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  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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  • C07K16/2854—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72 against selectins, e.g. CD62
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Definitions

• the invention relates to the field of immunology, and in particular to the production of antibodies. More specifically, it concerns producing such antibodies by a process which includes the step of immunizing a transgenic animal with an antigen to which antibodies are desired.
• the transgenic animal has been modified so as to produce human, as opposed to endogenous, antibodies.
• mice specifically, are described which, when administered immunogens, produce antibodies with human variable regions, including fully human antibodies, rather than murine antibodies that are
• immunoglobulins bearing the characteristic structures of antibodies produced by nonhuman hosts. Such antibodies tend to be immunogenic when used in humans.
• the invention is directed to methods to produce human antibodies by a process wherein at least one step of the process includes immunizing a transgenic nonhuman animal with the desired antigen.
• the modified animal fails to produce endogenous antibodies, but instead produces B-cells which secrete fully human immunoglobulins.
• the antibodies produced can be obtained from the animal directly or from immortalized B-cells derived from the animal. Alternatively, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the
• antibodies such as, for example, single chain F v molecules.
• the invention is directed to a method to produce a fully human immunoglobulin to a specific antigen or to produce an analog of said immunoglobulin by a process which comprises immunizing a nonhuman animal with the antigen under conditions that stimulate an immune response.
• the nonhuman animal is characterized by being substantially incapable of producing endogenous heavy or light
• the animal produces B cells which secrete immunoglobulins that are fully human and specific for the antigen.
• the human immunoglobulin of desired specificity can be directly recovered from the animal, for example, from the serum, or primary B cells can be obtained from the animal and immortalized.
• immortalized B cells can be used directly as the source of human antibodies or, alternatively, the genes encoding the antibodies can be prepared from the immortalized B cells or from primary B cells of the blood or lymphoid tissue (spleen, tonsils, lymph nodes, bone marrow) of the immunized animal and expressed in recombinant hosts, with or without
• immunoglobulins produced by the immunized animal can be used to generate a library of immunoglobulins to permit screening for those variable regions which provide the desired
• Clones from the library which have the desired characteristics can then be used as a source of nucleotide sequences encoding the desired variable regions for further manipulation to generate antibodies or analogs with these characteristics using standard recombinant techniques.
• the invention relates to an immortalized nonhuman B cell line derived from the above described animal.
• the invention is directed to a recombinant host cell which is modified to contain the gene encoding either the human immunoglobulin with the desired specificity, or an analog thereof which exhibits the same specificity.
• the invention is directed to antibodies or antibody analogs prepared by the above- described methods and to recombinant materials for their production.
• the invention is directed to antibodies which are immunospecific with respect to particular antigens set forth herein and to analogs which are similarly immunospecific, as well as to the recombinant materials useful to production of these antibodies.
• Figure 1 is a schematic of the construction of the yH1C human heavy chain YAC.
• Figure 2 is a schematic of the construction of the yK2 human kappa light chain YAC.
• Figure 3 shows the serum titers of anti-IL-6 antibodies from a XenoMouseTM immunized with human L-6 and which antibodies contain human k light chains and/or human ⁇ heavy chains.
• Figure 4 show the serum titers of anti-TNF ⁇ antibodies from a XenoMouseTM immunized with human TNF- ⁇ and which antibodies contain human k light chains and/or human ⁇ heavy chains.
• Figure 5 shows serum titers of anti-CD4 antibodies from a XenoMouseTM immunized with human CD4 and which
• antibodies contain human k light chains and/or human ⁇ heavy chains.
• Figure 6 shows the serum titers of a XenoMouseTM immunized with 300.19 cells expressing L-selectin at their surface. In the ELISA assay used, these antibodies are detectable if they carry human ⁇ constant region heavy chains.
• Figure 7 shows the serum titers of a XenoMouseTM immunized with 300.19 cells expressing L-selectin at their surface. In the ELISA assay used, these antibodies are detectable only if they carry human k light chains.
• Figure 8 shows a FACS Analysis of human neutrophils incubated with serum from a XenoMouseTM immunized with human L-selectin and labeled with an antibody immunoreactive with human light chain k region.
• Figure 9 shows a diagram of a plasmid used to transfect mammalian cells to effect the production of the human protein gp39.
• Figure 10 represents the serum titration curve of mice immunized with CHO cells expressing human gp39.
• the antibodies detected in this ELISA must be immunoreactive with gp39 and contain human heavy chain ⁇ constant regions of human k light chains.
• FIG 11 is a titration curve with respect to monoclonal antibodies secreted by the hybridoma clone D5.1.
• This clone is obtained from a XenoMouseTM immunized with tetanus toxin C (TTC) and contains human k light chain and human ⁇ constant region in the heavy chain.
• Figure 12 DNA sequence of the heavy chain of anti tetanus toxin monoclonal antibody D5.1.4 (a subclone of D5.1). Mutations form germline are boxed.
• Figure 13 DNA sequence of the kappa light chain of anti-tetanus toxin monoclonal antibody D5.1.4. Mutations form germline are boxed.
• Figure 14 shows the serum titers of anti-IL-8 antibodies of XenoMouseTM immunized with human IL-8 and which antibodies contain human k light chains and/or human ⁇ heavy chains.
• Figure 15 Inhibition of IL-8 binding to human neutrophils by monoclonal anti-human-IL-8 antibodies.
• Figure 16 DNA sequences of the heavy chain and kappa light chain of the anti-IL-8 antibodies D1.1 (16A- B), K2.2 (16C-D), K4.2 (16E-F), and K4.3 (16G-H).
• the methods of the invention include administering an antigen for which human forms of
• immunospecific reagents are desired to a transgenic nonhuman animal which has been modified genetically so as to be capable of producing human, but not endogenous, antibodies.
• the animal has been modified to disable the endogenous heavy and/or kappa light chain loci in its genome, so that these endogenous loci are incapable of the
• the animal will have been provided, stably, in its genome, at least one human heavy chain locus and at least one human light chain locus so that in response to an administered antigen, the human loci can rearrange to provide genes encoding human variable regions immunospecific for the antigen.
• YACs for the present invention can be found in, for example, Green et al . Nature Genetics 7:13-21 (1994).
• the human heavy chain YAC, yH1C (1020 kb), and human light chain YAC, yK2 (880 kb) are used.
• yH1C is comprised of 870 kb of the human variable region, the entire D and J H region, human ⁇ , ⁇ , and ⁇ 2 constant regions and the mouse 3' enhancer.
• yK2 is comprised of 650 kb of the human kappa chain proximal variable region (VK), the entire JK region, and CK with its flanking sequences that contain the Kappa deleting element (Kde). Both YACs also contain a human HPRT selectable marker on their YAC vector arm.
• yH1C and yK2 were accomplished by methods well known in the art.
• YAC clones bearing segments of the human immunoglobulin loci were identified by screening a YAC library (Calbertsen et al, PNAS 87:4256 (1990))
• yK2 was constructed from the clones A80-C7, A210- F10 and A203-C6 from the Olson library, disclosed in, for example, Burke et al., Science 236:806-812 (1987), Brownstein et al., Science 244:1348-1351 (1989), and Burke et al.,
• the first step is administration of the antigen.
• Techniques for such administration are conventional and involve suitable
• formulations which contain adjuvants and/or to administer multiple injections and/or to vary the route of the immunization, and the like are standard and optimization of them will depend on the characteristics of the particular antigen for which immunospecific reagents are desired.
• immunospecific reagents includes immunoglobulins and their analogs.
• analogs has a specific meaning in this context. It refers to moieties that contain the fully human portions of the immunoglobulin which account for its immunospecificity. In particular, complementarity determining regions (CDRs) are required, along with sufficient portions of the framework (Frs) to result in the appropriate three dimensional
• Typical immunospecific analogs of antibodies include F(ab") 2 , Fab', and Fab regions. Modified forms of the variable regions to obtain, for example, single chain F v analogs with the appropriate immunospecificity are known. A review of such F v construction is found, for example, in
• immunospecificities is also possible by coupling the variable regions from one antibody to those of second antibody.
• the moieties including the fully human variable regions produced by the methods of the invention include single-chain fusion proteins, molecules coupled by covalent methods other than those involving peptide linkages, and aggregated molecules.
• Examples of analogs which include variable regions coupled to additional molecules covalently or noncovalently include those in the following nonlimiting illustrative list. Traunecker, A. et al . Int. J. Cancer Supp (1992) Supp 7:51-52 describe the bispecific reagent janusin in which the F v region directed to CD3 is coupled to soluble CD4 or to other ligands such as OVCA and IL-7.
• the fully human variable regions produced by the method of the invention can be constructed into F v molecules and coupled to alternative ligands such as those illustrated in the cited article. Higgins, P.J. et al J. Infect Disease (1992)
• heteroconjugate antibody composed of OKT3 cross-linked to an antibody directed to a specific sequence in the V3 region of GP120.
• heteroconjugate antibodies can also be constructed using at least the human variable regions contained in the immunoglobulins produced by the invention methods. Additional examples of bispecific antibodies include those described by Fanger, M.W. et al. Cancer Treat Res (1993) 68:181-194 and by Fanger, M.W. et al. Crit Rev Immunol (1992) 12:101-124.
• Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or
• immunotoxins containing protein toxin portions can be
• immunotoxins produced as fusion proteins.
• the analogs of the present invention can be used in a corresponding way to obtain such immunotoxins.
• Illustrative of such immunotoxins are those described by Byers, B.S. et al. Seminars Cell Biol (1991)
• immunoglobulins and analogs of the invention will have agonist activity with respect to antigens for which they are immunospecific in the cases wherein the antigens perform signal transducing functions.
• a subset of antibodies or analogs prepared according to the methods of the invention which are immunospecific for, for example, a cell surface receptor will be capable of eliciting a response from cells bearing this receptor corresponding to that elicited by the native ligand.
• antibodies or analogs which are immunospecific for substances mimicking transition states of chemical reactions will have catalytic activity.
• a subset of the antibodies and analogs of the invention will function as catalytic antibodies.
• the genes encoding the immunoglobulins produced by the transgenic animals of the invention can be retrieved and the nucleotide sequences encoding the fully human variable region can be manipulated according to known techniques to provide a variety of analogs such as those described above.
• the immunoglobulins themselves containing the human variable regions can be modified using standard coupling techniques to provide conjugates retaining immunospecific regions.
• immunoglobulin “analogs” refers to the moieties which contain those portions of the antibodies of the invention which retain their human characteristics and their immunospecificity. These will retain sufficient human variable regions to provide the desired specificity.
• all of the methods of the invention include administering the appropriate antigen to the transgenic animal.
• the recovery or production of the antibodies themselves can be achieved in various ways.
• polyclonal antibodies produced by the animal and secreted into the bloodstream can be recovered using known techniques.
• Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, preferably including affinity chromatography with Protein A, anti- immunoglobulin, or the antigen itself. In any case, in order to monitor the success of immunization, the antibody levels with respect to the antigen in serum will be monitored using standard techniques such as ELISA, RIA and the like.
• antiserum with suitable reagents so as to generate Fab', Fab, or F(ab") 2 portions results in compositions retaining fully human characteristics.
• Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portions of immunoglobulins to detecting reagents such as radioisotopes.
• immunoglobulins and analogs with desired characteristics can be generated from immortalized B cells derived from the transgenic animals used in the method of the invention or from the rearranged genes provided by these animals in response to immunization.
• the B cells can be obtained, typically from the spleen, but also, if desired, from the peripheral blood lymphocytes or lymph nodes and immortalized using any of a variety of techniques, most commonly using the fusion methods described by Kohler and Milstein Nature 245:495 (1975)
• the resulting hybridomas (or otherwise immortalized B cells) can then be cultured as single colonies and screened for secretion of antibodies of the desired specificity.
• the screen can also include a confirmation of the fully human character of the antibody. For example, as described in the examples below, a sandwich ELISA wherein the monoclonal in the
• hybridoma supernatant is bound both to antigen and to an antihuman constant region can be employed.
• the desired antibodies can be recovered, again using conventional techniques. They can be prepared in quantity by culturing the immortalized B cells using conventional methods, either in vitro or in vivo to produce ascites fluid. Purification of the resulting monoclonal antibody preparations is less burdensome that in the case of serum since each immortalized colony will secrete only a single type of antibody. In any event, standard purification techniques to isolate the antibody from other proteins in the culture medium can be employed.
• the immortalized cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation.
• Rearranged antibody genes can be reverse transcribed from appropriate mRNAs to produce cDNA. If desired, the heavy chain constant region can be exchanged for that of a
• variable regions can be linked to encode single chain F v regions.
• F v regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed. Once the genetic material is available, design of analogs as described above which retain both their ability to bind the desired target, and their human characteristics, is straightforward.
• the coding sequences including those that encode, at a minimum, the variable regions of the human heavy and light chain, can be inserted into expression systems contained on vectors which can be transfected into standard recombinant host cells.
• host cells As described below, a variety of such host cells may be used; for efficient processing, however, mammalian cells are preferred. Typical mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells.
• the production of the antibody or analog is then undertaken by culturing the modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences.
• the antibodies are then recovered from the culture.
• expression systems are preferably designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also
• the primary B cells can be used directly as a source of DNA.
• the mixture of cDNAs obtained from B cells e.g., derived from spleens, is used to prepare an expression library, for example, a phage display library transfected into E . coli .
• the resulting cells are tested for immunoreactivity to the desired antigen.
• Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths, A.D., et al., EMBO J (1994) 13:3245-3260; by Nissim, A., et al . ibid, 692-698, and by Griffiths, A.D., et al . , ibid, 12:725-734.
• clones from the library are identified which produce binding affinities of a desired magnitude for the antigen, and the DNA encoding the product responsible for such binding is recovered and manipulated for standard recombinant
• Phage display libraries may also be constructed using previously manipulated nucleotide sequences and
• the cDNAs encoding heavy and light chain are independently supplied or are linked to form F v analogs for production in the phage library.
• the phage library is then screened for the antibodies with highest affinity for the antigen and the genetic material recovered from the appropriate clone.
• XenoMouseTM offers a significant advantage over previous applications of phage display.
• a combinatorial antibody library is prepared either from human bone marrow or from peripheral blood lymphocytes as described by Burton, D.R., et al., Proc. Natl. Acad. Sci. USA (1991) 88:10134- 10137.
• Using this approach it has been possible to isolate high affinity antibodies to human pathogens from infected individuals, i.e. from individuals who have been "immunized” as described in Burton, D.R., et al., Proc. Natl. Acad. Sci.
• the modified or unmodified rearranged loci are manipulated using standard recombinant techniques by constructing expression systems operable in a desired host cell, such as, typically, a Chinese hamster ovary cell, and the desired immunoglobulin or analog is produced using standard recombinant expression techniques, and recovered and purified using conventional methods.
• a desired host cell such as, typically, a Chinese hamster ovary cell
• the desired immunoglobulin or analog is produced using standard recombinant expression techniques, and recovered and purified using conventional methods.
• compositions for use in analysis, diagnosis, research, and therapy are compositions for use in analysis, diagnosis, research, and therapy.
• the particular use will, of course, depend on the immunoglobulin or analog prepared.
• the particular use will, of course, depend on the immunoglobulin or analog prepared. In general, the
• compositions of the invention will have utilities similar to those ascribable to nonhuman antibodies directed against the same antigen.
• utilities include, for example, use as affinity ligands for purification, as reagents in
• immunoassays as components of immunoconjugates, and as therapeutic agents for appropriate indications.
• variable regions by manipulating the genes encoding the amino acid sequences that form the framework regions does not provide the desired result since the CDRs, typically of nonhuman origin, cannot be manipulated without destroying immunospecificity.
• the methods of the present invention provide, for the first time, immunoglobulins that are fully human or analogs which contain immunospecific regions with fully human characteristics.
• leukocyte markers such as CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a,b,c, CD13, CD14 , CD18 , CD19, CD20, CD22, CD23, CD27 and its ligand, CD28 and its ligands B7.1, B7.2, B7.3, CD29 and its ligand, CD30 and its ligand, CD40 and its ligand gp39, CD44, CD45 and isoforms, Cdw52 (Campath
• CD56, CD58, CD69, CD72, CTLA-4 , LFA-1 and TCR histocompatibility antigens such as MHC class I or
• adhesion molecules including the integrins, such as VLA-1, VLA-2, VLA-3 , VLA-4 , VLA-5 , VLA-6, LFA-1, Mac-1, ⁇ V ⁇ 3, and p150,95; and the selectins, such as L-selectin, E-selectin, and P-selectin and their counterreceptors VCAM-1, ICAM-1, ICAM-2, and LFA-3; interleukins, such as IL-1, IL-2, IL-3, IL-4 , IL-5,
• interleukin receptors such as IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-llR, IL- 12R, IL-13R, IL-14R and IL-15R; chemokines, such as PF4, RANTES, MIP1 ⁇ , MCP1, IP- 10, ENA-78, NAP-2, Gro ⁇ , Gro ⁇ , and IL-8; growth factors, such as TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGF family, FGF, PDGF family, endothelin, Fibrosin (F g F -1 ), Laminin, and gastrin releasing peptide
• interleukin receptors such as IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8
• GFP growth factor receptors
• RGFbetaR RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R and other hematopoietic receptors
• interferon receptors such as IFN ⁇ R, IFN ⁇ R, and
• Igs and their receptors such as IGE, FceRI, and FceRII
• tumor antigens such as her2-neu, mucin, CEA and endosialin
• allergens such as house dust mite antigen, lol p1 (grass) antigens, and urushiol
• viral proteins such as CMV glycoproteins B, H, and gCIII, HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins, EBV envelope glycoproteins, VZV, envelope glycoproteins, HPV envelope glycoproteins, Hepatitis family surface antigens
• toxins such as pseudomonas endotoxin and osteopontin/uropontin, snake venom, spider venom, and bee venom
• blood factors such as complement C3b, complement C5a, complement C5b-9, Rh factor, fibrinogen, fibrin, and myelin associated growth inhibitor
• enzymes such as cholesterol ester transfer protein, membrane bound matrix metalloproteases
• immunoglobulins and analogs are those immunospecific with respect to human IL-6, human IL-8, human TNF ⁇ , human CD4 , human L-selectin, human PTHrp and human gp39.
• Antibodies and analogs immunoreactive with human TNF ⁇ and human IL-6 are useful in treating cachexia and septic shock as well as autoimmune disease.
• Antibodies and analogs immunoreactive with GP39 or with L-selectin are also effective in treating or preventing autoimmune disease.
• anti-gp39 is helpful in treating graft versus host disease, in preventing organ transplant rejection, and in treating glomerulonephritis.
• Antibodies and analogs against L-selectin are useful in treating ischemia associated with reperfusion injury.
• Antibodies to PTHrp are useful in treating bone disease and metastatic cancer.
• human antibodies against IL-8 may be used for the treatment or prevention of a pathology or condition associated with IL-8. Such conditions include, but are not limited to, tumor metastasis, reperfusion injury, pulmonary edema, asthma, ischemic disease such as myocardial
• infarction inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), encephalitis, uveitis, autoimmune diseases (such as rheumatoid arthritis, Sjögren's syndrome, vasculitis), osteoarthritis, gouty arthritis, nephritis, renal failure, dermatological conditions such as inflammatory dermatitis, psoriasis, vasculitic urticaria and allergic angiitis, retinal uveitis, conjunctivitis,
• neurological disorders such as stroke, multiple sclerosis and meningitis, acute lung injury, adult respiratory distress syndrome (ARDS), septic shock, bacterial pneumonia, diseases involving leukocyte diapedesis, CNS inflammatory disorder, multiple organ failure, alcoholic hepatitis, antigen-antibody complex mediated diseases, inflammation of the lung (such as pleurisy, aveolitis, vasculitis, pneumonia, chronic
• bronchitis bronchiectasis, cystic fibrosis
• Behcet disease Wegener's granulomatosis
• vasculitic syndrome bronchitis, bronchiectasis, cystic fibrosis
• Behcet disease Behcet disease, Wegener's granulomatosis, and vasculitic syndrome.
• Typical autoimmune diseases which can be treated using the above-mentioned antibodies and analogs include systemic lupus erythematosus, rheumatoid arthritis,
• psoriasis Sjogren's scleroderma
• mixed connective tissue disease dermatomyositis, polymyositis, Reiter's syndrome, Behcet's disease, Type 1 diabetes, Hashimoto's thyroiditis, Grave's disease, multiple sclerosis, myasthenia gravis and pemphigus.
• the antibodies may be administered in a pharmaceutically acceptable dosage form. They may be administered by any means that enables the active agent to reach the desired site of action, for example, intravenously as by bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous,
• the antibodies may be administered as a single dose or a series of treatments.
• the antibodies may be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
• the formulation may contain suitable additives such as, for example, starch, cellulose, silica, various sugars, magnesium carbonate, or calcium phosphate.
• Suitable vehicles are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
• the appropriate dosage of antibody will depend upon known factors such as the pharmacodynamic characteristics of the particular antibody, its mode and route of administration, the age, weight, and health of the recipient, the type of condition to be treated and the severity and course of the condition, frequency of treatment, concurrent treatment and the
• mice designated XenoMouseTM
• XenoMouseTM mice, designated XenoMouseTM
• PCT PCT
• HRP horseradish peroxidase
• the antigen was coated using plate coating buffer (0.1 M carbonate buffer, pH 9.6); the assay blocking buffer used was 0.5% BSA, 0.1% Tween 20 and 0.01% thimerosal in PBS; the substrate buffer used in color development was citric acid 7.14 g/1; dibasic sodium phosphate 17.96 g/1; the developing solution (made
• OPD OPD plus 5 ml hydrogen peroxide
• the stop solution (used to stop color development) was 2 M sulfuric acid.
• the wash solution was 0.05% Tween 20 in PBS.
• mice Three to five XenoMouseTM aged 8-20 weeks were age- matched and immunized intraperitoneally with 50 ⁇ g human IL-6 emulsified in incomplete Freund' s adjuvant for primary immunization and in complete Freund' s adjuvant for subsequent injections. The mice received 6 injections 2-3 weeks apart. Serum titers were determined after the second dose and following each dose thereafter. Bleeds were performed from the retrobulbar plexus 6-7 days after injections. The blood was allowed to clot at room temperature for about 2 hours and then incubated at 4°C for at least 2 hours before separating and collecting the sera.
• Example 2 Immunization and serum preparation were conducted as described in Example 1 except that human recombinant TNF ⁇ (at 5 ⁇ g per injection) was substituted for human IL-6.
• ELISAs were conducted as described in Example 1 except that the initial coating of the ELISA plate employed 100 ⁇ l/well recombinant human TNF ⁇ at 1 ⁇ g/ml in coating buffer.
• Serum titers for h ⁇ , h ⁇ , and hk after one and two immunizations of the XenoMouseTM are shown in Table 1.
• the XenoMouseTM switches isotypes from a predominant IgM response in the first immunization to an immune response with a large IgG component in the second immunization.
• the human CD4 antigen was prepared as a surface protein using human CD4 ⁇ on transfected recombinant cells as follows.
• Human CD4 ⁇ consists of the extracellular domain of CD4, the transmembrane domain of CD4, and the cytoplasmic domain corresponding to residues 31-142, of the mature ⁇ chain of the CD3 complex.
• RBL- 2H3 cells at 10 6 cells per well were cultured in 750 ⁇ l DMEM low + 20% FBS (Gibco) and 16 ⁇ g/ml polybrene with an equal volume of proviral supernatant for 2 hours at 37°C, 5% CO 2 .
• One ml of medium was removed and 750 ⁇ l of infection medium and retroviral supernatant were added to each well and the cultures incubated overnight.
• the cells were washed and expanded in DMEM low + 10% FBS until sufficient cells were available for sorting.
• the CD4 zeta transduced RBL-2H3 cells were sorted using the FACSTAR plus (Becton Dickinson). The cells were stained for human CD4 with a mouse antihuman CD4 PE antibody and the top 2-3% expressing cells were selected.
• Example 1 using 1 X 10 7 cells per mouse except that the primary injection was subcutaneous at the base of the neck. The mice received 6 injections 2-3 weeks apart. Serum was prepared and analyzed by ELISA as described in Example 1 except that the initial coating of the ELISA plate utilized 100 ⁇ l per well of recombinant soluble CD4 at 2 ⁇ g/ml of coating buffer. The titration curve for serum from
• XenoMouseTM after 6 injections is shown in Figure 5. Titers of human anti-CD4 reactivity were shown at concentrations representing greater than those of 1:1,000 dilution. Example 4
• the antigen was prepared as a surface displayed protein in C51 cells, a high expressing clone derived by transfecting the mouse pre-B cell 300.19 with LAM-1 cDNA
• LAM-1 is the gene encoding L-selectin
• Tedder, et al ., J. Immunol (1990) 144:532 or with similarly transfected CHO cells.
• the transfected cells were sorted using fluorescent activated cell sorting using anti-Leu-8 antibody as label.
• the C51 and the transfected CHO cells were grown in
• Sera were collected as described in Example 1 and analyzed by ELISA in a protocol similar to that set forth in Example 1.
• the transfected cells were plated into 96 well plates and cell monolayers grown for 1-2 days depending on cell number and used for ELISA when confluent.
• the cells were fixed by first washing with cold 1 x PBS and then fixing solution (5% glacial acetic acid, 95% ethanol) was added.
• the plates were incubated at -25°C for 5 minutes and can be stored at this temperature if sealed with plate sealers.
• the ELISA is begun by bringing the plates to room temperature, flicking to remove fixing solution and washing 5 times with DMEM medium containing 10% FCS at 200 ⁇ l per well.
• the wells were treated with various serum dilutions or with positive or negative controls.
• Positive control wells contained murine IgGl monoclonal antibody to human L- selectin.
• the wells were incubated for 45 minutes and monolayer integrity was checked under a microscope.
• the wells were then incubated with antihuman k chain antibody or antihuman ⁇ chain antibody conjugates with HRP described in Example 1.
• the plates were then washed with 1% BSA/PBS and again with PBS and monolayer integrity was checked. The plates were developed, stopped, and read as described above.
• the results for serum from XenoMouseTM are shown in Figures 6 and 7; human antibodies both to L-selectin and control 3T3 cells were obtained.
• the serum titers are higher for the L-selectin-expressing cells as compared to parental 3T3 cells.
• These results show the XenoMouseTM produces antibodies specific for L-selectin with human ⁇ heavy chain regions and human k light chains.
• the antisera obtained from the immunized XenoMouseTM were also tested for staining of human neutrophils which express L-selectin.
• Human neutrophils were prepared as follows:
• peripheral blood was collected from normal volunteers with 100 units/ml heparin. About 3.5 ml blood was layered over an equal volume of One-step Polymorph Gradient (Accurate
• the neutrophils were then stained with either;
• XenoMouseTM immunized with cells expressing human gp39.
• Human Antibodies Against Human gp39 gp39 (the ligand for CD40) is expressed on activated human CD4 T cells.
• the sera of XenoMouseTM is expressed on activated human CD4 T cells.
• immunized with recombinant gp39 according to this example contained fully human antibodies immunospecific for gp39.
• the antigen consisted of stable transfectants of
• CHO cells were split i:io prior to transfection in DMEM 4.5 g/l glucose, 10% FBS, 2 mM glutamine, MEM, NEAA supplemented with additional glycine, hypoxanthine and thymidine.
• the cells were cotransfected with the gp39 vector at 9 ⁇ g/10 cm plate (6 x 10 5 cells) and the DHFR expressing vector pSV2DHFRs (Subranani et al., Mol Cell Biol (1981)
• Example 1 The ELISA procedure was conducted substantially as set forth in Example 1; the microtiter plates were coated with CHO cells expressing gp39 grown in a 100 mm dish in DMEM, 4.5 g/1 glucose, 10% FCS, 4mM glutamine, and nonessential amino acid (NEAA) solution for MEM (100X). On the day preceding the ELISA assay, the cells were trypsinized and plated into well filtration plates at 10 5 cells/200 ⁇ l well and incubated at 37°C overnight. The positive controls were mouse antihuman gp39; negative controls were antisera from mice immunized with an antigen other than gp39. 50 ⁇ l of sample were used for each assay. The remainder of the assay is as described in Example 1.
• the antibodies prepared in this example were secreted by hybridomas obtained by immortalizing B cells from xenomice immunized with tetanus toxin.
• the immunization protocol was similar to that set forth in Example 1 using 50 ⁇ g tetanus toxin emulsified in complete Freund's adjuvant for intraperitoneal primary immunization followed by subsequent intraperitoneal injections with antigen incorporated into incomplete Freund's adjuvant. The mice received a total of 4 injections 2-3 weeks apart.
• anti-TTC antitetanus toxin C
• the spleen cells were fused with myeloma cells
• pen/strep for culture at 37°C and 10% CO 2 .
• the cells were plated in microtiter plates and maintained in HAT- supplemented medium for two weeks before transfer to HAT- supplemented medium.
• Supernatants from wells containing hybridomas were collected for a primary screen using an ELISA.
• the ELISA was conducted as described in Example 1 wherein the antigen coating consisted of 100 ⁇ l/well of tetanus toxin C (TTC) protein at 2 ⁇ g/ml in coating buffer, followed by incubation at 4°C overnight or at 37°C for two hours.
• TTC tetanus toxin C
• HRP-conjugated mouse antihuman IgM was used as described in Example 1.
• Two hybridomas that secreted anti-TTC according to the ELISA assay, clone D5.1 and clone K4.1 were used for further analysis.
• clone D5.1 secretes fully human anti-TTC which is detectable using HRP-conjugated antihuman ⁇ chain antibody and HRP-conjugated antihuman k chain antibody. This is confirmed in Figure 11.
• the antibody secreted by D5.1 did not immunoreact in ELISAs using TNF ⁇ , IL-6, or IL-8 as immobilized antigen under conditions where positive controls (sera from xenomice immunized with TNF ⁇ , IL-6 and IL-8 respectively) showed positive ELISA results.
• polyA mRNA was isolated from about 10 6 hybridoma cells and used to generate cDNA using random hexamers as primers. Portions of the product were amplified by PCR using the appropriate primers.
• the cell line was known to provide human k light chains; for PCR amplification of light chain encoding cDNA, the primers used were HKP1 (5'-CTCTGTGACACTCTCCTGGGAGTT-3') for priming from the constant region terminus and two oligos, used in equal amounts to prime from the variable segments; B3 (5'-GAAACGACACTCACGCAGTCTCCAGC-3').
• PTHrp 1-34 conjugated with BTG, as described by Ratcliffe et al ., J. Immunol. Methods 127:109 (1990), or with PTHrp (1-34) synthesized as a 4 branched-MAP (multiple antigenic peptide system).
• the antigens were emulsified in CFA (complete Freunds adjuvant) and injected i.p. at a dose of 25 ⁇ g per animal at 2 week intervals, and bled after two injections. The sera obtained from this bleed were analyzed by ELISA as described supra .
• Serum titers for h ⁇ , h ⁇ , and hk after one immunization of the XenoMouseTM are shown in Table 2.
• the XenoMouseTM showed low serum titers in 5 of 7 mice on the first bleed, but when PTHrp-MAP is used, 7 of 7 mice show high serum titers on the first bleed.
• Immunization and serum preparation were as described in Example 1 except that human recombinant IL-8 was used as an immunogen.
• ELISA assays were performed with respect to the recovered serum, also exactly as described in Example 1, except that the ELISA plates were initially coated using 100 ⁇ l/well of recombinant human IL-8 at 0.5mg/ml in the coating buffer.
• the results obtained for various serum dilutions from XenoMouseTM after 6 injections are shown in Figure 14. Human anti-IL-8 binding was again shown at serum dilutions having concentrations higher than that represented by a
• XenoMouseTM Groups of 4 to 6 XenoMouseTM aged between 8 to 10 weeks old were used for immunization and for hybridoma generation.
• XenoMouseTM were immunized intraperitoneally with 25 ⁇ g of human recombinant-IL-8 (Biosource International, CA, USA) emulsified in complete Freund's adjuvant (CFA, Sigma) for the primary immunization. All subsequent injections were done with the antigen incorporated into incomplete Freund's adjuvant (IFA, Sigma).
• IFA incomplete Freund's adjuvant
• Serum titers of immunized XenoMouseTM were first analyzed after a secondary dose of antigens, and from there after, following every antigen dose. Test bleeds were performed 6 to 7 days after the injections, by bleeding from the retro- bulbar plexus. Blood was allowed to clot at room temperature for about 2 hours and then incubated at 4oC for at least 2 hours before separating and collecting the sera.
• Spleen cells obtained from XenoMouseTM previously immunized with antigen were fused with the non secretory NSO myeloma cells transfected with bcl-2 (NSO-bcl2) as described in Galfre G, et al., Methods in Enzymology 73, 3-46, (1981). Briefly, the fusion was performed by mixing washed spleen cells and myeloma cells at a ratio of 5:1 and gently
• the BIAcore instrument uses plasmon resonance to measure the binding of an antibody to an antigen-coated gold chip.
• the BIAcore instrument, CM5 sensor chips, surfactant P20, and the amine coupling kit containing N- hydroxysuccinimide (NHS), N-ethyl-N 1 -(3-diethylaminopropyl)- carbodimide (EDC), and ethanolamine were purchased from Pharmaicia Biosensor. Immobilization of human recombinant IL-8 onto the sensor surface was carried out at low levels of antigen density immobilized on the surface and was performed according to the general procedures outlined by the
• HEPES buffer HBS; 10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20, pH 7.4
• the sensor surface was activated with 5 ⁇ l of a mixture of equal volumes of NHS (0.1 M) and EDC (0.1 M) injected at 10 ⁇ l/min across the surface for activation, then 5 ⁇ l of the ligand (human recombinant IL-8) at 12 ⁇ g/ml in 5 mM maleate buffer, pH 6.0 was injected across the activated surface, and finally non-conjugated active sites were blocked with an injection of 35 ⁇ l of 1 M ethanolamine. The surface was washed to remove non-covalently bound ligand by injection of 5 ⁇ l 0.1 M HCl. All the immobilization procedure was carried out with a continuous flow of HBS of 10 ⁇ l/min.
• B is immobilized on the surface and A is injected at a constant concentration C.
• the response is a measure of the concentration of the complex [AB] and all concentration terms can be expressed as Response Units (RU) of the BIAcore: dR/dt-kaC(Rmax-R) - kdR where dR/dt is the rate of change of the signal, C is the concentration of the analyte, Rmax is the maximum analyte binding capacity in RU and R is the signal in RU at time t.
• dR/dt is the rate of change of the signal
• C is the concentration of the analyte
• Rmax is the maximum analyte binding capacity in RU
• R is the signal in RU at time t.
• the values of ka and kd are independent of the concentration of immobilized ligand on the surface of the sensor.
• the dissociation rates (kd) and association rates (ka) were determined using the software provided by the manufacturers, BIA evaluation 2.1.
• the dissociation rate constant was measured during the dissociation phase that extended for 10 minutes at a constant buffer flow rate of 45 ul/min, after the completion of the injection of the
• affinities were determined to be very, ranging from 7 X 10 -11 to 2 X 10 -9 M. This compares vary favorably with the
• IL-8 The primary in vivo function of IL-8 is to attract and activate neutrophils. Neutrophils express on their surface two distinct receptors for IL-8, designated the A receptor and the B receptor. In order to determine whether the fully human antibodies could neutralize the activity of
• IL-8 two different in vitro assays were performed with human neutrophils. In one assay, the ability of the antibodies to block binding or radiolabelled IL-8 to neutrophil IL-8 receptors was tested. In a second assay, the antibodies were tested for their ability to block an IL-8-induced neutrophil response, namely the upregulation of the integrin Mac-1 on the neutrophil surface. Mac-1 is composed of two polypeptide chains, CD11b and CD18. Typically, anti-CD11b antibodies are used for its detection.
• Human neutrophils are isolated from either freshly drawn blood or buffy coat. Human blood is collected by venipuncture into sterile tubes containing EDTA. Buffy coats are obtained from Stanford Blood Bank. They are prepared by centrifuging anticoagulated blood (up to 400 ml) in plastic bags at 2600 xg for 10 min at 20°C with the brake off. The plasma supernatant is aspirated out of the bag and the buffy coat, i.e., the upper cell layer (40-50 ml/bag) is collected. One unit of buffy coat (40-50 ml) is diluted to final volume of 120 ml with Ca 2+ , Mg 2+ -free PBS.
• the cell pellet containing neutrophils and neutrophils was carefully withdrawn. To completely remove the mononuclear cells, the cell pellet containing neutrophils and
• erythrocytes is resuspended with 5 ml of PBS and transferred into clean 50-ml tubes.
• the cells are washed in Ca 2+ , Mg 2+ - free PBS (300 xg for 5 min at 4°C).
• the erythrocytes are then lysed with ammonium chloride.
• the cells are resuspended in 40 ml of an ice-cold solution containing 155 mM NH 4 Cl and 10 nM EDTA, pH 7.2-7.4.
• the tubes are kept on ice for 10 min with occasional mixing and then centrifuged at 300 xg for 5 min at 4°C.
• the pellet is resuspended in PBS and washed once (300 xg for 5 min at 4°C). If erythrocyte lysis appears incomplete, the treatment with ammonium chloride is repeated.
• the neutrophils are again washed and finally suspended either in assay medium (RPMI-1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 5x10 -5 2-mercapthoethanol, 1X non- essential amino acids, 1 mM sodium pyruvate and 10 mM Hepes) at a density of 3x10 7 cells/ml or in a binding buffer (PBS containing 0.1% bovine serum albumin and 0.02% NaN 3 ), at a density of 6x10 6 cells/ml.
• assay medium RPMI-1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 5x10 -5 2-mercapthoethanol, 1X non- essential amino acids, 1 mM sodium pyruv
• Multiscreen filter plates (96-well, Millipore, MADV
• N6550 were pretreated with a PBS binding buffer containing 0.1% bovine serum albumin and 0.02% NaN 3 at 25°C for 2 hours.
• Human IL-8 at a final concentration of 10 nM was preincubated with varying concentrations of monoclonal antibodies at 4°C for 30 minutes and at 37°C for an
• Neutrophils (4x10 5 /well) were exposed to IL-8 in the presence or absence of antibodies at 4°C for 90 min, and incubated with PE-conjugated mouse-anti-human-CD11b (Becton Dickinson) for 45 min at 4°C. The cells were washed with ice-cold PBS containing 2% fetal calf serum.
• a mouse monoclonal antibody against human CD11b obtained from R&D System, Inc. was used as a positive control while the purified myeloma human IgG2 (Calbiochem) was used as a negative control in the experiments.
• the expression levels of CD11b on neutrophils were measured and expressed as the mean fluorescence channel.
• the mean fluorescence channel derived form the negative control antibody was subtracted from those of experimental samples.
• CD11b expression in the presence of 10 nM of human IL-8 is 771. Sequence analysis of Immunoglobulin transcripts derived from anti-hIL-8 hybridomas.
• the heavy chain transcript from hybridoma D1.1 has a human V H 4-21(DP-63) variable region (7 point mutations were observed compared to the germline sequence), a human 21-10rc D segment, a human J H 3 joining region and a human gamma 2 constant region. See Figure 16A.
• the kappa light chain transcript from hybridoma D1.1 is comprised of a human kappa variable region with homology to V k 08/018 (DPK1) (16 point mutations were observed when compared to the germline sequence) a human J k 3 joining region, and a human kappa constant region. See Figure 16B.
• the heavy chain transcript from hybridoma K2.2 has a human V H 3-30 variable region (3 point mutations were observed compared to the germline sequence), a human IR3rc D segment, a human J H 4 joining region and a human gamma 2 constant region. See Figure 16C.
• the kappa light chain transcript from hybridoma The kappa light chain transcript from hybridoma
• K2.2 is comprised of a human kappa variable region with homology to V k IV (B3; DPK24) (9 point mutations were observed when compared to the germline sequence) , a human J ⁇ 3 joining region, and a human kappa constant region. See Figure 16D.
• the heavy chain transcript from hybridoma K4.2 has a human V H 4-34 variable region (8 point mutations were observed compared to the germline sequence) , a human K1 D segment, a human J H 4 joining region and a human gamma 2 constant region. See Figure 16E.
• the kappa light chain transcript from hybridoma K4.2 is comprised of a human kappa variable region with homology to V k 08/018 (DPK1) (6 point mutations were observed when compared to the germline sequence) , a human J k 4 joining region, and a human kappa constant region. See Figure 16F.
• the heavy chain transcript from hybridoma K4.3 has a human V H 5-51 (DP-73) variable region, a human M5-a/M5-b D segment, a human J H 4 joining region and a human gamma 2 constant region. See Figure 16G.
• the kappa light chain transcript from hybridoma K4.3 is comprised of a human kappa variable region with homology to V k 02/012 (DPK9) (9 point mutations were observed when compared to the germline sequence) , a human J k 4 joining region, and a human kappa constant region. See Figure 16H. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
• yH1C contained in S . cerivisiae was deposited with the American Type Culture Collection ("ATCC"), 12301 Parklawn Drive, Rockville MD 20852, USA, on April 26, 1996, and given ATCC accession no. ____________. The deposit of this YAC is for exemplary purposes only, and should not be taken as an admission by the Applicant that such deposit is necessary for enablement of the claimed subject matter.
• ATCC American Type Culture Collection

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