Reconstructed human anti-hm.24 antibody, dna encoding the h and l chain of a human anti-hm.24 antibody, a vector, a method of obtaining a reconstructed human anti-hm.24 antibody pharmaceutical composition for the treatment of myeloma

 

(57) Abstract:

The invention relates to the field of molecular biotechnology. Presents the reconstructed human anti-NM 1.24 antibody, which is a plot of H and L-chains and V-phase H and L-chains include FR man, a CDR derived from an anti-1.24 NM monoclonal antibodies. This CDR has low antigenicity. Get reconstructed human anti-NM 1.24 antibody by transformation of the host cell expressing the vector that contains DNA encoding the antibody. Human anti-NM 1.24 antibody is used as an active ingredient for the treatment of myeloma. 12 C. and 5 C.p. f-crystals, 40 ill., 6 table.

The scope of the invention

The invention relates to a reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody, to the coding of their genes, to methods of producing these antibodies and use of these antibodies. Reconstructed human antibodies and chimeric antibodies according to the present invention is applicable as therapeutic agents, for example for the treatment of myeloma.

Background of the invention

Human b cells go through various processes, which are classified on the base is s. At this final stage of their differentiation In cells, on the one hand, acquire the ability to produce cytoplasmic immunoglobulins, and, on the other hand, disappear associated with In-cell antigens, such as antibodies cell surface HLA-DR, CD20, Fc-receptors, C3-receptors of the complement system, etc. (Ling, N. R. et al., Leucocyte Typing III (1986), p. 320, Oxford, UK, Oxford).

To date, there are reports of such monoclonal antibodies as anti-PCA-1 (Anderson, K. S. et al., J. Immunol. (1983) 130, 1132), anti-SS-1 (Anderson, K. S. et al., J. Immunol. (1983) 132, 3172), anti-MM4 (Tong, A. W. et al., Blood (1987) 69 238), etc. that recognize antigens on the cell membrane and plasma cells. However, anti-D38 monoclonal antibody is still used for determination of plasma cells and myeloma cells (Epstein, J. et al. , N. Engl. J. Med. (1990) 322, 664, Terstappen, L. W. M. M. et al., Blood (1990) 76, 1739, Leo, R. et al., Ann. Hematol. (1992) 64, 132, Shimazaki, C. et al. , Am. J. Hematol. (1992) 39, 159, Hata, H. et al., Blood (1993) 81, 3357, Harada, H. et al., Blood (1993) 81, 2658, Billadeau, D. et al., J. Exp. Med. (1993) 178, 1023).

However, anti-D38 monoclonal antibody is an antigen associated rather with activation of T-cells, rather than the antigen associated with the differentiation of b-cells and is expressed on various cells in addition to b-cells. In addition, although not expressed CD38 on the rank consider what anti-D38 monoclonal antibody is not suitable for studies of differentiation and maturation of human b-cells or for treating diseases associated with plasma cells.

Goto, T. et al. reported murine anti-1.24 NM monoclonal antibody that recognizes the antigen with a molecular weight of from 29 to 33 kDa, which is expressed specifically on the lines b-cells (Blood (1994) 84, 1922-1930). On the basis of the fact that the antigen recognized by the anti-1.24 NM monoclonal antibody is associated with the final differentiation of b-cells (Goto, T. et al., Jpn. Clin. Immun. (1992) 16, 688-691), and that the introduction of anti-1.24 NM monoclonal antibody to mice transplanted with plasmacytomas leads to specific accumulation of the antibody in the tumor (Shuji Ozaki et al., The Program of General Assembly of the 19thJapan Myeloma Study Meeting, general presentation 3), it has been suggested that labeled with a radioisotope anti-1.24 NM monoclonal antibody, can be used to diagnose a tumor, for targeted therapies, such as radioimmunotherapy, etc.

In addition, in the above article, published in Blood, described that anti-1.24 NM monoclonal antibody has complementability cytotoxic activity on otoolefan, characterized by the accumulation of monoclonal plasmacytes (myeloma cells) in the bone marrow. Myeloma is a disease in which finally differentiated b cells that produce and secrete immunoglobulins or plasma cells, monoclonal proliferate mainly in the bone marrow, and accordingly, monoclonal immunoglobulins or their constituent components, the L-chain or H-chain, determined in serum (Masaaki Kosaka et al., Nippon Rinsho (1995) 53, 91-99).

Typically, for the treatment of myeloma used homeotherapeutics funds, but has not been found effective therapeutic agents, which could lead to remission in myeloma patients and prolong the life of myeloma patients. So long there is a need to develop drugs that would have a therapeutic effect on myeloma.

Mouse monoclonal antibodies have high immunogenicity (sometimes called "antigenicity") in humans. Accordingly, medical therapeutic value of mouse monoclonal antibodies for use in humans is limited. For example, a mouse antibody, is introduced to a person, can be metabolized as a foreign substance, so that the term gender is to taravati the expected effect. In addition, human antimurine antibodies that are produced against the introduced mouse antibodies can trigger immunological reactions adverse and dangerous for patients, such as disease, serum, other allergic reactions, or so on, So mouse monoclonal antibody cannot often enter people.

To solve these problems, developed a method of reducing the immunogenicity of antibodies derived from humans, such as obtained from mice monoclonal antibodies. One such example is a method of obtaining a chimeric antibody in which the variable plot (V-phase) antibodies obtained from the original mouse and his constant section (C-section) obtained from a suitable human antibody.

As thus obtained chimeric antibody contains a variable part of source mouse antibodies in intact form, it is expected that it will bind to the antigen with specificity identical to the specificity of the original mouse antibody. In addition, the chimeric antibody, the number of amino acid sequences obtained from an organism other than human are significantly reduced, and therefore it is expected that takeital can bind to the antigen in the same way, as the original murine monoclonal antibody, and may include immunological reaction against the variable segment of murine antibodies, despite the fact that its reduced immunogenicity (LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. USA, 86, 4220-4224, 1989).

The second way to reduce the immunogenicity of murine antibodies, although much more complex, is designed to further reduce potential immunogenicity of mouse antibodies. In this way only the plot, complementarity determining (CDR) of the variable segment of murine antibodies transplanted in the variable area of the human antibodies, receiving variable plot "reconstructed" human antibodies.

However, in order to make the structure of the variable segment of the reconstructed human antibodies as much as possible to the structure of the original mouse antibody, it is necessary that the portion of the amino acid sequence of frame plot (FR), which supports CDR, you could transplant it from the variable segment of murine antibodies in the variable area of the human antibody. Then, the V-phase gumanitarnogo reconstructed human antibodies bind with constant plot Chelopechene reconstructed humanitariannet the antibody is a CDR, and is only part of FR. The CDR consists of hypervariable amino acid sequence that does not contain species-specific sequences. Thus, humanitariannet antibody containing murine CDR should not be more immunogenic than the natural human antibody containing the CDR of a human antibody.

Relatively gumanitarnogo antibodies see Riechmann, L. et al., Nature, 332, 323-327, 1988; Verhoeye, M. et al., Science, 239, 1534-1536, 1988; Kettleborough, C. A. et al., Protein Engng., 4, 773-783, 1991; Meada, H. et al., Human Antibodies and Hybridoma, 2, 124-134, 1991; Groman, S. D. et al. , Proc. Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest, P. R. et al., Bio/Technology, 9, 266-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA, 88, 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154, 1992; and Sato, K. et al. Cancer Res., 53, 851-856, 1993.

Queen et al. (publication of the international application WO 90-07861) discloses a method of obtaining gumanitarnogo antibodies from antibodies against the receptor for IL-2 Anti-Tac. However, it is difficult to fully humanize all antibodies, even following the way opened in WO 90-07861. Thus, WO 90-07861 does not disclose a General method for the humanization of antibodies, but simply describes how the humanization of the antibody Anti-Tac, which is one of the antibodies against the receptor for IL-2. In addition, even if you completely follow the way WO 90-07862, turns out the second activity of the parent murine antibody.

In General, the amino acid sequence of CDR/FR specific antibodies differ. Accordingly, the determination of amino acid residue that should be replaced to create gumanitarnogo antibodies, and the choice of amino acid residue, which should replace the specified amino acid residue change for each individual antibody. Therefore, the method of obtaining humanized antibodies described previously in WO 90-07861, cannot be used for humanization of all antibodies.

Queen et al. Proc. Natl. Acad. Sci. USA (1989) 86, 10029-10033 made the same discovery as described in WO 90-07861. In this reference indicates that only one third of the activity of the parent murine antibody is achieved for gumanitarnogo antibodies obtained by the method described in WO 90-07861. In other words, it shows that the way WO 90-07861 by itself cannot lead to the production of fully gumanitarnogo antibodies, which would have activity equal to the activity of the parent murine antibody.

With et al., Cancer Research (1996) 56, 1118-1125 published by the group above Queen et al. In this reference indicates that humanitariannet antibody with activity equal to the activity of the parent murine antibody, it is impossible to construct even a str method WO 90-07861 by itself may not result in fully gumanitarnogo antibodies with activity equal to the activity of the parent murine antibody, but that this way of constructing gumanitarnogo antibodies, as described further in WO 90-07861, cannot be applied to the humanization of all antibodies.

Ohtomo et al., Molecular Immunology (1995) 32, 407-416 describes the humanization of mouse ONS-M21 antibody. From this link you that amino acid residue, which was offered for the humanization of the anti-TAC antibody in WO 90-07861, has no relation to the activity, and the method is as described in WO 90-07861, not applicable.

Kettleborough et al. Protein Eng. (1991) 4, 773-783 indicates that it was constructed several humanized antibodies of murine antibodies in the replacement of amino acid residues. However, this requires the substitution of a larger number of residues than is offered in the way of humanization of the anti-TAC antibody, as described in WO 90-07861.

In the above references indicate that the method of obtaining humanized antibodies, as suggested later in WO 90-07861, is a technique, which is applicable only to the anti-TAC antibody, and that even the use of the above methods will not lead to the achievement of the activity equal to the activity of the parent murine antibody.

Recognize the sequence of the anti-TAC antibody, described in WO 90-07861. Accordingly, this method of constructing gumanitarnogo antibodies, which can be applied to anti-TAC antibody, cannot be applied to other antibodies. Similarly, as the mouse anti-1.24 NM monoclonal antibody of the present invention has an amino acid sequence differing from the sequence of the anti-TAC antibody, it is impossible to apply the method of constructing gumanitarnogo antibodies for anti-TAC antibodies. In addition, successfully designed humanitariannet antibody of the present invention has an amino acid sequence that differs from the sequence gumanitarnogo anti-TAC antibodies described in WO 90-07861. This fact also indicates that the same method cannot be used for humanization of antibodies with different CDR-FR sequences.

Thus, even if the original humanitariannet murine antibody is known, the identity of the CDR-FR sequence gumanitarnogo antibodies with activity, confirmed only by trial and error. In WO 90-07861 no mention of FR sequence, which merged in humanitariannet the antibody constructed in the present sobremesa CDR sequence.

As mentioned above, humanized antibodies, as expected, should be useful for therapeutic purposes, but humanized anti-1.24 NM monoclonal antibodies are not known and even not suggested. In addition, there is no available standard method that could be applied to any of the antibody to obtain gumanitarnogo antibodies, and require different tricks for designing and humanization of antibodies, which would demonstrate sufficient activity link, activity of inhibiting the binding and neutralizing activity (e.g., Sato, K. et al., Cancer Res., 53, 851-856, 1993).

Description of the invention

In the present invention proposed reconstructed antibodies anti-NM 1.24 antibody. Further, in the present invention proposed a human/mouse chimeric antibodies that can be used in the design process of these reconstructed antibodies. In the present invention the proposed reconstructed fragments of antibodies. Further, in the present invention proposed expression system for producing chimeric antibodies, reconstructed antibodies and their fragments. In the present invention the methods of obtaining chimeric antibodies from the body and its parts.

More specifically, the present invention proposed chimeric antibodies and reconstructed antibodies that specifically recognize a polypeptide with the amino acid sequence set forth in Sequence ID 103 cDNA, which encodes the specified polypeptide was inserted between the XbaI cleavage sites of the vector pUC19, and thus, was obtained as a plasmid pRS38-pUC19. Escherichia coli containing this plasmid pRS38-pUC19, was international deposited on 5 October 1993, the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) as Escherichia coli DH5 (pRS38-pUC19) under registration number FERM BP-4434 in accordance with the Budapest agreement (see Japanese patent publication without examination (Kokai) 7-196694).

As one of the accomplishments of such chimeric antibodies or reconstructed antibodies mentioned chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody. A detailed description of the chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody below.

Thus, the present invention also proposed a chimeric L-chain comprising a constant section (C-section) constantly the area of the human heavy (H) chain and V-phase heavy (H) chain anti-NM 1.24 antibody.

Further, in the present invention proposed chimeric antibodies, including:

(1) L-chain comprising a C-section human L-chain V-section L-C circuit, anti-NM 1.24 antibody; and

(2) H-chain comprising a C-section human H chain and the V-phase H-chain anti-NM 1.24 antibody.

Further, in the present invention proposed a V-phase reconstructed human L-chain anti-NM 1.24 antibody, including:

(1) frame area (FR) V-area of the human L-chain, and

(2) CDR V-phase L-chain anti-NM 1.24 antibody; and V-phase reconstructed human H-chain anti-NM 1.24 antibody, including:

(1) FR V-area of the human H-chain, and

(2) CDR V-phase H-chain anti-NM 1.24 antibody.

Further, in the present invention proposed a reconstructed human L-chain anti-NM 1.24 antibody, including:

(1)-part of the human L-chain, and

(2) V-phase L-chain including FR human L-chain and the CDR of the L chain of anti-NM 1.24 antibody; and

reconstructed human H-chain anti-NM 1.24 antibody, including:

(1)-part of the human H-chain, and

(2) V-phase H-chain including FR human H-chain and the CDR of the H chain of anti-NM 1.24 antibody.

Further, in the present invention proposed recons the current human L-chain; and

(2) V-phase L-chain including FR human L-chain and the CDR of the L chain of anti-NM 1.24 antibody; and

(B) N-chain, including:

(1)-part of the human H-chain, and

(2) V-phase H-chain including FR human H-chain and the CDR of the H chain of anti-NM 1.24 antibody.

The present invention further proposed that the DNA encoding the V-section L-C circuit, anti-NM 1.24 antibody and DNA encoding the V-phase H-chain of an anti-HM 1.24 antibody.

Further, the present invention provides DNA encoding chimeric L-chain, including:

(1)-part of the human L-chain; and

(2) V-section L-C circuit, anti-NM 1.24 antibody and DNA encoding chimeric H-chain, including:

(1)-part of the human H-chain; and

(2) V-phase H-chain anti-NM 1.24 antibody.

The present invention further proposed that the DNA encoding the V-phase reconstructed human L-chain anti-NM 1.24 antibody, including:

(1) FR V-area of the human L-chain; and

(2) CDR V-phase L-chain anti-NM 1.24 antibody; and DNA encoding the V-phase reconstructed human H-chain anti-NM 1.24 antibody, including:

(1) FR V-area of the human H-chain; and

(2) CDR V-phase H-chain anti-NM 1.24 antibody.

In the present invention proposed DNA encoding ri

(2) V-phase L-chain including FR human L-chain and the CDR of the L chain of anti-NM 1.24 antibody; and

DNA encoding the reconstructed human H-chain anti-NM 1.24 antibody, including:

(1)-part of the human H-chain; and

(2) V-phase H-chain including FR human H-chain and the CDR of the H chain of anti-NM 1.24 antibody.

Further, in the present invention proposed a vector comprising any of the above DNA.

In the present invention proposed a host cell, transformed with the above vector.

In the present invention the methods of obtaining chimeric antibodies anti-NM 1.24 antibody comprising the stage of culturing the host cell, which was cotransformation expressing a vector comprising DNA encoding the indicated chimeric L-chain, and with an expression vector comprising DNA encoding the indicated N-chain, and highlight the desired antibodies.

Further, in the present invention the methods of obtaining a reconstructed human antibodies anti-NM 1.24 antibody comprising the stage of culturing the host cell, which was cotransformation expressing a vector comprising DNA encoding specified reconstructed chelsey N-chain, and highlight the desired antibodies.

Further, in the present invention proposed a pharmaceutical composition, particularly a therapeutic agent for treatment of myeloma comprising the indicated chimeric antibody or reconstructed human antibody.

Further, in the present invention proposed pharmaceutical compositions which contain as active ingredient a chimeric antibody specifically recognizing a polypeptide with the amino acid sequence presented in the Sequence ID 103, and pharmaceutical compositions which contain as the active ingredient reconstructed human antibody specifically recognizing a polypeptide with the amino acid sequence presented in the Sequence ID 103. As pharmaceutical compositions specifically proposed therapeutic agent for treatment of myeloma.

A brief explanation of drawings

Fig. 1 represents a graph showing that in FCM analysis using cell lines of human myeloma CRM, the fluorescence intensity of chimeric anti-NM 1.24 antibody is shifted similarly shift to murine anti-NM 1.24 antibody, when compared with controlrelated, chimeric anti-NM 1.24 antibody by analogy with mouse anti-NM 1.24 antibody dose-dependently inhibited the binding of biotinylated mouse anti-NM 1.24 antibody with WISH-cells.

Fig.3 represents a graph showing that control human IgG1 or murine anti-NM 1.24 antibody does not possess cytotoxicity, whereas chimeric anti-NM 1.24 antibody shows increased cytotoxicity against RPMI 8226 cells with increasing relations E/So

Fig. 4 is a diagram of a method of constructing L-chain reconstructed human anti-NM 1.24 antibody in the transplantation CDR PCR.

Fig. 5 is a diagram of a method of Assembly oligonucleotides RVH1, RVH2, RVH3 and RVH4 PCR when creating N-chain reconstructed human anti-NM 1.24 antibody.

Fig. 6 is a diagram of a method of constructing a V-phase H-chain of human/murine hybrid anti-NM 1.24 antibody and PCR.

Fig. 7 is a diagram of a method of constructing a V-phase H-chain of mouse/human hybrid anti-NM 1.24 antibody and PCR.

Fig.8 represents a graph showing that option and L-chain reconstructed human is and. -1 and -2 indicate that this is a different group.

Fig.9 is a graph showing antigennegative activity reconstructed human anti-NM 1.24 antibody, in which a L-chain and the option a, b, f or h N-chains were combined, and chimeric anti-NM 1.24 antibody.

Fig. 10 is a graph showing the binding activity of the reconstructed human anti-NM 1.24 antibody, in which variant b L-chain and the option a, b, f or h N-chains were combined, and chimeric anti-NM 1.24 antibody.

Fig. 11 is a graph showing the activity of inhibiting the binding of reconstructed human anti-NM 1.24 antibody, in which a L-chain and the option a, b, f or h N-chains were combined, and chimeric anti-NM 1.24 antibody.

Fig. 12 is a graph showing the activity of inhibiting the binding of reconstructed human anti-NM 1.24 antibody, in which variant b L-chain and the option a, b, f or h N-chains were combined, and chimeric anti-NM 1.24 antibody.

Fig. 13 is a graph showing the binding activity of the antigen variants a, b, C and d the H chain of the reconstructed human anti-NM 1.24 antibody, and x is ntigen options a and e H-chain reconstructed human anti-NM 1.24 antibody and Kemerovo anti-NM 1.24 antibody. -1 and -2 indicate that they belong to different groups.

Fig. 15 is a graph showing the activity of inhibiting the binding of the variants a, C, R and r H-chain reconstructed human anti-NM 1.24 antibody, and a chimeric anti-NM 1.24 antibody.

Fig. 16 is a graph showing the binding activity of the antigen of human/murine hybrid anti-NM 1.24 antibody, a mouse/human hybrid anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 17 is a graph showing the binding activity of the antigen variants a, b, C, and f N-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 18 is a graph showing the binding activity of the antigen variants a and g N-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 19 is a graph showing the activity of inhibiting the binding of options a and g N-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 20 is a graph showing the activity of tie and NM 1.24 antibody.

Fig. 21 is a graph showing the binding activity of the antigen variants f, h and j H-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 22 is a graph showing the activity of inhibiting the binding of options h and i H-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 23 is a graph showing the activity of inhibiting the binding of the variants of f, h and j H-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 24 is a graph showing the binding activity of the antigen variants h, k, l, m, n and o H-chain reconstructed human l-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 25 is a graph showing the binding activity of the antigen variants a, h, p and q N-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 26 is a graph showing the activity of inhibiting the binding WISH-cell variants h, k, 1, m, n and o N-chain reconstructed human anti-NM 1.24 antibody and himem the statements options and, h, p and q N-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 28 is a graph showing the binding activity of the antigen variants a, C, p and r H-chain reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody.

Fig. 29 is a graph showing that option's reconstructed human anti-NM 1.24 antibody has antigennegative activity equal to the activity of the variant r reconstructed human anti-NM 1.24 antibody.

Fig. 30 is a graph showing that option's reconstructed human anti-NM 1.24 antibody has an activity of inhibiting the binding of equal activity options r reconstructed human anti-NM 1.24 antibody.

Fig. 31 is a graph showing that purified reconstructed human anti-NM 1.24 antibody has the binding activity of the antigen equal to activity of chimeric anti-NM 1.24 antibody.

Fig. 32 is a graph showing that purified reconstructed anti-NM 1.24 antibody has an activity of inhibiting the binding, equal antimirova anti-NM 1.24 antibody causes prolongation of life compared with the introduction of a control human IgG1 in mice transplanted with cells of the myeloma person.

Fig.34 is a graph showing that if cells derived from the peripheral blood of a healthy person, used as effector cells, the control human IgG1 does not demonstrate cytotoxicity against cells CRM, and that murine anti-NM 1.24 antibody also has a weak cytotoxicity, whereas the reconstructed human anti-NM 1.24 antibody shows high cytotoxicity against cells CRM.

Fig.35 is a graph showing that when using cells derived from peripheral blood of a healthy person as effector cells, the control human IgG1 does not demonstrate cytotoxicity against cells ARH-77, and that murine anti-NM 1.24 antibody also has a weak cytotoxicity, whereas the reconstructed human anti-NM 1.24 antibody shows high cytotoxicity against cells ARH-77.

Fig. 36 is a graph showing that if the effector cells using cells derived from the bone marrow of SCID mice, the control human IgG1 does not demonstrate cytotoxicity against cells CRM, whereas reconstructed chelonian antibody concentrations.

Fig. 37 is a graph showing that mice transplanted with cells of the myeloma person's level of human IgG in the serum increased after the introduction of a control human IgG1 compared to the level prior to the injection, whereas the introduction of a reconstructed human anti-NM 1.24 antibody inhibits the increase of human IgG in the serum.

Fig. 38 is a graph showing that mice with transplanted myeloma cells introduction reconstructed human anti-NM 1.24 antibody causes prolongation of life compared with the period after the introduction of a control human IgG1.

Fig. 39 is a graph showing that mice with transplanted myeloma cells human human IgG in the serum increased after the introduction of melphalan and control human IgG1 compared to the level prior to the injection, whereas the introduction of a reconstructed human anti-NM 1.24 antibody inhibits the increase of human IgG in the serum.

Fig. 40 is a graph showing that mice with transplanted myeloma cells introduction rivers in the period after the introduction of melphalan or control human IgG1.

The method of carrying out the invention

1. Construction of chimeric antibodies

(1) cloning a DNA that encodes a V-phase mouse anti-1.24 NM monoclonal antibodies

Obtaining mRNA

For cloning of the DNA encoding the V-phase mouse anti-1.24 NM monoclonal antibodies have total RNA from selected hybridoma using a known method such as a method using guanidine-ultracentrifugation (Chirgwin, J. M. et al., Biochemistry (1979), 18, 5294-5299), the AGPC method (Chomczynski, P. et al, (1987), 162, 156-159), and so on, and mRNA receive, using junction-a column of oligo(dt)-cellulose supplemented with cleaning kit mRNA (mRNA Purification Kit, Pharmacia), etc. in Addition, using QuickPrep mRNA Purification Kit (Pharmacia), mRNA can be obtained without the stage of allocation of total RNA.

Receiving and amplification of cDNA

From the mRNA obtained in the above stage of obtaining mRNA, each cDNA for V-sections L-chain and H-chain is synthesized using reverse transcriptase cDNA V-phase L-chains are synthesized using a kit for the synthesis of the first chain cDNA AMV reverse transcriptase (AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit). For amplification of the synthesized cDNA using the appropriate primer, which hybridizes leader with a consistent Sequence ID 29-39, and primer ISS nucleotide sequence of which is represented by Sequence ID 40).

Synthesis and amplification of cDNA V-phase H-chain can be performed by PCR (polymerase chain reaction) using 5'-RACE (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989), using a set of 5'-Ampli FINDER RACE (CLONTECH). With the 5'-end of the previously synthesized cDNA Ampli FINDER, Anchor, and as a primer for amplification of the V-phase H-chain, you can use a primer that specifically hybridizes with Anchor primer (Sequence ID 77) and constant plot (C-plot) mouse H-chain (e.g., primer Mnsa, the nucleotide sequence of which is represented by sequence ID 42).

DNA isolation and determination of its nucleotide sequence

The PCR product is subjected to agarose gel electrophoresis, using known techniques, to cut the desired DNA fragment, and DNA extracted from him and cleaned, and then are ligated with a vector DNA.

DNA can be cleaned using a commercial kit (e.g., GENECLEAN II; BI0101). Known vector DNA (e.g., pUC19, Bluescript, and so on) can be used to store DNA fragments.

The above is Holocene recombinant vector. Then the obtained recombinant vector is introduced into Escherichia coli JM109, then resistant to ampicillin colonies are selected, and the vector DNA get on the basis of a known method (J. Sambrook, et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989).

After splitting the above vector DNA with enzymes, in a known manner to determine the nucleotide sequence of the desired DNA (for example, "dideoxy" method) (J. Sambrook, et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989). In accordance with the present invention can be used automatic sequencing (DNA Sequencer A; manufacturer ABI Co. Ltd.).

The area that defines complementarity

V-phase H-chain and V-phase L-chain form antigennegative website, and their complete structures have similar properties. Thus, each of the four frame sections (FR) was Legerova with three hypervariable sites, i.e. sites complementarity determining (CDR). Amino acid sequences of FRs were relatively stable conservative, whereas among the amino acid sequences of CDR plots the variations are extremely common (Kabat, E. A. et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).

Many CDR can also be part of a structure list. Three CDR steric remain in close proximity to each other, and form antigennegative site with three CDR plots mating.

Based on these facts, the amino acid sequence of the variable segment of mouse anti-NM 1.24 antibody fitted to the data for the amino acid sequences of the antibodies obtained Kabat et al. "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983), to study the homology and, thereby, to determine the CDR plots.

(2) Construction of expression vectors for chimeric antibodies

After the cloned DNA fragment encoding the V-sections of mouse L-chain and H chain of the mouse monoclonal antibodies can be obtained chimeric anti-NM 1.24 antibody by binding these murine V-plots with DNA encoding the constant area of the human antibodies, and their subsequent expression.

The main method of constructing chimeric antibody comprises linking the mouse leader sequence and the sequence V-phase present in the cloned cDNA sequence that encodes a C-section human antibodies already present in expressing vector for mammalian cells. In another embodiment, it VK is cloned cDNA, sequence that encodes a C-section human antibodies, which then bind with the expression vector for mammalian cells.

S-area of the human antibodies can be C-section any N-chain and C-section any L-chain. So, you can, for example, be mentioned C1, C2, C3, or C4 in human H-chain, or C or C L-chain.

To obtain chimeric antibody construct two types expressing vectors: they are expressing a vector comprising DNA encoding the V-plot of mouse L-chain and s-area of the human L-chain under the control plot, regulating the expression of such as system enhancer/promoter and expressing a vector comprising DNA encoding the V-part of the mouse H-chain, and the area of the human H-chain under the control plot, regulating the expression of such as system enhancer/promoter. Then, using these expressing vectors, is subjected to cotransformation such host cells, like mammalian cells, and these transformed cells are cultivated in vitro or in vivo, getting a chimeric antibody (e.g., WO 91-16928).

In another embodiment, DNA encoding the mouse leader sequence and the V-phase L-chain and s-area of the human L-chain, and DNA, built in the cloned cDNA, enter one expressing vector (see publication of the international application WO 94-11523), and cell-host transform, using the specified vector. Then this transformed cell host cultivated in vitro or in vivo, receiving targeted chimeric antibody.

1) Construction of chimeric H-chain

The expression vector for N-chain chimeric antibody can be obtained by introducing a cDNA that encodes a V-phase mouse H-chain in the appropriate expressing the expression vector containing the genomic DNA or cDNA encoding a C-section H-chain of a human antibody. As With parcels N-chain you can specify, for example, C1, C2, C3, or C4.

Designing expressing vector for chimeric H-chain containing genomic DNA for C1

As the expressing vector containing the genomic DNA for C1 as C-section H-chain, can be used, for example, HFE-PMh-g1 (publication of the international application WO 92/19759), or DHFR-E-RVh-PM1f (publication of the international application WO 92/19759).

To embed a cDNA that encodes a V-phase mouse H-chain in expressing these vectors by PCR to introduce a suitable nucleotide sequence. These are suitable nucleotide sequence can CC the ability for a suitable restriction enzyme 5'-end, and a consensus Kozak sequence immediately before the start codon, and PCR primer is designed so that it includes the 3'-end recognition sequence for the corresponding restriction enzyme, and splicing donor site, where the primary transcript of the genomic DNA is subjected to appropriate splicing, for transformation into mRNA.

Designed cDNA encoding the V-part of the mouse H-chain, treated with appropriate restriction enzymes, and inserted into the above expressing vector, and it is possible to construct a chimeric H-chain-expressing vector comprising DNA C1.

Design expressroute vector for cDNA chimeric H-chain

Expressing the vector containing the cDNA C1 as C-section H-chain, can be constructed as follows. Thus, it is possible to construct, obtaining mRNA from Cho cells, which were integrated expressing vector DHFR-E-RVh-PM1f (publication of the international application WO 92/19759) encoding the genomic DNA of the V-phase H-chain gumanitarnogo RM antibodies and C-section C1 N-chains of human antibodies (N. Takahashi, et al., Cell 29, 671-679, 1982), and expressing the vector RVl-PM1a (publication of the international application WO 92/19759), to whom nitela, and-plot-chain L-chain of a human antibody; cDNA cloning, including V-phase H-chain gumanitarnogo RM antibodies and C-section C1 N-chains of human antibodies by the method of RT-PCR; and ligera expressing in a suitable vector for animal cells, using suitable sites for enzymes.

For direct ligation of cDNA encoding V-part of the mouse H-chain cDNA containing a C-section C1 N-chains of human antibodies, you can enter the appropriate nucleotide sequence by PCR.

For example, those suitable nucleotide sequence can be entered by PCR using the PCR primer is designed such that it contains a recognition sequence for a suitable restriction enzyme to the 5'-end and a consensus Kozak sequence immediately before the start codon, and PCR primer is designed such that it contains a recognition sequence for a suitable restriction enzyme used for direct ligation of C-section C1 N-chains at the 3'-end.

Expressing the vector containing the cDNA chimeric H-chain, you can design, processing designed DVS is happening With the plot C1 N-chain, and embedding in expressing this vector, as pCOS1 or pCHOl.

2) construction of the L-chain chimeric antibodies

Expressing the vector for the L-chain of the chimeric antibody can be obtained by linking cDNA encoding V-plot of mouse L-chain with genomic DNA or cDNA that encodes a C-section L-chains of a human antibody, and then introducing it into a suitable expressing vector. As a C-section L-chain, you can specify, for example, chain or chain.

Designing expressing vector for a-chain cDNA chimeric L-chain

To construct expressing vector containing cDNA encoding the V-plot of mouse L-chain, you can enter the appropriate nucleotide sequence using PCR. So, for example, those suitable nucleotide sequence can be entered by PCR using the PCR primer is designed to contain the recognition sequence for a suitable restriction enzyme and a consensus Kozak sequence at the 5'-end, and PCR primer is designed to contain the recognition sequence for a suitable restriction enzyme 3'-end.

With-plot-chain of human L-chain to associate with V-uchastie genomic DNA. The expression vector for the-chain L-chain cDNA chimeric antibodies can be constructed by introducing recognition sequences of suitable enzymes with 5'-end or 3'-end of DNA that encodes a C-section-chain L-chain PCR, ligera designed V-plot of mouse L-chain C-plot-chain L-chain, and then embedded in expressing this vector, as pCOS1 or pCHOl.

2. Constructing a reconstructed human antibodies

(1) the Design of the V-phase reconstructed human anti-NM 1.24 antibody

For constructing a reconstructed human antibodies in which CDR of mouse monoclonal antibody has been transplanted into a human antibody, it is desirable that there be gomologichnosti between FR murine monoclonal antibodies and FR of the human antibody. Thus, the V-sections of the L-chain and H chain of the mouse anti-NM 1.24 antibody compared to the V-plots of all known antibody structures which have been established using the data Bank Protein Data Bank.

V-part of the L-chain of murine anti-NM 1.24 antibody is closest to a consensus sequence of subgroup IV, V-phase L-chains of a human antibody (HSGIV) homologically 66,4%. With each L-chain of murine anti-NM 1.24 antibody when compared with V-section L-chain known human antibodies demonstrates gomologichnosti 67,0% V-section L-chain of the human antibody REI, one of the subgroups IV-section L-chains of a human antibody. Therefore, FR from REI used as source material for constructing V-phase L-chain reconstructed human anti-NM 1.24 antibody.

Design option a V-phase L-chain reconstructed human anti-NM 1.24 antibody. In this embodiment, the FR of the human antibody are doing is identical to the FR-based REI, present in reconstructed human SAMRAT-1H antibody (see Riechmann, L. et al., Nature 322, 21-25 (1988), FR is contained in option a V-phase L-chain reconstructed human PM-1 antibody, as described in published international application WO 92/19759), and murine CDR doing identical with CDR in the V-section L-chain of murine anti-NM 1.24 antibody.

V-phase H-chain of murine anti-NM 1.24 antibody is closest to the consensus sequence of the V-phase H-chain of human antibodies (HSGI) homologically 54,7%. On the other hand, it demonstrates gomologichnosti in 34,6% and 48.1% HSGII and HSGIII respectively. When comparing the V-phase H-chain of murine anti-NM 1.24 antibody V-block H-chain known human antibodies, it appears that FR1 to FR3 are the closest V-block H-chain of the human antibody HG3, one of the subgroups I V-human antibody HG3 used as source material for constructing V-phase H-chain reconstructed human anti-NM 1.24 antibody.

However, since the amino acid sequence of FR4 of the human antibody HG3 was not described as use FR4 amino acid sequence of FR4 of the human antibody JH6 (Ravetch, J. V. et al., Cell, 27, 583-591), which shows the highest gomologichnosti with FR4 mouse anti-NM 1.24 antibody. FR4 JH6 has the same amino acid sequence as FR4 H-chain of murine anti-NM 1.24 antibody, with the exception of only one amino acid.

In the first embodiment and the V-phase H-chain reconstructed human anti-NM 1.24 antibody, FR1-FR3 make identical FR1-FR3 of a human antibody HG3, except that the amino acid at position 30 in human FR1 and in position 71 human FR3 make identical amino acids of murine anti-NM 1.24 antibody and CDR do identical CDR in the V-block H-chain of murine anti-NM 1.24 antibody.

(2) the Design of the V-phase L-chain reconstructed human anti-NM 1.24 antibody.

L-chain reconstructed human anti-NM 1.24 antibody construct using transplantation CDR PCR. This method is schematically shown in Fig. 4. Eight PCR primers used to construct the reconstructed chelovechestva.novelly ID 47) and H (Sequence ID 48) create for hybridization with the DNA sequence HEF expressing vector HEF-VL-g.

CDR-transplanted primers L1S (Sequence ID 49), L2S (Sequence ID 50) and L3S (Sequence ID 51) contain semantic DNA sequence. CDR transplanted the time duration L1A primers (Sequence ID 52), L2A (Sequence ID 53) and L3A (Sequence ID 54) contain the antisense DNA sequence, and each contains a DNA sequence (20-23 p. N.), complementary to the DNA sequence 5'-end primers L1S, L2S and L3S, respectively.

In the first stage PCR carried out four reactions: A-the time duration L1A, L1S-L2A, L2S-L3A and L3S-H, and each PCR product clean. Four PCR products from the first PCR provide an opportunity to connect with each other in accordance with their own complementarity (see WO 92-19759). Then add the external primers a and H for amplification of the full length DNA encoding the V-part of the L-chain reconstructed human anti-NM 1.24 antibody (second PCR). In the above PCR as a matrix you can use the plasmid HEF-RVL-M21a (publication of the international application WO 95-14041), encoding a V-phase L-chain reconstructed human ONS-M21 antibody-based FR derived from a human antibody REI.

In the first stage PCR using the clean, using a 1.5% agarose gel with a low melting point, and connect the second PCR. In the second PCR using each product of the first PCR and each external primer (a and N).

The DNA fragment length 516 p. N., obtained in the second PCR, purified, using a 1.5% agarose gel with a low melting point, split BamHI and HindIII and the resulting fragments clone in HEF expressing vector HEF-VL-g. After determining the DNA sequence of a plasmid containing the DNA fragment with the correct amino acid sequence of the V-phase L-chain reconstructed human anti-NM 1.24 antibody, called a plasmid HEF-RVLa-AHM-g. Amino acid sequence and nucleotide sequence of the V-phase L-chain contained in this plasmid HEF-RVLa-AHM-g, presented in Sequence ID 9.

Option b V-phase L-chain reconstructed human anti-NM 1.24 antibody can be constructed using mutagenesis using PCR. The mutagenesis primers FTY-1 (Sequence ID 55) and FTY-2 (Sequence ID 56) designed to carry out the mutation of the phenylalanine at position 71 on tyrosine.

After the above primers amplified with the genomic DNA of clone in HEF expressing vector HEF-VL-g to obtain plasmid HEF-RVLb-AHM-g. Amino acid sequence and nucleotide sequence of the V-phase L-chain contained in this plasmid HEF-RVLb-AHM-g, presented in Sequence ID 10.

(3) the Design of the V-phase H-chain reconstructed human anti-NM 1.24 antibody

3-1. Design options a-e V-phase H-chain reconstructed human anti-NM 1.24 antibody

DNA encoding the V-phase H-chain reconstructed human anti-NM 1.24 antibody, can be constructed as follows. Linking a DNA sequence encoding a FR 1-3 human antibody HG3, and FR4 of the human antibody JH6 with a DNA sequence that encodes a CDR V-phase H-chain of murine anti-NM 1.24 antibody, it is possible to construct DNA full length, encoding a V-phase H-chain reconstructed human anti-NM 1.24 antibody.

Then HindIII recognition site/consensus KOZAK sequence and a BamHI recognition site/splicing donor sequence, respectively, attached to the 5'-end and 3'-end of the DNA sequence, in order to ensure the embedding expressing vector HEF.

Designed DNA sequence shared by the will eraut computer analysis with respect to secondary structure.

Sequences of the four oligonucleotides RVH1-RVH4 forth in Sequence ID 57-60. The length of these nucleotides is 119-144 grounds, and they have overlapping plot 25-26 grounds. Among these nucleotides RVH2 (Sequence ID 58) and RVH4 (Sequence ID 60) have a sense DNA sequence, a RVH1 (Sequence ID 57) and RVH3 (Sequence ID 59) have antisense sequence. The method of Assembly of these four oligonucleotides by PCR is shown in Fig.5.

PCR carried out using four oligonucleotide and RHP1 (Sequence ID 60) and RHP2 (Sequence ID 62) as external primers.

Amplificatory DNA fragment length 438 grounds clean, split HindIII and BamHI, and then clone in expressing vector HEF-VH-gl. After determining the nucleotide sequence of the plasmid containing the DNA fragment encoding the correct amino acid sequence of the V-phase N-chain is called HEF-RVHa-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHa-AHM-gl, presented in Sequence ID 11.

Each of the options b, C, d and e V-teaching is Rurouni each of the options b and next V-phase H-chain reconstructed human anti-NM 1.24 antibody three-dimensional structural model of the V-phase mouse anti-NM 1.24 antibody can be constructed to predict the position of amino acid residue that should be replaced in the molecule antibodies.

Using primer mutagenesis BS (Sequence ID 63) and VA (Sequence ID 64), designed to implement the mutation of the arginine at position 66 in lysine, and as a matrix DNA the plasmid HEF-RVHa-AHM-gl using the PCR method, option b is amplified to obtain plasmid HEF-RVHb-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHb-AHM-gl, presented in sequence ID 12.

Using primer mutagenesis CS (Sequence ID 65) and SA (Sequence ID 66), designed to implement the mutation of threonine at position 73 in lysine, and as a matrix DNA the plasmid HEF-RVHa-AHM-gl PCR, the variant with amplified to obtain plasmid HEF-RVHc-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHc-AHM-gl, presented in Sequence ID 13.

Using primer mutagenesis DS (Sequence ID 67) and DA (Paleologena 73 in lysine, and as a matrix DNA the plasmid HEF-RVHa-AHM-gl by PCR, option d amplified to obtain plasmid HEF-RVHo-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHd-AHM-gl, presented in Sequence ID 14.

Using as primer mutagenesis ES (Sequence ID 69) and EA (Sequence ID 70), designed to implement the mutation of valine at position 67 in alanine and methionine in position 69 in leucine, and as a matrix DNA the plasmid HEF-RVHa-AHM-gl, option e is amplified to obtain plasmid HEF-RVHe-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHe-AHM-gl, presented in Sequence ID 15.

3-2. Design of hybrid V-phase H-chain

Designing hybrid V-phase H-chain, you can determine which FR V-phase gumanitarnogo antibodies contributes to the binding activity and the activity of inhibiting the binding. Of the two constructed sequences, amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody, whereas FR3 and FR4 derived from options and V-uchastnitsa) in one, and amino acid sequences of FR1 and FR2 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody, and FR3 and FR4 derived from a murine anti-NM 1.24 antibody (human/mouse hybrid anti-1.24 NM antibody) in the other. All amino acid sequences of CDR plots derived from the murine anti-NM 1.24 antibody.

Two hybrid V-phase H-chain construct by PCR. This method is schematically shown in Fig.6 and 7. To construct two hybrid V-parcel N-chain you can use the four primers. External primers (Sequence ID 71) and h (Sequence ID 72) provide for hybridization with the DNA sequence of the vector expressing vector HEF HEF-VH-gl. Primer HYS hybrid construction H-chain (Sequence ID 73) design so that he had a sense DNA sequence, and the hybrid primer HYA N-chain (Sequence ID No. 74) had the antisense DNA sequence so that these DNA sequences were complementary to each other.

To construct a hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody, a FR3 and FR4 receiving the plasmid HEF-1.24-gl as a matrix, external primer a, and the hybrid primer H-chain HYA, and PCR using the plasmid HEF-RVHa-AHM-gl as a matrix, hybrid primer HYA N-chain, external primer h, carry out the first stage of PCR, and each PCR product clean.

Two products of the first PCR provide an opportunity to connect at the expense of self-complementarity (publication of the international application WO 92-19759). Then, by adding the external primers a and h, the full length DNA encoding the hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody, amplified in the second stage PCR.

To construct a hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from a murine anti-NM 1.24 antibody, PCR using the plasmid HEF-RVHa-AHM-gl as a matrix, the external primer a, and the hybrid primer H-chain HYA, PCR using the plasmid HEF-1.24-gl as a matrix, hybrid primer HYS N the untreated products of the first PCR provide an opportunity to connect through their own complementarity (publication of the international application WO 92-19759). Then, by adding the external primers a and h, the resulting DNA full length, encoding a hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from a variant of a N-chain reconstructed human anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from a murine anti-NM 1.24 antibody, amplified in the second stage PCR.

Methods the first PCR, purification of PCR products Assembly, the second PCR and cloned into the HEF expressing vector HEF-VH-gl carried out in accordance with the method shown in example 9 Design V-phase L-chain reconstructed human anti-NM 1.24 antibody". After determining the DNA sequence of a plasmid which contains a DNA fragment encoding the correct amino acid sequence of the hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody, called HEF-MH-RVH-AHM-gl.

Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-MH-RVH-AHM-gl, presented in the Sequence is to be hybrid V-phase H-chain, in which the amino acid sequences of FR1 and FR2 derived from options and reconstructed human anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from the V-phase H-chain of murine anti-NM 1.24 antibody, called HEF-HM-RVH-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-HM-RVH-AHM-AHM-gl, presented in sequence ID 76.

3-3. Design options f-s V-phase H-chain reconstructed human anti-NM 1.24 antibody

Each of the options f, g, h, i, j, k, 1, m, n, o, p, q, r and s V-phase H-chain reconstructed human anti-NM 1.24 antibody construct as follows. When designing each of the options f and next V-phase H-chain reconstructed human anti-NM 1.24 antibody is possible to construct three-dimensional structural model of the V-phase mouse anti-NM 1.24 antibody, as described previously, in order to predict the position of amino acid residue that need to substitute in the molecule antibodies.

Using primer mutagenesis FS (Sequence ID 78) and FA (Sequence ID 79), designed to carry out the mutation of the threonine at position 75 on serine, have to obtain plasmid HEF-RVHf-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHf-AHM-gl, presented in Sequence ID 16.

Using primer mutagenesis GS (Sequence ID 80) and GA (Sequence ID No. 81), designed to carry out the mutation of the alanine at position 40 on arginine, and as a matrix DNA the plasmid HEF-RVHa-AHM-gl, version g amplified to obtain plasmid HEF-RVHg-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHg-AHM-gl, presented in sequence ID 17.

Using primer mutagenesis FS and FA, and as a matrix DNA the plasmid HEF-RVHb-AHM-gl, version h amplified to obtain plasmid HEF-RVHh-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHh-AHM-gl, presented in Sequence ID 18.

Using primer mutagenesis IS (Sequence ID 82) and IA (Sequence ID 83), designed to carry out the mutation of the arginine at position 83 by alanine and serine at position 84 on phenylalanine, and as a matrix DNA the plasmid HEF-RVRh-AHM-gl, variant i ampli shall be V-phase H-chain, contained in the plasmid HEF-RVHi-AHM presented in Sequence ID 19.

Using primer mutagenesis JS (Sequence ID 84) and JA (Sequence ID 85), designed to carry out the mutation of the arginine at position 66 in lysine, and as a matrix DNA the plasmid HEF-RVHf-AHM-gl, option j amplified to obtain plasmid HEF-RVHj-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHj-AHM-gl, presented in Sequence ID 20.

Using primer mutagenesis S (Sequence ID 86) and KA (Sequence ID No. 87), designed to implement the mutation of glutamic acid at position 81 in glutamine, and as a matrix DNA the plasmid HEF-RVHh-AHM-gl, version k amplified to obtain plasmid HEF-RVHk-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHk-AHM-gl, presented in sequence ID 21.

Using primer mutagenesis LS (Sequence ID 88) and LA (Sequence ID 89), designed to implement the mutation of glutamic acid at position 81 the GLA is t to obtain plasmid HEF-RVH1-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHI-AHM-gl, presented in Sequence ID 22.

Using primer mutagenesis MS (Sequence ID 90) and MA (Sequence ID 91), designed to implement the mutation of glutamic acid at position 81 in glutamine, serine at position 82 in isoleucine and threonine at position 87 in serine, and as a matrix DNA the plasmid HEF-RVHh-AHM-gl, a variant of the m amplified to obtain plasmid HEF-RVHm-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHm-AHM-gl, presented in Sequence ID 23.

Using primer mutagenesis NS (Sequence ID 92) and NA (Sequence ID 93), designed to implement the mutation of serine at position 82 in isoleucine, and as a matrix DNA the plasmid HEF-RVHh-AHM-gl, a variant of the n amplified to obtain plasmid HEF-RVHn-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHn-AHM-gl, presented in Sequence ID 24.

Using primer of mutagenesis O mutation of threonine at position 87 in series, and as a matrix DNA the plasmid HEF-RVHn-AHM-gl option on amplified to obtain plasmid HEF-RVHo-AHM-gl. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHo-AHM-gl, presented in Sequence ID of 25.

Using primer mutagenesis PS (Sequence ID 96) and RA (Sequence ID 97), designed to implement the mutation of valine at position 78 in alanine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, option R amplified by PCR to obtain plasmid HEF-RVHp-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHp-AHM-g1, presented in Sequence ID 26.

Using primer mutagenesis QS (Sequence ID 98) and QA (Sequence ID 99), designed to carry out the mutation of the threonine at position 75 in serine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, option q amplified by PCR to obtain plasmid HEF-RVHq-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHq-AHM-g1, presented in sequence ID to matrix DNA the plasmid HEF-RVHp-AHM-g1, option r amplified by PCR to obtain plasmid HEF-RVHr-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHr-AHM-g1, presented in Sequence ID 28.

Using primer mutagenesis SS (Sequence ID 100) and SA (Sequence ID 101), designed to carry out the mutation of the methionine at position 69 in salicin, and as a matrix DNA the plasmid HEF-RVHr-AHM-g1, option s amplified to obtain plasmid HEF-RVHs-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in the plasmid HEF-RVHs-AHM-g1, presented in Sequence ID 102.

Designed amino acid sequences of the V-phase L-chain is presented in table. 1, and the amino acid sequence of V-phase N-chain is presented in table. 2-4.

3. Obtaining chimeric antibodies and reconstructed human antibodies.

To obtain a chimeric antibody or a reconstructed human antibodies for each construct two expressing vector that includes expressing a vector comprising DNA encoding V we plot the ser/promoter system, and DNA encoding the V-plot of mouse L-chain and s-area of the human L-chain under the control of regulating the expression of the site, such as an enhancer/promoter system, or expressing a vector comprising DNA encoding the V-part of the humanized H-chain and s-area of the human H-chain under the control plot regulating expression, such as an enhancer/promoter system, and DNA encoding the V-part of the humanized L-chain and s-area of the human L-chain under the control plot regulating expression, such as an enhancer/promoter system.

Then the cell host, such as a mammal cells, subjected to cotransformation using these vectors, and transformed cells are cultivated in vitro or in vivo to obtain a chimeric antibody or a reconstructed human antibodies (see, for example, a publication of the international application WO 91-16928). Moreover, the gene of the antibody is administered to a mammal, such as a goat, to obtain a transgenic animal, the milk of which it is possible to obtain a chimeric antibody or a reconstructed human antibody.

In addition, genes V-phase H-chain, C-block H-chain V-region L-chain and C-section L-chain are ligated into a single vector for transformation glue the left mouse leader sequence and the V-phase H-chain and s-area of the human H-chain, present in the cloned cDNA, and DNA encoding the mouse leader sequence and the V-phase L-chain and s-area of the human L-chain is introduced into one expressing vector (a publication of the international application WO 94-11523).

For the expression of a reconstructed human antibodies DNA encoding the V-part of the humanized H-chain and s-area of the human H-chain and DNA encoding the V-part of the humanized L-chain and s-area of the human L-chain is introduced into one expressing vector (a publication of the international application WO 94-11523). Cell owners, transform, using the specified vector, and transformed cell hosts are cultivated in vivo or in vitro, getting the desired chimeric antibody or reconstructed human antibody.

The transformant that has been transformed, as described above, the gene coding for the desired chimeric antibody or reconstructed human antibody, cultivate, and the resulting chimeric antibody or reconstructed human antibody can be distinguished, either inside or outside cells and purified to homogeneity.

Isolation and purification of the desired protein of the present invention chimeric antibodies or reconstructed caloocan And, for example, you can specify HyperD, POROS, Sepharose F. F, etc. In another embodiment, it is possible to use conventional methods of isolation and purification, used for proteins, and the choice of method is not restricted. For example, a combination of various chromatographic techniques, ultrafiltration, vysalivaniya, dialysis, etc. can afford to isolate and purify the chimeric antibody or reconstructed human antibody.

To obtain chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody of the present invention can use any method of expression, including, for example, such eukaryotic cells, like animal cells, the system is stable cell lines of mammalian, insect cells, the system fungal cells and the system of yeast cells, and prokaryotic cells such as bacterial cells, such as cells of Escherichia coli, etc., Preferably, a chimeric antibody or a reconstructed human antibody of the present invention, it was possible to Express in cells, COS cells, Cho cells, Hela, Vero cells, myeloma cells or cells KSS.

In these cases, you can use the regular promoters, which are used for NGO-early promoter (HCMV). Examples expressing vectors containing the HCMV promoter, include V-V n-HC1, HCMV-VL-HC, etc., and those derived from pSV2neo (publication of the international application WO 92/19759).

In addition, as a promoter for expressi gene in mammalian cells for use in the present invention can be used viral promoters, such as retrovirus, virus polyoma, adenovirus, monkey virus 40 (SV40), and so on, and promoters derived from mammalian cells, such as elongation factor polypeptide 1(HTF-1), and so on, for example, when using the SV40 promoter, the expression can easily be done using the method of Mulligan et al. (Nature 277, 108(1979)), and if you use the promoter HTF-1, you can use the method of Mizushima, S. et al. (Nucleic Acids Research, 18, 5322, 1990).

As the replication source, you can use the source, derived from SV40, a virus polyoma, adenovirus, papilloma virus in cattle (BPV), etc., and to amplify the number of copies of the gene in the cellular system of the host expressing the vector may include, as a selective marker gene aminoglycosidetherapy (ARN), gene timedancing (TS), gene contingenciesliabilities E. coli (Ecogpt) gene digidrofolatreduktazy (DHRF), etc.

4
(1) Measurement of antibody concentrations

The concentration of purified antibodies can be measured by using ELISA, or by measuring the absorption.

Tablets ELISA to determine the concentration of the antibodies can be prepared as follows. Each well of 96-well plate to ELISA (for example, Maxisorp, manufacturer NUNC) immobilized 100 μl of goat antibodies against human IgG at a concentration of 1 µg/ml.

After blocking with 100 µg/ml diluted buffer (50 mm Tris-HCl, 1 mm MgCl2, 0.15 M NaC1, 0.05% of Tween 20, 0.02% of NaN3, 1% bovine serum albumin (BSA), pH 8.1), in each cell type serial dilution of the supernatant culture fluid of cells that expressed a chimeric antibody, a hybrid antibody or reconstructed human antibody, for example, the culture supernatant of COS cells or cells SNO, or purified chimeric antibodies, hybrid antibodies or reconstructed human antibodies. Then add 100 μl conjugated with alkaline phosphatase goat antibodies against human IgG, was added 1 mg/ml substrate solution (Sigma 104, p-nitrophenylphosphate, SIGMA production), and then measure the absorption at 405 nm using a plate reader tablets for Eoisode The Binding Site). The concentration of purified antibodies receive by measuring the absorption at 280 nm and taking the value of 1 mg/ml as 1,35 OP.

(2) the Activity of binding

The activity of binding can be measured using Cell-ELISA using human amniotic cell line WISH (ATCC CCL25). Tablets for cell ELISA can be obtained as follows. Prepared cells WISN in the appropriate concentration in PRMI 1640 medium, supplemented with 10% serum, amniotic calf, add in 96-well plates, incubated overnight, and after two rinses of PBS(-), fixed with 0.1% glutaraldehyde (production Nakalai tesque).

After blocking add 100 µl of serial dilutions of culture supernatant environment of cells in which the chimeric anti-NM 1.24 antibody, a hybrid anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody expressed, for example, the culture supernatant of COS cells or cells SNO, or purified chimeric anti-NM 1.24 antibody, a hybrid anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody to each well, incubated at room temperature for two hours, and then add peroxidase labeled rabbit th temperature add the substrate solution and again incubated. Then the reaction is stopped by adding 50 μl of 6 N. sulfuric acid, and then measure the absorbance at 490 nm using a MICROPLATE reader READER Model 3550 (BioRad).

(3) Measurement of activity of inhibiting the binding

The activity of inhibiting the binding of biotinylated mouse anti-NM 1.24 antibody is determined using cell ELISA using human amniotic cell line WISH (ATCC CCL25). Tablet for cell ELISA can be prepared according to the above (2). Cells WISH, prepared at the appropriate concentration in PRMI 1640 medium, supplemented with 10% serum, amniotic calf placed in 96-well plates, incubated overnight, and after twice washing PBS(-) fixed with 0.1% glutaraldehyde (Nacalai tesque).

After blocking in each well add 50 µl of serial dilutions of culture supernatant environment of cells in which the chimeric anti-NM 1.24 antibody, a hybrid anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody expressed, for example, the culture supernatant of COS cells or cells SNO, or purified chimeric anti-NM 1.24 antibody, a hybrid anti-NM 1.24 antibody or reconstructed human anti-1.24 NM anticell room temperature for two hours and after washing, add peroxidase labeled streptavidin (production DAKO).

After incubation at room temperature for 1 h and after wash add substrate solution, and then incubated. Then the reaction is stopped by adding 50 μl of sulfuric acid, and then measure the absorption at 490 nm using a MICROPLATE reader READER Model 3550 (BioRad).

Measurement of ADCC activity

ADCC activity of chimeric antibodies or reconstructed human antibodies of the present invention can be measured as follows. First, mononuclear cells isolated from human peripheral blood or bone marrow by centrifuging in the density gradient and prepare as effector cells. The myeloma cells are prepared as target cells, through the introduction of51SG label in RPMI 8226 cells (ATSC CCL155). Then chimeric antibody or reconstructed human antibody, which is to be determined on ADCC activity add to the labeled target cells, incubated, and then add the appropriate number of effector cells to target cells and incubated.

After incubation, the supernatant is taken for measuring radio is th emitted radioactivity. Cytotoxicity (%) can be calculated as (a-C)/(b-C)x 100, where a represents the emitted radioactivity (counts/min) in the presence of antibodies, is emitted radioactivity (counts/min), selected due NP-40, and represents the radioactivity (counts/min) emitted by only one culture fluid without antibodies.

If ADCC activity or CDC activity is expected to register With the site antibodies, as With the plot antibodies can be used in human or C1 human C3. Moreover, adding, changing or modifying a part of the amino acids-phase antibody, it is possible to induce a higher ADCC activity or CDC activity.

For example, there IgM-like polymerization IgG by substitution of amino acids (Smith, R. I. F. & Morrison, S. L, BIO/TECHNOLOGY (1994) 12, 683-688), IgM-like polymerization IgG by adding amino acids (Smith, R. I. F. et al., J. Immunol. (1995) 154, 2226-2236), expression by tandem binding genes encoding the L-chain (Shuford, W. et al., Science (1991) 252, 724-727), dimerization of IgG by substitution of amino acids (Sagap, R. C. et al., J. Exp. Med. (1992) 176, 1191-1195, Shopes, B. J. Immunology (1992) 148 2918-2922, dimerization of IgG as a result of chemical modification (Wolff, E. A. et al., Cancer Res. (1993) 53, 2560-2565), and the introduction of effector functions in staravina by mutagenesis oligomers, using the primers by adding a base sequence using the cleavage sites of the enzymes, and chemical properties, which induce covalent binding.

In vivo diagnosis of myeloma

Chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody of the present invention can be used as in vivo diagnostic agents for myeloma by associating it with a labeled compound, such as a radioisotope, etc.

In addition, fragments of chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody, such as Fab, F(ab')2, Fv or single-chain Fv (scFv), where Fv or Fv H-chain and L-chain are connected with a suitable linker, which was associated with such a labeled compound, as a radioisotope, etc., can be used as in vivo diagnostic tools for myeloma.

Specifically, these antibody fragments can be obtained by designing the gene encoding these antibody fragments, introducing them in expressing vector, and then expressive in suitable host cells, or splitting chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody, a suitable enzyme.

Wysokicska composition and drug treatment for myeloma

To confirm therapeutic effect of chimeric anti-NM 1.24 antibody or gumanitarnogo anti-NM 1.24 antibody of the present invention, the aforementioned antibody is administered to an animal, which transplanted myeloma cells, and evaluate the antitumor effect.

As myeloma cells that you want to transplant an animal, preferably a human myeloma cells, and can as an example be specified CRM (publication of the patent application of Japan without examination (Kokai) 7-236475), RPMI8226 (ATSS CCL 155), ARH-77 (ATCC CRL-1621) and S6B45 (Suzuki, H. et al., Eur. J. Immunol. (1992) 22, 1989-1993). As animals, which can be transplanted these cells, preferred animals with weakened immune functions, or animals without immune functions, and as an example, you can specify nude mice, SCID mice, mice beige and nude rats.

In addition, the antitumor action that you want to evaluate, you can confirm the change in the number of human immunoglobulins in serum by changing the volume and/or weight of the tumor, weight change of the proteins Vase Jones in the urine, the duration of survival of animals or etc.

The pharmaceutical composition or the medicinal product for l the new human anti-NM 1.24 antibody of the present invention can be systemically or topically to enter parenteral. For example, you can choose intravenous injection such as drip, intramuscular injection, intraperitoneal injection or subcutaneous injection, and to choose the appropriate dose mode depending on the age and health of the patient.

Effective dose is chosen in the range from 0.01 mg to 1000 mg/kg body weight/dose. In another embodiment, it is possible to select a dose of 5 mg/kg body weight, preferably from 50 to 100 mg/kg body weight.

The pharmaceutical composition or therapeutic agents for the treatment of myeloma, which contain as the active ingredient chimeric anti-NM 1.24 antibody or reconstructed human anti-NM 1.24 antibody of the present invention may contain pharmaceutically acceptable carriers or additives depending on the selected method of administration.

As examples of such carriers or additives you can specify the water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, natiolialist, sodium alginate, water-soluble dextran, natrocarbonatite, pectin, methylcellulose, ethylcellulose, xanthangum, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surface-active agents, etc., Possible to use additives can be selected from the above or combinations thereof (without limitation).

EXAMPLES

Hereinafter the present invention will be explained in more detail.

Example 1. Cloning of cDNA encoding V-phase mouse anti-NM 1.24 antibody

1. The allocation of messenger RNA (mRNA)

Using the dial to highlight the Fast Track mRNA Isolation Kit Version 3.2 (manufacturer Invitrogen) in accordance with the attached instructions mRNA extracted from 2108hybridoma cells (FERM BP-5233) that produce mouse anti-NM 1.24 antibody.

2. Amplificate gene encoding variable plot antibodies PCR

PCR carried out using thermoreactor Thermal Cycler (production Perkin Elmer Cetus).

2-1. Amplification and fragmentation of the gene encoding the V-plot of mouse L-chain

Of the selected thus mRNA synthesize single-stranded cDNA using a kit for the synthesis of AMV Reverse Trans-criptase First-strand cDNA Synthesis Kit (manufactured by Life Science), and used for PCR. As primers on the values ID 29-39, which hybridize with the leader sequence of mouse L-chain Kappa-type.

100 μl of PCR solution containing 10 mm Tris-Hcl (pH 8.3), 50 mm KS1, 0.1 mm dNTP (dATP, DSTF, dCTP, dTTP), 1.5 mm MgCl2, 5 Units of DNA polymerase Ampli Taq (manufactured Perkin Elmer Cetus), 0.25 mm primers MKV presented in Sequence ID 29-39, 3 mm primer ISS presented in Sequence ID 40, and 100 ng of single-stranded cDNA cover 50 μl of mineral oil, and then heated at a temperature of 94oC for 3 min, then 94oC for 1 min, and 55oC for 1 min, and 72oC for 1 min in order. After repeating this cycle 30 times, the reaction mixture was incubated at 72oC for 10 minutes Amplificatory DNA fragment allocate using agarose gel with a low melting point (production Sigma), and split the XmaI (New England Biolabs) and SalI (manufactured by Takara Shuzo) at 37oC.

2-2. Amplification and fragmentation of cDNA encoding V-part of the mouse H-chain

The gene encoding the V-part of the mouse H-chain, amplified by 5'-RACE (Rapid Amplification of cDNA ends; Forhman, M. A. et al., Proc. Natl. Acad. Sci. USA, 85 8998-9002, (1988), Edwards, J. B. D. M., et al, Nucleic Acids Res. , 19, 5227-5232, (1991)). After contenstants plot murine IgG2a, cDNA encoding the V-part of the mouse H-chain, amplified using a set of 5'-AmpliFINDER RACE KIT (production CLONTECH), using primer Mnsa (Sequence ID 42), which specifically hybridizes with constant plot murine IgG2a and anchor primer (Sequence ID 77) attached to the kit. Amplificatory DNA fragment purified using the agarose gel with a low melting point (production Sigma) and digested EcoRI (manufactured by Takara Shuzo) and XmaI (New England Biolabs) at 37oC.

3. Ligation and transformation

The DNA fragment containing the gene encoding the V-plot of mouse L-chain Kappa-type, obtained as described above are ligated with the vector pUC19, resulting from cleavage SalI and XmaI, by reacting in a reaction mixture containing 50 mm Tris-Hcl (pH 7,6), 10 mm MgCl2, 10 mm dithiothreitol, 1 mm ATP, 50 mg/ml of polyethylene glycol (8000) and Ed. T4-ligase (manufactured by GIBCO-BRL) with the 16oC for 2.5 h Similarly, the DNA fragment containing the gene encoding the V-part of the mouse H-chain is subjected to interaction and are ligated with the vector pUC19, resulting from cleavage EcoRI and XmaI at a temperature of 16oWith over three hours.

Then 10 μl of the above mixture is inutu: 42oWith, and again on ice for 1 min.

Then add 400 ál of 2 x YT medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)), incubated at 37oC for 1 h, and then E. coli plated on ordinary 2 x YT medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) containing 50 μg/ml ampicillin and then incubated overnight at 37oWith getting in the E. coli-transformant.

This transformant is cultivated over night at 37oWith 10 ml of 2 x YT medium containing 50 μg/ml of ampicillin, and then from this culture gain plasmid DNA using the alkaline method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)).

Thus obtained plasmid containing the gene encoding the V-plot of mouse L-chain Kappa-type derived from hybridoma, which produces anti-NM 1.24 antibody, called pUCHMVL9. Obtained by the above method plasmid containing the gene encoding the V-part of the mouse H-chain derived from hybridoma, which produces anti-NM 1.24 antibody, called pUCHMVHR16.

Example 2. Determination of nucleotide sequence of DNA

The nucleotide sequence of cDNA coding section in the above plasmids, determine the COI is izgotovitelem.

The nucleotide sequence of the gene encoding the V-part of the L-chain of murine anti-NM 1.24 antibody contained in the plasmid pUCHMVL9 presented in Sequence ID 1. The nucleotide sequence of the gene encoding the V-phase H-chain of murine anti-NM 1.24 antibody contained in the plasmid pUCHMVHR16 presented in Sequence ID 2.

Example 3. The definition of CDR

Complete structure of the V-sections of the L-chain and H-chain similar to each other that the four frame section are connected by three hypervariable sites, i.e. sites complementarity determining (CDR). Amino acid sequence of frame portion relative to the highly conservative, but the variation in the amino acid sequence of CDR is extremely high (Kabat, E. A., et al. , "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).

Based on these facts, the amino acid sequence of the variable segment anti-NM 1.24 antibody compared with the amino acid sequence of the antibody in the database for the study of gomologichnosti, and the plot CDR define, as shown in table. 5.

Example 4. Confirmation of expression of the cloned cDNA (Construction of chimeric anti-1.24 NM antibody)

1. Construit chimeric anti-NM 1.24 antibody cDNA clones PUCHMVL9 and pUCHMVHRl6 encoding V-sections of the L-chain and H chain of the mouse anti-NM 1.24 antibody, respectively modify by PCR, and then injected into expressing vector HEF (publication of the international application WO 92-19759).

Reverse primer ONS-L722S (Sequence ID 43) for V-phase L-chain and the reverse primer VHR16S (Sequence ID 44) for V-phase H-chain design in such a way that they hybridizations with DNA that encodes the beginning of the leader sequence of the V-phase of each, and that they had a consensus Kozak sequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950, (1987)), and site recognition for the restriction enzyme HindIII. Reverse primer VL9A (Sequence ID 45) for V-phase L-chain and the reverse primer VHR16A (Sequence ID 46) for V-phase H-chain design in such a way that they hybridisable with a DNA sequence that encodes the end of the J-phase, and so they have a splicing donor sequence and site recognition for restriction enzyme BamHI.

100 μl PCR reaction mixture containing 10 mm Tris-Hcl (pH 8.3), 50 mm KC1, 0.1 mm dNTP, 1.5 mm MgCl2, 100 PM of each primer, 100 ng DNA templates (pUCHMVL9 or pUCHMVHR16), and 5 Units. Ampli Taq enzyme cover 50 μl of mineral oil and zalecenia 1 min, and when 72oC for 1 min, and so 30 cycles, and finally incubated at 72oC for 10 min.

The PCR product purified by 1.5% agarose gel with a low melting point and decompose HindIII and BamHI, and then clone in HEF-VL-g V-phase L-chain, and HEF-VH-g1 for V-phase N-chain. After determining the DNA sequence, the plasmid containing the DNA fragment, which contains the correct DNA sequence, called HEF-1.24 L-g and F-1.24 H-g1, respectively.

Lots encoding the corresponding variable site of the above plasmid HEF-1.24 L-g and F-1.24 H-g1, break down the enzymes HindIII and BamHI to generate restriction fragments that are inserted into the HindIII site and BamHI sites of plasmid vector pUC19, and they are called pUC19-1.24 L-g and pUC19-1.24 H-g1, respectively.

Escherichia coli containing the appropriate plasmid pUC19-1.24 L-g and pUC19-l. 24H-g1, called Escherichia coli DH5(pUC19-1.24 L-g) and Escherichia coli DH5(pUC19-1.24Hg1); they were international deposited on 29 August 1996, the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under registration numbers FERM-5646 and FERM-5644, respectively, in accordance with the Budapest Treaty.

2. Transfection of cells COS-7

For there is owani in cells COS-7 (ATCC CRL 1651). HEF-1.24 L-g and HEF-1.24 H-g1 cotransformation cells COS-7 using electroporation using the apparatus Gene Pulser (manufactured by BioRad). Each DNA (10 μg) are added to 0.8 ml aliquot 1107cells/ml in PBS, and treated with pulses at 1500 V and a capacitance of 25 μf.

After a recovery period of 10 min at room temperature and subjected to electroporation of cells added to 30 ml of DMEM culture liquid (GIBCO) containing 10% serum amniotic calf without gamma-globulin. After incubation for 72 h in an incubator with CO2BNA120D (manufactured by TABAI), the supernatant culture fluid is harvested, and fragments of cells removed by centrifugation, which is then used in further experiments.

3. Running cytometrics analysis (FCM)

The binding activity of the antigen chimeric anti-NM 1.24 antibody explore using FCM (flow cytometry), using cells CRM. After 4,7105cells CRM (publication of the patent application of Japan without examination (Kokai) 7-236475) washed with PBS(-), add 50 ál of cell culture COS-7, which produce the above-mentioned chimeric anti-NM 1.24 antibody and 50 μl of FACS buffer (PBS ( -) containing 2% amniotic calf serum and 0.1% sodium azide), or 5 μl of 500 μg/ml purified the ve control add 50 μl of 2 μg/ml chimeric SK2 (publication of the international application WO 94-28159) and 50 μl of FACS buffer, or 5 μl of 500 μg/ml purified mouse IgG2a (UPC10) (manufactured by CAPPEL) instead of purified mouse anti-NM 1.24 antibody, and 95 μl of FACS buffer, and incubated similarly. After washing FACS buffer add 100 ál of 25 ág/ml conjugated with FITZ goat antibodies against human antibodies(GAH) (manufactured by CAPPEL), or 10 μg/ml conjugated with FITZ goat antibodies against mouse antibody (GAM) (manufactured by Becton Dickinson), and incubated at ice temperature for 30 minutes After washing FACS buffer all of this is suspended in 1 ml FACS buffer, and measure the intensity of fluorescence of each well using a FACScan (manufactured by Becton Dickinson).

As can be seen in Fig.1, it is revealed that chimeric anti-NM 1.24 antibody binds to cells CRM as peak fluorescence intensity shifted to the right for the cells to which you have added chimeric anti-NM 1.24 antibody, compared with control, similarly to the case when add mouse anti-NM 1.24 antibody. This confirms the fact that the cloned cDNA encodes variable plot murine anti-NM 1.24 antibody.

Example 5. The establishment of a Cho cell line that stably produces chimeric anti-NM 1.24 antibody

1. The design of expressing and BamHI fragment of about 2.8, etc., N., containing EF1 promoter and DNA encoding the V-phase H-chain of murine anti-NM 1.24 antibody, allocate, using a 1.5% agarose gel with a low melting point, then the above DNA fragment embed in a fragment length of approximately 6 T. p. N., obtained by splitting expressing vector used for expressing the vector in human H-chain, DHFR-E-Rvh-PM1f (publication of the international application WO 92/19759) containing the DHFR gene and the gene encoding the constant area of the human H-chain with PvuI and BamHI, to construct expressing vector DHFR-E-HEF-1.24 H-g1 for N-chain chimeric anti-NM 1.24 antibody.

2. The introduction of the gene into the cells SNO

To create a system that consistently produces chimeric anti-NM 1.24 antibody, the genes expressing the above vector HEF-1.24 L-g and DHFR-E-HEF-1.24 H-g1, which had been linearized by cleavage PvuI, introduced simultaneously in Cho DXB11 cell (obtained from the Medical Research Council Collaboration Center) by the method of electroporation under conditions similar to the above, the above transfection into cells COS-7.

3. Gene amplification using MTX

Among cells SNO with the introduced gene, only those cells SNO, which were introduced as expressing the vector of the L-chain and Expro-BRL), to which is added 500 μg/ml G418 (manufactured GIBCO-BRL) and 10% serum amniotic calf, and thus produce a selection. Then this culture liquid, add 10 nm MTX (manufactured Sigma). Among the growing clones, select those that produce chimeric anti-NM 1.24 antibody in large quantities. The result clones 8-13, which demonstrate the efficiency of production of about 20 μg/ml chimeric antibodies, and called the cell lines producing chimeric anti-NM 1.24 antibody.

Example 6. Construction of chimeric anti-NM 1.24 antibody

Chimeric anti-NM 1.24 antibody construct as follows. The above cells SNO, producing chimeric anti-NM 1.24 antibody, continuously cultured for 30 days, using a medium Dulbecco, modified Lawsuits (production GIBCO-BRL) containing 5% newborn calf serum not containing gamma globulin (production GIBCO-BRL), using the device for cell culture of high density Verax system 20 (production CELLEX BIOSCIENCE Inc.).

13, 20, 23, 26 and 30, the day after the start of cultivation, the culture fluid is extracted using a filter device under pressure SARTOBRAN (production Sartorius), and then hiswebsite Nippon Gaishi) and Super column Protein A (layer volume: 100 ml production Nippon Gaishi) using PBS as absorbent/wash buffer and 0.1 M natriciteres buffer (pH 3) as the eluting buffer in accordance with the attached manual. pH buervenich fractions was adjusted to 7.4, immediately adding 1 M Tris-model HC1 (pH 8.0). The antibody concentration was measured by absorption at 280 nm and calculated by taking the value of 1.35 OD corresponding to 1 µg/ml.

Example 7. The determination of the activity of chimeric anti-NM 1.24 antibody.

The content of the chimeric anti-NM 1.24 antibody is assessed according to the following activity of inhibiting the binding.

1. Measurement of the activity of inhibiting the binding

1-1. The design of the biotinylated anti-NM 1.24 antibody

After murine anti-NM 1.24 antibody diluted with 0.1 M bicarbonate buffer up to 4 mg/ml 4 ál add 50 mg/ml Biotin-N-hydroxysuccinimide (production EY LABS Inc.), and the reaction is carried out at room temperature for 3 hours then add 1.5 ml of 0.2 M solution of glycine, incubated at room temperature for 30 min to stop the reaction, and then biotinylated IgG fraction is collected using a PD-10 column (manufactured Pharmacia Biotech).

1-2. Measurement of the activity of inhibition with the t using cell ELISA, using human amniotic membrane cell line WISH cells (ATSC CCL 25). Tablets for cell ELISA prepared as follows. In a 96-well plate add 4105cells/ml, prepared in PRMI 1640 medium, supplemented with 10% amniotic calf serum, incubated overnight, and after twice washing PBS(-) immobilized 0.1% glutaraldehyde (manufactured by Nakalai tesque).

After blocking, 50 μl of serial dilutions of chimeric anti-NM 1.24 antibody or mouse anti-NM 1.24 antibody, obtained by affinity purification, add to each well, and simultaneously add 50 μl of 2 μg/ml biotinylated mouse anti-NM 1.24 antibody, incubated at room temperature for 2 h, and then add peroxidase labeled streptavidin (manufactured by DACO). After incubation at room temperature for 1 h followed by rinsing add the substrate solution. After the reaction is stopped by adding 50 μl of 6 N. sulfuric acid, measuring the absorption at 490 nm using a MICROPLATE reader READER Model 3550 (manufactured BioRad).

Presented on Fig. 2 the result shows that chimeric anti-NM 1.24 antibody has an activity of inhibiting the binding Vet, what chimeric antibody has the same V-phase, and mouse anti-NM 1.24 antibody.

Example 8. Measurement USDA-activity of chimeric anti-NM 1.24 antibody

ADCC (cellular cytotoxicity-dependent antibodies) activity was measured according to the method described in Current Protocols in Immunology, Chapter 7. Immunologic Studies in humans, Editor. John E, Coligan et al., John Wiley & Sons, Inc., 1993.

1. Getting effector cells

Monocytes isolated from peripheral blood or bone marrow of healthy people and patients with multiple myeloma by centrifugation in a density gradient. Thus, an equal amount of PBS(-) are added to the peripheral blood or bone marrow of healthy people and patients with multiple myeloma, which is placed on Ficoll (manufactured by Pharmacia) Conrey (manufactured by Daiichi Pharmaceutical Co. Ltd.) (specific gravity of 1.077) and centrifuged at 400 g for 30 minutes, the Layer of monocytes harvested, washed twice RPMI 1640 (manufactured by Sigma), supplemented with 10% serum, amniotic calf (manufacturer Witaker), and prepared at a density of cells 5106/ml in the same culture fluid.

2. Obtaining target cells

In a cell line of human myeloma RPMI 8226 (ATSS CCL 155) enter the tag by incubation in RPM/SUP>CR-sodium chromate at 37oC for 60 minutes After the introduction of the RFID tags of the cells washed three times balanced solution Hanks (Hanks) (HBSS) and adjusted to a concentration of 2105/ml.

3. ADCC analysis

In a 96-well plate with a U-shaped bottom (manufactured by Corning) was placed 50 μl 2105target cells/ml, 1 μg/ml affinity purified chimeric anti-NM 1.24 antibody and mouse anti-NM 1.24 antibody, or control human IgG (manufactured by Serotec), and the reaction is carried out at 4oC for 15 minutes

Then to this add 100 ál 5106effector cells/ml, and cultured in an incubator with CO2within 4 h, when the ratio (E:T) of effector cells (E) to Milenium cells (T) is set equal 0:1, 5:1, 20:1 or 50:1.

100 µl of the supernatant is taken and the radioactivity emitted from the culture supernatant fluid measured by the meter gamma radiation (ARC361, manufacturer Aloka). To measure the maximum radioactivity using 1% NP-40 (manufactured by BRL). Cytotoxicity (%) was calculated according to the formula (a-C)/(b-C) x 100, where a represents the radioactivity (counts/min) emitted in the presence of antibodies, represents the radioactivity (counts/min) emitted due to the NP-the AK is shown in Fig.3, if chimeric anti-NM 1.24 antibody is added compared with the control IgG1, cytotoxicity increases with increasing ratio of E:T, which indicates that it is a chimeric anti-NM 1.24 antibody has ADCC activity. In addition, since cytotoxicity is not observed, even if add mouse anti-NM 1.24 antibody, it was shown that the Fc-part of human antibodies necessary to achieve the ADCC activity, if the effector cells are cells derived from human.

Example 9. The design of reconstructed human anti-NM 1.24 antibody

1. The creation of the V-phase reconstructed human anti-NM 1.24 antibody

For constructing a reconstructed human antibodies, which was transplanted CDR of mouse monoclonal antibody, preferably, that there is a high gomologichnosti between FR mouse antibodies and FR of the human antibody. Thus, the V-sections of the L-chain and H chain of the mouse anti-NM 1.24 antibody compared to the V-plots of all known antibodies, whose structure was established using the data Bank Protein Data Bank.

V-part of the L-chain of murine anti-NM 1.24 antibody is closest to V-site consensus sequences for the 56,9%, 55,8% and 61.5% with HSGI, HSGII and HSGIII respectively.

If the V-part of the L-chain of murine anti-NM 1.24 antibody compared to the V-section L-chain known human antibodies, it was shown that there is gomologichnosti 67,0% V-area REI human L-chain, one of the subgroups I V-area of the human L-chain. So FR REI used as source material for constructing V-phase L-chain reconstructed human anti-NM 1.24 antibody.

Create a V-phase L-chain reconstructed human anti-NM 1.24 antibody. In this embodiment, the human FR make identical FR-based REI, present in reconstructed human SAMRAT-1H antibody (see Riechmann, L. et al., Nature 322, 21-25 (1988), FR contained in option a reconstructed human PM-1, is described in published international application WO 92-19759), and murine CDR doing identical CDR in the V-section L-chain of murine anti-NM 1.24 antibody.

V-phase H-chain of murine anti-NM 1.24 antibody is closest to V-site consensus sequences HSGI human H-chain homologically 54,7%. On the other hand, it was shown that it is homologous by 34.6%, and 48.1% HSGII and HSGIII respectively. If V-phase H-chain of murine anti-is scimi to the V-block H-chain of the human antibody HG3, one of the subgroups I V-area of the human H-chain (Rechavi, G. et al., Proc. Natl. Acad. Sci. USA, 80, 855-859) homologically 67,3%.

Therefore, the FR of the human antibody HG3 used as source material for constructing V-phase H-chain reconstructed human anti-NM 1.24 antibody. However, since the amino acid sequence of FR4 of human HG3 was not described, using the amino acid sequence of FR4 of the human antibody JH6 (Ravetch, J. V. et al., Cell, 27, 583-591), which shows the highest gomologichnosti with FR4 H-chain of murine anti-NM 1.24 antibody. FR4 of JH6 has the same amino acid sequence as FR4 H-chain of murine anti-NM 1.24 antibody, except for one amino acid.

In the first embodiment and the V-phase H-chain reconstructed human anti-NM 1.24 antibody FR1-FR3 make identical FR1-FR3 human HG3, and CDR do identical CDR V-phase H-chain of murine anti-NM 1.24 antibody, except that the amino acid at position 30 in human FR1 and in position 71 in the human FR3 make identical amino acids in the murine anti-NM 1.24 antibody.

2. The design of the V-phase L-chain reconstructed human anti-NM 1.24 antibody

L-CG method is shown in Fig.4. Eight PCR primers used for constructing a reconstructed human anti-NM 1.24 antibody (a) containing a FR derived from a human antibody REI. External primers (Sequence ID 47) and H (Sequence ID 48) design for hybridization with a DNA sequence expressing vector HEF-VL-g.

Primers transplantation CDR L1S (Sequence ID 49), L2S (Sequence ID 50) and L3S (Sequence ID 51) have a sense DNA sequence. Primers transplantation CDR time duration L1A (Sequence ID 52), L2A (Sequence ID 53) and L3A (Sequence ID 54) have antisense DNA sequence, and each contains a DNA sequence (20-23 p. N.), complementary to the DNA sequence 5'-end primers L1S, L2S and L3S, respectively.

In the first stage PCR spend four reactions A-time duration L1A, L1S-L2A, L2S-L3A and L3S-H for purification of PCR product. Four products from the first PCR allow the Assembly to each other due to their complementarity (publication of the international application WO 92-19759). Then add the external primers a and H for amplification of the full length DNA encoding the V-part of the L-chain reconstructed chelovecheski), encoding option a V-phase L-chain reconstructed human ONS-M21 antibody-based FR derived from a human antibody REI, used as a matrix.

In the first stage PCR using the PCR mixture containing 10 mm Tris-model HC1 (pH 8.3), 50 mm KS1, 0.1 mm dNTP, 1.5 mm MgCl2, 100 ng matrix DNA, 100 pmol of each primer and 5 Units. Ampli Taq. Each PCR vial cover 50 ál of mineral oil. Then after the first denaturation by heating at 94oTo conduct a cycle of reactions at 94o1 min, at 55o1 min and at 72o1 min, and then incubated at 72oC for 10 min.

PCR products A-time duration L1A (215 p. N.), L1S-L2A (98 p. N.), L2S-L3A (140 p. N.) and L3S-H (151 p. N.) clean using a 1.5% agarose gel with a low melting point, and assemble in the second PCR. In the second PCR 98 μl PCR mixture containing 1 μg of each of the products of the first stage PCR and 5 Ed. Ampli Taq, incubated for 2 cycles at 94o2 min, 55o2 min and 72oWith 2 minutes, and then add 100 pmol of each of the outer primers (a and N). PCR vials covered with 50 μl of mineral oil and perform 30 cycles of PCR under the same conditions as before.

The DNA fragment in 516 p. N., obtained in the second PCR, purified, the clone fragments in HEF expressing vector HEF-VL-g. After determining the DNA sequence of a plasmid containing the DNA fragment with the correct amino acid sequence of the V-phase L-chain reconstructed human anti-NM 1.24 antibody called plasmid HEF-RVLa-ASM-g. Amino acid sequence and nucleotide sequence of the V-phase L-chain contained in this plasmid HEF-RVLa-AHM-g are presented in Sequence ID 9.

Option b V-phase L-chain reconstructed human anti-NM 1.24 antibody construct using mutagenesis using PCR. The mutagenesis primers FTY-1 (Sequence ID 55) and FTY-2 (Sequence ID 56) designed to expose mutations of the phenylalanine at position 71, replacing it by tyrosine.

After the above primers amplified using the plasmid HEF-RVLa-AHM-g as a matrix, the final product was then purified and digested BamHI and HindIII. Thus obtained DNA fragments clone in HEF expressing vector HEF-VL-g, obtaining the plasmid HEF-RVLb-AHM-g. Amino acid sequence and nucleotide sequence of the V-phase L-chain contained in this plasmid HEF-RVLb-AHM-g, presented in Sequence ID 10.

3. The design of V-part is STCA N-chain reconstructed human anti-NM 1.24 antibody

DNA encoding the V-phase H-chain reconstructed human anti-NM 1.24 antibody construct as follows. Linking a DNA sequence encoding FR1 to 3 human antibody HG3, and FG4 human antibodies JH6 with a DNA sequence that encodes a CDR V-phase H-chain of murine anti-NM 1.24 antibody, the design of the full length DNA encoding the V-phase H-chain reconstructed human anti-NM 1.24 antibody.

Then to the 5'-end and 3'-end of the DNA sequence attached HindIII recognition site/KOZAK consensus sequence and a BamHI recognition site/splicing donor sequence, respectively, thus, to provide the embedding HEF expressing vector.

Designed DNA sequence is divided into 4 of the oligonucleotide. Then the oligonucleotides that potentially complicate the Assembly of these oligonucleotides are subjected to computer analysis in relation to the secondary structure. Sequences of the four oligonucleotides RVH1-RVH4 presented in Sequence ID 57-60. These oligonucleotides have a length of from 119 to 144 basis and contain 25-26 p. N. overlapping areas. Among these oligonucleic is 1 (Sequence ID 57) and RVH3 (Sequence ID 59) have antisense sequence. The method of Assembly of these four oligonucleotides by PCR is shown in Fig.5.

The PCR mixture (98 μl) containing 100 ng each of the four oligonucleotides and 5 Ed. Ampli Taq first denatured by heating at 94oC for 2 min, and then perform 2 cycles of incubation with 94oC for 2 min, 55oC for 2 min and 72oC for 2 min. After 100 pmol each of RHP1 (Sequence ID 61) and RHP2 (Sequence ID 62) add as external primers, PCR vials cover 50 ml of mineral oil. Then first perform denaturation by heating at 94oC for 1 min, and followed by 38 cycles consisting of 94oC for 1 min, 55oC for 1 min and 72oC for 1 min, and then incubated at 72oC for 10 min.

The DNA fragment 438 p. N. cleaned using a 1.5% agarose gel with a low melting point, split BamHI and HindIII, and then clone in HEF expressing vector HEF-VH-g1. After determining the nucleotide sequence of the plasmid that contains the DNA fragment that encodes the amino acid sequence of the correct V-phase H-chain, called HEF-RVHa-AHM-g1. Amino acid sequence and n is the ID sequences 11.

Each of the options b, C, d, and e V-phase H-chain reconstructed human anti-NM 1.24 antibody construct as follows.

Using the mutagenesis primers BS (Sequence ID 63) and VA (Sequence ID 64), designed to implement the mutation of the arginine at position 66 of lysine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, PCR option b amplified receiving plasmid HEF-RVHb-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHb-AHM-g1, presented in Sequence ID 12.

Using the mutagenesis primers CS (Sequence ID 65) and SA (Sequence ID 66), designed to implement the mutation of threonine at position 73 for lysine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, PCR variant with amplified receiving plasmid HEF-RVHc-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHc-AHM-g1, presented in Sequence ID 13.

Using the mutagenesis primers DS (Sequence ID 67) and DA (Sequence ID 68), designed to implement the mutation of arginine is m PCR option d amplified getting plasmid HEF-RVHd-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHd-AHM-g1, presented in Sequence ID 14.

Using the mutagenesis primers ES (Sequence ID 69) and EA (Sequence ID 70), designed to implement the mutation of valine at position 67 on alanine and methionine in position 69 for leucine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, option e amplified receiving plasmid HEF-RVHe-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHe-AHM-g1, presented in Sequence ID 15.

3-2. Design of hybrid V-phase H-chain

Design two hybrid V-phase N-chain. One is a mouse/human hybrid anti-NM 1.24 antibody in which amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody and the other is a human/mouse hybrid anti-NM 1.24 antibody in which amino acid sequences of FR1 and FR2 received FR4 derived from murine anti-NM 1.24 antibody. All amino acid sequences of CDR plots derived from the murine anti-NM 1.24 antibody.

PCR construct two hybrid V-phase N-chain. This method is schematically shown in Fig.6 and 7. To construct two hybrid V-parcel N-circuit using four primer. External primers (Sequence ID 71) and h (Sequence ID 72) is designed in such a way that they hybridizations with DNA sequence HEF expressing vector HEF-VH-g1. Primer HYS hybrid construction H-chain (Sequence ID 73) is designed so that it contains semantic DNA sequence, and the hybrid primer H-chain HYA (Sequence ID No. 74) has the antisense DNA sequence, so that these DNA sequences complementary to each other.

To construct a hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody and the sequence of FR3 and FR4 of options and V-phase H-chain reconstructed human anti-NM 1.24 antibody, carry out PCR using as template a plasmid F-1.24 H-g1, external primer a, and the hybrid primer H-chain HYA, and PCR, using as Matelot ID 72), carry out the first stage of PCR, and each PCR product clean. Two products of the first PCR allow the Assembly due to self-complementarity (publication of the international application WO 92-19759).

Then, by adding the external primers (Sequence ID 71) and h (Sequence ID 72) in the second stage PCR amplified full length DNA encoding the hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody.

To construct a hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody and the sequence of FR3 and FR4 of the murine anti-NM 1.24 antibody, conduct PCR, using as template the plasmid HEF-RVHa-AHM-g1, external primer a, and the hybrid primer HYA N-chain, and PCR using the plasmid HEF-1.24 H-g1 as a matrix, hybrid primer HYS H-chain, and external primer h, carry out the first stage of PCR, and each PCR product clean. The two purified PCR products give V).

Then, by adding the external primers a and h, the full length DNA encoding the hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody and the sequence of FR3 and FR4 of the murine anti-NM 1.24 antibody, amplified in the second stage PCR.

Ways of implementation of the first PCR, purification of PCR products, Assembly, implementation of the second PCR and cloned into the HEF vector HEF-VH-g1 is carried out in accordance with the methods provided in example 9. "Construction of the V-phase L-chain reconstructed human anti-NM 1.24 antibody".

After determining the DNA sequence of a plasmid which contains a DNA fragment encoding the correct amino acid sequence of the hybrid V-phase N-chain in which the amino acid sequences of FR1 and FR2 derived from murine anti-NM 1.24 antibody and the sequence of FR3 and FR4 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody, called HEF-MH-RVH-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-MH-RVH-AHM-g1, presented in Ponovo V-phase H-chain, in which the amino acid sequences of FR1 and FR2 derived from options and V-phase H-chain reconstructed human anti-NM 1.24 antibody and the sequence of FR3 and FR4 of the murine anti-NM 1.24 antibody, called HEF-HM-RVH-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-MH-RVH-AHM-g1, presented in Sequence ID 76.

3-3. Design options f-s V-phase H-chain reconstructed human anti-NM 1.24 antibody

Each of the options f, g, h, i, j, k, 1, m, n, o, p, q, r, and s V-phase H-chain reconstructed human anti-NM 1.24 antibody construct as follows.

Using the mutagenesis primers FS (Sequence ID 78) and FA (Sequence ID 79), designed to implement the mutation of threonine at position 75 on serine and valine at position 78 to alanine, and as a matrix DNA the plasmid HEF-RVHe-AHM-g1, using PCR amplified variant f, obtaining the plasmid HEF-RVHf-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHf-AHM-g1, presented in Sequence ID 16.

Using the mutagenesis primers GS (Polozenie 40 arginine, as a matrix DNA the plasmid HEF-RVHa-AHM-g1, option g amplified receiving plasmid HEF-RVHg-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHg-AHM-g1, presented in Sequence ID 17.

Using the mutagenesis primers FS (Sequence ID 78) and FA (Sequence ID 79), and as a matrix DNA the plasmid HEF-RVHb-AHM-g1, amplified version h, obtaining the plasmid HEF-RVHh-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHh-AHM-g1, presented in Sequence ID 18.

Using the mutagenesis primers IS (Sequence ID 82) and IA (Sequence ID 83), designed to implement the mutation of the arginine at position 83 by alanine and serine at position 84 on phenylalanine, and as a matrix DNA the plasmid HEF-RVHh-AHM-g1, variant i amplified receiving plasmid HEF-RVHi-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHi-AHM-g1, presented in Sequence ID 19.

Using the mutagenesis primers JS (Sequence ID 84) and JA (Posledovatelnostj lamido HEF-RVHf-AHM-g1, option j amplified receiving plasmid HEF-RVHj-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHj-AHM-g1, presented in Sequence ID 20.

Using the mutagenesis primers S (Sequence ID 86) and KA (Sequence ID No. 87), designed to implement the mutation of glutamic acid at position 81 glutamine, and as a matrix DNA the plasmid HEF-RVHh-AHM-g1, option k amplified receiving plasmid HEF-RVHk-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHk-AHM-g1, presented in Sequence ID 21.

Using the mutagenesis primers LS (Sequence ID 88) and LA (Sequence ID 89), designed to implement the mutation of glutamic acid at position 81 glutamine and serine at position 82B for isoleucine, and as a matrix DNA the plasmid HEF-RVHh-AHM-g1, option 1 amplified receiving plasmid HEF-RVHI-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVH1-ASM-g1, presented in Sequence ID 22.

Using PR is of mutation of glutamic acid at position 81 glutamine, serine at position 82B for isoleucine, and threonine at position 87 on serine, and as a matrix DNA the plasmid HEF-RVHh-AHM m option amplified receiving plasmid HEF-RVHm-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHm-AHM-g1, presented in Sequence ID 23.

Using the mutagenesis primers NS (Sequence ID 92) and NA (Sequence ID 93), designed to implement the mutation of serine at position 82B for isoleucine, and as a matrix DNA the plasmid HEF-RVHh-AHM-g1, a variant of the n amplified receiving plasmid HEF-RVHn-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHn-AHM-g1, presented in Sequence ID 24.

Using the mutagenesis primers OS (Sequence ID 94) and OA (Sequence ID 95), designed to implement the mutation of threonine at position 87 in the series, and as a matrix DNA the plasmid HEF-RVHh-AHM-g1, a variant of amplified receiving plasmid HEF-RVHo-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHo-AHM-g1, presented in Polnost ID 97), designed to implement the mutation of valine at position 78 to alanine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, option p amplified by PCR, obtaining the plasmid HEF-RVHp-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHp-AHM-g1, presented in Sequence ID 26.

Using the mutagenesis primers QS (Sequence ID 98) and QA (Sequence ID 99), designed to implement the mutation of threonine at position 75 on serine, and as a matrix DNA the plasmid HEF-RVHa-AHM-g1, option q amplified by PCR, obtaining the plasmid HEF-RVHq-AHM-g1. Amino acid sequence and nucleotide

sequence V-phase H-chain contained in this plasmid HEF-RVHq-AHM-g1, presented in Sequence ID 27.

Using the mutagenesis primers CS (Sequence ID 65) and SA (Sequence ID 66), and as a matrix DNA the plasmid HEF-RVHp-AHM-g1, option r amplificare have PCR, obtaining the plasmid HEF-RVHr-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHr-AHM-g1, presented in Sequence ID 28.

mutagenesis, using PCR. The primers of mutagenesis SS (Sequence ID 100) and SA (Sequence ID 101) designed to implement the mutation of the methionine in position 69 for isoleucine.

After the above primer amplified using as template the plasmid HEF-RVHr-AHM-g1, the final product was then purified, digested BamHI and HindIII, and the resulting DNA fragment clone in HEF expressing vector HEF-VH-g1, obtaining the plasmid HEF-RVHs-AHM-g1. Amino acid sequence and nucleotide sequence of the V-phase H-chain contained in this plasmid HEF-RVHs-AHM-g1, presented in Sequence ID 102.

Lots encoding the variable area of each of the above plasmids HEF-RVLa-AHM-g and HEF-RVHr-AHM-g1, split to obtain restriction fragments by restriction enzymes HindIII and BamHI.

They build into HindIII and BamHI sites of plasmid vector pUC19. Each plasmid is called pUC19-RVLa-AHM-g and pUCl 9-RVHr-AHM-g1.

Eschericia coli, which contain each of the plasmids pUC19-RVLa-AHM-g and pUC19-RVHr-AHM-g1, referred to as Eschericia coli DH5 (pUC19-RVLa-AHM-g) and Eschericia coli DH5 (pUC19-RVHr-AHM-g1), respectively, and they were international deposited on 29 August 1996, the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) CLASS="ptx2">

Lots encoding variable plot above plasmids HEF-RVHs-AHM-g1, split to obtain restriction fragments by restriction enzymes HindIII and BamHI. They build into HindIII and BamHI sites of plasmid vector pUC19. The resulting plasmid is called pUC19-RVHs-AHM-g1.

Eschericia coli, which contains the plasmid pUC19-RVHs-AHM-g1, referred to as Eschericia coli DH5 (pUC19-RVHs-AHM-g1), and it was international deposited on 29 September 1997, the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under the registration number FERM BP-6127, in accordance with the Budapest Treaty.

4. The design of reconstructed human anti-NM 1.24 antibody, chimeric anti-1.24 NM antibodies and hybrid antibodies with N-capio

To assess each of the chains reconstructed human anti-1.24 NM antibodies provide an opportunity to be expressed reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody as a positive control. When designing each of the options b and next V-phase H-chain reconstructed human anti-NM 1.24 antibody H chain hybrid antibodies provide an opportunity to be expressed in order to determine the chimeric H-chain, in order to evaluate the option and the L-chain reconstructed human anti-NM 1.24 antibody.

4-1. Expression of reconstructed human anti-NM 1.24 antibody

10 μg of each of expressing vector (HEF-RVHa-AHM-g1 to HEF-RVHr-AHM-g1 for N-chain reconstructed human anti-NM 1.24 antibody, and expressing the vector (F-RVLa-ASM-g or HEF-RVLb-ASM-g for L-chain reconstructed human anti-NM 1.24 antibody cotransporter in COS-7 cells using electroporation using the apparatus Gene Pulser (manufactured by BioRad). Each DNA (10 μg) are added to 0.8 ml of the aliquot 1107cells/ml in PBS, and treated with pulses at 1500 V and a capacitance of 25 μf.

After a 10 minute recovery period at room temperature and subjected to electroporation of cells added to 30 ml of DMEM culture medium (manufactured by GIBCO) containing 10% serum amniotic calf without gamma-globulin. After 72 h incubation in the incubator CO2BNA120D (manufactured by TABAI) in terms of the 37oC and 5% CO2the supernatant culture fluid harvested fragments of cells removed by centrifugation at 1000 rpm for 5 min in a centrifuge 15PR-22 (manufactured by HITACHI) equipped with centrifuge rotor 03 (manufactured by HITACHI), and Mitel BECKMAN), equipped with a centrifuge rotor JA-20.1 (manufactured by BECKMAN), and used for cell-ELISA.

Expression of reconstructed human anti-NM 1.24 antibody (2)

10 μg of each of expressing vector (HEF-RVHs-AHM-g1) for option "s" H-chain reconstructed human anti-NM 1.24 antibody, and expressing the vector (HEF-RVLa-AHM-g) for the L-chain reconstructed human anti-NM 1.24 antibody cotransporter in COS-7 cells using electroporation using the apparatus Gene Pulser (manufactured by BioRad). Each DNA (10 μg) are added to 0.8 ml of the aliquot 1107cells/ml in PBS and treated with pulses at 1500 V and a capacitance of 25 μf.

After a 10 minute recovery period at room temperature, and subjected to electroporation of cells added to 30 ml of DMEM culture medium (manufactured by GIBCO) containing 10% serum amniotic calf without gamma-globulin. After 72 h incubation in the incubator CO2BNA120D (manufactured by TABAI) in terms of the 37oC and 5% CO2the supernatant culture fluid harvested fragments of cells removed by centrifugation at 1000 rpm for 5 min in a centrifuge 05PR-22 (manufactured by HITACHI) equipped with centrifuge rotor 03 (manufactured by HITACHI), and microconcentration BECKMAN), equipped with a centrifuge rotor JA-20.1 (manufactured by BECKMAN), and sterilized by filtration using a filter Millex GV13mm (manufactured by Millipore) and used for cell-ELISA.

4-2. Expression of chimeric anti-NM 1.24 antibody

Using 10 μg of each of ekspressiruyushchikh vectors F-1.24 H-g1 for N-chain chimeric anti-NM 1.24 antibody and expressing the vector HEF-1.24 L-g L-chain chimeric anti-NM 1.24 antibody, chimeric anti-NM 1.24 antibody, which should be used for cell-ELISA, prepared in accordance with the above method for the expression of reconstructed human anti-NM 1.24 antibody.

4-3. Expression of anti-NM 1.24 antibody comprising a humanized L-chain, and chimeric H-chain

Using 10 μg of each of ekspressiruyushchikh vector HEF-1.24 H-g1 for N-chain chimeric anti-NM 1.24 antibody and expressing the vector HEF-RVLa-AHM-g option and the L-chain reconstructed human anti-NM 1.24 antibody, anti-NM 1.24 antibody, which comprises a humanized L-chain, and chimeric H-chain, which should be used for cell-ELISA are in accordance with the above method for the expression of reconstructed human anti-NM 1.24 antibody.

AHM-g1 or HEF-HM-RVH-AHM-g1) to V-phase hybrid H-chain and expressing vector HEF-RVLa-AHM-g for L-chain reconstructed human anti-NM 1.24 antibody the hybrid antibody H-chain, which should be used for cell-ELISA are in accordance with the above method for the expression of reconstructed human anti-NM 1.24 antibody.

4-5. Determining the concentration of antibodies

The concentration of the obtained antibodies was determined by ELISA. Each of the holes 96-hole tablet ELISA (isorp, manufacturer NUNC) immobilized by adding 100 μg of goat anti-human IgG antibody (manufactured by BIO SOURCE) prepared at a concentration of 1 μg/ml in covering buffer (0.1 M Panso3, of 0.02% NaN3pH to 9.6) and incubated at room temperature for 1 h After blocking, 100 μl of dilution buffer (50 mm Tris-HCl, 1 mm MgCl2, 0.15 M NaC1, 0.05% of Tween 20, 0.02% of NaN3, 1% bovine serum albumin (BSA), pH of 8.1), 100 μl of each of the dilutions of the culture supernatant of cells COS-7 secreting reconstructed human anti-NM 1.24 antibody, chimeric anti-NM 1.24 antibody or hybrid antibody H-chain, which were concentrated by ultrafiltration, add to each well and incubated at room temperature for one hour. Then after washing add 100 ál of goat antibodies against human IgG, macandog 1 h and washing add 100 ál of substrate solution in a concentration of 1 µg/ml (Sigma 104, p-nitrophenylphosphate, SIGMA) dissolved in substrate buffer (50 mm Panso3, 10 mm MgCl2pH 9,8), and then measure the absorption at 405 nm using a MICROPLATE reader READER Model 3550 (manufactured by BioRad). As a standard for measuring concentrations using human IgG1 (the manufacturer of The Binding Site).

5. The establishment of a Cho cell line that stably produces a reconstructed human anti-NM 1.24 antibody.

5-1. Designing expressing vector for N-chain reconstructed human anti-NM 1.24 antibody

Splitting plasmid HEF-RVHr-AHM-g1 enzymes Pvul and BamHI, the fragment of about 2.8, etc., ad containing DNA encoding the promoter EFI V-phase H-chain reconstructed human anti-NM 1.24 antibody, allocate, using a 1.5% agarose gel with a low melting point. Then, the above DNA fragment is inserted into the approximately 6 T. p. N. fragment, which is obtained by splitting expressing vector used for expressing the vector in human H-chain, DHFR-E-RVh-PM1f (publication of the international application WO 92-19759) containing the DHFR gene and the gene encoding the constant area of the human H-chain, with Pvul and BamHI to construct expresser the cells SNO

To create a system that consistently produces the reconstructed human anti-NM 1.24 antibody, the genes expressing the above vectors, DHFR-E-HEF-RVHr-AHM-g1 and HEF-RVLa-AHM-g, which had been linearized in the decomposition of Pvul, simultaneously introducing into cells Cho DXB-11 method of electroporation under conditions similar to the above (transfection of the above cells COS-7).

5-3. Gene amplification using MTX

Among cells SNO with the introduced genes only those cells SNO, which were introduced expressing vectors as L-and H-chains, can survive in a-MEM culture medium without nucleoside (manufactured by GIBCO-BRL), to which was added 500 μg/ml G418 (manufactured by GIBCO-BRL) and 10% serum amniotic calf, and it allows you to make a selection. Then 10 nm MTX (manufactured by Sigma) is added to the above culture medium. Among the growing clones, select those clones that produce a reconstructed human anti-NM 1.24 antibody in large quantities. As a result, clone 1, which demonstrates the efficiency of production of about 3 mcg/ml of reconstructed human anti-NM 1.24 antibody, and it is called the cell line producing the reconstructed human is body

Reconstructed anti-NM 1.24 antibody construct as follows. The above cells SNO, which produce a reconstructed human anti-NM 1.24 antibody, cultured for 10 days, using as environment-MEM culture medium without nucleoside (manufactured by GIBCO-BRL), to which was added 500 μg/ml G418 (manufactured by GIBCO-BRL) and 10% serum amniotic calf, using the CO2incubator BNAS120D (manufactured by TABAI) in terms of the 37oC and 5% CO2. 8 and 10 days after the start of cultivation, the culture fluid extract, cell debris removed by centrifugation for 10 minutes at 2000 rpm using a centrifuge RL-500SP (manufacturer Tomy Seico) equipped with a rotor TS-9, and then sterilized by filtration, using a filter with flask (manufactured by FALCON) with a membrane with a hole diameter of 0.45 μm.

After an equal amount of PBS(-) are added to the culture fluid of cells SNO, producing a reconstructed human anti-NM 1.24 antibody, the reconstructed human anti-NM 1.24 antibody is affinity purified using high-speed system for the purification of antibodies ConSep LC100 (manufactured by MILLIPORE) and column Hyper D Protein A (manufactured by Nippon Gaishi) using PBS the EPA in accordance with the attached instructions. pH buervenich factions brought about to 7.4, immediately adding 1 M Tris-model HC1 (pH 8.0), and then carried out using ultrafiltration concentrator with centrifuge Centriprep-10 (manufactured by MILLIPORE), concentrated and substitution on PBS(-), sterile filter using a membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 μm, receiving cleared reconstructed human anti-NM 1.24 antibody. The antibody concentration was measured by absorption at 280 nm, assuming a concentration of 1 mg/ml corresponding to 1.35 OP.

Example 11. The determination of the activity of reconstructed human anti-NM 1.24 antibody

Evaluating the binding activity of the antigen and activity of inhibiting the binding of reconstructed human anti-NM 1.24 antibody.

1. The method of measuring the activity of binding antigen and activity of inhibiting the binding

1-1. Measurement of the activity of the antigen binding

The binding activity of the antigen is measured by cell ELISA using WISH cells. Tablets, cell ELISA prepared as described above in example 7.1-2.

After blocking in each well add 100 µl of serial dilutions of the reconstructed chelovecheskogo selected from the culture supernatant of cells SNO. After 2 h incubation at room temperature and washing add rabbit antibody against human IgG labeled with peroxidase (manufactured by DAKO). After incubation for 1 h at room temperature and rinsing each well add 100 ál of substrate solution. After incubation the reaction is stopped with 50 μl of 6 n sulfuric acid, and the absorbance at 490 nm was measured using MICROPLATE reader READER Model 3550 (manufactured by BioRad).

1-2. Measurement of the activity of inhibiting the binding

The activity of inhibiting the binding of mouse anti-NM 1.24 antibody labeled with Biotin, measured by cell ELISA using WISH cells. Cell ELISA prepared as described above in example 7.1-2. After blocking in each well add 50 µl of serial dilutions of the reconstructed human anti-NM 1.24 antibody, which was obtained from the concentrate of the culture supernatant of cells COS-7, or selected from the culture supernatant of cells SNO, and simultaneously add mouse anti-NM 1.24 antibody labeled with Biotin. After 2 h incubation at room temperature and washing, add peroxidase labeled streptavidin (manufactured by DAKO). After inkubirovanie the Le incubation the reaction is stopped with 50 μl of 6 N. sulfuric acid, and the absorption at 490 nm was measured using MICROPLATE reader READER Model 3550 (manufactured by BioRad).

2. Evaluation of reconstructed human anti-NM 1.24 antibody

2-1. L-chain

Option a L-chain reconstructed human anti-NM 1.24 antibody appreciate, as specified for measuring the activity of binding antigen. As is shown in Fig.8, if option a L-chain is expressed in combination with a chimeric H-chain, it demonstrates a similar level of activity of binding antigen. However, subject to further improve the activity and compatibility with H-chain was designed variant b L-chain. Options a and b L-chain were evaluated together to determine the binding activity of the antigen and activity of inhibiting the binding when combined with options a, b, f, or h N-chain. As can be seen in Fig.9, 10, 11 and 12 option a L-chain has a higher activity than option b in respect of both activities for all alternatives a, b, f and h N-chain. Therefore, for the following experiments we used a variant of the L-chain reconstructed human anti-NM 1.24 antibody.

2-2. Options a-e N-chain

Options a-e N-chain reconstructed human anti-NM 1.24 antibody was evaluated in socetanny. The results show, as seen in Fig.11, 13, 14 and 15 that all options were weaker against both activities compared with chimeric anti-NM 1.24 antibody, which suggests the need for further substitution of amino acids.

2-3. The hybrid antibody H-chain

The hybrid antibody H-chain evaluated as described for measuring the activity of binding antigen. The results, as seen in Fig.16, show that the human/murine hybrid anti-NM 1.24 antibody shows activity similar to the activity of chimeric anti-NM 1.24 antibody against the binding activity of the antigen, whereas murine/human hybrid anti-NM 1.24 antibody has weaker activity than chimeric anti-NM 1.24 antibody. This indicates that for constructing a reconstructed human anti-NM 1.24 antibody having activity of binding antigen, similar to the activity of chimeric anti-NM 1.24 antibody, we need to convert the amino acids included in FR3 or FR4, among those contained in the V-block H-chain.

2-4. Options f-r H-chain

Options f N-chain reconstructed human anti-NM 1.24 antibody appreciate, as specified for measuring actinogen reduced in comparison with the activity of chimeric anti-NM, 1.24 antibody, but increased compared to the above options, which suggests that any of the four amino acids at position 67, 69, 75 and 78, which have been newly developed in this version, responsible for the activity of reconstructed human antibodies.

Option g H-chain reconstructed human anti-NM 1.24 antibody appreciate, as specified for measuring the activity of binding antigen. The result, as shown in Fig.18 and 19 shows that this option demonstrates the level of activity at most the same level of activity above option and that implies that, as shown previously for the H-chain of human/murine hybrid antibodies, the amino acid at position 40, which was converted in this embodiment, is not responsible for the activity of reconstructed human antibodies.

Options h-j H-chain reconstructed human anti-NM 1.24 antibody appreciate, as indicated for activity measurement of antigen binding activity and inhibition of binding. The result, as shown in Fig.20, 21, 22 and 23, indicates that all options were weaker against both activity compared with the activity of chimeric anti-NM 1.24 years 67 and 69 among the four newly modified amino acids in the variant f, not responsible for the increased activity of the reconstructed human antibodies.

Options k-p N-chain reconstructed human anti-NM 1.24 antibody appreciate, as indicated for activity measurement of antigen binding activity and inhibition of binding. The result, as shown in Fig.24, 25, 26 and 27 indicates that all options were weaker against both activity compared with the activity of chimeric anti-NM 1.24 antibody and were similar to the activities of the above options h, suggesting that the amino acid at position 80 and then after those that have been newly developed in these six ways, is not responsible for the increased activity of the reconstructed human antibodies.

Option q N-chain reconstructed human anti-NM 1.24 antibody appreciate, as indicated for activity measurement of antigen binding activity and inhibition of binding. The result, as shown in Fig.25 and 27, indicates that this option was weaker against both activities compared with the activities of the above options h or variant R, and was similar to the activity above option and that assumes that the substitution of amino acids in p is Ariant r N-chain reconstructed human anti-NM 1.24 antibody appreciate the above. The result, as shown in Fig.15 and 28 indicates that the variant r has the activity levels of binding of antigen and activity of inhibiting the binding, similar to the activities of chimeric anti-NM 1.24 antibody.

The above results indicate that the minimum transformation necessary to ensure that the reconstructed human anti-NM 1.24 antibody would have the level of activity of binding antigen to a similar level of activity of murine anti-NM 1.24 antibody or chimeric anti-NM 1.24 antibody, is the transformation of the amino acids at positions 30, 71 and 78, and in addition 73.

The binding activity of the antigen and activity of inhibiting the binding for options a-r N-chain reconstructed human anti-1.24 NM antibodies are summarized in table. 6.

2-5. Option s N-chain

Option s N-chain reconstructed human anti-1.24 NM antibodies evaluated in combination with the above option and the L-chain, as indicated for activity measurement of antigen binding activity and inhibition of binding. The result, as shown in Fig.29 and 30 shows that the s option has the activity of binding antigen and activity of inhibiting the binding, similar ako the present invention retains the ability to bind antigen even after as one or more of amino acid residues has been substituted by other amino acids. Accordingly, the present invention includes a reconstructed human anti-NM 1.24 antibody in which amino acid residues have been substituted by other amino acids in the variable section H-chain or L-chain, as long as it retains its original properties.

3. Evaluation of purified reconstructed human anti-NM 1.24 antibody

Purified reconstructed human anti-NM 1.24 antibody assess in respect of the above binding activity of the antigen and activity of inhibiting the binding. The result, as shown in Fig.31 and 32 shows that the reconstructed human anti-NM 1.24 antibody has an activity level of binding of the antigen and activity of inhibiting the binding of a similar level for chimeric anti-NM 1.24 antibody. This fact shows that the reconstructed human anti-NM 1.24 antibody has an activity of binding the same antigen, and mouse anti-NM 1.24 antibody.

Example 12. Antitumor activity of chimeric anti-NM 1.24 antibody against human myeloma model in mice

1. Obtaining antibodies, Podles, the received previously in example 6, concentrated, and buffer solution replace PBS(-) using centrifugal ultrafiltration concentrator Centriprep 10 (manufactured by Amicon). It is sterilized on the filter using a membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 μm. Prepare a concentration of 200 μg/ml, using sterilized filter PBS(-), and used in further experiments. The antibody concentration was measured by absorption at 280 nm and calculated by taking the optical density 1.35 OD corresponding to 1 mg/ml.

1-2. Cleaning the control human IgG

Human IgG, which is going to be used as control for chimeric anti-NM 1.24 antibody, purified as follows. After an equal amount of PBS(-) are added to Either IgG1 purification Kappa (the manufacturer of the BINDING SITE), its affinity purified using high-speed system for the purification of antibodies ConSep LC100 (manufactured by Millipore) and column Hyper D Protein And (the manufacturer Nippon Gaishi) using PBS(-) as the absorbent buffer, and 0.1 M natriciteres buffer (pH 3) as the eluting buffer in accordance with the attached instructions. pH buervenich fractions adjusted to approximately 7.4, immediately adding 1M Tris-model HC1 (pH 8.0), and for atovital Amicon) and replace the buffer in PBS(-). It is sterilized on the filter using a membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 μm. Prepare a concentration of 200 μg/ml, using sterilized filter PBS(-), and used in further experiments. The antibody concentration was measured by absorption at 280 nm and calculated by taking the optical density 1.35 OD corresponding to 1 mg/ml.

2. The method of quantitative determination of human serum IgG in mouse serum

The content of human IgG in mouse serum, quantitatively determined using the following ELISA. 100 μl of goat anti-human IgG, diluted to a concentration of 1 μg/ml in 0.1 M bicarbonate buffer (pH 9,6), placed in a 96-well plate (manufactured by NUNC) and incubated at 4oWith during the night to mobilitat antibody. After blocking add 100 µl of serially diluted mouse serum or human IgG as a standard, and incubated at room temperature for 1 h After washing add 100 ál of 2000-fold diluted anti-human IgG labeled with alkaline phosphatase (manufactured by CAPPEL), and incubated at room temperature for one hour. After wash add substrate solution and the 3. Antitumor activity of chimeric anti-NM 1.24 antibody against mice, which transplanted cells, human myeloma

3-1. The creation of mice transplanted with cells of the myeloma man

Mice transplanted with cells of the myeloma person create the following way. Cells CRM after passage in vivo using mice SCID (from Nichon CLEA) prepared at a concentration of 3107cells/ml in RPMI 1640 medium, supplemented with 10% amniotic calf serum (manufactured by GIBCO BRL). 200 μl of the suspension of the above cells CRM administered by injection into the tail vein of SCID mice (males age 8 weeks from Nichon CLEA), which before was intravenously injected 100 μl antiasian GM1(antiasialo GM1 manufacturer Wako Pure Chemical Industries Co., Ltd.).

3-2. The antibodies

On the 12th day after the transplantation of cells CRM from mice transplanted with cells of the myeloma person receive the serum, and the content of human IgG in the serum quantitatively determined using ELISA as described previously 2. Capture cells CRM in the bone marrow confirmed by increased levels of human IgG in the serum. On 14, 21 and 28 days after transplantation CRM cells in 100 µl of each antibody obtained previously in 1, enter these mice vnutrinomernye cell myeloma man

Antitumor activity of chimeric anti-NM 1.24 antibody judged by the survival period of the mice. As can be seen in Fig.33, mice that were injected chimeric anti-NM 1.24 antibody, demonstrating prolonged survival time compared with mice that were injected control human IgG. Thus, it is confirmed that the chimeric anti-NM 1.24 antibody has antitumor activity against mice transplanted with myeloma cells of a person.

Example 13. The determination of the activity of reconstructed human anti-NM 1.24 antibody

ADCC activity (dependent cellular cytotoxicity antibody) is determined according to the method presented in Current Protocols in Immunology, Chapter 7, Immunologic studies in humans. Editor, John E, Coligan et al., John Wiley & Sons, Inc., 1993).

1. Getting effector cells

Mononuclear cells isolated from peripheral blood of healthy individuals by centrifuging. Thus, an equal amount of PBS(-) are added to the peripheral blood of healthy people, which is deposited on a Ficoll-Plaque PLUS (manufactured by Pharmacia), and centrifuged at 400 g for 40 minutes Mononuclear cell layer is collected and washed four times RPMI 1640 medium, supplemented with 10% serum, amniotic calf (LASS="ptx2">

LAK (Limphokine Activated Killer Cell-lymphokine activated killer cells) induce cells from the bone marrow of SCID mice (breeding Nichon CLEA). Thus, bone marrow cells isolated from the femur of mice and washed twice in RPMI 1640 medium (manufactured by GIBCO BRL), supplemented with 10% amniotic calf serum (manufactured by GIBCO BRL), and are produced when the cell density 2105cells/ml in the same culture medium. Their incubated with 50 ng/ml recombinant human IL-2 (manufactured by R & D SYSTEMS) and 10 ng/ml recombinant murine GM-CSP (manufacturer R & D SYSTEMS) in CO2incubator (manufactured by TABAI) within 7 days. The number of cells was adjusted to 2106cells/ml in the same culture medium.

2. Obtaining target cells

In a cell line of human myeloma CRM (publication of the patent application of Japan without examination (Kokai) 7-236475) or plasmocytoma-leukemic ARH-77 (ATCC CCL-1621) to impose a radioactive label by incubation in RPMI 1640 medium (manufactured by GIBCO BRL), supplemented with 10% amniotic calf serum (manufactured by GIBCO BRL), together with 0.1 µci 51CR-sodium chromate (manufactured by ICN) at 37oC for 60 min After injection of radioactive label the cells washed three times with the same cool is t with a U-shaped bottom (manufactured by Becton Dickinson) add 50 ál 2105target cells/ml, 50 ál of reconstructed human anti-NM 1.24 antibody, mouse anti-NM 1.24 antibody, the control human IgG (manufactured by THE BINDING SITE) or control mouse IgG2a (UPC10, manufacturer CAPPEL), and put together in 4oC for 15 minutes

Then 100 μl of effector cells cultivated in CO2incubator for 4 h, and the ratio (E:T) of effector cells (E) to target cells (T) is 0:1, 3,2:1, 8:1, 20:1 or 50:1.

100 µl of the supernatant is taken and measured the radioactivity released into the culture supernatant, using a count of gamma radiation (ARC-300, the manufacturer Aloka). To measure the maximum radioactivity using 1% NP-40 (manufactured by Nakalai). Cytotoxicity (%) was calculated as (a-C)/(b-C) x 100, where a represents the radioactivity (counts/min) emitted in the presence of antibodies, represents the radioactivity (counts/min) emitted due NP-40, and represents the radioactivity (counts/min) emitted only in culture medium without antibodies.

In Fig.34 shows the result obtained when cells derived from the peripheral blood of a healthy person, used as e the config in case when cells derived from the peripheral blood of a healthy person is used as effector cells, a ARH-77 is used as target cells. If you add a reconstructed human anti-NM 1.24 antibody cytotoxicity increases with increase in concentration compared with the control human IgG, which indicates that the reconstructed human anti-NM 1.24 antibody has ADCC-aktivnosti.

In addition, if you add a reconstructed human anti-NM 1.24 antibody cytotoxicity significantly increased as compared with the murine anti-NM 1.24 antibody, which indicates that the reconstructed human anti-NM 1.24 antibody has higher ADCC activity than the murine anti-NM 1.24 antibody. Moreover, if CRM used as target cells, adding reconstructed human anti-1.24 NM antibody concentration of 0.1 μg/ml or higher does not cause changes in cytotoxicity, indicating that the concentration of 0.1 μg/ml or higher has sufficient ADCC-aktivnosti. If the target cells used ARH-77, adding reconstructed human anti-1.24 NM antibody concentration of 1 μg/ml or higher are not ADCC activity.

In Fig. 36 shows the result obtained when cells derived from the bone marrow of SCID mice are used as effector cells. If you add a reconstructed human anti-NM 1.24 antibody cytotoxicity increases with the concentration of the antibodies, as compared with the control human IgG, which indicates that the reconstructed human anti-NM 1.24 antibody has ADCC-aktivnosti. In addition, adding reconstructed human anti-1.24 NM antibody concentration of 0.1 μg/ml or higher does not cause changes in cytotoxicity, indicating that the concentration of 0.1 μg/ml or higher has sufficient ADCC activity.

These results show that the reconstructed human anti-NM 1.24 antibody has ADCC activity, even if the effector cells obtained from humans or mice.

Example 14. Antitumor activity reconstructed human anti-NM 1.24 antibody against human myeloma model in mice

1. Obtaining intended for the introduction of antibodies

Reconstructed human anti-NM 1.24 antibody, obtained by introducing the plasmid HEF-RVLa-AHM-g and plasmid HEF-R control human IgG, obtained in example 12.1-2, prepared at a concentration of 200 μg/ml, using a sterilized filter PBS(-), which is used as a subject to antibodies.

2. Antitumor activity reconstructed human anti-NM 1.24 antibody against mice transplanted with cells of the myeloma man

2-1. The creation of mice transplanted with cells of the myeloma man

Mice transplanted with cells of the myeloma person receive in accordance with example 12.3-1. Use SCID mice (5 weeks of age, from Nichon CLEA).

2-2. The antibodies

On the 9th day after the transplantation of cells CRM in mice transplanted with cells of the myeloma person received by 2-1, collect the serum, and the amount of human IgG in the serum determined quantitatively using ELISA as described in 12.2. Capture cells CRM on the bone marrow confirmed higher levels of human IgG in the serum. On the 10th day after transplantation of the cells CRM 100 μl of the antibody, prepared as described previously 1, intravenously injected these mice.

2-3. Evaluation of the antitumor action of reconstructed human anti-NM 1.24 antibody against m the tion of human anti-NM 1.24 antibody appreciate the change in the amount of human IgG in mouse serum and the survival period of the mice.

The change in the amount of human IgG in mouse serum to quantify serum collected at 35 days after transplantation CRM cells, determining the content of human IgG using ELISA, as described and example 12.2. The results presented in Fig.37 shows that in the control group, which was administered to human IgG1, the amount of human IgG in the serum at 35 days after transplantation of cells CRM increased about 1000 times in comparison with the quantity on the 9th day (the day before the introduction of antibodies), whereas in the group, which was introduced reconstructed human anti-NM 1.24 antibody, this number was almost equal to or even lower than that on the 9th day, for any dose that indicates what a reconstructed human anti-NM 1.24 antibody inhibits the growth of cells CRM. On the other hand, survival period, as shown in Fig.38, the prolongation is observed for the group introduced a reconstructed human anti-NM 1.24 antibody compared with the group with a control human IgG1. Outlined shows that the reconstructed human anti-NM 1.24 antibody has antitumor activity against mice transplanted with CL the human anti-NM 1.24 antibody and existing drugs melphalan against myeloma person in the model in mice

1. Getting subject to drug administration

1-1. Getting to be antibodies

Reconstructed human anti-NM 1.24 antibody, obtained by introducing the plasmid HEF-RVLa-AHM-g and plasmids HTF-RVHr-AHM-g1 cells SNO prepared at a concentration of 40 and 200 mcg/ml using a sterilized filter PBS(-) and control human IgG obtained in example 12.1-2, prepared at a concentration of 200 μg/ml, using a sterilized filter PBS(-), which is used as a subject to antibodies.

1-2. Getting melphalan

Melphalan (production SIGMA), which is an existing drug for the treatment of myeloma, prepared at a concentration of 0.1 mg/ml using 0.2% of carboxymethylcellulose (CMC) manufacturer Daicel Chemical Industries, Ltd.).

2. Antitumor activity reconstructed human anti-NM 1.24 antibody and melphalan in respect of mice transplanted with cells of the myeloma man

2-1. The creation of mice transplanted with cells of the myeloma man

Mice transplanted with cells of the myeloma person create by way of example 14.2-1.

2-2. Medication

On the 9th day after transplantado, and the amount of human IgG in the serum determined quantitatively using ELISA as described in 12.2. Capture cells CRM on the bone marrow confirmed higher levels of human IgG in the serum. On the 10th day after transplantation of the cells CRM 100 µl of each antibody, prepared as described previously in 1-1, intravenously injected these mice. In addition, 200 μl of 0.2% CMC solution orally administered once a day for 5 days starting from day 10 after transplantation. On the other hand, for the group, which is administered melphalan, mellanby solution, prepared as described previously in 1-2, orally administered in amounts of 100 μl per 10 g of body weight (1 mg/kg per melphalan) once daily for 5 days, starting 10 days after transplantation of cells CRM.

2-3. Evaluation of the antitumor action of reconstructed human anti-NM 1.24 antibody against mice transplanted with cells of the myeloma man

Antitumor activity reconstructed human anti-NM 1.24 antibody appreciate the change in the amount of human IgG in mouse serum and the survival period of the mice.

The change in the amount of human IgG in mouse serum quantitatively obradovo IgG using ELISA, as indicated in example 12.2. The results presented in Fig.39 shows that in the control group, which was administered to human IgG1, the amount of human IgG in the serum at 35 days after transplantation of cells CRM increased about 1000 times in comparison with the number 9 day (the day before the introduction of antibodies), whereas it is seen that cells CRM grew in the body data of mice. In the group which was administered melphalan, also the amount of human IgG in the serum increased a higher level than it was before drug administration, although not as much as in the control group, which was administered to human IgG. This result indicates that the introduction of melphalan not enough inhibits the growth of cells CRM. On the other hand, the amount of human IgG in the serum of the group, which was introduced reconstructed human anti-NM 1.24 antibody, 35 day was less than 9 days after transplantation for all doses, which indicates that the reconstructed human anti-NM 1.24 antibody inhibits the growth of cells CRM.

On the other hand, survival period, as shown in Fig.40, also there is prolongation for the group, which was introduced reconstructed human anti-is then reconstructed human anti-NM 1.24 antibody has an antitumor effect in respect of mice transplanted with cells of the myeloma person, and that antitumor activity of the antibodies of the present invention is stronger than the action of existing drugs melphalan.

The above results show that when using derived from human effector cells, mouse anti-NM 1.24 antibody has low cytotoxicity against myeloma cells, whereas the reconstructed human anti-NM 1.24 antibody and chimeric anti-NM 1.24 antibody have high cytotoxicity. This fact demonstrates the importance of humanizing antibodies, and gives hope for the usefulness of reconstructed human anti-NM 1.24 antibody for people.

Reconstructed human anti-NM 1.24 antibody demonstrates a very strong antitumor activity against human SCID mice transplanted with cells of the myeloma person. As people effector cells receive from people and usually contain lymphocytes, it is expected an even stronger antitumor activity reconstructed human anti-NM 1.24 antibody.

In the model with myeloma reconstructed human anti-NM 1.24 antibody shows strong antitumor activity in comparison with the existing medication and PvE drugs for the treatment of myeloma.

Comparative example 1. The design hybridoma, which produces a murine anti-1.24 NM monoclonal antibody

Hybridoma, which produces a murine anti-1.24 NM monoclonal antibody, get in the way, opened the Goto, T. et al., Blood (1994) 84, 1992-1930.

Plasma cell lines CRS-32, negative nuclear antigen of Epstein-Barr (EBNA) (1107cells) derived from bone marrow of a patient with multiple myeloma (Goto, T. et al., Jpn. J. Clin. Hematol. (1991) 32, 1400) was injected intraperitoneally twice BALB/C mice (from Charles River) every six weeks.

To further assess the titer production of antibodies 1,5106CRS-32 cells injected into the spleen of the mice three days prior to sacrifice of the animals (Goto, T. et al. , Tokushima J. Exp. Med. (1990) 37, 89). After killing spleen extract, and spleen cells removed by way Groth, de St. & Schreidegger (Cancer Research (1981) 41, 3465) are subjected to cell fusion with myeloma cells SP2/0.

Antibodies in hybridoma supernatant culture sceneroot using ELISA (Posner, M. R. et al., J. Immunol. Methods (1982) 48, 23), using the tablets coated with cells of cattle-32. 5104cells CRS-32 is suspended in 50 ml of PBS, and aliquots placed in a 96-well plate (U-shaped bottom. Corning, the manufacturer Iwaki who we are and incubated for 2 h at 4oC. Then react with goat antibody against mouse IgG labeled with peroxidase (manufactured by Zymed) at 4oC for 1 h, once washed and react with the substrate solution of o-phenylenediamine (manufactured by Sumitomo Bakelite) at room temperature for 30 minutes

After reaction stop 2 N. sulfuric acid, measuring the absorption at 492 nm using ELISA reader Reader (manufactured by BioRad). To remove hybridoma, which produces antibody against human immunoglobulin, positive hybridoma supernatant culture fluid pre-adsorb on serum human and sceneroot reactivity against other subcellular components. Positive hybridoma selected, and investigate their reactivity towards different cell lines and human samples using flow cytometry. The chosen hybridoma clones twice clone, injection in the abdominal cavity treated will pristane BALB/c mice, and then they get ascitic fluid.

Monoclonal antibody is purified from mouse ascites by precipitation ammoniumsulfate and affinity chromatography Protein A (Ampure PA, is ugali Quick Tag FITC (manufacturer Boechringer Mannheim).

In the monoclonal antibodies produced 30 hybridoma clones that react with cattle-32 cells and RPMI 8226. After cloning investigate the reactivity of the supernatant fluids of these hybrid with other cell lines and mononuclear cells obtained from peripheral blood.

Among them there are three clones that produce monoclonal antibodies that specifically interact with the plasma cell. Among these three clones selected hybridoma clone that produces a monoclonal antibody which is most suitable for cytometrical analysis in the stream and which has the complement-dependent cytotoxicity against cells RPUI 8226, and called 1.24 NM. The subclass of the monoclonal antibody producing this hybridoma, determined by ELISA, using subclass-specific rabbit antimurine antibody (manufactured by Zymed). Anti-NM 1.24 antibody refers to the IgG2a subclass. Hybridoma, which produces anti-NM 1.24 antibody was international deposited on September 14, 1995 the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under registration number FERM BP-5233 in accordance with the Budapest is/BR> 1. Construction of cDNA library

1) Obtaining total RNA

cDNA, which encodes NM 1.24 antigen, which is a polypeptide specifically recognized by mouse anti-1.24 NM monoclonal antibody allocate as follows.

From human cell lines scattered myeloma CRM total RNA receive according to the method of Chirgwin et al. (Biochemistry, 18, 5294 (1979)). So, 2,2108CRM cells completely homogenized in 20 ml of 4 M guanidinoacetate (manufactured by Nakalai tesque).

The homogenate was applied to the layer of 5.3 M cesium chloride in a centrifuge tube and then centrifuged at 31000 rpm at 20oC for 24 h to precipitate RNA, using the rotor Beckman SW40. The precipitated RNA was washed with 70% ethanol and dissolved in 300 μl of 10 mm Tris-model HC1 (pH 7.4) containing 1 mm EDTA and 0.5% VAT. After adding to this pronase (manufactured by Boehringer) to a concentration of 0.5 mg/ml all incubated at 37oC for 30 minutes the mixture is extracted with phenol and chloroform to precipitate RNA. Then the precipitated RNA was dissolved in 200 μl 10 mm Tris-model HC1 (pH 7.4) containing 1 mm EDTA.

2) Obtaining a poly(A)+RNA

Using 500 µg total RNA obtained as described above as a raw material, produce poly(A)+RNA using the set for konstruirovanie cDNA library

Using 10 μg of the above poly(A)+RNA as raw material, synthesize double-stranded cDNA using the kit for cDNA synthesis TimeSaver cDNA Synthesis Kit (manufactured by Pharmacia) according to the manufacturer's instructions that came with the set, and using Directional Cloning Toolbox (manufactured by Pharmacia), it attributed the EcoRI adaptor in accordance with the manufacturer's instructions attached to the kit. Cinelu and treated with restriction enzyme NotI EcoRI adapter is carried out in accordance with the instructions attached to the kit. Next, double-stranded cDNA with attached adapter size of about 500 p. N. or above is isolated and purified using a 1.5% agarose gel with a low melting point (production SIGMA), receiving about 40 μl of double-stranded cDNA with attached adapter.

Thus obtained double-stranded cDNA with attached adapter associated with vector pCOS1 (publication of the patent application of Japan without examination (Kokai) 8-255196), who had previously been treated with restriction enzymes EcoRI and NotI and alkaline phosphatase (manufactured by Takara Shuzo) using T4 for constructing DNA ligase (manufactured by GIBCO BRL) to construct a cDNA library. The constructed cDNA library transducer in Eschericia coli strain D.

2. Cloning due to direct expression

1) Transfection of cells COS-7

cDNA amplified cultivating about 5105clones above transductional Eschericia coli in 2-YT medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)) containing 50 μg/ml ampicillin, and plasmid DNA isolated from Eschericia coli alkaline method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989)). The obtained plasmid DNA transferout cells COS-7, using the apparatus Gene Pulser (manufactured by BioRad).

Then 10 μg of purified plasmid DNA is added to 0.8 ml of cells COS-7, which are suspended in PBS at a concentration of 1107cells/ml and treated with pulses at 1500 V and a capacitance of 25 μf. After a 10 minute recovery period at room temperature and subjected to electroporation cells were cultured in DMEM (manufactured by GIBCO BRL), supplemented with 10% serum, amniotic calf in the conditions of the 37oC and 5% CO2within three days.

2) Preparation of the cell for penning

Cuvette for penninga coated with mouse anti-NM 1.24 antibody prepared according to the method of C. Seed et al. (Proc. Natl. Acad. Sci. USA, 84, 3365-3369 (1987)). So, mouse anti-NM 1.24 antibody is added to 50 mm Tris-HCl, pH 9,5, to a concentration of 10 μg/ml. Three ml dissolve the at room temperature for 2 hours After triple washing 0.15 M NaC1 solution and blocking PBS containing 5% amniotic calf serum, 1 mm EDTA and 0.02% NaN3these plates are used for subsequent cloning.

3) Cloning of cDNA-libraries

Cells COS-7, transfetsirovannyh as stated above, free with PBS containing 5 mm EDTA, and then washed once with PBS containing 5% serum amniotic calf. Then these cells are suspended in PBS containing 5% serum amniotic calf of 0.02% NaN3concentrations up to about 1106cells/ml, which are then added to the cuvette for penninga, prepared as above, and incubated at room temperature for 2 hours After three careful washing PBS containing 5% amniotic calf serum, and 0.2% NaN3, plasmid DNA isolated from the cells associated with the cuvette for panning using a solution containing 0,6% VAT and 10 mm EDTA.

The selected plasmid DNA again transducer in schericia coli strain DH5. After amplification of plasmid DNA, as described above, it secrete alkaline method. The selected plasmid DNA transferout cells COS-7 method of electroporation and plasmid DNA isolated from the associated cells, as described above. This is the result confirms the concentration of inserts with a size of about 0.9, etc., N. Eschericia coli, transductional with a part selected plasmid DNA inoculant in agrarian plate 2-YT containing 50 μg/ml ampicillin. After culturing overnight plasmid DNA isolated from the same colony. Her break down the enzymes EcoRI and NotI, and get a clone of R. 3.19, containing an insert of 0.9 T. p. N.

The nucleotide sequence of this clone is determined in reactions using a set of PRISM Dye Terminator Cycle Sequencing Kit (manufactured Perkin Elmer) in accordance with the attached to the kit instructions, and carry out sequencing using an ABI sequencing machine A DNA Sequencer (manufactured Perkin Elmer). Amino acid sequence and the sequence of bases is presented in Sequence ID 103.

cDNA encoding the polypeptide with the amino acid sequence presented in the sequence ID 103 is inserted into the XbaI cleavage site of the vector pUC19 and receive in the form of plasmids pRS38-pUC19. Eschericia coli containing this plasmid pRS38-pUC19, was international deposited on 5 October 1993, as Eschericia coli DH5 (pRS38-pUC19), the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) as under the registration number FERM BP-4434 in accordance with the Budapest agreement (see the how to chimeric anti-NM 1.24 antibody consists of the variable segment of mouse anti-NM 1.24 antibody and the constant area of the human antibody, and reconstructed human anti-NM 1.24 antibody consists of a plot, mouse anti-NM 1.24 antibody complementarity determining, frame area of the human antibody and a constant area of the human antibodies, it has a weak antigenicity against humans, and therefore, as expected, can be used as pharmaceutical compositions, particularly in the treatment of myeloma.

1. V-phase reconstructed human L-chain anti-NM.24 antibodies (FERM BP-5233), including alternately spaced frame sections (FR) V-area of the human L-chain and the CDR of the V-phase L-chain anti-NM.24 antibodies (FERM BP-5233) and presented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where CDR1, CDR2 and CDR3 have amino acid sequences shown in SEQ ID NO : 3, 4 and 5, respectively.

2. V-phase reconstructed human L-chain anti-NM.24 antibodies (FERM BP-5233) under item 1, where the specified FR derived from the human subgroup 1 (HSG1).

3. V-phase reconstructed human L-chain anti-NM.24 antibodies (FERM BP-5233) under item 1, where the specified FR obtained from REI.

4. V-phase reconstructed human L-chain anti-NM.24 antibodies (FERM BP-5233) where the specified V-phase contains amino acid Posay anti-NM.24 antibodies (FERM BP-5233), includes alternately spaced frame sections (FR) V-area of the human H-chain and the CDR of the V-phase H-chain anti-NM.24 antibodies (FERM BP-5233) and presented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where CDR1, CDR2 and CDR3 have amino acid sequences shown in SEQ ID NO : 6, 7 and 8 respectively.

6. V-phase reconstructed human H-chain anti-NM.24 antibodies (FERM BP-5233) under item 5, where the specified FR derived from the human subgroup 1 (HSG1).

7. V-phase reconstructed human H-chain anti-NM.24 antibodies (FERM BP-5233) under item 5, where the specified FR obtained from the HG3 and/or JH6.

8. V-phase reconstructed human H-chain anti-NM.24 antibodies (FERM BP-5233) under item 7, where the amino acid at position 30 is a threonine, the amino acid at position 71 is an alanine, and the amino acid at position 73 is a lysine.

9. V-phase reconstructed human H-chain anti-NM.24 antibodies (FERM BP-5233) where the specified V-phase contains the amino acid sequence represented in SEQ ID NO : 108 or 128.

10. Reconstructed human L-chain, consisting of V-phase reconstructed human L-chain anti-NM.24 antibodies (FERM BP-5233) according to any one of paragraphs. 1-4 and to the ro, consisting of V-phase reconstructed human H-chain anti-NM.24 antibodies (FERM BP-5233) according to any one of paragraphs. 5-9 and constant area of the human gamma-chain (C1-plot).

12. Reconstructed human anti-HM.24 antibody (FERM BP-5233), consisting of a reconstructed human L-chain according to p. 10 and reconstructed human H-chain under item 11.

13. DNA encoding the amino acid sequence of the V-phase reconstructed human L-chain anti-NM.24 antibodies (FERM BP-5233) according to any one of paragraphs. 1-4 and includes the nucleotide sequence shown in SEQ ID NO : 9 or 10.

14. DNA encoding the amino acid sequence of the V-phase reconstructed human H-chain anti-NM.24 antibodies (FERM BP-5233) according to any one of paragraphs. 5-9 and comprising the nucleotide sequence shown in SEQ ID NO : 9-28, 75,76 or 102.

15. Expressing a vector comprising DNA under item 13 or 14, adapted for transformation of a host cell.

16. The method of obtaining a reconstructed human antibodies under item 12, which includes stages of culturing the host cell transformed by expressing vector for p. 15, and the selection of the indicated antibodies.

17. Pharmaceutical 12, which specific recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO : 103.

 

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The invention relates to the field of medicine and relates to a composition for inhibition of angiogenesis, monoclonal antibodies, polypeptide and method of inhibiting tumor growth

The invention relates to biotechnology, in particular to recombinant IL4-antibodies used for treating disorders associated with the activity IL4

The invention relates to a monoclonal antibody having the ability to inhibit homing hematopoietic stem cells and to identify surface antigen stromal cells, having the ability to maintain homing hematopoietic stem cells, as well as to hybridoma producing monoclonal antibody

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8

The invention relates to the field of medicine and biotechnology, namely to new proteins, which factors in the growth and development of megakaryocytes (MGDFs; mostly labeled Mp1-ligands), the biological activity of which is to stimulate the growth of megakaryocytes and their differentiation or maturation, which ultimately leads to the formation of platelets

The invention relates to biotechnology and can be used in the selection of organisms, for example plants

The invention relates to genetic engineering

The invention relates to recombinant DNA, intended in particular for use in genetic engineering of plants

The invention relates to biotechnology

The invention relates to the field of molecular biology, molecular genetics and biotechnology and can be used for the purposes of gene therapy in medicine and in agriculture and in industrial biotechnology for gene-specific silencing of those genes, the expression of which contributes to the development of certain diseases or commits a process of manufacturing a desired product

The invention relates to biotechnology, immunology and medicine and can be used to direct cellular immune response to an infectious agent

The invention relates to biotechnology, in particular genetic engineering, and can be used for typing strains of the plague microbe various natural focal toiletries

The invention relates to new anti-EGFR antibody and single-chain Fv (оцFv) fragments, which can be derived from phage-antibody libraries constructed from cells of the immunized mammal, preferably a mouse
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