Humanized antibodies to .gamma.-interferon

The invention provides humanized immunoglobulins that bind to and neutralize .gamma.-interferon. The antibodies are useful for treatment of diseases of the immune system, particularly autoimmune diseases.

FIELD OF THE INVENTION
 The present invention relates generally to the combination of recombinant
 DNA and monoclonal antibody technologies for developing novel biologics
 and, more particularly, for example, to the production of non-immunogenic
 (in humans) immunoglobulins specific for gamma-interferon (.gamma.-IFN)
 and their uses in vitro and in vivo. The present invention also relates
 more specifically to humanized monoclonal antibodies against .gamma.-IFN,
 polynucleotide sequences encoding the antibodies, a method of producing
 the antibodies, pharmaceutical compositions comprising the antibodies as
 an active ingredient, and therapeutic agents for suppressing undesired
 immune responses comprising the antibodies as an active ingredient.
 BACKGROUND
 The mammalian immune response is mediated by several types of cells that
 interact specifically with foreign material, i.e., antigens. One of these
 cell types, B cells, is responsible for the production of antibodies.
 Another cell type, T cells, include a wide variety of cellular subsets
 that destroy cells infected with virus or control the in vivo function of
 both B cells and other hematopoietic cells, including T cells. A third
 cell type, macrophages, process and present antigens in conjunction with
 major histocompatibility complex (MHC) proteins to T cells. Communication
 between these cell types is mediated in a complex manner by lymphokines,
 such as interleukins 1-6 and .gamma.-IFN (see, generally, Paul, W. E.,
 ed., Fundamental Immunology, 3rd ed., Raven Press, New York (1993), which
 is incorporated herein in relevant part by reference.)
 One important lymphokine is .gamma.-IFN, which is secreted by some T cells.
 In addition to its anti-viral activity, .gamma.-IFN stimulates natural
 killer (NK) cells and T helper 1 (Th1) cells, activates macrophages, and
 stimulates the expression of MHC molecules on the surface of cells (Paul,
 op. cit., pp. 764-766). Hence .gamma.-IFN generally serves to enhance many
 aspects of immune function, and is a logical candidate for a therapeutic
 drug in cases where such enhancement is desired, e.g., in treating cancer.
 Conversely, in disease states where the immune system is over-active,
 e.g., autoimmune diseases and organ transplant rejection, antagonists of
 .gamma.-IFN can be useful to treat the disease by neutralizing the
 stimulatory effects of .gamma.-IFN.
 Mouse monoclonal antibodies that bind to and neutralize .gamma.-IFN have
 been reported (see, e.g., Van der Meide et al., J. Gen. Virol, 67, 1059
 (1986)). Such anti-.gamma.-IFN antibodies have been reported to delay or
 prevent rejection in vitro and in vivo mouse models of transplants,
 (Landolfo et al., Science 229, 176 (1985) and Rosenberg et al., J.
 Immunol. 144, 4648 (1990)), both of which are incorporated herein by
 reference). Treatment of mice prone to develop a syndrome like systemic
 lupus erythematosus (SLE) with a monoclonal antibody to .gamma.-IFN was
 reported to delay onset of the disease (Jacob et al., J. Exp. Med. 166,
 798 (1987)). An anti-.gamma.-IFN antibody has also been reported to
 alleviate adjuvant arthritis in rats (Jacob et al., J. Immunol. 142, 1500
 (1989))and colitis in mice. (Powrie et al., Immunity 1, 553-562 (1994)).
 Queen et al., WO 92/11018 discuss the mouse AF2 antibody to .gamma.-IFN,
 certain humanized immunoglobulins, and use of the same for treating
 inflammatory disease.
 The use of non-human monoclonal antibodies such as AF2 has certain
 drawbacks in human treatment, particularly in repeated therapeutic
 regimens as explained below. Mouse monoclonal antibodies, for example,
 have a relatively short circulating half-life in humans, and lack other
 important immunoglobulin functional characteristics when used in humans.
 Perhaps more importantly, murine monoclonal antibodies contain substantial
 amino acid sequences that will be immunogenic when injected into a human
 patient. Numerous studies have shown that, after injection of a foreign
 antibody, the immune response elicited by a patient against the injected
 antibody can be quite strong, essentially eliminating the antibody's
 therapeutic utility after an initial treatment. Moreover, if mouse or
 other antigenic (to humans) monoclonal antibodies are used to treat
 various human diseases, subsequent treatments with unrelated mouse
 antibodies may be ineffective or even dangerous in themselves, because of
 cross-reactivity.
 Thus, there is a need for improved forms of humanized immunoglobulins
 specific for .gamma.-IFN antigen that are substantially non-immunogenic in
 humans, yet easily and economically produced in a manner suitable for
 therapeutic formulation and other uses. The present invention fulfills
 these and other needs.
 OBJECTS AND SUMMARY OF THE INVENTION
 It is the object of the present invention to provide humanized monoclonal
 antibodies against .gamma.-IFN; polynucleotide sequences encoding the
 antibodies; a method for producing the antibodies; a pharmaceutical
 composition comprising the antibodies as an active ingredient; a
 therapeutic agent for treating diseases, particularly autoimmune diseases,
 and for immune system suppression comprising the antibody as an active
 ingredient; and a method for treating such diseases.
 The invention provides humanized immunoglobulins that are humanized
 versions of the mouse AF2 immunoglobulin. The mouse AF2 immunoglobulin is
 characterized by a light chain variable region designated SEQ ID No:2 and
 a heavy chain variable region designated SEQ ID No:4. The humanized
 immunoglobulins of the invention comprise humanized heavy and light
 chains. Position 11 of the humanized heavy chain variable region framework
 is occupied by the amino acid present in the equivalent position of the
 mouse AF2 heavy chain variable region framework. A preferred humanized
 immunoglobulin of the invention comprises a humanized light chain variable
 region designated SEQ ID No:6 and a humanized heavy chain variable region
 designated SEQ ID No:8.
 The humanized immunoglobulins specifically bind to the .gamma.-IFN antigen
 and neutralize .gamma.-IFN. The humanized immunoglobulins are also capable
 of blocking the binding of the CDR-donating mouse monoclonal antibody to
 .gamma.-IFN. --IFN. Preferred humanized immunoglobulins have two pairs of
 light chain/heavy chain complexes, at least one chain comprising one or
 more mouse complementarity determining regions (CDRs) functionally joined
 to human framework region segments. For example, mouse CDRs, with or
 without additional naturally-associated mouse amino acid residues, can be
 introduced into human framework regions to produce humanized
 immunoglobulins capable of binding to the antigen at affinity levels
 stronger than about 10.sup.7 M.sup.-1.
 The immunoglobulins, including binding fragments and other derivatives
 thereof, of the present invention can be produced readily by a variety of
 recombinant DNA techniques, with ultimate expression in transfected cells,
 preferably immortalized eukaryotic cells, such as myeloma or hybridoma
 cells. Polynucleotides comprising a first sequence coding for humanized
 immunoglobulin framework regions and a second sequence coding for the
 desired immunoglobulin CDRs can be produced synthetically or by combining
 appropriate cDNA and genomic DNA segments.
 The humanized immunoglobulins can be utilized in substantially pure form
 and can be prepared in a pharmaceutically accepted dosage form, which
 varies depending on the mode of administration.

DEFINITIONS
 The phrase "substantially identical," in the context of two nucleic acids
 or polypeptides (e.g., DNAs encoding a humanized immunoglobulin or the
 amino acid sequence of the humanized immunoglobulin) refers to two or more
 sequences or subsequences that have at least about 80%, most preferably
 90-95% or higher nucleotide or amino acid residue identity, when compared
 and aligned for maximum correspondence, as measured using the following
 sequence comparison method and/or by visual inspection. Such
 "substantially identical" sequences are typically considered to be
 homologous. Preferably, the "substantial identity" exists over a region of
 the sequences that is at least about 50 residues in length, more
 preferably over a region of at least about 100 residues, and most
 preferably the sequences are substantially identical over at least about
 150 residues, or over the full length of the two sequences to be compared.
 As described below, any two antibody sequences can only be aligned in one
 way, by using the numbering scheme in Kabat. Therefore, for antibodies,
 percent identity has a unique and well-defined meaning.
 Amino acids from the variable regions of the mature heavy and light chains
 of immunoglobulins are designated Hx and Lx respectively, where x is a
 number designating the position of an amino acid according to the scheme
 of Kabat, Sequences of Proteins of Immunological Interest (National
 Institutes of Health, Bethesda, Md., 1987 and 1991). Kabat lists many
 amino acid sequences for antibodies for each subgroup, and lists the most
 commonly occurring amino acid for each residue position in that subgroup
 to generate a consensus sequence. Kabat uses a method for assigning a
 residue number to each amino acid in a listed sequence, and this method
 for assigning residue numbers has become standard in the field. Kabat's
 scheme is extendible to other antibodies not included in his compendium by
 aligning the antibody in question with one of the consensus sequences in
 Kabat by reference to conserved amino acids. The use of the Kabat
 numbering system readily identifies amino acids at equivalent positions in
 different antibodies. For example, an amino acid at the L50 position of a
 human antibody occupies the equivalent position to an amino acid position
 L50 of a mouse antibody.
 The basic antibody structural unit is known to comprise a tetramer. Each
 tetramer is composed of two identical pairs of polypeptide chains, each
 pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70
 kDa). The amino-terminal portion of each chain includes a variable region
 of about 100 to 110 or more amino acids primarily responsible for antigen
 recognition. The carboxy-terminal portion of each chain defines a constant
 region primarily responsible for effector function. The variable regions
 of each light/heavy chain pair form the antibody binding site. Thus, an
 intact antibody has two binding sites.
 Light chains are classified as either kappa or lambda. Heavy chains are
 classified as gamma, mu, alpha, delta, or epsilon, and define the
 antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within
 light and heavy chains, the variable and constant regions are joined by a
 "J" region of about 12 or more amino acids, with the heavy chain also
 including a "D" region of about 10 more amino acids. (See generally,
 Fundamental Immunology, Paul, W., ed., 3rd ed. Raven Press, N.Y., 1993,
 SH. 9 (incorporated by reference in its entirety for all purposes)).
 From N-terminal to C-terminal, both light and heavy chain variable regions
 comprise alternating framework and complementarity determining regions
 (CDRs): FR, CDR. FR, CDR. FR, CDR and FR. The assignment of amino acids to
 each region is in accordance with the definitions of Kabat (1987) and
 (1991), supra, and/or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);
 Chothia et al., Nature 342:878-883 (1989).
 Preferably, analogs of exemplified humanized immunoglobulins differ from
 exemplified immunoglobulins by conservative amino acid substitutions. For
 purposes of classifying amino acids substitutions as conservative or
 nonconservative, amino acids may be grouped as follows: Group I
 (hydrophobic sidechains): met, ala, val, leu, ile; Group II (neutral
 hydrophilic side chains): cys, ser, thr; Group III (acidic side chains):
 asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V
 (residues influencing chain orientation): gly, pro; and Group VI (aromatic
 side chains): trp, tyr, phe. Conservative substitutions involve
 substitutions between amino acids in the same class. Non-conservative
 substitutions constitute exchanging a member of one of these classes for a
 member of another.
 The term epitope includes any protein determinant a capable of specific
 binding to an immunoglobulin. Epitopic determinants usually consist of
 chemically active surface groupings of molecules such as amino acids or
 sugar side chains and usually have specific three dimensional structural
 characteristics, as well as specific charge characteristics.
 As used herein, the term "immunoglobulin" refers to tetrameric antibodies
 as well as a variety of forms besides antibodies; including, for example,
 Fv, Fab, and F(ab').sub.2 as well as bifunctional hybrid antibodies (e.g.,
 Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains
 (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988)
 and Bird et al., Science 242, 423-426 (1988), which are incorporated
 herein by reference). (See, generally, Hood et al., Immunology, Benjamin,
 N.Y., 2.sup.ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory
 Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood,
 Nature, 323, 15-16 (1986), which are incorporated herein by reference.).
 As used herein, the term "framework region" refers to those portions of
 immunoglobulin light and heavy chain variable regions that are relatively
 conserved (i.e., other than the CDRs) among different immunoglobulins in a
 single species, as defined by Kabat, et al., op. cit. As used herein, a
 "human framework region" is a framework region that is substantially
 identical (about 85% or more) to the framework region of a naturally
 occurring human antibody.
 As used herein, the term "humanized immunoglobulin" refers to an
 immunoglobulin comprising a human framework, at least one CDR from a
 non-human antibody, and in which any constant region present is
 substantially identical to a human immunoglobulin constant region, i.e.,
 at least about 85-90%, preferably at least 95% identical. Hence, all parts
 of a humanized immunoglobulin, except possibly the CDRs, are substantially
 identical to corresponding parts of one or more native human
 immunoglobulin sequences. For example, a humanized immunoglobulin would
 not encompass a chimeric mouse variable region/human constant region
 antibody.
 The term "patient" includes human and veterinary subjects.
 The term "substantially pure" or "isolated" means an object species is the
 predominant species present (i.e., on a molar basis it is more abundant
 than any other individual species in the composition), and preferably a
 substantially purified fraction is a composition wherein the object
 species comprises at least about 50 percent (on a molar basis) of all
 macromolecular species present. Generally, a substantially pure
 composition comprises more than about 80, 90, 95 or 99% percent by weight
 of all macromolecular species present in the composition. Most preferably,
 the object species is purified to essential homogeneity (contaminant
 species cannot be detected in the composition by conventional detection
 methods) wherein the composition consists essentially of a single
 macromolecular species.
 DETAILED DESCRIPTION
 The invention provides humanized immunoglobulins that specifically bind to
 .gamma.-IFN, and methods of using the same for suppressing undesired
 immune responses.
 I. Humanized Antibodies Specific for .gamma.-IFN
 Humanized immunoglobulins of the invention have variable framework regions
 substantially from a human immunoglobulin (termed an acceptor
 immunoglobulin), preferably the human acceptor antibody EU, and CDRs
 substantially from a mouse immunoglobulin termed AF2 (referred to as the
 donor immunoglobulin). The constant region(s), if present, are also
 substantially from a human immunoglobulin. The humanized antibodies
 exhibit a specific binding affinity for .gamma.-IFN of at least 10.sup.7,
 10.sup.8, 10.sup.9, or 10.sup.10 M.sup.-1. Usually the upper limit of
 binding affinity of the humanized antibodies for human .gamma.-IFN is
 within a factor of 3, 4, 5 or 10 of that of AF2. Often the lower limit of
 binding affinity is also within a factor of 3, 4, 5 or 10 of that of AF2.
 Preferred humanized immunoglobulins compete with AF2 for binding to
 .gamma.-IFN and prevent .gamma.-IFN from binding to and thereby
 transducing a response through a .gamma.-IFN receptor. The humanized
 antibodies preferably neutralize 80,90, 95 or 99% of .gamma.-interferon
 activity at 1, 2, 5, 10, 20, 50 or 100-fold molar excess.
 The mouse AF2 antibody is described by Queen et al., WO 92/11018, and has
 heavy and light chain variable regions designated SEQ ID Nos: 2 and 4. The
 mouse antibody has IgG2b isotype and a kappa light chain. The heavy and
 light chain variable regions of the preferred human acceptor antibody EU,
 and those of other possible human acceptor antibody are described by
 Kabat, Sequences of Proteins of Immunological Interest (National
 Institutes of Health, Bethesda, Md., 1987 and 1991). The human acceptor
 antibody is chosen such that its variable regions exhibit a high degree of
 sequence identity with those of the mouse AF2 antibody. The heavy and
 light chain variable framework regions can be derived from the same or
 different human antibody sequences. The human antibody sequences can be
 the sequences of naturally occurring human antibodies or can be consensus
 sequences of several human antibodies.
 The design of humanized immunoglobulins can be carried out as follows. When
 an amino acid falls under the following category, the framework amino acid
 of a human immunoglobulin to be used (acceptor immunoglobulin) is replaced
 by a framework amino acid from a CDR-providing non-human immunoglobulin
 (donor immunoglobulin):
 (a) the amino acid in the human framework region of the acceptor
 immunoglobulin is unusual for human immunoglobulins at that position,
 whereas the corresponding amino acid in the donor immunoglobulin is
 typical for human immunoglobulins in that position;
 (b) the position of the amino acid is immediately adjacent to one of the
 CDRs; or
 (c) the amino acid is capable of interacting with the CDRs (see, Queen et
 al., op. cit., and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869 (1991),
 respectively, both of which are incorporated herein by reference). For a
 detailed description of the production of humanized immunoglobulins see,
 Queen et al., op. cit., and Co et al., op. cit.
 Queen et al., WO 92/11018 report certain humanized forms of AF2, comprising
 CDR regions from AF2 and variable region frameworks from EU in which
 certain positions are substituted. The present humanized immunoglobulins
 preferably contain the same substitutions as described by Queen et al.,
 supra. However, additional substitutions are also present. Specifically,
 position H11 is substituted with the amino acid occupying the equivalent
 position of the mouse AF2 heavy chain.
 Position H11 does not fulfill the criteria for substitution given above,
 but nevertheless makes a significant contribution to neutralizing activity
 in humanized immunoglobulins incorporating this substitution. The
 desirability of substituting at this position was determined by
 substitution of various positions in a chimeric AF2 antibody (i.e., having
 mouse variable domains and human constant regions) with amino acids from
 equivalent positions in the human EU antibody (SEQ ID NOS:12 and 13).
 Substitution of position H11 caused a significant reduction in the
 neutralizing activity of the chimeric antibody for .gamma.-IFN.
 Usually the CDR regions in humanized antibodies are substantially
 identical, and more usually, identical to the corresponding CDR regions in
 the mouse antibody from which they were derived. Although not usually
 desirable, it is sometimes possible to make one or more conservative amino
 acid substitutions of CDR residues without appreciably affecting the
 binding affinity of the resulting humanized immunoglobulin. Occasionally,
 substitutions of CDR regions can enhance binding affinity.
 Other than for the specific amino acid substitutions discussed above, the
 framework regions of humanized immunoglobulins are usually substantially
 identical, and more usually, identical to the framework regions of the
 human antibodies from which they were derived. Of course, many of the
 amino acids in the framework region make little or no direct contribution
 to the specificity or affinity of an antibody. Thus, many individual
 conservative substitutions of framework residues can be tolerated without
 appreciable change of the specificity or affinity of the resulting
 humanized immunoglobulin.
 Analogs of HuZAF show substantial amino acid sequence identity with HuZAF.
 Heavy and light chain variable regions of analogs are encoded by nucleic
 acid sequences that hybridize with the nucleic acids encoding the heavy or
 light chain variable regions of HuZAF, or degenerate forms thereof, under
 stringent conditions. Phage-display technology offers powerful techniques
 for selecting such analogs of HuZAF with retaining binding affinity and
 specificity (see, e.g., Dower et al., WO 91/17271; McCafferty et al., WO
 92/01047; and Huse, WO 92/06204 (each of which is incorporated by
 reference in its entirety for all purposes).
 The variable segments of humanized antibodies produced as described supra
 are typically linked to at least a portion of an immunoglobulin constant
 region (Fc), typically that of a human immunoglobulin. Human constant
 region DNA sequences can be isolated in accordance with well-known
 procedures from a variety of human cells, but preferably immortalized
 B-cells (see Kabat et al., supra, and WO87/02671). Ordinarily, the
 antibody contains both light chain and heavy chain constant regions. The
 heavy chain constant region usually includes CH1, hinge, CH2, CH3, and,
 sometimes, CH4 regions.
 The humanized antibodies include antibodies having all types of constant
 regions, including IgM, IgG, IgD, IgA and IgE, and any isotype, including
 IgG1, IgG2, IgG3 and IgG4. When it is desired that the humanized antibody
 exhibit cytotoxic activity, the constant domain is usually a
 complement-fixing constant domain and the class is typically IgG.sub.1.
 When such cytotoxic activity is not desirable, the constant domain can be
 of the IgG.sub.2 class. The humanized antibody may comprise sequences from
 more than one class or isotype.
 Having conceptually selected the CDR and framework components of humanized
 immunoglobulins, a variety of methods are available for producing such
 immunoglobulins. Because of the degeneracy of the genetic code, a variety
 of nucleic acid sequences encode each immunoglobulin amino acid sequence.
 The desired nucleic acid sequences can be produced by de novo solid-phase
 DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the
 desired polynucleotide. All nucleic acids encoding the antibodies
 described in this application are expressly included in the invention.
 Once expressed, the whole antibodies, their dimers, individual light and
 heavy chains, or other immunoglobulin forms of the present invention can
 be purified according to standard procedures in the art, including
 ammonium sulfate precipitation, affinity columns, column chromatography,
 gel electrophoresis and the like (see, generally, Scopes, R., Protein
 Purification, Springer-Verlag, N.Y. (1982), which is incorporated herein
 by reference). Substantially pure immunoglobulins of at least about 90 to
 95% homogeneity are preferred, and 98 to 99% or more homogeneity most
 preferred, for pharmaceutical uses. Once purified, partially or to
 homogeneity as desired, the polypeptides may then be used therapeutically
 (including extracorporeally) or in developing and performing assay
 procedures, immunofluorescent stainings, and the like. (See, generally,
 Immunological Methods, Vols. I and II, Lefkovits and Pernis, eds.,
 Academic Press, New York, N.Y. (1979 and 1981).
 II. Therapeutic Methods
 Pharmaceutical compositions comprising immunoglobulins of the present
 invention are useful for parenteral administration, i.e., subcutaneously,
 intramuscularly and particularly, intravenously. The compositions for
 parenteral administration commonly comprise a solution of the antibody or
 a cocktail thereof dissolved in an acceptable carrier, preferably an
 aqueous carrier. A variety of aqueous carriers can be used, e.g., water,
 buffered water, 0.4% saline, 0.3% glycine and the like. These solutions
 are sterile and generally free of particulate matter. The compositions may
 contain pharmaceutically acceptable auxiliary substances as required to
 approximate physiological conditions such as pH adjusting and buffering
 agents, toxicity adjusting agents and the like, for example sodium
 acetate, sodium chloride, potassium chloride, calcium chloride, sodium
 lactate, histidine and arginine. The concentration of the immunoglobulins
 in these formulations can vary widely, i.e., from less than about 0.01%,
 usually at least about 0.1% to as much as 5% by weight and are selected
 primarily based on fluid volumes, and solubilities in accordance with the
 particular mode of administration selected.
 Thus, a typical pharmaceutical composition for injection could be made up
 to contain 1 ml sterile buffered water, and 1-100 mg of immunoglobulin. A
 typical composition for intravenous infusion can be made up to contain 250
 ml of sterile Ringer's solution, and 10 mg of immunoglobulin. Actual
 methods for preparing parentally administerable compositions are known or
 apparent to those skilled in the art and are described in more detail in,
 for example, Remington's Pharmaceutical Science (15th Ed., Mack Publishing
 Company, Easton, Pa., 1980), which is incorporated herein by reference.
 The immunoglobulins of this invention can be frozen or lyophilized for
 storage and reconstituted in a suitable carrier prior to use. This
 technique has been shown to be effective with conventional immunoglobulins
 and art-known lyophilization and reconstitution techniques can be
 employed. Lyophilization and reconstitution can lead to varying degrees of
 immunoglobulin activity loss (e.g., with conventional immunoglobulins, IgM
 antibodies tend to have greater activity loss than IgG antibodies) and
 that use levels may have to be adjusted to compensate.
 The compositions can be administered for prophylactic and/or therapeutic
 treatments. In therapeutic application, compositions are administered to a
 patient already suffering from an undesired immune response in an amount
 sufficient to cure or at least partially arrest the condition and its
 complications. An amount adequate to accomplish this is defined as a
 "therapeutically effective dose." Amounts effective for this use depend
 upon the severity of the condition and the general state of the patient's
 own immune system, but generally range from about 0.01 to about 100 mg of
 antibody per dose, with dosages of from 0.1 to 50 mg and 1 to 10 mg per
 patient being more commonly used. Single or multiple administrations on a
 daily, weekly or monthly schedule can be carried out with dose levels and
 pattern being selected by the treating physician. It must be kept in mind
 that the materials of this invention may generally be employed in serious
 disease states, that is life-threatening or potentially life-threatening
 situations. In such cases, in view of the minimization of extraneous
 substances and the lower probability of "foreign substance" rejections
 which are achieved by the present humanized immunoglobulins of this
 invention, it is possible and may be felt desirable by the treating
 physician to administer substantial excesses of these immunoglobulins.
 In prophylactic applications, compositions are administered to a patient
 who is at risk of developing an inappropriate immune response in an amount
 sufficient to suppress the response. Such an amount is defined to be a
 "prophylactically effective dose." In this use, the precise amounts again
 depend upon the patient's state of health and general level of immunity,
 but generally range from 0.1 to 100 mg per dose, especially 1 to 10 mg per
 patient.
 The methods are effective on a variety of disease states associated with
 undesired immune response mediated by HLA class II antigens and/or Th1
 cells. Such disease states include graft versus host disease and
 transplant rejection in patients undergoing an organ transplant, such as
 heart, lung, kidney, and liver, and autoimmune diseases, such as Type I
 diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus
 erythematosus, Hashimoto's thyroiditis, psoriasis primary biliary
 cirrhosis, and inflammatory bowel disease, e.g., Crohn's disease.
 The humanized immunoglobulins can be utilized alone in substantially pure
 form, or together with a chemotherapeutic agent such as a non-steroidal
 anti-inflammatory drug, a corticosteroid, or an immunosuppressant. The
 agents can include non-steroidal anti-inflammatory agents (e.g., aspirin,
 ibuprofen), steroids (e.g., prednisone) and immunosuppressants (e.g.,
 cyclosporin A, methotrexate cytoxan)
 Humanized immunoglobulins of the present invention can also be used in
 combination with other antibodies, particularly humanized antibodies
 reactive with other lymphokines or lymphokine receptors. For example,
 suitable antigens to which a cocktail of humanized immunoglobulins may
 react include interleukins 1 through 18 and the p55 and p75 chains of the
 IL-2 receptor (see, Waldmann, Annu. Rev. Biochem. 58, 875 (1989) and Queen
 et al., Proc. Natl. Acad. Sci. USA 86, 10029 (1989), both of which are
 incorporated herein by reference). Other antigens include those on cells
 responsible for the disease, e.g., the so-called "Clusters of
 Differentiation" (Leucocyte Typing III, ed. by A. J. McMichael, Oxford
 University Press 1987), which is incorporated herein by reference).
 Diagnostic Methods
 Humanized anti-.gamma.-IFN antibody is also useful in diagnostic methods.
 Humanized anti-.gamma.-IFN antibody is useful for measuring expression of
 .gamma.-IFN, and consequent development of an immune response. Methods of
 diagnosis can be performed in vitro using a cellular sample (e.g., blood
 sample,lymph node biopsy or tissue) from a patient or can be performed by
 in vivo imaging. Humanized anti-.gamma.-IFN antibody is also useful for
 purifying human .gamma.-IFN.
 In particular embodiments, compositions comprising humanized immunoglobulin
 of the present invention can be used to detect .gamma.-IFN, for example,
 by radioimmunoassay or ELISA. Thus, a humanized immunoglobulin of the
 present invention, such as a humanized immunoglobulin that binds to the
 antigen determinant identified by the AF2 antibody can be labeled and used
 to identify anatomic sites that contain significant concentrations of
 .gamma.-IFN. For example but not for limitation, one or more labeling
 moieties can be attached to the humanized immunoglobulin. Exemplary
 labeling moieties include, but are not limited to, radiopaque dyes,
 radiocontrast agents, fluorescent molecules, spin-labeled molecules,
 enzymes, or other labeling moieties of diagnostic value, particularly in
 radiologic or magnetic resonance imaging techniques.
 The following examples are offered by way of illustration, not by
 limitation. It will be understood that although the examples pertain to
 the humanized AF2 antibody, producing humanized antibodies with high
 binding affinity for the .gamma.-IFN antigen it is also contemplated using
 CDRs from other monoclonal antibodies that bind to an epitope of
 .gamma.-IFN.
 All publications mentioned herein are incorporated herein by reference for
 the purpose of describing and disclosing, for example, the constructs, and
 methodologies that are described in the publications which might be used
 in connection with the presently described invention. The publications
 discussed above and throughout the text are provided solely for their
 disclosure prior to the filing date of the present application. Nothing
 herein is to be construed as an admission that the inventors are not
 entitled to antedate such disclosure by virtue of prior invention.
 EXAMPLES
 1. Production of Humanized Immunoglobulins Cloning and Sequencing of Mouse
 AF2 Variable Region cDNAs
 Cloning of cDNA sequences encoding the variable regions of the light and
 heavy chains of the mouse AF2 antibody is described by Queen et al., WO
 92/11018. The sequences of these cDNAs are shown in FIG. 1.
 Two plasmid vectors were prepared for construction and expression of a
 chimeric antibody comprising the variable domains of the mouse AF2
 antibody linked to human constant regions. The plasmid pVg1-dhfr (Queen et
 al., supra contains a human cytomegalovirus IE1 promoter and enhancer (M.
 Boshart et al., Cell 41, 521 (1985)), the human genomic Cg1 segment
 including part of the preceding intron, and a dihydrofolate reductase
 (dhfr) gene (Simonsen et al., Proc. Natl. Acad. Sci. USA 80, 2495 (1984),
 which is incorporated herein by reference) for selection. The plasmid pVk
 (Queen et al., supra) is similar to pVg1-dhfr but contains the human
 genomic Ck segment and the gpt gene. Derivatives of the AF2 heavy and
 light chain variable regions were prepared from the cDNAs by polymerase
 chain reaction. The 5' primers hybridized to the V regions starting at the
 ATG codons and contained XbaI sites; the 3' primers hybridized to the last
 15 nucleotides of the J regions and contained splice donor signals and
 XbaI sites (see, Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029
 (1989), which is incorporated herein by reference). The modified V regions
 were cloned into the XbaI sites of the respective plasmid vectors between
 the CMV promoter and the partial introns of the constant regions.
 For expression of the chimeric antibody, the heavy chain and kappa chain
 plasmids were transfected into Sp2/0 mouse myeloma cells by
 electroporation and cells selected for gpt expression. Clones secreting a
 maximal amount of complete antibody were detected by ELISA. Chimeric AF2
 antibody was shown to bind to human .gamma.-IFN by ELISA.
 Design of Humanized AF2 Variable Regions
 To retain the binding affinity of the mouse antibody in the humanized
 antibody, the general procedures of Queen et al. were followed (Queen et
 al. Proc. Natl. Acad. Sci. USA 86: 10029 (1989) and U.S. Pat. Nos.
 5,585,089 and 5,693,762). The choice of framework residues can be critical
 in retaining high binding affinity In principle, a framework sequence from
 any human antibody can serve as the template for CDR grafting; however, it
 has been demonstrated that straight CDR replacement into such a framework
 can lead to significant loss of binding affinity to the antigen (Tempest
 et al., Biotechnology 9: 266 (1992); Shalaby et al., J. Exp. Med. 17: 217
 (1992)). The more homologous a human antibody is to the original murine
 antibody, the less likely will the human framework introduce distortions
 into the mouse CDRs that could reduce affinity. Based on a sequence
 homology search against an antibody sequence database, the human antibody
 Eu was chosen as providing good framework homology to the mouse AF2
 antibody. Other highly homologous human antibody chains would also be
 suitable to provide the humanized antibody framework, especially kappa
 light chains from human subgroup I and heavy chains from human subgroup I
 (as defined by Kabat et al., Sequences of Proteins of Immunological
 Interest, 5th ed., U.S. Department of Health and Human Services, 1991).
 The computer programs ABMOD and ENCAD (Levitt et al., J. Mol. Biol. 168:
 595 (1983)) were used to construct a molecular model of the AF2 variable
 domain, which was used to locate the amino acids in the AF2 framework that
 are close enough to the CDRs to potentially interact with them. To design
 the humanized HuZAF heavy and light chain variable regions, the CDRs from
 the mouse AF2 antibody were grafted into the framework regions of the
 human Eu antibody. At framework positions where the computer model
 suggested significant contact with the CDRs, the amino acids from the
 mouse antibody were substituted for the original human framework amino
 acids. For the humanized form of AP2 designated HuZAF, this was done at
 residues 27, 28 (within Chothia CHR H1), 30, 38, 48, 67, 68, 70, 72, 74,
 98 and 107 of the heavy chain and at residues 48, 63, and 70 of the light
 chain. Furthermore, framework residues that occurred only rarely at their
 positions in the database of human antibodies were replaced by a human
 consensus amino acid at those positions or by the corresponding mouse
 antibody amino acids. For HuZAF this was done at residues 93, 95, 98, 107,
 108, 109, and 111 of the heavy chain and at residue 48, 63 and 70 of the
 light chain.
 In addition, in HuZAF,position H11 was substituted with the amino acid
 occupying the equivalent position of the heavy chain of mouse antibody
 AF2. H11 was identified as being a candidate for substitution by
 substitution of various positions in a chimeric AF2 antibody (i.e., having
 mouse variable domains except at substituted positions) with amino acids
 from equivalent positions in the human EU antibody and testing each
 variant for reduced neutralizing activity. The final sequences of the
 HuZAF light and heavy chain variable domains incorporating all of the
 above substitutions are shown in FIGS. 2A and 2B.
 Other humanized immunoglobulins were designed also containing mouse AF2 CDR
 regions and human EU variable regions but containing various subsets of
 the above substitutions (see FIG. 3). Haf25 is the same as HuZAF except
 that the antibody lacks substitutions at positions H11 and H38. HuXAF is
 the same as huZAF except that the former antibody lacks a substitution at
 position H38.
 However, there are many potential CDR-contact residues that are also
 amenable to substitution and that may still allow the antibody to retain
 substantial affinity to the antigen. For example, the first four
 N-terminal amino acid residues in the humanized AF2 light chain can
 alternatively be substituted with the sequence from the murine antibody
 because of its contacts with the CDRs.
 Likewise, many of the framework residues not in contact with the CDRs in
 the humanized anti .gamma.-IFN heavy and light chains can accommodate
 substitutions of amino acids from the corresponding positions of the human
 EU antibody, from other human antibodies, from the mouse AF2 antibody, or
 from other mouse antibodies, without significant loss of the affinity or
 non-immunogenicity of the humanized antibody.
 Various alternative amino acids can be selected to produce versions of
 humanized anti-.gamma.-IFN that have varying combinations of affinity,
 specificity, non-immunogenicity, ease of manufacture, and other desirable
 properties. Thus, the examples are offered by way of illustration, not of
 limitation.
 For the construction of genes for the humanized antibodies, nucleotide
 sequences were selected that encode the protein sequences of the humanized
 heavy and light chains, plus typical immunoglobulin signal sequences,
 generally utilizing codons found in the mouse sequence. Several degenerate
 codons were changed to create restriction sites or to remove undesirable
 ones. The nucleotide sequences also included the same splice donor signals
 used in the chimeric genes and an XbaI site at each end. Certain genes
 were constructed from four overlapping synthetic oligonucleotides. For
 each variable domain gene, two pairs of overlapping oligonucleotides on
 alternating strands were synthesized that encompassed the entire coding
 sequences as well as the signal peptide and the splice donor signal. The
 oligonucleotides were synthesized on an Applied Biosystems 380B DNA
 synthesizer. Each oligo was about 110-140 bases long with about a 15 base
 overlap. Double stranded DNA fragments were synthesized with Klenow
 polymerase from each pair of oligonucleotides, digested with restriction
 enzymes, ligated to the pUC18 vector and sequenced. Two fragments with the
 respectively correct half-sequences were then ligated into the XbaI sites
 of the pvg1-dhfr or pVk expression vectors in the appropriate orientations
 to produce the complete heavy and light chain genes. Certain of the genes
 for the humanized AF2 variants were generated by PCR mutagenesis of
 previous genes.
 The heavy chain and light chain plasmids were transfected into Sp2/0 mouse
 myeloma cells by electroporation and cells selected for gpt expression.
 Clones were screened by assaying human antibody production in the culture
 supernatant by ELISA, and antibody was purified from the best-producing
 clones. Antibody was purified by passing tissue culture supernatant over a
 column of staphylococcal protein A-Sepharose CL-4B (Pharmacia). The bound
 antibody was eluted with 0.2 M. Glycine-HCl, pH 3.0 and neutralized with 1
 M Tris pH 8.0. The buffer was exchanged into PBS by passing over a PD10
 column (Pharmacia) or by dialysis.
 2. Assay for Neutralizing Activity Against .gamma.-IFN
 .gamma.-IFN increases the level of expression of MHC molecules on
 responsive cell lines. Hs294T is a human melanoma cell line that
 upregulates the amount of MHC class II molecules expressed on the surface
 when incubated with .gamma.-IFN for 48-72 hr. (Zarniecki et al., J.
 Immunology, 140, 4217-4223 (1988)). This enhancement can be detected using
 a monoclonal antibody specific for the upregulated molecule and indirect
 immunofluorescence and subsequent flow cytometry. An antibody can be
 assayed for .gamma.-IFN neutralizing activity by measuring whether the
 antibody inhibits the upregulation of MHC class II molecules on this cell
 line. .gamma.-IFN for use in the assay was purchased from R&D Systems, 614
 McKinley Place, N.E., Minneapolis, Minn. 55413.
 Increasing concentrations of antibody were added to a fixed amount of
 .gamma.-IFN that had previously been shown to upregulate the level of MHC
 class II molecules on HS294T cells. The cells were incubated for 48-72 hr
 with the antibody-.gamma.-IFN mixture and examined for the level of MHC
 class II molecules by indirect immunofluorescence using a mouse monoclonal
 antibody specific for human MHC class II molecules. Analysis by flow
 cytometry allowed for the determination of the median fluorescence
 intensity of the cell population, which was then plotted against antibody
 concentration to show the neutralizing capacity of the antibody.
 As seen in FIG. 4, HuZAF has significantly better neutralizing activity
 than haf25, i.e., substitutions at positions H11 and H38 improved
 neutralizing activity. HuXAF also had better neutralizing activity than
 haf25, indicating that substitutions at H11 alone made an important
 contribution to neutralizing activity.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 13
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 381
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Mus sp.
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (1)..(381)
 &lt;223&gt; OTHER INFORMATION: AF2 VL
 &lt;400&gt; SEQUENCE: 1
 atg gaa tca cag act ctg gtc ttc ata tcc ata ctg ctc tgg tta tat 48
 Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu Leu Trp Leu Tyr
 1 5 10 15
 ggt gct gat ggg aac att gtt atg acc caa tct ccc aaa tcc atg tac 96
 Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Tyr
 20 25 30
 gtg tca ata gga gag agg gtc acc ttg agc tgc aag gcc agt gaa aat 144
 Val Ser Ile Gly Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn
 35 40 45
 gtg gat act tat gta tcc tgg tat caa cag aaa cca gag cag tct cct 192
 Val Asp Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro
 50 55 60
 aaa ctg ctg ata tat ggg gca tcc aac cgg tac act ggg gtc ccc gat 240
 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp
 65 70 75 80
 cgc ttc acg ggc agt gga tct gca aca gat ttc act ctg acc atc agc 288
 Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser
 85 90 95
 agt gtg cag gct gaa gac ctt gca gat tat cac tgt gga cag agt tac 336
 Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr
 100 105 110
 aac tat cca ttc acg ttc ggc tcg ggg aca aag ttg gaa ata aag 381
 Asn Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
 115 120 125
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 127
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mus sp.
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: AF2 VL
 &lt;400&gt; SEQUENCE: 2
 Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu Leu Trp Leu Tyr
 1 5 10 15
 Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Tyr
 20 25 30
 Val Ser Ile Gly Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn
 35 40 45
 Val Asp Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro
 50 55 60
 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp
 65 70 75 80
 Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser
 85 90 95
 Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr
 100 105 110
 Asn Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
 115 120 125
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 408
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Mus sp.
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (1)..(408)
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequenceAF2 VH
 &lt;400&gt; SEQUENCE: 3
 atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48
 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
 1 5 10 15
 gtc ctc tcc cag gtc caa ctg cag cag cct ggg gct gac ctt gtg atg 96
 Val Leu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Asp Leu Val Met
 20 25 30
 cct ggg gct cca gtg aag ctg tcc tgc ttg gct tct ggc tac atc ttc 144
 Pro Gly Ala Pro Val Lys Leu Ser Cys Leu Ala Ser Gly Tyr Ile Phe
 35 40 45
 acc agc tcc tgg ata aac tgg gtg aag cag agg cct gga cga ggc ctc 192
 Thr Ser Ser Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Arg Gly Leu
 50 55 60
 gag tgg att gga agg att gat cct tcc gat ggt gaa gtt cac tac aat 240
 Glu Trp Ile Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn
 65 70 75 80
 caa gat ttc aag gac aag gcc aca ctg act gta gac aaa tcc tcc agc 288
 Gln Asp Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
 85 90 95
 aca gcc tac atc caa ctc aac agc ctg aca tct gag gac tct gcg gtc 336
 Thr Ala Tyr Ile Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
 100 105 110
 tat tac tgt gct aga gga ttt ctg ccc tgg ttt gct gac tgg ggc caa 384
 Tyr Tyr Cys Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln
 115 120 125
 ggg act ctg gtc act gtc tct gca 408
 Gly Thr Leu Val Thr Val Ser Ala
 130 135
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 136
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Mus sp.
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: AF2 VH
 &lt;400&gt; SEQUENCE: 4
 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
 1 5 10 15
 Val Leu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Asp Leu Val Met
 20 25 30
 Pro Gly Ala Pro Val Lys Leu Ser Cys Leu Ala Ser Gly Tyr Ile Phe
 35 40 45
 Thr Ser Ser Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Arg Gly Leu
 50 55 60
 Glu Trp Ile Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn
 65 70 75 80
 Gln Asp Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
 85 90 95
 Thr Ala Tyr Ile Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
 100 105 110
 Tyr Tyr Cys Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln
 115 120 125
 Gly Thr Leu Val Thr Val Ser Ala
 130 135
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 384
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial
 Sequencehuman-mouse
 transgenic construct HuZAF VL
 &lt;400&gt; SEQUENCE: 5
 atg gag acc gat acc ctc ctg cta tgg gtc ctc ctg cta tgg gtc cca 48
 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
 1 5 10 15
 gga tca acc gga gat att cag atg acc cag tct ccg tcg acc ctc tct 96
 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser
 20 25 30
 gct agc gtc ggg gat agg gtc acc ata acc tgc aag gcc agt gaa aat 144
 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn
 35 40 45
 gtg gat act tat gta tcc tgg tat cag cag aag cca ggc aaa gct ccc 192
 Val Asp Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
 50 55 60
 aag ctt cta att tat ggg gca tcc aac cgg tac act ggg gta cct tca 240
 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser
 65 70 75 80
 cgc ttc agt ggc agt gga tct ggg acc gat ttc acc ctc aca atc agc 288
 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
 85 90 95
 tct ctg cag cca gat gat ttc gcc act tat tac tgc gga cag agt tac 336
 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr
 100 105 110
 aac tat cca ttc acg ttc ggt cag ggg acc aag gtg gag gtc aaa cgt 384
 Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg
 115 120 125
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 128
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial
 Sequencehuman-mouse
 transgenic construct HuZAF VL
 &lt;400&gt; SEQUENCE: 6
 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
 1 5 10 15
 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser
 20 25 30
 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn
 35 40 45
 Val Asp Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
 50 55 60
 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser
 65 70 75 80
 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
 85 90 95
 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Tyr
 100 105 110
 Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg
 115 120 125
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 409
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial
 Sequencehuman-mouse
 transgenic construct HuZAF VH
 &lt;400&gt; SEQUENCE: 7
 atg gga tgg agc tgg atc ttt ctc ttc ctc ctg tca ggt acc gcg ggc 48
 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
 1 5 10 15
 gtg cac tct cag gtc cag ctt gtc cag tct ggg gct gaa ctc aag aaa 96
 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys
 20 25 30
 cct ggg agc tcc gtg aag gtc tcc tgc aaa gct tct ggc tac atc ttt 144
 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe
 35 40 45
 act agc tcc tgg ata aac tgg gta aag cag gcc cct gga cag ggt ctc 192
 Thr Ser Ser Trp Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu
 50 55 60
 gag tgg att gga agg att gat cct tcc gat ggt gaa gtt cac tac aat 240
 Glu Trp Ile Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn
 65 70 75 80
 caa gat ttc aag gac aag gct aca ctt aca gtc gac aaa tcc acc aat 288
 Gln Asp Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn
 85 90 95
 aca gcc tac atg gaa ctg agc agc ctg aga tca gag gac act gca gtc 336
 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
 100 105 110
 tat tac tgt gca aga gga ttt ctg ccc tgg ttt gct gac tgg ggc caa 384
 Tyr Tyr Cys Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln
 115 120 125
 gga acc ctg gtc aca gtc tcc tca g 409
 Gly Thr Leu Val Thr Val Ser Ser
 130 135
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 136
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial
 Sequencehuman-mouse
 transgenic construct HuZAF VH
 &lt;400&gt; SEQUENCE: 8
 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
 1 5 10 15
 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys
 20 25 30
 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe
 35 40 45
 Thr Ser Ser Trp Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu
 50 55 60
 Glu Trp Ile Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn
 65 70 75 80
 Gln Asp Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn
 85 90 95
 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
 100 105 110
 Tyr Tyr Cys Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln
 115 120 125
 Gly Thr Leu Val Thr Val Ser Ser
 130 135
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 114
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequencehumanized
 immunoglobulin huXAF
 &lt;400&gt; SEQUENCE: 9
 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser
 1 5 10 15
 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser
 20 25 30
 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
 35 40 45
 Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn Gln Asp Phe
 50 55 60
 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr
 65 70 75 80
 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
 85 90 95
 Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln Gly Thr Leu
 100 105 110
 Val Thr
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 114
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequencehumanized
 immunoglobulin huZAF
 &lt;400&gt; SEQUENCE: 10
 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser
 1 5 10 15
 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser
 20 25 30
 Trp Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
 35 40 45
 Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn Gln Asp Phe
 50 55 60
 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr
 65 70 75 80
 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
 85 90 95
 Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln Gly Thr Leu
 100 105 110
 Val Thr
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 114
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequencehumanized
 immunoglobulin haf25
 &lt;400&gt; SEQUENCE: 11
 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
 1 5 10 15
 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser
 20 25 30
 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
 35 40 45
 Gly Arg Ile Asp Pro Ser Asp Gly Glu Val His Tyr Asn Gln Asp Phe
 50 55 60
 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr
 65 70 75 80
 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
 85 90 95
 Ala Arg Gly Phe Leu Pro Trp Phe Ala Asp Trp Gly Gln Gly Thr Leu
 100 105 110
 Val Thr
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 12
 &lt;211&gt; LENGTH: 107
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Variable region of the human Eu antibody
 light
 chain.
 &lt;400&gt; SEQUENCE: 12
 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
 1 5 10 15
 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Thr Trp
 20 25 30
 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Met
 35 40 45
 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ile Gly
 50 55 60
 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
 65 70 75 80
 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Asp Ser Lys
 85 90 95
 Met Phe Gly Gln Gly Thr Lys Val Glu Val Lys
 100 105
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 13
 &lt;211&gt; LENGTH: 117
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Variable region of the human Eu antibody
 heavy
 chain.
 &lt;400&gt; SEQUENCE: 13
 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
 1 5 10 15
 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Arg Ser
 20 25 30
 Ala Ile Ile Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
 35 40 45
 Gly Gly Ile Val Pro Met Phe Gly Pro Pro Asn Tyr Ala Gln Lys Phe
 50 55 60
 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr
 65 70 75 80
 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys
 85 90 95
 Ala Gly Gly Tyr Gly Ile Tyr Ser Pro Glu Glu Tyr Asn Gly Gly Leu
 100 105 110
 Val Thr Val Ser Ser
 115