HK1186479B - Antibodies specific for human cd22 and their therapeutic and diagnostic uses - Google Patents

Description

Human CD 22-specific antibodies and therapeutic and diagnostic uses thereof

The application is a divisional application of an invention patent application with the patent application number of 200710085290.4, the application date of 5/2/2003 (according to the application number of PCT/GB2003/001934) and the same name of the invention.

Technical Field

The present invention relates to an antibody molecule specific for an antigenic determinant of the B lymphocyte antigen (CD 22). The invention also relates to the therapeutic use of said antibody molecules and to a method for producing said antibody molecules.

Background

In a natural antibody molecule, there are two heavy chains and two light chains. Each heavy chain and each light chain has a variable region at its N-terminus. Each variable region consists of 4 Framework Regions (FR) and alternating 3 Complementarity Determining Regions (CDR). Typically, the residues in the variable domains are numbered according to the system of Kabat et al. This system was proposed in "sequence of proteins of immunological interest" in 1987 by Kabat equivalent to nihsa (hereinafter referred to as "Kabat et al (vide supra)") of the united states department of health and human services. Unless otherwise stated, this numbering system is used in this specification.

Kabat residue designations often do not coincide with linear numbering of amino acid residues. The actual linear amino acid sequence, whether framework regions or CDRs, may contain fewer or additional amino acids than the strict Kabat numbering, which correspond to truncated or inserted structural components in the basic variable region structure. For a given antibody, the correct Kabat numbering of residues can be determined by alignment with homologous residues in the antibody sequence having the "standard" Kabat numbering sequence.

The CDRs of the heavy chain variable region are located at residues 31-35(CDR-H1), residues 50-65(CDR-H2) and residues 95-102(CDR-H3) according to Kabat numbering.

The CDRs of the light chain variable region are located at residues 24-34(CDR-L1), residues 50-56(CDR-L2) and residues 89-97(CDR-L3) according to Kabat numbering.

The construction of CDR-grafted antibodies (CDR-grafted antibodies) is described in European patent application EP-A-0239400, which discloses ｃA method in which the CDRs of ｃA mouse monoclonal antibody are grafted to the framework regions of the human immunoglobulin variable regions by site-directed mutagenesis using long oligonucleotides. The CDRs determine the antigen-binding specificity of the antibody and are relatively short peptide sequences carried on the framework regions of the variable regions.

The earliest work on humanized (humanizing) monoclonal antibodies by CDR grafting was performed on monoclonal antibodies recognizing synthetic antigens such as NP. However, Verhoeyen et al (Science,2391534-1536,1988) and Riechmann et al (Nature,332323-324,1988) describe examples in which a mouse monoclonal antibody recognizing lysozyme and a rat monoclonal antibody recognizing an antigen on human T cells were humanized by CDR-grafting, respectively.

Riechmann et al found transfer of individual CDRs in CDR grafts (as defined by Kabat et al (see above) and Wu et al, J.Exp.Med.,132211-250,1970) is insufficient to provide satisfactory antigen-binding activity. It has been found that many framework residues must be altered in order to make them correspond to those of the donor framework region. International patent application No. WO90/07861 describes proposed criteria for selecting framework residues that require alteration.

Numerous reviews have been published discussing CDR-grafted antibodies, including Vaughan et al (Nature Biotechnology,16,535-539,1998)。

malignant lymphoma is a highly variable class of tumors. Most of the cases occur in the elderly. Currently, 200,000 to 250,000 patients in the united states suffer from Non-hodgkin lymphoma (NHL). The disease is the second fastest growing cancer in the united states, increasing at a new rate of about 55,000 cases per year. The increased incidence of the disease has not been explained simply by the growing population of the elderly and exposure to known risk factors.

The classification of lymphomas is complex and has progressed in the last decade. The revised european-american lymphoma (REAL) classification was adopted in 1994. This classification has classified B-cell lymphomas (most commonly recognized), T-cell lymphomas, and primary lymphomas that cannot be classified into recognized subtypes. In common practice, the classification of NHLs into low, moderate and high grade according to their general histological appearance reflects their clinical characteristics well.

NHL mainly affects lymph nodes, but in individual patients tumors can involve other anatomical sites, such as liver, spleen, bone marrow, lung, intestine and skin. The disease is usually manifested as an indolent enlargement of the lymph nodes. Extranodal lymphomas (extranodalymphomas) most commonly affect the gut, although in practice primary lymphomas have been described for every organ. Systemic symptoms include fever, night sweats, weakness and weight loss.

Until recently, the annorbor classification system, which is based entirely on the anatomical extent of the disease, became the major determinant of NHL treatment. This information can be refined by incorporating additional prognostic indicators, including age, serum lactate dehydrogenase levels, and performance. Even so, knowledge of the ann arbor classification system, together with the histological and immunological subtype of the tumor, remains a major determinant of treatment.

Low-grade NHL has an indolent course, with patients surviving 8-10 years on average. Currently available treatments have little impact on survival, although radiation therapy of localized disease and chemotherapy of systemic symptoms improve the quality of life of patients. Combination chemotherapy may be prepared for recurrent disease. Moderate diseases and especially high-grade diseases are very aggressive and easily spread. This level of disease requires urgent treatment. In patients with very severe disease, radiation therapy can be an effective component of treatment. Many different chemotherapeutic regimens have been used and long-term disease-free survivors can be obtained in more than half of the patients. For patients with relapsed or refractory disease, stem cell-supported high dose therapy was initially used, but now there is room for first-line therapy for patients with low risk disease. In recent years, with the trend toward aggressive treatment, the general elderly and relatively infirm of many NHL patients must be balanced and there is a need to match the toxicity of the treatment to the individual prognosis of each patient's disease.

There is a need for improved treatments that are more effective and more tolerable. The most recently used drugs include new cytotoxic drugs that are gradually added to the combination, and antibody-based therapies are employed.

Non-hodgkin's lymphomas include a wide variety of B cell lymphomas. Thus, B cell antigens are suitable targets for antibody therapy.

CD22 is a 135kDa membrane glycoprotein and belongs to a family of sialic acid binding proteins called sialoadhesins. It is detected in the cytoplasm early in B cell development, appears on the cell surface simultaneously with IgD, and is present on most mature B cells. Expression increases with B cell activation. CD22 is lost with terminal differentiation and is generally reported to be absent on plasma cells. Thus, the internalizing antigen is present on the surface of pre-B cells and mature B cells, but not on stem cells or plasma cells.

There are two isoforms of CD22 in humans. Its major form (CD22 β) contains 7 immunoglobulin-like (Ig-like) domains in the extracellular region. The CD22 a variant lacks Ig-like domain 4 and may have a truncated cytoplasmic domain. Antibodies that block the adhesion of CD22 to monocytes, neutrophils, lymphocytes and erythrocytes have been shown to bind within the 1 st or 2 nd Ig-like domain.

Once the B cell antigen receptor is linked and associated with Lyk, Syk and phosphatidylinositol 3-kinase, the cytoplasmic domain of CD22 is phosphorylated for tyrosine. The function of CD22 is to down-regulate the threshold for B cell activation. It may also mediate cell adhesion by interacting with cells bearing appropriate sialylglycoconjugates (sialoglycoconjugates).

CD22 is expressed in most B cell leukemias and lymphomas including NHL, acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL) and especially acute non-lymphocytic leukemia (ANLL).

Monoclonal antibodies against CD22 have been described in the prior art. WO98/41641 describes the use of a catalyst at V_H44 and V_L100 having a cysteine residue. WO96/04925 describes V of anti-CD 22 antibody LL2_HRegion and V_LAnd (4) a zone. US5686072 describes combinations of anti-CD 22 and anti-CD 19 immunotoxins. WO98/42378 describes the use of naked anti-CD 22 antibodies in the treatment of B cell malignancies.

Recently, many antibody-based therapies are either licensed, such as Rituxan (an unlabeled CD 20-specific chimeric human γ 1(+ m γ 1V region)), or clinical trials are underway for the disease. These rely on either complement-mediated or ADCC-mediated B cell killing or radionuclides (e.g., for clinicians and patients)¹³¹I or⁹⁰Y), has involved difficulties in preparation and use. There is a need for antibody molecules that treat NHL and that are reusable and can be easily and efficiently prepared. There is also a need for antibody molecules with high affinity for CD22 and low immunogenicity in humans.

Disclosure of Invention

In a first aspect, the present invention provides an antibody molecule specific for human CD22, the antibody molecule comprising a heavy chain wherein the variable region comprises CDRs (as defined by Kabat et al (supra)) having the sequences given below: h1 in FIG. 1 for CDR-H1 (SEQ ID NO: 1); h2 in FIG. 1 for CDR-H2 (SEQ ID NO:2), or H2 from which a potential glycosylation site has been removed, or H2 from which the lysine residue at position 60 (according to the numbering system of Kabat) has been substituted with other amino acids, or H2 from which the glycosylation site and the active lysine at position 60 have been removed; or H3 in FIG. 1 for CDR-H3 (SEQ ID NO: 3).

The antibody molecule of the first aspect of the invention comprises at least one CDR selected from H1, H2 and H3(SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3) of the heavy chain variable region. Preferably the antibody molecule comprises at least two and more preferably all three CDRs in the heavy chain variable region.

In a second aspect of the invention, there is provided an antibody molecule specific for human CD22, the antibody molecule comprising a light chain wherein the variable region comprises CDRs (as defined by Kabat et al (supra)) having the sequences given below: l1 in FIG. 1 for CDR-L1 (SEQ ID NO:4), L2 in FIG. 1 for CDR-L2 (SEQ ID NO:5) or L3 in FIG. 1 for CDR-L3 (SEQ ID NO: 6).

The antibody molecule of the second aspect of the invention comprises at least one CDR selected from L1, L2 and L3(SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6) of the variable region of the light chain. Preferably the antibody molecule comprises at least two and more preferably all three CDRs in the light chain variable region.

Preferably the antibody molecules of the first and second aspects of the invention have a complementary light chain or a complementary heavy chain, respectively.

Preferably the antibody molecule of the first or second aspect of the invention comprises a heavy chain in which the variable region comprises CDRs (as defined by Kabat et al (vide supra)) having the sequences given below: h1 in FIG. 1(SEQ ID NO:1) for CDR-H1, H2 in FIG. 1(SEQ ID NO:2) for CDR-H2, or H2 from which a potential glycosylation site has been removed, or H2 from which the lysine residue at position 60 (according to the numbering system of Kabat) has been substituted with another amino acid, or H2 from which an active lysine at glycosylation site and position 60 has been removed, or H3 in FIG. 1(SEQ ID NO:3) for CDR-H3; and a light chain in which the variable region comprises CDRs (as defined by Kabat et al (vide supra)) having the sequences given below: l1 in FIG. 1 for CDR-L1 (SEQ ID NO:4), L2 in FIG. 1 for CDR-L2 (SEQ ID NO:5) or L3 in FIG. 1 for CDR-L3 (SEQ ID NO: 6).

The CDRs shown in SEQ ID NOs: 1-6 above and FIG. 1 are derived from mouse monoclonal antibody 5/44.

The complete sequence of the variable region of mouse 5/44 antibody is shown in FIG. 2 (light chain) (SEQ ID NO:7) and FIG. 3 (heavy chain) (SEQ ID NO: 8). This mouse antibody is also referred to below as a "donor antibody" or a "murine monoclonal antibody".

A first alternative preferred embodiment of the first or second aspect of the invention is mouse monoclonal antibody 5/44 having a light chain variable region sequence and a heavy chain variable region sequence as shown in FIG. 2(SEQ ID NO:7) and FIG. 3(SEQ ID NO:8), respectively. 5/44 the light chain constant region is kappa and the heavy chain constant region is IgG 1.

In a second alternative preferred embodiment, the antibody according to any one of the first and second aspects of the invention is a chimeric mouse/human antibody molecule, herein referred to as a chimeric 5/44 antibody molecule. The chimeric antibody molecule comprised the variable region of mouse monoclonal antibody 5/44 (SEQ ID NOs: 7 and 8) and a human constant region. Preferably, the chimeric 5/44 antibody molecule comprises a human ck domain in the light chain (Hieter et al, Cell,22197- "207, 1980; genebank accession No. J00241) and contains the human gamma 4 domain in the heavy chain (Flanagan et al, Nature,300709-713,1982), optionally the serine residue at position 241 is replaced by a proline residue.

Preferably the antibody of the invention comprises a heavy chain in which the variable region comprises H2 'as CDR-H2 (as defined by Kabat et al (see above)), in which the potential glycosylation site sequence has been removed and the affinity of the chimeric 5/44 antibody for the CD22 antigen has unexpectedly been increased, and preferably it has a sequence as CDR-H2 as given in H2' (SEQ ID NO: 13).

In addition, the antibody of the invention may comprise a heavy chain in which the variable region comprises H2 "as CDR-H2 (as defined by Kabat et al (vide supra)), wherein the lysine residue at position 60, which is at an exposed position in CDR-H2 and which is believed to be likely to react with conjugating agents (conjugation agents) to result in a decrease in binding affinity of the antigen, is substituted with another amino acid resulting in a conservative substitution. Preferably, CDR-H2 has a sequence as given in H2 "(SEQ ID NO: 15).

In addition, the antibody of the invention may comprise a heavy chain wherein the variable region comprises H2' "as CDR-H2 (as defined by Kabat et al (vide supra)), wherein the potential glycosylation site sequence and the lysine residue at position 60 are substituted with other amino acids. Preferably, CDR-H2 has a sequence as given in H2' (SEQ ID NO: 16).

In a third alternative preferred embodiment, the antibody according to any one of the first and second aspects of the invention is a CDR-grafted antibody molecule. The term "CDR-grafted antibody molecule" as used herein refers to an antibody molecule in which the heavy and/or light chain contains one or more CDRs (including modified CDRs, if desired) from a donor antibody (e.g., a murine monoclonal antibody) grafted onto the heavy and/or light chain variable region framework of an acceptor antibody (e.g., a human antibody).

Preferably, such CDR-grafted antibodies have a variable region comprising a human acceptor framework region and one or more of the above-described donor CDRs.

When grafting CDRs, any suitable acceptor variable region framework sequence may be used, including murine, primate, and human framework regions, with respect to the class/type of donor antibody from which the CDRs are derived. Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al (supra)). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences may be used. The framework region of the light chain is preferably a human germline subtype sequence (DPK9+ JK1) as shown in FIG. 5(SEQ ID NO: 17). The framework region of the preferred heavy chain is the human subtype sequence (DP7+ JH4) as shown in FIG. 6(SEQ ID NO: 21).

In the CDR-grafted antibody of the present invention, an antibody having a chain homologous to that of the donor antibody is preferably used as the acceptor donor. The acceptor heavy and light chains need not be derived from the same antibody and may, if desired, comprise mixed chains having framework regions derived from different chains.

In addition, in the CDR-grafted antibody of the present invention, the framework region does not necessarily have sequences identical to those of the acceptor antibody. For example, a rare residue may become a more frequently occurring residue of that class or type of receptor chain. Alternatively, the residues selected in the acceptor framework regions may be altered so that they correspond to residues present at the same position in the donor antibody or to conservatively substituted residues of residues present at the same position in the donor antibody. These changes necessary to restore the affinity of the donor antibody should be kept to a minimum. It is proposed in WO91/09967 that it may be desirable to alter the choice of residues in the acceptor framework regions.

Preferably in a CDR-grafted antibody molecule of the present invention, if the acceptor light chain has the human subtype DPK9+ JK1 sequence (as shown in FIG. 2) (SEQ ID NO:17), the acceptor framework region of the light chain comprises donor residues at positions 2, 4, 37, 38, 45 and 60 and may additionally comprise donor residues at position 3 (according to Kabat et al (vide supra)).

Preferably in a CDR-grafted antibody molecule of the present invention, if the acceptor heavy chain has the human DP7+ JH4 sequence (as shown in FIG. 3) (SEQ ID NO:21), the acceptor framework region of the heavy chain comprises donor residues in addition to one or more donor CDRs at positions 1, 28, 48, 71 and 93, and may additionally comprise donor residues at positions 67 and 69 (according to Kabat et al (vide supra)).

The donor residues are residues from the donor antibody, i.e., the antibody from which the CDRs were originally derived.

Preferably the antibody of the invention comprises a heavy chain wherein the variable region comprises H2 'as CDR-H2 (as defined by Kabat et al (see above)), wherein potential glycosylation site sequences have been removed in order to increase the affinity of the chimeric 5/44 antibody for the CD22 antigen, and preferably has a sequence as given by H2' (SEQ ID NO:13) as CDR-H2.

In addition, the antibody of the invention may comprise a heavy chain in which the variable region comprises H2 "as CDR-H2 (as defined by Kabat et al, (vide supra)) wherein the lysine residue at position 60, which is at an exposed position within CDR-H2 and which is believed to be likely to react with a conjugation agent, is substituted with another amino acid, resulting in a decrease in antigen binding affinity. Preferably, CDR-H2 has a sequence as given in H2 "(SEQ ID NO: 15).

In addition, the antibody of the invention may comprise a heavy chain wherein the variable region comprises H2' "as CDR-H2 (as defined by Kabat et al, (vide supra)), wherein the potential glycosylation site sequence and the lysine residue at position 60 are substituted with other amino acids. Preferably, CDR-H2 has a sequence as given in H2' (SEQ ID NO: 16).

The antibody molecule of the invention may comprise: a fully antibody molecule having a full-length heavy chain and a full-length light chain; fragments thereof, e.g. Fab, modified Fab, Fab ', F (ab')₂Or an Fv fragment; light or heavy chain monomers or dimers; single chain antibodies, such as single chain Fv in which the heavy chain variable region and the light chain variable region are linked by a peptide linker. Likewise, the heavy chain variable region and the light chain variable region may be combined with other antibody domains as appropriate.

The antibody molecules of the invention may have effector or reporter molecules attached thereto. For example, macrocycles for chelating which may have heavy metal atoms or toxins (e.g., ricin) attached thereto by covalent bridging structures. Alternatively, antibody molecules in which the Fc fragment (CH2, CH3, and hinge regions), CH2 and CH3 regions, or CH3 region of a fully immunoglobulin molecule has been replaced with a functional non-immunoglobulin molecule (e.g., an enzyme or toxin molecule) or linked thereto by peptide bonds can be prepared using recombinant DNA technology methods.

The binding affinity of the antibody molecule of the invention is preferably at least 0.85 × 10^-10M, more preferably at least 0.75 × 10^-10M, most preferably at least 0.5 × 10^-10M。

Preferably, the antibody molecule of the invention comprises the light chain variable region 5/44-gL1(SEQ ID NO:19) and the heavy chain variable region 5/44-gH7(SEQ ID NO: 27). These light chain variable region sequences and heavy chain variable region sequences are shown in FIGS. 5 and 6, respectively.

The invention also relates to variants of the antibody molecules of the invention having improved affinity for CD 22. These variants can be obtained by a number of affinity maturation protocols including: CDR mutations (Yang et al, J.mol.biol.,254392-,10779-783,1992), the use of E.coli (E.coli) mutants (Low et al, J.mol.biol.,260359-368,1996), DNA shuffling (Pattern et al, curr. opin.Biotechnol,8724-733,1997), phage display (Thompson et al, J.mol.biol.,25677-88,1996) and sexual PCR (Crameri et al, Nature,391,288-291,1998). Vaughan et al (see above) discuss these methods of affinity maturation.

The invention also provides DNA sequences encoding the heavy and/or light chains of the antibody molecules of the invention.

Preferably, the DNA sequence encodes the heavy or light chain of an antibody molecule of the invention.

The DNA sequences of the present invention may also comprise synthetic DNA (e.g., chemically prepared), cDNA, genomic DNA, or any combination thereof.

The invention also relates to cloning or expression vectors comprising one or more DNA sequences according to the invention. Preferably, the cloning or expression vector comprises two DNA sequences encoding the light and heavy chains, respectively, of the antibody molecule of the invention.

Conventional methods, transfection methods and culture methods by which vectors can be constructed are well known to those skilled in the art. In this regard, reference may be made to "Current protocols molecular biology", 1999, F.M. Ausubel (eds.), WileyInterscience, New York and Maniatis Manual published by ColdSpringHarbor publishing.

The DNA sequence encoding the antibody molecule of the invention may be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of an antibody may be synthesized as desired based on the determined DNA sequences or based on the corresponding amino acid sequences.

DNA encoding acceptor framework sequences are readily available to those skilled in the art and can be readily synthesized based on their known amino acid sequences.

DNA sequences encoding the antibody molecules of the invention may be prepared using standard molecular biology techniques. The desired DNA sequence may be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and Polymerase Chain Reaction (PCR) techniques may also be used, as appropriate.

Any suitable host cell/vector system may be used to express the DNA sequence encoding the antibody molecule of the invention. Bacteria (e.g., E.coli) and other microbial systems can be used in part to express Fab and F (ab')₂Fragments, in particular antibody fragments such as Fv fragments and single-chain antibody fragments (e.g., single-chain Fv). Eukaryotic host cell (e.g., mammalian host cell) expression systems can be used to produce larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells.

The invention also provides a method of making an antibody molecule of the invention, the method comprising: host cells containing the vectors of the invention are cultured under conditions suitable to result in the expression of the protein from the DNA encoding the antibody molecule of the invention, and the antibody molecule is then isolated.

The antibody molecule may comprise only a heavy chain polypeptide or a light chain polypeptide, in which case only the heavy chain polypeptide or light chain polypeptide coding sequence need be used to transfect the host cell. To prepare a product comprising both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide. Alternatively, a vector may be used that includes sequences encoding a light chain polypeptide and a heavy chain polypeptide.

The invention also provides a therapeutic or diagnostic composition comprising an antibody molecule of the invention and a pharmaceutically acceptable excipient, diluent or carrier.

The invention also provides a method of preparing a therapeutic or diagnostic composition comprising mixing together an antibody molecule of the invention and a pharmaceutically acceptable excipient, diluent or carrier.

In the therapeutic or diagnostic composition, the antibody molecule may be the only active ingredient, or may simultaneously comprise other active ingredients, including other antibody ingredients (e.g., anti-T cell, anti-IFN γ or anti-LPS antibodies) or non-antibody ingredients (e.g., xanthine).

Preferably, the pharmaceutical composition comprises a therapeutically effective amount of an antibody of the invention. The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent required to treat, ameliorate or prevent a target disease or disorder, or to exhibit a detectable therapeutic or prophylactic effect. For any antibody, a therapeutically effective dose can be estimated initially in a cell culture assay or in an animal model (usually rodent, rabbit, dog, pig or primate). Animal models can also be used to determine appropriate concentration ranges and routes of administration. These data can then be used to determine effective dosages and routes of administration for humans.

The exact effective amount for a human patient will depend upon the severity of the disease state, the general health of the patient, the age, weight and sex, diet, time and frequency of administration, drug combination, response sensitivity and tolerance/response to treatment. Such effective amounts can be determined by routine experimentation and are within the judgment of the clinician. In general, an effective dose is from 0.01mg/kg to 50mg/kg, preferably from 0.1mg/kg to 20mg/kg, more preferably about 15 mg/kg.

The compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.

The dosage at which the antibody molecule of the invention is administered depends on the nature of the condition to be treated, the grade of the malignant lymphoma or leukemia and whether the antibody molecule is used for prophylaxis or treatment of an existing condition.

The frequency of administration will depend on the half-life of the antibody molecule and its duration of action. If the half-life of the antibody molecule is short (e.g. 2-10 hours), it may be necessary to administer 1 or more doses per day. On the other hand, if the half-life of the antibody molecule is long (e.g., 2-15 days), it may only be necessary to administer 1 dose daily, weekly, or even every 1 or 2 months.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier for administration of the antibody. The carrier itself should not induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactivated viral particles.

Pharmaceutically acceptable salts may be used, for example, inorganic acid salts (such as hydrochloride, hydrobromide, phosphate and sulfate) or organic acid salts (such as acetate, propionate, malonate and benzoate).

The pharmaceutically acceptable carrier in the therapeutic composition may additionally comprise liquids such as water, saline, glycerol and ethanol. In addition, these compositions may contain adjuvants such as wetting agents, emulsifying agents, or pH buffering substances. These carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.

Preferred forms of administration include forms suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take such forms as suspensions, solutions or emulsions in oily or aqueous media and may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antibody molecule may be in dry form for reconstitution with a suitable sterile liquid immediately prior to use.

Once formulated, the compositions of the present invention can be administered directly to the patient. The patient to be treated may be an animal. However, it is preferred that the composition is suitable for administration to a human patient.

The pharmaceutical compositions of the present invention may be administered by any route including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal (see, e.g., WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. The pharmaceutical compositions of the invention may also be administered using Hypospray. These therapeutic compositions are generally formulated in injectable forms as liquid solutions or suspensions. Solid forms suitable for solution in a liquid vehicle or suspension may also be formulated prior to injection.

Direct administration of the composition is generally accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or to the interstitial space of a tissue. The composition may also be administered to the site of injury. The administration therapy may be a single dose schedule or a multiple dose schedule.

It is understood that the active ingredient in the composition is an antibody molecule. As such, it is susceptible to degradation in the gastrointestinal tract. Thus, if the composition is administered by a route that uses the gastrointestinal tract, the composition will need to contain an agent that protects the antibody from degradation, but once absorbed by the gastrointestinal tract, it will release the antibody.

Pharmaceutically acceptable carriers are discussed in detail in Remington's pharmaceutical sciences (MackPublining company, NJ, 1991).

It is also contemplated that the antibodies of the invention may be administered by using gene therapy. To achieve this, DNA sequences encoding the heavy and light chains of the antibody molecule and under the control of suitable DNA components are introduced into the patient such that the antibody chains are expressed from the DNA sequences and assembled in situ.

The invention also provides antibody molecules of the invention for use in the treatment of diseases mediated by cells expressing CD 22.

The invention also provides the use of an antibody molecule of the invention in the manufacture of a medicament for the treatment of a disease mediated by cells expressing CD 22.

The antibody molecules of the invention may be used in any therapy where it is desired to reduce the level of cells expressing CD22 present in a human or animal body. These CD 22-expressing cells may circulate in the body or be localized at specific sites in the body at undesirably high levels. For example, in B cell lymphomas and leukemias, the level of cells expressing CD22 is elevated. The antibody molecules of the invention are useful for treating diseases mediated by cells expressing CD 22.

Preferably the antibody molecules of the invention are used for malignant lymphomas and leukemias, most preferably for the treatment of NHL.

The invention also provides a method of treating a human or animal patient suffering from or at risk of a cell mediated disease which expresses CD22, said method comprising administering to said patient an effective amount of an antibody molecule of the invention.

The antibody molecules of the invention may also be used in diagnosis, for example for in vivo diagnosis and imaging of disease states involving cells expressing CD 22.

Drawings

The invention is further described by way of the following examples only and with reference to the following description of the drawings in which:

FIG. 1 shows the amino acid sequence of the CDR of mouse monoclonal antibody 5/44 (SEQ ID NOs: 1-6);

FIG. 2 shows the complete sequence of the variable region of the light chain of mouse monoclonal antibody 5/44;

FIG. 3 shows the complete sequence of the heavy chain variable region of mouse monoclonal antibody 5/44;

FIG. 4 shows the removal strategy of glycosylation sites and active lysine in CDR-H2;

FIG. 5 shows a graft design of 5/44 light chain sequence; wherein DPK-9 is a human germline acceptor framework sequence. The vertical lines indicate that there is a difference between murine and human residues. The underlined sequence indicates the donor residues remaining in the graft. CDRs are indicated in blue (not shown for DPK-9). Graft gL1 has 6 donor framework residues and gL2 has 7.

FIG. 6 shows a graft design of 5/44 heavy chain sequences; wherein DP7 is a human germline acceptor framework sequence. The vertical lines indicate that there is a difference between murine and human residues. The underlined sequence indicates the donor residues remaining in the graft. CDRs are indicated in blue (not shown for DP 7). Grafts gH4 and gH6 have 6 donor framework residues, and grafts gH5 and gH7 have 4.

FIG. 7 shows vectors pMRR14 and pMRR10.1;

FIG. 8 shows the results of Biacore assay of chimeric 5/44 mutants;

FIG. 9 shows oligonucleotides for assembly of the 5/44gH1 and gL1 genes;

FIG. 10 shows the intermediate vectors pCR2.1(544gH1) and pCR2.1(544gL 1);

FIG. 11 shows oligonucleotide cassettes for preparing other grafts;

FIG. 12 shows a competition assay between fluorescently labeled mouse 5/44 antibody and graft variant; and

FIG. 13 shows the overall DNA sequence and protein sequence of the grafted heavy and grafted light chains.

Detailed Description

Example 1: candidate antibodies of each generation

A series of anti-CD 22 antibodies were selected from the hybridomas using the following selection criteria: binding to Daudi cells, internalization on Daudi cells, binding to Peripheral Blood Mononuclear Cells (PBMC), internalization on PBMC, affinity (greater than 10)^-9M), mouse γ 1 and yield. 5/44 are selected as preferred antibodies.

Example 2: gene cloning and expression of chimeric 5/44 antibody molecules

5/44 preparation of hybridoma cells and RNA preparation thereof

Hybridoma 5/44 was generated by conventional hybridoma technology following immunization of mice with human CD22 protein. RNA was prepared from 5/44 hybridoma cells using the RNEasy kit (Qiagen, Crawley, UK; Cat. No. 74106). The obtained RNA was reverse transcribed into cDNA as described below.

Distribution of CD22 on NHL tumors

Immunohistochemistry studies were performed using 5/44 anti-CD 22 monoclonal antibody to check the incidence and distribution of staining. Control anti-CD 20 and anti-CD 79a antibodies were included in this study to determine the B cell region of the tumor.

A total of 50 tumors were studied and classified using the WorkingFormulation and REAL classification system as follows:

7B lymphoblastic leukemia/lymphoma (high/I)

4B-CLL/Small lymphocytic lymphoma (Low/A)

3 lymphoplasmacytoma (lymphoplasmacytoma)/Immunocytoma (Low/A)

1 mantle cell lymphoma (Medium/F)

14 follicular central cell lymphoma (low to medium/D)

13 diffuse large cell lymphoma (moderate to high/G, H)

6 unclassifiable lymphomas (K)

2T cell lymphoma

With 5/44 antibody at 0.1. mu.g/ml, 40B cell lymphomas were positive for CD22 antigen and 6 were positive when the concentration was increased to 0.5. mu.g/ml. For the remaining 2B cell tumors, negative at 0.1. mu.g/ml, the remaining tissue was not sufficient to be tested with higher concentrations. However, a parallel experiment with another Celltech anti-CD 22 antibody 6/13 gave a stronger staining than 5/44, with all 48B-cell lymphomas staining positive for CD 22.

Thus, it can be concluded that the CD22 antigen is widely expressed on B-cell lymphomas and therefore provides a suitable target for immunotherapy of NHL.

5/44V_HAnd V_LPCR cloning of

The cDNA sequences encoding 5/44 heavy chain variable region and light chain variable region were synthesized using reverse transcriptase to generate single-stranded cDNA copies of the mRNA present in the total RNA. The cDNA was then used as a template for amplification of murine V region sequences by Polymerase Chain Reaction (PCR) using specific oligonucleotide primers.

a) cDNA Synthesis

cDNA was synthesized in a 20 μ l reaction volume containing the following reagents: 50mM Tris-HCl (pH8.3), 75mM KCl, 10mM dithiothreitol, 3mM MgCl₂0.5mM each of deoxyribonucleoside triphosphates, 20 units of RNAsin, 75ng of random hexanucleotide primer, 2. mu.g of 5/44RNA and 200 units of Moloney murine leukemia virus reverse transcriptase. After incubation at 42 ℃ for 60 minutes, the reaction was terminated by heating at 95 ℃ for 5 minutes.

b)PCR

Aliquots of the cDNA were subjected to PCR using primer combinations specific for the heavy and light chains. A degenerate primer pool designed to anneal with the conserved sequence of the signal peptide was used as the forward primer. All these sequences in turn contain a restriction site starting 7 nucleotides from their 5' end (V)_LSfuI；V_HHindIII), GCCGCCACC (SEQ ID NO:50) for optimal translation of the obtained mRNA, an initiation codon and 20-30 nucleotides based on the leader peptide sequence of known mouse antibodies (Kabat et al, sequence of proteins of immunologicalinterest, 5 th edition, 1991, U.S. department of health and Humanservices, public health service, national institutes of health).

The 3' primer was designed to span the framework 4J-C junction of the antibody and contain a restriction site for the enzyme BsiWI, such that V_LCloning of the PCR fragment was easy. The heavy chain 3' primer is a mixture designed to span the antibody J-C junction. The 3' primer includes an ApaI restriction siteTo facilitate cloning. The 3' region of the primer contains a mixture of sequences based on those found in known mouse antibodies (Kabat et al, 1991, supra)

The above primer combination enables V_HAnd V_LThe PCR products of (a) were directly cloned into suitable expression vectors (see below) to produce chimeric (mouse-human) heavy and light chains and used for these genes expressed in mammalian cells to produce chimeric antibodies of the desired isotype.

Incubation for PCR (100. mu.l) was performed as follows: each reaction contained 10mM Tris-HCl (pH8.3), 1.5mM MgCl₂50mM KCl, 0.01% w/v gelatin, deoxyribonucleoside triphosphates each 0.25mM, 10pmole5 'primer mix, 10pmole 3' primer, 1. mu. lcDNA and 1 unit Taq polymerase. The reaction was incubated at 95 ℃ for 5 minutes and then cycled through the following cycles: 94 ℃ for 1 minute, 55 ℃ for 1 minute and 72 ℃ for 1 minute. After 30 cycles, aliquots of each reaction were analyzed by agarose gel electrophoresis.

For the heavy chain V region, amplified DNA products are only obtained when the pool of primers annealing within the initiation site of framework I replaces the pool of signal peptide primers. The fragments were cloned into a DNA sequencing vector. The DNA sequence was determined and translated to give the deduced amino acid sequence. The deduced sequence can be confirmed by reference to the experimentally determined N-terminal protein sequence. FIGS. 2 and 3 show the DNA/protein sequences of the mature light chain V region and heavy chain V region, respectively, of mouse monoclonal antibody 5/44.

c) Molecular cloning of PCR fragments

The murine V region sequences were then cloned into the expression vectors pmrr10.1 and pMRR14 (fig. 7). These are vectors for light and heavy chain expression containing DNA encoding human kappa light chain and human gamma-4 heavy chain constant regions, respectively. The V was ligated to the sequencing vector by restriction digestion using SfuI and BsiWI restriction sites_LThe region was subcloned into an expression vector, resulting in plasmid pMRR10(544 cL). Introduction of the Signal peptide by PCR amplification of the heavy chain DNA Using the 5' primer, since this is done in the clonesStrategy-no leader sequence was obtained using the mouse heavy chain antibody from a different inbred (in-house) hybridoma (named 162). The 5' primer has the following sequence:^5’GCGCGCAAGCTTGCCGCCACCATGGACTTCGGATTCTCTCTCGTGTTCCTGGCACTCATTCTCAAGGGAGTGCAGTGTGAGGTGCAGCTCGTCGAGTCTGG^3’(SEQIDNO:51)。

reverse primer and primer for initial V_HThe primers for gene cloning were identical. The resulting PCR product was digested with the enzymes HindIII and ApaI, then subcloned, and its DNA sequence confirmed, resulting in plasmid pMRR14(544 cH). Both expression vectors were transiently co-transfected into CHO cells to generate chimeric c5/44 antibody. This was done using Lipofectamine reagent according to the manufacturer's protocol (InVitrogen: Life technology, Groningen, the Netherlands. catalog No. 11668-027).

Removal of glycosylation sites and active lysine

A potential N-linked glycosylation site sequence was observed in CDR-H2, having the amino acid sequence N-Y-T (FIG. 3). SDS-PAGE, Western blotting and gel sugar staining of 5/44 and fragments thereof (including Fab) indicated that the site was indeed glycosylated (not shown). In addition, lysine residues were observed at exposed positions within CDR-H2, which makes it possible to reduce the binding affinity of the antibody by providing additional sites for conjugation with an agent that can be conjugated to the antibody.

One PCR strategy was to introduce amino acid substitutions in the CDR-H2 sequence to remove glycosylation sites and/or active lysine, as shown in figure 4. The glycosylation sites were removed using the forward primer encoding the mutations N55Q, T57A, or T57V (fig. 4) and the 4 th forward primer containing the substitution K60R was generated to remove the active lysine residues (fig. 4). In each of these PCR amplifications, a framework 4 reverse primer was used. The PCR product was digested with the enzymes XbaI and ApaI and inserted into pMRR14(544cH) (also cut with XbaI and ApaI) to generate expression plasmids encoding these mutants. The N55Q, T57A and T57V mutations remove the glycosylation site by altering the amino acid sequence away from the consensus sequence N-X-T/S, while the K60R mutation results in the substitution of the potentially active lysine with the equally positively charged residue arginine. The resulting cH variant plasmid is co-transfected with the cL plasmid to generate an expressed chimeric antibody variant.

Evaluation of Activity of chimeric Gene

After transient transfection into CHO cells, the activity of the resulting chimeric gene was evaluated.

c) Determination of affinity constants by BiaCore analysis

The affinity of binding of chimeric 5/44 or a variant thereof to a CD22-mFc construct was investigated using BIA technology, wherein the glycosylation site or active lysine of chimeric 5/44 or a variant thereof had been removed. The results are shown in FIG. 8. All binding assays were on BIAcore^TM2000 apparatus (pharmacia BiosensorAB, Uppsala, Sweden). This assay was performed by capturing CD22mFc through immobilized anti-mouse Fc. The antibody is present in the soluble phase. Samples, standards and controls (50 μ l) were injected onto immobilized anti-mouse Fc and then onto antibodies in the soluble phase. After each cycle, the surface was regenerated with 50 μ l of 40mM HCl at 30 μ l/min. Kinetic analysis was performed using BIAevaluationionon 3.1 software (Pharmacia).

Removal of the glycosylation site in construct T57A resulted in a slightly faster association rate (on-rate) and a significantly slower off-rate (off-rate) with an approximately 5-fold improvement in affinity compared to chimeric 5/44. The N55Q mutation had no effect on affinity. This result was unexpected because it suggests that removal of the sugar itself had no significant effect on binding (as did the N55Q change). Improved affinity was only observed in the T57A modification. One possible explanation is that threonine at position 57 negatively affects binding, which is removed when threonine is converted to alanine, whether or not sugars are present. The hypothesis that alanine is smaller and that the negative impact of threonine is related to its size, can be supported from the results obtained using the T57V mutation: substitution of valine at position 57 is not good (results not shown).

The removal of lysine by the K60R mutation had no effect on affinity, i.e. the introduction of arginine eliminated the potential active site without affecting affinity.

Thus, mutations for removal of glycosylation sites and for removal of active lysine are both included in the humanization design.

Example 2: 5/44 CDR grafting

Molecular cloning of the 5/44 antibody heavy and light chain variable region genes and their use to make chimeric (mouse/human) 5/44 antibodies have been described above. Mouse 5/44V_LRegion and V_HThe nucleotide and amino acid sequences of the regions are shown in FIGS. 2 and 3(SEQ ID NO:7 and SEQ ID NO:8), respectively. This example describes the CDR grafting of the 5/44 antibody onto human frameworks to reduce possible immunogenicity in humans, following the method of Adair et al (WO 91/09967).

5/44 CDR grafting of light chain

Alignment of the protein sequence with a consensus sequence from the human subtype I kappa light chain V region indicated 64% sequence identity. Thus, to construct CDR-grafted light chains, acceptor framework regions were selected that correspond to those of human VK subtype I germline O12, DPK9 sequences. The framework 4 acceptor sequence was derived from the human J region germline sequence JK 1.

A comparison of the amino acid sequence of the murine 5/44 framework region with the acceptor sequence is given in FIG. 5 and shows that there are 27 differences between the donor and acceptor chains. At each position, the murine residues were stacked by pairs (packing) or at V_H/V_LThe possibility of the interface influencing to directly or indirectly contribute to antigen binding is analyzed. A murine residue is retained if it is considered important and differs significantly in size, polarity or charge from a human residue. Based on this analysis, two CDR-grafted light chains having the sequences given by SEQ ID NO:19 and SEQ ID NO:20 (FIG. 5) were constructed.

5/44 CDR grafting of heavy chain

The CDR grafting of the 5/44 heavy chain was accomplished using the same strategy as described for the light chain. The V region of the 5/44 heavy chain was found to be homologous (70% sequence identity) to human heavy chains belonging to subtype I, thus the sequences of the human subtype I germline framework VH1-3, DP7 were used as acceptor framework. The framework 4 acceptor sequence is derived from the human J-region germline sequence JH 4.

5/44A comparison of the heavy chain and framework regions is shown in FIG. 6, where it can be found that the 5/44 heavy chain differs from the acceptor sequence at position 22. Analysis of the contribution of any of these sequences to antigen binding was possible, resulting in the construction of 5 CDR-grafted heavy chains with the sequences given below: SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27 (FIG. 6).

Construction of genes for transplantation sequences

The genes were designed to encode the graft sequences gH1 and gL1, and a series of overlapping oligonucleotides were designed and constructed (FIG. 9). PCR assembly technology was used to construct CDR-grafted V region genes. Prepare 100 μ l of reaction volume containing the following components: 10mM Tris-HCl (pH8.3), 1.5mM MgCl₂50mM KCl, 0.001% gelatin, 0.25mM each of deoxyribonucleoside triphosphates, 1pmole each of the "inner" primers (T1, T2, T3, B1, B2, B3), 10pmole each of the "outer" primers (F1, R1), and 1 unit of Taq polymerase (AmpliTaq, applied BioSystems, Cat. No. N808-0171). PCR cycling parameters were 94 ℃ for 1 minute, 55 ℃ for 1 minute, and 72 ℃ for 1 minute, 30 cycles. The reaction products were then electrophoresed on a 1.5% agarose gel, and the electrophoretic band was excised and recovered using a QIAGEN spin column (QIAquick gel extraction kit, cat # 28706). The DNA was eluted with a volume of 30. mu.l. Aliquots (1. mu.l) of gH1 and gL1DNA were then cloned into the InVitrogen TOPOTA cloning vector pCR2.1TOPO (catalog number K4500-01) according to the manufacturer's instructions. The non-expression vector is used as a cloning intermediate to facilitate sequencing of a large number of clones. Correct clones containing gH1 and gL1 were identified by DNA sequencing using vector-specific primers, yielding plasmids pcrr 2.1(544gH1) and pcrr 2.1(544gL1) (fig. 10).

Produced using oligonucleotide cassette displacementThe green humanized grafts gH4, gH5, gH6 and gH7 and gL 2. FIG. 11 shows the design of the oligonucleotide cassette. To construct each variant, the vector (pCR2.1(544gH1) or pCR2.1(544gL1)) was excised with the restriction enzymes indicated (XmaI/SacII for the heavy chain and XmaI/BstEII for the light chain). The large vector fragment was purified by agarose gel electrophoresis and used for ligation to oligonucleotide cassettes. These cassettes consisted of 2 complementary oligonucleotides (as shown in FIG. 11) in a volume of 200. mu.l (12.5mM Tris-HCl (pH7.5), 2.5mM MgCl₂25mM NaCl, 0.25mM dithioerythritol) was mixed at a concentration of 0.5 pmole/. mu.l. Annealing was completed by heating to 95 ℃ in a water bath (500ml volume) for 3 minutes, then allowing the reaction to cool slowly to room temperature. The annealed oligonucleotide cassette was diluted 10-fold with water and then ligated into a suitable cleavage vector. DNA sequencing was used to confirm the correct sequence, resulting in plasmids pCR2.1(5/44-gH4-7) and pCR2.1(5/44-gL 2). These confirmed graft sequences were then subcloned into the expression vectors pMRR14 (heavy chain) and pMRR10. l (light chain).

CD22 binding Activity of CDR-grafted sequences

In various combinations, vectors encoding the graft variants were co-transfected into CHO cells along with the original chimeric antibody chains. In a competition assay, the binding activity of the binding pair of the original mouse 5/44 antibody was compared for competition with binding of Ramos cells (obtained from ATCC, a burkitt lymphoma lymphoblastic human cell line expressing surface CD 22). This assay is considered to be the best method to compare the capacity of the grafts to bind to cell surface CD 22. The results are shown in FIG. 8. As can be seen, there were very small differences between any of the grafts, all of which were shown to be more effective than the chimeras in competing against the murine parent. The incorporation of 3 additional human residues at the end of CDR-H2(gH6 and gH7) did not significantly affect binding.

The graft, gL1gH7, was selected to bind the least number of murine residues. The light chain graft gL1 had 6 donor residues. Residues V2, V4, L37 and Q45 are potentially important packing residues. Residue H38 at V_H/V_LAnd (6) an interface. Residue D60 is a surface residue adjacent to CDR-L2 andcan directly contribute to antigen binding. Of these residues, V2, L37, Q45 and D60 are present in germline sequences of human kappa genes from other subtypes. The heavy chain graft gH7 has 4 donor framework residues (residues R28 is considered to be part of the CDR-H1 according to the structural definition used in CDR grafting (see Adair et al (1991, WO 91/09967).; residues E1 and A71 are surface residues near the CDRs. residue I48 is a potential stacking residue; residue T93 is present in V93. fig._H/V_LAnd (6) an interface. Of these residues, E1 and a71 are present in other germline genes of human subtype I. Residue I48 is present in human germline subtype 4, whereas T73 is present in human germline subtype 3.

The overall DNA sequence and protein sequence of both the light and heavy chains, including the approximate positions of introns within the constant region genes provided by the vector, are shown in fig. 13 and are given in seq id no:29 and seq id no:28 (for the light chain) and in seq id no:31 and seq id no:30 (for the heavy chain), respectively.

The DNA encoding these light and heavy chain genes was excised from these vectors. Heavy chain DNA was digested at the 5 'HindIII site and then treated with the Klenow fragment of E.coli DNA polymerase I to generate a 5' blunt end. Cleavage at the 3' EcoRI site resulted in a heavy chain fragment which was then purified by agarose gel. Likewise, a light chain fragment was generated, blunt ended at the 5 'SfuI site and with a 3' EcoRI site. These two fragments were cloned into DHFR-based expression vectors and used to generate stable cell lines in CHO cells.

Claims (22)

1. An antibody molecule specific for CD22, the antibody molecule comprising a heavy chain wherein the variable region comprises CDRs which are: CDR-H1 consisting of SEQ ID NO:1, CDR-H2 consisting of residues 50-66 of SEQ ID NO:23 and CDR-H3 consisting of SEQ ID NO: 3; and the antibody molecule comprises a light chain in which the variable region comprises CDRs which are: CDR-L1 consisting of SEQ ID NO. 4, CDR-L2 consisting of SEQ ID NO. 5 and CDR-L3 consisting of SEQ ID NO. 6.

2. The antibody molecule of claim 1 which is a CDR grafted antibody molecule.

3. The antibody molecule of claim 2, wherein the variable region comprises human acceptor framework regions and non-human donor CDRs.

4. The antibody molecule of claim 3, wherein the human acceptor framework region of the heavy chain variable region is based on SEQ ID NO 21 and 22 and comprises donor residues at positions 1, 28, 48, 71 and 93 corresponding to residues at positions 1, 28, 48, 72 and 97, respectively, of SEQ ID NO 8 according to the Kabat numbering.

5. The antibody molecule of claim 4, further comprising donor residues at positions 67 and 69, which donor residues correspond to residues at positions 68 and 70, respectively, of SEQ ID NO 8 according to the Kabat numbering.

6. The antibody molecule according to any one of claims 3 to 5, wherein the human acceptor framework region of the light chain variable region is based on SEQ ID NO 17 and 18 and comprises donor residues at positions 2, 4, 37, 38, 45 and 60 corresponding to residues at positions 2, 4, 42, 43, 50 and 65, respectively, of SEQ ID NO 7 according to the Kabat numbering.

7. The antibody molecule of claim 6, further comprising a donor residue at position 3, which donor residue corresponds to the residue at this position in SEQ ID NO. 7 according to the Kabat numbering.

8. The antibody molecule of claim 3, wherein the human acceptor framework region of the heavy chain variable region is based on SEQ ID NO 21 and 22 and comprises donor residues at positions 1, 28, 48, 71 and 93 corresponding to residues at positions 1, 28, 48, 72 and 97, respectively, of SEQ ID NO 8 according to the Kabat numbering; wherein the human acceptor framework region of the light chain variable region is based on seq id nos. 17 and 18 and comprises donor residues at positions 2, 4, 37, 38, 45 and 60 corresponding to residues at positions 2, 4, 42, 43, 50 and 65, respectively, of seq id No. 7 according to Kabat numbering.

9. The antibody molecule of claim 3, wherein the human acceptor framework region of the heavy chain variable region is based on SEQ ID NO 21 and 22 and comprises donor residues at positions 1, 28, 48, 71, 93, 67 and 69 corresponding to residues at positions 1, 28, 48, 72, 97, 68 and 70, respectively, of SEQ ID NO 8 according to the Kabat numbering; wherein the human acceptor framework region of the light chain variable region is based on seq id nos. 17 and 18 and comprises donor residues at positions 2, 4, 37, 38, 45 and 60 corresponding to residues at positions 2, 4, 42, 43, 50 and 65, respectively, of seq id No. 7 according to Kabat numbering.

10. The antibody molecule of any one of claims 1-5, 8 and 9, comprising seq id No. 19 (light chain variable region 5/44-gL 1) and seq id No. 23 (heavy chain variable region 5/44-gH 1).

11. A DNA sequence encoding the antibody molecule of any one of claims 1-10.

12. A cloning or expression vector comprising a DNA sequence according to claim 11.

13. A host cell comprising the cloning or expression vector of claim 12.

14. The host cell of claim 13, wherein the host cell is a mammalian cell.

15. Use of an antibody molecule specific for human CD22 according to any one of claims 1-10 in the manufacture of a medicament for the treatment of a disease mediated by cells expressing CD22, wherein the disease is a B-cell lymphoma or leukemia.

16. The use of claim 15, wherein the disease is malignant B-cell lymphoma.

17. The use of claim 16, wherein the malignant B-cell lymphoma is non-hodgkin's lymphoma.

18. A therapeutic or diagnostic composition comprising an antibody molecule according to any one of claims 1to 10.

19. The therapeutic or diagnostic composition of claim 18, comprising a pharmaceutically acceptable excipient, diluent or carrier.

20. The therapeutic or diagnostic composition of claim 18 or 19, further comprising an anti-T cell, anti-IFN γ or anti-LPS antibody, or a non-antibody component.

21. A method of producing an antibody molecule according to any one of claims 1to 10, said method comprising culturing a host cell according to claim 13 or 14 under conditions suitable to result in expression of the protein by the DNA encoding said antibody molecule and isolating said antibody molecule.

22. A process for the preparation of a therapeutic or diagnostic composition according to any one of claims 18 to 20, which process comprises admixing an antibody molecule according to any one of claims 1to 10 with a pharmaceutically acceptable excipient, diluent or carrier.