EP1474682A2

EP1474682A2 - Production of human antibodies in immunodeficient, non-human, mammalian hosts

Info

Publication number: EP1474682A2
Application number: EP03710962A
Authority: EP
Inventors: Anke Kretz-Rommel; Hong Xin
Original assignee: Alexion Pharmaceuticals Inc
Current assignee: Alexion Pharmaceuticals Inc
Priority date: 2002-02-11
Filing date: 2003-02-10
Publication date: 2004-11-10
Also published as: CA2476232A1; JP2005517401A; AU2003215145A1; WO2003068915A3; EP1474682A4; WO2003068915A2

Abstract

Human antibodies are produced by grafting tissue from one or more human immune organs into an immunodeficient, non-human, mammalian host such as, for example, a SCID mouse.

Description

PRODUCTION OF HUMAN ANTIBODIES IN IMMUNODEFICIENT, NON-HUMAN, MAMMALIAN HOSTS

BACKGROUND

Technical Field

This disclosure relates to the creation of a functional human immune system in non-human, mammalian recipients, especially immunodeficient mice such as SCID mice. Methods for the production of human antibodies by grafting tissue from a human immune organ into non-human, mammalian recipients, especially immuπodθficient mice such as SCID mice are also described.

Background of Related Art

The use of monoclonal antibodies (mAbs) as therapeutics has expanded dramatically over the past few years. The vast majority of mAbs are of non-human (largely rodent) origin. However non-human mAbs pose the problem of immunogenicity in humans into which the rodent mAbs are introduced. Anti-rodent antibodies produced by the introduction of rodent mAbs can result in enhanced clearance of the serum, blocking of the therapeutic effect of the rodent mAb and hypersensitivity reactions. These limitations have prompted the development of "humanizatioπ" technologies to alter rodent antibodies to make them more related to human antibodies. Nonetheless, the use of fully human antibodies is most desirable.

SClD-hu mice have been designed as an animal model to study the human immune system under experimental conditions. The original SClD-hu mouse model injecting human primary blood lymphocytes into SCID mice (Mosier et al., Nature 1988, 325, 256) was followed by many studies transplanting hematopoietic organs (reviewed in Seminars in Immunology 1996, Vol. 8, 207). The SCID-hu-PBL model has been successfully used to elicit secretion of human antibodies into the serum after immunization with viral antigens (e.g., Ducosal et al., Nature 1992, 355, 258). However, antibody production in the SCID-hu-PBL mouse is limited to the mature, biased immune repertoire of the donor. Similarly, use of splenocytes primed in vitro followed by transfer into SCID mice and immunization as disclosed by Brams and Morrow (see pending application 770,057.) relies. on the mature, biased immune system. Therefore, it would be desirable to design a system that allows for the development Of a broad repertoire of T and B cells including all components of the immune system.

SUMMARY

The lymphoid system comprises organs where immune cells originate, mature or reside. The principle organs of the immune system are thymus, spleen, bone marrow, skin, lymph nodes and in the fetus also in the liver. It has now been found that engraftment of all these organs into an immunodeficient, non-human, mammalian host such as, for example, a SCID mouse, establishes a human immune system capable of mounting an immune response against a variety of immuπogens. In addition to grafting immune organs, providing an adequate growth factor and cytokine environment assists in obtaining an antibody response. Once a detectable immunogeπ-specific response has been mounted, cells from the grafted immune organs can be harvested and the cells or genetic material from the cells can be immortalized and/or cloned as desired

Thus, in accordance with this disclosure, human antibodies can be produced by grafting tissue from a human immune organ into a non-human, mammalian recipient. In particularly useful embodiments, the recipient is a SCID mouse. The method of producing a human antibody begins with the step of grafting human tissue into an immunodeficient, non-human, mammalian recipient. The human tissue grafted into the host includes sufficient portions of one or more human immune organ to produce antibodies. The development of a functional human immune system is enabled by the injection of appropriate cytokines and growth factors, such as, for example, provided in methocult and matrigel. The immunodeficient, non-human, mammalian recipient is immunized with an immunogen, preferably after the presence of human antibodies is detected. Cells that produce human antibodies in response to the immunogen are then harvested.

The step of harvesting cells that produce human antibodies can be accomplished by removing from the recipient at least a portion of the human tissue previously grafted therein or mouse immune organs to which human cells have migrated. RNA from the removed human tissue can then be isolated using conventional techniques. Once the RNA is isolated, it can be used to construct an antibody library which can be screened to identify one or more antibodies that interact with the immunogen. Alternatively, human B cells are harvested and fused to a hybridoma partner. In another aspect, a new and useful hybrid SCID mouse having human tissue grafted therein is described. The human tissue implanted into the mouse includes at least a sufficient portion of at least one human immune organ to produce a human antibody.

In yet another aspect, antibody libraries derived from human cells that developed from human tissue implanted within an immunodeficient, non-human, mammalian recipient are described.

In yet another aspect, methods for testing of the effects of drugs on the human immune system are described in which a drug is administered to a non-human mammalian having at least a sufficient portion of thymus, spleen, bone marrow, skin, lymph nodes and fetal liver grafted therein to produce a human antibody and any effects of the drug on human antibody production is observed.

Detailed Description Of Preferred Embodiments

Human antibodies are produced by human tissue grafted into a non-human, mammalian recipient. The grafted tissue includes sufficient portions of one or more human immune organs to produce a fully human antibody. The term "human immune organ" is intended to include human tissue from any organ that produces one or more molecules involved in the production of a human antibody. Thus, for example, tissue containing or capable of developing cell types that actually produce antibodies are included in the graft. In addition, tissue involved in the recognition, breakdown or presentation of an antigen to an antibody producing cell may also be included in the grafted tissue. Suitable tissue types which fall within the category of "human immune organ" include of thymus, spleen, bone marrow, skin, lymph nodes and liver. Other suitable types of tissue will be readily apparent to those skilled in the art. The tissue to be grafted is preferably obtained from a fetal source, having a gestational age of at least four months, more usually in the range of 18 to 24 weeks and ranging up to neonate tissue, depending upon the nature of the tissue or organ. Tissue from a single donor is preferred. The use of fetal instead of adult tissues is preferred because it allows development of the immune system directly within the recipient. As the fetal cells develop they are exposed to proteins native to the recipient, and the immune organs become tolerant to the environment within the recipient.

In a particularly useful embodiment, thymus, spleen, bone marrow, skin, lymph nodes and fetal liver are all grafted into the recipient to ensure that all aspects of antibody production are provided. Transplanting bone, fetal liver and thymus allows the development of T and B cells, thereby providing a broad repertoire of potential antigen specificity. Addition of skin provides the site of immunogen injection (as described in more detail below) with the highest probability of human antigen presenting cells picking up the immunogen. Lymph nodes can be important for T/B cell interaction and the spleen is a potential resource of B cells to construct a display library.

The amount of tissue grafted into the recipient should be an amount sufficient to produce antibodies. The amount of tissue grafted can be as little as 1 mm³ of each type of tissue. On the other hand, grafts of 100 mm³ or more can be used, depending on the specific recipient chosen. The tissue may be fresh tissue, obtained within about 48 hours of death, or cryopreserved in a manner that maintains viability of the tissue.

The recipient can be any type of animal into which human tissue can be grafted and remain viable. Genetically immunocompromised mice are a particularly preferred - recipient, especially NOD-SCID mice. Where possible, it is preferred to irradiate the recipient before the human tissue is grafted therein. As those skilled in the art will appreciate, irradiation of the recipient will eliminate the native immune function of the recipient. The specific parameters for irradiation will depend on the particular type of recipient, especially the volume of the recipient. It should be understood, of course, that the conditions for irradiation should not be sufficiently severe as to kill the recipient. Irradiation conditions for various types of recipients are known to those skilled in the art. In general, the lowest dose of radiation necessary to cause loss of immune function should be applied. By starting low and applying progressively higher doses of radiation, an effective dosage of radiation can be determined without undue experimentation. SCID mice can be irradiated with gamma radiation in an amount ranging from 100 to 2000 RAD. Grafting can be accomplished by simply making an incision in the skin of the recipient and placing the human tissue under the skin of the recipient. If desired, the grafted tissue can be secured to a desired location within the recipient's body such as, for example by sutures or staples. The location at which the human tissue is grafted is not critical. Considerations in choosing a location for the graft include the likelihood that the grafted tissue will be vascularized, the ease of implantation and the ease of retrieval of the implanted tissue. Preferably the grafting site is a highly vascularized location. The site of grafting can be downstream from a convenient site in the blood or lymphatic system for introduction of an antigen as described below. In particularly useful embodiments, primary lymph organs (e.g., bone, liver and thymus) are grafted at a location remote from the secondary lymph organs (e.g., spleen, lymph nodes and skin). Once the tissue is placed and/or secured within the recipient, the incision in the recipient's skin can be closed (e.g., by suturing, stapling or adhesive). While the human skin can be placed below the recipient's skin, it is preferred to remove a section of the recipient's skin and provide a surface graft of the human skin to allow injection of an immunogen directly through the human skin graft. Optionally, the mononuclear fraction of cord blood can be injected into the recipient's blood stream at or around the time of grafting.

At or after the placement of the graft, the immediate environment surrounding the graft is optionally treated to enhance lymphocyte development and antibody production. Suitable treatments include the application of compositions containing components known to enhance vascularization and cell growth, the differentiation of cells and/or the production of antibodies. Suitable compositions include gelling agents (such as, for example, methylcellulose or agar) containing growth factors (such as, for example, Stem Cell Factor, Granulocyte acrophage-Stimulatiπg Factor, IL-3, IL-6, Graπulocyte- Colony Stimulating Factor, and erythropoietin). Commercially available products that can be used for this purpose include METHOCULT® GF H4435 available from Stemcell Technologies Inc., Vancouver, British Columbia. Details regarding METHOCULT® products are disclosed in the following articles: Conneally E, Bardy P, Eaves CJ, Thomas T, Chappel S, Shpall EJ, Humphries RK: Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer. Blood 87: 456, 1996; Eaves CJ: Assays of hemopoietic progenitor cells. Williams Hematology, 5 (eds. E Beutler, MA Lichtman, BS Coder, TJ Kipps), McGraw-Hill, Inc., pp L22-6, 1995; Eaves C, Lambie K: Atlas of Human Hematopoietic Colonies, StemCell Technologies Iπc, 1995; Eaves AC, Eaves CJ: Current Therapy in Hematology-Oncology 4: 159, 1992; Helgason CD, Sauvageau G, Lawrence HJ, Largman C, Humphries RK: Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. Blood 87: 2740, 1996; Hogge DE, Lansdorp PM, Reid D, Gerhard B, Eaves CJ: Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor. Blood 88: 3765, 1996; Hough MR, Chappel MS, Sauvageau G, Takei F, Kay R, Humphries RK: Reduction of early B lymphocyte precursors in transgenic mice overexpressing the murine heat-stable antigen. J Immunol 156: 479, 1996; Lemieux ME, Rebel VI, Lansdorp PM, Eaves CJ; Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lympho-myeloid differentiation in long-term marrow "switch" cultures. Blood 86: 1339, 1995; Mayani H, Dragowska W, Lansdorp PM: Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells. Blood 81 : 3252, 1993; Petzer AL, Hogge DE, Lansdorp PM, Reid DS, Eaves CJ: Self-renewal of primitive human hematopoietic cells (long-term culture-initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci USA 93: 1470, 1996. The disclosure of each of these references is incorporated herein by reference.

Another useful composition that can be applied to the grafted human tissue is a composition capable of supporting cell growth. Suitable compositions in this category include basement-membrane-derived compositions containing a biologically active polymerizable extract containing lamiπin, collagen IV, nidogen, heparan sulfate proteoglycan and entactin. The term "biologically active" as used in connection with this basement-membrane-deriyed composition means capable of supporting normal growth and differentiation of various cell types when cultured including epithelial cells. One such composition is commercially available under the tradename MATR1GEL® Basement Membrane Matrix available from Becton Dickinson Labware, Bedford MA. Details regarding MATRIGEL® products are disclosed in U. S. Patent No. 4,829,000, the disclosure of which is incorporated herein by reference. A combination of such materials can advantageously be employed.

The composition(s) can be applied directly to the location of grafted organ at the time of grafting and/or subsequent to grafting by injection to the site of grafting. The amount of the composition applied is not critical and will depend on such factors as the type(s) and amount of human tissue grafted, the specific composition employed, the specific recipient, and the location of the graft. Typically from about 100 μl to about 500 μl of the composition will be applied at the time of grafting. In particularly useful embodiments, the composition is re-applied after grafting at intervals of between seven and ten days until immunization, as discussed in more detail below.

After the grafting is accomplished, the recipient is monitored to ascertain whether human antibodies are being produced. Techniques for detection of human antibodies are within the purview of one skilled in the art. Kits for detecting the presence of human antibodies in an animal's serum such as, for example, Easy-Titer® human IgG assay kit are commercially available from Pierce (Rockford, IL). Normally, by the point in time when human antibodies are detected, the grafted tissue remains viable, has vascularized and is believed to have lymphatic vessels connected thereto. Generally, at least one week will transpire, however anywhere from 2 to 20 weeks or more may be allowed to pass before the next step (immunization) is performed.

Once it is detected that human antibodies are present in the recipient, the recipient is immunized with one or more immunogen. Suitable immunogeπs include both haptens and antigens, where the haptens are modified to provide for an immune response. Compounds of interest may include small synthetic organic molecules, generally of less than about 5 kD (kilodaltons), usually less than about 2 kD, polypeptides and proteins, lipids, saccharides, and combinations thereof. The compounds may be synthetic or naturally occurring, including drugs, hormones, cytokines, surface membrane proteins, enzymes, sugar side groups, toxins, etc. Cells or tumor tissue can also be used as the immunogen. The immunogen may be combined with a wide variety of adjuvants, such as alim, RIBI, CpG oligodeoxyπucleotide, complete Freund's adjuvant, specol, B. pertussis or its toxin, etc.

In an alternative embodiment, an immunizing composition is prepared by culturing the mononuclear fraction of cord blood to grow dendritic cells. The dendritic cells are then stimulated with an immunogen. The resulting composition is then injected into the recipient to accomplish immunization. The immunogen can be administered systemically to the recipient of the graft or injected locally at the site of the graft. Administration will normally be by injection, either subcutaneous, intramuscular, intraperitoπeal or intravascular, preferably directly into the site of the grafted human tissue. In particularly useful embodiments, the recipient has received a human skin graft and the immunogen is injected through the grafted skin or adjacent to it Considerations in determining how much immunogen should be administered include the nature of the immunogen, the amount of tissue grafted into the recipient and the desired degree and swiftness of the immune response sought. One or more booster injections maybe made, usually within 1 to 6, more usually 2 to 4 weeks of the previous injection. The booster injection may have the same composition or different composition than the prior injection. Immunization may also be accomplished by the RIMMS technique which is well known to those skilled in the art.

The recipient is then monitored to ascertain whether an immunogen-specific response has been mounted. Techniques for detection of antibodies specific to any given immunogen are within the purview of one skilled in the art. One suitable method of detecting the presence of human immunogen-specific antibodies in an animal's serum is disclosed in Current Protocols in Immunology, Coligaπ et al., Chapter 2.1 (John Wiley & Sons, 2000 ed.), the disclosure of which is incorporated herein by reference.

Once the immunogen-specific immune response is detected, at least a portion of the grafted tissue is removed from the recipient. Mouse tissue into which human cells may have migrated may also be extracted to maximize the amount of antibody producing cells collected. Cells or genetic material from the tissue can be immortalized and/or cloned as desired. Thus, for example, RNA from the removed tissue is isolated using techniques well known to those skilled in the art. The recovered RNA is used to generate one or more antibody libraries and the libraries are screened to identify antibodies that bind to the antigen or a component thereof. In preferred embodiments, the library is screened to identify antibodies that have either an agonistic or antagonistic effect on the antigen. Techniques for producing and screening antibody libraries are well known to those skilled in the art. See, for example, U.S. Patent No. 6,291 ,161 to Lerner et al. and copending U.S. Provisional Application Nos. 60/323,455 and 60/323,400, the disclosures of which are incorporated herein in its entirety by this reference.

As another example, antibody-producing cells can be selected and fused with non-antibody producing cells such as, for example, immortalized cell lines. These fusion partners are typically transformed human cells such as human myeloma cells. One suitable human myeloma cell line is disclosed by Karpas, et al., PNAS, vol. 94, no. 4, pages 1799-1804, 2001. After fusion, fused cells are segregated into individual cultures and propagated, and hybridoma lines which express immunogen-specific monoclonal antibodies are selected. These cell lines can be maintained in culture or cryopreserved using techniques well known to those of ordinary skill in the art. The hybridomas may then be introduced into host animals, e.g. mice or rats, to produce ascites fluid or mechanically expanded, using spinner flasks, roller bottles, etc, The host will be immunocompromised, so as to be able to accept the neoplastic graft. The resulting antibodies may be used in a variety of ways, both diagnostic and therapeutic. However, since other antibodies which are normally more easily obtained, such as non-human antibodies can be used in in vitro diagnostics, for the most part the subject antibodies will be used for in vivo diagnostic and therapeutic use in humans. Thus, the subject antibodies may be used in the treatment of disease, neutralizing viruses or other pathogens, for in vivo diagnoses, for targeted toxicity against neoplastic cells or precursors to such cells, passive immunization, in conjunction with transplantation, and the like. The subject antibodies may be modified by radiolabeling, conjugation to other compounds, such as biotin, avidin, enzymes, cytotoxic agents, e.g. ricin, diphtheria toxin, arbin, etc., and the like.

In addition to using the present methods for the production of human antibodies, the non-human recipients of the grafted human immune organ can be used in methods for testing of the effects of drugs on the human immune system. In these methods, a drug is administered to the non-human mammalian having at least a sufficient portion of thymus, spleen, bone marrow, skin, lymph nodes and fetal liver grafted therein to produce a human antibody. After a period of time, any effects of the drug on human antibody production is observed. Techniques for determining the effects of a drug on a human immune system are known to and within the purview of those skilled in the art. The following non-limiting example is provided to illustrate one embodiment of the methods described herein. EXAMPLE

NOD/LtSz-scid/scid mice were irradiated with 250 RAD to completely eliminate any residual immune function. Fetal tissue from gestation week 18-24 was obtained and small pieces of liver, spleen and thymus cortex and thymus medulla were prepared. Also, bone was processed by cutting off approximately 0.5 cm at the end on both sides of the femur and tibia. Lymph nodes from Peyefs patch were collected and skin pieces of approximately 1 cm² were prepared. One incision was made above each leg and bone, liver and thymus pieces were placed under the skin. After initial experiments yielding a recall, but not a new immune response, the system was improved by injecting methocult into the area containing the grafted organs to provide an appropriate cytokine environment for immune cell engraftment. A 1 cm2 patch of skin was removed from the back of the mouse and the human skin was grafted into the deficit. An incision was made adjacent to the human skin graft and lymph node and spleen tissue was placed under the recipient's skin. After initial experiments yielding a recall, but not a new immune response, the system was improved by injecting 200 μl of MATRIGEL® was injected into the grafted spleen area to provide enhanced blood vessel formation.

The presence of human T cells, B cells and dendritic cells in blood, bone marrow and spleen one month, two or three months after grafting was confirmed by FACS analysis. Serum was collected at multiple time-points and human immunoglobulin titers were determined. 25 out of 27 grafted mice developed human IgG titers of at least 2500 ng/ml within one month. Once human IgG was detected in the mouse serum, immunization with immunogen was initiated. Initially, mice received 10 μg Hepatitis B\ surface antigen or tetanus toxoid in complete Freund's adjuvant subcutaneously adjacent to the grafted skin. Immunizations were repeated after 3 weeks and 6 weeks using 10 μg antigen in complete Freund's adjuvant. Some mice were immunized using CpG oligodeoxyπucleotides. 100 μg CpG oligodeoxynucleotides and 10 μg antigen were injected subcutaneously at one to three week intervals. Serum IgG titers specific for tetanus toxoid or Hepatitis B surface antigen were determined by ELISA. A weak response to tetanus toxoid, but no response to hepatius B surface antigen was observed. To improve the system and enable a new immune response and not just a recall response, matrigel and methocult were introduced as described above. With this improvement, two out of three hepatitis B and six out of seven tetanus toxoid immunized mice showed specific human antibodies to the respective antigen after the third immunization. Also, three out of five mice developed an anti-lgE response with substantial titers (up to 1 :5000) after immunization with human IgG Fc. Two out of four mice immunized with LF showed a moderate response to that antigen.

Mice were immunized one more time and the immune organs are extracted to isolate RNA. RNA is isolated from the collected immune organs and an antibody library is produced. The library is panned using the immunogen with which the mice were immunized. Fully human antibodies which bind to or otherwise interact with the immunogen are thus identified.

While the above description contains many specific details of methods in accordance with this invention, these specific details should not be construed as limitations on the scope of the invention, but merely as exemplifications of preferred embodiments thereof. Those skilled in the art will envision many other possible variations that all within the scope and spirit of the invention as defined by the claims appended hereto.

Claims

We claim:

1. A method of producing a human antibody comprising the steps of: a) grafting human tissue into an immunodeficient, non-human, mammalian host, the human tissue comprising at least a sufficient portion of thymus, spleen, bone marrow, skin, lymph nodes and fetal liver to produce a human antibody; b) applying a composition to the grafted human tissue, the composition including at least one growth factor and a material to support cell growth; c) immunizing the host with an immunogen; and d) harvesting at least a portion of the grafted tissue that includes cells that produce human antibodies.

2. A method as in claim 1 where the immunodeficient, non-human, mammalian host is a rodent.

3. A method as in claim 1 where the immunodeficient, non-human, mammalian host is a SCID mouse.

4. A method as in claim 1 wherein the immunodeficient, non-human, mammalian host is a NOD/LtSz-scid/scid mouse.

5. A method as in claim 1 further comprising the step of irradiating the immunodeficient, non-human, mammalian host prior to said grafting step.

6. A method as in claim 1 further comprising the step of testing for the presence of human antibodies prior to said immunizing step.

7. A method as in claim 1 wherein said step of harvesting cells comprises removing at least a portion of the human tissue from the host, isolating RNA from the removed human tissue, constructing an antibody library and identifying one or more antibodies that bind to the immunogen. .

8. A human antibody produced in accordance with the method of claim 1.

9. An immunocompromised mammal having grafted therein at least a sufficient portion of thymus, spleen, bone marrow, skin, lymph nodes and fetal liver to produce a human antibody.

10. An immunocompromised mammal as in claim 9 which is a SCID mouse.

11. An antibody library derived from human cells that developed from human thymus, spleen, bone marrow, skin, lymph nodes and fetal liver implanted within an immunodeficient, non-human, mammalian host.

12. An antibody library as in claim 11 wherein the immunodeficient, non-human, mammalian host is a SCID mouse.

13. An antibody library as in claim 11 wherein the immunodeficient, non-human, mammalian host is a NOD/LtSz-scid/scid mouse.

14 A method of testing the effect of drugs comprising: administering a drug to a non-human mammalian having at least a sufficient portion of thymus, spleen, bone marrow, skin, lymph nodes and fetal liver grafted therein to allow the development of one or more types of human immune cells selected from the group consisting of T cells, B cells and dendritic cells; and observing any effects of the drug on human antibody production.

15. A method comprising. ^• isolating antibody producing cells from an immunodeficient, non-human, mammalian host into which at least a sufficient portion of human thymus, spleen, bone marrow, skin, lymph nodes and fetal liver has been grafted to produce a human antibody; and fusing one or more of the isolated cells to a hybridoma.