or substantially complete human immunoglobulin locus. These genetically transformed mice are capable of synthesizing high affinity human immunoglobulins to weak immunogens. A breeding pair of such transformed mice are then introduced into an antigen-free environment. These genetically transformed mice can then propagate and their progeny maintained in the antigen-free environment. Ultimately (typically after two-five generations), such a process will result in antigen-free, genetically transformed mice whose immune system is highly sensitive to weak immunogens. The highly resultant sensitive immune system is able to produce, for example, high affinity, human antibodies to weak immunogens. Such weak immunogens, if presented to a non-antigen-free mouse would most likely result in the production of a low affinity antibody, if one at all. In another embodiment, the process of producing such mice is reversed. Antigen-free mice raised in an antigen-free environment, are genetically transformed with a human antibody producing immune system and then reintroduced and maintained in the antigen-free environment. Such mice will also have the ability to produce high affinity human immunoglobulins when challenged with a weak immunogen.

EXAMPLE 1 - Production of a Antigen-Free Colony

An antigen-free animal is referred to as a "gnotobiote." A gnotobiote is free from all demonstrable associated forms of life, including bacteria, viruses, fungi, protozoa, and other saprophytic or parasitic forms of life. Gnotobiotes are derived by aseptic cesarean section or sterile hatching of eggs that are reared and continuously maintained with germfree techniques under isolator conditions and in which the composition of any associated fauna and flora, if present, is fully defined by accepted current methodology.

An antigen-free (or germfree) system includes barriers against entry of unwanted microbial invaders. In addition to the physical barriers such as plastic, metal rubber and glass which enclose the animal, the system requires operational barriers for air filtration, food and water sterilization, and manipulation by gloves, which form an integral part of the barrier system. Additionally, the entry of supplies to the isolater are preferably performed under sterile conditions. Such supplies, including nutrients, are ideally treated to substantially reduce or eliminate sources of antigenic contamination.

In the present invention, any antigen-free animal can be utilized. For example, commonly used antigen-free animals include, but are not limited to, mouse, rabbit, pig, rat, guinea pig, goat, sheep, primate and poultry. In this invention, a mouse is preferably used due to its convenient size relatively low housing cost (when compared to other animals) and ability to rapidly generate progeny. The parameters for establishing an antigen-free environment and principles of cesarean surgery are set forth in US Patent No. 5,223,410 to Gargan et al., and US Patent No. 5,721,122 to Gargan et al., the teachings of which are herein incorporated by reference in their entirety.

In the present invention, it is preferable that the antigen-free animal is bred on a chemically defined (CD), low molecular weight, water-soluble, ultrafiltered diet. It is surmized that such a diet permits one to obtain complete control of nutrient and antigen intake by the animal. Such a diet is generally made up entirely of ingredients that are capable of chemical definition, for example, amino acids, simple sugars, lipids, vitamins and minerals. For the purpose of the subject invention a chemically defined diet comprises amino acids, simple sugars, lipids, vitamin and minerals and no components having a molecular weight greater than about 10,000 daltons. Thus, all of the components of a chemically defined diet are of low molecular weight and are naturally circulating nutrients in animals and, therefore, it is believed that such components will not stimulate an immune response. An example of a chemically defined diet is set forth in US Pat. No. 5,223,410. Not wishing to be bound by theory, it is thought oral tolerance induced by limiting the antigenic effect of food intake may at least be partially responsible for the increased sensitivity to antigens of low immunogenicity.

It is also preferred in the instant invention to utilize a filter paper bedding to reduce or eliminate the antigen-free animal from eating the bedding, which action could result in an immune response. The consumption of filter paper is not believed to elicit an immune response. The antigen- free system can be tested for microbial contamination according to guidelines set out in Wostmann, B.S. (ed.) 1970, Gnotobiotics Standards and guidelines for the breeding care and management of laboratory animals, National Research Council, National Academy of Sciences, Washington, DC, the entire teachings of which are herein incorporated by reference. Wetted swabs using diet and water from inside the isolator housing the animal(s) can be used to obtain fecal smears obtained fresh from a mouse and from the accumulated waste under each cage isolator. Smears can also be taken from the walls of the isolator particularly around the entry ports. Microbiological cultures can be established and examined for the presence of microbes in order to monitor the sterility of the environment. The usual brother X sister mating system employed in conventional breeding colonies can also be used in gnotobiotic colonies. True random breeding includes some matings of sibilings and of first cousins. Although such matings are normally desired to limit in breeding, in the present circumstances, the introduction of additional gene diversity is preferably avoided in order to maximize the stability of the modified genome. Other systems of minimal inbreeding can be used and reference is made to Falconer, DC, 1967, Genetic aspects of breeding methods, p 72-96, In The UFAW handbook on the care and management of laboratory animals, 3^rd ed. E and S Livingstone, Ltd., London; and to National Research Council, Institute for Laboratory Animal Resources, 1969, A guide to genetic standards for laboratory animals, National Academy of Sciences, Washington, DC, the teachings of which are incorporated herein by reference in their entirety.

EXAMPLE 2 - Genetic Transformation of the Antibody Producing Animal

The current invention contemplates the creation of genetically transformed animals having a near complete human immunoglobulin locus, including both a human heavy chain locus and a human kappa light chain locus such that a substantially complete human antibody can be produced by the animal. Most preferably, the animal's innate immune system is functionally eliminated by either genetic removal or other manipulation which effectively prevents transcription and translation of the endogenous immune genome. In a preferable embodiment, these transformed animals comprise a heavy chain locus which includes greater than about 20%, more preferably greater than about 40%, still more preferably greater than about 50%, still more preferably greater than about 60% of the human heavy chain variable region. With respect to the human kappa light chain, the locus preferably comprises greater than about 20%, more preferably greater than about 40%, still more preferably greater than about 50%, and even more preferably greater than about 60% of the human kappa light chain variable region.

In the current invention, the genetically transformed animals include the entire D_H region, the entire J_H region, the human mu constant region, and can additionally be equipped with nucleotide sequences encoding other human constant regions for the generation of additional isotypes. Such immunoglobulin isotypes include Y_l5 Y₂, Y₃, α, ε, β, and other constant region encoding genes. Alternative constant regions can be included on the same transgene. In this invention genetically transformed animals include, but are not limited to, mouse, primate, pig, sheep, goat, guinea pig, foul, and rat. Preferably, the animal is a mammal. The techniques for producing genetically transformed animals, in particular, genetically transformed animals having a near complete human immunoglobulin locus is well known to those skilled in the art and is ably described in WO 98/24893, and Green et al., Nature Genetics, 1, 13-21 (1994), (the entire teachings of which are incorporated herein by reference in their entirety) such that those procedures need not be repeated here. An important key feature to the present invention is the ablation of the endogenous immune system of the host genetically transformed animal. This can be facilitated by homologous recombination that replace or delete the region as described in WO 98/24893. By replacing the endogenous immune system with a human immunoglobulin locus, the genetically transformed animal will, when presented with an immunogen, synthesize and elaborate a human immunoglobulin in response to such a challenge.

In one embodiment of the present invention, a non-human mammal, such as a mouse, possess a genome which comprises an endogenous immunoglobulin locus that is functionally eliminated so, in that the animal can not raise an immune response using its endogenous immunological repertoire when presented with an immuno logical challenge; a human heavy chain immunoglobulin locus in substantially germline configuration, the human heavy chain immunoglobulin locus comprising a human mu constant region and regulatory and switch sequences thereto, a plurality of human J_H genes, a plurality of human D_H genes, and a plurality of human V_H genes; and a human kappa light chain immunoglobulin locus in substantially germline configuration, the human kappa light immunoglobulin locus comprising a human kappa constant region, a plurality of J_κ genes, and a plurality of V_κ genes, wherein the number of V_H and V_κ genes are selected to substantially restore normal B-cell development in the animal host. In this embodiment, preferably the heavy chain immunoglobulin locus comprises a second constant region selected from the group consisting of human gamma- 1, human gamma-2, human gamma-3, human gamma-4, alpha, delta, and epsilon. Preferably, the number of V_κ genes is greater than about 15, the number of D_H genes is greater than about 25, the number of J_H genes is greater than about 4, and the number of V_H genes is greater than about 15. It is preferred that, in a population of animals, B-cell function is reconstituted on average to greater than 50% as compared to the wild type. The Washington University (Browstein et al., 1989, the entire teachings of which are incorporated herein by reference) and the CEPH (Albertsen et al., 1990, the entire teachings of which are incorporated herein by reference) human- Y AC libraries can be conveniently screened for YACs containing sequences from the human heavy and kappa light chain loci as previously described by Mendez et al., 1995, the entire teachings of which are also incorporated herein by reference. Cloning and characterization of 1H and IK YACs was described by Mendez et al. 3H and 4H YACs can be identified from the Washington University library using a V_H3 probe (0.55 kb Pstl/Ncol, see Berman et al., 1988, the entire teachings of which are incorporated herein by reference). The 17H YAC may be conveniently cloned from the GM1416 YAC library and determined to contain 130 kb of heavy chain variable sequences and a 150 kb chimeric region at its 3' end pursuant to the method of Matsuda et al., 1993, the entire teachings of which are incorporated herein by reference. 2K and 3K YACs can be recovered from the CHEF library using V_κII-specifιc primer (Albertsen et al., 1990).

Standard methods for yeast growth, mating, sporulation, and phenotyping can be employed (Sherman et al., 1986, the entire teachings of which are incorporated herein by reference). Targeting of YACs and YAC vector arms with yeast and mammalian selectable markers can be facilitated by lithium acetate transformation (Scheistl and Geitz, 1989, the entire teachings of which are incorporated herein by reference).

YAC-containing spheroplasts can be fused with cells, such as E14.TG3B1 ES cells (Jakoboviys et al., 1993, and Green et al., 1994, the entire teachings of which are incorporated herein by reference). HAT-resistant colonies can be expanded for analysis. YAC integrity can be evaluated by Southern Blot analysis using protocols and probes described in Berman et al. (1998) and Mendez et al. (1994), and hybridization conditions as described in Gemmil et al. (1991, the entire teachings of which are incorporated herein by reference). Chimeric animals can be generated by microiηjection of ES cells into the blastocysts pursuant to conservative genetically transformed animal production techniques. YAC-containing offspring can be identified by PCR analysis of tail DNA (Green et al.,

1994).

EXAMPLE 3 - Monoclonal Antibody Production

Kohler and Milstein received a Noble Prize for their work on the production of monoclonal antibodies. They established a technique that successfully results in the formation of monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein, 1975, Nature 256, 495-497, the teachings of which are herein incorporated by reference in its entirety). In brief review, the technique involves fusing using, for example, polyethylene glycol or Sendai virus, antibody producing plasma cells with myeloma cells, thus creating a hybrid cell line called a hybridoma. The plasma cells are obtained from the spleen of an animal (including the genetically transformed, gnotobiotics of the present invention) which has been challenged with a particular immunogen of choice. The cells of the spleen are dissociated and isolated into individual cells. An individual plasma cell is then fused with a myeloma cell, are typically malignant cells obtained from a primary tumor of the bone marrow. The hybridoma that is created can synthesize and secrete homogenous antibodies typically referred to as monoclonal antibodies. The techniques for producing monoclonal antibodies are well known to those of ordinary skill in the art and are described in numerous references including Current Protocols In Molecular Biology, Ausbel, F.M. et al. (eds.) 1991, vol. 2, section 11, the entire teachings of which are herein incorporated by reference in their entirety.

A means for detecting the desired hybridoma is desirable. Generally, the hybridomas are cultured in selective media, for example HAT medium, which contains hypoxanthine, aminopterin and thymidine. HAT medium permits the proliferation of hybrid cells and prevents growth of unfused myeloma cells which normally would continue to divide indefinitely. Aminopterin blocks de novo purine and pyrimidine synthesize by inhibiting the production of tetrahydrofolate. The addition of thymidine bypasses the block in pyrimidine synthesis, while hypoxantine is included in the media so that inhibited cells can synthesis purine using the nucleotide salvage pathway. The myeloma cells preferably employed are mutants lacking hypoxanthine phosphoribosyl transferase (HPRT) and thus cannot utilize the salvage pathway. In the surviving hybrid, the plasma cell supplies genetic information for production of this enzyme. Since plasma cells themselves have a limited life span in culture (approximately two weeks), the only cells which can proliferate in HAT media are hybrids formed from myeloma and spleen cells.

The mixture of fused myeloma and plasma cells is diluted in HAT medium and cultured in multiple wells of a microtiter plate in order to facilitate the screening of antibody secreted by the hybrids and to prevent individual hybrids from overgrowing. In two to three weeks, the supernatant of the individual wells containing hybrid clones is assayed for specific antibody production.

Once the desired fused cell hybrids have been selected and cloned into individual antibody-producing cell lines, each cell line can be propagated. A sample of the hybridoma can be injected into a histocompatible animal of the type that was used to provide the somatic (plasma cell) and myeloma cells for the original fusion. The injected animal develops tumors elaborating the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration. Alternatively, the individual cell lines can be propagated in vitro in laboratory culture vessels. The culture medium, containing high concentrations of a single specific monoclonal antibody, can be harvested by decantation, filtration or centrifugation.

The skilled practices will readily recognize that numerous alterations and modifications of the procedures and examples described herein may be made without departing from the spirit or scope of the principles of the present invention. What is claimed is:

Claims

1. An antigen-free non-human mammal, comprising a genome having a human immunoglobulin locus capable of producing a substantially human antibody upon stimulation of the mammal with an antigen.

2. The mammal of claim 1 wherein said mammal's endogenous immune system is functionally eliminated so that it does not produce an antibody to said stimulation with an antigen.

3. The mammal of claim 2 wherein said mammal is a mouse.

4. A non-human gnotobiotic capable of producing a substantially human antibody.

5. A method for producing an antigen- free genetically transformed non-human mammal capable of producing a substantially human antibody, comprising the steps of:

(a) providing a breeding pair of mammals which have had (i) their innate immune systems functionally removed and (ii) introduced into their genome the genes for producing a human antibody;

(b) introducing said breeding pair of mammals from step (a) into an antigen-free environment;

(c) permitting said breeding pair to reproduce and produce progeny; and

(d) rearing said progeny in an antigen-free environment.

6. The method of claim 5 wherein said genes for producing a human antibody comprise the human heavy chain locus and the human light chain locus.

7. The method of claim 6 wherein both said heavy chain locus and said light chain locus include genes for both the human constant and variable antibody regions.

8. The method of claim 7 wherein said mammal is a transgenic mouse.

9. A human antibody produced by the mammal of claim 3.

10. A hybridoma cell generated using a splenocyte obtained from the mammal of claim 3.

11. A human antibody produced by the hybridoma cell of claim 10.