WO1993005796A1 - Procede de production d'anticorps humains dans un animal non humain, et animaux utilises a cet effet - Google Patents

Procede de production d'anticorps humains dans un animal non humain, et animaux utilises a cet effet Download PDF

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Publication number
WO1993005796A1
WO1993005796A1 PCT/US1992/008005 US9208005W WO9305796A1 WO 1993005796 A1 WO1993005796 A1 WO 1993005796A1 US 9208005 W US9208005 W US 9208005W WO 9305796 A1 WO9305796 A1 WO 9305796A1
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pbl
human
lymphocytes
cells
mouse
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PCT/US1992/008005
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English (en)
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Michel A. Duchosal
Sabine A. Eming
Patricia J. Mcconahey
Frank J. Dixon
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The Scripps Research Institute
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0271—Chimeric animals, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • the present invention relates to a method for producing human antibodies in a non-human animal by reconstituting a human immune system in the animal.
  • the invention also relates to a non-human animal having a reconstituted human immune system.
  • mice have also been used. These mice are deficient in B and T cell(s) (functions) (Bosma et al. Nature, 301:527-530, 1983; and Custer et al. Am. J. Pathol.. 120(3) :464-477, 1985) , and have been proven to sustain the differentiation of mature human B and T cells from human fetal lymphoid xenografts (McCune et al, Science, 241:1632-1639, 1988).
  • C.B-17-scid/scid mice lack B and T cells and their associated functions and, therefore, suffer from a severe combined immunodeficiency (SCID) disease .
  • SCID severe combined immunodeficiency
  • Bosma et al Nature, 301:527-530 (1983).
  • SCID-hu human fetal lymphoid cells
  • hu-PBL-SCID human peripheral blood leukocytes
  • a method has now been discovered that provides for more complete population of a SCID mouse recipient such that the reconstituted mouse exhibits human IgG serum levels of typically 2 to 5 grams per liter in the absence of human antibodies immunoreactive with Epstein-Barr virus antigens.
  • the reconstituted hu- PBL-SCID mouse produced by the present invention can respond immunologically and produce substantial specific human IgG upon immunization with a preselected antigen.
  • the invention contemplates a method for producing human antibodies that immunoreact with a preselected antigen in a severe combined immunodeficiency (SCID) mouse comprising the steps of isolating a suspension of viable human peripheral blood lymphocytes (PBL) from a human donor, contacting the viable PBL in a medium in which lymphocytes are viable with the preselected antigen in an amount sufficient for the antigen to induce an immune response in the PBL, intraperitoneally introducing the PBL into a mouse having severe combined immunodeficiency, thereby forming a hu-PBL-SCID mouse having a reconstituted human immune system, and immunizing the said hu-PBL- SCID mouse with the antigen in an amount sufficient to induce an immune response wherein human antibodies immunoreactive with the antigen are formed in the hu- PBL-SCID mouse.
  • PBL peripheral blood lymphocytes
  • the invention also contemplates a reconstituted hu-PBL-SCID mouse containing human lymphocytes, such that the hu-PBL-SCID mouse has a plasma concentration of 2 to 5 grams per liter of human immunoglobulin and plasma that is seronegative for Epstein-Barr virus.
  • a further embodiment contemplates a method for producing a hu-PBL-SCID mouse having a reconstituted human immune system that is defined by a plasma concentration of human immunoglobulin of 2 to 5 grams per liter comprising the steps of lymphopheresing blood from a human donor to produce lymphopheresed cells containing lymphocytes, red blood cells and plasma, centrifuging the lymphopheresed cells through a medium in which lymphocytes are viable at a gravity force and for a time sufficient to pellet the lymphocytes but not the red blood cells and plasma present in the lymphopheresed cells and form pelleted lymphocytes and a RBC/plasma-containing supernatant, separating the pelleted lymphocytes from the supernatant to form isolated PBL, resuspending the isolated PBL in a medium in which lymphocytes are viable to form the suspension of isolated viable PBL, and introducing the isolated viable PBL into a mouse having severe combined immunodeficiency, thereby forming a hu-PBL
  • the invention contemplates a method for producing a hu-PBL-SCID mouse as above comprising the steps of centrifuging heparinized whole blood from the donor through a high density ficoll medium of density 1.119 gram per illiliter to form upper, lower and inter layers, recovering the inter layer containing lymphocytes, red blood cells and plasma, diluting the inter layer with an equal volume of medium in which lymphocytes are viable, centrifuging the diluted inter layer as above to pellet the lymphocytes but not the red blood cells or plasma and form pelleted lymphocytes and a RBC/plasma-containing supernatant, separating the pelleted lymphocytes from the supernatant to form isolated lymphocytes, resuspending the isolated lymphocytes in medium in which lymphocytes are viable to form the suspension of isolated viable PBL, and introducing the isolated viable PBL into a mouse having severe combined immunodeficiency, thereby forming a hu-PBL-SCID mouse having a re
  • Figure 1 illustrates the time course evolution of mean human IgG serum levels in 66 SCID mice populated with 15 x 10 6 PBL from an EBV " donor [ ] , 9 mice irradiated to 250 rads 12 hours before receiving 15 x
  • Figures 2A through 2E illustrate the immunization with TT antigen.
  • Figure 2A shows a comparison of TT serum titer for a donor (#4) not boosted for 20 years (open circles) with that (+SEM) obtained by boosting the donor's PBL in 6 hu-PBL-SCID mice (closed circles) .
  • These 6 hu-PBL-SCID mice had, 29 days post cell transfer, 2.86 + 1.19, and 6.36 + 3.34 IU of mean absolute-, and normalized-IgG anti-TT/ml, respectively.
  • the mean values for 3 similarly populated mice but not immunized is represented (closed triangles) .
  • This ELISA did not show any reactivity when sera from non-populated SCID, BALB/c, or C57BL/6 mice were applied.
  • Figures 2B and 2D show the IgG anti-TT serum response in hu-PBL-SCID mice non-immunized with TT.
  • Figures 2C and 2E show the anti-TT serum response in hu-PBL-SCID mice immunized with TT. Mice were populated
  • the present invention is founded on two discoveries. First, it is shown that a method of introducing human lymphocytes into an immunodeficient non-human animal according to the present invention produces a non-human animal having a reconstituted human immune system which is capable of producing higher plasma levels of human immunoglobulin than previously reported in the absence of human antibodies immunoreactive with EBV antigens. Second, it is shown that a method of immunizing the reconstituted human immune system according to the present invention induces a specific immune response to the preselected immunizing antigen.
  • the present invention contemplates a non-human animal having a reconstituted human immune system.
  • the reconstituted human immune system is comprised of a xenogenic transplant of human peripheral blood lymphocytes (PBL) and thus the reconstituted animal is referred to herein as a hu-PBL animal.
  • the non-human animal is a mammal such as a mouse, hamster, rat, dog, cat, goat, monkey or the like mammal, and is more preferably a mouse.
  • the recipient non-human mammal must be an immunodeficient animal in order that the xenogenic transplant succeed.
  • Immunodeficiency in a mammal can be provided by a variety of means such as by genetic deficiency or by irradiation of the recipient animal. Immune-impairing genetic deficiencies are known for a variety of the above recited species, for example the beige/nude/xid mouse (Kamel-Reid et al, Science F 242:1706-1709, 1988) or the severe combined immunodeficiency (SCID) mouse. Particularly preferred is the homozygous C.B-17-SCID/SCID (SCID) mouse (Bosma et al, Nature, 301:527-530, 1983).
  • SCID severe combined immunodeficiency
  • a preferred irradiation-induced immunodeficiency is the lethally- irradiated BALB/c mouse described by Lubin et al, (Science, 252:427-431, 1991). SCID mice are available from commercial vendors and the research community in their mouse breeding facility.
  • a human immune system-reconstituted non-human animal that is described as exemplary herein that is particularly preferred is the SCID mouse, and is referred to herein as a hu-PBL-SCID mouse.
  • a hu-PBL-SCID mouse of this invention is defined by a plasma concentration of human gamma globulin (HGG) of 1 to 8 grams per liter (g/1) , preferably 2 to 5 g/1.
  • HGG human gamma globulin
  • Assays to determine plasma levels of HGG are well known in the art.
  • An exemplary assay for HGG is described by Duchosal et al, in J. Exp. Med. f 172:985- 988 (1990) .
  • a hu-PBL-SCID mouse of this invention is seronegative for human antibodies immunoreactive with antigens of the Epstein-Barr Virus (EBV) .
  • EBV serology can be determined by any of the well established blood bank protocols that are commercially available.
  • the donor does not contain latent EBV infection, as can be determined by nucleic acid hybridization. It is well established by earlier work by Mosier and others using PBL from EBV seropositive (EBV " ) or EBV seronegative (EBV * ) donors that human B cell lymphomas can develop in the reconstituted mouse when the donor is EBV + , particularly when the SCID mouse has received the higher published numbers of PBL in the transplant.
  • the present invention provides a hu-PBL non-human animal containing plasma human gamma globulin levels of 1 to 7 g/1, and more typically levels of 2 or 3 to 5 g/1 without the requirement for PBL from a EBV + donor.
  • one benefit to the invention is the ability to obtain PBL from a human donor who is a patient in need of an antibody supplement, such as a passive immunization, but who is EBV " .
  • the EBV " donated PBL can be transplanted and the resulting hu-PBL-SCID mouse can be immunized according to the present invention to yield 1 to 8 g/1 of plasma HGG which contains substantial levels of immunoglobulin specific for the immunizing antigen.
  • the preparation of therapeutic antibodies from sources free from EBV is preferred for health safety reasons.
  • the invention contemplates a method for producing a hu-PBL non-human animal having a reconstituted human immune system.
  • the method generally involves the steps of isolating PBL from a human donor according to the methods described below, and introducing the isolated PBL into an immunodeficient non-human animal to form a hu-PBL non-human animal.
  • a reconstituted human immune system By a reconstituted human immune system is meant that the animal contains human lymphocytes and exhibits a plasma concentration of human immunoglobulin from 1 to 8 g/1, and more typically from 2 to 5 g/1.
  • a hu-PBL non-human animal containing a reconstituted human immune system has the capacity to respond immunologically to immunization as described herein and produce human antibodies immunospecific for the preselected immunizing antigen and described herein.
  • Methods for determining the presence of human lymphocytes are well known and involve the use of immunological reagents specific for human lymphocyte markers such as human leukocyte common antigen CD45, 2B11 and PD7/26, CD4, or other human specific lymphocyte markers.
  • hu-PBL non-human animal The contemplated methods for preparing a hu-PBL non-human animal are exemplified herein by the detailed descriptions for preparing a hu-PBL-SCID mouse. However, the basic observations of PBL manipulations and immunization protocols described herein may be applied to the other non-human animals described. 1. Isolation of Human PBL A hu-PBL-SCID mouse having a reconstituted human immune system is produced by first isolating PBL from a human donor, preferably an EBV seronegative donor.
  • the method of isolating the PBL can be accomplished by a variety of methods as discussed further below, but generally involves manipulations designed 1) to remove the red blood cells (RBC) and associated human plasma from the donor lymphocyte sample because immunodeficient non-human recipients do not tolerate human plasma and RBC, 2) to minimize the trauma to the lymphocytes being isolated from the donor, and 3) to minimize the time the lymphocytes spend outside the body of either the donor or the recipient.
  • RBC red blood cells
  • Two exemplary and preferred PBL isolation methods are described herein, which utilize either whole, heparinized blood or lymphopheresed blood products, namely lymphopheresed cells.
  • the isolation of PBL from whole blood comprises the steps of first adding a clotting inhibitor such as heparin, citrate or the like as is well known in the hematological arts.
  • a clotting inhibitor such as heparin, citrate or the like as is well known in the hematological arts.
  • Exemplary is the heparinized whole blood described in the examples.
  • separation of the blood constituents is effected by differential centrifugation on a cell separating medium to form isolated lymphocytes. Typically this involves separation on a high density ficoll medium, as is well known.
  • a preferred ficoll medium for preparing human PBL has a density of 1.119 gram per milliliter (gm/ml) because the resulting inter layer contains macrophages, polymorphonuclear cells (PMN) , lymphocytes, human plasma, and trace amounts of residual red blood cells.
  • Previous ficoll mediums utilized for preparing PBL have a density of 1.077, which separates the lymphocytes away from the macrophages and PMN.
  • the method for isolating PBL from whole blood comprises the steps of centrifuging heparinized whole blood from a human donor through a high density ficoll medium of density 1.119 gm/ml to form an upper, lower and inter layer. Centrifugation conditions for effecting the formation of the layers is dependent on the geometry of the medium, as is well known.
  • a preferred ficoll medium of density 1.119 gm/ml is prepared using Histopaque-1119 according to the manufacturer's specifications (Sigma Chemical Co., St.Louis, MO) .
  • the inter layer is collected (recovered) and diluted with a medium compatible with lymphocyte cell viability to allow washing of the collected lymphocytes.
  • a medium that is compatible with lymphocyte cell viability is a medium in which lymphocytes are viable, and can be subsequently shown to remain viable in culture.
  • the medium is isotonic to minimize any adverse affects that in vitro culturing may have on the viability of the lymphocytes.
  • the degree of dilution is designed to facilitate a centrifugation step for removing residual red blood cells present in the inter layer. Volumes of diluent of about 0.5 to 2 times the inter layer volume are used, although equal volumes are preferred.
  • a medium compatible with lymphocyte cell viability is any culture medium formulation designed for lymphocyte stabilization or culturing. Cell viability can be monitored by a variety of means including integrity of dye exclusion over time after exposure to the medium. Exemplary is Earle's medium described herein having 1 % bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • the diluted inter layer is subjected to centrifugation conditions comprising a gravity force and a time period sufficient to sediment (pellet) the lymphocytes without pelleting the RBC, forming pelleted lymphocytes and a supernatant containing the RBC and plasma.
  • centrifugation conditions sufficient for separation of RBC and plasma from lymphocytes in suspension are to subject the diluted inter layer to 1200 rotations per minute (RPM) on a Damon IEC EPR-6000 centrifuge at 4 degrees Centigrade (4C) for 10 minutes (min) , using a swinging bucket rotor fitted with 50 ml buckets having a distance from center axis to tube bottom of about 12 inches.
  • RPM rotations per minute
  • 4C degrees Centigrade
  • Other configurations could readily be substituted to preferentially pellet the lymphocytes.
  • the pelleted lymphocytes are then separated from the RBC/plasma-containing supernatant, typically by decanting the supernatant, to form isolated lymphocytes in pellet form
  • the isolated lymphocytes (in pellet form) are resuspended in a medium in which lymphocytes are viable, defined as before, and preferably of the same composition to form a suspension of isolated viable peripheral blood lymphocytes (PBL) .
  • the final volume of PBL in the suspension can vary widely, depending on the amount of PBL to be introduced into the recipient non-human animal, and the volume of cells that can be conveniently injected.
  • typical injection volumes are from 0.2 to 2.0 ml, preferably about 1.0 ml.
  • Typical cell concentrations in the isolated PBL suspension can be from about 4 x 10 5 to 4 x 10 7 cells per ml, preferably are about 10 6 to 250 x 10 6 , and more preferably are about 50 x 10 6 cells per ml.
  • the isolation of viable PBL from lymphopheresed blood products comprises the steps of first obtaining fresh human lymphopheresed cells from a donor.
  • Fresh cells are those which are collected from a human donor using an automated blood cell separator over a period of less than 6 hours, preferably less than 4 hours.
  • Automated blood cell separators useful for conducting lymphopheresis include the Cobe 2997 unit (Cobe, Denver, CO) or the Fenwall CS 3000 (Fenwall, Deerpark, IL) used according to the manufacturer's instructions for effecting lymphopheresis to produce lymphopheresed cells, including PBL, human plasma and RBC.
  • lymphopheresed cells are washed as before to remove RBC/plasma by centrifugation and resuspension as above to form a suspension of isolated viable PBL.
  • a hu-PBL non-human animal is produced by introducing the isolated PBL into a recipient immunodeficient non-human animal, thereby reconstituting the animal with a human immune system.
  • the recipient immunodeficient non-human animal used to exemplify the present invention is the SCID mouse described before.
  • Introduction of the previously isolated PBL is accomplished by injection of the suspension of isolated viable PBL intraperitoneally into a living, recipient SCID mouse, thereby forming a hu-PBL-SCID mouse having a reconstituted human immune system.
  • the recipient SCID mouse is from about 4 to 12 weeks old, and more preferably is 6 to 8 weeks old.
  • the amount of PBL to be injected can vary from 2 to 50 x 10 6 cells, preferably from 10 to 20 x 10 6 cells, and more preferably is about 50 x 10 6 cells.
  • the quality of the isolated viable PBL is important for the higher levels of effective xenogeneic transplant, measured as plasma levels of HGG, it is preferred if the injections occur after the minimum possible amount of time after removal of the PBL from the donor. Typical times range from about 1 to 6 hours, but are preferably within 2 to 4 hours.
  • a preferred PBL for introduction is obtained from an EBV " seronegative donor.
  • the present invention also contemplates methods for producing human antibodies in the reconstituted human immune system present in an immunodeficient non- human animal, such as in a hu-PBL-SCID mouse described herein.
  • the invention describes methods for inducing the human immune system to produce human antibodies that immunoreact with a preselected antigen, by exposing (contacting) the PBL of the reconstituted human immune system at particular times in the method to a preselected antigen, thereby "immunizing" the reconstituted human immune system. Thereafter, the "immunized” system produces human antibodies that immunoreact with the preselected antigen.
  • the resulting human antibodies produced by the present methods have a preselected immunoreactivity similar to the reactivity obtained by conventional immunization of a mammal, with the exception that the human donor is not exposed to the antigen in the course of the method of this invention.
  • the present methods for producing human antibodies in a hu-PBL immunodeficient animal of this invention comprise the steps of first isolating a suspension of viable human PBL as previously described.
  • the donor has a history of contact with the preselected antigen such that the population of isolated human PBL will contain memory B cells, and members of the population will have the potential, upon "immunization" (first and subsequent contacts with the preselected antigen outside the body of the donor) to respond immunologically in the form of a secondary immune response, expressing IgG class immunoglobulin immunospecific for the preselected antigen.
  • the second step in the method for producing human antibodies is to contact the isolated human PBL with a preselected antigen as to induce the secondary response, in a manner similar to in vivo immunization, with the exception being that the contact is not in the donor. Rather, the contact is before or at the time of xenogenic transplantation.
  • the isolated viable human PBL are cultured in vitro under in vitro immunizing conditions in the presence of the preselected antigen in an amount of antigen and for a time period sufficient for the antigen to induce an immune response in the PBL.
  • in vitro immunizing conditions are human PBL culturing conditions, i.e., tissue culture at 37 degrees Centigrade in medium in which lymphocytes are viable, where the culture medium contains for at least a portion of the culturing time period an amount of the preselected antigen sufficient to induce an immune response.
  • composition of the in vitro immunization conditions can include additional reagents designed to modulate the human B cell response as is well known, including growth hormones, stabilizing proteins, cytokines and the like. See, for example, the teachings in "Therapeutic Monoclonal Antibodies", Borrebaeck et al, Eds., Stockton Press, New York, NY, p.1-15, (1990).
  • Concentrations of the antigen and the human PBL to be induced (“immunized") in vitro can vary widely, but typically the lymphocyte concentration varies from 0.4 x 10 6 to 40 x 10 6 lymphocytes per ml of culture medium, preferably about 1 x 10 6 to 10 x 10 6 lymphocytes per ml, and more preferably about 4 x 10 6 lymphocytes per ml.
  • the concentration of antigen can be from about 0.1 to 20 micrograms (ug) per ml, preferably about 1 to 5 ug/ml.
  • the time period for in vitro contacting (incubation) of the preselected antigen with the human PBL can vary also, although consideration of the integrity of the isolated PBL requires that the contacting be less that 6 hours, typically about 0.5 to 6 hours, and preferably about 4 hours. Exemplary in vitro culture medium, lymphocyte and antigen concentrations, and incubation time periods are described herein.
  • the contacting can be accomplished by admixture of the isolated viable human PBL and the preselected antigen at the time of introduction of the PBL into the recipient. That is, by either admixture of the antigen and the PBL immediately (within minutes) prior to injection, or by co-injection into the intraperitoneal cavity of the recipient, the first "immunizing" contact may be afforded.
  • This latter embodiment is also referred to as “substantially simultaneously" carrying out the contacting and introducing steps of the method.
  • one or more in vivo immunizations are conducted to boost the secondary immune response, manifest in the form of the production of human IgG in the blood of the recipient.
  • the timing of the immunizations, the amount of antigen and the carrier the antigen is delivered in, and the sites for immunization on the recipient can generally be varied as for conventional immunizations. However, typically a first immunization in complete Freund's adjuvant (CFA) followed by a second immunization in normal saline has produced the highest values of specific antibody production, as shown in the examples.
  • CFA complete Freund's adjuvant
  • the amounts of preselected antigen utilized in the in vivo immunizations is typically in the range of 1 to 100 ug, preferably about 5 to 50 ug, and more preferably about 10 ug, although these amounts can vary depending on the molecular weight and immUnogenicity of the antigen.
  • the time periods for immunizations in vivo can range from about days 0 to 21 for the first, and about days 7 to 28 for the second. Additional immunizations may also be conducted to further boost the response. Exemplary combinations of first and second immunization are at days 2 and 21 or 22 or 23.
  • a single in vivo immunization after introduction can be administered where the antigen is contacted with the PBL by co-injection into the peritoneal cavity.
  • the human antibodies so produced in hu-PBL immunodeficient animal can then be harvested by a variety of means including 1) conventional collection of sera containing the human antibodies, 2) preparation of monoclonal antibodies by immortalizing the antibody producing human B cells of the hu-PBL immunodeficient animal, or 3) molecular cloning of the messenger RNA (mRNA) present in the human B cells of the hu-PBL immunodeficient animal and in turn preparing "repertoires" of molecular clones that each express a functional antibody molecule.
  • mRNA messenger RNA
  • Populations of vectors are so constructed to form a repertoire of cloned immunoglobulin genes derived from a single mouse having human B cells responding to the single preselected immunizing antigen.
  • the vectors are then inserted into a bacterial host, typically E.coli, and the bacteria are cultured under conditions for expressing the cloned immunoglobulin genes to produce functional heterodimeric antibody molecules having assembled heavy and light chains.
  • the population of antibody molecule-expressing clones are screened for the production of antibody molecules that immunoreact with a preselected antigen.
  • the antigen selected was tetanus toxin (TT) .
  • TT tetanus toxin
  • Those antibody-expressing clones producing preselected antigen-immunoreactive antibodies are selected for further growth, cultured in a clonal fashion and under conditions for expressing the selected antibody, and the resulting expressed antibody is collected from the culture medium of the clone, thereby yielding harvested human antibody.
  • PBL Peripheral Blood Lymphocytes
  • the aim of the transfer method was to minimize the number of manipulations required for PBL isolation, as well as to reduce the time between blood drawing, purification of PBL and PBL injection into SCID mice. Briefly, undiluted heparinized blood obtained from consenting donors was purified by lymphopheresis after which the product was centrifuged for 15 minutes at 250 x g. Lymphopheresis was conducted at the Green Hospital of the Scripps Clinic and Research Foundation's Pheresis Unit on a Fenwall CS 3000 Blood Cell Separator according to the manufacturer's instructions for isolating lymphopheresed blood products. The resultant pellet was resuspended in Earle's medium (Flow Laboratories, Inc.) and the number of lymphocytes in the PBL cell suspension was determined.
  • Earle's medium Flow Laboratories, Inc.
  • Resuspended PBL prepared in Example 1 diluted to selected concentrations depending on the experimental design were injected intraperitoneally (i.p.) in C.B-17-scid/scid (SCID) mice (day 0) .
  • PBL were usually injected into SCID mice within 2-3 hours, and never more than 6 hours, after blood drawing.
  • the range of PBL concentrations tested went from 2.6 X 10 6 up to 50 X 10 6 cells/ml.
  • the preferred PBL cell concentration was determined to be 50 X 10 6 cells/ml.
  • the SCID mice used for reconstitution experiments received the PBL injections at 2-3 months of age after being tested and determined not to be leaky.
  • SCID mice were then injected with 15 x 10 6 PBL. This amount of cells was chosen for the analysis as it was consistently associated with peak serum levels of human IgG higher than 2,000 ⁇ g/ml with a cell aliquot size that permitted large groups of SCID mice to be derived from the same donor.
  • the mean human IgG serum level in hu-PBL-SCID mice increased for the first 2 months to a peak of approximately 3,500 ⁇ g/ml as shown in Table 1 below, a level approximately 1/5 to 1/2 that of a normal human donor (normal human IgG serum level 7,000-17,000 ⁇ g/ml). In the few mice surviving beyond 2 months, human IgG serum levels decreased over the next year to a level approximately 1/10 that of the peak, but were still detectable for as long as two years as shown in Figure 1.
  • SCID mice were populated with 15 x 10 6 PBL, either from 3 donors (4 experiments) wit
  • Manipulations of the recipient or the cells described above affected the timing, but not the magnitude, of human IgG formation.
  • Human IgG among the groups of mice that were either irradiated, injected with cells kept frozen, or controls, reached similar maximal levels (2443, 2547, and 2723 ⁇ g/ml, respectively) as shown in Figure 1.
  • PBL transfer method resulted in only 3 of 14 SCID mice populated with 10-50 x 10 6 PBL from tetanus toxoid immune donors had detectable anti-TT antibody serum levels.
  • PBL from donors who were not exposed to TT for more than one year were evaluated.
  • mice When normalized for the IgG serum level of the respective donors, these three groups of hu-PBL-SCID mice had corrected mean levels of anti-TT antibody of 11.19, 1.15, and 0.26 IU/ml (the respective serum levels of the human donors were 3.40, 0.63, and 0.35 IU/ml).
  • HRE was performed on sera derived from 115 hu-PBL-SCID mice. Only 11 of 62 SCID mice populated with 15 or 50 x 10 6 PBL from EBV + donors had electrophoretically restricted serum immunoglobulin migration pattern (mig) before day 30. A long term analysis involving 127 sera from 53 SCID mice populated with 15 x 10 6 PBL showed 16 of 25 EBV " hu-PBL-SCID mice, and 16 of 28 EBV + hu-PBL-SCID mice developing mig.
  • EBV + hu-PBL-SCID mice had mig prior to day 60, while 14 EBV + hu-PBL-SCID mice had mig at day 43. Seventeen of 32 mig * hu-PBL-SCID mice had multiple mig at HRE, with comparable frequencies in recipients of EBV + and EBV " cells. Mig in EBV " hu-PBL-SCID mice differed from mig in EBV + hu-PBL-SCID mice in their lower intensity, the absence of associated lymphoproliferative disease, and their high proportion of lambda light chain utilization (ten of 18 EBV " versus 2 of 9 EBV + mig analyzed) . E. Survival of hu-PBL-SCID Mice
  • lymphoproliferative diseases in virtually all autopsied SCID recipients of PBL from EBV + donors. These lymphoproliferative diseases were associated with the high mortality of EBV + hu-PBL-SCID mice. The fifty percent mortality of such reconstituted mice was at about 60 days.
  • GvH disease is in part mediated through the invasion of host tissue organs by cells originating from the graft.
  • dot blot analysis was performed using a human-specific ALU repeat family probe.
  • the BLUR2 human ALU repeat family probe
  • the probe was subcloned in the Bam HI restriction site of the pGEM-7Zf(+) vector (Promega Biotec, Madison, WI) using standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ED, Cold Spring Harbor Laboratory Press, New York, (1989)).
  • the probe was labeled with 32 P using the random hexamer priming method as described by Feinberg et al.. Anal. Biochem.. 132:6-13 (1983).
  • radiolabeled 35 S-RNA transcripts were generated after linearization of the plasmid construct with EcoRI or Sad, using T3 or T7 RNA polymerase, respectively.
  • the transcription reaction conditions have been described by Pardoll et al., J. Exp. Med. , 165:1624-1638 (1987).
  • the 32 P or 35 S radiolabeled probes were extracted by phenol/chloroform, purified using a G-50 Sephadex column (Quick Spin column, Boehringer Manheim Biochemicals, Indianapolis, IN)", filtered through a 0.22 um filter, and stored in TE at -80°C until use.
  • in situ hybridizations were performed using a specific human ALU repeat family probe as described in Example 3F.
  • the in situ hybridization procedure was derived from previously described methods. See, Pardoll et al., supra; Jeong et al., J. Immunol.. 140:2436-2441 (1988); and Mueller et al., J. Exp. Med..
  • mice were sacrificed and autopsied when moribund or when scheduled for histologic analysis. Pieces of tissues were fixed in Bouin's, paraffin embedded, and 4 um sections were prepared.
  • Tissue sections were then deposited on precleaned glass slides, deparaffinized, and digested with 1 ⁇ g/ml of proteinase K (Beckmann) for 20 minutes. Following post-fixation in Bouin's for 20 minutes, the slides were acetylated in a 0.1 M triethanolamine, pH 8.0, solution containing 0.25% of acetic anhydride, and then transferred for 30 minutes into a solution containing 0.1 M Glycine. After dehydration in graded ethanol, the slides were air dried. Forty ⁇ l of hybridization solution containing 50% deionized formamide (Sigma, St.
  • the slides were washed 3 times with 3 changes of 2x SSC, 50% formamide solution containing 0.2 M beta-mercaptoethanol (Sigma) at 5 °C. Between these washing cycles, the tissue sections were digested with RNase A (100 ⁇ g/ml, Worthington Biochemical) , and RNase Tl (1 u/ml, BRL) in 2x SSC for 30 minutes at 37°C. The slides were dehydrated in graded concentrations of ethanol, air dried, coated with a nuclear emulsion (Ilford K5.D) , exposed at 4°C for 9 days, and developed and fixed (D-19, Rapid fix, Kodak International Biotechnologies Inc.).
  • RNase A 100 ⁇ g/ml, Worthington Biochemical
  • RNase Tl (1 u/ml, BRL
  • mice were maintained for 1 hour at 37°C, and overnight at 4°C, with either a monoclonal mouse anti-human leukocyte common antigen (CD45, 2Bll,and PD7/26, Dako Corp.), mouse anti-human T cell (UCHL1, Dako Corp.) , mouse anti-human B cell (BI, Coulter Immunology) , or polyclonal rabbit anti-human IgG or anti-human IgM (Dako Corp.), antibodies. Controls included incubation with non-immune mouse and rabbit sera.
  • a monoclonal mouse anti-human leukocyte common antigen CD45, 2Bll,and PD7/26, Dako Corp.
  • mouse anti-human T cell UCHL1, Dako Corp.
  • mouse anti-human B cell BI, Coulter Immunology
  • polyclonal rabbit anti-human IgG or anti-human IgM Dako Corp.
  • the sections were further processed for indirect immunoperoxidase visualization using diaminobenzidine (DAB) as the chromogen accordingly to manufacturer's instructions (Vectastain Elite ABC kit, Vector Laboratories) using as secondary antibodies either a horse anti-mouse, or a goat anti-rabbit IgG conjugated to avidin (Vector Laboratories) .
  • DAB diaminobenzidine
  • Human cell origin of the positive cells with in situ hybridization as described in Example 3G was confirmed by their surface reactivity with antibody directed against human leukocyte common antigen.
  • Human T cells were found in the center portion of the white pulp of the spleen at day 30 or later, and included the majority (>95%) of cells infiltrating tissues described above.
  • Human B cells defined by their reactivity with antibody against human CD20, human IgG, or human IgM, made up ⁇ 5% of the human cells infiltrating most tissues.
  • B cells were of variable proportion of the total " human cell population (up to 40% in 2 hu-PBL-SCID mice) and clustered primarily at the periphery of the white pulp, and in the red pulp.
  • the shape and Ig pattern staining of these B cells suggest that the majority were plasma cells.
  • the human immune system reconstituted in SCID mice using a transfer method that minimizes both the time and manipulations required to transfer PBL was characterized.
  • This procedure described above in Examples 1-3, permitted 27% of SCID mice to acquire significant human IgG serum levels when injected with as few as 2.6 x 10 6 PBL, and 100% developed 2-5 g/1 of human IgG in their sera when injected with 15 x 10 6 PBL.
  • These human IgG levels were previously associated with higher numbers of PBL transferred, or such levels were lower with similar numbers of cells used. See, Mosier et al., Nature, 335:256-259 (1988) and Mosier, J. Clin. Immunol. , 10:185-191 (1990).
  • This method employs relatively few cells to achieve a significant reconstitution of a human immune system, and will be particularly useful in studies of patients with low peripheral lymphocyte counts, such as AIDS or other lymphopenic patients, as well as in studies requiring large numbers of recipient mice.
  • hu-PBL-SCID mice had relatively low serum levels of human IgM compared to IgG, an observation similar to that reported with SCID mice populated with heavily manipulated PBL. Interestingly, hu-PBL-SCID mice with the highest IgG serum levels also had the highest ratio of human IgM relative to IgG, suggesting a distribution of these two isotypes more similar to that of human donors in individuals showing the greatest B cell activity.
  • EBV " hu-PBL-SCID mice developed mig at the same frequency as EBV + hu-PBL-SCID mice, although later. All these mlgs were clearly of human origin because they were immunofixed with anti-human specific Ig antibodies and observed in SCID mice, the large majority of which were not leaky at the time of analysis.
  • SCID mice populated with EBV ' donor PBL displayed a high proportion (>50%) of clinical signs compatible with GvH disease, and had a high mortality (>40%) although not suffering from lymphoproliferative disease.
  • EBV ' hu-PBL-SCID mice were sick, and some started to die.
  • the observations presented herein definitively rule out the possibility of an underlying lymphoproliferative disease secondary to EBV presence as being responsible for the sickness of the reconstituted animals.
  • the differences in GvH expression may be explained by quantitative and/or qualitative variations in the human cell population engrafted in hu-PBL-SCID mice.
  • the GvH disease probably influenced the mortality rate in EBV + hu-PBL-SCID mice, and explained the absence of lymphoproliferative disease recorded at autopsy of some sick EBV + hu-PBL-SCID mice.
  • HBc hepatitis B core
  • HBc antigen was prepared as follows: E. coli bacteria (JM109 strain) were transformed with the pFS14 expression plasmid, induced with IPTG and lysed according to Stahl et al. , Proc. Natl. Acad. Sci. USA. 79:1606-1610 (1982). Following DNAsel digestion, the protein was further purified by selective centrifugation, gel filtration (BIO-Gel-A-50m, Biorad) , and finally hydroxylapatite chromatography (Bio-Gel-HT, Biorad) .
  • Ly phophereses as described in Example 1 were performed at Scripps Clinic and Research Foundation from donors giving their informed consent.
  • the cells were washed in Earles medium (Flow Laboratories, Inc.) to eliminate human plasma, highly toxic when injected i.p. into SCID mice, but no attempt was made to eliminate the erythrocytes and platelet contaminants.
  • mice were subsequently injected subcutaneously (s.c.) with 10 ⁇ g of HBc in CFA at the base of the tail on day 2, and with the same dose of antigen s.c. in saline on day 22 or 23.
  • Control groups were processed in parallel and consisted of SCID mice populated with 50 x 10 6 PBL incubated without antigen, injected on day 2 with CFA, and on day 22/23 with saline only. Additional experimental groups were evaluated as described below in the results.
  • Hu-PBL-SCID mice were bled sequentially, and human IgG serum levels determined as described by Duchosal et al., J. Exp. Med.. 172:985-988 (1990).
  • IgG anti-HBc titers were determined by indirect ELISA. Microtiter flat-bottom plates (Costar) were coated with 0.5 ug/ml of purified recombinant HBc in PBS overnight at 4°C. Non-specific binding was prevented by maintenance with PBS-5% non-fat dairy milk. Serial dilutions of collected sera starting at 1/200 from hu-PBL-SCID mice, the respective donor, a donor known for high IgG anti-HBc activity (standard) , as well as from humans with no history of hepatitis, were further maintained in duplicate.
  • mice Decreased T-B cell cooperation for this antigen in these mice, and production of human anti-mouse antibodies secondary to GvH, could explain the decrease in absolute and even more in normalized IgG anti-HBc titers with time, respectively.
  • these mice showed no increase in IgG anti-HBc titers after booster injection at three weeks, and the magnitude of their anti-HBc antibody response was lower.
  • the specificity of the anti-HBc antibody response was indicated by the fact that the mean antibody levels against another protein antigen (TT) of non-immunized and HBc immunized hu-PBL-SCID mice groups were similar.
  • TT protein antigen
  • results are summarized as follows: a) specific antibody production in the absence of antigenic exposure of cells or recipients is observed only when the donor has intermediate or higher levels of IgG anti-HBc; b) three of 12 HBc-immunized hu-PBL-SCID mice derived from a donor with no specific serum titer display low human anti-HBc antibody titers peaking at day 15 limited to the IgM isotype; c) the titer of specific IgG in the donor is correlated with the proportion of responder mice in the group rather than with the level of the immune response in an animal. The level of this response can be of far greater magnitude that the level of the donor; and d) the IgG anti-HBc response was specific.
  • HBc-Pre-Sl and Pre-S2 Antigens Three connected surface glycoproteins, called large, middle, and major proteins, are present at the surface of the HBV. These proteins are produced by three alternate translation initiation sites defining the pre-Sl, pre-S2, and S regions, respectively (Tiollais et al., Nature, 317:489-495, 1985) . The proteins derived from these regions are far less immunogenic than the HBc antigen (Milich et al., Science. 234:1938-1401, 1986).
  • the plasmid HBc-pre-Sl is composed of the full-length sequence of the gene for the HBc protein with an upstream sequence corresponding to the pre-Sl 12-47 amino-acid region [Tiollais et al., supra: Valenzeula et al.. Nature, 280:815-819 (1979); Galiber et al., Nature, 281:646-650 (1979); and Pasek et al., Nature, 282:575-579 (1979)] inserted in the pKK-223 expression vector (Pharmacia LKB) .
  • Recombinant HBc-pre-Sl protein was purified as described by Milich et al..
  • the pre-S2 peptide is a mixture of amino-acid sequences 148-174 (T cell epitope) linked to 131-143 (B cell epitopes) of the adw and (ayw, adr) subtypes of the pre-S2 region.
  • SCID mice were populated with PBL as described in Examples 2 and 3, immunized, and the control group processed as described above for Hbc. The following modifications were made to the immunization protocol: SCID mice were populated with PBL in vitro incubated either with 3 ⁇ g/ml of HBc-pre-Sl antigen or 20 ⁇ g/ml of pre-S2 peptide antigen, and injected at day 2 and 22 with 20 ⁇ g of HBC-pre-Sl antigen, or 50 ⁇ g of pre-S2 peptide antigen, respectively.
  • IgG anti-pre-Sl, and IgG anti-pre-S2 titers were determined by ELISA, as described for HBc above, with the following modification: microtiter plates were coated overnight at 37°C with 10 ⁇ g/ml of a mixture of peptides corresponding to the adw2, and ayw subtypes of either the HBV amino-acid sequence 12-47 (pre-Sl) , or 120-145 (pre-S2) , and the starting serum dilutions were 1/50. The specificity of the assay was assessed as described above for HBc.
  • the immunogenicity of the recombinant HBc-pre-Sl protein antigen was established by the fact that one hundred percent of hu-PBL-SCID mice derived from donor #3, and immunized with this antigen mounted a brisk IgG antibody response against HBc. These mice immunized with HBc-pre-Sl had significantly higher IgG anti-pre-Sl titers than controls, with peak median normalized IgG anti-pre-Sl titer at day 15 corresponding to a 4-fold increase relative to the donor, with one individual having an 11-fold increase. No correlation could be observed between the level of IgG anti-HBc, and IgG anti-pre-Sl response within an individual.
  • mice were immunized with a peptide containing 2 overlapping B cell epitopes, and a T cell epitope of the pre-S2 region (pre-S2 peptide) .
  • pre-S2 peptide a T cell epitope of the pre-S2 region
  • Non-immunized hu-PBL-SCID mice derived from donor #3 (who had intermediate levels of IgG anti-pre-S2 titers) have generally such titers in their sera again with individual variability.
  • mice immunized with the pre-S2 peptide had median normalized IgG antibody serum levels between day 15 and day 29 more than twice, with five of 12 individuals more than 3 times, those of the controls.
  • Tetanus toxoid (TT) , a thymus-dependent antigen, was utilized in the model because a) the kinetics of human antibody responses to a challenge with this antigen are well characterized and provide a reference for consideration of the antibody response in the hu-PBL-SCID mouse model; b) the time between the last contact with this antigen (immunization) and the PBL transfer to the SCID mouse can be accurately determined.
  • mice were handled as described above for HBc with the following modification: in the immunized group, PBL were maintained in vitro with 15 ⁇ g/ml of TT antigen (Wellcome) , and 50 ⁇ g of TT were injected into mice at day 2, and 23. IgG anti-TT levels were determined as described below with the exception that an Fc-specific mouse-anti-human anti-Ig antibody was used to detect anti-TT antibodies that immunoreacted with the TT-coated wells. The threshold of the assay was 0.003 IU/ml.
  • TT immunized hu-PBL-SCID mice derived from donors #4 ( Figure 2B) and #5 ( Figure 2D) had median normalized serum IgG anti-TT levels 8-60 and 3-12 times higher than their corresponding time-matched non-immunized controls, respectively.
  • Hu-PBL-SCID mice populated with PBL from donor #4 had median normalized IgG anti-TT levels approximately 9 times higher, with some individuals having up to a 40- to 60- fold increase, over the level recorded in the donor which level at day 0 was 0.65 and at day 37 at 7.60 [IU/ml].
  • I mulon were coated with TT (Wellcome) in carbonate buffer, and non-specific binding prevented by incubation with PBS-0.1% BSA.
  • Donor or hu-PBL-SCID mouse serum dilutions were applied in duplicate, and maintained for 1 hour at 37°C and 12 hours at 4°C.
  • monoclonal mouse anti-human IgGl HP6001 , IgG2 (HP6014) , IgG3 (HP6050) , or IgG4 (HP6025) were added at low dilution to assure saturation, and maintained for 1 hour at 37°C, and 12 hours at 4°C.
  • mice 50 X 10 6 PBL, antigen-exposed and saline-exposed, were injected into two groups of SCID mice as described in Example 2.
  • TT-exposed PBL received a booster of TT (50 ⁇ g/ml) in CFA followed by a second booster at day 23 of TT (50 ⁇ g/ml) in saline.
  • the control group of ten mice received similar injections that lacked the TT antigen.
  • Serum samples were collected from the mice at day 3, 15, 23, 30, 37 and 49. The amount of human Ig as well as specific anti-TT antibody were measured as described above. The results of these experiments are shown below in Table 2 and Table 3.
  • Table 2 contains the levels of non-specific human Ig and specific anti-TT antibody for mice that received PBL exposed to TT in vitro and boosters of TT.
  • Table 3 contains the levels of the control animals. The determined levels of both non-specific human Ig in milligrams/liter (mg/L) and specific anti-TT antibody in IU/ml are presented in a column format based on the days the serum was collected. The ten mice injected in each group are numerically designated.
  • HGG mg/L (Human gamma globulin, i.e., non-specific Ig)
  • ⁇ TT IU/ml
  • DB day mouse dead by the day indicated.
  • N.A. sample not available because the mouse expired.
  • mice in Table 2 where PBLs. were exposed to TT in vitro exhibited a marked rise in anti-TT antibody levels.
  • the levels in this group of mice continued to rise throughout the time course evaluated and in some mice levels of anti-TT antibody reach 40.4 IU/ml.
  • the non-specific Ig levels were significantly higher and surpassing 6 mg/L in some of the mice receiving TT-exposed PBL and TT boosters compared to those that received saline-exposed PBL and saline boosters.
  • mice reconstituted with PBL that had been exposed in vitro to TT and subsequently boosted with TT compared to no response in mice reconstituted with PBL that were exposed in vitro to saline and subsequently boosted with saline.
  • mice injected with TT simultaneously with PBL that had not been exposed to TT in vitro were compared against control mice that did not receive any TT antigen at all, against mice that received PBL exposed to TT in vitro for four fours with 15 ⁇ g/ml TT and subsequent injections of TT (50 ⁇ g/ml) subcutaneously at days 0 and 15, and against mice that received PBL exposed to TT in vitro for four hours with 15 ⁇ g/ml TT and subsequent injections of TT (50 ⁇ g/ml) i.p. at days 0 and 15.
  • TT was admixed with CFA while for the day 15 injections, TT was admixed in saline.
  • TT was admixed in saline for both injections.
  • hu-PBL-SCID mice can be immunized with protein and peptide antigens to mount a low primary (IgM) or a large secondary (IgG) antibody response.
  • IgM primary
  • IgG large secondary
  • the assessment of the functionality of the transferred human immune system in SCID mice has evident consequences in the study of the model.
  • PBL derived from patients with localized or generalized autoimmune diseases could be immunized with cross-reactive antigens supposed to trigger the proliferation of autotoxic clones, and assessment of their pathogenicity studied in vivo.
  • the importance of cells and/or cell products on the evolution of an (auto)immune response could be assessed in vivo.
  • the competitive ELISA assay, and nucleic acid sequencing were performed as described (Persson et al., supra). This library was screened using labelled TT. Two positive clones were plaque purified and phagemids excised and used to transform E.coli. These transformants produced Fabs with apparent binding affinities of 10 8 -10 9 M "1 as measured in a competition ELISA. The clones were sequenced at the nucleic-acid level.

Abstract

Procédés de production d'anticorps humains dans un animal non humain immunodéficitaire, tel qu'une souris immunodéficitaire. On décrit également des procédés de production d'un animal non humain présentant un système immunitaire humain reconstitué utile dans la production d'anticorps humains.
PCT/US1992/008005 1991-09-19 1992-09-18 Procede de production d'anticorps humains dans un animal non humain, et animaux utilises a cet effet WO1993005796A1 (fr)

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0539970A2 (fr) * 1991-10-30 1993-05-05 Idemitsu Kosan Company Limited Procédés de production des lymphocytes humains et des anticorps humains, et anticorps humains ainsi produits
WO1994005779A1 (fr) * 1992-09-09 1994-03-17 O'brien, Caroline, Jane Procede de verification de reponse immune individuelle a un anticorps a l'aide d'une souris a scid
US5663481A (en) * 1993-08-06 1997-09-02 Mount Sinai Hospital Corporation Animal model of the human immune system
US5958765A (en) * 1995-06-07 1999-09-28 Idec Pharmaceuticals Corporation Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
WO1999060846A1 (fr) * 1998-05-26 1999-12-02 Innogenetics N.V. Procede permettant d'augmenter des lymphocytes b de primates chez la souris immunodeprimee, et de produire de grands nombres de lymphocytes b specifiques de l'antigene pour la production d'anticorps monoclonaux de primates
US6063983A (en) * 1995-09-05 2000-05-16 The General Hospital Corporation Monoclonal lymphocytes and methods of use
US7704505B2 (en) 1994-08-15 2010-04-27 Medimmune, Llc Human-murine chimeric antibodies against respiratory syncytial virus
WO2010075475A1 (fr) 2008-12-23 2010-07-01 Abbott Laboratories Anticorps tyrosine kinase du type fms soluble (sflt-1) et composition associée, trousse, procédés d'utilisation, matériaux et procédé de fabrication
WO2011116023A1 (fr) 2010-03-18 2011-09-22 Abbott Laboratories Procédé permettant d'analyser la lipocaline urinaire associée à la gélatinase des polynucléaires neutrophiles (ungal) dans le pronostic de la fonction de transplantation rénale cadavérique chez un patient, y compris un patient chez lequel a été diagnostiquée une reprise retardée de la fonction du greffon (dgf), procédé permettant d'analyser la ungal dans l'évaluation du risque de dgf chez un patient chez lequel a été diagnostiquée une reprise avancée de la fonction du greffon (egf), et kits afférents
WO2011163558A1 (fr) 2010-06-25 2011-12-29 Abbott Laboratories Matériaux et procédés pour le dosage d'anticorps anti-virus de l'hépatite c (hvc)
WO2012064674A1 (fr) 2010-11-09 2012-05-18 Abbott Laboratories Matériels et méthodes pour un immunoessai permettant d'analyser les ptérines
EP2508608A1 (fr) 2003-06-09 2012-10-10 Alnylam Pharmaceuticals Inc. Procédé de traitement d'une maladie neurodégénérative
WO2013102149A1 (fr) 2011-12-31 2013-07-04 Abbott Laboratories Protéine de liaison de la vitamine d humaine tronquée et mutation et fusion de celle-ci, et matériaux associés et procédés d'utilisation
US8541646B2 (en) 2009-10-06 2013-09-24 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
WO2014078280A1 (fr) * 2012-11-13 2014-05-22 Genentech, Inc. Enrichissement de plasmoblastes spécifiques d'antigènes
US8986686B2 (en) 2002-06-14 2015-03-24 Medimmune, Llc Stabilized liquid anti-RSV antibody formulations
US9283274B2 (en) 2009-10-06 2016-03-15 Medimmune Limited RSV specific binding molecule
US9321831B2 (en) 2007-06-01 2016-04-26 Medimmune Limited RSV-specific binding molecules and means for producing them
US9655352B2 (en) 2011-02-15 2017-05-23 Regeneron Pharmaceuticals, Inc. Humanized M-CSF mice
US9820476B2 (en) 2012-09-07 2017-11-21 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US9901082B2 (en) 2012-11-05 2018-02-27 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US10123518B2 (en) 2015-04-13 2018-11-13 Regeneron Pharmaceuticals, Inc Genetically modified non-human animals and methods of use thereof
US10463028B2 (en) 2014-05-19 2019-11-05 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals expressing human EPO

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ENGLEMAN et al., eds, "Human Hybridomas and Monoclonal Antibodies", Published 1985, by PLENUM PRESS (NEW YORK), pages 149-161 and 162. *
J. EXP. MED., Volume 170, issued December 1989, KRAMS et al., "Generation of Biliary Lesions After Transfer of Human Lymphocytes Into Severe Combined Immunodeficient (SCID) Mice", pages 1919-1930. *
NATURE, Volume 335, issued 15 September 1988, MOSIER et al., "Transfer of a Functional Human Immune System to Mice with Severe Combined Immunodeficiency", pages 256-259. *
PROC. NATL. ACAD. SCI. USA, Volume 87, issued October 1990, MULLINAX et al., "Identification of Human Antibody Fragment Clones Specific for Tetanus Toxoid in a Bacteriophage (Lambda) Immunoexpression Library", pages 8095-8099. *
PROGRESS IN IMMUNOLOGY, Volume 7, issued 1989, MOSIER et al., "Elements of the Human Immune System: Studies of Mature Lymphoid Cells Following Xenotransplantation to SCID Mice", pages 1264-1271. *
SCIENCE, Volume 246, issued 08 December 1989, HUSE et al., "Generation of a Large Combinatorial Library of the Immunoglobulin Repertovire in Phage Lambda", pages 1275-1281. *

Cited By (49)

* Cited by examiner, † Cited by third party
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US5681729A (en) * 1991-10-30 1997-10-28 Idemitsu Kosan Company Limited Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby
US5869268A (en) * 1991-10-30 1999-02-09 Idemitsu Kosan Company Limited Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby
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US5958765A (en) * 1995-06-07 1999-09-28 Idec Pharmaceuticals Corporation Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
US6413771B1 (en) 1995-06-07 2002-07-02 Idec Pharmaceuticals Corporation Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
US6537809B2 (en) 1995-06-07 2003-03-25 Xenerex Biosciences Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
US6063983A (en) * 1995-09-05 2000-05-16 The General Hospital Corporation Monoclonal lymphocytes and methods of use
AU751647B2 (en) * 1998-05-26 2002-08-22 Innogenetics N.V. Method for expanding primate B cells selectively in immunocompromised mice and producing large numbers of antigen-specific B lymphocytes for the production of primate monoclonal antibodies
WO1999060846A1 (fr) * 1998-05-26 1999-12-02 Innogenetics N.V. Procede permettant d'augmenter des lymphocytes b de primates chez la souris immunodeprimee, et de produire de grands nombres de lymphocytes b specifiques de l'antigene pour la production d'anticorps monoclonaux de primates
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US10059757B2 (en) 2007-06-01 2018-08-28 Medimmune Limited RSV-specific binding molecules and means for producing them
US9321831B2 (en) 2007-06-01 2016-04-26 Medimmune Limited RSV-specific binding molecules and means for producing them
WO2010075475A1 (fr) 2008-12-23 2010-07-01 Abbott Laboratories Anticorps tyrosine kinase du type fms soluble (sflt-1) et composition associée, trousse, procédés d'utilisation, matériaux et procédé de fabrication
US9283274B2 (en) 2009-10-06 2016-03-15 Medimmune Limited RSV specific binding molecule
US10035843B2 (en) 2009-10-06 2018-07-31 Medimmune Limited RSV-specific binding molecule
US11051499B2 (en) 2009-10-06 2021-07-06 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
US8541646B2 (en) 2009-10-06 2013-09-24 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
US8692052B2 (en) 2009-10-06 2014-04-08 Regeneron Pharmaceuticals Genetically modified mice and engraftment
US9301509B2 (en) 2009-10-06 2016-04-05 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
US9554563B2 (en) 2009-10-06 2017-01-31 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
US10723786B2 (en) 2009-10-06 2020-07-28 Medimmune, Limited RSV-specific binding molecule
US10278374B2 (en) 2009-10-06 2019-05-07 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
WO2011116023A1 (fr) 2010-03-18 2011-09-22 Abbott Laboratories Procédé permettant d'analyser la lipocaline urinaire associée à la gélatinase des polynucléaires neutrophiles (ungal) dans le pronostic de la fonction de transplantation rénale cadavérique chez un patient, y compris un patient chez lequel a été diagnostiquée une reprise retardée de la fonction du greffon (dgf), procédé permettant d'analyser la ungal dans l'évaluation du risque de dgf chez un patient chez lequel a été diagnostiquée une reprise avancée de la fonction du greffon (egf), et kits afférents
US9551714B2 (en) 2010-06-25 2017-01-24 Abbott Laboratories Materials and methods for assay of anti-hepatitis C virus (HCV) antibodies
WO2011163558A1 (fr) 2010-06-25 2011-12-29 Abbott Laboratories Matériaux et procédés pour le dosage d'anticorps anti-virus de l'hépatite c (hvc)
WO2012064674A1 (fr) 2010-11-09 2012-05-18 Abbott Laboratories Matériels et méthodes pour un immunoessai permettant d'analyser les ptérines
US10785966B2 (en) 2011-02-15 2020-09-29 Regeneron Pharmaceuticals, Inc. Humanized M-CSF mice
US9655352B2 (en) 2011-02-15 2017-05-23 Regeneron Pharmaceuticals, Inc. Humanized M-CSF mice
WO2013102149A1 (fr) 2011-12-31 2013-07-04 Abbott Laboratories Protéine de liaison de la vitamine d humaine tronquée et mutation et fusion de celle-ci, et matériaux associés et procédés d'utilisation
US9820476B2 (en) 2012-09-07 2017-11-21 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US10433527B2 (en) 2012-09-07 2019-10-08 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US11026408B2 (en) 2012-09-07 2021-06-08 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US9986724B2 (en) 2012-11-05 2018-06-05 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US9901082B2 (en) 2012-11-05 2018-02-27 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US10785968B2 (en) 2012-11-05 2020-09-29 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US11778995B2 (en) 2012-11-05 2023-10-10 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
WO2014078280A1 (fr) * 2012-11-13 2014-05-22 Genentech, Inc. Enrichissement de plasmoblastes spécifiques d'antigènes
US10463028B2 (en) 2014-05-19 2019-11-05 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals expressing human EPO
US11766032B2 (en) 2014-05-19 2023-09-26 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals expressing human EPO
US10561126B2 (en) 2015-04-13 2020-02-18 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof
US10123518B2 (en) 2015-04-13 2018-11-13 Regeneron Pharmaceuticals, Inc Genetically modified non-human animals and methods of use thereof
US11576356B2 (en) 2015-04-13 2023-02-14 Regeneron Pharmaceuticals, Inc. Genetically modified non-human animals and methods of use thereof

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