CA2206471A1 - Monoclonal antibody fragments having immunosuppressant activity - Google Patents

Abstract

The invention comprises monovalent mAb fragments (Fab) of mAb which have the ability of downregulate HLA-DR expression on antigen presenting cells. The Fab fragments can downregulate such HLA-DR expression without the cytotoxicity of the parent mAb or of bivalent fragments (F(ab)'2) of the mAb. The Fab fragments of the invention are therefore potent, class II MHC-specific immunosuppressive compounds without cytotoxic side effects.

Description

CA 02206471 1997-0~-29 WO 96/17874 P~,1i~l 9~/04648 Monoclonal Antibody Fragments havin~ Immunosuppressant Activity s Class II major histocompatibility complex (MHC) molecules bind antigenic peptide fragments, and display them to helper (CD4+) T cells ("Th" cells) (Ref. 1). Monoclonal antibodies (mAb) specific for class II
MHC molecules have been shown to be ex-tremely potent selective inhibitors of Th cell responses in vitro (Ref. 2). Since their discovery, l 0 they have been considered as potential drugs for selective immuno-suppressive treatment of autoimmune disorders, such as rheumatoid arthritis. Initial in vivo studies demonstrated the beneficial influence of these mAbs on Th-cell mediated hetero- and autoimmune responses (Refs. 3-6). However, in some cases, the experimental in 1S vivo application of class II MHC-specific mAbs was associated with unexpected complications resulting in death of laboratory primates (Refs. 7, 8). The latter observation suppressed the interest in further studies of immunomodulation by MHC-specific mAbs. A recent publication has described that a 10-fold reduction of class II MHC
20 expression in transgenic mice causes Th cell nonresponsiveness due to inefficient antigen presen-tation (Ref. 19). This demonstrates that the reduction in the expression of class II MHC correlates with immunosuppression in an in vivo model.

2s In accordance with the invention, it has been surprisingly found that monovalent mAb fragments (Fab) of mAb which have the ability to downregulate HLA-DR expression can them-selves downregulate such HLA-DR expression without the cytotoxicity of the mAb, itself, or of bivalent fragments (F(ab)'2) of the mAb . The Fab fragments of the 30 invention are therefore potent class II MHC-specific immuno-suppressive compounds without cytotoxic side effects.

Before the present invention is described in more specific terms a short description of the figures is given in the following:
3s SUBSTITUTE SHEET (RULE 26) -CA 02206471 1997-0~-29 W O96/17874 P~~ 95~ 61 Fig. 1 Effects of a DR binding competitor peptide and a DR-specific mAb on the EBV transformed B cell line Priess.

Fig. 2 Time course of modulatory and cytotoxic effects of mAb 6 L243 on LG2.

Fig. 3 Duration of modulatory and cytotoxic effects of L243 on LG2 after removal of mAb.

o Fig. 4 Downregulation of HLA-DR expression on different APC
populations after co-culture with DR specific mAb L243 and its fragments.

Fig. S Effects of increasing concentrations of DR specific Fab fragment on the EBV transformed B cell line LG2.

Fig. 6 Effects of prolonged co-culture of L243 and its fragments on LG2 cells.

Fig. 7 Dependency of EBV-LCL cytotoxicity on DR-crosslinking.

Fig. 8 Selectivity of DR downregulation on resting B cells and monocytes/macrophages.
~i Fig. 9 Selectivity of DR downregulation on B cell blasts.

Fig. 10 Selectivity of DR downregulation on LG2 cells.

Fig. 11 Allotype non-selectivity of DR downregulation by mAb.

Fig. 12 Pan-class II downregulation on TS- 10 cells by 1-1 C4 Fab.

Fig. 13 Lack of TNFa secretion increase upon co-culture of LG2 and Priess cells with L243 and its fragments.

CA 02206471 1997-0~-29 WO g6/17874 P~ ;l 9S/04W8 Fig. 14 Antibody concentration requirement for Th cell inhibition and DR downregulation.

Fig. 15 Effect of anti-DR mAb and Fab fragments on antigen 5 presentation by fixed APC.

Fig. 16 Effect of antigen load on the potency of mAb, Fab, and peptide antagonists.

10 Fig. 17 Relative effects of Fab and peptide on antigen dose-response curves.

Fig. 18 Effect of class II antagonists on ongoing Th cell response.
L~ .
Certain monoclonal antibodies (mAbs) which are specific for HLA-DR (Human Leukocyte Antigen of the type "DR"; a class II
Major Histocompatibility Complex ("MHC") molecule) can downregulate the expression of HLA-DR molecules- on the surface ao of leukocytes which are antigen presenting cells ("APC") by about 90%. The same mAbs also inhibit the activation of human Th cell clones that require antigen presentation by HLA-DR molecules for activation. The inhibitory potency of such mAbs is several 100 to several lOOO fold higher than that of the currently available peptide antagonists (see Table 1, below). This downregulation of the expression of HLA-DR and the inhibition of the activation of Th cells is a pharmacological activity which results in immunosuppression. This immunosuppression would be useful in the treatment of autoimmune diseases, especially rheumatoid 3~ arthritis.

In accordance with the invention, it has been surprisingly found that monovalent, antigen-binding mAb fragments (Fab) of mAbs having the ability to downregulate HLA-DR expression can 36 themselves downregulate such HLA-DR expression without the cytotoxicity of the mAb, itself, or of the bivalent fragments (F(ab)'2) of the mAb . The Fab fragments of the invention are therefore potent, class II MHC-specific immunosuppressive CA 02206471 1997-0~-29 W O96/17874 rCTn~S/~U~8 compounds without cytotoxic side effects. Thus, the present invention comprises a Fab fragment of an anti-HLA-DR mAb wherein said intact mAb is cytotoxic to antigen presenting cells and downregulates HLA-DR expression on the remaining antigen 5 presenting cells. Such a mAb inhibits Th cell activation. The mAbs from which the Fab fragments of the present invention are derived all bind to the first domain of HLA-DR.

Examples of three downregulating mAbs useful in accordance 0 with the invention are:

LB3.1 (mouse IgG2b, pan-DRa 1-specific; refs. 9-10);

L243 (mouse IgG2a, pan-DRal-specific; refs. 10-11; ATCC
6Accession No. HB55); and SFR3-DR5 (rat IgG2b, DRBl*llOX-specific; ref. 13; ATCC
Accession No. HB-151).

In addition, an HLA-DR downregulating mAb, 1-lC4 (mouse IgG2a~
I~-chain specific; ref. 14), which additionally downregulates HLA-DQ
and -DP, was generated by conventional means as described in Example 22. Thus, the Fab fragments of the above mAbs are encompassed by the present invention.
~;
Consistent with the ability of downreg~ ting mAbs to inhibit Th cell activation, five non-downregulating mAbs, CCCL20 (mouse IgG2b, specific for three DRB1 (~-chain) allelic forms (DRB1*0101, DRB1*0401, DRB1*0404); ref. 12) and 8D1, 9F1, 9F2, lOF12 (all four 30 mouse IgG1), inhibit Th cell activation only very weakly or not at all.

The active mAbs are cytotoxic to B lymphoblastoid cells and to a small proportion of normal activated B cells. Like the mAbs, 35 the bivalent F(ab)'2 fragments of these mAbs mediate downregulation but are also cytotoxic. However, in accordance with the invention, the monovalent Fab fragments of these mAbs CA 02206471 1997-OF.-29 WO 96117M4 PCTIEl'95/04648 _ S _ loose cytotoxicity, but surprisingly retain the downregulating property of the parent mAb.

The anti-HLA-DR mAbs used to obtain the Fab fragments of 5 the invention may be produced by any conventional means, e.g., generally by the procedure first described by Kohler and Milstein.
By injection of an antigen into a mouse or rat, rabbit, sheep or the like (preferably a mouse), monoclonal antibodies can be prepared by recovering antibody producing cells from such an immunized 10 ~nim~l and immortalizing said cells obtained in conventional fashion like fusion with myeloma cells, e.g., PAI mouse myeloma cells, SP2/0- or SP2/0-Agl4-cells [ATCC No. CRL 1581; ATCC No.
CRL 8287][for a general guideline for producing antibodies see, e.g., "Antibodies-A Laboratory Manual", Harlow & Lee, ed. Cold Spring 5 Harbor Laboratory Press (1988)]. Supernatants of cultures of such hybridomas can then be screened for monoclonal antibodies (mAbs) by conventional procedures like radioimmuno- or enzymimmuno- or dot-immunobinding or immunofluoresence assays. mAbs can be purified from hybridoma supernatants by a~ conventional chromatographic procedures like, for example, ion-exchange chromatography, affinity chromatography on protein A, anti-immunoglobulin-antibodies, or the antigen or a part thereof bound to a solid support, HPLC and the like. The production of a mAb useful in accordance with the invention is shown in Fx~mple 25 22.

For the production of large quantities of mAbs, in accordance with methods well known in the art, hybridomas secreting the desired antibody can be injected intraperitoneally into mice which 30 have been pretreated with, for example, pristane before injection.
Up to around 100 mg of a mAb can be produced by the resulting ascites tumors in one mouse. mAbs can be purified from the ascites fluid produced by such tumors by the methods described above.
~5 mAbs can be characterized according to their subclass by known methods, such as by Ouchterlony immunodiffusion. It is also known in the art that mAbs can be modified for various uses, CA 02206471 1997-0~-29 WO 96117M4 P~:l/~9~104648 or fragments thereof can be generated, which are still capable of binding antigen. Such fragments can be generated, for example, by enzymatic digestion of mAbs with papain, pepsin, or the like.

Immunogen for producing mAb which can be used in accordance with the invention is preferably HLA-DR ~-chain [see e.g. WO92/10589; J. Biol. Chemistry 262, 8748-8758 (1987);
sequence information can be obtained also from sequence data bases, for example like Genbank (Intelligenetics, California, USA), 0 European Bioinformatics Institute (Hinxton Hall, Cambridge, GB), NBRF (Georgetown University, Medical Centre, Washington DC, USA) and Vecbase (University of Wisconsin, Biotechnology Centre, Medision, Wisconsin, USA); such sequences can than be used by methods known in the state of the art to produce the antigen for 5 the preparation of the monoclonal antibodies for use in the present invention] which has been purified by conventional means, such as SDS-PAGE.

The identification of the above-described anti-HLA-DR mAbs ao which are cytotoxic to APC and also downregulate HLA-DR
expression may be performed by any conventional means.
Preferably, this identification of anti-HLA-DR mAb is performed in accordance with Example 3 where an Epstein-Barr Virus transformed human B-Lymphoblastic Cell Line (EBV-LCL) is used 25 as a model of an APC (particularly, activated B cells). Cultures of at least 1 ml containing about 10~ EBV-LCL cells per milliliter are preferably used. The culture is incubated with 20 nM of the anti-HLA-DR mAb for 16 hours, and the number of dead cells and the HLA-DR expression by the rem~ining viable cells is determined by 30 conventional means. For the purposes of this invention, cytotoxicity and downregulation are defined as follows: 1) the mAb is cytotoxic to antigen presenting cells if, under the above conditions, at least 25%, preferably 40%, of the EBV-LCL antigen presenting cells are killed by the intact mAb; and 2) the mAb 35 downregulates HLA-DR expression on antigen presenting cells if, under the above conditions, it reduces the number of HLA-DR
molecules on the surface of the EBV-LCL antigen presenting cells which remain alive by at least an average of 50%, .

CA 02206471 1997-0~-29 W O96/17874 1~ 9~1W648 The EBV-LCL antigen presenting cells are labeled to detect the dead cells and to measure the HLA-DR express ion of the rem~ining viable cells by conventional 5 immunofluoresence. The cells are analyzed using a flow cytometer (e.g., FACScan, Becton-Dickinson, San Jose, California), and the percent dead cells and reduction of HLA-DR expression on the rem~ining cells is calculated by conventional means, preferably using the software normally used with the flow cytometer (e.g., 0 LYSIS II software with the FACScan cytometer).

The EBV-LCL used to screen for monoclonal antibodies having the desired properties are not critical. Any conventional EBV-LCL
may be used in accordance with the invention. F.x~mples of EBV-5 LCL useful in accordance with the invention are Priess (ECACC(Salisbury, UK~ Accession No. 86052111), LG2 (Istituto Nazionale Per La Ricerca Sul Cancro (Genova, Italy) Accession No. G201 12301), and TS-10 (ECACC (Salisbury, UK) Accession No.
85 1 029 1 1 ).
aD
The Fab fragment of the above-described mAbs may be produced by any conventional means. The Fab fragment may be produced by digestion of the parent mAb by pepsin and isolating the Fab fragments by means known in the art (e.g., Andrew and 25 Titus, "Fragmentation of imm~lnoglobulin G", Curren~ Protocols in Immunology, Coligan et al., eds. (Greene & Wiley 1994)).
Accordingly such a process and a Fab fragment whenever prepared by such a process are also an object of the present invention.
Preferably, the Fab fragments of the invention are produced by a 30 recombinant cell line which expresses a gene which encodes the desired Fab fragment. Such recombinant cell lines may be produced by any conventional means. For example, the portion of the gene encoding the Pab fragment would be cloned by conventional means from the hybridoma which secretes the parent 3~ mAb of the Fab fragment. The Fab fragment cDNA may then be incorporated by conventional means into an expression vector, which in turn, is used to transfect an appropliate cell line.

CA 02206471 1997-0~-29 W 096/17874 P~l/~195~ 6 The preferred Fab fragments of the invention are "humanized" so that they are less antigenic when ~ministered to humans than a Fab fragment that is produced directly from an ~nim~l, preferably murine, mAb. The method by which h~-m~ni7.ed 5 Fab fragments of the invention are produced is not critical. Any conventional means known in the art may be used. Such methods utilize the fact that any immunoglobulin (Ig), such as a monoclonal antibody, consists of a constant domain and of a variable domain where the antigen binding occurs. The variable domain, in turn, o consists of six complementarity-determining regions ("CDR's") embedded in a framework region (three CDR's on each of the light chain and the heavy chain of the Ig). (Ref. 42). It is the CDR's which are responsible for the specificity of the mAb. Since mAbs are of murine or other ~nim~l species origin, hllm~ni7~tion of mAbs is 5 performed essentially by-replacing at least one, but preferably all six, of the CDR's of a human immunoglobulin with the corresponding CDR's of an ~nim~l mAb which has the desired specificity. Thus, the human Ig serves as the framework for the ~nim~l CDR's. In such procedures, the ~nim~l mAb, usually murine, ao is described as the "donor" and the human Ig is described as the "acceptor."

Further "fine tuning" of the amino acid sequence of the hllm~ni7ed mAb as described in the art may also be performed to 25 optimize antigen specificity of the hllm~ni7ed mAb. For example, International Patent Application Publication No. WO 90/7861 discloses that a panel of 10-20 human Ig's should be screened, and the human Ig whose variable region has the greatest degree of homology with the variable region of the murine donor mAb, 30 typically 65-70% or higher homology, should be used as the acceptor. Further substitutions of donor amino acids for acceptor amino acids (typically about three substitutions) may be made outside the CDR's based upon the four criteria disclosed in WO
90/7861 at pages 12- 15.
3~
In another example of hllm~ni7.~tion known in the art, EP 0 620 276 discloses a hierarchy of particular substitutions which may be made to the acceptor Ig outside the engrafted donor CDR's CA 02206471 1997-0~-29 _ 9 _ in order to increase the specificity of the hllm~ni7e~ mAb. Such substitutions are disclosed as obviating the need to select a human acceptor Ig whose variable region has a high degree of homology with the variable region of the donor mAb. A specific protocol for B hllm~ni7~tion iS provided in EP 0 620 276 at pages 8-9.

Methods for generating DNA sequences for the expression in a host cell of a hllm~ni7ecl intact mAb useful for obt~ining the Fab fragments of the invention, or for the expression of a hnmani7ed 10 Fab fragment of the invention, are known in the art and are not critical. Such methods include, e.g., site-directed mutagenesis, constructing the whole variable region using overlapping oligonucleotides which incorporate the ~nim~l CDR's on a human framework, and using PCR grafting. (WO 90/7861, EP 0 620 276, 1~ Ref. 43).

Thus, the invention may be further described as a Fab fragment comprising a immunoglobulin Fab fragment and six complementarity-determining regions which are contained within ao said immunoglobulin Fab fragment, wherein from one to six of said complementarity-determining regions are the complementarity-determining regions of a monoclonal antibody having the following properties:

1) the monoclonal antibody binds to the first domain of HLA-DR, 2) the monoclonal antibody is cytotoxic to antigen presenting cells which express HLA-DR, 3) the monoclonal antibody downregulates HLA-DR expression on the antigen presenting cells.

The preferred embodiment of the invention is a hnm~ni7ed 35 Fab fragment wherein, in accordance with the above, the immunoglobulin Fab fragment is human and the monoclonal antibody having properties 1-3 is ~nim~l, preferably murine. This humanized Fab fragment would be a human immunoglobulin Fab CA 0220647l l997-0~-29 W O96/17874 P~l/rl9~ 51e fragment in which from one to six of the CDR's contained therein had been replaced by the corresponding CDR's of the anim~l mAb.
Thus, the preferred Fab fragment of the invention is a Fab fragment comprising a human immunoglobulin Fab fragment and 5 six complementarity-determining regions which are contained within said human immunoglobulin Fab fragment, wherein from one to six of said complementarity-determining regions are the complementarity-determining regions of a monoclonal antibody having properties 1-3, above.
0 It is preferred that this h~lm~ni7ed Fab fragment of the invention contain all six of the animal mAb CDR's.

In the case where the immunoglobulin Fab fragment is of anim~l origin, it is contemplated that the Fab fragment of the invention can be a Fab fragment of the monoclonal antibody, itself, which has properties 1-3, above. Such a Fab fragment would be useful as an intermediate for use in obtaining the anim~l CDR's that will be contained in the preferred hllmani7ed Fab fragment of the invention.

The preferred Fab fragments of the invention have properties simil~r to Fab fragments obtained from monoclonal antibodies LB3.1, L243, SFR3-DR5 and 1-lC4, described above.
Since the Fab fragments of the invention inhibit Th cell activation, 25 they may be used in the treatment of various diseases in which activated Th cells are a source of disease damage or symptoms. One such disease is rheumatoid arthritis. (Ref. 33). Downregulating HLA-DR on APC of a patient with rheumatoid arthritis, and thereby inhibiting Th cell activation in such a patient, would slow or stop 30 the progression of the disease. Inhibiting Th cell activation in a patient with rheumatoid arthritis would also relieve symptoms, such as pain and infl~mmation, by decreasing or halting the release of mediators which are the cause of those symptoms.

Thus, the invention also comprises Fab fragment as described herein and their use as a therapeutically active agent, especially as an immunsuppressive agent and more specifically for the treatment of rheumatoid arthritis and a method of suppressing the CA 02206471 1997-0~-29 WO 96/17874 1 ~ 9S/04648 immune response of a patient comprising ~iministering a therapeutically effective amount of a Fab fragment of the invention to a patient in need of such treatment. The invention further comprises a method of treating rheumatoid arthritis in a patient by lministering a therapeutically effective amount of a Fab fragment of the invention to a patient in need of such treatment. The amount of Fab fragment to be ~iministered may be determined by any conventional means. Also, the ~lmini stration of the Fab fragment of the invention may be performed by any conventional o means.

A-lministration of a Fab fragment of the invention is preferably accomplished using a pharmaceutical composition of the invention (described below). Administration is preferably performed parenterally (subcutaneously, intramuscularly, or intravenously), especially intravenously. The dosage required to inhibit Th cell activation in a patient, and thereby treat rheumatoid arthritis, may be determined by any conventional means, e.g., by dose-limiting clinical trials. However, a dosage of-about 1-10 ~o mg/day i.v., especially about 3-7 mg/day, particularly about 5 mg/day, is preferred (Refs. 34, 35), preferably delivered as a bolus.
Treatment is preferably once daily for one week or less, however daily treatment may be continued for up to three weeks, if necessary.
zj The Fab fragments of the invention may be formulated as a fluid pharmaceutical composition, e.g. for parenteral a-lministration comprising the Fab fragment of the invention dissolved in a conventional pharmaceutically acceptable fluid carrier material.
30 The composition may further comprise other pharmacologically a~tive substances. Preferably, the composition contains about 0.5-5 mg/ml of a Fab fragment of the invention, especially about 1-2 mg/ml. The preferred fluid carrier is sterile, physiological saline.

The results described below in Examples 1-7 indicate that inhibition of Th cell activation by HLA-DR-specific mAb can operate by multiple actions: (a) elimin~tion of available APC by direct cytotoxicity, (b) reduction of available HLA-DR molecules on CA 0220647l l997-0~-29 W 096tl7874 P~ l95~'~1618 the rem~ining viable APC by downregulation of cell surface expression, (c) hindrance of class II MHC-TcR interaction. The Th cell inhibition may also occur by mAb blocking the peptide binding groove on DR molecule, rendering it inaccessible for antigen (30). It 5 is therefore understandable why mAbs are superior Th cell inhibitors to the current peptide antagonists, the effect of the latter being exclusively based on blocking the antigen binding site of HLA-DR molecules.

The mech~ni~ms underlying cytotoxicity and class II
downregulation by antibody remain to be investigated. However, it is clear that cytotoxicity requires crosslinking, whereas downregulation is also achieved with monovalent Fab fragments of the invention without the cytotoxicity of the parent mAb. This difference permits the separation of these two properties by antibody fragmentation. Assuming that the previously observed side effects of anti-class II Ab (Refs. 7, 8) were, at least in part, associated with direct cytotoxicity, the Fab fragments of the invention provide for the therapeutic use of these antibodies as ao immunosuppressants, without adverse effects.

Examples Example 1 Isotype specificity of anti-DR mAb.

The precise specificities of mAbs were determined by their ability to stain mouse cell lines transfected with human HLA class 30 II genes.

Methods: Mouse fibroblast lines transfected with indicated human class II genes (15), as well as untransfected host cells (Lmtk-) were stained by the standard indirect immunofluoresence using DR-35 specific mAb and FITC-conjugated goat-anti mouse Ig (Southern, Birmingham, Alabama) as the primary and secondary reagents, respectively. Samples were analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, California). Results are shown in Table W O96/17874 P~l/r~9S104648 1. "+" and "-", presence and absence of antibody binding, respectively; "NT", not tested.

W O96/17874 P~ r9StO4648 Conclusion: Antibodies 8Dl, 9Fl, 9F2 and lOF12 are pan-DR
specific, whereas l-lC4 can recognize all three human class II
isotypes (DR, DP and DQ).

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SUBSTITUTE SHEET (RULE 26) CA 02206471 1997-0~-29 WO 96/178'74 P~ r9S/04648 Example 2 Chain and domain specificity of anti-HLA-DR mAbs.

5 Methods: Using the standard gene cloning, recombination and transfection technology (10), two mouse B cell lines expressing chimeric human/mouse class II molecules were generated.
Transfectant M 12.C3.25 was derived from a mouse class II-negative host line M12.C3 (15), and expresses MHC product 0 composed of ~1 and 1~1 domains derived from a human HLA-DRA*0101/DRBl*0401 molecule, and a2 and 1~2 domains of a mouse I-Ed protein, to which DR-specific reagents do not bind.
Transfectant CH27.105 was derived from a mouse class II-positive host line CH27 (16), and expresses the original mouse El~k chain 5 associated with the chimeric human/mouse a chain described above. Standard indirect immunofluoresence staining was performed using indicated IgG2 and IgGl antibodies, in conjunction with protein A-FITC (Boehringer, Mannheim, Germany) and goat anti-mouse IgGl-FITC (Southern, Birmingham, Alabama), ao respectively. Samples were analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, California). Results are shown in Table 2 (presentation is as in Table 1).

Conclusion: The results confirm the previously reported DRal 25 specificity of LB3.1 and L243 (10). Antibodies 8Dl and l-lC4 are ~l-specific, whereas the epitope recognized by CCCL20 appears to be ~2 domain-dependent. Rem~ining reagents (9Fl, 9F2, lOF12) seem to bind determinant(s) expressed by the second HLA-DR
domain(s). Corresponding epitope of anti-DRB 1 * l l OX mAb could 30 not be mapped using these transfectants, since SF~3-DR5 does not recognize DRBl*0401 allotype.

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CA 02206471 1997-0~-29 W O96/17874 P~ 19~/04648 Effect of anti-DR mAbs and their fragments on HLA-DR
expression and cell viability The DR expression and viability of antigen presenting cells (APC) pre-cultured with either a DR-binding competitor peptide aXA (17), or DR-specific mAb was ex~mined at respective concentrations that, in the case of aXA and two mAb (LB3.1, 1-lC4), blocked antigen presentation to Th cells. As APC, an Epstein-Barr virus transformed human B-lymphoblastic cell line (EBV-LCL) 0 was used .

Example 3 Effects of a DR binding competitor peptide and a DR-specific mAb 5 on an EBV-LCL

Methods: EBV-LCL (Priess, ECACC (Salisbury, UK) Accession No.
86052111) (105 cells/ml) were cultured in the presence of DR-specific rnAb (LB3.1, 1-lC4, CCCL20) or peptide aX-A
aD (aXAAAKTAAAAa-NH2; ref. 17) at the concentrations indicated in Fig. 1 for 16 hours. Cells were subsequently washed and stained by the standard indirect immunofluoresence using DR-specific mAb [LB3.1 (a), CCCL20 (b), 1-lC4 (c)], and FITC-conjugated goat-anti mouse Ig (Southern Biotechnology Associates Inc., Birmingham, 25 Alabama) as the primary and secondary reagents, respectively.
Samples were analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, California). The results are shown in Fig. 1.
The X-axis represents HLA-DR expression, and the Y-axis fluorescence of propidium iodide-stained dead cells, both in 30 arbitrary fluorescence units. "Background" represents control cell population cultured for 16 hours in normal medium and subsequently labeled without primary reagent, respectively.

Conclusion: Co-culture with peptide aXA (Fig. la) did not 35 significantly affect either cell viability or DR expression, whereas DR specific mAb LB3.1 (Fig. la) and 1-lC4 (Fig. lc) induced high cytotoxicity, as well as reduced DR expression on the rem~ining viable cells. However, these properties were not shared by all CA 02206471 1997-0~-29 WO96/17874 P~ l9'1'~16l~

anti-DR mAbs, since CCCL20 affected neither viability nor DR
expression, even at increased concentration (Fig. lb).

DR downmodulation and cytotoxicity on EBV-LCL could be 5 induced with two more DR specific mAb, L243 and SFR-DR5, whereas four other anti-DR mAb (8D1, 9Fl, 9F2 and lOF12) were neither cytotoxic, nor reduced DR expression (data not shown).

Example 4 Time course of modulatory and cytotoxic effects of mAb L243 on LG2.

Methods: EBV-LCL LG2 (Istituto Nazionale Per La Ricerca Sul L~ Cancro (Genova, Italy) Accession No. G201 12301) (105 cells/ml) were cultured in the presence of DR-specific mAb L243 (Becton-Dickinson) at the concentration of 10 nM for the indicated period of time. Cells were subsequently washed and stained by the standard indirect immunofluoresence using DR-specific mAb L243 and FITC-aD conjugated goat-anti mouse Ig (Southern, Birmingham, Alabama) as the primary and secondary reagent, respectively. Dead cells were stained with propidium iodide, and samples were analyzed on a FACScan flow cytometer. The results are shown in Fig. 2.
Histograms represent relative number of dead cells (light) and live 25 cells expressing decreased amounts of HLA-DR (dark).

Conclusion: Kinetic in vitro studies demonstrated detectable cytotoxicity of mAb after 2 hours, and plateau toxicity after 8 hours. Downregulation of DR became detectable after 4 hours, 30 reaching its peak after 8 hours.

Example S

Duration of modulatory and cytotoxic effects of L243 on LG2 after 35 removal of mAb.

Methods: EBV-LCL LG2 (105 cells/ml) were cultured in the - presence of DR-specific mAb L243 (Becton-Dickinson) at the W O96/17874 1~ 195~1~18 concentration of 10 nM for the indicated period of time. Antibody was removed by 3x washing and cell culture was resumed in the same volume of the fresh medium, as indicated. Cells were subsequently washed and stained by the standard indirect 6 immunofluoresence using DR-specific mAb L243 and FITC-conjugated goat-anti mouse Ig (Southern, Birmingham, Alabama) as the primary and secondary reagent, respectively. Dead cells were stained with propidium iodide, and samples were analyzed on a FACScan flow cytometer. The results are shown in Fig. 3.
o Histograms represent relative number of dead cells (light) and live cells expressing decreased amounts of HLA-DR (dark).

Conclusion: Antibody induced DR downregulation lasts for 16 hours after removal of mAb.
.
Example 6 Downre~ulation of HLA-DR expression on different APC populations after co-culture with DR specific mAb L243 and its fragments.
aD
The effects of anti-DR mAb on different, untransformed MHC
class II positive cell subpopulations: resting and activated B cells, monocytes/macrophages and activated Th cells was further examined Methods: Isolation of B cells and monocytes/macrophages from fresh peripheral blood was done by removing T cells plus monocytes or B cells (where applicable) prior to culture, using magnetic beads (Dynal) precoated with mAb specific for CD3 (SK7) 30 and CD14 (M~P9) or CDl9 (4G7), respectively (Becton-Dickinson).
Th and B cell blasts were generated by 3-5 days in vitro stimulation of peripheral blood mononuclear cells with phytohemagglutinin (1 ~lg/ml; Sigma) and pokeweed mitogen (2.5 llg/ml; Sigma), respectively. L243 was digested by pepsin and 35 papain in order to isolate F(ab)'2 and Fab fragments, respectively as per ref. 18. Undigested antibodies and their Fc fragments were removed by affinity chromatography on protein G columns. APC
( 105 cells/ml) were cultured in the presence of equivalent CA 02206471 1997-0~-29 W 096/17874 1~11rl9~/04648 concentrations of complete L243 antibody (10 nM) or its F(ab)'2 (10 nM) and Fab (20 nM) fragments for 16 hours, as indicated in Fig. 4. Cells were subsequently washed and stained as in Example 3. Results are shown in Fig. 4. The X-axis represents HLA-DR
5 expression, and the Y-axis fluorescence of propidium iodide-stained dead cells, both in arbitrary fluorescence units. Numbers indicate percentages of cells in the corresponding quadrants. "DR-FITC staining" and "Background FITC staining" represent control cell populations cultured for 16 hours in normal medium and 0 subsequently labeled with and without primary reagent, respectively .

Conclusion: The cell surface expression of DR molecules decreased in all populations in the presence of a complete mAb, as well as its 6 bi- and monovalent fragments F(ab)'2 and Fab, respectively. The extent of DR reduction was high in B cells (80-90%), intermediate in monocytic APC (50-65%), and low (less than 50%) in Th cell blasts.
Co-culture of EBV-LCL and preactivated B cell blasts with bivalent anti-DR reagents [complete mAb and F(ab)'2] resulted in high (60-aD 70%) and marginal (5-15%) cytotoxicity, respectively, whereas monovalent fragments (Fab) only induced downregulation without significant cell death. The cytotoxic effect was completely absent in subpopulations of resting B cells, activated Th lymphocytes and monocytes/macrophages preincubated with any form of DR-25 specific mAb used in these experiments.

Similar profiles (partly shown in further sections) wereobtained using complete mAb 1-1 C4 and LB3.1 and their Fab fragments. [F(ab)'2 fragment of LB3. 1 antibody cannot be generated 30 because its isotype does not permit papain digestion].

Example 7 Effects of increasing concentrations of DR specific Fab fragment on 36 the EBV transformed B cell line LG2.

The difference in terms of cytotoxicity between mono- and bivalent mAb reagents was explored to determine if it was only CA 02206471 1997-0~-29 W O96/17874 P~ 19~/W648 quantitative, or if it was due to a qualitatively different mech~ni~m of action.

Methods: As in Fx~mples 3 and 6. Results are shown in Fig. 5.
5 Presentation is as in Fig. 1.

Conclusion: Increased concentration of L243-Fab failed to induce any significant decrease in APC viability.

0 Example 8 Effects of prolonged co-culture of L243 and its fragments on LG2 cells.

5 Methods: APC (105/ml) were cultured in the presence of equivalent concentrations of complete L243 antibody (10 nM) or its Fab fragment (20 nM) for the indicated period of time. Cells were subsequently washed and stained as in F.x~mrle 6. Results are shown in Fig. 6. Presentation is as in Fig. 2. -aDConclusion: Prolonged co-culture period failed to induce any significant decrease in APC viability.

Example 9 Cytotoxicity of EBV-LCL depends on DR-crosslinking.

Methods: EBV-LCL (Priess) (105 cells/ml) were cultured for 16 hours in the presence of L243 Fab (20 nM) fragments and goat 30 anti-mouse IgG precoated magnetic beads ("anti-Ig"; Dynal), in order to crosslink cell membrane bound Fab fragments, as indicated. Cells were subsequently washed and stained as in Example 6. Results are shown in Fig. 7. Presentation is as in Fig. 1.

35 Conclusion: Crosslinking of HLA-DR-bound Fab generates cytotoxic effect on EBV-LCL.

From the results described thus far, it was concluded that:

CA 0220647l l997-0~-29 W Og6117874 }~ 19 (a) HLA-DR expressed on any mononuclear blood cell type can be downregulated upon its ligation by specific mAb;

(b) unexpectedly, DR ligation by monovalent reagents (Fab) can also lead to DR downregulation;

(c) since cytotoxicity is apparent only on EBV, a model of activated B cells, and mitogen preactivated B cells, it is 0 probably dependent upon the activated stage of B cells; and (d) cytotoxicity is the consequence of DR-crosslinking by bivalent ligands [complete mAb and F(ab)'2, crosslinked Fab], and does not require the Fc portion of antibody.

Previous studies have shown that ligation of class II
molecules with whole mAb or T-cell receptors signals a series of changes in APC such as cytokine secretion (refs. 19-20), modulation of cell growth and immunoglobulin secretion (refs. 21-28), :~ upregulation of costimulatory (ref. 15) and cell adhesion molecules (refs. 20, 29), and downregulation of CD23 (ref. 25). It was therefore important to establish whether the decrease of DR
expression upon pre-culture with mAb was restricted to the class II molecule ligated by the antibody, or it was part of a globally 25 induced downregulation of numerous cell surface proteins. The expression of HLA class II isotypes DP and DQ, HLA class I
molecules, and an MHC unrelated adhesion protein CD18, was analyzed on different APC subpopulations after co-culture with F(ab)'2 or Fab fragments of L243 .
Example 10 Selectivity of DR downregulation on resting B cells and monocytes/macrophages .

Methods: As in Example 6. Standard indirect immunofluoresence staining by mAb specific for HLA-DP (clone B7/21), -DQ, (SK10), CD18 (L130) (all of IgGl subclass, Becton-Dickinson) and anti-HLA

CA 02206471 1997-0~-29 W O96/17874 PCTn~5/04648 class I (W6-32 IgG2a, Accurate, Westbury, New York) reagents was performed in conjunction with goat anti-mouse-IgGl-FITC and FITC-labeled protein A (Boehringer), respectively. This procedure excludes additional indirect FITC-labeling of residual membrane-5 bound DR-specific antibody fragment. Results are shown in Fig. 8.
Presentation is as in Fig. 4. "Control" represents cell population cultured for l 6 hours in normal medium and subsequently labeled with Protein A and FITC-conjugated goat anti-mouse IgG1, together.

Conclusion: While DR molecules exhibited reduced expression, DP, DQ, class I, and CD18 molecules remained unaffected in both resting B cells and monocytes/macrophages precultured with F(ab)'2 fragments.

Example 1 1 Selectivity of DR downregulation on B cell blasts.

a~ Methods: As in Fx~mple lO. Results are shown in Fig. 9.
Presentation is as in Fig. 4.

Conclusion: While DR molecules were downregulated, the expression of DP, DQ, class I, and CDl8 molecules remained 25 unaffected in B cell blasts precultured with F(ab)'2 fragments.

Example 12 Selectivity of DR downregulation on LG2 cells.

Methods: As in F.x~mrle lO. Results are shown in Fig. 10.
Presentation is as in Fig. 4.

Conclusion: While the expression of DR molecules appeared to be 35 selectively reduced upon ligation by Fab, bivalent F(ab)'2 fragments induced a significant downregulation of other class II
isotypes, class I molecules and CD18. Since the level of non-selective modulation is only two-fold and DR reduction is ten-fold, CA 02206471 1997-0~-29 WO 96/17874 PCT/~;l 9~16 l~

it is reasonable to assume that the decrease in expression of other cell surface molecules is associated with a general noxiousness of bivalent reagents on EBV-LCL.

Example 13 Allotype non-selectivity of DR downregulation by mAb.

Modulatory mAb LB3.1, L243 and 1-lC4 are pan-DR specific, i.e. they do not distinguish between different allelic forms of HLA-DR. Therefore, they are not suitable for testing allotype specificity of DR downregulation. However, this question could be addressed using downregulatory mAb SFR3-DR5, which is exclusively specific for DRBl*llOX (formerly DR5; ref. 13).
.
Method: B cells (isolated from fresh peripheral blood of a heterozygous DRBl*O101/IIOIX donor as in Example 6) were cultured in the presence of DRBl*llOX-specific mAb SFR3-DR5 (20% hybridoma supernatant fluid; ref 14). Cells were ao subsequently washed and stained by standard indirect immunofluoresence with either DRBl*llOX-specific mAb SFR3-DR5, or DRBl*llOl-specific mAb CCCL20 (12), in conjunction with goat anti-mouse & rat Ig (Southern), as indicated. Results are shown in Fig. 11. Presentation is as in Fig. 4.
~i Conclusion: DRBl*llOX-specific reagent SFR3-DR5 induced downregulation of both DR allomorphs expressed by heterozygous resting B cells, affecting DR allotype not recognized by the mAb, too. Thus, the DR downregulation does not appear to be allotype 30 specific.

Example 14 Pan-class II downre~ulation on TS- 10 cells by 1-1 C4 Fab.

Since mAb l-lC4 can recognize 1~ chain of all three HLA class II isotypes (DR, DP, DQ) it was important to test whether it is capable of reducing their expression accordingly.

CA 02206471 1997-0~-29 WO 96/17874 PCTIEP95tO4648 Methods: EBV-LCL TS-10 (ECACC (Salisbury, UK) Accession No.
85102911) were cultured in the presence of 1-lC4 Fab (20 nM), anti-DP mAb (10 nM) or anti-DQ mAb (10 nM) for 16 hours, as 5 indicated. Cells were subsequently washed and stained as in F.x~mples 6 and 10. Results are shown in Fig. 12. Histogram st~ining profiles of live gated cells are shown. The X-axis represents fluorescence intensity in arbitrary units, and the Y-axis relative cell number. Open histograms represent control cell 0 populations labeled with the respective secondary reagents.

Conclusion: 1- l C4 Fab downregulated expression of all three class II isotypes (DR, DP, DQ) with the reduction intensity comparable to the one achieved by isotype-specific mAb (anti-DP, anti-DQ). This L~ pan-class II downmodulation by 1-lC4 Fab was selective, since HLA class I and CD18 molecules remained unaffected. Therefore, in addition to being useful for treating HLR-DR-linked indications such as rheumatoid arthritis, Fab fragments of the invention obtained from pan-class II mAbs such as l-lC4 w~uld be useful a~ for treating diseases linked to HLA-DP and -DQ expression, such as multiple sclerosis, type I diabetes mellitus, myasthenia gravis, erythematosus, organ transplant rejection, and graft versus host disease. (Refs. 36-41) z~ Example 15 Lack of TNFa secretion increase upon co-culture of LG2 and Priess cells with L243 and its fragments.

Previous studies have shown that crosslinking of DR
molecules by L243 on EBV-transformed B cell line JY increased the secretion of TNFa (20). Since this could be a possible mech~nism for cytotoxicity, the TNFa release by Priess and LG2 cells co-cultured with the same mAb L243 and its fragments was 35 measured.

Methods: APC ( 105 cells/ml) were cultured in the presence of equivalent concentrations of complete L243 antibody (10 nM) or CA 02206471 1997-0~-29 its F(ab)'2 (10 nM) and Fab (20 nM) fragments for 16 hours, as indicated. TNFa concentration in culture supernatants was measured using internally controlled standard ELISA kit (T cell Diagnostics, Cambridge, Massachusetts). Cytotoxic and DR-5 modulatory effects of monoclonal reagents were confirmed byimmunofluoresence as in Fx~mple 4 (data not shown). Results are shown in Fig. 13.

Conclusion: No significant TNFa increase was detected in these 0 cultures. Therefore, the mech~ni~m of cytotoxicity, as well as that of selective DR downmodulation remain to be investigated.

Inhibition of Th cell response with anti~DR mAbs and their fragments Example 16 Inhibition of Th cell response with anti-DR mAb~ their fragments and DR-binding peptides.

Methods: CD4 positive Th cell clones NBHAC25, KMHA25 and DSHABB10 prepared as per Ref. 32 respond to influenza virus hemagglutinin (HA) peptide HA307-319 (PKYVKQNTLKLAT) presented by DRA/DRBl*0101, DRA/DRBl*0401 and 25 DRA/DRB 1 *0401, respectively. Th cells were incubated with mitomycin C-treated APC, antigen (134 nM), and inhibitors.
Antigen specific Th cell proliferation was measured by the standard 3H-thymidine incorporation assay. Results are shown in Table 3.
Conclusion: The mAbs LB 3.1, L243 and 1-lC4, i.e., those inducing downregulation of DR molecules, were also potent inhibitors of Th helper cell activation. Conversely, mAb that did not downregulate DR (CCCL20, 8D1, 9F1, 9F2, lOF12), either failed to affect Th cell 3~ responses, or induced marginal inhibitory effect (Table 3). Thus, the capability of DR downregulation correlates with the inhibitory activity. In addition, these results indicate that downregulatory WO 96/17874 I ~ 9S/04648 mAb tend to recognize epitopes located on peptide-binding (a 1 and B1) domains of class II molecule.

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a ~ 5 u ~ 3~ 4~-SUBSTITUTE SHEET (RULE 26) CA 02206471 1997-0~-29 W Og6/17874 1~lirl9S/04G48 Example 17 Antibody concentration requirement for Th cell inhibition and DR
downre ~ulation .

To further explore the correlation demonstrated in Example 16, the antibody concentration requirement for downregulation and inhibition was determined.

0 Methods: Th cell clones KMHA25 and DSHABB10 (see in Table 3) were incubated with mitomycin C-treated Priess cells as APC, HA307-319 peptide as antigen (330 nM), and the indicated concentrations of mAb LB3. 1. Antigen specific Th cell proliferation (upper panel) was measured by 3H-thymidine incorporation 3 days ~5 later. Flow cytometry of Priess cells with mAb LB3.1 (lower panel) was performed as in Example 3. Results are shown in Fig. 14.

Conclusion: A near complete inhibition of Th responses was achieved with mAb concentration which induced 90%
ao downregulation of DR in about 2/3 of APC (without killing them), suggesting a similar concentration requirement for both phenomena.

Example 18 Effect of anti-DR mAb and Fab fragments on antigen presentation by fixed APC.

It was investigated whether DR downregulation is the only 30 mech~nism involved in inhibition of Th cell response.

Methods: APC (LG-2) were fixed with glutaraldehyde (Fluka Chemie, Buchs, Switzerland). Response of Th cell clone NBHAC25 to mitomycin treated ("My") or fixed ("Fix") LG-2 plus HA307-319 85 (134 nM) in the presence of mAb or Fab was measured as in Example 17. Results are shown in Fig. 15.

CA 02206471 1997-0~-29 W O96/17874 l~ 19S/~U~8 Conclusion: Anti-class II mAbs and their Fab fragments can also inhibit Th cell response to peptide antigen presented on fixed (i. e., dead) APC, where downregulation of class II molecules is not possible. Thus, at least one additional mechanism, most likely steric hindrance of class II- Th cell antigen receptor (TCR) interaction, plays a role in Th cell inhibition.

Example 19 10 Effect of antigen load on the potencv of mAb. Fab~ and peptide antagonists.

The relative efficiency of anti-class II mAbs, their fragments, and peptide binding site antagonists in inhibition of Th cell L~ response was compared.

Methods: As in Example 15. Th clone NBHAC25; APC LG-2. Upper panel HA 307-319 13.4 nM, lower panel 330 nM. Peptides as in Table 3. Results are shown in Fig. 16.
ao Conclusion: Antibodies and Fab fragments are comparable in inhibitory capacity, the latter being only marginally less efficient than the former. Both were, however, several hundred fold more efficient than peptides. It is important to note that increasing the 25 antigen concentration (30 fold higher in the lower than in the upper panel of Fig. 16) rendered the peptide antagonist less efficient, but did not affect the potency of mAb. This observation is explained by differences in the mechanism of inhibition: whereas peptides directly compete with the antigen for class II binding 30 sites, antibodies downregulate class II expression as well as hinder MHC-TCR interaction.

CA 02206471 1997-0~-29 WO96/17874 P~ 19S/04648 Example 20 Relative effects of Fab and peptide on antigen dose-response curves.

The ability of Fab fragments and peptides to inhibit Th cell response to a wide range of antigen concentrations was compared.

Methods: As in F.x~mple 17. Th clone, NBHAC25; APC, LG-2;
o HA307-319 from 13.4 pM to 134 nM; Fab, 100 nM; peptide aXA, 100 ~lM. Results are shown in Fig. 17.

Conclusion: It is clear from the data that Fab fragments have the ability to inhibit Th cell response at zlO00 fold higher antigen L~ concentrations than peptides, and this difference remains constant over the whole range of antigen concentrations.

Example 21 ao Effect of class II antagonists on ongoing Th cell response.

It was important to establish whether different kinds of class II antagonists can interfere with an ongoing Th cell response.
Although this question cannot be properly investigated within the 25 short time frame of in vitro Th cell response, it was attempted to make a comparison between Fab fragments and peptides added at different time points to the APC-antigen-Th cell system.

Methods: As in Example 17. Th clone, NBHAC25; APC, LG-2;
30 HA307-319, 4 nM. Incubation of APC with HA from -2 hours on.
Clone added at 0 hour. Fab of LB3.1 was used. Results are shown in Fig. 18.

Conclusion: Delayed addition of a peptide antagonist resulted in a 35 gradual, time dependent abrogation of inhibitory activity, which could not be restored by increasing the concentration of competitor. In contrast, Fab fragments caused a near complete inhibition even when added 2 hours after the Th cells, and the CA 02206471 1997-0~-29 WO 96/17874 P- -lihl 951'~ 16 l~

decreased inhibitory potency upon delayed addition could be compensated by increased Fab concentrations. Thus, Fab fragments seem to be more efficient in interfering with ongoing Th cell response than the currently available peptide competitors.

Example 22 Production of mAb 1-lC4 0 HLA-DR was immunoprecipitated from EBV-LCL Priess using mAb L243, and the HLA-DR a and B chains were separated by SDS-PAGE.
A 28k electrophoretic band containing DR-~ chain was cut from the gel and used to immunize a BALB/c mouse. Mouse immune B cells were subsequently immortalized by fusion with myeloma line PAI-~5 0 [Stocker, W. et al., Research Disclosure, 217:155-157 (1982)] in order to obtain mAb secreting hybridomas. Culture supernatants of such hybridomas were screened for their capability to inhibit the activation of HA/DRB1*0401 Th cell clone KMHA25 (see Table 3) in the presence of antigen HA 307-319 and Priess as the APC.
aD Hybridoma 1-lC4 was identified to be secreting a mAb having inhibitory capacity.

CA 02206471 1997-0~-29 W O96/17874 l~ l9S/W648 REFERENCES

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Claims (14)

Claims

1. A Fab fragment comprising an immunoglobulin Fab fragment and six complementarity-determining regions which are contained within said Fab fragment, wherein from one to six of said complementarity-determining regions are the complementarity-determining regions of a monoclonal antibody having the following properties:

1) the monoclonal antibody binds to the first domain of HLA-DR, 2) the monoclonal antibody is cytotoxic to antigen presenting cells which express HLA-DR, 3) the monoclonal antibody downregulates HLA-DR expression on the antigen presenting cells.

2. The Fab fragment of claim 1 wherein the monoclonal antibody is a murine monoclonal antibody.

3. The Fab fragment of claim 2 wherein the immunoglobulin is a human immunoglobulin.

4. The Fab fragment of claim 3 wherein the six complementarity-determining regions of said Fab fragment are the six complementarity-determining regions of said monoclonal antibody.

5. A fluid pharmaceutical composition comprising:

1) a pharmaceutically acceptable fluid carrier; and 2) a therapeutically effective amount of a Fab fragment as claimed in any one of claims 2 to 4.

6. The composition of claim 5 wherein the amount of the Fab fragment is from 0.5 to 5 mg/ml of the fluid composition.

7. The composition of claim 6 wherein the amount of the Fab fragment is from 1 to 2 mg/ml of the fluid composition.

8. A process for the preparation of a Fab fragment as defined in any one of claims 1 to 4 characterized in that a monoclonal antibody as defined in claims 1 or 2 is cleaved by pepsin and the Fab fragments are isolated by methods known in the state of the art.

9. A Fab fragment whenever prepared by the process of claim 8.

10. A Fab fragment according to any one of claims 1 to 4 as a therapeutically active agent, especially as an immunsuppressive agent.

11. Use of a Fab fragment according to any one of claims 1 to 4 for suppressing the immune response of a patient.

12. Use of a Fab fragment according to any one of claims 1 to 4 for the treatment of rheumatoid arthritis.

13. The invention as hereinbefore described.

14. A method for suppressing the immune response of a patient comprising administering a therapeutically effective amount of a Fab fragment comprising a Fab fragment as defined in claim 3 or 4.