Antiidiotypic antibodies, which induce an immune response against the receptor of the epidermal growth factor

 

(57) Abstract:

The invention relates to immunology and concerns antiidiotypic antibodies, which induce an immune response against the receptor for epidermal growth factor. The invention includes a monoclonal antibody Ab2, simulating the mental image of the EGFR antigen, which is derived from a mouse antibody mAb 425 and produced by the cell line deposited under the number of ATSS HB 9629, and the method of its production by immunization of an animal corresponding idiotypical antibody and a pharmaceutical composition based on it. The technical result consists in obtaining a new product, which is a good candidate for idiotypical vaccine used for the inhibition of tumor growth. 3 S. and 3 C.p. f-crystals, 6 ill., 5 table.

The invention relates to antiidiotypic antibodies, which induce an immune response against tumors bearing as an antigen receptor of the epidermal growth factor (EGFR). The present invention mainly relates to antiidiotypic antibody having an "internal image" of the antigen, and are able to simulate the external domain of the human EGFR. In a preferred embodiment of the present izobreteniya options. Antibodies of the present invention can be used for immunotherapy and immunoprophylaxis of tumors.

In the description of the present invention uses some of the abbreviations and technical terms have the following meanings:

"FR" (wireframe plots) represent four subdistrict variable regions of the light or heavy chain that support at the CDR site.

"CDR (sections defining complementarity) represent three subdistrict variable regions of the light and heavy chains, which are hypervariable sequences, and form a loop structure, which is mainly responsible for the implementation of direct contact with the antigen.

"EGF" and "EGFR" refers to epidermal growth factor and its receptor.

"PCR" means polymerase chain reaction.

"ScFV" means single-stranded FV- a fragment of an antibody.

"VLmeans variable region light chain.

"VKmeans the variable region of the Kappa light-chain.

"VHmeans variable region of the heavy chain.

"Chimeric" or "partially humanized" antibodies are sasti (including CDR), derived from antibodies derived from other sources, non-human, for example, from mouse antibodies.

"Humanized or fully humanized antibodies are antibodies that contain a constant region and frame sections (FR) derived from a human immunoglobulin; and hypervariable sites (CDR) derived from other sources not related to the person.

"Ab1" means "first" antibody or parent antibody that induces, upon immunization, the cascade produced antibodies.

"Ab2" means typical antiidiotypic antibody (=antiidiotype) directed against idiotypes Ab1.

"Ab3" means antiidiotypic antibody directed against idiotypes Ab2, and therefore, by its specificity comparable to Ab1.

PBS means phosphate buffered saline.

FCS - mean fetal calf serum.

HBSS means balanced salt solution Hanks.

F1TC - means fluoresceinisothiocyanate.

MTC - means a mixed cell culture.

KLH - means hemocyanin lymph snails (chromoprotein derived from mollusk Medathura crenulata, strong peroxidase person.

Antiidiotypic antibodies (antiidiotype) are antibodies directed against epitopes (called idiotype), localized in antigennegative region or variable region of the molecule other antibodies. Interaction between idiotype (Ab1) and antiidiotype (Ab2) play an important role in maintaining immune homeostasis. theory of the network idiotypes and antiidiotype led to the explanation of the phenomena of immunoregulation (Jerne F. G. 1974, Arn. Immunol. 125C: 373). Antiidiotypic antibodies bearing the "internal image" of the antigen mimic the three-dimensional structure of the antigen recognized by the antibody Ab1. Introduction antibody Ab2 can cause the production of antibodies Ab3 against the original antigen. Idiotypical vaccines have been successfully used for the treatment of certain vector-borne diseases, and to treat cancer (see, for example,

Uytdehaag, F. G. &., C. M. H. Osterhans, 1986, J. Immunol. 134: 1125; Kennedy, P. C. et al. 1986, Science 232: 220; Hiernaux, J. R., 1988; 56: 1407; Strein K. E. & T. Soder-strom, 1984, J. Exp. Med. 160: 1001).

In tumor immunology idiotypical vaccination was used to modulate tumor growth in experimental in vivo and in vitro systems (Smorodinsky, N. I. et al., 1988, Eur J. Immunol. 18: 1713; Viale G, et al. , 1987, J. Immunal. 139: 1438). Herlyn and others (1987, Proc. Natl. Acad. Sci. USA, 76: 1438) reported the patients, suffering from progressive carcinoma of the colon, and in which there was a cessation of development of metastases and partial clinical remission in patients treated only one antiidiotype. Antiidiotypic antibodies and their use in cancer therapy are also described, for example, in U.S. patents 4918164 and EP 0141783.

Epidermal growth factor (EGF) is a polypeptide hormone that is mitogenic for epidermal and epithelial cells. When interacting with sensitive cells, EGF binds to membrane receptors (EGFR). The receptor for EGF is a transmembrane glycoprotein of about 170 kDa, and is the gene product of the proto-oncogene c-erb-B.

Mab 425 is a mouse monoclonal antibody produced against a well-known cell line carcinoma human A431 (ATCC CRL 1555); and the specified antibody binds to the external domain of the human EGFR and inhibits the binding of EGF. It was found that MAb 425 (ATCC HB 9629) mediates tumor cytotoxicity in vitro, and inhibits in vitro growth of tumor cells epidermoid and cell lines derived from carcinomas of the colon (Rodeck et al., Cancer Res. 1987, 47: 3692). "Ocelote biochemical processes, which leads to DNA replication and cell division (Cargenter G., 1987, Annu. Rev. Biochem. 56: 881). Recently it was found that the system EGFR is involved in the oncogenic transformation of cells (Di Fiore, P. P. et al., 1987, Cell 51: 1063.10), it has Been suggested that the growth of some tumors that Express EGFR and secrete EGF or TGF is due to the proliferation autostimulatory cells (Ennis, B. W. et al., 1989, Mol. Endo. 3: 1830: with increased expression of EGFR on the cell surface was detected in tumors originating from different tissues, for example, in breast tumors, bladder carcinomas, melanomas, brain tumors, not derived from nerve cells (Neal D. E. et al., 1985; Lancet, 1, 366; Libermann, T. A. et al., 1984, Cancer Res 44: 753; Herlyn M. et al., 1982, J. Clin. Immunol 2: 135). EGFR is a therapeutic target because it is directly involved in cell proliferation of some tumors. It has been shown that over-expression of EGFR has a poor predictive value, because it is correlated with increased invasive ability of tumor (Sainsbury J. R. et al., 1985, Lancet I:364).

Passive or active immunization of cancer patients against EGFR induces in the blood of these patients specific antibodies, which act as antagonization for idiotypical vaccine since it can be isolated from tumor cells, although insufficient for therapeutic purposes the number.

Several groups of researchers have been studied and discussed antiidiotypic antibodies, and, mainly, the structural similarity of these antibodies with their antigens and the "internal images" (see, for example, Tsjisaki, M. et.al., 1993, J. Immunol, 150-508; Raychandhuri S., et.al., 1990, J. Immunol 145-760; Bruck C., et al. 1986, Proc. Natl. Acad. Sci. USA, 83: 6578).

The present invention relates mainly to new antiidiotypic antibody (antiidiotype), which induces an immune response against the receptor for epidermal growth factor. In particular, the invention relates, at least two such antiidiotype (marked A and 3B6 that mimic a region of the antigen on the surface of the receptor for human epidermal growth factor. Thus, antibodies of the present invention, and in particular, antibodies that are able to simulate the EGFR can be used to induce and enhance the immune response against all types of human tumors expressing the receptor of epidermal growth factor on their cell surface, such tumors like melanoma, glioma and carcinoma. These vivo assessment.

Thus, the aim of the present invention to provide a new antibody that is a monoclonal antiidiotypic (Ab2) antibody inducing an immune response against the receptor for epidermal growth factor (EGFR).

More specifically, the present invention relates to monoclonal antiidiotypic antibody that mimics the "internal image" of EGFR antigen, recognized the corresponding murine, humanized or chimeric monoclonal idiotypical (Ab1) antibody. This antibody (Ab1) may not occur from a person, that is, it may be a murine, humanized or chimeric monoclonal antibody defined above. Because this antibody is "figuratively reproducible", i.e., essentially, there are several antibodies Ab1 directed against EGFR epitope, the present invention also relates to antiidiotypic antibodies that can recognise this group of antibodies Ab1 against EGFR.

In addition, the present invention relates to antiidiotypic antibodies against EGFR, which can be derived from the corresponding antibodies Ab1 against EGFR, for example, by immunization.

Thus, in order nastojasih owano by immunization of an animal using the appropriate mouse, "humanized, or chimeric, idiotypical (Ab1) antibody.

Antiidiotypic antibodies of the present invention receive, preferably, by immunization of an animal by introducing him monoclonal anti-EGFR antibody 425, or "humanized and chimeric variants of this antibody. MAb 425 is produced by a known cell line deposited under the number of admission ATCC HB 9629.

Thus, the aim of the present invention to provide a monoclonal antiidiotypic antibodies, where idiotypical antibody (Ab1) is mAb 425 (renowned for HB 9629) or the antibody derived from mAb 425 by "humanization" or chimerization using the techniques described, for example, in WO 92/15683.

The present invention also relates to specific antiidiotypic antibodies, which have well-known amino acid sequences in the hypervariable (CDR) and variable (FR) regions of antibodies, and which is shown in Fig. 5A-F.

Another aim of the invention is to obtain antiidiotypic monoclonal antibodies, where the CDR region and the FR-region of the specified antibodies have the amino acid sequence shown in Fig. 5A-is giving antiidiotypic antibodies having the amino acid or nucleotide sequence shown in Fig. 5A-F.

In accordance with the present invention, the described sequence may also be modifications and changes that occur in well-defined sequences, imaging Fig. 5, due to spontaneous or chemically or physically induced mutations, insertions, deletions, and substitutions of single amino acid or nucleotide residues, provided that these modifications and changes do not affect the biological activity and properties of the final antibody.

The term "antiidiotypic antibody" also refers to the parts or fragments of the indicated antibodies, such as single-stranded Fv-fragments or F(ab)'2and Fab' - fragments are well known in the art (Skerra & Plyckthum, Science, 1988, 240: 1038; Better et al., Science, 1988, 240: 1041). Single-stranded Fv- fragments (where VLand VHsections are connected together) is also described in the literature (e.g., Bird et al., Science 1988, 242: 423; Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85: 5879).

In addition, the present invention relates to a method for antiidiotypic antibodies defined in the description and the formula nastojalo antibodies (Ab1), which binds to the antigen directly with their hypervariable sites (CDR), with subsequent isolation and purification of the synthesized antiidiotypic antibodies (Ab2), which binds to idiotype antibodies Ab1, by standard methods.

Another objective of the present invention to provide a pharmaceutical composition containing antiidiotypic monoclonal antibody defined in the present description of the invention; and optionally, a pharmaceutically acceptable carrier.

Finally, the invention also relates to the use of the specified antiidiotypic antibodies for the manufacture of a medicinal product with antitumor activity.

In the application described the three monoclonal antiidiotypic antibodies (A, B and N) that recognize idiotype, overlapping with antigennegative center (protopam) antibodies mAb 425. Conducted serological and immunological studies have shown that antibodies A and B are "internal image" of the epitope recognized by the antibody mAb 425 inducyruya thus the humoral response in syngeneic (mouse Ba1b/c) and allogeneic (B6 mouse-2F1) system. Anti is in animals, however, these antiidiotype were ineffective against generation Ab1-like response in rabbits and rats. Cloning and sequencing of their V - regions revealed the presence of amino acid homology between both CDR (light and heavy) chains and some remnants of EGFR localized in the region of the binding ligand that suggests about imitation antigen. The study of antigenicity homologous regions allowed to give an acceptable explanation for the absence of a biological effect in a heterogeneous system, and confirmed the potential efficacy of antiidiotypic antibodies in immune therapy.

Fig. 1 shows the inhibition of binding of 425 with EGFR-antiidiotype A, V, 15H8 or unrelated mb. The percentage of inhibition was calculated as described in "Materials and methods" using the OD values obtained from ELISA.

Fig. 2 - inhibition curves by antiidiotype A () V (), N () and mouse immunoglobulin IgG1 (Sigma) binding of mouse (a) and "humanized" version of (In) antibody 425 with GFR-expressing A431 cells. The results were expressed as the mean OD value for the two duplicate samples. X axis: competitor (μg/ml); y-axis: OD.

Fig. 3 - competitive ELISA analysis on Ab3-Ab phase or unrelated Ab2 mab 15, pre-incubated with A (), B (), and 15H8 as control was used mab 15 (a) and normal mouse serum. A: rat Ab3-containing serum against 5A6; B: rat serum Ab3-serum against 3B6; C: rat AB3-serum against mAb15; D: control rat serum against mAb15; X-axis: OD; Y-axis: serial dilution 10-3.

Fig. 4 - solid phase ELISA: inhibition by antiidiotype binding of Ab3 Balb/c and B6D2F1 received from 5A6 and 3B6 - immunized mice with purified EGF-receptor.

Fig. 5 - complete sequence of the variable regions (including the leader sequence) heavy and light chains of murine monoclonal antibodies 15H8, 5A6 and 3B6, shows nucleotidase and amino acid sequences. A leader sequence (which has a label marked in bold; CDR sequences are shown in italics; and the sequence used for amplification of the variable regions, highlighted by a solid line. Heavy chain 15H8, 5A6 and 3B6 have a characteristic structure of the group IIID. Light chain 15H8 and 5A6 have a characteristic structure of Kappa-group Y, whereas 3B6 has a structure characteristic of Kappa group III according to the classification CABAC:

A: a heavy chain 15H8

/BR> Fig. 6 - comparison of sequences CDR2H, and CDR3H CDR21 antibody Ab2 and external domain EGF-receptor. Identical amino acids are shown in solid frames. In the dotted part shows similar amino acids. Potential antigenic region shown in the shaded part.

Table I. Constants affinity 5A6, 3B6 and 15H8 in relation to 425. Dissociation constants were calculated from graphs of Scatchard describing the binding of Ab2 to 425, based on the OD values obtained by ELISA, which was performed as described below, in the Chapter "Materials and methods".

Table II. Inhibition of Ab1-Ab2 binding by Ab3 antibodies and detection of cross-reacting idiotypes to Ab2. Twofold dilution of antisera Ab3 obtained from mice (A), rabbits (B) and rats (C) immunized with antiidiotype or unrelated mAb, and analyzed for inhibition, binding immunizing Ab2, other antiidiotype, and mouse isotype and allotype, the respective mAbs. Indicated titers of serum giving 100% inhibition.

Table III. Immune response against EGFR in Balb/c mice, determined by indirect immunofluorescence assay against EGFR-positive (A431) or negative (SA) flow cells and by ELISA - anichini IgM + IgG with FITC (Fluorescein-isothiocyanate).

b) Detection of rabbit artemisinin IgG using FITC.

c) Positive animals immunized animals.

5. Detailed description of the invention

Biological materials and General methods

Microorganisms, cell lines, plasmids, family, promoters, markers of resistance, the sites of initiation of replication, or other parts of the vectors, which are referred to in this application have been purchased or obtained by other available means. It should be noted that if this application is not given any other data, all these materials are used only for illustrative purposes, i.e., is not decisive for the present invention and may be replaced by other appropriate means, and biological materials, respectively.

The methods used to implement the present invention, described in detail below. In the proposed application is not carried out detailed descriptions of standard methods, well known to any specialist or described in detail in the cited sources, patent applications and well-known works (e.g., Harlow E & D. Lane, 1988, Anfibodies, a laborafory mannal. Cold Spring Harbor Laboratory, N. Y.).

For in vivo assays were used the e new Zealand rabbits (Biocentre).

A431 is ploscockletocnuu (epidermoid) carcinoma (ATCC CRL 1555), expressing EGFR. C33A (ATCC HTB31) is a carcinoma of the cervix, and is receptor-negative. As a "partner" to merge when receiving hybridoma used HL1-Friendly-myeloma-653 (Ventrex, bioventures group). All cell lines were maintained in RPMI medium 1640, to which was added 10% fetal calf serum and 2 mm glutamine.

Antibody mAb 425 (AbI) against EGFR was produced according to the method Rodeck and others (see above). In various analyses as control was used a few mouse mAb unrelated specificity, namely: mab 15 (IgGI, k) against the gp85/45 small cell lung cancer; R24 (IgG3,k) against GD3; 14F9 (IgG3,k) against GD3, and Me36I (IgG2f) against GD2, which have been described previously (Kamma H. et. al. , 1989, Cancer Res. 49(8): 5118; Dippold, W. G., 1980, Proc. Natl. Acad. Sci. USA. 77:6114, Masso O. et.al., 1991, Immunologia 10:36; Ihurin I. et.al., 1987, Cancer Res. 47:1229).

FIII (IgGl, k) was obtained by PEG-induced fusion of splenocytes derived from immunized by RNase Balb/c mice and Friengly-myeloma.

Getting idiotypical mAb against mAb 425

Mouse hybridoma was produced using splenocytes from Balb/c mice immunized antibody mAb 425, and fused with cells Friendlu-653 myeloma. With the result of two mergers, that gave 2,8% and 10.5% specific effectiveness, respectively. After three cloned by the method of serial dilution, was established, their antibodies belong to a class of IgGI,k.

Characterization 5A6, 3B6 and 15HB

For characterization Ab2 used crude supernatant or purified protein samples A. Antiidiotypic nature of this antibody was confirmed by its specific binding to mAb 425 when testing the panel with an unrelated mAb with different antigenic specificity (FIII, 361, 15 and 14F9).

To determine the localization 425-idiotope recognizable by antiidiotype, they were tested against sEGFR by ELISA analysis on the inhibition of binding of 425 with EGFR. In the result, there was complete inhibition of EGFR reactivity (Fig. 1), which indicates that antiidiotype recognize idiotope inside antigennegative site.

To identify antiidiotype 5A6, 3B6 and 15HB as Ab2b analyses were conducted in order to determine quickly if idiotope recognized by mAb 425, with CDR or antigennegative site. For such analyses by indirect ELISA test using A431 cells were constructed curves of inhibition of binding of d is to link both options 425; each of Ab2 had similar curves of inhibition of binding, which suggests that idiotope 425 recognized by the antibody 5A6, 3B6 and 15H8, are directly involved in antigen recognition. The inhibition of binding of cells 425 with A431 cells antibody 15H8 found efficiency, which is 10 times the efficiency 5A6 and 3B6. To determine the degree of compliance with the Ab2 paratope 425 calculated dissociation constant (KD) of antiidiotype. Dissociation constants of each of Ab2 are presented in table. I. 15H8 has a higher affinity to 425, which correlates with its higher ability to inhibit the binding of EGFR to 425. These results allow to assume that at least two different idiotope on mAb 425 recognized antiadiotipiceskih antibodies.

Induction of syngeneic (Balb/c), allogeneic (B6D 2FI) and xenogeneic (rat, rabbit) antibody Ab3

The suitability of these Ab2 for use as idiotypical vaccines was determined by evaluation of their immunogenicity. Antibody Ab2, bearing true "internal image" of the antigen must have the ability to induce antibodies Ab3 with Ab1-like specificity, beyond the species barrier. Ab3 were produced from thinking the antibodies were detected 2 weeks after the first dose and reached a maximum level after 6-8 weeks after the first dose. In rats and rabbits, specific response against the immunizing Ab2 was maintained for almost 12 months, whereas in mice, this response was shorter, and after 6 months, the title Ab3 decreased. All rabbits were produced antimachine Ig; and the titer of the antisera Ab3 against immunizing Ab2 was 5-10 times higher than the titers detected against izotopicheskii and alltimecase Ig, suggesting that the immune response against the constant region Ab2 does not violate the immune response against the v-region.

To determine the effectiveness of immunization against those determinants Ab2, which Konstantinou with paratopes mAb 425 (where should be the antigen-mimicking idiotope), an analysis was conducted on the inhibition of binding in which the binding 5A6, 3B6 or 15H8 (antibody Ab2) with mAb 425 was measured in the presence of serial dilutions of Ab3-containing serum taken from animals immunized antibody Ab2, or unrelated murine mAb. In table. 2-4 breeding Ab3-containing serum, which give 100% inhibition of binding of Ab2-Ab1 obtained from Balb/c mice (table. 2), rabbits (table. 3) and rats (table. 4). As can be seen from the table. 2-4 all antisera Ab3 against 5A6 produced in these 3 species, inhibited the binding of mAb 425 with 5A6 and 3B6 what you can expect, what both of these antiidiotype may have a common epitopes. In addition, it was found that some rat and mouse antisera against 15H8 inhibit the binding 5A6 and 3B6 with 425 that allows to make a conclusion about their partial idiotypical identity. This reactivity was not observed in the case of rabbit antisera against 15H8, which probably indicates that Ab3 response produced in rabbit, can be directed mainly against other, more immunodominant idiotopes; and this supposition is confirmed by the detection of higher inhibitory titers for the rabbit Ab3 - containing serum in comparison with rat and mouse antisera.

Antigenic identity between 5A6 and 3B6 was investigated using a competitive ELISA test, where serial dilutions (10-3-10-5) rabbit and rat antisera (Ab3) were analyzed for the binding of Ab2 in the presence of an equal volume (1 mg/ml) Ab2 or control mAb. Curves of inhibition of binding to the rat Ab3-containing sera is shown in Fig. 3. 5A6 and 3B6 generate identical range Ab3 in rats and rabbits: anticavity against 5A6 with the same effectiveness react with the antibody 5A6 and 3B6 deposited on ELISA-tablets and equal Scania, it is shown in Fig. 3 for rat serum.

Identification of Ab3 antibodies against EGFR

Antibodies to the receptor were detected in Ab3-containing antigen by direct immunofluorescence using A431 cells and C33A. The results obtained are presented in table. 5.

Antiidiotypic vaccination in a murine system induced the formation of antibodies against EGFR, but Ab3-containing sera from rats and rabbits should not detect antibodies to EGFR. The production of antibodies to EGFR mice B6D 2FI confirms the fact that Ab2-vaccination genetically not limited to mouse strain Balb/c. When testing against SEGFR using ELISA titers of specific antibodies reached values of 1/50 - 1/1600.

Inhibition of binding of Ab3 with EGFR antiadiotipiceskih antibody 5A6, 3B6 and 15H8

These analyses were carried out to determine, imitate if antiidiotype three-dimensional structure of EGFR. Various breeding Ab3-containing serum against EGFR, taken from Balb/c mice and B6D2F1, mixed with antiidiotype and left for reaction for 4 h at room temperature, after which they were analyzed for antibodies to the receptor using SEGFR-coated tablets, and on the basis of values OP, vychislyat about these antiidiotype imitate identical three-dimensional structure of the original antigen. The percentage of inhibition of anti-EGFR Ab3-idiotype 15H8 was 15 - 35% compared to A or 3B6.

It was stated that antiidiotype, bearing the true internal image of the antigen, can mimic the immunogenic properties of the antigen and induce a specific response in various animal species, except A and 3B6, which are not able to induce anti-EGFR antibodies in rats and rabbits; however, performed serological tests suggest that antibodies to the receptors found in mice were induced idiotypical determinants A and 3B6, similar to EGFR.

The primary structure of Ab2 and the comparison of their amino acid sequences with EGFR

To identify structurebased features underlying the immunogenic properties of Ab2, conducted DNA sequencing. VHand VLsequence A, 3B6 and 15H8 shown in Fig. 5. Amino acid sequence of VHareas were classified in accordance with the criteria Kabata as belonging to the subgroup IIID. According Cabatu VLarea A and 15H8 belong to the group of KV, and 3B6 - kIII. CDR sequences sravnivala in Fig. 6, where an identical amino acid shown in the solid part, and the equivalent amino acids shown in dotted frames. Maximum homology was observed between residues 77-84 from CDR2H and remains 125-129. from CDR3H, and residues 70-76 from A and 74-80 from CDR2L, and region of EGFR that has the highest homology with CDR3H, was located in a hypothetical ligand-binding domains of the receptor protein described A. Ullrich and others (30). 15H8 has an identical sequence CDR2H, but different sequence CDR3H and CDR2L. To determine the role of each homologous CDR-plot in the formation of an "internal model" of EGFR, were compared with sequences 5A6 and 3B6 with known VHand VLthe areas described by Kabata. In accordance with the present invention it was found that some VH-chains have a high degree of homology with A and 3B6, in particular, they have similar CDR1H and CDR2H, which suggests that the sequence of CDR3H play a major role in the recognition of a 425-paratope and simulation EGFR. It was found that CDR3H Ig 63 (mainly murine or human origin) have partial homology (50 - 87%) with CDR3H from A and 3B6. Homologous amino acids, obviously, is localized to residues homology with EGFR. However, it was found that none of the antibody is murine or human origin had no balance V in position 100D except A and 3B6. VLA and 3B6 found homology with murine Kappa-chains, and completely different sequence VL-chain have the same specificity. Obviously, the role of CDR2 is not associated with a strong amino acid identity. A, 3B6 have different sequences, forming an identical internal structure of the image EGFR, however, amino acid homology with EGFR, obviously, are accumulated in this area.

To assess the immunological effects of these antiidiotypic as vaccines, were analyzed availability and antigenic properties CDR2H-, CDR3H and CDR2D-areas. Antigenic index (a measure of antigenic probability) was calculated using the program ANTIGENIC, and to determine availability was found homologous sequences was determined using the program DSSP.

Analysis of secondary and primary structure showed that these homologous residues are available, and, moreover, coincide with areas having positive antigenic index, and therefore, they can participate directly in the production of the th and 11024 and 11603 of Angstroms accessible surface of the protein, accordingly, methods to form antigenic isotopes; however, both of these antibodies generate identical Ab3 response in mice, rabbits and rats. The results suggest that anti-antiidiotypic the response observed in this system, aimed solely at idiotype defined VH-chains A and 3B6.

Because immunological and structural analyses antibioticheskih antibodies A and 3B6 showed that they bear the internal image of the external domain of the EGFR that studies have been conducted to determine why the response is not induced in other species. Positive antigenic CDR3H region GYVGYAIDY bearing EGFR-equivalent segments GYVGY, compared with CDR3H sequences of mouse, rat and rabbit antibodies. A large group of mouse antibodies has predicted antigenic region on the CDR3H sequence YAIDY; and it was expected that the mouse's own immunogenic idiotype determine residues GYVC. It was found that Ig does not have the sequence YAIDY, and, in all probability, rabbit anti-anti-idiotypic response directed against other idiotypes defined more major antigenic regions, which prevents the production response against vnutrennih bsor.

For the active immunotherapy of cancer, we used different methods. To obtain specific antitumor response, regulated by the interaction of the "idiotype-antiidiotype", cancer patients were injected antiidiotypic antibodies that mimic the immunological effect of tumour markers.

Antiidiotypic mAb against mAb 425 were obtained in the development of specific vaccines for EGFR. To do this, we chose three antibodies (A, 3B6 and N), recognize setassociative idiotope on mAb 425, based on their CDR sequences, as illustrated by inhibiting binding "humanized" version of mAb 425 with EGFR.

Immunological and serological tests showed that A and 3B6 act as "interior designs" antigen induces specific humoral response in mice. Ab1-like antibodies of the Ab3-serum contact membraneassociated EGF-receptor on the cells A331, and react against sEGFR. This reaction is completely inhibited by both antibodies A and 3B6 (regardless of antiidiotype used for immunoassay), which suggests that 5A6 and 3B6 are one and the same "internal image" of EGFR. Immunological data allow to suppose Oliounine idiotopes from 15H8; and in addition, serological analyses showed partial inhibition of the binding of anti-A and anti-3B6 mouse Ab3 with Ab2 or EGFR in the presence 15H8. Immunological studies have revealed the absence of biological effects, that are induced antibodies (IgM-reactive cells A, but does not recognize sEGFR), which, however, is not inhibited by the antibody 15H8. The results obtained suggest that in the process of immunization were activated nonspecific clones.

In order to find out what kind of structure in antiidiotypic involved in the induction response against EGFR, studies have been conducted structural correlation between idiotype and antigen.

After sequencing A, 3B6 and 15H8, and comparative assessment of amino acid homology with respect to EGFR was detected homologous series and the equivalent amino acid in antigenic and available areas CDR2H, CDR3H and CDR2L. The obtained data were matched with several existing information, indicating the presence of homologous sequences in the CDR-sections (or contiguous parcels) VL- VH-chains, which are involved in the structural formation of idiotypes, bearing the "internal image" of the antigen (31-urologicheskim action was the reason for analysis in order to identify the role of the hypervariable and framework regions in the induction of Ab3 response and structure formation "internal image" of EGFR.

Obviously, CDR2L, CDR2H and CDR3H from A and 3B6 participate in the construction of the "internal image" of the antigen, since it is in these areas was found maximum homology and amino acid according to the external domain of the EGFR. Three-dimensional simulation, obviously, plays a very important role, as evidenced by the following data:

a) CDR21 from A and B are different, but induce the same response against EGFR.

b) Homologous residues in CDR2H were found in unrelated antibodies, and therefore, their participation in the formation of patterns "internal image" does not depend on the exclusive line of identity.

C) Set the affinity between CDR-sites and remains 148-154, 437-452 and 492-502 in EGFR based, mainly, on the equivalence of amino acids.

d) is Equivalent amino acids are localized exactly in the CDR, and not in other areas.

Idiotypical analyses of several families of antibodies (anti-PC, andextremely, antigalactic, anti-N1P antibodies) conducted Davie and others (34), p is H and CDR3H, and Association with specific L-chains. The data obtained by the authors of this application as a result of immunological analysis and sequencing suggest about producing cascade Ab1-Ab2-Ab3 Id, restricted to a limited set of areas defined 425-paratope

and Ab2-paratope. The most important fact that deserves attention, is getting two Ab2, namely A and B possessing similar immunological properties, and hence idiotypical determinants originating from identical VHcircuits connected to different VL-chains.

Biological efficiency antiidiotypic antibodies correlates with their affinity for the set idiotypical determinants in susceptible species. Analysis of the residues involved in the formation of patterns "internal image" of an antibody A and B showed that these residues are located in the larger antigenic region (residues 100F, 101 H-chain), having, as has been established, the homology with other murine antibodies (from a database of proteins NBPF), but not with rabbit antibodies. Thus, the structure of the "internal image" defines the idiotype, "see" the mouse immune system, but zamaslennymi immunological analysis; namely, the titers of the antisera Ab3 against 5A6 and 3B6-paratope in rabbits is higher than in mice. It was also found that antibodies in human (from the database NBPF) are homologous residues 100F -101 that provide a theoretical basis for assumptions about the effectiveness of 5A6 and 3B6, bearing the "internal image" of the antigen in the human system.

Efficiency antiidiotypic antibodies bearing the "internal image" of the antigen as a vaccine has been illustrated in various animal models and in clinical trials. About obtaining antiidiotype that recognize anti-EGF antibodies, recently told Snarez (Snarez E. et al., 1993, Immunologia 12:122), which received 4 mAb, not bearing the internal image. 5A6 and 3B6 are the first antiadiotipiceskih mAb, bearing the "internal image" of the antigen, which became known that they mimic the external domain EGP-receptor of human rights. Biological properties of mAb 425 was possible to determine that 5A6 and 3B6 mimic the ligand-binding site of the EGF-receptor. Serological studies of biological activity in animal models, and structural analysis of these antiidiotypic antibodies allow to draw a conclusion about the possibility of their effective use for the vaccination of man.

6. Therapeutic whitesky purposes. Therefore, the aim of the present invention to provide a pharmaceutical composition containing as active ingredient at least one antibody or its fragment as defined in the description and the claims, in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

Antibodies of the present invention is usually administered by intravenous injection or parenteral. Basically, the dose range of input fragments of antibodies varies widely so that this dose was sufficient to achieve the desired suppression of tumor growth and tumor-lytic effect. These doses depend on age, health, gender and severity of illness of the patient, and may vary from 0.1 to 200 mg/kg, and preferably from 0.1 to 100 mg/kg per dose per day, which can be entered as a single or divided dose, for one or several days.

Preparations for parenteral administration are sterile aqueous or anhydrous solution or suspension or emulsion. Examples of anhydrous solvents that can be used for these purposes are propylene glycol, polyethylene glycol, vegetable oils, the local solvent. Antibodies of the present invention can be used as ingredients in the composition containing a physiologically acceptable carrier. Examples of suitable carriers are saline, a phosphate buffer solution (PBS), ringer's solution or the lactate-containing solution Riegner. In the pharmaceutical compositions may also contain preservatives, and other additives, such as antibiotics, antioxidants, and hepatoblastoma agents.

The antibody (or, optionally, its fragment) may also be conjugated with known methods with cytokines, such as 1L-2, in order to maintain their cytotoxicity.

The pharmaceutical compositions of the present invention can be used to treat tumors of all types, including melanoma, glioma and carcinoma, and tumours of the circulatory system, and solid tumors. For diagnostic purposes, the antibodies can be conjugated, for example, impervious to x-ray dye, or they can be subjected to radioactive tagging. The preferred method of labeling is a method for iodination. For diagnostic purposes, it is preferable if the antibody will be put in the form F(ab')2- Popravko the background.

Example (1). "Humanized" mAb 425

Getting a "humanized" mAb 425 was described in the literature (Kettleborough, C. A. et al., 1991, Protein Eng. 4: 773). The reconstructed form of mAb 425 contains the murine CDR inserted into a human variable regions while retaining structure antigennegative center. This was done to more accurately determine the location of idiotypes 425 recognized by the antibody Ab2.

Example (2). Production antiidiotypic mAb anti-EGFR mAb 425

Balb/c mice were primiraly by intraperitoneal (i.p.) injection of 75 mg of purified mAb 425, conjugated to KLH (Sigma), fixed with glutaraldehyde, and mixed with CFA (Difco). Repeated injections, the animals were injected after 7, 14, 21, 28, and 38 days after the first injection using the same immunogen, emulsified in IFA. The cell fusion was performed 3 days after the last injection. The fusion of spleen cells from immunized mice with cells HL1-Friendly-653 myeloma using polyethylene glycol 1450 (Boehringer Mannheim) was carried out in accordance with known circuit (Carceller A., 1988, Sangre 33 (3):220). Hybrid cells were cultured at 39% RPMI 1640, 39% hybridoma medium (Gibro), 20% fetal calf serum and 2% environment GAT. Positive hybrids were subjected to tre

Antiidiotypic antibodies were detected using a sandwich-ELISA test conducted using polystyrene tablets for micrometrology (Nunc, Maxisorb) coated with 1 mg/ml of purified mAb 425. These plates were blocked with 2% BSA-PBS and untreated supernatant or serum, diluted in 0.2% BSA-PBS, incubated overnight at 4oC. After washing, PBS - 0.05% Tween 20, was added (1 h, 37o(C) biotinylated mAb 425, obtained by the method of Harlow and Lane (see above), then the tablets three times washed and added HRPO-streptavidin (Dako), diluted in the ratio of 1/1000 in 2% SA-PBS. As the substrate was used a solution of 3,3,5,5-tetramethylbenzidine (Sigma). The optical density (OD) was measured at 450 nm.

Example (4). The dissociation constant (KD) for antiidiotypic Ab2, 5A6, 3B6 and 15H8

Determination of dissociation constants was performed by the method of Friguet (Friguet B. , et al. , 1985, J. Immunol Methods 77:306), and in short, a serial dilution of mAb 425 (2 of 10-8M - 2 10-11) were incubated with a fixed concentration of mAb2 until then, until a balance was achieved. The concentration of free Ab2 were measured by indirect ELISA using tablets for micrometrology coated with 1 mg/ml mAb 425 and conjugated with HRPO rabbit artemisinin immunodeficiency, the MENA EGFR person and immunoaffinity chromatography EGFR, secreted by cells A431 described in the literature (W. Weber et al., 1984, J. Biol. Chem 259 (23):14631). This external domain of the EGFR was used to confirm the specificity Ab3 methods indirect and competitive analyses.

Example (6). Analysis to determine the localization of idiotypes recognized by the antibodies mAb 425

To determine the localization of idiotypes 425, specific antibody 5A6, 3B6 and 15H8, was tested for the ability of these antibodies to inhibit the binding of mAb 425 with EGF-receptor. First, this test was performed using purified sEGFR. The crude supernatant and 50 mg/ml of purified Ab2) was mixed with 125 ng of mAb 425. After 4-hour incubation at 37oC, 425-activity was analyzed using ELISA using tablets for micrometrology coated with 2.5 mg/ml of sEGFR. Tablets, three times washed with a mixed solution of PBS-1% BSA, and added conjugated with alkaline phosphatase (AP) rabbit antimurine Ig (Dako). As the substrate used 3,3,5,5-tetramethylbenzidine, and the OD at 405 nm was recorded tablet reader Tifertek Multiscan.

These analyses were supplemented by a comparison of curves of their inhibition of the binding built for mouse and reconstructed 425: 100 ng/ml murine or reconstructed 425 smedslund murine IgGI antibodies. The residual activity was estimated using indirect cellular ELISA using A431 cells, pre-fixed with 0.1% glutaraldehyde tablets for micrometrology (5 of 105per well). After 60-minute incubation at 37oC, cells 3 times washed with PBS - 1% BSA - 0.05% Tween-20. For detection of bound AbI used conjugated with HRPO goat antimurine IgG2 (Dianova) and conjugated with HRPO goat anti-human IgG, respectively, and the substrate used orthophenylene (Sigma).

Example (7). The production of Ab3 in syngeneic (mouse Balb/c), allogeneic (F6D2F1-mouse) and xenogeneic (rat Wistar and rabbits NZW) systems, and detection Ab3

Purified Ab was made with KLH (Calbiochem) using glutaraldehyde (Sigma), and were emulsiable complete or incomplete adjuvant's adjuvant (CFA or IFA), and was administered to the animals at 0, 15, 30 and 60 day. In some analyses to study the stability of Ab3 response conducted additional injections after 8-12 months. Each animal received a dose of 75 mg (mouse), 150 mg (rats) and 300 mg (rabbits) full of immunoglobulins by intraperitoneal inoculation (mouse) or subcutaneously in several different areas (rats and rabbits). The control group received the same dose of non-specific the memory after each injection and one week after injection. In tablets for micrometrology, pre-coated with 1 mg/ml Ab2 or other mouse immunoglobulins were added in serial dilution of serum (in a mixture of PBS and 0.15% dry milk). Related anti-antiidiotype were detected using biotinylated Ab2 (mouse Ab3-serum); conjugated to HRPO rabbit anticrimine immunoglobulins (Dako), not able to cross-reaction with mouse Ig; and conjugated with HRPO swine anti-rabbit Ig (Dako). Antiidiotypic and alltimecase antibody produced in rats and rabbits, have been eliminated extensively adsorption 1/50-1/100-diluted serum samples murine IgGI antibodies after incubation overnight at 4oC.

Example (8). Incubation Ab2 to Ab1-anticorodal Ab3

This analysis was performed in order to identify antibodies that react with those areas Ab2, which are in contact with paratopes mAb 425. To do this, 8-16 ng/ml of purified 5A6, 3B6, 15H8 and FIII was mixed with serial dilutions of Ab3-containing serum, and after incubation overnight at 4oC was estimated activity Ab2 by indirect ELISA using 1 mg/ml 425 deposited on polystyrene plates. Related Ab2 were detected using conjuly identity Ab2 through mutual competition for the binding of Ab3

To determine idiotypical identity between all antiadiotipiceskih antibody Ab2 analyzed for inhibition of binding of the antisera Ab3 with Ab2-coated tablets. Serial cultivation of rat antisera Ab3 (10-3- 10-5) tested on the binding of Ab2 in the presence of an equal volume (1 mg/ml) Ab2 or control mAb, pre-incubated overnight at 4oC.

Example (10). Detection of anti-EGFR antibodies with Ab3 response

Mouse, rat, and rabbit antisera Ab3 analyzed for Ab1-like activity by cell ELISA using A431 cells, as described above, or by direct ELISA using sEGFR (2.5 mg/ml). As a "second" antibodies used were conjugated with HRPO rabbit antimurine Ig (Dako), rabbit anticrimine Ig (Dako) and swine anti-rabbit Ig (Dako), respectively. Antibodies to the receptor were also analyzed by direct immunofluorescence analysis using a flow of living cells A-431, cultivated in tablets Terasaki.

Example (11). Inhibition of binding EGFR and Ab3-antiidiotype 5A6, 3B6 and 15H8

Receptor and antiidiotype were investigated by competitive analysis on 1/25 - 1/50-dilutions of antisera Ab3 against EGFR, and then using ELISA, cell ELISA and direct immunofluorescent analysis tested protivozachatocnuu activity. The percentage of inhibition was calculated as follows:

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Example (12). Obtaining RNA and cDNA

Total RNA was isolated from 5A6-, 3B6 - 15H8-hybridoma cell lines. Cells washed with PBS, suspended in a solution of the thiocyanate guanidine, after which RNA was isolated by the method of ultracentrifugation in a gradient of cesium chloride (see Chirgwin et al., 1979, Biochemistry 18, 5294). The first chain cDNA was synthesized using a set of Pharmacia. The synthesis was carried out in 15 µl of 5 µg RNA for 1 h at 37oC. First circuit cDNA was used directly for amplification without prior cloning.

Example (13). PCR amplification

To amplify copies of the variable regions of the light chain of mouse immunoglobulin and variable regions of the heavy chain of mouse immunoglobulin used a series of PCR primers. 5'- Primers were designed for hybridization with a leader sequence. These primers were a mixture of 61 of the oligonucleotide for the variable domain of the heavy chain and of the 405 oligonucleotides for variableswere primers, which is specific to IgGI and Kappa-chain of the mouse.

To implement amplification using a thermostable DNA polymerase, 25 ál reaction mixture containing: 1 μl of hybrid cDNA-RNA", 250 nm corresponding to a mixture of 5'- and 3'-primers, 200 mm of each dNTP, 1 mm MgCl2(for light chain), 2 mm MgCl2(for the heavy chain), and 1 unit of Tag polymerase (Cetus), covered the top with mineral oil, and subjected to hybridization at a temperature ranging from 60oC to the annealing temperature 55oC. 1/10 of the PCR reaction were subjected to electrophoresis on 1% agarose TAE gel, and then stained with ethidiumbromid to visualize obtained PGR products.

Example (14). Molecular cloning and sequencing

1 µl of each PCR product ligated into the TA vector (Invitrogen, San Diego). DNA reaction products of ligation for areas VHand VL-circuits each Ab2 transformed by heat shock into competent cells of E. coli TGI. The result was the establishment of six DNA libraries: one for the variable regions of the light chain and the other for the variable regions of the heavy chain of each mAb. Colonies were selected on plates with LB-medium containing 100 mg/ml carbenicillin, and some of them were collected using somocista hydrolysis as described Gussov (Gussov D., Clackson, T., 1989. Nucleic Acids Res.,17: 4000). Double-stranded plasmid DNA for sequencing was obtained (Wizarol preps., Promega Corp.) from transformants. Then, in accordance with the methodology of Sanger have dideoxy-sequencing by chain termination using Tag polymerase (Sanger F. et al., 1977, Proc Nat Arad. Sci USA 74: 5463). The primers used in the sequencing reactions were annealed at TA-vector,

Example (15). Computer analysis and comparison of sequences

To determine the secondary structure of Ab2 and balances available used system software DSSP (Sybil). For the preliminary determination of antigenic sites on the method Kalaskar and Tongaonkar (Kalaskar A. S. et al., 1990, FEBS-Lett 276 (1-2):172) used the program ANTIGENIC. The homology between Ab2 and EGFR was determined by the BESTFIT program using disk Gencore to lookup symbols. Compare the sequence of human EGFR was previously described in the literature (Vllrich et al., Nature 309: 418). Ab2 was compared with other known antibodies using Protein data Bank Identification Resourel (PIR ), compiled by the National Foundation for biomedical research National Riomedical Ressarch Fundation (NBRE)) and program PBPSIS.

1. Monoclonal antiidiotypic antibody (Ab2) that simulates nutrem idiotypical antibody Ab1, characterized in that idiotypical antibody or is derived from a mouse antibody mAb 425, which is produced by the cell line deposited under the number of ADS on 9629.

2. Monoclonal antiidiotypic antibody under item 1, obtained by immunization of an animal corresponding murine, humanized or chimeric idiotypical antibody.

3. Monoclonal antibody under item 1 or 2, in which the CDR region and the FR-region of the specified antibodies containing the amino acid sequence shown in Fig. 5A-F.

4. Monoclonal antibody under item 1 or 2, containing the nucleotide sequence shown in Fig. 5A-F, which encodes a CDR region and the FR-region indicated antibodies.

5. The method of obtaining antiidiotypic antibody defined in any of paragraphs. 1-3, characterized in that the animal is subjected to immunization relevant idiotypical antibody which is or derived from a murine antibody mAb 425, which is produced by the cell line deposited under the number of ATSS HB 9629, which binds to a polypeptide epitope of the external domain of the EGFR person directly using the CDR regions, and then synthesized antiidiotypic antibody, which bound the ical composition, containing antiidiotypic monoclonal antibody defined in any of paragraphs.1-3, and, optionally, a pharmaceutically acceptable carrier.

 

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