IE84118B1 - Recombinant antibodies for human therapy - Google Patents

Description

PATENTS ACT, 1992 92/2437 RECOMBINANT ANTIBODIES FOR HUMAN THERAPY IDEC PHARMACEUTICALS CORPORATION Field of the Invention The invention relates to recombinant antibodies useful for human therapy, and to methods for production of such antibodies.

Background oi the Invention Murine monoclonal antibodies are used in diagnosis of human disease, and in solving basic biological research problems. These reagents are also used in clinical trials as therapeutics for both acute and chronic human diseases, including leukemias, lymphomas, solid tumors (g;g;, colon, breast, hepatic), Albs and autoimmune diseases.

Mouse/human chimeric antibodies have been created, and shown to exhibit the binding characteristics of the parental mouse antibody, and effector functions associated with the human constant regioni see e.g., Cabill& et al., U.s. Patent 4,816,557; Shoemaker et al., U.S. Patent ,978,745; Beavers et al., U.S. Patent 4,975,369; and Boss et al., U.S. Patent 4,816,397.

Generally these chimeric anti— bodies are constructed by preparing a genomic gene library from DNA extracted from pre—existing murine hybridomas.

Nishimura et al., 47 ,Cancer Research 999, 1987. The library is then screened for variable region genes from both heavy and light chains exhibiting the correct anti- body fragment rearrangement patterns. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then expressed in a cell line of choice, usually a murine myeloma line.

Such chimeric antibodies have been used in human therapy. Antibodies to these chimeric antibodies, how- have been produced by the human recipient in a Such anti-chimeric antibody antibodies ever, number of cases. are detrimental to continued therapy with the chimeric antibody.

Erlich et al., 34 Clinical Chemistry 1681, 1988, Erlich et al., 7 Hybridoma 385, 1988, Erlich et al., 6 Hybridoma 151, 1987, and Erlich et al., 1 Human Antibody Hybridomas 23, 1990 (not admitted to be prior art to the present application) state that human monoclonal anti- bodies are expected to be an improvement over mouse monoclonal antibodies for in vivo human therapy. They also postulate that non—human primate antibodies, e. ., chimpanzee monoclonal antibodies, to be tolerated in humans because they are structurally similar to human antibodies. Since human antibodies are non-immunogenic in Rhesus monkeys (2&g;’ do not induce an antibody response), they predict that primate antibodies will be non- immunogenic in humans. They indicate that the testing of antibodies in humans is unnecessary if a primate antibody has a constant region structure identical to that of a human immunoglobulin or, at least, a structure no more different from a human immunoglobulin than the different human antibodies differ from. each other. Thus, they suggest that chimpanzee antibodies may be useful in human therapy.

Summary of the Invention Accordingly, the invention provides a chimeric antibody that specifically binds to a human antigen, wherein the antibody is not immunogenic in a human, is not the same as a human or chimpanzee antibody and comprises the whole of the variable region of an immunoglobulin of an Old World monkey selected from rhesus monkeys, cynomolgus monkeys and baboons and the constant regions of a human or chimpanzee immunoglobulin, wherein said chimeric antibody is obtainable by a method comprising the steps of: A raising an Old World monkey antibody to a human target antigen in an Old World monkey selected from rhesus monkeys, cynomolgus monkeys, and baboons, isolating an Old World monkey nucleic acid encoding the whole variable region of said Old World monkey antibody, providing a human or chimpanzee nucleic acid encoding a human constant region of a human antibody, ligating said Old World monkey nucleic acid and said human or chimpanzee nucleic acid to form a recombinant nucleic acid, and expressing said recombinant nucleic acid to produce said chimeric antibody.

The invention also provides a method for producing a chimeric antibody to a human target antigen, said antibody being not immunogenic in a human, comprising the steps of: raising an Old World monkey antibody to said antigen in an Old World monkey selected from rhesus monkeys, cynomolgus monkeys, and baboons, isolating an Old World monkey nucleic acid encoding the whole variable region of said Old World monkey antibody, providing a human nucleic acid encoding a human constant region of a human antibody, . ligating said Old World monkey nucleic acid and said human nucleic acid to form a recombinant nucleic acid, and expressing said recombinant nucleic acid to produce said chimeric antibody.

Unlike some prior antibodies used for human therapy, the antibodies of the present invention do not suffer from several drawbacks, _e;._g._, 1) immunogenicity and induction of human anti-antibody (HAA) response upon repeated administration necessary to treat chrome conditions, 2) relatively shortly half-life compared to human antibodies, and 3) lack of effector functions with human cells or complement.

The lack of these drawbacks is a significant advantage for human therapy with antibodies made by the present invention. For example, in the case of chronic human diseases, including auto~irnmune diseases, or any disease where prolonged administration of an antibody is necessary, one of the major obstacles to repetitive antibody therapy is the host response to the therapeutic antibody.

HAA responses are often unpredictable from one patient to another. such responses are predominantly, though not exclusively, directed against the constant region of the antibody molecule, and once they occur they often pre- clude, or reduce the effectiveness of, any further therapy with that antibody, or another antibody of the same isotype.

Potentially, the problems of HAA could be circum- vented by the use of human monoclonal antibodies. This approach, however, suffers from the ethical, clinical, and immunological limitations on immunization of human sub- jects with many antigens of choice (gpgp, human antigens, which phrase includes antigenic or immunogenic portions of any proteins, polypeptides or their equivalent present in a human) for antibody generation. Applicants’ approach to circumvent this problem includes generation of antibodies of the appropriate specificity and desired effector func- tion, and their use in production of recombinant anti- bodies. These recombinant antibodies generally include an appropriate portion of the variable region of an antibody derived from an immunized monkey, and the constant region of an antibody from a human or chimpanzee. Thus, the specificities and high affinities of monoclonal antibodies are retained, and the appropriate human or chimpanzee con- stant region displaying the desired effector functions can be readily chosen.

The present invention is further based on a method for amplification of monkey immunoglobulin genes, e.g., by the polymerase chain reaction (PCR), from RNA extracted from monkey lymphocytes using synthetic oligonucleotide primers specific for heavy and light chain variable region gene families. The amplified genes or appropriate por- tions (e.g., complementarity’ determining region (CDR)- coding regions; see Winter, No. GB2l88638A) British Patent Application are cloned into an expression vector containing a human or chimpanzee constant region gene for the production of a monkey/human recombinant antibody These antibodies represent immunotherapeutic agents capable of localizing and/or killing appropriate target cells (e;g;, tumor cells) after in vivo administration.

An antigen—recognizing portion of the variable region of a monkey antibody gene may be cloned by providing nucleic acid, e;g;, RNA from the monkey, forming cDNA to the RNA (using reverse transcriptase), providing a primer complementary to the cDNA sequence encoding a 5' leader sequence of the antibody gene, contacting that CDNA and the primer to form a hybrid complex and amplifying the cDNA to produce nucleic acid encoding the variable region of the monkey antibody gene.

By "antigen—recognizing portion" is meant a portion of a variable region of a monkey antibody which is responsible for binding and/or recognizing the target antigen (or epitope or idiotype) of the antibody. For example the whole variable region.

The phrases "variable region", "leader sequence", "constant region" and "framework" are used in their common art recognized manner, examples of which are provided below and in the art cited above. .the desired antibody.

In preferred embodiments, the leader sequence is a human, chimpanzee or monkey leader sequence of approxi- mately 60 bases, examples of which are provided in FIG. 1.

Applicant has discovered that the monkey, chimpanzee and human ‘variable region. leader sequences are suffi- ciently similar that primers constructed to one are suit- able for amplification of the other. In the method, the RNA is amplified sufficiently to produce enough nucleic acid to place that nucleic acid within a vector for later cloning.

Antibody to a human antigen, which antibody is not immunogenic in humans may be produced by raising in a monkey a monkey antibody to the human antigen, and isolating monkey nucleic acid encoding an antigen-recognizing portion of a variable region of the monkey antibody. Human nucleic acid encoding 21 human constant region of an antibody is provided, and ligated to the monkey nucleic acid to form recombinant nucleic acid.

This recombinant nucleic acid is then expressed to produce Alternatively, chimpanzee constant region-encoding nucleic acid can be used to form the recombinant antibody. There are few, if any, differ- ences in the amino acid sequence of human and chimpanzee constant regions (i.e., they are homologous), and those differences present between human and monkey can be read- ily altered by standard techniques if the nucleic acid encoding the monkey constant region is used to form a recombinant antibody. All that is critical in the inven- tion is that an antibody be produced that is less immuno- genic than the monkey constant region so that no signi- ficant immune response ensues when the recombinant anti- (Such antibody regions Thus, the zee antibody constant region. In summary, the antibody is as human an antibody as is necessary to reduce the chance of an undesired immunological response to the antibody, and contains an antigen-binding portion of a monkey .antibody.

By "not immunogenic" is meant that the antibody does not raise an antibody response of sufficient magnitude to reduce the effectiveness of continued administration of the antibody in the majority of humans for sufficient time to achieve therapeutic efficacy, g;g;, as compared to a murine or murine-human chimeric antibody. Preferably, no antibody response is observed.

In preferred embodiments, the method includes immor- talizing a cell of the monkey which is responsible for producing the monkey antibody, gpgp, by hybridoma fusion, viral transformation with Herpes papio, single B-cell cloning (also termed "transient immortalization"), and production of a library of recombinant immunoglobulins.

In other preferred embodiments, the method includes selecting a B-cell from the monkey from either a periph- eral blood leukocyte, the spleen, bone marrow or a lymph node; selecting a clone which produces the appropriate antibody; rescuing the immunoglobulin genes encoding that antibody from the immortalized cell line; and reexpressing the genes in a producer cell line (iigp, a cell line which causes sufficient production of the antibody to be useful for human therapy).

The invention features a recombinant antibody formed from either a human or chimpanzee constant region and an antigen recognizing portion of a monkey variable region.

The antibody of the invention may have attached to it an effector or reporter molecule. For instance, an antibody of the invention may have a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridg- ing structure. In addition, the Fc fragment or CH3 domain of a complete antibody molecule may be replaced by an enzyme or toxin molecule, and a part of the immunoglobulin chain may be bonded with a polypeptide effector or reporter molecule. Bispecific antibodies can also be con- structed by standard procedure.

In another aspect, the invention features pharmaceu- tical compositions in which antibodies of the present invention are provided for therapeutic or prophylactic Such antibodies can also be provided as immuno- molecules which are characterized by two USES . toxins, i.e., components and are particularly useful for killing sel- ected cells in vitro or in vivo. one component is a cytotoxic agent which is usually fatal to a cell when attached or absorbed. The second component, known as the "delivery vehicle" provides a means for delivering the toxic agent to a particular cell type, such as carcinoma cells. The two components are commonly chemically bonded together by any of a variety of well—known chemical or genetic procedures. For example, when the cytotoxic agent is a protein and the second component is an intact immuno- globulin, the linkage may be by way of heterobifunctional crosslinkers, e_._g_._, carbodiimide, glutaraldehyde, and the like. Production of various immunotoxins is well-known in the art.

In another related aspect, nucleic acid encoding a human/monkey recombinant antibody.

In preferred embodiments, the nucleic acid encodes a human or chimpanzee constant region and an antigen recognizing the invention features portion of monkey variable region; and the nucleic acid is purified, i;e;, separated from biological components with which it naturally occurs, or more preferably, is provided as a homogeneous solution.

In a further aspect, the invention features a method for treating a human having a particular antigen, e;g;, one associated with disease. The method includes administering a therapeutically effective amount of a recombinant antibody specific for the particular antigen, wherein the recombinant antibody is one having either a human or chimpanzee constant region and an antigen recognizing portion of a monkey variable region.

In preferred embodiments of the above aspects, the antigen is a tumor antigen, an antigen involved in an immune disorder, an antigen involved in an autoimmune response, a receptor expressed on a host cell, or an antigen selected from the human antigens CD58, VLA4 (d4B1 integrin), CD2, LFA3, CD4, CD19, CD20, human T—cell receptor, CD3, CD8, ELAM, LAM, CD25, CD23, CD41, CD44, CD45, CD71, TNFd, TNFB, Tn antigen, IL-1, IL-8, C5a, adhesion molecules, e.g., VCAM, CD54, CD28, CD11a, CD1lc, CD18, and CD1lb, the neu oncogene product, MDR—1 (P—glycoprotein), TGFd and its receptor, and PDGF; and the recombinant antibody is active to either deplete (kill or eliminate) undesired cells (e;g;, anti—CD4) by acting with complement, or killer cells, or is active as a cytotoxic agent or to cause Fc—receptor binding by a phagocyte. Alterna- tively, the antibody blocks or stimulates receptor func- tions, or neutralizes active soluble products, such as one or more of the interleukins, TNF and C5a.

In other aspects, the invention features pharmaceutical compositions of the above antibodies.

Compositions or products according to the inven- tion may conveniently be provided in the form of solutions suitable for parenteral or nasal or oral administration.

Appropriate preparations of antibody may be mixed with appropriate preparations of other agents, resulting in increased clinical utility. other features and advantages of the invention will be apparent from the following description of the pre- ferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments The drawings will first briefly be described.

Drawings FIG. nine different Ig heavy chain leader sequences and ten is a tabular representation of 20 codons in monkey Ig heavy chain leader sequences; FIG. 2 is a diagrammatic representation of the struc- ture of various Ig chains showing_the relative position of leader, variable, and constant regions with the positions of restriction sites and primers used for amplification; FIG. 3 is a diagrammatic representation of a heavy chain cassette vector for expression of human or chimeric antibodies; FIG. 4 is a diagrammatic representation of a light chain cassette vector designed for expression of human or chimeric antibodies; FIGS. 5 and 6 are diagrammatic representations of vectors designed for expression of immunoglobulin from kappa or lambda light chain cDNA, respectively. In these vectors, the immunoglobulin genes are arranged in a tandem configuration. using" neomycin phosphotransferase as the selectable marker; FIGS. 7—1, 7-2, and 8 show the nucleic acid sequence of various leader sequence primers useful in the invention (these primers in FIG. 8 correspond to those listed as SEQ. ID Nos. 1 - 12 igfigg; FIGS. 9A through 9H are comparisons of human and.mon- key regions in the VH1, VH2, VH3, VH4, and VHS sequences, and VKI and VKII, and VlambdaIII sequences, respectively; is a comparison of human and monkey VH3 with one comparison to the human VH2 sequence; FIG. sequences, FIG. 11 is a graphical representation of the binding of an antibody of the invention to a human CD4 antigen; FIG. 12 is a graphical representation of the inhibi- tion of binding of lF3 by the antibody shown in FIG 10; 13 and 14 are portions of the nucleotide of anti-CD4 VH, and VL regions respectively; is a graph showing anti-CD54 activity; and 16 is a histogram comparing plasmid expression FIGS. sequences FIG.

FIG. features.

Monkey Antibodies Old World monkeys include those referred to as baboon and macaque monkeys (including Rhesus monkey and cynomol— This invention provides details of use of These gus monkey). the claimed invention with various monkey genes. examples are not limiting in the invention and may be readily applied to other Old World Monkeys.

Referring to Fig. 2, there is shown in diagrammatic form the general structure of genes encoding immunoglobu— lin heavy, kappa light, and lambda light chains. Each of ATG start codon followed by a region these chains is formed with an a leader sequence of approximately 60 bases, riable region of the immunoglobulin, and a Examples of dif- of heavy and their encoding a va constant region of that immunoglobulin. or signal peptides, These sequences, ferent leader sequences, chains are shown in FIG. 1. equivalent in monkey, can be determined by standard tech- niques well known to those of ordinary skill in the art, and as described below.

The sequences shown in the lower portion of FIG. 1 are human leader sequences. Applicants have discovered that construction of primers complementary to these leader regions allows amplification of Ig genes from monkeys.

Similarly, primers homologous to monkey leader sequences (sgg gggé, upper portion of FIG. 1) can be used in ampli- fication of ‘monkey immunoglobulin genes, and also for amplification of human immunoglobulin genes.

By’ use of such primers in standard amplification procedures, genes encoding various monkey immunoglobulin genes, can be readily isolated, and the sequences encoding the variable regions of the antibodies determined. Exam- ples of such procedures are provided below. The results of the analysis provided below are presented in FIGS. 9A through 9H, and in FIG. 10. Surprisingly, applicant found that, despite the ability to produce antibodies to rela- tively conserved human antigens in the monkeys, the vari- able regional framework sequences of the antibodies so produced were indistinguishable from those—of human anti- bodies. That is, the amount of variability in immunoglo- bulin sequence observed for the monkeys was similar to that observed for humans, and it was impossible to deter- mine which antibody was derived from a human or monkey without analysis of the source itself.

Thus, for example, referring to FIG. 10, acid sequence of the VH3 region of human was compared with monkey. The human antibodies showed a range of homology among themselves from 83 - 98%, while those of the monkey were from 90 - 95% homologous with the human VH3 region.

In contrast, the human VH2 region was homologous to the human VH3 by 60%. [In this drawing, as in the other the presence of the same amino acid at any the amino drawings, location is shown by a dash, different amino acid is shown by the standard one letter while the presence of a Positions to which no consensus amino acid can be assigned are shown as an X.] Similarly, homology for VH1, VH2, VH3, VH4, and VHS, and for VKI and VKII and Vlambda III is shown in FIGS. 9A — 9H, respectively. Again, sig- nificant homology between the.monkey immunoglobulin region and that of the human was observed in each variable region including immunoglobulin J regions sequences. such high that observed among human code.‘ homology is similar to antibodies.

The methodology by which the sequences were deter- mined is presented below in the examples. Those of ordi- nary skill in the art will recognize that these examples are not limiting in this invention and equivalent results and monoclonal and chimeric antibodies can be obtained by similar procedures well known to those of ordinary skill in the art. See e.g., U.S. patents 4,816,567; 4,978,745; 4,975,369; 4,816,397, ggpra. For example, after cloning a gene encoding a monkey variable region, such a gene is readily ligated to one encoding a monkey or human constant region, and the fused genes expressed in a producer cell line to produce the desired antibody. Below are provided examples of such procedures.

In the following examples, method involves the isolation of total RNA from monkey B cells from immunized the first step in the peripheral blood or spleen cells. monkeys may be obtained from peripheral blood or lymph nodes and used directly or, they may be preferentially expanded. Expansion may involve Herpes papio virus transformation, fusion to a heterologous myeloma cell with subsequent selection, or cloning of single B cells under limiting dilution in the presence of stimulated human T cells.

Total RNA is then converted to single stranded (ss) CDNA by reverse transcriptase using non-specific (oligo-dT or random hexamers) or specific (immunoglobulin CH1 or CK or Clambda constant region) oligonucleotide primers.

Sing1e—stranded cDNA produced by this reaction is ampli- fied using the polymerase chain reaction in which ss cDNA, together with deoxynucleotide triphosphates, a DNA poly- merase (gpgé, a thermostable polymerase) and specific primers, are used to amplify heavy or light chain variable region immunoglobulin genes; The primers used are single stranded synthetic oligonucleotides ranging from 20 to 40 bases, containing some degenerate bases which bind to the Six different 5' -1 incorporating a leader sequences. leader sequence primers FIG. immunoglobulin 5' (se_e.

Other sets of primers can be used in order to incor- porate different, unique restriction sites to allow direc- tional cloning of PCR-amplified DNA into an appropriate expression vector possessing the same restriction site.

A set of primers binding to the 3‘ end of the antibody heavy chain leader sequence incorporating a ulgl site, or a set of primers binding to the first 23 bases of frame- work one incorporating a XQQI site can also be used and are described in Fig. 7-1. heavy and light chain variable region genes may be cloned into a shuttle vector directly after PCR amplification to allow further molecular manipulations if necessary or, cloned directly into an expression vector that contains human heavy or light chain constant region genes. The molecular configuration of the immunoglobulin genes in the expression vector may be genomic, in which immunoglobulin promoter/enhancer and other regulatory regions are present The monkey immunoglobulin as well as splice donor/acceptor sequences at the intron/ exon junctions. Alternatively, chimeric immunoglobulin genes can be inserted in a CDNA configuration using heter- ologous viral promoter/enhancer sequences.

Example 1: Seggence of Monkev Antibodies FIGS. 7 and 8 show the primers with or without restriction sites respectively, used in the PCR amplifica- from monkey and/or human tion of immunoglobulin genes cDNAs. Details of the procedures used are provided below.

RNA was isolated from the spleen, peripheral blood and lymph node cells of monkeys using the standard guanidinium isothiocyanate method. The total RNA fraction isolated by this method was then used as a template for subsequent amplification reactions. An aliquot of the RNA was incu- bated in the presence of 200 units of Moloney murine leu~ kemia virus reverse transcriptase and non-specific (oligo-dT or random hexamers) or specific (immunoglobulin IgG CH1 region or kappa chain constant region CK) oligo- nucleotide primers (50-100 picomoles) to generate a non- coding sense single CDNA strand. Single stranded cDNA produced by this reaction was then amplified using the polymerase chain reaction (PCR). An aliquot of single stranded cDNA was incubated together with deoxynucleotide tri—phosphates (20pM), a thermostable DNA polymerase (2-5 units) and human-derived synthetic oligonucleotide primers (50 picomoles), to amplify either heavy or light chain variable region immunoglobulin genes.

Using pairs of primers shown in FIG. 8, representative cynomolgus immunoglobulin heavy and light several chain variable region sequences from a number of gene families were amplified. These amplified sequences were cloned into the EcoRV site of the plasmid vector p—Blue- available from Stratagene, CA) and used for DNA sequencing was performed using the script (pBS, DNA sequencing. plasmid DNA containing’ the cloned. insert as a double stranded DNA template and the standard chain termination sequencing method.

Representative cynomolgus monkey immunoglobulin sequences are shown in FIGS. 9A - 9H. Included in FIGS. 9A - 9H are the consensus amino acid sequence for human variable region genes representing each of the major vari- able region gene families. The percentage homology of each monkey sequence with the human consensus sequence, excluding the constant domain regions and the CDR regions, is shown. The level of homology between human and monkey sequences for a given family is as high as between two human sequences within that family. It is impossible therefore to distinguish between variable region immuno- globulin sequences originating from Old World monkeys, based on sequence and those originating from humans comparisons alone.

Isolation of 35A Monkey antigen—specific B cells were obtained in several ways: by fusion of immunized monkey lymph node cells to the human/mouse heteromyeloma fusion partner cell line KSH6/B5, and subsequent screening of the hybridoma by virally transformed B cells, or by in vitro In the.latter case lines, single B cell cloning techniques. growth of a single monkey B cell was supported in vitro by co-cultivation with human T cells that were stimulated by antibody. A single B cell was placed in each well of a 96 well tissue culture plate together Awith approximately 150,000 anti-CD3-stimulated mytomycin C—treated human T cells. After a two week incubation period the single B cell expanded to at least 200 differentiated plasma cells.

Culture supernatant from these wells were assayed for the presence of immunoglobulin by ELISA technique using a cap- ture antibody of goat anti-monkey immunoglobulin.

Cells either from antigen-specific virally trans- formed cells or hybridomas were grown up in sufficient numbers for extraction of RNA.

Wells from the in vitro single B cell cloning technique that were positive for immunoglobulin were removed, washed twice with cold phos- phate buffered saline pH 7.5 and centrifuged (1000 x g 10 The washed cells were suspended in loopl of lysis 25mM sodium min). solution (4 M: guanidinium isothiocyanate, 0.1M 2-mercapto- citrate pH 7.0, 0.5% sodium sarcosine, ethanol). lopl of 2M sodium acetate, pH 4.0, was added and mixed. Protein was removed by adding 100 pl water saturated phenol, mixing and adding 20ml of chloroform/ isoamyl alcohol (4921). After vortexing and incubation on ice for 15 min, the samples were centrifuged at 10,000 X g for 20 minutes. The aqueous phase was transferred to a new tube, mixed with an equal volume of isopropanol and incubated for 1 hr at —20°C. The precipitate was collected by centrifugation at 10,000 x g for 15 minutes, washed with 70% ethanol, recentrifuged and the pellet dried in a Speedivac (Savant). The dried. RNA. was redissolved a second time in 100 pl of lysis buffer. isopropanol was added and incubated at —20°C for one hour. ate was collected by centrifugation at 10,000 The pel- -2 0°C An equal volume is The precipit X g for 15 minutes and washed with 70% ethanol. let was dried in a Speedivac (Savant) and stored at in 70% ethanol until use.

Synthesis of single stranded cDNA The total RNA extracted from the cells originating was dissolved in 32ml of double dis- of from a single well tilled water to which is added lpl (50-100 picomoles) primer (either random hexamers, oligo dT or 3' immuno- globulin-specific primers) and lopl of 5X reverse tran- scriptase buffer (0.25 Tris-Hcl pH 8.3, 0.375M Kcl, 15mM Mgclz, 50mM dithiothreitol). The mixture was heated at 65°C for 5 minutes after which it was placed in ice for 2 minutes. After heating, lul RNAsin (Promega), Spl of 5mM deoxynucleotide triphosphates, and lpl (200units) of Moloney murine leukemia virus reverse transcriptase (BRL) was added, and the mixture incubated at 37°C for 1.5 hours.

After completion of the reverse transcriptase reaction, the single stranded CDNA/RNA mixture was extracted with phenol/chloroform and passed through a lml G-25 SEPHADEX spin column. The material passing through this column was used as template ss cDNA for PCR amplification.

Amplification of ss cDNA 3-lopl of the ss cDNA was mixed with lopl 10X PCR buffer (500mM KCl, 100mM Tris-HCl pH 3.3, l5mM Mgclz), l.6ul of 1.25mM deoxynucleotide triphosphates, 50 pico- moles of specific immunoglobulin 5' primer, 50 picomoles of specific immunoglobulin 3' primer, and 2 - 5 units of thermostable DNA polymerase (Synthetic Genetics). The reaction volume was brought to loopl with water and over- laid with loopl of mineral oil. The reaction mixture was incubated at the following temperatures for the specified times.

°C for 1 minute °C for 2 minutes °C for 2 minutes This cycle was repeated 30-35 times. The amplified prod- ucts were examined by agarose gel electrophoresis using a 1.2% agarose gel and molecular weight standards. The amplified immunoglobulin variable region genes ran approx- imately between 350-500 bp markers. The PCR amplified products were then used for cloning into the appropriate plasmid vector.

Example g: Cloning Monkey Antibody genes Since monkey variable region gene sequences, at the cDNA level, are indistinguishable from human members of the equivalent gene family, immune responses to monkey/ human chimeric antibodies, if any, are unlikely to be any different than those mounted against human antibody molecules.

PCR technology can be used to introduce specific restriction enzyme sites, including (but not limited to) expression vector. Alternatively, these primers were used to amplify directly from cellular RNA.

Expression vectors which have been constructed are of two types. The first (see FIGS. 3 and 4) allows cloned cDNA immunoglobulin variable regions from monkeys to be inserted into a cassette vector, using unique restriction sites, in which the immunoglobulin gene elements are arranged in a genomic configuration. This type of vector incorporates an immunoglobulin promoter, the two exons making up the immunoglohulin leader sequence, two cloning sites gggl and final, downstream splice donor sequences, an immunoglobulin enhancer region, a human constant region gene (heavy or light chain) and downstream polyadenylation In addition, they include a bacterial origin of a beta—lactamase gene for bacterial selec- signals. replication, tion, and a neomycin phosphotransferase gene for G418 selection, or a xanthine-guanine phosphoribosyl transfer- ase (gpt) gene for mycophenolic acid selection in mammal- ian cells. The heavy and light chain expression vectors use neomycin phosphotransferase (Neo) and xanthine—guanine phosphoribosyl transferase (Gpt) genes respectively as the selectable marker.

The second type of expression system (see FIGS. 5 and 6) uses immunoglobulin genes in a cDNA configuration.

That is, no introns or splice sites are present between leader sequence and the 3' constant region This type of vector utilizes heterologous the 5' sequences. viral promoter/enhancer sequences, driving immunoglobulin heavy and light chain genes arranged in a tandem fashion, polyadenylation sequences and a selectable mammalian cell marker (Neo). The Neo gene can be modified to weaken its translation, e;g44_ by changing the codon upstream and adjacent the start site of the gene from ACC to TCT. In addition, a dihydrofolate reductase (dhfr) gene is present for subsequent gene amplification with methotrexate. Mon- key immunoglobulin variable region genes to be cloned into -CDNA configured expression vectors were amplified either chain, light chains. however are not excluded.

Chimeric heavy and light chain immunoglobulin genes were introduced separately or sequentially (for genomic- ally configured constructs), or on the same vector (for cDNA configured constructs), Electroporation was used to introduce by electroporation into a producer cell line. linearized MDNA constructs into either Chinese hamster ovary (CHO) cells, followed by single cell cloning of the transfectants into 96 well Electroporation conditions using or mouse myeloma cells, tissue culture plates. a BTX-100 (BTX, San Diego) electroporation device and a lml disposable plastic cuvette gave optimal numbers of transfectants from a given amount of vector DNA. CHO cells that were adapted to grow in suspension in serum- free medium (CHO-S SFM II minus hypoxanthine and thymi- Gihco) were used in constructs containing viral regulatory elements. dine, Subclonin I variable re ion e es The products of the PCR reaction were extracted with phenol/chloroform and passed through a lml SEPHADEX G—25 spin column. If the DNA fragment was to be blunt end cloned into a plasmid lul 1M Mgclz, 0.5ml of 1.25mM deoxy— nucleotide triphosphates and lul of Klenow DNA polymerase (5 units) was added to the total PCR reaction mixture (looul) and incubated at 37°C for 15 minutes to fill in any 5' overhangs. Before blunt end cloning into a plas- mid, the 5' ends were phosphorylated as follows; Sul of lOmM ATP, lul of T4 polynucleotide kinase (10 units) were added to the total reaction mix and incubated at 37°C for minutes. Amplified fragments that contained internal restriction sites were first cut with the appropriate restriction enzyme and used directly for ligation without phosphorylation. In both cases the fragment to be cloned was extracted with phenol/chloroform before ligating into the appropriate vector. used. or pGenex-H or pGenexL, enzymes was used. lpl of 10X ligation buffer (500mM Tri-HCl pH 7.6, l00mM MgCl2, lOmM ATP, lOmM dithiothrei- tol), lul T4 DNA ligase (1 unit) was added and the reac- The ligated E. coli HB10l tion allowed to proceed at 14°C overnight. material was used to transform competent cells using the standard calcium chloride method of trans- The transformed bacteria were selected by Indivi- formation. growth on LB ampicillin—containing agar plates. dual colonies were selected and grown up overnight in LB ing ampicillin and plasmid DNA extracted After restric- medium contain using the standard alkaline lysis method. tion analysis to determine which clones contained immuno- globulin inserts, the DNA was prepared for sequencing.

Seggencigg cloned genes Cloned immunoglobulin. variable region genes were sequenced using a standard chain termination method.

Double stranded plasmid DNA containing the cloned insert Before sequencing, DNA was was used as the sequencing template. the double stranded DNA was chemically denatured. sequenced using T7 DNA polymerase SEQUENASE (United States Biochemical Corporation, OH), radiolabeled alpha deoxyATP, and the following sequencing primers: (5' CAGAGCTGGGTACGTCCTCA 3') and (5' GCCCCCAGAGGTGCTCTTGG 3') for immunoglobulin G heavy chain variable region in 5' to 3' and 3' to 5' directions respectively. (5' CAGAGCTGGGTA CGTGAACC 3') and (5' GGCTTGAAGCTCCTCAGAGG 3') for immuno- globulin lambda light chain variable region in 5' to 3' and 3' to 5' directions, respectively. Reaction products Cleveland, were separated on 6% polyacrylamide gels and read.

The results of sequencing a number of Old World mon- key immunoglobulin heavy and light variable region genes are summarized in FIGS. 9A - 9H. Cloning and sequencing cynomolgus immunoglobulin genes has not been previously described in the literature. has the degree of homology between human and cynomolgus V region genes been possible to define. The homology between a single chimpanzee variable lambda gene and its human genomic counterpart has been described showing only a 2% difference in the framework regions.

Nor, therefore, Transfection and selection After sequencing cloned heavy and light chain varia- they were sub-cloned into appropriate These may be vectors constructed that subcloning is unnecessary.

Electropgration Electroporation was used to either co—transfect heavy and light chain genomic constructs or sequentially trans- fect heavy and light chain genomic constructs into Sp2/0 cells. In sequential transfections electroporation of the chimeric light chain construct was followed by selection in mycophenolic acid. screening culture supernatants from clones, grown in 96 well plates, for light chain produc- tion. with antisera against human light chain constant region using an ELISA technique, allowed selection of the highest light chain expressing clones. Subsequent elec- troporation of light chain transfectants with a vector containing the monkey/human chimeric heavy chain immuno- globulin construct allowed the selection of transfectomas expressing chimeric antibody of the desired specificity and isotype.

The light chain construct pGENEX-L (FIGS. 3 and 4) was transfected into the murine myeloma cell line Sp2/0 by electroporation as follows. SP2/0 cells at a concentra- tion of 1 X 107/ml in transfection buffer (272 mM sucrose, 7mM sodium phosphate pH 7.4, 1mM Mgclz) were mixed with 50 ug of pGENEX-L containing the appropriate cloned light chain gene, which had previously been linearized by diges- tion with the restriction enzyme gygll. The cells were placed into a lml disposable plastic spectrophotometry cuvette and plate electrodes 3.5 mm apart inserted into the cuvette. Using a BTX—100 (BTX, Inc.) transfection apparatus the cells were given a pulse of current for 500 microseconds such that approximately 50% cell death occurred. This value was determined prior to the trans- fection by pulsing the cells, in the absence of DNA, with increasing voltages and measuring the numbers of cells surviving 24 hours later. The voltage versus cell viabil- ity was plotted graphically and the voltage corresponding to 50% cell death used for all subsequent electroporation experiments. Using the BTX-100 apparatus, the optimal value was found to be a pulse at an amplitude of 200 for microseconds. After pulsing the cells, they were allowed to recover on ice for 15 minutes before being transferred to 96-well tissue culture plates in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum and 10% Sp2/0 conditioned medium. Cells were plated at a concentration at which cell growth was seen in approximately 1 in 3 of the wells after selection with the appropriate drug. This parameter was determined for each electroporation experiment by plating varying numbers of electroporated cells per well (1000 — 10,000) and select- ing for cells which have incorporated the plasmid. The number of wells on each plate which showed cell growth was counted after 2-3 weeks of selection. Thus, the appropri- ate number of cells that gave 1 out of 3 positive wells with a given concentration of a particular plasmid was determined for use in future experiments.

Directly after electroporation cells were placed in medium without drug. Fresh medium was added two days after electroporation containing either G418 or mycophe- nolic acid for cells exhibiting neomycin phosphotransfer- ase or guanasyl phosphotransferase activity respectively.

Cells were fed every 2 days for the first week and then twice a week thereafter. The concentration of drug to use was determined by incubating cells in the presence of increasing concentrations of drug and monitoring cell The concentration of drug used was twice that For Spz/0 approximately lug viability. which gave 100% killing. mycophenolic acid/ml was required and for G418 approxi- mately aooug/ml.

Cells were electroporated in several ways, (pGenex-H and either heavy and light chain genomic vectors pGenex-L) were co—electroporated, or the light chain was electroporated alone. In the latter case, clones were screened for high level expression of chimeric immunoglo- bulin light chain using an ELISA technique. These clones were then grown up and electroporated with heavy chain containing vector. If cDNA tandem gene constructs were used, the expression vector (TCAE5.2 or TCAE6) was first linearized by digestion with the restriction enzyme NotI.

A single electroporation was sufficient to achieve inte- gration of both heavy and light chain genes. After 2-3 weeks supernatants from wells which continue to grow in the presence of appropriate drug were assayed for the secretion of chimeric immunoglobulin light chain or whole immunoglobulin using an ELISA technique. Immunoglobulin genes in a CDNA configuration were electroporated into either Spz/0 cells, as described above, or into Chinese Hamster Ovary (CHO) cells adapted to grow in suspension in CHO cells were electroporated using a set at conditions for serum-free medium.

BTX 600 electroporation apparatus, achieving maximal numbers of G418 resistant colonies.

These were 210 volts, 400 pF and 13 ohms. After electro- poration, cells were counted, washed in transfection buf- fer, resuspended in the same buffer and placed on ice for minutes. Cells were adjusted to 1 x 107 live cells/ml and 400 pl of cell suspension placed in a 0.4 ml sterile disposable cuvette (BTX Inc.). 25 pg of Not 1 linearized TCAE 5.2 or TCAE 6 vector DNA, containing cloned macaque immunoglobulin variable region genes, were resuspended in TE buffer (10 mM Tris, 1 mm EDTA, pH 8.0) at 1 pg/ml and Electroporation was carried tus using the automatic added to the cell suspension. out by discharging the appara charge and pulse button. The cuvette was placed on ice for 15 minutes, the cells diluted into 120 mls of serum- free medium and placed into six 96-well plates (200 pl per well containing approximately 6,667 electroporated or ,333 live cells per well). Independent electroporation parameters were established for this cell line and selec- tion by G418 was at 400 pg/ml. screening for production of antibody The presence of human, monkey or chimeric antibody secreted by transfectants was assayed by an ELISA tech- nique as follows: 96-well flat bottom plates (Dynatech) were coated with goat anti-human IgG or kappa at 200 ng/well in Coating Buffer (sodium carbonate 0.8 mg/ml, sodium bicarbonate 1.55 mg/ml, pH9.6) and incubated for at least 16hr at 4°C. The coating buffer was removed and the wells blocked with 120 pl of Blocking Buffer (1% bovine serum albumin in phosphate buffered saline containing 0.2% sodium azide) and incubated at 37°C for 1hr. Up to 125 pl of cell culture supernatant was added to the wells con- taining blocking buffer and incubated 2 hrs at 37°C. The plates were then washed five times with PBS. 100 pl of horse radish peroxidase-labeled goat anti-human IgG (or kappa) diluted 1:1000 — 1:5000 in Dilution Buffer (1% bovine serum albumin, 0.05% Tween-20, 0.02% sodium azide in PBS) was added. Plates were incubated at 37°C for 1 hr then washed five times with PBS. Chimeric antibody was detected with 100 #1 of hydrogen peroxide and the sub- strate, 3,3',5,5'-tetramethylbenzidine (1:1 v/v) per well.

The color reaction was terminated after 2 to 5 min. with 100 pl of 2M sulfuric acid per well. provide a transient immortalization, as described by Amaroso and Lipske 145 J. Immunology 3155, 1990, or (c) by use of a recombinant immunoglobulin bacteriophage lib- raries, as described by Huse et al., 246 ggigggg 1275, and Hccafferty et al., 348 Nature 552, 1990. Screen- ing for appropriate antibody containing" clones can be performed by those techniques described above, or equiva- lent techniques well known to those of ordinary skill in the art, and the desired immunoglobulin gene rescued from the immortalized cell line. In addition, the antibody produced by isolated monkey B cells can be used in human therapy, without manipulation to form a chimeric antibody.

The human constant region may be obtained by standard techniques, with any desired isotype well known to those of ordinary skill in the art, and the variable region of a monkey antibody ligated with that human constant region.

Particularly useful chimeric antibodies against specific cell surface receptors which can be used in immunotherapy of humans include CD4, ICAMS, CD19, CD20, CD8, CD11a, CDllb, CD28, CD18, CD45, CD71, and TCR.

Example 3: Cloning and Expressing a MonkevjHuman Chimeric Antibody with Specificity for CD4 The following is a specific example of the methods and antibodies of this invention.

Generation of Monkey Immortalized B-cell Lines An adult cynomolgus monkey (White Sands New Mexico Primate Center) was immunized intramuscularly, at.multiple sites, with 150—300pg of soluble CD4 (SCD4) or cell mem- branes (1 x 108 cells) from the CD4 positive cell line supT1 using a standard adjuvant. Immunization was repeated every 2-3 weeks a total of six times. The monkey was boosted by injection of loopg of sCD4 into the inguinal region of one thigh and one week later the drain- ing lymph node from the same thigh surgically removed.

Lymphocytes were removed from the lymph node by slicing the tissue and rinsing with sterile DMEM medium. The cell suspension was passed through a nylon gauze and collected by centrifugation at 1000 x g for 10 minutes.

Approximately 1 x 103 lymphocytes were suspended in Tris—ammonium chloride buffer (16mM, pH 7.5) and warmed to 37°C for 5 minutes to lyse the erythrocytes. Lymphocytes were collected by centrifugation and resuspended in L-leu- cine methyl ester (LME) and incubated at 37°C for 45 min- The LME treated cells were filtered through a nylon 1ml of fetal calf serum was utes. screen and centrifuged. added, the cells suspended and washed twice in serum-free RPMI. The cells were counted and mixed into a single 50ml conical centrifuge tube together with an equal number of K6H6/B5 heteromyeloma cells, prewashed twice in serum free medium. Cells were gently suspended in 1 ml of 50% PEG (polyethylene glycol) added slowly with gentle stirring over a 1 minute period. The cells were then resuspended by the addition of 20ml of serum-free medium over a 5 min- ute period, with gentle mixing to dilute out the PEG.

After washing twice with serum-free medium cells were resuspended at a concentration of 5 x 105/0.1 ml in RPMI medium, containing 20% fetal calf serum and gentamycin and placed into 96 well micro tissue culture plates at 0.1 ml per well. An equal volume of HAT medium (0.1 ml) was added to each well and the hybrids allowed to grow for -17 days before screening.

Screening of Fused Cell Hybrids for the Production of Anti-CD4 The assay to determine anti-CD4 specificity was as follows: ELISA plates were coated with recombinant sCD4 at a concentration of loong per well and blocked with 1% bovine serum albumin in PBS. 50 pl aliquots of hybridoma supernatant were removed from each well and allowed to incubate with the sCD4 coated plates for 60 minutes.

Binding was detected by incubation with ‘El labeled goat anti—human or goat anti-monkey Ig for 60 minutes. After washing four times with distilled water, the wells were counted in a gamma counter. Positive wells were re- assayed in duplicate and the hybridoma cells from those wells subcloned three times, first at 5 cells per well then twice at 1 cell per well. At this stage anti-SCD4 positives were screened for the ability to bind to cell surface CD4. This was done by inhibition of binding of an anti-CD4 murine monoclonal, termed 1F3, to the CD4 posi- tive cell line supT1. Briefly this was done by co-incu- bating different amounts of monkey anti—CD4 and long of "51-labeled 1F3 with 3 x 105 supT1 cells/well in a 96 well plate. After incubation for 1 hour at room temperature (about 20-25°C) cells were removed by vacuum onto glass fiber filters. After extensive washing with PBS the fil- ters were counted in a gamma counter to determine the inhibition of 1F3 binding to supT1 cells by the monkey hybridoma supernatants.

A candidate clone was chosen which produced an anti- body that showed strong inhibition against lF3. The clone we chose was isotyped using human isotyping reagents and found to be an IgG2 possessing a lambda light chain. This cell line was grown up to larger numbers for cloning of its immunoglobulin genes.

Cloning of Heayy and Light Chain Variable Re ion Genes om Mo ke Immorta ‘zed B-cells Total RNA was isolated from 1 x 107 monkey immortal- ized B—cells using the guanidinium isothiocyanate method described above. One tenth of the total RNA was used to make single stranded cDNA using an oligo-dT oligonucleo- tide primer and reverse transcriptase, also as described One tenth of the amount of ss cDNA was used to set The six PCR reactions each included one above. up PCR reactions. of six 5' V; family specific oligonucleotide primers con- taining a gal I restriction site together with an IgG 3' constant region oligonucleotide containing an Egg I site, both shown in Figure 7-1. Similarly, five PCR reactions, utilizing one of five 5' lambda leader sequence oligonu- cleotide primers containing a fig; II site and a 3' lambda constant region prime containing an gyr II site, were run.

Reaction conditions were as described above. Each PCR reaction was run in triplicate. The products of each of the heavy chain and light chain amplification reactions were run on 1.2% agarose gels. The VH4 heavy chain primer (SEQ. ID. NO.: 13: 5' ACTAAGTCGACATGAAACACCTGTGGTTCTT 3') and lambda primer (SEQ. ID. NO.: 14: (5' ATCACAGATCTCTCAC CATGACCTGCTCCCCTCTCCTCC 3') gave strong bands on agarose gel electrophoresis. The products of these reactions were used for cloning into the vector TCAE 6, which contains human IgG1 and human lambda constant region sequences.

Cloning of the two variable region genes into the expression vector TCAE 6 was done sequentially. First, the heavy chain PCR product and the vector TCAE 6 were digested with the restriction enzymes gal I and Egg I, the products extracted with phenol/chloroform, and passed through a SEPHADEX G-25 spin column. The PCR product was ligated to the cut vector overnight at 14°C in the pres- ence of T4 DNA ligase. Approximately 500ng total DNA was ligated in.a volume of lopl with an insert/vector molar ratio of 10:1. Ligated material was used to transform XL-1 Blue competent cells (Stratagene) and the transformed cells plated onto LB agar plates containing Soug/ml ampi- Colonies of ampicillin resistant bacteria were Plasmid DNA was cillin. picked and grown as Sml minicultures. extracted from each of these cultures by a standard alka- line lysis method cut with the restriction enzymes gal I and flhg I and the products run on a 1.2% agarose gel.

Plasmids with inserts of approximately 450bp were used as templates for the subsequent cloning of light chain vari- able regions. The products of the light chain PCR reac- tion as well the plasmid containing the heavy chain insert were cut with the restriction enzymes ggl II and Ag; II and ligated together. Plasmid minicultures were screened by cutting with ggl II and Ag; II. Digests giving an insert of approximately 400-450 bp were scored positive.

Plasmids containing both gal I/flag I and ggl II/Ag; II inserts were grown up in larger quantities for DNA sequencing.

The tandem chimeric antibody expression vectors TCAE .2 and TCAE 6 were derived from the vector CLDN, which itself is a derivative of the vector RLDN10b (253 Science 77—79,1991). RLDNl0b in turn is a derivative of the expression vector TND (7 Qgg 651-661, 1988) RLDN10b differs from the vector TND in the following ways. The dihydrofolate reductase (DHFR) transcriptional cassette (promoter, CDNA, and polyadenylation region) was placed inbetween the tissue plasminogen activator cassette (t-PA expression cassette) and the neomycin phosphotrans— ferase (NED) cassette so that all three cassettes are in tandem and in the same transcriptional orientation. In addition, the DHFR.gene promoter in CLDN has been replaced by the mouse beta globin major promoter (3 gal. Cell Biol. 1246-54, 1983) and the t-PA CDNA replaced by a polylinker.

All three eukaryotic transcriptional cassettes (Expression , DHFR, NEO) can be separated from the bacterial plasmid DNA (pUC9 derivative) by digestion with the restriction endonuclease NotI.

CLDN differs from RLDN10b because the Rous LTR in front of the polylinker has been replaced by the human cytomegalovirus immediate early gene promoter enhancer (41 Cell, 521, 1985).

The expression vectors TCAE 5.2 and TCAE 6 differ from CLDN in that: 1) They contain four transcriptional (instead of three), in tandem order: (a) A.human immunoglobulin light chain constant region derived via amplification of CDNA by a polymerase In TCAE 5.2 this is the human immunoglo- cassettes chain reaction. bulin light chain kappa constant region (Kabat numbering amino acids 108-214, allotype Km3), and in TCAE 6 the human immunoglobulin light chain lambda constant region (Kabat numbering amino acids 108-215, genotype Oz minus, Mcg minus, Ke minus allotype) (b) A human immunoglobulin heavy chain constant region; in both constructs the human immunoglobulin heavy chain was a gamma 1 constant region (Kabat numbering amino acids 114-478 allotype Gmla, Gmlz), which was derived via amplification of cDNA by a polymerase chain reaction. (c) DHFR; containing its own eukaryotic pro- moter and polyadenylation region. (d) NEO; also containing its own eukaryotic promoter and polyadenylation region.

) The human immunoglobulin light and heavy chain cassettes contain synthetic signal sequences for secretion of the immunoglobulin chains ) The human immunoglobulin light and heavy chain cassettes contain specific DNA linkers which allow for insertion of light and heavy immunoglobulin variable regions which maintain the translational reading frame and do not alter the amino acids normally found in immunoglo- bulin chains. The incorporation of the changes described, led to the construction of the vectors TCAE 5.2 and TCAE 6. The cloning of the immunoglobulin light and heavy variable region genes, from the anti-CD4 heterohybridoma cell line E9.1, into TCAE 6 led to the construct which is deposited in the ATCC. The construct, which has been deposited, contains the cynomolgus monkey immunoglobulin heavy chain variable region and cynomolgus monkey immuno- globulin light chain variable region, whose sequences are shown in Figures 13 and 14 respectively, cloned from the anti-CD4 hybridoma cell line E9.1. The heavy chain con- stant region is of human origin of the gamma 1 isotype and Gmla, Gmlz allotype. The lambda light chain constant region is also of human origin, of the Oz minus, mcg minus genotype and Ke minus allotype. The immunoglobulin genes are cloned into the mammalian expression vector TCAE 6, shown in Figure 6, which, when electroporated into the mammalian cell line CHO produced a monkey/human anti-CD4 chimeric antibody. The DNA construct described herein, has been used to transform the bacterial strain XL-1 Blue, selected in the antibiotic ampicillin and deposited as a bacterial cell suspension in sterile LB medium containing % glycerol.

Another useful expression system is one in which the gene encoding a selective marker is modified to enhance yield of recombinant systems encoding a desired sequence.

For example, translation initiation impairment of a domi- nant selectable marker results in fewer drug resistant colonies compared to a non-impaired vector, but each indi- vidual colony expresses significantly higher levels of colinked gene product than in the unimpaired vector. For example, translation initiation is the first step in pro- tein synthesis. The translation initiation site of the neomycin phosphotransferase gene (G418 resistance gene) was changed from a consensus Kozak (sequence — ccAccATGG) to a poor Kozak (sequence — ccTccATGC). Translational initiation impairment of the G418 resistance gene resulted in: 1) a significant (5 fold) reduction in the number of G418 resistant colonies obtained from a same amount of plasmid DNA transfected per cell, and 2) a significant .increase in the amount of colinked product gene expressed in each clone. In the clones containing the consensus Kozak 73% of the colonies screened produced less than 25ng/ml, with only 3% producing greater than 100 ng/ml.

For clones with the altered, poorer Kozak, 8% of the colonies screened produced less than 25 ng/ml, compared with 63% of colonies producing greater than 100 ng/ml.

Specifically, referring to Fig. 16 (where TCAE 5.2 has a consensus Kozak, and TCAE 12 has a poorer Kozak), 258 colonies were derived from 2 electroporations of 25 pg of DNA which contains a neomycin phosphotransferase gene with a consensus (unchanged) translation start site. 201 of these colonies (78%) did not express any detectable gene product (i.e., <25 ng/ml of chimeric immunoglobulin), and only 8 colonies (3%) expressed more than 100 ng/ml. 98 colonies were derived from 6 electroporations of 25 pg of DNA which contains the neomycin phosphotransferase gene with an altered translation start site. 63% of these colonies were expressing more than 100 ng/ml, and only 8% of these colonies expressed less than 25 ng/ml.

DNA Seguencigg Plasmid DNA was prepared from 100ml cultures. further purified by precipitating (1 volume) with a mix- ture of 2.5M sodium chloride and 20% polyethylene glycol After centrifugation It was (6 volumes) on ice for 15 minutes. at 10,000 x g for 20 minutes, the pellet was washed with 70% in a Speedivac (Savant). water at a concentration of 150-250 pg/ml. carried out on Spg of double stranded DNA using the tech- ethanol, recentrifuged and dried The pellet of DNA was resuspended in deionized Sequencing was nique of Sanger. Sequencing primers which were homologous to sequences within the expression vector upstream and downstream of either the light chain or heavy chain The inserts were sequenced in both 5' Two clones of anti—CD4 inserts were used. to 3' and 3'_to 5' directions. light chain and two clones of anti-CD4 heavy chain each generated from separate PCR reactions were sequenced in parallel in order to determine whether any nucleotide changes had been introduced during the PCR reaction. Both of the chosen heavy chain and both light chain clones were found to be identical over their entire length, confirming that no errors had been introduced during the amplifica- tion process. The sequence of the anti-CD4 heavy and light chains are shown in Figures 13 and 14 and Sequence Listing Nos.: 15 and 16.

Expression of Monkey(fluman Chimeric Anti-CD4 The expression vector TCAE 5.2 and TCAE 6 are not only able to be used for stable integrated expression into the cell lines Spz/0 and CH0 but, because it includes the is also able to be expressed transiently in COS cell expression was performed as SV40 origin, the cell line COS. follows: COS cells were seeded one day before the trans- fection so that they would be 50-70% confluent the follow- Culture medium was removed and the cells washed ing day. twice with Transfection Buffer (TB — 140m.M Nacl, 25mM Tris, 5mM Kcl, O.5mM Naguxr 1mM Mgcly 1mM Caclfl. 30 ug of cesium chloride purified TCAE 6 plasmid containing the anti-CD4 monkey/human chimeric heavy and light immunoglo- bulin chains were mixed with 3ml of DEAE dextran per dish The DNA was allowed to incubate with the DNA solution was removed and (1 mg/ml in TB). cells for 1 hour at 37°C. replaced with 3ml of 20% glycerol for 1.5-2.5 minutes, after which the cells were twice washed with TB. Cells were incubated in Sml of fresh medium containing lOouM chloroquine for 3-5 hours at 37°C, after which they were washed twice with medium and incubated with normal DMEM for 72 hours. Supernatant (loopl) from the transfected COS cells was assayed at various dilutions for the pres- ence of antibody by an ELISA-based technique. Goat anti- human lambda was used to coat 96 well assay plates and a peroxidase—labeled goat anti-human IgG as the detection antibody, under standard ELISA conditions. cos cells were found to produce between 10 and 40 ng/ml of monkey/human chimeric antibody. Larger volumes of supernatant were concentrated 10 fold and used in a direct binding RIA to CD4 positive supT1 cells. The parental whole monkey anti- body and an irrelevant human immunoglobulin were used as a positive and negative controls respectively (Fig. 11) .

Furthermore, the monkey anti-CD4 and the monkey/human chimeric anti-CD4 were used to inhibit the binding of a high affinity mouse anti-CD4 (1F3) antibody (Fig. 12). It can be seen that the monkey/human recombinant antibody not only binds to CD4 positive cells but is able to inhibit the binding of lF3 to CD4 positive cells in approximately the same concentrations of wholly monkey antibody or lF3 itself.

The following is an example of the methods and antibodies of this invention.

An adult cynomolgus monkey (White Sands New Mexico Primate Center) was immunized intramuscularly, at multiple sites, with 5 x 108 whole CD54 positive human lymphocytes.

SB cells (human B lymphoid acti- Cells used were alternately, line) and activated peripheral human lymphocytes, vated by pre—incubation with a mixture of pokeweed mitogen (2.5mg/ml), phorbol monoacetate (40 nM) and phytohemagglu- tinin (4mg/well) with the inclusion of a standard adju- Immunization was repeated every 2-3 weeks over a Sera from the immunized animals were vant. period of 8 months. screened at various times by inhibition of binding of a murine antibody, 84H10, known to bind to ICAM-1. A satur- ating amount of 84Hl0 was bound to Chinese hamster ovary cells (CHO), previously transfected with an expression vector containing human CD54 c-DNA and selected for high expression of cell surface CD54, together with increasing dilutions of monkey serum. The inhibition of 84Hl0 binding is shown in FIG. 15. other murine monoclonal antibodies which recognize other human lymphocyte antigens were tested by inhibition using monkey sera obtained by the same immunization methods.

Antibodies produced in the manner described above, or by equivalent techniques, can be purified by a combination of affinity and size exclusion chromatography for charac- terization in functional biological assays. These assays include determination of specificity and binding affinity as well as effector function associated with the expressed isotype, e.g., ADCC, or complement fixation. such anti- bodies may be used as passive or active therapeutic agents against a number of human diseases, including B cell infectious diseases including AIDS, and transplantation. autoimmune The lymphoma, and inflammatory diseases, antibodies can be used either in their native form, or as part of an antibody/chelate, antibody/drug or antibody/toxin complex. Additionally, whole antibodies may be used as imaging reagents or as potential vaccines or immunogens in active immunotherapy for the generation of anti—idiotypic responses.

The amount of antibody useful to produce a therapeu- tic effect can be determined by standard techniques well known to those of ordinary skill in the art. The anti- bodies will generally be provided by standard technique within a pharmaceutically acceptable buffer, and may be administered by any desired route. Because of the effi- cacy of the presently claimed antibodies and their toler- ance by humans it is possible to administer these anti- bodies repetitively in order to combat various diseases or disease states within a human.

The anti—CD4 recombinant antibodies of this invention are also useful for inducing immunosuppression, ;;e;, inducing a suppression of a human's or animal's immune system. fore relates to a method of prophylactically or therapeu- This invention there- tically inducing immunosuppression in a human or other animal in need thereof by administering an effective, non- toxic amount of such an antibody of this invention to such human or other animal.

The ability of the compounds of this invention to e immunosuppression may be demonstrated in standard for example, a mixed lympho- ibition of induc tests used for this purpose, cyte reaction test or a ‘test. measuring inh T—cell proliferation measured by thymidine uptake.

The fact that the antibodies of this invention have n means that they are utility in inducing immunosuppressio n of resistance to or useful in the treatment or preventio rejection of transplanted organs or tissues (g;g;, kidney, bone marrow, skin, cornea, gtg.); the treat- mmune, inflammatory, prolifera- and of cutaneous heart, lung, ment or prevention of autoi tive and hyperproliferative diseases, manifestations of immunologically medicated diseases (elgé, rheumatoid arthritis, lupus erythematosus, systemic Hashimotos thyroiditis, multiple type 1 diabetes, lupus erythematosus, sclerosis, myasthenia gravis, nephrotic syndrome, psoriasis, atopical dermatitis, con- dermatitides, uveitis, tact dermatitis and further eczematous seborrheic dermatitis, Lichen planus, Pemplugus, bullous pemphigus, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythema, cutaneous eosinophilias, Alopecia the treatment of reversible obstructive intestinal inflammations and allergies proctitis, eosinophilia gastro- Crohn's disease and ulcerative one skilled in the art would be able, experimentation, to determine what an effective, non-toxic amount of antibody would be for the purpose of inducing an effective by routine immunosuppression. Generally, however, dosage will be in the range of about 0.05 to 100 milli- grams per kilogram body weight per day.

The antibodies of this inven- tion should also be useful for treating tumors in a mam- mal. More specifically, they should be useful for reduc- ing tumor size, inhibiting tumor growth and/or prolonging the survival time of tumor-bearing animals. Accordingly, this invention also relates to a method of treating tumors in a human or other animal by administering to such human or animal an effective, non-toxic amount of an antibody.

One skilled in the art would be able, by routine experi- mentation, to determine ‘what an effective, non-toxic amount of antibody would be for the purpose of treating carcinogenic tumors. Generally, however, an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day.

The antibodies of the invention may be administered to a human or other animal in accordance with the afore- mentioned methods of treatment in an amount sufficient to produce such effect to a therapeutic or prophylactic degree. Such antibodies of the invention can be adminis- tered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administra- tion and other well—known variables.

The route of administration of the antibody of the invention may be oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. The subcutane- ous and intramuscular forms of parenteral administration are generally preferred.

The daily parenteral and oral dosage regimens for employing compounds of the invention to prophylactically or therapeutically induce immunosuppression, or to thera- peutically treat carcinogenic tumors will generally be in the range of about 0.05 to 100, but preferably about 0.5 to 10, milligrams per kilogram body weight per day.

The antibody of the invention may also be adminis- By "inhalation" is meant intranasal Appropriate dosage tered by inhalation. and oral inhalation administration. forms for such administration, such as an aerosol formula- tion or a metered dose inhaler, may be prepared by conven- tional techniques. The preferred dosage amount of a com- pound of the invention to be employed is generally within the range of about 10 to 100 milligrams.

The antibody of the invention may also be adminis- tered topioally. By topical administration is meant non- systemic administration and includes the application of an antibody compound of the invention externally to the epidermis, to the buccal cavity and instillation of such an antibody into the ear, eye and nose, and where it does not significantly enter the blood stream. By systemic administration is meant oral, intra- venous, intraperitoneal and intramuscular administration.

The amount of an antibody required for therapeutic or prophylactic effect will, of course, vary with the anti- body chosen, the nature and severity of the condition being treated and the animal undergoing treatment, and is ultimately at the discretion of the physician. A suitable topical dose of an antibody of the invention will gener- ally be within the range of about 1 to 100 milligrams per kilogram body weight daily.

Formulations While it is possible for an antibody to be The topical formulations of the present invention, comprise an active ingredient together with one or more therefor and optionally any other The carrier(s) must be The active acceptable carrier(s) therapeutic ingredients(s). "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. 7 Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treat- ment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suit- able aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and prefer- ably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and steril- ized by autoclaving or maintaining at 90°-100°C for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those.for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as Castor oil or arachis oil.

Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingre- dient for external application. They may be made by mix- ing the active ingredient in finely—divided or powdered form, alone or in solution or suspension in an aqueous or with the aid of suitable machinery, non-aqueous fluid, The basis may comprise with a greasy or non-greasy basis. hydrocarbons such as hard, soft or liquid paraffin, gly- a metallic soap; a mncilage; an oil of arachis, cerol, beeswax, natural origin such as almond, corn, olive oil; wool fat or its derivatives, or a fatty acid castor or such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols. The formulation may incorporate any suitable surface active agent such as. an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof.

Suspending agents such as natural gums, cellulose deriva- tives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included . ' It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of an antibody or fragment thereof of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular animal being treated, and that such opti- mums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of the invention given of an antibody per day for a defined number of days, by those skilled in the art using conventional course of can be ascertained treatment determination tests.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following are, therefore, to be construed as merely illus- trative examples and.not a limitation of the scope of the present invention in any way.

Capsule Composition A pharmaceutical composition of this invention in the form of a capsule is prepared by filling a standard two- piece hard gelatin capsule with 50 mg. of an antibody of the invention, in powdered form, 100 mg. of lactose, mg. of talc and 8 mg. of magnesium stearate.

Injectable Parenteral Composition A pharmaceutical composition of this invention in a form suitable for administration by injection is prepared by stirring 1.5% by weight of an antibody of the invention in lO% by volume propylene glycol and water. The solution is sterilized by filtration.

Ointment Composition Antibody of the invention 1.0 g.

White soft paraffin to 100.0 g.

The antibody of the invention is dispersed in a small volume of the vehicle to produce a smooth, homogeneous product. Collapsible metal tubes are then filled with the dispersion.

Topical Cream Composition Antibody of the invention l.O g.

Polawax GP 200 20.0 g.

Lanolin Anhydrous 2.0 g.

White Beeswax 2.5 g.

Methyl hydroxybenzoate 0.1 g.

Distilled Water to 100.0 g.

The polawax, beeswax and lanolin are heated together at 60°C. A solution of methyl hydroxybenzoate is added and homogenization is achieved using high speed stirring.

The temperature is then allowed to fall to 50°C. The antibody of the invention is then added and dispersed throughout, and the composition is allowed to cool with slow speed stirring.

Topical Lotion Composition Antibody of the invention 1.0 g.

Sorbitan Monolaurate 0.6 g.

Polysorbate 20 0.6 g.

Cetostearyl Alcohol 1.2 g.

Glycerin 6.0 g.

Methyl Hydroxybenzoate 0.2 g.

Purified water 3.9. to 100.00 ml. (3.9. = British Pharmacopeia) The methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of the water at 75°C. The sorbitan monolaurate, polysorbate 20 and cetostearyl alcohol are melted together at 75°C and added to the aqueous solution. The resulting emulsion is homogenized, allowed to cool with continuous stirring and the antibody of the invention is added as a suspension in the remaining water.

The whole suspension is stirred until homogenized.

Eye Drop Composition Antibody of the invention 0.5 g.

Methyl Hydroxybenzoate 0.01 g.

Propyl Hydroxybenzoate 0.04 g.

Purified Water B.P. to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml. purified water at 75°C and the resulting solu- The antibody or fragment thereof and the solution is ster- tion is allowed to cool. of the invention is then added, ilized by filtration through a membrane filter (0.022 um pore size), and packed aseptically into suitable sterile containers.

Composition for Administration by Inhalation For an aerosol container with a capacity of 15-20 ml: of an antibody of the of a lubricating agent, such as mix 10 mg. invention with 0.2-0.5% polysorbate 85 or oleic acid, and disperse such mixture in such as freon, preferably in a combination and difluorochloro- a propellant, of (1,2 dichlorotetrafluoroethane) methane and put into an appropriate aerosol container either intranasal or oral inhalation adapted for administration.

Composition for Adminstration by Inhalation For an aerosol container with a capacity of 15-20 ml: dissolve 10 mg. of an antibody of the invention in ethanol (6-8 ml.), add 0.1-0.2% of a lubri- cating agent, such as polysorbate 85 or oleic acid; and disperse such in a propellant, such as freon, preferably in combination of (1.2 dichlorotetrafluoroethane) and difluorochloromethane, and put into an appropriate aerosol container adapted for either intranasal or oral inhalation administration.

The antibodies and pharmaceutical compositions of the invention are particularly useful for parenteral adminis- i.e., subcutaneously, intramuscularly or intra- The compositions for parenteral administration antibody tration, venously. will commonly of the invention solved lJ1 an acceptable carrier, A variety of aqueous carriers may be employed, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions may be ster- ilized by conventional, well-known sterilization tech- The compositions may contain pharmaceutically comprise a solution of an cocktail thereof dis- preferably an aqueous Or a carrier. -3.-_, Water. niques. acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffer- ing agents, etc. The concentration of the antibody or fragment thereof of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about .5%, usually at or at least about 1% to as much as 15 or % by weight, and will be selected primarily based on fluid volumes, viscosities, etc., according to the parti- cular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and 50 mg. of an antibody or fragment thereof of the invention. similarly, a pharma- ceutical composition of the invention for intravenous The antibodies of the inven- tion can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilization and reconstitution techniques can be employed.

Depending on the intended result, the pharmaceutical composition of the invention can be administered for pro- phylactic and/or therapeutic treatments. In therapeutic application, compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance. single or multiple administrations of the pharmaceu- tical compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical composition of the invention should provide a quantity of the altered antibodies of the invention sufficient to effectively treat the patient.

It should also be noted that the antibodies of this invention may be used for the design and synthesis of either peptide or non-peptide compounds (mimetics) which would be useful in the same therapy as the antibody. fieg, g;g;, Saragovi et al., Science, ggg, 792-795 (1991). (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: CCATGGACTG GACCTGG (3) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: ATGGACATAC TTTGTTCCAC (4) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: LENGTH: 20 TYPE: NUCLEIC ACID STRANDEDNESS: single TOPOLOGY: Linear (A) (B) (C) (13) (xi) SEQUENCE DESCRIPTION : SEQ ID NO: CCATGGAGTT TGGGCTGAGC (5) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: LENGTH: 20 TYPE: NUCLEIC ACID STRANDEDNESS: Single TOPOLOGY: Linear (A) (B) (C) (D) (xi) SEQUENCE DESCRIPTION : SEQ ID NO: ATGAAACACC TGTGGTTCTT : : : (6) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: NUCLEIC_ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO:-5: ATGGGGTCAA CCGCCATCCT 20 (7) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 6: ATGTCTGTCT CCTTCCTCAT 20 (8) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: 7: TTGGGGCGGA TGCACT 16 (9) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 8: GATGGGCCC TTGGTGGA ~ 17 (10) INFORMATION FOR SEQ ID NOE 9; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: 9: GATGACCCAG TCTCCAGCCTC 21 K (11) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: 10: CTCACTTGCT GCACAGGGTCC 21 Y VR M (12) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION : SEQ ID N0: 11: AAGACAGATG GTGCAGCCA 19 (13) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: 12: GGAACAGAGT GACCGAGGGG 20 (14) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 13: ACTAAGTCGA CATGAAACAC CTGTGGTTCT T 31 (15) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 14: ATCACAGATC TCTCACCATG ACCTGCTCCC CTCTCCTCC 39 (16) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 423 (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: 15: GAC ATG AAA CAC CTG TGG TTC TTC CTC CTC CTG GTG GCA GCC CCC AGA 48 TGG GTC TTG TCC CAG GTG CAG CTG CAG GAG GCG GGC CCA GGA CTG GTG 96 AAG CCT TCG GAG ACC CTG TCC CTC ACC TGC AGT GTC TCT GGT GGC TCC 144 ATC AGC GGT GAC TAT TAT TGG TTC TGG ATC CGC CAG TCC CCA GGG AAG 192 GGA CTG GAG TGG ATC GGC TAC ATC TAT GGC AGT GGT GGG GGC ACC AAT 240 TAC AAT CCC TCC CTC AAC AAT CGA GTC TCC ATT TCA ATA GAC ACG TCC 288 AAG AAC CTC TTC TCC CTG AAA CTG AGG TCT GTG ACC GCC GCG GAC ACG 336 GCC GTC TAT TAC TGT GCG AGT AAT ATA TTG AAA TAT CTT CAC TGG TTA 3 TTA TAC TGG GGC CAG GGA GTC CTG GTC ACC GTC TCC TCA (17) INFORMATION FOR SEQ ID NO: (i) SEQUENCE CHARACTERISTICS: (Xi) SEQUENCE DESCRIPTION : SEQ ID NO: 16: ACC ATG GAC TCT TCC CCA AGG AAA CTG GTC TTC TCT GTC GAG ACT GCT (A) (B) (C) (D) TGG GCC CAG GTA TAT TCC GGG LENGTH: TYPE: NUCLEIC ACID STRANDEDNESS: Single TOPOLOGY: GCT TCC ACG CAG GCT AAC GAT CTG TAT GCC TGG GAC TCA GAG CTG GAG GGG TAC AGC GGG GCT CTC TTG TTC CAG GAA AAC GAC Linear CTC AGT ACC CAG CGG ACC TAT GGC CTC CTT CAG CCT CGC TGT GGG GGA AAG CCA CCG CCC TCA GGG GCC ACC CTG TAC TGT CAG GGG ACC CGG GCT CAC TCA GTG GAC AAC CAG GCC ATC CCT ACC ATC GTG TGG CTG ACC TCC GTT CCT GCG AGC GAC ACA GTG GGA GTG CGA GGG AGT 192 240 288 336 384

Claims (5)

1. A chimeric antibody that specifically binds to a human antigen, wherein the antibody is not immunogenic in a human, is not the same as a human or chimpanzee antibody and comprises the whole of the variable region of an immunoglobulin of an Old World monkey selected from rhesus monkeys, cynomolgus monkeys and baboons and the constant regions of a human or chimpanzee immunoglobulin, wherein said chimeric antibody is obtainable by a method comprising the steps of: raising an Old World monkey antibody to a human target antigen in an Old World monkey selected from rhesus monkeys, cynomolgus monkeys, and baboons, isolating an Old World monkey nucleic acid encoding the whole Variable region of said Old World monkey antibody, providing a human or chimpanzee nucleic acid encoding a human constant region of a human antibody, ligating said Old World monkey nucleic acid and said human or chimpanzee nucleic acid to form a recombinant nucleic acid, and expressing said recombinant nucleic acid to produce said chimeric antibody.

2. A chimeric antibody according to claim 1, that is therapeutically effective when administered at a dosage of from 0.05mg to 100mg/kg body weight.

3. A chimeric antibody according to claim l or 2,V comprising constant regions of immunoglobulin heavy and light chains of~a human antibody and variable regions of immunoglobulin heavy and light chains of an Old World monkey antibody specific to a human antigen.

4. A chimeric antibody according to any one of the preceding claims, which specifically binds to a human antigen chosen from CD58, VCAM, VLA4, CD2, LFA3, ELAM, LAM, CD25, CD4, CD19, CD20, CD23, CD41, CD44, CD54, TNFO, TNFB, Tn antigen, IL-1, IL-8, human T cell receptor, CD3, CD28, cos, CDl1a, CDllb, CDl1c, CD18, CD5a, c045, neu oncogene product, MDR—l, TGFd, TGFd receptor, PDGF and CD71.

5. A chimeric antibody according to claim 4, that specifically binds CD4 and which comprises immunoglobulin variable light and heavy chains comprising the peptide sequences shown in