AU724562B2 - Anti-EGFR single-chain Fvs and anti-EGFR antibodies - Google Patents

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Applicant(s): Address for Service: Invention Title: Merck Patent GmbH Frankfurter Strasse 250 D-64293 Darmstadt

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DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Anti-EGFR single-chain Fvs and anti-EGFR antibodies The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 1995 MRCIGOD.DOC j^ -1- Anti-EGFR Single-Chain Fvs and Anti-EGFR Antibodies TECHNICAL FIELD OF THE INVENTION This invention relates to new anti-EGFR antibodies and antibody fragments, preferrably single-chain Fvs (scFvs) which can be obtained from phage-antibody libraries constructed from cells of an immunized 15 mammalian, preferably a mouse. The antibody fragments isolated from the phage-antibody libraries can be engineered to create partially humanized whole antibody molecules. These chimeric anti-EGFR antibodies contain constant regions of human immunoglobulins, and 20 can be,used as well as their fragments as agents for the diagnosis and therapy of human tumors.

Furthermore, the invention demonstrates that phage-antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mammalians in comparison with the standard hybridoma technology.

The invention relates, moreover, to pharmaceutical compositions comprising said antibodies or fragments for the purposes of treating tumors like melanoma, glioma or carcinoma. The said antibodies or fragments can be used also for diagnostic applications regarding locating and assessing the said tumors in vitro or in vivo.

1995 MRCIGOD.DOC -2- The specification relates to several technical terms which are herewith defined as follows: "FRs" (framework regions) mean the four subregions of the light or heavy chain variable regions that support the three CDRs.

"CDRs" (complementarity determining regions) mean the three subregions of the light or heavy chain variable regions which have hypervariable sequences and form loop structures that are primarily 10 responsible for making direct contact with antigen.

"Chimeric" or partially humanized antibodies mean antibodies comprising constant regions deriving from human sources and variable regions (CDRs included) deriving from non-human sources, e.g. from the mouse.

"Humanized" or fully humanized antibodies mean antibodies comprising constant regions and FRs deriving from human sources whereas the CDRs derive from non-human sources.

"EGF" and "EGFR" mean the epidermal growth factor ant its receptor.

"PCR" means the polymerase chain reaction.

"scFv" means single-chain Fv which is an antibody fragment.

means light chain variable region.

Wk" means kappa light chain variable region.

W"V" means heavy chain variable region.

PBS means phosphate buffered saline FCS means fetal calf serum HBSS means Hanks balanced salt solution FITC means fluoresceineisothiocyanate MTC means mixed cell culture -2a Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

**ee *9 0* o *9o9 9 9 9 r I-'iS-AIj^/l./ L/k/^ -3- BACKROUND OF THE INVENTION Epidermal growth factor (EGF) is a polypeptide hormone which is mitogenic for epidermal and epithelial cells. When EGF interacts with sensitive cells, it binds to membrane receptors (EGFR). The EGFR is a transmembrane glycoprotein of about 170 kD and is a gene product of the c-erb-B proto-oncogene.

*i MAb 425 is a murine monoclonal antibody raised against the well known human A431 carcinoma cell line (ATCC CRL 1555), binds to a 15 polypeptide epitope of the external domain of the human EGFR, and inhibits the binding of EGF. MAb 425 (ATCC HB 9629) was found to mediate tumor cytotoxicity in vitro and to suppress tumor cell growth of epidermoid and colorectal carcinoma-derived cell lines in vitro (Rodeck et al., Cancer Res. 1987. 47: 3692). Humanized and chimeric versions of MAb 425 have been disclosed in WO 92/15683.

Over the last few years, methods have been described (Skerra and PlOckthun, Science 1988. 240: 1038; Better et al., Science 1988. 240: 1041) with which functional antibody fragments can be produced in eukaryotic host cells, such as E. coli. These include the Fv fragment and the Fab fragment, whereby the Fv fragment is of special interest.

Single-chain Fvs (wherein the V L and the VHChain are linked together) have been also described (Bird et al., Science 1988. 242: 423; Huston et al., Proc. Natl. Acad. Sci. USA 1988. 85:5879).

Phage-antibody libraries offer an alternative technology to hybridoma technology in the isolation of antibodies from immunized animals.

Hybridoma technology works by immortalizing the cells that produce -4the antibodies. Phage-antibody technology works by immortalizing the genes that code for the antibodies (Winter, G. and Milstein, Nature 1991. 349: 293). In phage-antibody technology, the antibody heavy chain variable region (VH) and light chain variable region (VL) genes are PCR-amplified, the variable regions are randomly combined and expressed as antibody fragments on the surface of phage particles, and libraries of phage antibodies are screened for antibodies that bind to antigens of interest.

10 Hybridoma technology has been very successful at isolating mouse monoclonal antibodies when it has been possible to generate a strong 15 immune response in the spleens of the animals. For example, mouse MAbs against human epidermal growth factor receptor (EGFR) have been isolated from the spleens of mice immunized intraperitoneally with human A431 tumor cells (Murthy et al., Arch. Biochem. Biophys.

1987. 252: 549). The potential advantage of phage-antibody "technology over hybridoma technology is that virtually any source of antibody-expressing cells can be used as starting material and that large numbers of different antibodies can be rapidly screened. Another advantage of the phage-antibody technology is that the genes coding for the variable regions of the antibodies of interest have already been cloned and are immediately available for further genetic engineering.

In one report, an anti-tetanus toxoid Fab fragment isolated from a phage-antibody library was converted into a whole antibody molecule (Bender et al., Hum. Antibod. Hybridomas 1993. 4: 74).

During the last ten years, in vitro immunization has been used as an altemative technique to active immunization to generate monoclonal 3 antibodies (mAbs) against a wide variety of antigens from both human 1995 MRCIGOD.DOC and murine systems Vaux, Helenius, A. and Mellman, I.; Nature, 1988. 336: 36; Gathuru, J.K et al.; J. Immunol. Methods, 1991.

137:95; Borrebaeck, Immunol. Today, 1988.9:355). Advantages of this approach are that only small amounts of antigen are required and that the method is applicable for generating human hybridomas.

However, the generation of poor affinity IgM antibodies and the difficulty of immortalizing human lymphocytes after in vitro immunization have become persistent problems associated with this technology.

A new way of obtaining antibodies is by PCR amplification of repertoires Sof heavy (VH) and light (VL) chain variable region genes which are then randomly recombined and expressed as phage display libraries Antibody variable-region genes were cloned and fused to the minor coat protein (gene 3) as a single chain Fv fragment (scFv) The phage particle displays on its surface the antibody fragment and can be selected t by panning using the antibody's binding properties. This technology has 20 the advantage that random recombination of V genes may produce novel pairings with new specificities and affinities which could not be selected by natural processes. Moreover, such an approach makes possible the use of naive or in vitro immunized lymphocytes from murine or human sources.

Previous attempts to obtain mAbs against EGFR by murine B cells in vitro immunization and hybridoma technology rendered low-affinity, crossreacting antibodies. In order to overcome such handicaps, the 30 combination of in vitro immunization followed by PCR cloning technology was carried out.

995 MRCIGOD.DOC

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-6- SUMMARY OF THE INVENTION This invention compares mouse anti-EGFR antibodies isolated from three different phage-antibody libraries with a mouse MAb (425) isolated by standard hybridoma technology (Murthy et al., Arch.

Biochem. Biophys. 1987. 252:549; Kettleborough et al., Protein Eng.

1991. 4:773). Libraries were prepared, not only from the spleen of an immunized mouse, but also from the draining lymph node of an immunized mouse and from in vitro immunized mouse cells. Two of the single-chain Fvs (scFvs) that %ere isolated from the libraries were 20 engineered to create chimeric whole antibody molecules with the mouse variable regions joined to human constant regions.

In detail, the present invention relates to an anti-EGFR single-chain Fv obtainable from phage-antibody libraries constructed from cells, preferrably of the spleen or the draining lymph node of an immunized mammalian, preferably a mouse, or from in vitro immunized cells. In principal, the invention is not restricted to scFvs but extends also to 30 other anti-EGFR antibody fragments such as Fab or F(ab')2.

9* Some of the scFvs according to the invention have well-defined DNA and amino acid sequences. Therefore, in another aspect of the present invention there is provided a singe-chain Fv fragment, wherein the variable 1995 MRCIGOD.DOC -7regions of the heavy and light chain comprise a DNA and/or a amino acid sequence selected from one of the heavy and light chain sequences given in Sequence Id. Nos. 1 32, preferably in Fig. 5 8.

Because in many cases only completely functioning, whole antibodies can be used for diagnostic and therapeutic purposes it is in the interest of the invention to link the variable regions from single-chain Fvs with the constant regions of human immunoglobulins forming whole, partially or humanized anti-EGFR antibodies.

Therefore, in another aspect the present invention provides a whole anti-EGFR antibody constructed from DNA sequences deriving from antibody 1 fragments as defined above, below or as defined in the claims, and from DNA sequences deriving from constant regions of human immunogobulins, wherein, as a preferred embodiment, the heavy chain comprises the amino acid sequence of a gamma-1 chain, and the light 20 chain comprises the amino acid sequence of a kappa chain.

According to the present invention the anti-EGFR scFvs, are isolated by using the phage-antibody library technology. Therefore the E 2 invention relates to a process for the preparation of an anti-EGFR single-chain Fv comprising the following steps: isolating RNA from immunized mammalian cells, preferably mouse cells, 30 (ii) synthesizing first-strand cDNA, 30 .(iii) amplifying the VH and Vk genes in cDNAs from the immunized cells, (iv) cloning said genes together with suitable restriction sites into a phagemid vector, transforming prokaryotic cells with the ligation mixtures,

MRCIGOD.DOC

-8- (vi) screening the phage libraries for phage antibodies directed to EGFR using purified EGFR, and (vii) producing the desired single-chain Fv in prokaryotic host cells, preferably E. coil.

Additionally the present invention discloses a process for the preparation of a whole anti-EGFR antibody by cloning the DNA coding for the variable regions of anti-EGFR antibody fragments produced as indicated above or as defined in the claims into at least one eukaryotic expression vector containing genomic DNA which codes for the constant regions of human immunoglobulins, transforming eukaryotic cells with said vector(s) and expressing and isolating the antibody.

The anti-EGFR scFvs, and above all, the whole anti-EGFR antibodies S can be used in diagnosis and therapy of human tumors. Thus, the 20 invention relates to a pharmaceutical composition comprising an anti- S EGFR single chain Fv or a whole anti-EGFR antibody as defined above or as defined in the claims.

9 The results and advantages of the present invention can be summarized as follows: Novel mouse anti-EGFR antibodies were isolated from phage-antibody libraries. The new antibodies represented at least four different V subgroups and four different VKsubgroups (Kabat et al., Sequences of proteins of immunological interest. 5th Eds., U.S. Dept. of Health and Human Services, Bethesda 1991). They showed different pairings and sequences from those used by a mouse MAb isolated using hybridoma technology. Mouse 425 MAb has a vH2b and Vk4 pairing that was not 19 J) MRC1iGD.Doc -9observed in the phage antibodies. The VH of scFv L3 11D had the highest percent identity to 425 VH The majority of the differences were in the CDRs. The Vk of scFv S4 2D had the highest percent identity to 425 Vk Again the majority of differences were in the CDRs, particularly CDR3. In this invention, a variety of new anti-EGFR antibodies were isolated from the phage-antibody libraries and these antibodies all differ from 425 MAb with at least two of the scFvs recognizing a different epitope on EGFR from that recognized by 425 MAb. This is in contrast to a previous report where the antibodies isolated from combinatorial libraries were reported to be very similar to those isolated by hybridoma technology (Caton and Koprowski, Proc.

1 Nad. Acad. Sci. USA 1990. 87:6450).

Of the three phage-antibody libraries, the best library in terms of the .O number of selection steps required to get high affinity antibodies and in terms of the diversity of high affinity antibodies isolated was the library 20 generated from the draining lymph node. Lymph nodes were selected as a source of RNA for the construction of phage-antibody libraries for two reasons. First, previous work had demonstrated that a higher proportion of B cells producing high affinity IgG antibodies was obtained from the popliteal lymph nodes following immunization via the footpad than from spleens following immunization via the peritoneum (Venn and Dresser, J. Immunol. Methods 1987. 102: 95). Second, draining lymph nodes are considered to be a good source for the 30 isolation of human anti-tumor antibodies. Thus, the isolation of mouse anti-EGFR antibodies from the popliteal lymph node of a footpadimmunized mouse was a model for the isolation of human anti-EGFR antibodies from the axillary lymph nodes of a breast cancer patient.

The feasibility of preparing good size libraries from small amounts of 1995 'ARCIGOD.DOC lymph node material and of then isolating high affinity antibodies from the libraries was demonstrated.

Although mouse anti-EGFR antibodies were isolated from all three phage-antibody libraries, it is not clear that any of the newly isolated antibodies have higher affinities than the mouse 425 MAb isolated using hybridoma technology. In the first analyses, the phage-antibody derived scFvs appeared to bind to EGFR better than the scFv constructed from 425 MAb (Fig. In other experiments with chimeric whole antibody molecules, one of the chimeric antibodies (S4 2D) showed an affinity for EGFR that equalled that of the chimeric 425 antibody. The second chimeric antibody (L3 11D) had an affinity that was four-fold lower than that of chimeric 425 antibody (Fig. Binding data obtained using scFvs was misleading probably because preparations of scFvs can contain mixtures of monomers and dimers (Griffiths et al., EMBO J. 1993. 12: 725). In contrast, chimeric IgG 20 antibodies are not expected to form dimers and the chimeric L3 11D S and S4 2D antibodies were demonstrated to be the size expected for bivalent, monomeric chimeric IgG antibodies. Analysis of affinitypurified preparations of 425, L3 11D, and S4 2D scFvs, however, showed that these preparations of scFvs did contain monomeric, dimeric, and other multimeric forms. In addition, the relative proportions of monomeric and multimeric forms varied for each scFv.

The 425 scFv had the lowest percentage of dimeric forms. As 30 predicted, the dimeric and particularly the larger multimeric forms showed stronger binding to purified EGFR than the monomeric form. It appears that 425 scFv has a weaker tendency to dimerize than some of the newly isolated scFvs.

Although the expression of antibody fragments on the surface of phage particles forms the basis of a powerful method for rapidly selecting for 1995 MRCIGOD.DOC 11 antibodies with the desired specificities, neither phage antibodies nor the antibody fragments themselves (scFvs or Fabs) are likely to be the desired end product. Further it is demonstrated how the mouse scFvs isolated from phage libraries can be readily converted into whole antibody molecules. In this case, the mouse variable regions were joined to human constant regions to create partially humanized chimeric antibodies.

These results show that it is possible to use phage-antibody technology to isolate a variety of anti-EGFR antibody fragments from immunized mice. Whole antibody molecules with the desired constant regions can then be constructed from the antibody fragments. In some cases, hybridoma technology may still be the method of choice for isolating monoclonal antibodies from mice. If a highly immunogenic antigen is available and if a few hybridoma cell lines producing one or a few different anti-antigen antibodies are adequate, then there is probably little reason to consider phage-antibody technology. If, however, special immunization protocols such as footpad injections would be advantageous in generating high affinity antibodies, or if a large number of antibodies against a variety of epitopes on the antigen 25 are required, or if antibodies directed against a very discreet, and 25 possibly less immunogenic, epitope are required, then phage-antibody technology may be the method of choice. Also, if further genetic engineering of the antibodies is anticipated, then the phage-antibody 0 technology is advantageous in that the antibody genes have already been cloned.

The present approach of combining in vitro immunization with a particulate antigen and PCR-cloning technology has generated scFv fragments which reacted with EGFR and did not cross-react with other antigens. The immunization protocol reported here depends on the 1995 MRCIGOD.DOC -12antigen presentation, which is not soluble but is a membrane vesicle preparation, and on the culture medium itself, which is devoid of FCS.

Both methodologies have been reported as a means of increasing the efficiency of in vitro immrrization by making the antigen available to the antigen-presenting cells Brams, P. et al.; J. Immunol. Methods, 1987.

98:11).

The results obtained with MTC are in agree with previous papers g.

Borrebaeck, CAK and MOler, SA; J. Immunol., 1986.136: 3710; .Mller, S.A and Borrebaeck, C.AK, in Borrebaeck, CAK In Vitro Immunization in Hybridoma Technology, Elsevier Science Publishers B.V., Amsterdam 1988, p. which propose the use of MTC supematants as a source of lymphokines for improving the in vitro immunization process.

The membrane vesicle preparation should be envisaged as a polyantigen since many different antigenic determinants are present in such vesicles. For this reason, it would appear that they induce a certain level of polyclonal activation. We have ruled this out because the anti-EGFR specific response was clearly different from the response obtained after a standard polyclonal activator.

Instead of immortalizing the B-cells after in vitro immunizations, we have 25 used the molecular strategy of immortalizing the antibody VH and VL genes. These monoclonal antibody fragments were expressed and produced in bacteria. The phage display system is a powerful method to isolate antibody fragments against specific antigens. The presence of a stop codon between the antibody fragment and the g3p coat protein permits the switch between surface display and secretion as a soluble scFv fragment using suppressor or hon-suppresor strains (Hoogenboom et al., Nucl. Acids Res. 1991. 19: 4133).

Due to the increase of specific response and mRNA levels in in vitro antigen stimulated B-cells, in vitro immunization contributes to the 1995 MRCIGOD.DOC 13isolation of antibody fragments with high specificities to the antigen. After two rounds of selection, 100% of the clones were positive for binding EGFR. In contrast, clones derived from in vio immunization processes were 100 positive only after four rounds of selection (Kettleborough, et al., EP 94104160 and Eur. J. Immunol. 1994.24:952).

The use of phage display libraries from naive antibody genes might allow specific human antibody fragments to be made without immunization or after in vito immunization. Antibody fragments can be directly produced in bacteria, thus in a simple, fast and economic way.

BIOLOGICAL MATERIALS AND GENERAL METHODS Microorganisms, cell lines, plasmids, phagemids, promoters, resistance markers, replication origins or other fragments of vectors which are mentioned in this application are commercially or otherwise 2 generally available. Provided that no other information in the 20 application is given, they are used only as examples and are not essential according to the invention and can be replaced by other suitable tools and biological materials, respectively.

•i Bacterial hosts are preferably used for cloning the scFvs and for producing the scFv proteins. Examples for these hosts are: E. coli or Bacillus.

Eukaryotic hosts like COS, CHO or yeasts, for example, are preferred in order to produce the whole anti-EGFR-antibodies according to the invention.

1915 ~MRCIGOD.DOC -14- The techniques which are essential according to the invention are described in detail in the specification. Other techniques which are not described in detail correspond to known standard methods which are well known to a person skilled in the art, or are described more in detail in the cited references and patent applications and in the standard literature.

10 Brief Description of the Figures: Figure 1. Amino acid sequences of scFvs isolated from phage- 15 antibody libraries. scFvs from the lymph node library. scFvs from the spleen library. Complementarity determining regions (CDRs) and framework regions (FRs) are indicated.

Figure 2. Binding of scFvs to EGFR. The concentrations of scFvs 20 in bacterial supematants were estimated and the scFvs tested by o ELISA for binding to purified EGFR. scFvs from the lymph node library. scFvs from the spleen library. P1 (positive control) is the scFv derived from MAb 425. L1 and SI (negative controls) are nonbinding scFvs from the pre-selected lymph node and spleen libraries.

Figure 3. Intermediate vectors used to reconstruct the variable regions for expression in mammalian cells. VH vector.

VK

vector.

Fiure 4. Binding of chimeric whole antibodies to EGFR. The concentrations of antibodies in COS cell supematants were determined by ELISA and the antibodies tested by ELISA for binding to purified EGFR.

MKt.AtjtJD.IMC 15 Figure S. DNA and amino acid sequence of scFv No. L2 11 C.

Light chain; Heavy chain.

Amino acid positions: FR-I: 1 -23, CDR-1: 24 -34, FR-2: 35 49, CDR-2: 50 56, FR-3: 57 -88, CDR-3: 89 -97, FR-4: 98 -109.

FR-I1: 1 -30, CDR-1: 31 -351 FR-2: 36-49, CDR-2: 50-68, FR-3: 67 -98, CDR-3: 99-108f FR-4: 109 -119i.

Figue 6.DNA and amino acid sequence of scFv No. L2 12B.

9 Light chain; Heavy chain.

Amino acid positions: 0 FR-i: 1- 23, CDR-1: 24 -38, FR-2: 39-49, CDR-2: 50 -56, .FR-3: 57-88, CDR-3: 89- 97, 999'FR-4: 98-109.

FR-I: 1 -30, CDR-1:- 31 FR-2: 36-49, CDR-2: 50 -66, FR-3: 67-98, CDR-3- 99 108, FR-4: 109-119.

Figure 7. DNA and amino acid sequence of scFv No. L3 11 D.

Light chain; Heavy chain.

The amino acid posititions of the FRs and CDRs correspond to those 12 given in Fig. 6.

1995 MRCIGOD.DOC -16- Figure 8. DNA and amino acid sequence of scFv No. S4 2D Light chain; Heavy chain.

Amino acid positions: FR-1: 1- 23, CDR-1: 24 FR-2: 36-50, CDR-2: 51 -57, FR-3: 58 89, CDR-3: 90-98, FR-4: 99- 110 FR-1: 1-30, CDR-1: 31-35, FR-2: 36-49, CDR-2: 50-66, FR-3: 67-98, CDR-3: 99- 107, "i 15 FR-4: 108-118.

i The sequences of Figures 5 8 are also given in the attached Sequence Listing which is part of the disclosure of this invention.

DETAILED DESCRIPTION OF THE INVENTION (1)Construction and screening of phage-antibody libraries Three phage-antibody libraries were constructed, one from the spleen of a mouse immunized with human carcinoma cell line A431 (8.8 x 10 members), one from the popliteal lymph node of a mouse immunized in the footpad with purified EGFR (6.5 x 106 members), and one from mouse lymphocytes immunized in vitro with A431 vesicles (1.1 x 10 members), (details of construction of A431 vesicles and in vitroimmunization are given in Examples 1, Prior to selection, at least 46 clones from each library were analyzed by BstN fingerprinting 46 clones from each library were analyzed by BstNI fingerprinting 1995 MRCIGOD.DOC -17- (Clackson et al, Nature 1991. 352: 624) to determine the diversity of the repertoires. A wide range of digestion pattems was observed. Also prior to selection, scFvs from 96 clones from each library were tested by ELISA for binding to EGFR. None of the scFvs from the spleen and lymph node library bound to EGFR. One of the scFvs from the in vitro immunized library bound to EGFR. After one round of selection using EGFR-coated immunotubes, a cear enrichment for EGFR-binding scFvs was observed with the lymph node library and with the in vitro immunized library. A second round of selection was needed before any EGFR-binding scFvs were detected from the spleen library. By the third round of selection, the majority of the scFvs from the lymph node and in vitro immunized libraries were positive for binding to EGFR.

After a fourth round of selection with the spleen library, the majority of the scFvs were positive for binding to EGFR (Table 1).

a S 1995 MRCIGOD.DOC 18- Table 1.

selection.

Percent of EGFR-binding clones after each round of ii'.

S

9 9 9. 9 9 99 99 9 9 9 9 Lymph Splee In vitro Node n Immuniz Library Libra ed ry Cells Library Pre- 0 0 1 selection First 77 0 84 round Second 86 26 100 round Third 90 77 100 round Fourth not 97 not round tested tested Sequence analysis of EGFR-binding clones After each round of selection, scFv inserts from EGFR-binding clones were analyzed by BstNI fingerprinting (Clackson et al, Nature 1991.

352: 624). It became clear that there was an enrichment for certain digestion patterns. Clones with different BstNI fingerprints were chosen from the second and third rounds of selection of the lymph node library and from the third and fourth rounds of the spleen library for DNA sequencing of the VHS and VKS. Clones from later rounds of selection LYYJ i4LKkIU U i 19were analyzed because higher affinity antibodies were expected to be in the later rounds (Clackson et al, Nature 1991. 352: 624).

Sixteen clones from the lymph node library were sequenced and six different scFvs were obtained (Figure Five of these were pairings of unique VHS and VKs. The sixth was a variation of a previously occurring VH with six amino acid changes, five of which were in framework region (FR) 1. Two of these changes can be attributed to the use of the degenerate VH1 BACKSFI primer (Hoogenboom et al., Nucl. Acids Res. 1991. 19: 4133). The others may be a result of PCR errors. The VHs were classified into two subgroups, VH2b and VH3d, while the VKs fell into four subgroups, VK 3 VK4, Vc5, and VK 6 (Kabat 1 5 et al., Sequences of proteins of immunological interest. 5th Eds., U.S.

Dept. of Health and Human Services, Bethesda 1991). Ten individual clones from the spleen library were sequenced and four different scFvs were found. Three of these were pairings of unique VHS and VKS while 20 the fourth was similar to one of the previous pairings with only two amino acid differences in VH, one of which occurred in complementarity determining region (CDR) 2, and two amino acid differences in VK. Classification into subgroups revealed VHS from subgroups VH2a, VH2c, and VH3d and VKS from subgroups VK 3 and VK4. Comparison of the scFvs obtained from the lymph node and spleen libraries revealed only one scFv that was common to both libraries, scFv L3 10A/scFv S4 10H (Figure This clone appeared to 3 bind strongly to EGFR when tested by ELISA. While much care was taken to eliminate any cross-contamination between libraries, it is difficult to rule out minor contamination with a strongly-binding EGFR clone. However, considering the inbred nature of Balb/c mice, it is possible that the same scFv arose independently from two different libraries.

1995 MRCIGOD.DOC Analysis of the affinity and specificity of binding to EGFR Based on good binding to antigen and diversity in DNA sequences, several scFvs derived from the lymph node and spleen libraries were chosen for further analysis. These scFvs were analyzed by ELISA for binding to purified EGFR, binding to irrelevant antigens, and binding to tumor cell lines that did or did not express EGFR. As a positive control, scFvs were prepared from mouse 425 MAb As negative controls, scFvs were prepared from phage antibodies isolated from the lymph node and spleen libraries prior to selection (Li and S1, respectively).

The concentration of scFvs was determined by comparing dilutions of the scFvs to be tested with dilutions of a purified scFv of known 15 concentration in a Western blot.

The scFvs were tested by ELISA for binding to purified EGFR and the results plotted (Figure It was possible to rank the scFvs with respect to their binding to EGFR. These rankings were reproducible S 20 between experiments. The scFvs that bound most strongly to EGFR were L2 1C and L3 10A from the lymph node library and S4 10H from the spleen library. As described previously, scFvs 13 10A and $4 have the same DNA sequences. A scFv (S4 5A) that was very similar to scFv S4 10H, with two amino acid changes in VH and two in Vk, consistently gave a lower ranking than S4 10H. In contrast, the differences in sequence observed between L2 12B and L3 11D did not appear to have a pronounced effect on the binding. Of the scFvs isolated only two, L2 8C and L2 11C, appeared to bind less well than scFv 425.

The scFvs were tested by ELISA for binding to plastic and to a panel of unrelated proteins (ovalbumin, hen egg lysozyme, cytochrome c, glyceraldehyde 3-phosphate dehydrogenase, CBA albumin, and BSA).

None of the scFvs gave a signal above background.

AMKLR-rJU.LUUC -21- The scFvs were tested by ELISA for binding to three tumor cell lines.

Cell lines A431 and MDA MB 468 are EGFR-bearing tumor cells isolated from the vulva and breast, respectively. Cell line SK-MEL-23 Sis a ganglioside-bearing melanoma cell line and was included as a negative control. Of the ten scFvs tested, only four bound to both purified EGFR and EGFR-bearing tumor cells (L2 12B, L3 11D, L2 11 C, and S4 2D, Figures 5 No binding to SK-MEL-23 cells was detected. There are several possible explanations for this surprising result. One may be that the EGFR that was used for immunization, selection, and ELISA was secreted EGFR-related protein (Weber et al., Science 1984. 224: 294). This protein has an additional 17 amino acids at the C-terminus (GUnther et al., J. Biol. Chem. 1990. 265: 15 22082). The scFvs were tested by ELISA for binding to this 17 amino acid peptide and no binding was observed. It is possible that the secreted EGFR-related protein and the EGFR on the tumor cell surface have differences in conformation or glycosylation.

To further investigate binding to tumor cells, three scFvs (L2 11A, L3 11D and S4 2D) were purified and analyzed for binding to A431 tumor cells by flow cytometry. The 425 scFv was used as a positive control.

Of the three scFvs tested, only L3 11D and S4 2D bound to A431 cells.

These two scFvs had similar binding profiles to scFv 425.

Purified scFvs prepared from two of the isolates that bound to both EGFR and EGFR-bearing tumor cells (L3 11D and S4 2D) were tested in competition binding assays with mouse 425 MAb. While purified scFv 425 was able to inhibit mouse 425 MAb from binding to EGFR over a given concentration range, scFvs L3 11D and S4 2D did not inhibit mouse 425 MAb from binding to EGFR at these concentrations.

These two scFvs appear to recognize an epitope on EGFR that is different from that recognized by mouse 425 MAb.

1995 MRCIGOD.DOC -22- Chimeric whole antibodies derived from scFvs.

Two scFvs (L3 11D and S4 2D) were selected for conversion into whole antibody molecules. DNAs coding for the mouse VHs and Vs were cloned into intermediate vectors containing DNA sequences coding for immunoglobulin leader sequences and splice donor signals (Fig. The positioning of the cloning sites in the VH intermediate vector meant that the first residue of the VH was changed from aspartic acid to glutamic acid. From the intermediate vectors, DNA .fragments containing the VHs and VKs, now joined to leader and splice donor sequences, were cloned into mammalian cell expression vectors I15 containing DNAs coding for either human gamma-1 constant region or human kappa constant region (Maeda et al., Hum. Antibod. Hybridomas 1991. 2: 124). For each chimeric antibody, the heavy chain and light chain expression vectors were co-transfected into COS cells. As a 20 positive control, cells were also co-transfected with heavy and light oO*. chain expression vectors coding for chimeric 425 antibody (Kettleborough et al., Protein Eng. 1991. 4: 773). Medium was collected from the cells and analyzed by ELISA to determine the concentration of antibody present and the ability of the antibody to bind to EGFR (Fig. When the antibody concentration required to achieve half-maximum binding to antigen were compared, chimeric S4 2D antibody bound to EGFR equally as well as chimeric 425 antibody.

Chimeric L3 11D antibody, however, bound to EGFR approximately four-fold less well than chimeric 425 antibody. The affinity of chimeric 425 antibody (Kettleborough et al, Protein Eng. 1991. 4: 773) has been determined by competition binding analysis to be 1.9 x 108 M- 1 These results were surprising because previous data analyzing the scFvs had indicated that scFvs S4 2D and L3 11D both bound to EGFR better 1995 MRCIGOD.DOC -23than scFv 425 (Fig. Protein A-purified samples of chimeric L3 11D and S4 2D antibodies were analyzed by SDS-PAGE under reducing and non-reducing conditions. Chimeric L3 11D and S4 2D antibodies were also tested by flow cytometry for binding to A431 and SK-MEL-23 cells. Both chimeric antibodies bound well to the EGFR-expressing A431 cells and did not bind to the EGFR-negative SK-MEL-23 cells.

Therapeutic and diagnostic use The antibody fragments and whole antibodies according to the invention can be administered to human patients for therapy.

Therefore, it is an object of the invention to provide a pharmaceutical formulation comprising as active ingredient at least one antibody or antibody fragment as defined above and in the claims, associated with one or more pharmaceutically acceptable carrier, excipient or diluent therefore.

Typically the antibody of this invention will be injected intravenously or parenterally. Generally, the dosage ranges for the administration of the antibodies fragments are large enough to produce the desired tumor suppressing and tumor lysing effect. The dosage will depend on age, condition, sex and extent of the disease in the patient and can vary from 0.1 mg/kg to 200 mg/kg, preferably from 0.1 mg/kg to 100 mg/kg/dose in one or more doses administered daily, for one or several days.

Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oils, and injectable organic esters such as ethyl oleate and other solvents known in the art which are suitable for these 1995 MRCIGOI).DOC -24purposes. The antibodies of this invention can be used in a composition comprising a physiologically acceptable carrier. Examples of such suitable carriers are saline, PBS, Ringer's solution, or lactated Ringer's solution. Preservatives and other additives such as antibiotics, antioxidants, and chelating agents may also be present in the pharmaceutical formulations.

The antibody (fragment) can also be conjugated according to known methods to cytokines such as IL-2 in order to support their cytotoxicity.

The pharmaceutical formulations of the present invention are suitable for the treatment of all kinds of tumors, including melanomas, gliomas and carcinomas, as well as tumors of the circulating system and solid tumors.

For diagnostic purposes the antibody can be conjugated, for example, 20 to a radio-opaque dye or can be radiolabelled. A preferred labelling method is the lodogen method. Preferably the antibody will be administered as F(ab') 2 or scFv fragments for diagnostic purposes.

This provides superior results so that backround substraction is unnecessary.

30 EXAMPLE 1: A431 vesicles Shed membrane vesicle preparations were obtained as previously described by (Cohen et al., J. Biol. Chem. 1982. 257: 1523; Yeaton et al., J. Biol. Chem. 1983. 258: 9254) with some modifications. Confluent flasks containing A431 cells were washed with PBS containing calcium 1995 MRCIGOD.DOC and magnesium. Hypotonic PBS was added and flasks were shaken for 15 minutes. Cells were then washed with vesiculation buffer (100 mM NaCI, 50 mM Na 2

HPO

4 5 mM KCI, 0.5 mM MgSO 2 pH Vesiculation buffer was added and flasks were kept in agitation at room temperature and at 37°C. Then, buffer was decanted through metallic screen into 50 ml tubes in ice and centrifuged for 5 minutes at 150 x g at 4°C. The pellet was discarded and the supematant was ultracentrifuged at 39,000 rpm for 90 minutes. The final pellets were resuspended in 10 mM Hepes buffer (pH To analyze EGFR from vesicles, samples were precipitated with 9 volumes of ethanol resuspended with 0.08 M Tris, pH 6.8, and then SDS-PAGE was carried out with MAb 425 as standard.

The protein content of the preparations was quantitated by a modified Coomassie Plus method using BSA as a standard and read at 595 nm.

To analyze EGFR from vesicles, samples were precipitated with 9 20 volumes of ethanol (overnight at 4C). The pellet was resuspended with 20 Tris (0.08 M, pH 6.8) and then a SDS-PAGE was run stacking gel; 1h, 35 mA; 10% running gel; 2.5 h; 40 mA). Samples and standard were 1 in duplicate. One of them was stained with Coomassie Blue and the other S 25 was blotted onto nitrocellulose sheets (12 V; 16 h at 4 0 C) and treated with mouse mAb 425 (anti-EGFR) and anti-mouse IgG antibody conjugated to S" alkaline phosphatase.

Three media were used in the in vitro immunizations. Medium-1 (M1), 30 Medium-2 (M2) and Mixed Thymocyte Culture medium (MTC). M1 consisted of HL1 (Ventrex Laboratories, USA) supplemented with 50 mM 2-mercaptoethanol and 2 mM L-Glutdmine (Gibco). M2 consisted of HL1 supplemented with 50 mM 2-mercaptoethanol; 40 U/ml IL-2 (Genzyme); 20 mg/ml Adjuvant Peptide (Sigma); 2 mM L-glutamine; 100 U/ml penicillin (Gibco); 100 mg/ml streptomycin (Gibco). 4% or 20% of FCS 1995 MRCIGOD.DOC -26- (Biological Industries) was added to M2. MTC was prepared as described by Vaux Briefly single cell suspensions of thymuses of three-week-old Balb/c and C57/BL-1 mice were prepared by pressing the thymus glands through a sterile 50-mesh screen. The cell sus-pension was collected, washed twice in HBSS and the number of viable cells was determined by trypan blue exclusion. Thymocytes were then cultured at a density of 2.5x10 thymocytes of each strain per ml in HL1 medium containing 4% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. After 48 hours, the supematant was recovered, filtered through a 0.22 mm filter, and stored at -70 0

C.

A suspension of splenocytes from non-immunized eight-week old BALB/c mice was obtained as described for thymocytes. Viability was determined by trypan blue exclusion.

2 0. EXAMPLE 2: In vitro immunization and screening Three media were used in the in vitro immunizations. Medium-1 (M1), Medium-2 (M2) and Mixed Thymocyte Culture medium (MTC). M1 consisted of HL1 (Ventrex Laboratories, USA) supplemented with 50 mM 25 2-mercaptoethanol and 2 mM L-Glutamine (Gibco). M2 consisted of HL1 supplemented with 50 mM 2-mercaptoethanol; 40 U/ml IL-2 (Genzyme); 20 mg/ml Adjuvant Peptide (Sigma); 2 mM L-glutamine; 100 U/ml penicillin (Gibco); 100 mg/ml streptomycin (Gibco). 4% or 20% of FCS (Biological Industries) was added to M2, MTC was prepared as 30 described by Vaux Briefly single cell suspensions of thymuses of three-week-old Balb/c and C57/BL-1 mice were prepared by pressing the thymus glands through a sterile 50-mesh screen. The cell sus-pension was collected, washed twice in HBSS and the number of viable cells was determined by trypan blue exclusion. Thymocytes were then cultured at a density of 2.5x106 thymocytes of each strain per ml in HL1 medium

MRCIGOD.DOC

-27containing 4% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. After 48 hours, the supematant was recovered, filtered through a 0.22 mm filter, and stored at -70 0

C.

A suspension of splenocytes from non-immunized eight-week old BALB/c mice was obtained as described for thymocytes. Viability was determined by trypan blue exclusion.

Single cell suspensions from thymuses of three-week-old Balb/c and C57/BL-1 mice were obtained by pressing the thymus glands through a sterile 50-mesh screen. The cell suspension was collected, washed with HBSS and the number of viable cells was determined by trypan blue exclusion. Thymocytes were then cultured at a density of 2.5x10 6 thymocytes of each strain per ml in HLI-medium containing 4% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 pg/mi streptomycin.

After 48 hours the supematant was recovered, filtered and stored. A suspension of splenocytes from non-immunized eight-week-old 20 BALBIc mice was obtained as described for thymocytes. Viability was determined by trypan blue exclusion.

In vitro immunizations were performed in 6-well plates (Costar). Wells containing 107 splenocytes in 3.5 ml of Mi-medium (consisting of HLI-medium, Ventrex Laboratories, USA, supplemented with 50 pM 2mercaptoethanol and 2 mM L-glutamine (Gibco)) were incubated (370C, 5% CO2) with vesicles bearing EGFR at the desired concentration. Vesicles from cells not expressing EGFR or PBS were 30 added in control wells. After some hours, 3.5ml of M2-medium (consisting of HL1 supplemented with 50 pM 2-mercaptoethanol, U/ml IL-2 (Genzyme), 20 pg/ml adjuvant peptide (SIGMA), 2 mM Lglutamine, 100 U/ml penicillin (Gibco), 100 pg/ml streptomycin (Gibco)) containing 4% or 10% FCS (Biological Industries) was added to each well. In some experiments M2 was replaced by MTC-medium 1995 MRCIGOD.DOC -28- (mixed thymocyte culture medium (Vaux et al., Nature 1988. 336: 36) supplemented with adjuvant peptide (20pg/ml) and IL-2 (40 U/ml) (Note that the final concentration of FCS, IL2 and adjuvant peptide in culture is 50% reduced). Cells were incubated for 72, 96, 120 or 144 h in the same conditions and, finally, the cells were tested for the presence of specific immunoglobulin or processed for RNA isolation.

Screening was carried out with purified antigens or A431 fixed cells.

The procedure was essentially as previously described (Carroll et al., Hybrdoma 1990. 9: 81) with some modifications. Briefly, sterile 96well plates (Nunc, Maxisorb) were coated overnight with purified EGFR pg/ml), GD 3 ganglioside (2pg/ml), or RNase (10pg/ml) in PBS.

When A431 cells were used as antigen, cells were cultured in 96-well plates until confluent and fixed with 0.1 glutaraldehyde. In vitro immunized lymphocytes were washed and resuspended in HL1 medium supplemented with 2 FCS and 2 mM of L-glutamine at S 20 5x10 5 cells/ml and 1x10 5 cells were added to each well and i •0 incubated (370C, 5% CO 2 for 48 h. Sixteen duplicates of each group were done. Lymphocytes were then removed by washing 5 times in PBS containing 0.1 Tween-20. Specific immunoglobulins were 25 detected using peroxidase labelled rabbit anti-mouse immunoglobulin (Dako) (1 hour, 370C). 2,2'-Azino-bis(3-ethylbenz-thiazoline-6sulfonicacid)-diammonium salt (ABTS) (Sigma) in citrate-phosphate buffer (0.55 mg/ml) was used as substrate.

30 EXAMPLE 3: Library construction Three libraries were constructed from RNA prepared from the spleen of a mouse immunized intraperitoneally with A431 cells (Murthy et al., Arch. Biochem. Biophys. 1987. 252: 549) from the popliteal lymph node of a mouse immunized in the footpad with purified EGFR, and from mouse cells immunized in vitro with A431 vesicles. First-strand 1995 MRCIGOD.DOC -29cDNA was synthesized. The VH and V, genes were PCR-amplified and assembled (Clackson et al., Nature 1991. 352:624). Using PCR, Notl and Sfil restriction sites were appended and the scFvs cloned into the phagemid vector pHEN1 (Hoogenboom et al., Nucl. Acids Res.

1991.19: 4133). The ligation mixtures were electroporated into E. coli cells and the resulting colonies scraped into medium to generate library stocks (Marks et al., J. Mfo. Biol. 1991. 222:581).

EXAMPLE 4: Library screening Phage antibodies were rescued from the libraries using M13K07 helper phage (Promega, Madison, WI) (Marks et al., J. Mol. Biol. 1991.

222:581). Immunotubes (Nunc, Life Sciences, Paisley, UK) were coated with 4 ml of 2.5 ig/ml EGFR in PBS overnight. After three washes with PBS, tubes were incubated at 37°C for at least 1 h in PBS 20* containing 2% milk powder (PBSM). The phage (1012 to 1013) were .resuspended in 4 ml PBSM and incubated in the EGFR-coated tube for 1 h at room temperature. The tube was washed 20 times with PBS, 0.1 Tween and 20 times with PBS. Bound phage were eluted after a 10 min incubation in 1 ml of 0.1 M triethylamine with end-over-end mixing. The eluted phage were neutralized by the addition of 0.5 ml of 1 M Tris-HCI, pH 7.5 and used to infect log-phase E. coli TG1 cells.

Infected cells were plated and individual colonies picked for smallscale induction of scFvs. The remaining colonies were scraped into medium and an aliquot used to prepare phage for the next round of screening.

EXAMIPLE 5: Production and analysis of scFvs 1995 MRCIGOD.DOC Soluble scFvs were produced in E. coli HB2151 as previously described Kettleborough et al., I. The scFv concentrations in the bacterial supematants were estimated using a purified scFv preparation of known concentration as a standard. Supematants were filtered and sodium azide added to Serial dilutions of the supematants and of the standard were spotted onto Immobilon-PVDF filters (Millipore, Watford, UK) using a 96-well manifold. The filters were treated as for a Western blot (Towbin et al., Proc. Nal. Acad. Sci.

USA 1979. 76: 4350). The scFvs were detected using an antibody (9E10) directed against the C-terminal tag (Munro and Pelham, Cell 1986. 46:291) followed by a peroxidase-conjugated goat anti-mouse 5 IgG and IgM antibody (Jackson ImmunoResearch Lab Inc., West Grove, PA). The reactions were developed using the ECL system (Amersham, Aylesbury, UK). Pre-flashed autoradiographs were scanned using a densitometer. A standard curve was prepared and used to estimate the scFv concentrations in the supematants.

S Antigen-binding ELISAs were carried out with EGFR-coated plates g/ml). Supematants containing scFvs were diluted in PBSM and added to the plates. Bound scFvs were detected using 9E10 antibody as described above. Supematants were also tested for binding to a panel of unrelated proteins and plastic. ELISA plates were coated ovemight at 100 g/ml with ovalbumin, hen egg lysozyme, cytochrome c, glyceraldehyde 3-phosphate dehydrogenase, murine albumin (CBA strain), and BSA. Undiluted supematants containing 2% milk powder were added in duplicate to the coated plates and bound scFvs detected as described above.

Cell-binding ELISAs were carried out using tumor cell lines, A431 (ATCC CRL 1555), MDA MB 468 (ATCC HTB 132), and SK-MEL-23 (negative control). Cells were grown to confluency in poly-D-lysine- -31treated 96 well tissue culture trays (Nunc). Cells were washed with DMEM and blocked at 37 0 C for 2 h with PBS containing 2.5% BSA.

After aspiration, supematants were added to each well together with an equal volume of 2xYT media containing 4% milk powder and incubated at 4 0 C for 1 h. Bound scFvs were detected as described above.

A competition-based ELISA was carried out by pre-incubating EGFRcoated ELISA plates with 50 1 of purified scFv (100 pg/ml) for 10 min.

Mouse MAb 425 (50 1) was then added to give concentrations of 3.13 to 200 ng/ml. Following incubation and washing, bound mouse MAb 425 was detected using peroxidase-conjugated goat anti-mouse IgG 15 and IgM antibody.

EXAMPLE 6: DNA analysis For BstNI fingerprinting, the scFv inserts from individual clones were o* amplified by PCR and the products digested with BstNI (Clackson et al., Nature 1991. 352: 624). DNA was sequenced using a Sequenase kit (United States Biochemical, Cleveland, OH).

EXAMPLE 7: Purification of scvs Bacterial supematants were clarified by centrifugation and filtration through 0.2pm filters before loading onto a mi column of purified EGFR (5 mg) coupled to cyanogen bromide-activated Sepharose 4B (Pharacia, Uppsala, Sweden) The column was u uwashed with 30 ml of PBS followed by 5 mi 0.2o glycine, pH 5.0. The scFvs were eluted PBS followed by 5 ml 0.2M glycine, pH 5.0. The scFvs were eluted IY93 MRCIGOD.I)OC -32with 0.2M glycine/HCI, pH 2.8. The eluate was neutralized with PBS. Protein-containing fractions were pooled and the buffer changed by ultrafiltration (Amicon, Stonehouse, UK) to PBS containing 1% BSA S and 0.05% sodium azide.

EXAMPLE 8: FACS analysis of purified scFvs A431 cells were trypsinized and incubated in DMEM containing FCS. Cells were washed twice with cold DMEM and filtered through a pm screen. Cells (106) were incubated on ice for 30 min in 50 1 PBS, 1% BSA, with purified scFvs. After two washes with cold PBS, S bound scFvs were detected using 50 p FITC-conjugated 9E10 15 antibody (100 pg/ml). After 30 min on ice, cells were washed once with PBS, fixed in PBS containing 1% formaldehyde, and analyzed using a FACSCAN (Becton-Dickinson, Cowley, UK).

EXAMPLE 9: Construction, analysis and expression of whole chimeric antibodies Using Pstl and BstEII sites, DNAs coding for the VHS of the selected scFvs were subcloned into an intermediate VH vector containing a eukaryotic leader sequence derived from human antibody HG3 CL (Rechavi et al., Proc. Nal. Acad. Sci USA 1983. 80:855) and a splice donor site (Fig. The DNAs coding for the Vks were adapted for insertion into an intermediate Vk vector using PCR primers to incorporate Xhol and Sstl sites at the and ends (VkFor: 5'-CCG TTT CAG CTC GAG CTT GGT CCC-3' VkBack: 5'-GAC ATT GAG CTC ACC CAG TCT CCA-3').

The Sstl-Xhol fragments were cloned into the intermediate Vk vector containing a eukaryotic leader sequence derived from reshaped 1995 MRCIGOD.DOC -33human CAMPATH-1 light chain (Riechmann et al., Nature 1988. 332: 21) and a splice donor site (Fig. The DNAs coding for the variable regions plus eukaryotic flanking regions were cloned as Hindlll-BamHI fragments into mammalian cell expression vectors containing genomic DNAs coding for human gamma-1 constant region or human kappa constant region (Maeda et al., Hum. Antibod. Hybridomas 1991. 2: 124). The heavy and light chain expression vectors were 1 electroporated into COS cells. After 72 h, medium was collected and the chimeric anti-EGFR antibodies analyzed by ELISA (Kettleborough et al., Protein Eng. 1991. 4:773).

15 EXAMPLE 10: Production of sc Fvs derived from in vitro immunized cells.

The methods disclosed below are slight modifications of the methods .described above. Immunization, library construction and screening are given in Examples 1 4. The following steps are described in detail below: After screening the primary library and the clones derived from the three rounds of panning, some single ampicillin-resistant colonies were selected. Phagemid DNA was prepared by alkaline lysis and used to *transfect E.coli HB2151, a non-supressor strain, by heat shock. Colonies were inoculated into 2xTY-Amp-Glu and grown overnight at 30 0 C. A 5 ml aliquot was used to inoculate 50 ml of 2xTY broth containing 100 mg ampicillin/ml and 0.1% glucose and grown with shaking at 30 C for 1 h (until log-phase). Cells were harvested and expression of soluble scFv was induced by the addition of isopropyl -D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM (De Bellis, D. and Schwartz, I.; 1990. 1311). Cultures were grown oveight at 30 0

C

Nucleic Acids Res.: 1990. 18: 1311 Cultures were grown overnight at 30°C LYV.) &1RC1WiaD0C -34with shaking. Supematants containing scFv were taken, clarified by centrifugation and filtration through 0.22 mm filters and tested. Bacterial supematants were tested for binding to EGFR by ELISA, as described (Kettleborough, et al., EP 94104160 and Eur. J. Immunol. 1994. 24: 952).

The specificity of selected scFv fragments was checked by ELISA using plates coated with various proteins related and non-related to EGFR, as well as other antigens and plastic. The antigens used were: RNase, BSA, OVA, GD 3 ganglioside, vitronectin receptor (VNR), platelet glycoprotein Ilbilla (GPIIblla), and disialyl-lacto-N-tetraose (DSLNT). Coating was done ovemight at the optimum concentration for each antigen. Coated ELISA plates were blocked for 1 h at 37 0 C with 1.5% skimmed milk in PBS After washing, 100 ml of scFv supematants were added to the microtiter wells and incubated for 2 h at 37 0 C. Bound scFv were detected using the anti-c-myc antibody 9E10 (spent culture media from Myc 1- 9E10.2 hybrid) and an alkaline phosphatase-conjugated rabbit anti-mouse antibody (Dako).

Three EGFR-bearing tumor cell lines, A431, MDA MB 231 human breast adenocarcinoma (ATCC, HTB 26), and HT29 human colon adenocarcinoma (ATCC, HTB 38), and one non-expressing EGFR cell line, WM164, were used to test the ability of scFv to bind to EGFR on S: cells by mean FACS analysis and immunofluorescence with unfixed cells.

For the indirect immunofluorescence analysis, cells were plated into Terasaki plates (2x10 4 cells/well) and cultured for 24 h. Cells were them incubated with 20 ml of crude bacterial supematant containing the scFv fragments for 90 min at room temperature. Incubations with primary antibody (anti-cmyc) and secondary antibody were carried out for 60 min at room temperature. The secondary antibody, FICT-conjugated rabbit anti-mouse antibody (Dako) was diluted 1:20.

1995 MRCIGOD.DOC For FACS analysis, 5x105 cells were washed with PBS with 1% BSA and 0.1% sodium azide (PBS-BSA) and incubated at 40C for 20 min with ml of crude bacterial supematant. After two washes with cold PBS-BSA, bound scFv was detected using anti-c-myc antibody and FITC-conjugated goat anti-mouse antibody (Becton-Dickinson) diluted 1:25 in PBS-BSA.

Propidium iodide (PI) was added at a final concentration of 5 mg/ml. Flow cytometry analysis were performed in a EPICS Profile II equipped with an 1 air-cooled argon laser. The 488 nm line (15 mV) was used for the excitation. A 530 nm band pass filter was used to collect FITC emission and a 625 nm band pass filter was used to collect PI emission. Living cells were selected by setting a bitmap on forward and side scatter and by 1 exclusion of Pl-stained cells.

The diversity of the primary and selected libraries was determined by PCR amplification of cloned fragments (Gossow, D. Clackson, T; Nucleic Acids Res. 1989. 17: 4000) and analysis of the BstNI digestion pattern 20 Some clones were sequenced using a Sequenase kit (USB) by the S: dideoxy chain termination method (Sanger, F et al.; Proc. Nat.Acad. Sci., U.S.A. 1977. 74: 5463).

.I Crude bacterial supematants (10 ml) were subjected to SDS-PAGE using a 12.5% gel. Western blotting was performed essentially as described by Towbin (Towbin et al. J. Proc.NatAcad.Sci., U.S.A 1979. 76: 4350). Proteins were transferred by electroblotting to Immobilon-P (Millipore) or nitrocellulose (Bio-Rad). The blot was blocked with PBS containing 2% skimmed milk scFv fragments were detected using anti-c-myc antibody (9E10), peroxidase-conjugated anti-mouse antibody (Jackson), and an enhanced chemiluminiscence System (ECL, Amersham).

The quantitative analysis of the shed membrane vesicles revealed a total protein concentration of 2.5 mg/ml, of which only 10-14% corresponded to EGFR (Sato et al.; J. Natl. Cancer Inst. 1989.21: 1601; Yeaton, R et al., 1995 MRCIGOD.DOC -36- J. Biol. Chem., 1983. 258: 9254), 250 to 350 ng/ml. Electrophoretic analysis using PAGE-SDS followed by Coomassie-blue staining showed that the vesicles contained a rather complex mixture of proteins. No protein degradation was detected. Western blot analysis revealed that under our experimental conditions complete molecules of EGF receptor were present in the membrane vesicle preparation.

In order to determine the requirements for FCS and limpholdnes MTC and M2 containing 20% or 4% FCS were compared. Vesicles bearing EGFR and PBS were used as antigen and control respectively. Splenocytes were incubated in six well plates with or without antigen for 3 h in M1 (serum-free). MTC or M2 was then added and, after 72, 96, 120 or 144 h, s 15 screening was carried out using A431 fixed cells. In all experiments, the number of viable cells recovered was between 20 and 40% in agreement .with published results (Gavilondo-owley, J. et al.; In Vitro Immunization in Hybridoma Technology, Elsevier Science Publishers Amsterdam 1988, p.

r 20 131). The maximum specific response was obtained on day four with MTC; whereas, M2 at 4% or 20% FCS or 10% final concentration) delayed the maximum response until day six (Table However MTC and 10% FCS triggered a non-specific response, probably by polyclonal activation, as could be seen when the results were expressed as the ratio S of specific non-specific response. For further assays we decide to use M2 supplemented with 4% FCS and 6 d of culture.

The presence of EGFR in the suface of vesicles strongly enhanced the response to this antigen. Insimilar protocols as described above, vesicles from expressing and non-expressing EGFR cell lines were compared.

Lymphocytes were cultured with vesicles in M1 for 3 h. Afterwards M2 containing 4% FCS was added. After 6 d, lymphocytes from each group were cultured for 48 h in 96 well plates coated with EGFR, A431-fixed cells, RNase or GD3. As expected, the results of these assays showed a 1995 MRCIGOD.DOC -37multispecific pattern of response (Table The reactivity against EGFR was clearly increased in terms of optical density when EGFR-expressing vesicles were used as antigen.

Taken together, these results suggest that, although immature, there was a measurable antigen-dependent response after in vitro immunization which generated several pools of immunized lymphocytes against EGFR suitable for PCR-cloning of variable regions.

A library of 1,1x105 dones was obtained after cloning scFv fragments derived from in vibt immunization into the pHEN1 phagemid. This library was generated in parallel with two more libraries providing of in viv immunization. The construction of these phages libraries has been 15 described previously (Kettleborough, et al., EP 94104160 and Eur. J.

Immunol. 1994.24: 952).

To select the scFv fragments binding to EGFR, phage were panned using EGFR-coated immunotubes. Eluted phage were used to reinfect a SupE 2 strain of E.coi. In total, three rounds of selection were carried out. In each round, a tube without antigen was tested in paralel to calculate the background. In the first panning, 1.5x101' phage particles were applied to the immunotube and 6.6x10 4 were eluted from the coated immunotube; S 25 whereas, only 200 colonies were obtained from the background population. After the third panning, 1x10" phages were applied and 5.6x101 0 were eluted.

To further characterize the scFv fragments, we selected 22 clones from the phage populations, before selection and after each round of selection.

The diversity of the library was analyzed by the BstNI digestion patterns of the cloned fragments. Prior to selection the library appeared to be extremely diverse. Fingerprinting of binding clones derived after the first 1995 MRCIGOD.DOC -38round of selection indicated the presence of several groups withthe same restriction pattern.

Clones were selected from different rounds of selection based an their digestion patterns. DNA sequencing revealed the presence of drerent sequences in most of the selected clones. The length and composition of complementarity determining regions (CDRs) of dones 10D2, 5D3, 10E2, 183, 4B3 and 5E2 were different. The most variation was'observed in the CDR3s of VH and VL sequences. Clones 5D3 and 1E3 were derived from the third round of selection. They bound strongly to EGFR as analyzed by SELISA and flow cytometry and had the same sequence.

Soluble scFv fragments were obtained by growth of the non-suppressor 15 E.colistrain HB 2151 in presence of IPTG.

To verify scFv production, bacterial medium from individual clones, was analyzed by gel electrophoresis. Western blot analysis revealed a clear band around 35,000 kD.

Clones with binding activities to EGFR were identified by ELISA. To examine the cross-reactivity of selected clones, ELISA assays using different antigens were carried out. The antigens (EGFR, RNase, BSA, S KLH, OVA, GDa ganglioside, vitronectin receptor, platelet glycoprotein Ilbllla, and disialyl-lacto-N-tetraose) were coated into ELISA plates at the optimum concentration (Table No binding to non-EGFR antigens was detected. The scFvs were also tested for binding to three EGFR-bearing tumor cell lines (human epidermoid carcinoma A431, human breast adenocarcionma MDA MB 231 and human colon adenocarcinoma HT 29). WM 164 a human melanoma non-expressing EGFR was used as a negative control. Those that bound to tumor cell lines was tested by indirect immunofluorescence using unfixed cells and quantified by FACS analysis. The use of unfixed cells ensures the natural conformation of the membrane receptors. Positive clones showed a clear fluorescence using -39- A431 cells. Fluorescence with the others EGFR-bearing tumor cell lines was weak. No binding to the negative cell line was detected. The results were confirmed by flow cytometry. Seventeen positive clones and three negative clones were analyzed for binding to A431, MDA MB 231 and HT 29 cells by flow cytometry. WM 164 was used as the negative cell line.

The 425 scFv (P1 clone) was used as a positive control and the cloning vector (HEN) as a negative control. The results are summarized in Table 5. Two clones, 4B2 and 5E2, were positive for binding to EGFR, as analyzed by ELISA, but negative for binding to EGFR-expressing tumor cell lines.

0

S

1995 &MRCR1t)iyDC Table Effect of different media on in vito immunization-' Day of sc=eing against of A43 1 -3th day 4th day f th day 6th day rAmyl OrII .D.cl Ratiod) O.D. Ratio f 01. Ratio f 01. Rato 9 9=* .9 9. 9 9.

9 5 9 9 .9 95 9= 9 9= 9=.

9 9= 9.9=9= 1 Vesicles 0.393 2.11 10.801 3.76 10.784 3.90 0.951 10.3 PBS 0.186 0.213 0.201 0.092 2 Vesicles 0.527 2.50 0.852 1.76 0.863 2.75 1.168 3.94 PBS 0.210 0.482 0.3 13 0.296 3 Vesicles 0.763 1.48 1.169 2.01 1.089 2.07 1.115 1.91 PBS 0.5 13 0.581 0.525 0.581 Asmy 1: MI plus M2, 4% FCS (Final FCS: 2%) Assay 2: M1 plus W, 20% FCS (Final FCS: Assay 3- A-medium plus MTC, 4% FCS (Final FCS: 2%) a) BALB/c mouse spleen cells were incubated in 3.5 ml of M1 with 20 vesicles from A431 cells or PBS for 3 h in wells of 6 well plates. Afterwards ml of MTC or M2 containing 4% or 20%A FCS were added and the plates incubated. At 3, 4, 5 or 6 days in vfm immunized lymphocytes were removed from culture medium, washed in HBSS to remove vesicles and 25 seeded in 96 well plates coated with fixed A431 cells, and incubated for 48 h (see Methods).

b) Final concentration of FCS in culture medium.

c) 0.0. Optical densitly read at AO5nm. It represents the mean of sixteen wells.

d) Ratio of specific response (vesicles as antigen) unspecific response (PBS as antigen).

1995 MRCIGOD.DOC -41- Table 3. Multi-specificity of the response after in vitro iunizaionOa) Screening against Antigen A431 CELLS EGFR GD3 RNase group Assay 1 EGFR 0.512 0* 0.328 0.140 0.249 EGFR 0.427 0.070 0.123 0.304 Assay 2 EGFR 1.430 0.730 0.233 0.670 EGFR 0.789 0.195 0.118 0.561 a) Lymphocytes were in vdfo immounized usin ite GF-xresn vesicles (EGFR+) or non-EGFR expressing vesicles (EGFR-). After six days 15 of incubation, cells were removed from culture and screened against the above mentioned-antigens.

b) Response is expressed as optical density (405 nm).

Table 4. Cross-reactivity of selected scFv fragments against several antigens

S.

6 9 ~qq~

S

S.

6 6 66 6 69 6 66 .6 ~6 S 5-*6 9 6S .5 a 6~.

S

a S~.

ANTIGEN COATING lmu RESULT EGFR 2.5 RNase BSA 10 KLH OVA

GD

3 ganglioside 2 VNR 1 GPllbmaI DSLNT a) b) ELISA assays were performed as described.

Vrtronectn receptor (VNR) platelet glycoprotein lbi~a (Gpllbinla); disialyl-lacto- Letraose

(DSLNT).

1995 MRCIGOD.DOC

S

S.

15

S*

S2 S e se

S

25

S

S

ha..

-42- Table 5. Reactivity of scFv clones against EGFR. Comparative results between an ELISA method with purified soluble antigen and cytometric analysis of cell lines.

CLONES CYTOMETRIC ANALYSIS OF TUMOR CELL LINES ELSA (mean of anrita flaomeenc units) Postive WM164 A431 MDAAMB231 IT29 EGFR 7H1 1.5 112.9 16.4 2.6, 1,2 482 1.2 5.3 4.2 0.6 2 10D2 1.5 145.3 36.3 4.8 2 12D2 1,8 129.5 29.3 5.7 2 SE2 1.4 2.5 7.1 0.5 1.8 8E2 1.5 134.5 47.7 51 1.9 5M2 1.3 146.3 40.6 5.7 1.9 11H2 1.9 152.2 25.3 2 1.9 IB3 0.6 105.1 36.4 5.2 >2 4B3 0.5 78 15.8 2.3 2 3D3 1.2 94.3 25.1 4.8 1.9 5D3 0.5 112 22.2 5,5 >2 4F3 0.4 110.3 32.3 6.2 >2 4G3 0.4 76,5 20.4 2 >2 1E3 0.4 118.3 33.8 5.1 2 3H3 0.6 76.5 33.7 4.2 >2 Negative SF1 2.4 2.3 3,6 1.8 0.2 7G1 1.4 10.2 4 2.8 0.2 111 0.5 5 4 0.75 0.2 Table 5: coninuafion ContrOlS (b HEN 0.4 4.1 3.7 1 0.2 P1 0.6 85.5 21.3 2.5 1.9 a) Three EGFR-bearing cell lines (A431, MDAAMB231 and HT29) and one non-expressing cell line (WM164) were used to assay the ability of scFv to bind to tumor cells lines by cYtometric analysis as described.

b) Vectormwithout fragment (HEN) and scFv fragnent from 425-miAb (P1) were used as negative and positive controls, re-speciel.

205 iM~ NMkCi(jci.)oc -44- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Merck Patent GmbH STREET: Frankfurter Str. CITY: Darmstadt COUNTRY: Germany POSTAL CODE (ZIP): 64271 (G)_TELEPHONE: 49-6151-727022 TELEFAX: 49-6151-727191 (ii) TITLE OF INVENTION: Anti-EGFR Single-Chain Fvs and Aznti-EGFR Antibodies (iii) NUMBER OF SEQUENCES; 32 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk 15 COMPUTER: IBM PC compatible OPERATING-SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO.- 1: SEQUENCE CHARACTERISTICS: 20(A) LENGTH: 327 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:.

NO

FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAL STAGE: adult TISSUE TYPE:4 Lymph node (vii) IMMEDIATE SOURCE: CLONE: L2 11C (light chain) (ix) FEATURE: NAME/KEY: CDS LOCATION4:1. .327 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAO ATT GAG CTC ACC GAG TCT CCA GCC TCC CTG OT GCA TOT GTG GGA 48 Asp Ile Glu Leu Thr Gin Ser Pro Ala Ser Leu Ala Ala Ser Val Gly 1 5 10 1995 MRCIGOD.DOC .999 9 .9 i i .9 9.

9 9 9* 99 *9 99 99 9 9 9 25 99 99 9 9 C *99* GAA ACT GTC ACC ATC ACA TGT CGA GCA AGT GAG Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 25 TTA GCA TOG TAT CAG CAG AAG CAA GGG AAA TCT Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser 40 TAT AGT GCA AGO GCC TTG GMA GAT GOT GTC CCA Tyr Ser Ala Ser Ala Leu Glu Asp Gly Val Pro s0 55 AGT GGA TCT G ACA CAG TAT TCT TTA MAG ATC Ser Gly Ser Oly Thr Gin Tyr Ser Leu Lys Ile 70 75 GAR GAT ACC OCT ACT TAC TTC TOT AAA CAG ACT Giu Asp Thr Ala Thr Tyr Phe Cys Lys Gin Thr 90 ACG TTC GOT OGA GO ACC AAG CTG GAA ATA MA Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 INFORMATION FOR SEQ ID NO.- 2: SEQUENCE CHARACTERISTICS: LENGTH: 109 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24.

Asp Ile Glu Leu Thr Gin Ser Pro Ala Ser Leu 1 5 10 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu 20 25 Leu Ala Trp Tyr Gin Gin Lys Gin Giy Lys Ser 35 40 Tyr Ser Ala Ser Ala -Leu Glu Asp Gly Val Pro 50 -55 Ser Gly Ser Gly Thr Gin Tyr 8cr Leu Lys Ile 70 75 Glu Asp Thr Ala Thr Tyr Phe Cys Lys Gin Thr 85 90 Thr Phe Gly Gly Gly Thr Lys Leu Glu le Lys 100 105 INFORMATION FOR SEQ ID NO: 3: SEQUENCE

CHARACTERISTICS*.

LENGTH: 357 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear AAC ATT ASA le CCT CAG Pro Gin TCGO AGO Ser Arg OC MAC ASA Aen TAT GAO Tyr Asp OGG ocM Arg Ala Ala Ala Asn Ile Pro Gin Ser Arg ASA Ken Tyr Asp Arg Ala TAC TAT Tyr Tyr CTC OTO Lei Leu TC AGT Phe Ser ATG 0KG Met Gin GTT COG Val Pro

ACT

Ser

ATC

Ile

GGC

Gly

OCT

Pro

TG

Trp 96 144 192 240 288 327 Ser Tyr Leu Phe Met Val Val Tyr Leu Ser Gln Pro

~LL~J~

-46- (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iV) ANTI-SENSE: No FRAGMIENT TYPE:- N-terminal (vi) ORIGINAL SOURCE-.

ORGANISM: mouse STRAIN:SBalb/c D ELOpI4NTAL STAGE: adult TISSUE TYPE: Lymph Node (vii) IMMEDIATE

SOURCE:

CLONE: L2 11C (heavy chain) (ix) FEATURE:- NMNE/KEY: 005 LOC&TION:1. .357 (xi) SEQUENCE DESCRIPTIONI: SEQ ID NO: 3:.

9999 *9 i i 9* 9 Gin 110 GTG CAA 070 GAG Val Gin Lou Gin

GAG

Giu 115 TCA GGG CCT GAG Ser Gly Pro Glu

OTO

Leu 120 GTG AGG COT GG Val Arg Pro Gly

OT

Ala TCA GTG AAG ATO Ser Val Lys Met

TOO

Ser 130

ATG

Met TGO AAG GMT TCA Cys LYS Ala Ser AAA CAG AGG OCT Lys Gin Arg Pro 150

G

Gly 135 TAT ACC T70 ACT Tyr Thr Phe Thr ACC TAO The Tyr TGG ATA CAC Trp Ile His GGA CAA GO OTT Gly Gin Gly Lou 9~ 9 #9 99 9 *99* 9. #9-99 9".

GAG, TGG ATT Gin Trp Ile GAG h-AT 770 Gin Asn Phe GGC ATG ATT GAT COT TOO AAT Gly Met le Asp Pro Ser Asn 160

ACT

Ser 165 GAA ACT AGG TTA Giu Thr Arg Leu hAT hen 48 96 144 192 240 288 336 357 AGG GAO Arg Asp 175 hAG GCO ACA TTG Lys Ala Thr Lou AGT 0Th Ser Val I80 GAO AAA TOC Asp Lys Ser

TOO

8cr 185 AAT AAA 000 TAO hen Lys Ala Tyr

ATG

Met 190 GAG 070 AGO AGC Gin Lou 8cr Ser

CTG

Lou 195 ACA TOT GAG GAO The Ser Glu Asp

TOT

Ser 200 GCA ATO TAT TAO Ala Ile Tyr Tyr

TGT

Gys GCA AGA TOG GAO Ala Arg Trp Asp ACC AOG GTC Cc Thr Thr Val Thr 225

TAO-

Tyr 210 GOT AGT GGC Gly Ser oiy GAO TTT His Phe 215 QAC TAO TGG GOC Asp Tyr Trp Gly CAA GGO Gin Gly 220 GTC TOO TGA Val Scr Ser INFORMATION FOR SEQ ID NO: 4: A i) SEQUENCE

CHARACTERISTICS:

LENGTH: 119 amino acids TYPE: amino acid TOPOLOGY:- linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 1995 MRCIGOD-DOC 47- Gin Val Gin Leu Gin Glu Ser Gly Pro Glu Leu Val Arg Pro Gly Al& 1 5 10 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 25 Trp, Ile His Trp, Met Lye Gin Arg Pro Gly Gin Gly Leu Gin Trp Ile 35 40 Gly Met Ile Asp Pro Ser Aen Ser Glu Thr Arg Leu Asn Gin Asn Ph.

55 Arg Asp.Lys Ala Thr Leu Ser Val Asp Lys Ser Ser Asn Lye Ala Tyr 70 75 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 Ala Arg Trp, Asp Tyr Gly Ser Gly His Phe Asp Tyr Trp, Gly Gin Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 151 (iM SEQUENCE CHARACTERISTICS: LENGTH: 339 base pairs TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 20 (iii) HYPOTHTICAL: NO 9-9 ANTI-SENSE: NO FRAGMENT TYPE: N-terminal *99 (vi) ORIGINAL SOURCE: 99,9 ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAIL STAGE: adult (vii) IMMEDIATE SOURCE: (B3) CLONE: L2 12B (light chain) (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (xi) SEQUENCE DESCRIPTION:. SEQ ID NO: GAG ATT GAG CTC ACC CAG TCT CCA GCT TOT TTG GCT GTG TCT CTA GOG 48 Asp Ile Glu Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 120 125 130 135 GAG AGG GCC ACC ATC TCC TGC AGA GCC AGC GAA AGT GTT GAT AAT TTT 96 Gin Arg Ala Thr Ile Ser Cys Arg Al a Ser Glu Ser Val Asp Asn Phe 140 145 150 195 MRCIGODDOC -48- GOC ATT AGT TTT ATG AAC TOO TTC CAA CAG AAA CCA GGA CAG CCA CCC 144 Gly Ile Set Phe Met Asn Trp Phe Gin Gin Lys Pro Gly Gin Pro Pro 155 160 165 AAA CTC CTC ATC TAT GGT GCA TCC AAC CAA GGA TCC GOGTC CCT GOC 192 Lys Leu Leu Ile Tyr Gly Ala Ser Aen Gin Gly Ser Gly Val Pro Ala 170 17S 180 AGG TTT ACT GOC AGT GGG TCT 000 ACA GAC TTC AGC CTC AAC ATC CAT 240 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Lou sn Ile His 185 190 195 CCT CTG GAG GAG GAT GAT ACT OCA, ATO TAT TTC TOT CAG CAA ACT flAG 288 Pro Lou Glu Glu Asp Asp Thr Ala Met Tyr Ph. Cys Gin Gin Set Lys 200 205 210 21S SGAG OTT CO CTC ACG TTC GOT OCT GGG ACC flAG CTG, GAA ATA AAA COG 336 "0 GiU Val Pro Lou Thr Ph. Gly Ala Gly Thr Lys Lou Giu Ile Lys Arq 220 225 230 GCG 339 Ala INFORMATION FOR SEQ ID NO; 6: 15~ 15(i) SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids 9 i~ TYPE: amino acid TOPOLOGY: linear 5 (ii) MOLECULE TYPE-:-protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: :20 Asp Ile Glu Lou Thr Gin Ser Pro Ala Ser Leu Ala Val Set Leu Gly 10 Gin Arg Ala Thr Ile Set Cys Arg Ala Ser Giu Ser Val Asp Asn Phe 25 Gly Ile Set Ph. Met Asn Trp Ph. Gin Gin Lys Pro Gly Gin Pro Pro 40 Lys Leu Leu Ile Tyr OWy Ala Ser Aen Gin Gly Ser Gly Val Pro Ala -so9 55 60 .Arg Phe Set Gly Ser Gly Ser Oly Thr Asp Phe Set Leu Aen le His 70 75 Pro Leu Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gin Gin Set Lys 90 Glu Val Pro Leu Thr Phe Gly Ala Gly Thr Lye Leu Giu Ile Lye Arg 10105 110 Ala INFORMATION FOR SEQ ID NO: 7:.

SEQUENCE CHARACTERISTICS: LENGTH*. 357 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 1995 -MRCIGOD-DOC -49- (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terniinal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAL STAGE: adult TISSUE TYPE: Lymph node (vii) IMMIEDIATE SOURCE: CLONE: L2 12B (heavy chain) (ix) FEATURE: NAME/KEY: CDS I.OCATION:1. 357 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 9*9* 9 9. 9 9* 99* 9 9* 9. i 9~ 99 9 9 99 9 *999** *999 9 9~9 a *9e 99 9. 9 99 a.

9. 9 9 a..

9 a 9-9~.

9. 9-.

9* 9 a *9-9* 15 AG GTG 15 Gin Val 115 CAG CTG CAG GAG Gin Leu Gin Glu GGA CT GAG CTG Gly Pro Glu Leu

GTG

Val.

125 AAG CCT G00 GOT Lys Pro Giy Ala

TTA

Leu 130 GTG MAG ATA TCO Val Lys Ile Ser

TGO

Cy5 135 RG GCT TOT GOT Lys Ala Ser Gly

TAG

Tyr 140 ACC TTO ACC AGO Thr Phe Thr Ser

TAC

Tyr 145 -TOG ATG CAC TOG GOG AAG CAG AGG CCT GGA CAA 000 GTT GAG TGG ATC 20 Trp Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp le 150 155 160 GGA GAG ATT Gly Glu Ile RAG GGC AAO Lys Gly Lys 180

GAT

Asp 165 OCT TCT GAT AG? Pro Ser Asp Ser ACT MAC TAC AMT Thr Asn Tyr Asn CAR MAG TTC Gin Lys Phe 175 ACA GCC TAC Thr Ala Tyr 0CC ACA TTG ACT Ala Thr Leu Thr GAC AAA TCO TOO Asp Lye Ser Ser

ARC

Asn 190 144 192 240 288 336 ATG CAG met Gin 195 CTC AGC AGO CTG Leu Ser Ser Lou

ACA

Thr 200 TCT GAG GAC TCT Ser Olu Asp Ser

GCG

Ala 205 GTC TAT TAG TOT Val Tyr Tyr Cys

GCA

Ala 210 AGA TCG GAC TAC Arg Ser Asp Tyr

GOT

Gly 215 AGT AGO CRC TTT Ser Ser His Phe

GAC

ASP

220 TAG TOG GGC CAR Tyr Trp Gly Gln 000 Gly 225 ACC ACO GTC ACC Thr Thr Val Thr GTC TCO TCA Val Ser Ser 230 INFORMATION FOR SEQ ID NO: 8:.

SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid TOPOLOGY:. linear 19) MRCIGOL).L)C Gi L~e Tr Gi Ly 6 me Al] *0e* 9 15

CCC

C C C 9 9* I 9 9* 9 C. CC

C

20 9 C-C Ci 9 C 99C 9

C-C..

25 9* Ce 9 9 C-99 Th (2; (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:. SEQ ID NO: 8.

n Val Gln Leu Gin Glu Ser Gly Pro Giu Leu Val Lys Pro Gly Ala 10 u Val Lys le Ser Cys Lys Ala Ser Gly Tyr Thr Ph. Thr Ser Tyr 25 p Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile 40 y Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn GIn Lys Ph.

55 s Gly Lys Ala Thr Leu Thr Val Asp Lys 8cr 8Cr Asn Thr Ala Tyr 5 70 75 s0 t Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 90 a Arg Ser Asp Tyr Gly Ser Ser His Phe Asp Tyr Trp Gly Gin Gly 100 105 110 r Thr Val Thr Val 8cr 8cr 115 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 339 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:.

NO

FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN:. Balb/c DEVELOPMENgTAL STAGE: adult TISSUE TYPE: Lymph node (vii) IMMEDIATE SOURCE: CLONE: L3 11D (light chain) (ix) FEATURE: NAME/KEY: CDS LOCATION:1...339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATT GAG CTC ACC CAG TCT CCA GCT TCT TTG GCT GTG TCT CTA GG Ile Glu Lou Thr Gln Ser Pro Ala Set Leu Ala Val Ser Leu Gly 125 130 135 AGG GCC ACC ATC TCC TGC CGA GCC AGC GAA AGT OTT OAT AAT TTT Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 140 145 150

GAC

Asp 120

CAG

Gin -51-

C

Gly

AAA

Lys

AGO

Arg

OCT

Pro 200

GAG

Glu

GCG

Ala

ATT

Ile

CTC

Leu

TTT

Phe 185

TTG

Leu

OTT

Val AGT TTT Ser Phe 155 CTC ATC Leu Ile 170 AGT GGC Ser Gly GAG GAG Glu Giu CCG CTC Pro Leu

ATC

Mot

TAT

Tyr

ACT

Ser

GAT

Asp

ACO

Thr 220 AAC TG Asn Trp GOT OCA Oly Ala G00 TCT Gly Ser 190 OAT ACT Asp Thr 205 TTC GOT Phe Gly TTC CAA CAG Ph. Gln Gin 160 TCC AAC CAA Ser Asn Gin, 175 GO ACA GAC Gly Thr Asp GCA ATO TAT Ala Met Tyr OCT GO ACC Ala Gly Thr 225 AAkA CCA Lys Pro GGA TC Oly Ser TTC AGO Phe Ser 195 TTC TOT Phe Cys 210 AAG CTO Lys Leu GGA CAG CCA CCC Gly Gin Pro Pro 165 000 OTC CCT 0CC Oly Val Pro Ala 180 CTC AAC ATC CAT Lou Aen Ile His CAG CAA AGT AAG Gin Gln Ser Lys 215 GAG CTO AAA cG; OiU Lou Lys Arg 230 144 192 240 288 336 339 a-.

*0 0 000 9 0~ 000001 0 a. 0 0~00 9.

00 *0 a

C

9* a. 0 0* 15 INFORM4ATION FOR SEQ ID No:. SEQUENCE CHmrRiSTICS.- LENGTH: 113 amino aCids TYPE: amino acid TOPOLOGY.- linear (ii) MOLECULE TYPE; protein 20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO.- Asp Ile Glu Lou Thr Gin Ser Pro Ala Ser Leu Ala 10 Gin Arg Ala Thr Ile Ser Cys Arg Ala Ser GiU Ser 20 25 Gy Ile Ser Phe Met Aun Trp Phe Gin Gin Lys Pro 3540 Lys Leu Leu Ile Tyr Gly Aia Ser Aen Gin Gly Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser 70 75 Pro LeU Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys 85 90 Giu Vai Pro Leu Thr Phe Gly Aia Giy Thr Lys Leu 100 105 Ala Val Val Gly Gly Leu Gin Glu Ser Asp Gin Val Asn Gin Leu 110 Leaa Asn Pro Pro Ile Ser Lys Giy Phe Pro Ala His Lys Arg 1995 ILRCIGODDoc -52 INFORMATION FOR SEQ ID NO: ll: i)SEQUENCE CHARACTERISTICS: LENGTH: 357 base pairs (1B) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAL STAGE: adult TISSUE TYPE:. Lymph node (vii) IMMEDIATE SOURCE: CLONE: L3 11D (heavy chain) (iX) FEATURE: NAMS/KEY: CDS OCATION:1. .357 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- 11: 9 .9 99 9 *99 9 9 9.

9 99. i 9.

99 9 99 9 9 '.99 9 99 9 .9 9 9' 9 9 9 9 9 99 *9 9 9 9-0+S 9 GAG GTG Glu Val 115 CAG CTG CAG Gin Lou Gin CAG TCR Gin Ser 120 GGG GCT GAG CTT Gly Ala 01U Lou

GTG

Val 125 AAG CCT GGG GCT Lys Pro Gly Ala

TCA

Ser 130 CTO RAG CTG TCC Val Lys Leu Ser

TGC

Cys 135 RAG GCT TCT C Lys Ala Ser Gly

TAC

Tyr 140 ACC TTC ACC AGO Thr Phe Thr Ser

TAC

Tyr 145 TGG ATG CAC TG Trp Met His Trp

GTG

Val 150 AAG CAG AGG COT Lys Gin Arg Pro

GGA

Gly 155 CAR GGC CTT GAG Gin Gly Leu Glu TGG ATC Trp Ile 160 25 TAR GAG ATT Cly Glu Ile RAG GGC AAG Lye Gly Lys 180 ATG CAG CTC Met Gin Leu- 195 CCT TCT GAT AGT Pro Ser Asp Ser TAT ACT Tyr Thr 170 ARC TAC ART Asn Tyr Aen CAR AAG TTC Gin Lys Phe 175 ACA GCC TAO Thr Ala Tyr 48 96 144 192 240 288 336 CCC RCA TTG ACT Ala Thr Lou Thr

GTA

Val 185 GAC ARA TCC TCC Asp Lys Ser Ser

ROC

Ser 190 AGC RGC CTG Ser Ser Lou

ACA

Thr 200 TCT GAG GAC TCT Ser Glu Asp Ser

GCO

Ala 205 GTC TAT TAC TOT Val Tyr Tyr Cye

GCA

Ala 210 RGA TCG GAC TAC Arg Ser Asp Tyr

GGT

Gly 215 AGT AGC CRC TTT Ser Ser His Phe TAC TGG GGC CAR Tyr Trp Gly Gln

CG

Gly 225 ACC ACG GTC Thr Thr Val ACC GTC TCC TCA Thr Val Ser Ser 230 1995_ MRCIGOD.DOC -53 INFORM4ATION FOR SEQ ID No:. 12: Mi SEQUENCE CHARACTERISTICS: LENGTH: 119 amino aCids TYPE: amino acid TOPOLOGY:. linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:. SEQ ID NO: 12: Giu Val Gin Leu Gin Gin Ser Gly Ala Glu Lou Val Lys Pro Gly Ala 1 10 Ser Val Lys Lau Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 25 Trp Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile 35404 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Aen Gin Lys Phe 55 Lys Gly Lys Ala Thr Lou Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 9 4. 65 70 75 s0 Met Gln Lou Ser Ser Lou Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 90 *Aia Arg Ser Asp Tyr Giy Ser Ser His Phe Asp Tyr Trp, Gly Gin Giy 99100 10S 110 Thr Thr Val Thr Val 5cr Ser 115 20 INFORMATION FOR SEQ ID NO:- 13: SEQUENCE CHARACTERISTICS: LENGTH: 327 base pairs TYPE: nucleic acid STRANDEDNESS:. single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:- ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAL STAGE: adult TISSUE TYPE: Lymph node (vii) IMMEDIATE SOURCE: CLONE: S4 2D (light chain) (ix) FEATURE: NAME/KEY: CDS LOCATION:.1. .327 1995 MRC[GODOC -54- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GAC ATT GAG CTC ACC CAG TCT Asp 120

GA.

TAC

Tyr

ATT

Ile

OGC

Gly

GCT

Ala 15 200

CC

Arg Ile Glu Leu Thr Gin Ser AAG ATC Lys Ile TTG CAT Lou His TAT AGO Tyr Arg 170 AGT GG Ser Gly 185 GFIA GAT GlU Asp ACG, TTC Thr Ph.

ACT ATC Thr Ile 140 TGO TAT Trp, Tyr 155 ACA TCC Thr Ser TCT GOG Ser Gly OTT 0CC Val Ala GOA GG Gly Gly 220 125

ACC

Thr

CAG,

Gin

AAT

Aen

ACC

Thr

ACT

Thr 205

GC

Gly

TGC

Cys

CAG

Gin

CTG

Leu

TCT

Ser 190

TAC

Tyr

ACC

Thr CCA ACC ACC ATG Pro Thr Thr Met 130 AGT 0CC AGC TCA Ser Ala Ser Ser 145 AAG CC GGA TTC Lys Pro Gly Ph.

160 OCT TCT GGA GTC Ala Ser Gly Val 175 TAC TCT CTC ACA Tyr Ser Lou Thr TAC TG CAG cAG Tyr Cys Gin Gin -210 AAG CTG GAA ATC Lys Lou Olu Ile 22S

OCT

Ala

AOT

Ser

TCC

Ser

CCA

Pro

ATT

Ile 195

GT

Gly

AAA

Lys

OCA

Ala

ATA

Ile

CCT

Pro

OCT

Ala 180

GC

Gly

AGT

Ser

COG

?Arg TCT CCC Ser Pro AGT TCC Ser 8cr

ISO

AAA CTC Lye Lou 165 00C T Arg Phe ACC ATG Thr met AGT ATA Ser Ile

COO

Gly 13S

AAT

Aen

TTG

LOU

ACT

Ser

GAG

Oiu

CCA

Pro 215 48 96 144 192 240 288 *3 3* 0 .9 0

S

50 0 9.9 0* 0 0 09 0 03.0 t 00 9 3000 00 59 0 00 00

S

SOO

0 3390 0 5 0*50 0090 INFORMATION FOR SEQ ID NO:- 14: 20 Mi SEQUENCE CHARACTERISTICS: LENGTH: 109 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Asp le Giu Leu Thr Gin Ser Pro Thr Thr Met Ala Ala 1 5 10 Giu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser le 20 25 Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Phe Ser Pro 40 Ile Tyr Arg Thr Ser Asn Leu Ala Ser Giy Val Pro Ala s0 55 60 Gly Ser Gly Ser Giy Thr Ser Tyr Ser Leu Thr Ile Gly 70 75 Ala Giu Asp Val Ala Thr Tyr Tyr Cys Gin Gin Gly Ser 90 Arg Thr Ph. Oly Gly Oly Thr Lye Leu Glu Ile Lys Arg 100 105 Pro 1s Ser Leu Phe Met I le Gly Asn Leu Ser Glu Pro 1995 MRCIGOD.DOC 55 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 354 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGy:- linear (ii) MOLECULE TYPE:- CDNA (iii) HYPOTHETICAL: No ANTI-SENSE: No Mv FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAL STAGE: adult TISSUE TYPE:. Lymph node (Vii) IMMEDIATE

SOURCE:.

CLONE: S4 2D (heavy chain) (ix) FEATURE: NAME/KEY: CDS LOCATION:1. .354 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

S.

0* 0 C! C 9 9 S.

0S 9 900 S

C*

C C

S.

*0*9!C

S

059 9- S C .5 9 e.g.

99 C C. *e

S

C

GAG

GlU 110 GTC AAG cTG cAG Val Lys Leu Gin

CAG

Gin 115 TCA GGA CC GAG Ser Gly pro Glu GTA AAG CCT GG Val Lys Pro Gly

GCT

Ala TCA GTG AAG ATG Ser Val Lys Met

TCC

Ser 130 TGC MAG GCT TOT Oys Lys Ala Ser

GGA

Gly 135 TAO GCA TTC ATA Tyr Ala Phe Ile AGT TTT Ser Phe 140 GTT ATG CAC Val Met His GGA TTT ATT Gly Phe Ile 160

TGG

Trp, 145 GTG MAG cAG MAG Val LYS Gin Lys

OCT

Pro 150 GGG GAG GOC CTT Gly Gin Gly Leu GAG TOG ATT Glu Trp Ile 155 GAG MAG TTC Glu Lys Phe MAT CCT TAC MAT Aen Pro Tyr Asn

CAT

Asp 165 GOT ACT MAG TAO Gly Thr Lye Tyr 48 96 144 192 240 288 336 MAA GAO Lye Asp 175 MAG GCC ACA CTG Lye Ala Thr LeU TCA GAC AAA TCC Ser Asp Lys Ser

TCC

Ser 185 AGO ACA GCC TAC Ser Thr Ala Tyr

ATG

met 190 GAG CTC AGO AGO Glu Leu Ser Ser

OTO

Leu.

195 ACC TOT GAG GAC Thr Ser Glu Asp GOG GTC TAT TAC Ala Val Tyr Tyr

TOT

Cys GCA AGT GGG GAT Ala Ser Gly Asp

TAO

Tyr 210 GAO AGO GOT ATG-GAC Asp Arg Ala Met Asp 215 TAO TGG G00 CAA Tyr Trp Gly Gin 000 ACC Oly Thr 220 ACG GTO ACC Thr Val Thr GTO TOO TCA Val Ser Ser 225 1995 MRCIGOD.DOC -56 INFORMATION FOR SEQ ID No: 16:- SEQUENCE CHARACTERISTICS: LENGTH: 118 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Glu Val Lye Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ile Ser Phe 25 Val Met His Trp, Val Lys Gin Lys Pro Gly Gin Gly Leu Giu Trp Ile Gly Phe Ile Asn Pro Tyr AMn Asp Giy Thr Lys Tyr Aen Glu Lys Phe so 505 Lye Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr to 65 70 75 8o too 0 15 Met Glu Leu Ser Ser Lou Thr Ser Glu Asp Ser Ala Vai Tyr Tyr Cys as 8 90 Ala Ser Gly Asp Tyr Asp Arg Ala Met Asp Tyr Trp Gly Gin Giy Thr .0100 105 110 *Thr Vai Thr Val Ser Ser 115 INFORMATION FOR SEQ ID NO: 17:.

SEQUENCE CHARACTERSTICS: LENGTH: 717 base pairs TYPE: nucleic acid too. STRANDEDNESS: single too. TOPOLOGY: linear 0. to 25 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN:-Baib/c DEVELOPMENTAL STAGE: adult TISSUE TYPE: spienocytes (vii) IMIMEDIATE SOURCE: CLONE: 4 B 2 (ix) FEATURE: NAME/KEY: CDS LOCATION:1- 717 -57- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17.

GAG GTG Glu Val 120 AAG CO CAG GAG TOT GGG GGA GAO TTA GTG Lys Lau Gin Glu Ser Gly Gly Asp Lou Val 125 130 MAG COT GA

GGG

Lys Pro Gly Gly

TOO

Ser 135 CTG AAA CTC TCC Leu Lys Lau Ser TGT GOA GOC TOT Cys Ala Ala Ser 140 COG cAG ACT cCA Arqj Gin Thr Pro OGA TTC Gly Phe ACT TTC AGT

AGO

Thr Phe Ser Ser TAT G ATG TCT TOG Gly Met Ser Trp

OTT

Val 155

GAC

Asp AG( AGO 070

GAG

Lys Arg Leu Glu TOT GTC GCA ACC ATT Ala Thr Ile MAG GGG CGA Lys Gly Arg 185

AGT

Ser 170 ACT GOT GGT

GO-T

Ser Gly Gly Ala

TAO

Tyr ATO TAC TAT Ile Tyr Tyr OCA GAO ACT GTG Pro Asp Ser Val TTC ACC ATC TOO Phe Thr Ile Ser

AGA

Arg GAO RAT OC MAG ASP Asn Ala Lys Asn AOC 07u TAO OTG OAA Lau Gin 200 ATG AGOC AOT CTG Met Ser Ser Lau **99 i i.

*9e *i i i *9 9* 9* 9 S. .5 S 95.5 S. .5 555*

S

AAG

Lys TOT GAG GAC ACA Ser GlU Asp Thr 000 Ala ATG TAT TAO TOT Met Tyr Tyr Cys

GCA

Ala 215 AGA OTT GMA

ACC

Arg Leu Glu Thr

GGG

Gly GAO TAT GOT Asp Tyr Ala

TTG-GAC

Leu Asp TAO TOG GGC OAA Tyr Trp Gly Gin GGG ACC AOG OTC

ACC

Thr Thr Val Thr

GTC

Val 235 TOO TCA GOT GGC Ser Ser Gly Gly

GOT

Gly GGC TOG GGC GOT Gly Ser Gly Gly GOT G00 Gly Gly 48 96 144 192 240 288 336 384 432 480 528 576 624 672 717 20 TOG GOT GO Ser Gly Gly TTG OT OTC Leu Ala Val 265 OAA AGT GTT Gin Ser Val 280

GGC

Gly 250 GGA TOT GAC ATT Gly Ser Asp Ile GAG 070 Glu Lau ACC OAG

TOT

Thr Gin Ser C CT TOT Pro Ala Ser 260 MAG GAO AGO TOT OTA

GGG

Ser Leu Gly GAT TAT GAT Asp Tyr Asp CkG AGO GCO Gin Arg Ala ACC ATA TTO Thr le Phe

TOO

GOT

Giy GAT AGT TAT ATG Asp Ser Tyr Met

AAC

Agn TGG TAO CAA CkG Trp Tyr Gin Gin

MA

Lys 295 00K OGA CAG

COA

Pro Gly Gin Pro

COO

Pro AA 070 070 ATO Lys Lou Lou Ile GMA TOT 000 GC Giu Ser Cly Val TAT GOT

CGA

Tyr Al1a Arg 305 AGT GOO

TOT

Ser Gly Ser

OCT

Pro 315 CO AGG TTT AGT Ala Arg Phe Ser

GGO

Gly TOO AAT CTA Ser Asn Leu 310 GGG ACA GAO Gly Thr Asp 325 GOK ATG TAT Ala Met Tyr TTO AGO 070 Phe Ser Leu 770 TGT CAG Phe Cys Gin 345 ATO OAT COT Ile Hi's Pro GTG GAG Val Giu GAG GAT GAT

ATT

Giu Asp Asp Ile CMA ACT AGO RAG GTT COG Gin Ser Arg Lys Val Pro 350 TOO TOG TTC Trp Ser Phe GOT GGA GGG Gly Gly Gly 58 99 99 9 99 9 9 *99 9 9* 9 *99 9 .9 9 9 99 *9*999 9 9999 9.

*9 9 99 99 9.

.9 9 9 9 *9*9 9* *9 9 9 9999

G

A

L

15

L

Tf 20 Se Le 14

GI

25 Ly Gli Pht Phe 225 (2) INFORM4ATION FOR SEQ It) NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 239 amino acids TYPE: amino acid TOPOLOGY:- linear OLECULE TYPEZZ Protein (xi) SEQUENCE DESCRIPTION: SEQ ID IUu Val Lys Lou Gin Glu Ser Gly Gly As 1 1 oer Leu Lys Lou Ser Cys Ala Ala Ser Gi 25 liy Met Ser Trp, Val Arg Gin Thr Pro As 35 40 lia Thr Ile Ser Ser Gly Gly Ala Tyr Ii1 so 55 ys Gly Arg Phe Thr Ile Ser Arg Asp As 65 70 eU Gin Met Ser Ser Lou Lys Ser Glu Asj 85 la Arg Leu Glu Thr Gly Asp Tyr Ala Let 100 105 ir Thr Val Thr Val Ser Ser Gly Gly G1l 115 120 ~r Gly Gly Gly Gly Ser Asp Ile Glu Leu 130 135 u Ala Val Ser Leu Gly Gin Arg Ala Thr 5 150 n Ser Val Asp Tyr Asp Gly Asp Ser Tyr 165 170 a Pro Gly Gin Pro Pro Lys Leu Lou le 180 185 a Ser Gly Val Pro Ala Arg Ph. Ser Gly 195 200 SSer Leu Asn Ile His Pro Val GlU GlU 210 215 Cys Gin Gin Ser Arg Lys Val Pro Trp 230 INFORMATION FOR SEQ ID NO: 194 SEQUENCE

CHARACTERISTICS:

LENGTH: 732 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA NO: 18: 5p, Leu Val 0O *y Phe Thr p Lys Arg e Tyr Tyr 60 n Ala Lys 75 SThr Ala'~ 1Asp Tyr Gly Ser C Thr Gin s 140 Ile Ph. c 155 Met Asn T Tyr Ala A Ser Gly Si 24 Asp Asp IJ 220 Ser Phe G] 235 Ly Ph Le Pr Aar Wet ~er ~ys rp rg e5 Le -y 0 Pro Gly is e Ser Ser 30 u Giu Ser 5 SAsp Ser Thr Leu *Tyr Tyr Gly Gin4 110 Gly Gly Pro Ala S Lys Asp S

I

Tyr Gin G 175 Ser Asn L 190 Gly Thr A Ala Met Ti Gly Gly Gly Tyr Val Val Tyr Cys .iy ~er ~er in eu 1995 MRCIGOD-yY2 59 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Baib/c TISSUE Type: spienocytes (vii) IMMEDIATE SOURCE:- CLONE: 10 D 2 (single-chain Fv, heavy and light chain plus linker) (ix) FEATURE: NAME/KEY:- CDs LOCATION:1. .732 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GAG

Glu 240 G CAG MT CAG CMG TCT G GCT GAA Val Gin Lou Gin Gin Ser Gly Ala Glu 245 GTG AAG COT GGG Val Lys Pro Gly

GOT

Ala 96.i ii 99 9 9.

i 6 996 9 99 699 S 6.

6. 9 ii.

9 9999 9 99 9 9t99 6.

9 9i 9.

9 9 6 9 9-~9 i 9-9.9 *6 99 is 9 *99* 9 9-9w TCA GTG AAG TTG Ser Val Lys Leu

TCC

Ser 260 TGC AAG GOT TOO Cys Lys Ala Ser GGC TAO ACC TTC ACC Gly Tyr Thr Phe. Thr 265 AGO CAC Ser His TGG ATG CAC Trp, Met Hius GGA GAG TTT Gly Glu Phe 290

TGG

Trp 275 GTG MG CAG AMO Val Lys Gln Arg

GOT

Ala 280 GGA CMA 000 Gly Gln Gly OTT GAG TGG ATC Lou Glu Trp Ile 28S MAT CCC AGC AAC Asn Pro Ser Asn

GGC

Gly 295 CGT ACT MAC TAC Arg Thr Asn Tyr

MAT

PAsn 300 GAG MAA TTC Glu Lys Phe MAG AGC Lys Ser 305 MAG GCC ACA CTG Lys Ala Thr Leu 0Th GAC AMA TCC Val Asp Lys Ser

TOC

Ser 315 AGO ACA C TAO Ser Thr Ala Tyr

ATG

Met 320 CMA CTO AGO AGO Gin Leu Ser Ser

CTG

Lou 325 ACA TOT GAG GAO Thr Ser Glu Asp GOG GTC TAT- TAO Ala Val Tyr Tyr

TGT

Cys 48 96 144 192 240 288 336 384 432 480 528 GCC AGT COG GAO Ala Ser Arg Asp

TAT

Tyr 340 GAT TAO GAO GGA Asp Tyr Asp Gly

CGO

Arg 345 TAC TTT GAO TAO Tyr Phe Asp Tyr TGG G Trp Gly 350 CMA GGG ACC Gin Gly Thr GGT 000 TOG Gly Gly Ser 370

ACG

Thr 355 GTC ACC GTC TOO Val Thr Val Ser

TOA

Ser 360 GGT GGC GGT GCO Gly Gly Gly Gly TOG G00 GOT Ser Gly Cly 365 CAG TOT OCA Gin Ser Pro GGT GGC CCC GGA Gly Gly Gly Gly

TOT

Ser 375 GAO ATT GAG CTO Asp Ile Glu Leu

ACC

Thr 380 GOA ATO Ala Ile 385 ATG TOT GCA TOT Met Ser Ala Ser GGG GAG AAG GTC Cly Glu Lys Val

ACC

Thr 395 ATG ACC TGC AGT Met Thr Cys Ser

CO

Ala 400 AGO TOA ACT GTA Ser Ser Ser Val

ACT

Ser 405 TAO ATG TAO TGG Tyr Met Tyr Trp CAG CAG AMA COA Gin Gin Lys Pro

GGA

Gly 415 9)95 1MRCIGOD.DOC

TCC

Ser

GTC

Val

ACA

Thr

CAG

Gin 1o L: 480 (2) 15

TCC

Ser

CCT

Pro

ATC

Ile

TG

Trp 465

GAA

Glu

CCC

Pro

GTT

Val

AGC

Ser 450

AGT

Ser

ATA

Ile AGA CTC CTG Arg Leu Lea 420 CGC TTC AGT Arg Phe Ser 435 CGA ATG GAG Arg Met Giu AGT TAC CCA Ser Tyr Pro

AAA

Lys ATT TAT Ile Tyr GGC AGT Gly Ser OCT GAA Ala Gita 455 CCC ATO Pro met 470 ACA TCC AAC Thr Ser Asn 425 TCT GG ACC Ser Gly Thr OCT GCC ACT Ala Ala Thr ACG TTC GGA Thr Ph. Gly 475 CTG OCT TCT GGA Leu Ala Ser Gly 430 TCT TAC TCT CTC Ser Tyr Ser Leu 445 TAT TAC TGC CAG Tyr Tyr Cys Gin 460 GOG G00 ACC AAG Gly Gly Thr Lys 576 624 672 720 732 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH*. 244 amino acids TYPE: amino acid TOPOLOGY: -linear (ii) MOLECULE TYPE: protein (XI) SEQUENCE DESCRIPTION:. SEQ ID NO: Giu Val Gin Leu Gin Gin Ser Gly Ala GlU Lau Val Lys Pro Ci 9-C C

CC..

9 CC 9 9C C.

C

9CCC CC C-C 9. C

C

*C9#

C

*9C! 20 1er Trp Gly 25 Lys 65 met Ala Gin Gly Ala 145 Ala Val met Glu Ser Gin Ser Gly Gly 130 Ile Ser Lye His 35 Phe Lys Leu Arg Thr 115 Ser met Ser Lea Trp Asn Ala Ser Asp 100 Thr Gly Ser Ser 5 Ser Val Pro Thr Ser Tyr Val Gly Ala Val 165 Cys Lys Ser Leu 70 Leu Asp Thr Gly Ser 150 Ser Lys Gin Asn 55 Thr Thr Tyr Val Gly 135 Pro Tyr Ala Arg 40 Gly Val Set Asp Ser 120 Ser Gly Met Set 25 Ala Arg Asp Glu Gly 105 Set Asp Glu Tyr 10 Gly Gly Thr Lys Asp 90 Arg Gly Ile Lys Trp 170 Tyr Gin Aen Ser 75 Ser Tyr Gly Glu Val 155 Tyr Thr Gly Tyr Ser Ala Phe Gly Leu 140 Thr Gin Phe Leau Aen Ser Val Asp Gly 125 Thr met Gin Gly Seri Trp Lys Ala Tyr Trp Gly Ser Cys Pro 175 Ala His Ile Phe Tyr Cys Gly Gly Pro Ser 160 Gly MR'CKo[.Doc -61- Se Va Th Gi 22 Le (2 15 209 2. 5 9..AG ber *999G 9., 9. .A

GAG

Ls r Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly 180 185 190 1 Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu, 195 200 205 r Ile Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 210 215 220 ni Trp Ser Ser Tyr Pro Pro Met Tyr Thr Phe Gly G;ly Gly Thr Lys 5 230 235 240 u Glu Ile Lys )INFORMATION FOR SEQ ID NO: 21: Mi SEQUENCE

CHARACTERISTICS:

LENGTH: 732 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE:- No FRAGMENT TYPE: N-terminal (vi) ORIGINAL

SOURCE:

ORGANISM: mouse STRAIN: Balb/c TISSUE TYPE: spienocytes (vii) IMMEDIATE

SOURCE:

CLONE: 3 D 3 (single-chain Fv, heavy and light chain plus linker) (ix) FEATURE: NAME/KEY:

CDS

LOCATION..732 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GTC CAA CTG CAG CAG TCA GGG GCT GMA CTG GTG AAG CCT GGG GCT Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 250 255 260 GTG AAG TTG TCC TOC AAG GCT TCC GGC TAC ACC TTC ACC AGC CAC Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 265 270 275 ATG CAC TGG GTG AAG CAG AGG GCT GGA CAA GGC CTT GAG TGG ATC I Met His Trp Val Lys Gin Arg Ala Gly Gin Gly Leu Glu Trp Ile 280 285 290 GAG TTT AAT CCC AGC AAC GOC CGT ACT AAC TAC AAT GAG AAA ATC I Giu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Ile 295 300 305 AGC AAG GCC ACA CTG ACT GTA GAC AMA TCC TCC AGC ACA GCC TAC 2 Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 310 315 320 48 96 L44 .92 1995 MRCIGOD-DoC 62 ATG CAA CTC AGO AGO CTG AMA TCT GAG GAC TCT GOO GTC TAT TAC TOT 288 Het Gin Lou Scr Ser Lou Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 325 330 335 340 GCC AGT CGG GAO TAT GAT TAC GAC GGA COG TAC TTT GAC TAC TOG GOC 336 Ala Ser Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe Asp Tyr Trp Gly 345 350 355 CAA GGG ACC ACG OTC ACC OTC TOC TCA GOT GGC GGT GGC TCG 000 GOT 384 Gin Gly Thr Thr Val Thc Val Ser Ser Gly Gly Gly Gly 8cr Gly Gly 360 365 370 GOT 000 TCG GGT 000 GGC GGA TCT GAC AT? GAG CTC ACC CAG TOT OCA 432 Gly Gly Ser Gly Gly Gly Gly Sec Asp Ile Glu Lou Thr Gin Ser Pro 375 380 385 ACA ATC ATG TCT OCA TCT CCA 000 GAG AAG GTC ACC ATG ACC TOO AGT 480 Th Ile Met Ser Ala Ser Pro Gly Olu Lys Val Thr Met Thr Cys Ser 390 395 400 GAO AGO TCA ACT GTA AGT TAO ATO TAC TOG TAO CAG CAG AAG ACA 00k 528 Asp Ser Sor Ser Val Ser Tyr Met Tyr Trp Tyr Gin Gin Lys Thr Oly 405 410 415 420 :C C G T T T TAT GAC ACA TOO AAC CTG OC TOT OGA 576 Ser Ser Pro Arg Lou Lou Ile Tyr Asp Thr ser Asn Leu Ala Ser Gly 425 430 435 OTC COT GTT COO TC AG? 000 AGT G00 TOT G0O ACC TOT TAC TCT CTO 624 Val Pro Val Acg Phe Ser Oly Sor Gly Ser Gly Thr 8cr Tyr 8cr Lou 440 445 450 *ACA ATC AGO CGA ATG GAG OT GAA GAT OCT GCC ACT TAT TAO TOC CAG 672 20 Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala The Tyr -Tyr cys Gln 455 460 465 CAG TOG AGT AGT TAC CCA CCC ATG TAC AG TTC GGA GGG 000 ACC AAG 720 Gin Trp See Ser Tyr Pro Pro Met Tyr The Phe Gly Gly Gly The Lys *470 475 480 CTG GAA ATA AAA 732 Leu Glu Ile Lys 25 485 INFORMATION FORzSEQ ID NO: 22:.

SEQUENCE CHARACTERISTICS: LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Glu Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10o Ser Val Lye Lou Ser Cys Lys Ala Ser Gly Tyr The Phe Thr Ser His 25 Trp Met His Trp Val Lys Gin keg Ala Gly Gin Gly Lou Glu Trp Ile 40 1995f MRCIG01) L)OC 63 Gly Glu Phe Asn so Pro Ser P Lys Ser Met Gln Ala Ser ln Gly Gly Gly 130 Thr Ile 145 Asp Ser: Ser Ser Val Pro Thr= Ile 210 ln Trp 20 225 Leu Glul Lys Ala Leu Ser Arg Asp 100 Thsr Thr 115 Ser Gly Met Ser Ser Ser Pro Arg 180 Val Arg 195 Ser Arg Ser Ser Ile Lys Thr Leu 70 Ser Leu Tyr Asp Val Thr Gly ly Ala Ser 150 al Ser 165 Leu Leu Phe Ser Met Glu Tyr Pro 230

T

T

G

P

I

G

A

2

P:

oan Gly Arg 55 'hr Val Asp fhr Ser Glu .)rr Asp Gly 105 'al Ser Ser 120 :ly Ser Asp .35 'ro Gly Glu Yr met Tyr le Tyr Asp 185 ly Ser Gly 200 la Glu Asp 15 ro met Tyr Thr Asn LYS Ser 75 Asp Ser 90 Arg Tyr Gly Gly Ile Glu Lys Val 155 Trp Tyr 170 Thr Ser Ser Gly A~la Ala Thr Phe 235 Tyr 60 Ser Ala Phe Gly Leu 140 Gln Asn Thr Thr 220 Gly Asn Glu Ser Thr Val yr Asp Tyr 110 Gly Ser 125 Thr Gln Met Thr Glni Lys Leu Ala 190 Ser Tyr 205 Tyr Tyr Gly Gly Lys Ala Tyr Trp Gly Ser Cys Thr 1755 Ser Ser Cys Thr Ile Tyr so Cys Gly Gly Pro Ser 160 Glyt Gly Leu Gln Lys 240 r r i i i r c, rr i i ii i cr+ i i i c r r r i s i i c ii i ii c i r i r L INFOR1AT~ON FOR SEQ ID NO: 23.- Mi SEQUENCE CHARACTERISTICS: LENGTH: 738 base pairs TYPE! nucleic acid STRANDEDNbESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTEHETICAL: No (iv) ANTI-SENSE! No FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c TISSUE TYPE: splenocytes (ii) IMMEDIAE SOURCE: CLONE: 1 E (single-chain Fv, heavy and light chain plus linker) 1995 MPOCIGODDOC -64- (iX) FEATURE: NAME/KEY: CDS LOCATIoN:1. .738 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

GAG

Glu GTG CAG CTG CAG Val Gin Lau Gin cAG Gin 250 TOT 000 GOT GMA Ser Gly Ala Glu OTG GTG AAG OCT 000 GCT Lau Val. Lys Pro Gly Ala 255 260 TCA GTG MAG TTG Ser Val Lys Lau

TC

Ser 265 TGC AMG GOT TCC CyS Lys Ala Ser

C

Gly 270 TAC ACC TTC ACC Tyr Thr Phe Thr AGC CAC Ser His 275 TGG ATG CAC Trp Met Hig GGA GAG TTT Gly Glu Phe 295

TG

Trp 280 GTG MAG CAC- hOG Val Lys Gin Arg

OICT

Ala 285 GGA CMA GGC CTT Gly Gin Gly Leu MAT CCC AGO MAC Asn Pro Sor Aean

C

Gly 300 CGT ACT Am TAO Arg Thr Asn Tyr

AAT

Aen 305 GAG TOG ATC Glu Trp Ile 290 GAG AMA TTc Glu Lye Phe ACA OCT TAC Thr Ala Tyr 55 ~S S Sb S *59 S S. 055 S 5* 5 5* 9 S S5*S 55 S S *5 4~ 55 95 0 *0S*

S

555.

.9 .5 *0 0 5 '55.

a hAG AGO Lys Ser 310 MAG 0CC ACA CTG Lys Ala Thr Lou

ACT

Thr 315 GTh GAC AMA TC Val. Asp Lys Ser TCC AOC Ser Sir 320

ATG

Met 325 CAA OTC AGO AGO Gin Lau Ser Ser

CTG

Lau 330 ACA TOT GAG GAC Thr Ser Glu Asp TOT GOG GTC TAT TAO TGT Ser Ala Val Tyr Tyr Cys 335 340 GCC AGT COG GAC Ala Ser Arg Asp

TAT

Tyr 345 GAT TAC GAO GA Asp Tyr Asp Gly TAC TTT GAC TAC Tyr Phe Asp Tyr TGG 000 Trp Gly 355 20 OAA GGG AC Gin Gly Thr GGT GGG TOG Gly Gly Ser 375 TCT CCA ACA 25 Ser Pro Thr 390 hOG Thr 360 GTC ACC GTC TCC Val Thr Val Ser

TCA

Ser 365 GOT GGO GGT C Gly Gly Gly Gly TOG G00 GGT Ser Gly Gly 370 CTO ACC CAG Leu Thr Gln GGT 000 000 GGA Gly Gly Gly Gly

TCT

Ser 380 GGA TCT GAC ATT Gly Ser Asp Ile

GAG

Glu 385 144 192 240 288 336 384 432 480 528 576 624 672 720 ATO ATG TcT Ile Met Ser

GCA

Ala 395 TOT CCk GG GAG Ser Pro Cly Glu

AAG

Lys 400 GTC ACc ATG ACC Val Thr Met Thr

TOC

Cys 405 AGT GAC -AGC TCA Ser Asp Ser Ser hOT Ser 410 0Th AGT TAC ATO Val Ser Tyr Met

TAC

Tyr 415 TGG TAO CAG CAG Trp Tyr Gin Gin

AAG

Lys 420 CCA GGA TOC TC Pro Gly Ser Ser

CCC

Pro 425 AGA CTC CT0 ATT Arg Leu Leu Ile GAC ACA TOO AMC Asp Thr Ser hen CTG OCT Leu Ala 435 TCT GGA GTC Ser Gly Val TOT CTC ACA Ser Leu Thr 455

OCT

Pro 440 OTT CGC TTC AGT Val Arg Phe Ser AGT 000 TCT 000 Ser Gly Ser Gly ACC TOT TAO Thr Ser Tyr 450 ACT TAT TAC Thr Tyr Tyr ATC AGO CGA ATG Ile Ser Arg Met

GAG

Giu 460 OCT GAA GAT Ala Glu Asp OCT 000 Ala Ala 465 TOC CAG Cys Gin 470 CAG TOO AGT AGT Gin Trp Ser Ser CCA COO ATG TAO Pro Pro Met Tyr hOG Thr 480 TTC GGA 000 GGG Phe Gly Gly Gly 1995 MK&I~tO).~u0C ACC AAG CTG GAA ATA AAA Thr Lys Leu Glu Ile Lys 485 490 INFORMATION FOR SEQ ID NO: 24:- SEQUENCE

CHARACTERISTICS:.

LENGTH: 246 amiino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Val Gin Leu Gin Gin Ser Gly Aia Giu Lou Val 10 15 10 Ser Val Lys Leu Ser Cys Lys Ala 5cr Gly Tyr Thr 25 Trp Met His Trp Val Lys Gin Arg Ala Gly Gin Gly 35 40 Lys Pro Gly Phe Thr Ser Leu Glu Trp 09 9@

U.

U

U.

U.

*U*Us.

U

U.

U

U U. *U 9S U 9 es'' 9 9**U U. *U

U

U

"U.

U

15 G1Y Gu Phe Aen Pro 8cr Aen Gly Arg Thr Aen Lys 65 Met 20 Ala Gin Gly 25 ser 145 Cys Pro Ser Ser Cys 225 Ser Lys Ala Thr Leu Thr Gin Ser Giy Gly 130 Pro Ser Gly Gly Leu 210 Gin Leu Arg Thr Ser Thr Asp Ser Val 195 Thr Gin 5cr Asp 100 Thr Gly Ile Ser Ser 180 Pro Ile Trp 70 5cr Leu Thr 85 Tyr Asp Tyr Val Thr Val Giy Giy Gly 135 Met Ser Ala 150 Ser Ser Val 165 Pro Arg Leu Val Arg Phe Ser Arg Met 215 Val Ser Asp Ser 120 Ser Ser Ser Leu Ser 200 Glu Asp Giu Gly 105 5cr Gly Pro Tyr Ile 185 Gly Lys Asp 90 Arg Gly Ser Gly Met 170 Tr Ser Ser 75 Ser Tyr Giy Asp Giu 155 Tyr Asp Gly Tyr 60 Ser Ala Phe Gly le 140 Lys Trp Thr Ser Aen Ser Val Asp Gly 125 Glu Val Tyr Ser Gly 205 Giu Thr Tyr Tyr 110 Ser Leu Thr Gin Asn 190 Thr Lys Ala Tyr Trp Gly Thr met Gin 175 Leu Ser Ala His Ile Phe Tyr Cys Gly Gly Gin Thr 160 Lys Ala Tyr Ala Giu Asp Ala Ala Thr Tyr Tyr 220 Ser Ser Tyr Pro Pro Met 230 Tyr 235 Thr Phe Giy Giy Giy 240 Thr Lys Lou Glu Ile Lys 245

MRCIGOD.DOC

66- INFORM4ATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 726 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal

'C

9. 9 o C C

C

i 9 CC 9

C

9C*C

S

9* C CC C CC C 9

C

999.

9 CC CC

C

9 99 CC 9

''CC

(Vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/C TISSUE TYPE: splenocytes (Vii) IMMEDIATE SOURCE: CLONE: 5 F 1 (single-chain Fv, heavy, light chain, linker) (ix) FEATURE: NAME/KEY:. CDS LOATION:l. .726 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 CAG GTG AAA Gin Val Lys TCA GTG AAG Ser Val Lys 265 TGG ATG CAC 25 Trp Met His 280

CTG

Leu 250 CAG GAG TCT GG Gln Glu Ser Gly

OCT

Ala 255 GAA CTO GTG AAG Glu ILeu Val Lye CCT 000 GCT Pro Gly Ala 260 ACC AGC CAC Thr Ser His TTG TOC TGC AAG Leu Ser Cys Lys TCC GGC TAC ACC Ser Gly Tyr Thr

TTC

Phe 275 TGG GTG AAG Trp Val Lys

CAG

Gln 285 AGG GCT GGA Arg Ala Gly CAA GG4C Gln Gly 290 CTT GAG TGG ATC Leu Glu Trp Ile

GGA

Gly 295

AAG

Lys GAG ATT AAT CCC Glu Ile Asn Pro AGC AAG GCC ACA Ser Lys Ala Thr 315

AGA

Arg 300 AG GCG CCT ACT Thr Ala Pro Thr

AAC

Asn 305 TAC AAT GAG AAA Tyr Aen Glu Lys

TTC

Phe 310 48 96 144 192 240 288 336 384 CTG ACT GTA GAC Leu Thr Val Asp

AAA

Lys 320 TCC TCC AGC ACA Ser Ser Ser Thr GCC TAC Ala Tyr 325 ATG CAA CTC Met Gin Leu GCC AGT CG Ala Ser Arg 345

AGC

Ser 330 AGC CTG ACA TCT Ser Leu Thr Ser GAC TCT GCG GTC Asp Ser Ala Val TAT TAC TGT Tyr Tyr Cys 340 TAC TOG GGC Tyr Trp Gly GAC TAT GAT TAC ASP Tyr Asp Tyr

GAC

Asp 350 GGA CGG TAC TTT Gly Arg Tyr Phe

GAC

Asp 355 CAA 000 Gin Gly 360 ACA AG GTC ACC Thr Thr Val Thr

GTC

Val 365 TCC TCA GOT 000 Ser 5cr Gly Gly

GT

Gly 370 GGC TCG GGC GOT Gly Ser Gly Gly 1995 MRCIGOD.Ixoc -67 GGT GGG TCG GOT 000 000 GGA TOT GAC ATT GAG CTc ACC CAG TCT CCA -432 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Ginl Ser Pro 375 380 385 390 ACA ATC ATG TCT OCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC AGT 480 Thr Ile Met Ser Ala Ser Pro Gly Giu Lys Val Thr Met Thr Cys Ser 395 400 405 GAC AGO TCA AGT GTA AGT TAC AG TAC TOG TAC CAG GAG AAG AcA GOA 528 Asp Ser Ser Ser Val Ser Tyr Thr Tyr Trp, Tyr Gin Gin Lys Thr Gly 410 415 420 TCC TCC CCC AGA CTC CTG ATT TAT GAC ACh TCC AAcC TG OCT TCT OGA S76 Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Aen Leu Ala Ser Gly 425 430 435 -0 GTC CCT GTT COO TTC AGT 000 ACT 000 TOT GG0 ACC TCT TAC TOT CTC 624 Val Pro Val Arg Phe Ser (fly Sor Gly Ser Gly Thr Ser Tyr Ser Leu 440 445 450 ACA ATO AGC CGA ATG GAG GOT GAA GAT OCT GCC ACT TAT TAO TOO GAG 672 Thr IleSer Ag Met GluAlaGlu Asp AaAa T yrTyCysGin 455 460 465 470 *~15 CAG TOG AGT AGT TAO 000 OTC ACG TTC GOT GCT 000 ACC AAG CTG GAA 720 :.Gin Trp, Ser Ser Tyr Pro Leu Thr Ph. Gly Ala Gly Thr Lys Lou (flu 5.475 480 485 ATA AAA 726 Ile Lys 20(2) INFORMATION FOR SEQ XD NO: 26: SEQUENCE

CHARACTERISTICS:

LENGTH: 242 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID No.- 26: Gin Val Lye Leu Gin Giu Ser Gly Ala Giu Leu Val Lys Pro Gly Ala 1 5- 10 is Ser Val Lys Lou Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 25 Trp Met His Trp Val Lys Gin Arg Ala Gly Gin Gly Leu Giu Trp Ile 40 Giy Glu Ile Asn Pro Arg Thr Ala Pro Thr Asn Tyr Asn Giu Lys Phe 55 Lys Ser Lys Ala Thr Leu Thr Val Asp-Lys Ser Ser Ser Thr Ala Tyr 70 75 Met Gin Leu Ser Ser Leu Thr 5cr Giu Asp Ser Ala Val Tyr Tyr Cys 90 Ala Ser Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe Asp Tyr Trp Giy 100 105 110 -68 Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly GlY Gly Ser Gly Gly 115 120 125 GlY Gly Ser Gly GlY Gly Gly Ser Asp Ile Giu Leu Thr Gin Ser Pro 130 135 140 Thr Ile Met Ser Ala Ser Pro Gly Giu Lys Val Thr Met Thr Cys Ser 145 150 155 160 Asp Ser Ser Ser Val Ser Tyr Thr Tyr Trp Tyr Gin Gin Lys Thr Gly 165 170 175 Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Aen Leu Ala Ser Gly 180 185 190 Val Pro Val Arg Phe Ser Giy Ser Gly Ser Gly Thr Ser Tyr Ser Lou 195 200 205 Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 210 215 220 Gin Trp Ser Ser Tyr Pro Lou Thr Phe Gly Ala Gly Thr Lys Leu Giu *225 230 235 240 Ile Lys i. INFORMATION FOR SEQ ID NO:- 27: SEQUENCE

CHA*RACTER~ISTICS:

LENGTH: 726 base pairs TYPE: nucleic acid STRANDEDNESS:. single TOPOLOGY: linear 20 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

'9 FRAGMENT TYPE: N-terminal 25(vi) ORIGINAL

SOURCE:

ORGANISM: mouse STRAIN: Balb/c TISSUE TYPE: aplenocytes (vii) IMMEDIATE

SOURCE:

LIBRARY: 7 G I (single-chain Fv, heavy, light chain, linker) (ix) FEATURE: NAME/KEY:

CDS

LOGATION:1. .726 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GAG GTC AAG CTG CAG CAG TCA GO GCT GAA CTG GTG AAG CCT GGG GCT 48 Giu Val Lys Leu Gin Gin Ser Gly Ala Gilu Leu Val Lys Pro Gly Ala 245 250 255 TCA GTG AAG TTG TCC TGC AAG GCT TCC GGC TAC ACC TTC ACC AGC CAC 96 Ser Val Lys Leu Ser Cys Lye Ala Ser Gly Tyr Thr Phe Thr Ser His 260 265 270 1995 MRCIGOD.OOC 69 TTG GAT CAC TGG GTG AAG CAG AGG 000 TGG Lou Asp His Trp Val Lys Gin Arg Gly Trp, 275 280 GGC OTT GAG TG Gly Leu Glu Trp

ATC

Ile 290 GGA CAG TTT AAT Gly Gin Phe Asn ccc Pro 295 AGC AAC 000 CGT Ser Asn Gly Arg

ACT

Thr 300 MAC TAO MAT GAG Asn Tyr Asn Giu AAA TTC Lys Ph.

305 AAG AGO AAG Lys Ser Lys ATC GAA CTO 11e Glu Lou 325 GCC AGT CGG Ala Ser Arg 340 ACA OTG ACT GTA Thr Leu Thr Val

GAC

Asp 315 AAA TOO TOO AGO Lys Ser Ser Ser ACA GOC TAC Thr Ala Tyr 320 TAT TAO TOT Tyr Tyr Cys AGO AGC CTG ACA Ser Ser Leu Thr GAO TAT GAT TAO Asp Tyr Asp Tyr 34S

TOT

Ser 330 GAG GAO TGC TOG Giu Asp Oys Ser

GTC

Val 335 GAO GGA CGG TAC Asp Gly Arg Tyr

TTT

Phe 350 GAO TAO TOGG0 Asp Tyr Trp Oly Sec.

S

*5 C

S.

S S C S

C

5= 55 S

S.

S

S

5, C S S S S S-C

C-.

S 5= 6 6 C-C C-S

S

CAA

Gin 355 000 ACC AOG GTO Gly Thr Thr Val

ACC

Thr 360 OTC TOO TOA GGT Val Ser Ser Gly GOT GGC TOG 000 Gly Gly Ser Gly

GT

Giy 370 15 GOT 000 TOG GOT 15 Gly Oly Ser Gly 000 Oly 375 000 GOA TOT GAO Gly Oly Ser Asp

ATT

le 380 GAG CTO ACC OAG Glu Lou Thr Gin TOT OOA Ser Pro 385 ACA ATO ATG Thr le Met GAC AGO TCA 20 Asp Ser Ser 405

TOT

Ser 390 OCA TOT OOA 000 Ala Ser Pro Gly

GAG

Giu 395 MAG GTC ACC ATG Lys Val. Thr Met ACC TOO AGT Th~r Oys Ser 400 MAG ACA GWA Lys Thr Gly 240 288 336 384 432 480 528 576 624 672 720 726 AGT GTA AGT TAO Ser Val Ser Tyr TAO TGG TA CAG Tyr Trp Tyr Gin

OAG

Gin 415 TCO TCC Ser Ser 420 COO AGA OTT OTO Pro Arg Leu Lou

ATT

Ile 425 TAT GAO ACA TOO Tyr Asp Thr Ser

AAO

Agn 430 CTG GOT TOT GGA Lou Ala Ser Gly

GTO

Val 435 CCT OTT CGO TTO pro Val Arg Phe

AGT

Ser 440 000 AGT O0G TCT Oly Ser Gly Ser ACC TOT TAO TOT Thr Ser Tyr Ser ACA ATO AGO 00k Thr Ile Ser Arg

ATG

Met 455 GAG OT GMA OAT Glu Ala Oiu Asp

GOT

Ala 460 0CC ACT TAT TAC Ala Thr Tyr Tyr TOO CAG Cys Gin 465 CAG TGG AGT Gin Trp Ser

AGT

Ser 470 TAC 000 CTO AOG Tyr Pro Lou Thr

TTO

Phe 475 GGT OCT 000 ACC Gly Ala Gly Thr MAG OTG GMA Lys Lou Glu 480 ATA AAA Ile Lys INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 242 amino acids TYPE: aino acid TOPOLOGY: linear 1995 MRCIG{JD.I)OC (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Glu Val Lys Leu *9*9 9 *9 S

S

S. 5-9 99 5 9

S

9 5 1 Ser Lau Giy Lye 65 Ile Ala 15 Gin Gly Thr 145 20 Asp Ser Val 25 Thr Gin 225 Ile Val Asp Gin Ser Giu Ser Gly Giy 130 Ile Ser Ser Pro Ile 210 Trp Lys Lys His Phe Lys Leu Arg Thr 115 Ser met Ser Pro Val 195 Ser Lau Trp, Asn Ala Ser Asp 100 Thr Gly Ser Ser Arg 180 Arg Arg Gin Ser Val Pro Thr Ser Tyr Val Gly Ala Val 165 Lau Phe met Gin Ser Gly Ala Glu Leu Val Lys Pro Cys Lys Ser Lau 70 Lau Asp Thr Gly Ser 150 Ser Leu Ser Glu Pro 230 Lys Gin Asn S5 Thr Thr Tyr Val Gly 135 Pro Tyr Ile Gly Ala 215 Ala Arg 40 Gly Val Ser Asp Ser 120 Ser Gly Met Tyr Ser 200 Glu Ser 25 Gly Arg Asp Giu Giy 105 Ser Asp Giu Tyr Asp 185 Gly Asp Gly Trp Thr Lys Asp 90 Arg Gly Ile Lys Trp 170 Thr Set Ala Tyr Gin Aen Ser 75 Cys Tyr Gly Giu Val 155 Tyr Ser Gly Ala Thr Gly Tyr Set Ser Phe dly Lou 140 Thr Gin Aen Thir Thr 220 Phe Lau Asn Ser Val Asp Gly 125 Thr met Gin Leu Ser 205 Tyr Thr Glu Gin Thr Tyr 110 Set Gin Thr Lys Ala 190 Tyr Tyr Gly Ala Ser His Trp Ile Lye Phe Ala Tyr s0 Tyr Cl's Trp Gly Gly Gly Ser Pro Cys Ser 160 Thr Gly 175 Ser Gly Ser Lau Cys Gin Ser Set Tyr Lau Thr Phe Gly Ala Gly Thr Lys Lau Gin 240 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 726 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:. No 1995 NIRCIGOD.DOC -71 (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c DEVELOPMENTAL STAGE: adult TISSUE TYPE: spienocytes (vii) IMMEDIATE SOURCE: CLONE: 11 H 1 (single-chain Fv, heavy and light chain plus linker (ix) FEATURE: NAME/KEY: CDS LOCATION:l. .726 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: S

S

S.

S S 9*9 S 55 S j S*-S 95

S.

S

995 9 5555 S S *5 5 -S S

S

S-S 55 4 I S 59 *5 5 5 55=55

S

GAG GTO AAG Glu Val Lys 245 CTG CAG CAG TCA Leu Gin Gin 8cr

CG

Gly 250 GOT GAA CTG GTG Ala Glu Lou Val

AAG

LYS

255 OCT OGG OCT Pro Gly Ala TCA GTG Ser Val 260 AAG TTG TCC TGC Lys Lou Ser Cys

AG

Lys 265 OCT TCC GGC TAC Ala 5cr Gly Tyr

ACC

Thr 270 TTC ACC AGC CAC Ph. Thr 8cr His

TGG

Trp 275 ATG CAC TGG GTG Met His Trp, Val

AAG

Lys 280 CAG AGG GOT GGA Gin Arg Ala Gly

CAA

Gln 285 GGC TTG GAG TG Gly Lou Glu Trp

ATC

Ile 290 20 GGA GAG TTT AAT Gly Glu Phe An

CCC

Pro 29S AGO AAO GGC CGT Ser Ann Gly Arg

ACT

Thr 300 AAC TAO hAT GAG Aen Tyr Aen Glu AAA TTC Lys Phe 305 MAG AGC MAG Lyn 5cr Lye ATG CAA CTC 25 met Gin Leu 325 GOC AGT 000 Ala 5cr Arg 340 ACA CTG ACT OTA Thr Lou Thr Val

GAC

Asp 315 MAA TOO TCO AGC Lys Ser Ser Ser ACA GOC TAO Thr Ala ay 320 TAT TAC TGT Tyr Tyr Cys AGO AGO CTG ACA Ser Ser Lou Thr

TOT

5cr 330 GAG GAO TOT 000 Glu Asp Ser Ala

GTC

Val 335 48 96 144 192 240 288 336 384 432 480 528 GAC TAT GAT Asp Tyr Asp GAC GGA OGG TAO Asp Gly Arg Tyr

TTT

Phe 350 GAO TAO TGG GGC Asp Tyr Trp Gly

CAA

Gin 355 000 ACC hOG OTO Gly Thr Thr Val

ACC

Thr 360 OYTC TOO TCA GGT Val Ser Ser Gly GOT 000 TCG GGC Gly Gly Ser Gly

GGT

Gly 370 GOT GGG TCG GOT Gly Gly Ser Gly G00 Gly 375 000 GGA TCT GAO Gly Gly Ser Asp GAG OTC &CC CAG Glu ]Lou Thr Gin TOT OCA Ser Pro 385 TCA ATO ATG Ser Ile Met GAO AGO TOA Asp Ser Ser 405

TOT

Ser 390 OCA TOT CCA 000 Ala Ser Pro Gly

GAG

Glu 39S hAG GTC AOC ATG Lys Val Thr Hot AOC TOO AGT Thr Cys Ser 400 hAG ACA GGA Lye Thr Gly AGT GTA hOT TAO Ser Val 5cr Tyr

ATG

Met 410 TAO TOG TAO CAG Tyr Trp Tyr Gin

CG

Gin 415 kflt~iUtJLia.jj~.

TCC

Ser

GTC

Val 435

ACA

Thr

CAG

Gin

ATA

Ile 72- TCC CCC AGA CTC CTG AT? TAT GAC ACA TCC AAC CTG GCT TCT GGA 4er Pr 9r Leu Lou Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly 420425 -430 CCT GTT CGO TTC AGT 000 AG;T COG TOT COG ACC TCT TAC TCT CTC Pro Val Arg Phe Ser Gly 8cr Gly Sor Gly Thr 5cr Tyr Set Lou 440 445 450 ATC AGC CGA ATO GAG OCT GAA GAT OCT GCC ACT TAT TAC TOO CAG Ile Ser Arg Met Glu Ala GlU Asp Ala Ala Thr Tyr Tyr Cys Gin 455 460 465 TOG AGT ACT TAC CCA CAC ACG TTC GOGT OCT 000 ACC AAG CTG GAA Trp Ser Ser Tyr pro His Thc Ph. Gly Ala Gly Thr Lys Lou Glu 470 475 48

AAA

Lys 576 624 672 720 726

CCC

C

*tC.

C

C

C. CC

C

CCC.

~C CC CC C

C

C-

CC.;.

20 25

G

a

S

A

INFOR~MATION MOR SEQ ID NO: ()SEQUENCE

CHARACTERISTICS:

LENGTH: 242 amino acids TYPE: amino acid TOPOLOGY:- linear (i)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GiU Val Lye Lou Gin Gin Ser Gly Ala Glu Leu Val. Lys Pro 15 10 Ser Val Lys Lou Ser CYS LYS Ala Set Gly Tyr Thr Phe Thr 25 30 rrp, Met His Trp, Val Lye Gin Arg Ala Gly Gin Gly Leu Glu 40 -45 'ly Glu Phe Asn Pro Ser Asn Gly Arg Thr Aen Tyr Asn Glu 55 60 ~ys Ser Lye Ala Thr Leu Thr Val Asp Lys Set Ser Ser Thr 65 70 75 let Gin Lou Set Set Lou Thr Ser Giu Asp Ser Ala Val Tyr 90 l~a Ser Arg Asp Tyr Asp Tyr Asp Gly -Arg Tyr Phe Asp Tyr 100 105 110 In Gly Thr Thr Val Thr Val. Ser Ser Gly Gly Gly Gly Set 115 120 125 ly Gly Ser Gly Gly Gly Gly Ser Aeplle Glu Leu Thr Gin 130 135 140 er Ile Met Set Ala Set Pro Gly Glu Lye Val Thr Met Thr 150 155 ep Set Ser Set Val Ser Tyr Met Tyr Trp Tyr G].n Gin Lye 165 170 Giy Ala Ser His Trp Ile Lye Ph.

Ala Tyr Tyr Cys Trp, Gly Gly Gly Ser Pro CyS Ser 160 rhr Gly 175 1995, MRCIGODDot 73 Se, Va Thi Gi, 22! (2) 15 .:20 25 Se

GA

Gly

G

Lys §er Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly 180 185 190 L fto Val Arg Phe Ser Gly Set Gly Ser Gly Thr Ser Tyr Ser Leu 195 200 205 C le Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 21i0 215 220 1 ?rp Ser Ser Tyr Pro His Thr Phe Gly Ala Gly Thr Lys Leu, Giu 5230 235 240 SLys INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 732 base pairs TYPE: nucleic acid- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (1iii) HYPOTHETICAL: NO fiv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal ORIGINAL SOURCE: ORGANISM: mouse STRAIN: Balb/c TISSUE TYPE: splenocytes (V'ii) IMMEDIATE SOURCE: CLONE: 1 A 1 (single-chain Fv, heavy and light chain plus-linker) (ix) FEATURE: NAME/KEY: CDS LOCATION:1..732 SEQUENCE DESCRIPTION: SEQ ID NO: 31: GTG CAG CTG CAG CAG TCT GGG GCT GAA CTG GTG AAG COT GGG GCT Val Gin Leu Gin Gin Ser Gly Ala GlU Leu Val Lys Pro Giy Ala 245 250 255 §TG AAG TTG TCC TOO AAG GCT TCC GGC TAC AC TTC ACC AG-C CAC Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 260 -265 270 ATG CAC TGG GTG AAG CAG AGO GOT GGA CAA GGC CTT GAG TGG ATC blet His Trp Val Lys Gln Arg Ala Gly Gin Gly Leu Glu Trp Ile 280 285 290 GAG TTT AAT CCC AGC AAC GcC CGT ACT AAC TAO AAT GAG AAA TTC Olu Phe Asn Pro Ser Asn Giy Arg Thr Asn Tyr Asn Glu Lys Phe 295 300 305 hOC AAG GCC ACA CTG ACT GTA GAC AAA TOC TCC AGC ACA GOT TAC ger Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 310 315 320 48 96 144 192 240

I"

74- ATG CAA CTC Met Gin Leu 325 AGC AGc CTG ACA Ser Ser Leu Thr GAG GAO TCT CG Glu Asp Ser Ala GTC TAT TAC TGT Val Tyr Tyr Cys 335 GAO TAC TGG GGC Asp Tyr Trp Gly GCC AGT CGC GAC TAT Ala Ser Arg Asp Tyr 340 CAT TAO Asp Tyr 345 GAC GGA OGC TAO Asp Gly Arg Tyr

TTT

Phe

CA

Gin GGC ACC ACG GTC Gly Thr Thr Val

ACC

Thr 360 OTC TCC TCA GGT Val Ser Ser Gly

GCO

Gly GC? GO TOG; GO Gly Gly Ser Cly

GGT

Gly GGT GGG TOG GGT Gly Gly Ser Gly

GGC

Gly 375 GGC GGA TOT GAC Gly Gly Ser Asp GAG CTC ACC OAG GlU Leu Th~r (in TCT OCA Ser Pro ACA ATC Thr Ile GAO AGO Asp Ser

AGO

Met

TOT

Ser 390 GCA TOT cCA GG Ala Ser Pro Gly GAG AMG GTC Giu Lys Val 395 TAO TGG TAC Tyr Trp Tyr

S

StS 555

S

S 5- 9* 5 9 ~S* 9 5~ 99 9 S. 55 .5 TCA AGT GTA AGT Ser Ser Val Ser 405 0CC AGA CTC OTG Pro Arg Leu Leu ACC ATG, Thr Met CAG CAG Gin Gin ACC TGC AT Thr Cys Ser 400 AAG ACA GGA Lys Thr Gly TAO ATG Tyr Met 410 288 336 384 432 480 52a 576 624 672 720 B( TOO TOO *~Ser Ser 420

ATT

Ile 425 TAT GAO ACA TOC Tyr Asp Thr Ser

AAC

Asn CTG GT TT WA Leu Ala Ser ly

GTC

Vai 435 COT GTT 000 TTO Pro Vai Arg Phe

AGT

Ser 440 GO AGT COG TCT Gly Ser Gly Ser

CG

Gly ACC TOT TAO TOT Thr Ser Tyr Ser

CTC

Leu ACA ATC AGO OGA 2oThr Ile Ser Arg

ATG

met 455 GAG GOT GAA GAT GiU Ala Giu Asp

GOT

Ala GCC ACT TAT TAC Ala Th~r Tyr Tyr 450CA TC CGn CysGi CAG TGG AGT Gin Trp Ser

AGT

Ser 470 TAC OCA CCC ATG Tyr Pro Pro met

TAC

Tyr 475 ACG TTC GGA CG Thr.Phe Cly Gly GGG ACA AAG Gly Thr Lys 480 TTC GAA ATA AAA Leu Giu Ile Lys 485 INFORMATION FOR SEQ ID NO: 32: SEQUENCE

CHARACTERISTICS:

LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY:,linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Glu Val Gin Leu Gin Gin Ser Giy Ala Glu Lou Val 1 5 10 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr 25 Trp Met His Trp Val Lys Gin Arg Ala Gly Gin Gly 40 Lys Pro Gly Ala Phe Thr Ser His Leu GlU Trp Ile 1995 MRCIGOD.Doc 75 Gly Giu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Giu Lys Phe 55 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 70 75 Met Gin Leu Ser Ser Leu Thr Ser Giu Asp Ser Ala Val Tyr Tyr Cys 85 90 Ala Ser Arg Asp T!yr Asp Tyr Asp Gly Arg Tyr The Asp Tyr Trp Gly 100 105 110 Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gin Ser Pro 130 135 140 Thr Ile Met 8cr Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser 145 150 155 160 Asp Ser Ser Ser Val 5cr Tyr Met Tyr Trp Tyr Gin Gin Lys Thr Gly #9@165 170 175 Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Pro Va 180 185 190 VloVal Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 195 200 205 Thr Ile Ser Arg Met Giu Ala Giu Asp Ala Ala Thr Tyr Tyr Cys Gin 210 215 220 Gin Trp, Scr Ser Tyr Pro Pro Met Tyr Thr Phe Gly Gly Gly Thr Lys 2~ 20 225 230 235 240 Leu Glu le Lys

Claims (9)

1. An anti-EGF-R single-chain Fv obtainable from a phage- antibody library constructed from cells from an immunized mammal, wherein light chain and heavy chain comprises amino acid sequences selected from SEQ ID NO: 1 SEQ ID NO: 28.

2. An anti-EGF-R single chain Fv according to claim 1, wherein said library is constructed from cells of an immunized mouse.

3. An anti-EGF-R single-chain Fv according to claim 1, wherein said library is constructed from cells of: i) the lymph node; ii) the spleen; or iii) in vitro immunised cells.

4. A DNA molecule encoding a chimeric anti-EGF-R antibody comprising DNA sequences selected from SEQ ID NO: 1 SEQ ID NO: 28 encoding heavy chain and light chain variable region an anti-EGF-R scFv of claim 1 and a DNA sequence encoding a constant region of human immunoglobulin.

S S A chimeric anti-EGF-R antibody comprising a variable region S. 2 with heavy chain and light chain amino acid sequences according to claim 1, a human gammra- heavy chain constant region and a human kappa light chain constant region. -77- 9*t*

6. Process for the preparation of a whole anti-EGFR antibody by cloning the DNA coding for the variable regions of anti-EGFR antibody fragments as claimed in claim 1 into at least one eukaryotic expression vector containing genomic DNA which codes for the constant regions of human immunoglobulins, transforming eukaryotic cells with said vector(s) and expressing and isolating the antibody. S*

7. Pharmaceutical composition comprising and anti-EGFR antibody fragment according to any one of claims 1 to 3, a chimeric anti-EGFR antibody produced from the DNA molecule of claim 4, or a whole anti-EGFR antibody according to claim 5, in association with a pharmaceutically acceptable carrier and/or diluent. 0 kAVAOipatmtistWPDC.%RS\SS a7204+9.dm-3(95 -78-

8. Use of an anti-EGFR antibody fragment according to any one of claims 1 to 3, or a chimeric anti-EGFR antibody produced from the DNA molecule of claim 4, or a whole anti-EGFR antibody according to claim 5 for the manufacture of a drug directed to tumors or for the diagnostic location and assessment of tumor growth.

9. A method for the treatment or prophylaxis of tumors, or for the diagnostic location and assessment of tumor growth, comprising the step of administering to a subject a therapeutically effective amount of an anti-EGFR antibody fragment according to any one of claims 1 to 3, a chimeric anti-EGFR antibody produced from the DNA molecule of claim 4, or a whole anti-EGFR antibody according to claim An anti-EGF-R single-chain Fv, a DNA molecule encoding a chimeric anti-EGF-R antibody, a process for the preparation of a whole anti-EGFR antibody, a pharmaceutical composition comprising an anti-EGF-R antibody fragment, a 15 pharmaceutical composition comprising a whole anti-EGFR antibody, use of an anti-EGF-R antibody or fragment thereof or a method for the treatment or o prophylaxis of tumors, or the diagnostic location and assessment of tumor growth substantially as herein described with reference to the examples and accompanying figures. DATED this twenty ninth day of May, 2000. 25 MERCK PATENT GMBH By Its Patent Attoreys DAVIES COLLISON CAVE DAVIES COLLISON CAVE