Isolated human antibody or its antigen-binding fragment, isolated nucleic acid (variants), recombinant expression vector, using host-cells for expression of antibody and method for its synthesis, pharmaceutical composition and method for inhibition of activity of human tnfα

FIELD: biotechnology, immunology, molecular biology, medicine, pharmacy.

SUBSTANCE: invention describes the isolated human antibody or its antigen-binding fragment able to bind the human tumor necrosis factor (TNF-α). Amino acid sequence is given in the description. Invention discloses nucleic acid encoding heavy and light chain of isolated human antibody. Nucleotide sequences are given in the description. Invention describes recombinant vector expressing variable region of heavy and light chains of isolated human antibody, Chinese hamster ovary cells CHO dhfr- carrying vector. Invention discloses a method for synthesis of isolated human antibody. The isolated human antibody or its antigen-binding fragment can be used as an active component of pharmaceutical composition used in treatment of disturbances when activity of TNF-α is harmful. Using the invention allows neutralization of effect of TNF-α in case when its activity is harmful. Invention can be used in medicine.

EFFECT: valuable medicinal properties of antibody, improved method for synthesis.

17 cl, 11 dwg, 17 tbl, 4 ex

 

The technical field to which the invention relates.

The invention relates to medical pharmacology, and relates, in particular, human antibodies, which specifically bind or neutralize hTNFα and can be used in the manufacture of drugs for treatment of disorders in which the activity of TNFα is harmful.

The level of technology

The tumor necrosis factor α (TNFα) is a cytokine produced by many cell types, including monocytes and macrophages, which was originally identified based on its ability to induce necrosis of some murine tumors (see, for example, Old L. (1985) Science 230:630-632). Subsequently, it was shown that the factor called cachectin associated with cachexia is the same molecule that TNFα. TNFα participated in the generation of the shock (see, for example, Beutler Century and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518; Beutler Century and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655). Moreover, TNFα participated in the pathophysiology of a number of other diseases and disorders of human rights, including sepsis, infections, autoimmune diseases, rejection of the transplant and disease graft versus host disease (see, for example, Moeller, A., et al., (1990) Cytokine 2:162-169; U.S. Patent No 5231024 A. Moeller et al.; European Patent No 260610 B1 A. Moeller et al.; Vasilli P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).

Due to the harmful role of the human is a mini-TNFα (hTNFα) in a number of disorders in humans have been developed therapeutic ways to inhibit or counteract the activity of hTNFα. In particular, antibodies that bind or neutralize hTNFαconsidered as a means of inhibiting the activity of hTNFα. One of the earliest of these antibodies were mouse monoclinal antibodies (mAbs), secreted by hybridomas derived from lymphocytes of mice immunized with hTNFα (see, for example, Hahn T. et al. (1985) Proc. Natl. Acad. Sci. USA 82:3814-3818; Liang C-M. et al. (1986) Biochem. Biophys. Res. Commun. 137:847-854; Hirai M. et ai. (1987) J. Immunol. Methods 96:57-62; B.M. Fendly et al.(1987) Hybridoma 6:359-370; A. Moeller et al. (1990) Cytokine 2:162-169; U.S. Patent No 5231024 Moeller et al.; European Patent No 186833 B1 Wallach D.; European Patent Application No 218868 A1 Old et al.; European Patent No 260610 B1 A. Moeller et al.). Since these mouse antibodies against hTNFα often showed high affinity to hTNFα (e.g., Kd≤10-9M) and were able to neutralize the activity of hTNFαtheir application in vivo could limit the problems associated with administration of mouse antibodies to humans, such as short half-life in syvorotke, the failure to include certain functions of effectors of human rights and the appearance of unwanted immune response against murine antibodies in humans (reaction "human antibodies against mouse (HAMA)).

When you try to overcome the problems associated with IP is by the use of a fully murine antibodies in humans, mouse antibodies against hTNFα have been using genetic engineering made "humanoid". For example, were obtained chimeric antibodies in which the variable parts of the chain antibodies are murine in nature, and constant parts of the antibody chains - man (Knight D.M. et al. (1993) Mol. Immunol. 30:1443-1453; PCT Publication No WO 92/16553 P.E. Daddona et al.). In addition, they also received human-like antibodies in which the hypervariable domains of the variable regions of the antibodies are murine nature, but the other variable plots and constant portions of antibodies are human nature (PCT Publication No WO 92/11383 Adair J.R. et al.). However, because these chimeric and humanized antibodies still retain some mouse sequence, they can still cause unwanted immune reaction, human antichimeric antibodies (HACA), especially with the introduction of the long periods, for example, in chronic indications, such as rheumatoid arthritis (see, for example, Elliott M.J. et al. (1994) Lancet 344:1125-1127; Elliott M.J. et al. (1994) Lancet 344:1105-1110).

Preferred inhibitory hTNFα agent against murine mAbs and their derivatives (e.g., chimeric or human-like antibodies) would be a fully human antibody against hTNFαbecause such an agent would not cause reactions HAMA, even if used for a long is erodov. Human monoclonal antibodies against hTNFα were obtained by methods using human hybridoma (P. Boyle et al. (1993) Cell. Immunol. 152:556-568; P. Boyle et al. (1993) Cell. Immunol. 152:569-581; European Patent Application No 614984 A2 Boyle et al.). However, as reported, these monoclonal antibodies hybridoma nature had so low affinity against hTNFαthat it was difficult to compute by known methods, were not able to bind soluble hTNFα and were not able to neutralize hTNFαinduced cytotoxicity (see Boyle et al., supra). Moreover, the success of the method using human hybridoma depends on the natural presence in human peripheral blood lymphocytes that produce antibodies that are specific for hTNFα. In some studies in humans have found serum autoantibodies against hTNFα (Fomsgaard A. et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B:447-452), whereas in other studies they were not detected (Leusch H-G. et al. (1991) J. Immunol. Methods 139:145-147).

Alternative to the existing in vivo human antibodies against hTNFα would be recombinant antibodies against hTNFα. Described recombinant human antibodies that bind hTNFα with relatively low affinity (i.e. Kd˜10-7M) and fast speed, disco is IACEE (i.e. Tooff˜10-2with-1). However, due to the relatively fast kinetics of dissociation of these antibodies may not be applied for therapeutic purposes. In addition, have been described recombinant human antibodies against hTNFαthat does not neutralize the activity of hTNFαbut significantly enhance the binding of hTNFα surface cells and strengthen the implementation in the cytoplasm hTNFα (A. Lidbury et al. (1994) Biotechnol. Ther. 5:27-45; PCT Publication No WO 92/03145 Aston R. et al.).

Accordingly, human antibodies, such as recombinant human antibodies that bind soluble hTNFα with high affinity and low dissociation kinetics and who are able to neutralize the activity of hTNFαincluding hTNFαinduced cytotoxicity (in vitro and in vivo) and hTNFα-induced activation of cells, are still necessary.

The invention

This invention is a human antibodies, preferably recombinant human antibodies, that specifically bind human TNFα. Antibodies corresponding to the invention are characterized by binding hTNFα with high affinity and slow dissociation kinetics and neutralization activity of hTNFαincluding hTNFαinduced cytotoxicity (in vitro and in vivo) and hTNFα-induced activation of the cells. Antibodies which, corresponding to the invention, further characterized by binding hTNFαbut not hTNFβ (lymphotoxin) and have the ability to link, in addition to human TNFαalso factors tumor necrosis (TNFα) other primates and TNFα Narimanov.

Antibodies corresponding to the invention can be full length (for example, antibody lgG1 or lgG4) or can include only an antigen-binding fragment (e.g., Fab fragment, F(ab')2or scFv). The most preferred recombinant antibody corresponding to the invention, denoted by the D2E7 has domain CDR3 light chain containing the amino acid sequence of SEQ ID No 3 and CDR3 domain of the heavy chain containing the amino acid sequence of SEQ ID No 4. Preferably the antibody D2E7 has a variable area light chain (LCVR), containing the amino acid sequence of SEQ ID No 1 and variable plot heavy chain (HCVR), containing the amino acid sequence of SEQ ID No 2.

In one embodiment, the invention is selected human antibody or antigen-binding fragment that bind human TNFα Cd1×10-8M or less, dissociate with rate constant Koff1×10-3with-1or less (both values are determined using surface plasma resonance) and neutral the comfort cytotoxicity of human TNFα in the standard analysis using L929 in vitro with IC501×10-7M or less. More preferably, if the selected human antibody or antigen-binding fragment dissociate from the Association (complex) human TNFα Coffequal to 5×10-4with-1or less, and even more preferably Tooff1×10-4with-1or less. More preferably, if the selected human antibody or antigen-binding fragment neutralize the cytotoxicity of human TNFα in the standard analysis using L929 in vitro with IC501×10-8M or less and even more preferably with IC505×10-10M or less.

In another embodiment, the invention is a human antibody or antigen-binding fragment with the following characteristics:

a) dissociates from hTNFα Coff1×10-3with-1or less than that determined using surface plasma resonance;

b) has domain CDR3 light chain containing the amino acid sequence of SEQ ID No 3 or SEQ ID No 3, modified by replacing the single alanine at position 1, 4, 5, 7, or 8, or replacement of one to five conservative amino acid positions 1, 3, 4, 6, 7, 8 and/or 9;

(C) has a domain CDR3 of the heavy chain that contains linakis is now the sequence of SEQ ID No 4 or SEQ ID No 4, modified by replacement of one alanine in position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or changing from one to five conservative amino acid positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

More preferably, if the antibody or antigen-binding fragment dissociate from human TNFα Coff5×10-4with-1or less. Even more preferably, if the antibody or antigen-binding fragment dissociate from human TNFα Coff1×10-4with-1or less.

In yet another embodiment, the invention is a human antibody or antigen-binding fragment with a LCVR having a CDR3 domain containing the amino acid sequence of SEQ ID No 3 or SEQ ID No 3, modified by replacing the single alanine at position 1, 4, 5, 7, or 8, or HCVR having a CDR3 domain of the heavy chain containing the amino acid sequence of SEQ ID No 4 or SEQ ID No 4, modified by replacing the single alanine in position 2, 3, 4, 5, 6, 8, 9, 10 or 11. More preferably, if the LCVR further has a CDR2 domain containing the amino acid sequence of SEQ ID No 5, and the HCVR further has a CDR2 domain containing the amino acid sequence of SEQ ID No 6. Even more preferably if the LCVR further has CDR1 domain containing the amino acid sequence of SEQ ID No 7, and the HCVR has a CDR1 domain, containing the second amino acid sequence SEQ ID No 8.

In yet another embodiment, the invention is selected human antibody or antigen-binding fragment with LCVR containing the amino acid sequence of SEQ ID No 1, and HCVR containing the amino acid sequence of SEQ ID No 2. In some embodiments, the implementation of the antibody has a constant area lgG1 heavy chain or the constant plot lgG4 heavy chain. In other embodiments, implementation of the antibody is a Fab fragment, a fragment F(ab')2or Fv fragment of the same chain.

In other embodiments implementing the invention provides antibodies or antigen-binding fragments with LCVR having a CDR3 domain containing the amino acid sequence selected from the group consisting of SEQ ID No3, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21, SEQ ID No 22, SEQ ID No 23, SEQ ID No 24, SEQ ID No 25, SEQ ID No 26, or with a HCVR having a CDR3 domain containing the amino acid sequence selected from the group consisting of SEQ ID No 4, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 34, SEQ ID No 35.

In yet another embodiment, the invention is selected human antibody or antigen-binding fragment that neutralizes the activity of human TNFαbut not a human TNFβ (lymphotoxin). In a preferred variant implementation, the value of the human antibody or antigen-binding fragment neutralizes the activity of human TNFα , TNFα chimpanzees and TNFα at least one of priority, is selected from the group consisting of TNFα baboon, TNFα marmoset, TNFα cynomolgus and TNFα rhesus. Preferably, if the antibody also neutralizes the activity of TNFα at least one naramata. For example, in one embodiment, the selected human antibody or antigen-binding fragment neutralizes the activity of canine TNFα. In another embodiment, the selected human antibody or antigen-binding fragment neutralizes the activity of porcine TNFα. In yet another embodiment, the selected human antibody or antigen-binding fragment neutralizes the activity of mouse TNFα.

Another aspect of the invention relates to nucleic acid molecules encoding corresponding to the invention the antibody or antigen-binding fragments. Preferred nucleic acid corresponding to the invention encoding a LCVR D2E7, has the nucleotide sequence presented in Figure 7 and SEQ ID No 36. Another preferred nucleic acid corresponding to the invention encoding a HCVR D2E7, has the nucleotide sequence presented on Figure 8 and SEQ ID No 37. Recombinant expressing vectors bearing encoding antibodies nucleic acids is, corresponding to the invention, and cells of the host, which introduced such vectors, are also covered by the invention, as methods for producing antibodies, corresponding to the invention by cultivating host cells corresponding to the invention.

Another aspect of the invention relates to a method of inhibiting the activity of human TNFα the use of antibodies or antigen-binding fragment corresponding to the invention. In one embodiment, the method involves contacting the human TNFα with the antibody corresponding to the invention, or antigen-binding fragment, while inhibited the activity of human TNFα. In another embodiment, the method involves the introduction of antibodies, corresponding to the invention, or antigen-binding fragment of the person suffering from disorders in which the activity of TNFα is harmful, while in humans, inhibited the activity of TNFα. The disorder can be, for example, sepsis, autoimmune disease (e.g. rheumatoid arthritis, Allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and renal syndrome), infectious disease, cancer, graft rejection or disease graft-versus-host, pulmonary violations is s, bone violation, intestinal disorders or heart disorder.

List of drawings

In Figures 1A and 1B presents the nucleic acid sequences of the variable segment D2E7 light chain (D2E7 VL; also shown in SEQ ID No 1), alanine-sanatani D2E7 VL (LD2E7*.A1, LD2E7*.A3, LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 and LD2E7*.A8), variable area light chain such D2E7 antibodies 2SD4 (2SD4 VL; also shown in SEQ ID No 9) and other variable sites of the light chain, such D2E7 (EP B12, VL10E4, VL1009, VL100D2, VL10F4, LOE5, VLLOF9, VLLOF10, VLLOG7, VLLOG9, VLLOH1, VLLOH10, VL1B7, VL1C1, VL1C7, VL0.1F4, VL0.1H8, LOE7, LOE7.A and LOE7.T). Figure 1A presents the domains FR1, CDR1, FR2 CDR2 and. Figure 1B presents the domains FR3, CDR3 and FR4. The domain light chain CDR1 ("CDR L1"), CDR2 ("CDR L2) and CDR3 ("CDR L3) are in the rectangle.

In Figures 2A and 2B presents the amino acid sequence of the variable segment D2E7 (D2E7 VH; also shown in SEQ ID No 2), alanine-sanatani D2E7 VH (HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and HD2E7*.A9), variable plot heavy chain similar to the D2E7 antibody 2SD4 (2SD4 VH shown in SEQ ID No 10) and other variable sites of the heavy chain, such D2E7 (VH1B11, VH1D8, VH1A11, VH1B12, VH1-D2, VH1E4, VH1F6, VH1G1, 3S-H2, VH1-D2.N and VH1-D2.Y). Figure 2A presents the domains FR1, CDR1, FR2 CDR2 and. Figure 2B presents the domains FR3, CDR3 and FR4. The domains of the heavy chain CDR1 ("CDR H1), CDR2 ("CDR H2) and CDR3 ("CDR H3") are in the rectangle.

Figure 3 graphically presents ingabire is the use of TNFα -induced cytotoxicity of L929 human antibody D2E7 against hTNFα compared to the murine antibody MAK 195 against hTNFα.

Figure 4 graphically presents the inhibition of binding of rhTNFα with receptors hTNFα on the cells U-937 by using human antibody D2E7 against hTNFα no comparison with the murine antibody MAK 195 against DTNFα.

Figure 5 graphically presents the inhibition induced TNFα the expression of ELAM-1 on HUVEC by using human antibody D2E7 against hTNFα compared to the murine antibody MAK 195 against DTNFα.

The Figure 6 presents a graph in the form of columns of protection against TNFα-induced death sensitized D-galactosamine mice by introducing a human antibody against hTNFα D2E7 (black bars) compared to the murine antibody MAK 195 against hTNFα (hatched bars).

The Figure 7 presents the nucleotide sequence of the variable segment D2E7 light chain with the predicted amino acid sequence below the nucleotide sequence. Lots of CDR L1, CDR L2 and CDR L3 underlined.

The Figure 8 presents the nucleotide sequence of the variable segment D2E7 heavy chain with the predicted amino acid sequence below the nucleotide sequence. Participation is key CDR H1, CDR H2 and CDR H3 underlined.

Figure 9 shows a graph representing the effect of treatment with antibody D2E7 transgenic mice TD with an average size of joints as a model of arthritis.

Information confirming the possibility of carrying out the invention

This invention relates to the selected human antibody or antigen-binding fragments that bind TNFα with high affinity, low dissociation rate and high neutralizing activity. Various aspects of the invention relate to antibodies, fragments of antibodies and their pharmaceutical compositions, as well as nucleic acids, recombinant expressing vectors and host cells for making such antibodies and fragments. Uses of antibodies, corresponding to the invention, for detection of human TNFα or for inhibiting the activity of human TNFα either in vitro or in vivo are also covered by the invention.

With the purpose of easier understanding of the invention first defines some of the terms.

The term "human TNFα" (abbreviated here as hTNFα or simply hTNF), as used here, is intended to determine the human cytokine that exists as Sekretareva form with a molecular weight of 17 KD and associated with membanes form with a molecular mass of 26 KD, biologically AK the active form which contains a trimer ecovalence related molecules with a weight of 17 KD. The structure of hTNFα described in, for example, Pennica, D. et al. (1984) Nature 312:724-729; Davis J.M. et al. (1987) Biochemistry 26:1322-1326; Jones DURING et al. (1989) Nature 338:225-228. The term human TNFα involves the incorporation of recombinant human TNFα (rhTNFα), which can be obtained by standard methods of recombinant expression or purchased (R & D Systems, Catalog No. 210-TA, Minneapolis, MN).

The term "antibody", as used here, is intended for the determination of immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains)connected by disulfide bonds. Each heavy chain contains a variable plot heavy chain (abbreviated here as HCVR or VH) and constant plot heavy chain. Stuck-at plot heavy chain contains three domains CN, CH2 and CH3. Each light chain contains a variable area light chain (abbreviated here as LCVR or VL) and a constant area light chain. Constant area light chain contains one domain, CL. Areas of VH and VL can be further subdivided into areas of hypervariability called complementarity determining plots (CDR), surrounded by areas that are more conservative, called skeletal sites (FR). Each VH and VL is composed of three CDRs and four FR plots located from amino to carboxyl end in the following order: FR1, CDR1, FR2, CR2, FR3, CDR3, FR4.

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used here, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., hTNFα). It is shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibodies. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL domains, VH, CL and CN; (ii) the fragment F(ab')2, a bivalent fragment comprising two Fab fragment linked by a disulfide bridge in the area of the loop; (iii) a Fd fragment consisting of the VH domains and SN; (iv) a Fv fragment consisting of domains VL and VH one shoulder antibodies; (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546), which consists of a VH domain; and (vi) the selection (CDR), which defines complementarity. Moreover, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be recombinant methods involve using a synthetic linker that enables them to obtain a single protein chain in which the sections of the VL and VH coupled with the formation of monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). It is assumed that such is ntitle of one chain are also covered by the term "antigen-binding fragment" of an antibody. These include also other forms of antibodies from the same chain, such candiate. Diately are bivalent, bespecifically antibodies, in which the domains VH and VL are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing of the two domains on the same chain, forcing the domains to pair with complementary domains of another chain and create two antigen-binding site (see, e.g., Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; R.J. Poljak et al. (1994) Structure 2:1121-1123).

Further, the antibody or antigen-binding fragment may be part of larger molecules immunoadhesin, formed by covalent or non-covalent coupling of the antibody or antibody fragment with one or more protein or peptide. Examples of such molecules immunoadhesin include the use of the site core streptavidin to get tetramer scFv molecules (Kipriyanov S.M. et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine labels to obtain bivalent and reduced biomolecules scFv (Kipriyanov S.M. et al. (1994) Mol. Immunol., 31:1047-1058). Antibody fragments, such as Fab, F(ab')2can be obtained from whole antibodies using accepted methods, such as the decomposition of papain or pepsin, respectively, of whole antibodies. Balayage, antibodies, fragments of antibodies and molecules immunoadhesin can be obtained using standard methods with the use of recombinant DNA as described here.

The term "human antibody", as used here, includes antibodies having variable and constant parts, selected from the germline sequences of human immunoglobulin. Human antibodies corresponding to the invention may include amino acid residues not encoded by the germline sequences of the human immunoglobulin (e.g., mutations introduced omnidirectional or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, CDR and especially in CDR3. However, the term "human antibody", as used here, does not include antibodies in which CDR sequence selected from the embryos of other mammals, such as mice, were transplanted into human skeletal sequence.

The term "recombinant human antibody", as used here, includes all human antibodies that are obtained, expressed, created or selected recombinant means, such as antibodies expressed using a recombinant expressing the vector introduced into the cell-host (described further in Section II below), antibodies isolated from N. the boron-known recombinant, combinatorial human antibody (described further in Section III below), antibodies isolated from an animal (e.g. a mouse)that is transgenic in terms of genes of the human immunoglobulin (see, for example, Taylor, L.D. et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies obtained, expressed, created or selected by any other means that involve splicing of the gene sequence of human immunoglobulin to other DNA sequences. Such recombinant human antibodies have variable and constant parts, selected from the sequences of the human germline immunoglobulin. In some embodiments, implementation, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using an animal transgenic sequences lg human somatic mutagenesis in vivo and, thus, the amino acid sequence of plots VH and VL of the recombinant antibodies are sequences that, because they are selected from the sequences of the germline VH and VL of a human and close to them, not in natural conditions exist in embryonic set of human antibodies in vivo.

"Isolated antibody", as used here, is intended for the determination of antibodies, which is almost free of other antibodies having different antigenic specificities (e.g., highlighted an is Italo, which specifically binds hTNFαpractically does not contain antibodies that specifically bind antigens other than hTNFα). The selected antibody that specifically binds hTNFα, may, however, have cross-reactivity with other antigens, such as molecules TNFα from other species (discussed in more detail below). Moreover, the selected antibody can almost not contain other cellular material and/or chemical compounds.

"Neutralizing antibody", as used herein (or an "antibody that neutralized the activity of hTNFα)implies antibody binding with hTNFα leads to inhibition of the biological activity of hTNFα. This inhibition of the biological activity of hTNFα can be assessed by measuring one or more indicators of biological activity of hTNFαsuch as hTNFα-induced cell activation and binding hTNFα with receptors hTNFα. These indicators of biological activity of hTNFα can be assessed by one or more method various standard in vitro and in vivo, are known in the prior art (see Example 4). Preferably, the ability of antibodies to neutralize the activity of hTNFα is estimated by the inhibition of hTNFα-induced cytotoxicity of L929 cells. As an additional or alternative parameters is and activity of hTNFα may be assessed the ability of antibodies to inhibit hTNFα-induced expression of ELAM-1 on HUVEC, as the degree hTNFα-induced cell activation.

The term "surface plasma resonance", as used here, refers to an optical phenomenon, put in the basis of the analysis occurring at this time biospecific interactions by detection of alterations of the concentrations of protein in the biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Further described in Example 1, and Jönsson, U. et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U. et al. (1991) Biotechniques 11:620-627; Johnson C. et al. (1995) J. Mol. Recognit. 8:125-131 and Johnson C. et al. (1991) Anal. Biochem. 198:268-277.

The term "Koff"used here is designed to determine the rate constants of dissociation of antibodies from complex antibody/antigen.

The term "Kd"used herein is intended to denote the dissociation constants of the specific interaction of antibody-antigen.

The term "nucleic acid molecule", as used here, include DNA molecules and RNA molecules. The nucleic acid molecule can be admonitive or double, but preferably double-DNA.

The term "isolated molecule nucleic acid"as used here in relation to nucleic acids encoding antibodies or antibody fragments (e.g. the, VH, VL, CDR3)that bind hTNFα, is used to detect the nucleic acid molecule in which the nucleotide sequences encoding antibodies or antibody fragments that do not contain other nucleotide sequences encoding antibodies or fragments of antibodies that bind antigens other than hTNFαthese other sequences in natural conditions to join the nucleic acid in human genomic DNA. Thus, for example, isolated nucleic acid corresponding to the invention, the coding section VH antibodies against TNFαcontains no other sequences encoding other areas VH that bind antigens other than TNFα.

The term "vector" in the context of the description refers to a nucleic acid molecule capable of transporting another nucleic acid to which it was connected. One type of vector is a "plasmid," which means circular double-loop DNA, which can be legirovanyh additional segments of DNA. Another type of vector is a viral vector, into which additional DNA segments can be legirovanyh into the genome of the virus. Certain vectors are capable of Autonomous replication in a cell host, in which they are introduced (e.g., bacterial vectors having a bacterial origin (point is at the beginning) replication and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of the host cell by introducing into the cell the owner and therefore are replicated together with the genome of the host. Moreover, certain vectors can direct the expression of genes with which they are operatively linked. Such vectors are called in the context of application "expressing recombinant vectors" (or simply "expressing vectors"). Basically expressing vectors for use in the methods using recombinant DNA are often in the form of plasmids. In the present description "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention includes other forms expressing vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "recombinant a host cell" (or simply "a host cell") in the context of the application is designed to identify cells in which the introduced recombinant expression vector. It should be understood that such terms refer only certain this cell, but not the progeny of such cells. Because certain what odificatio in subsequent generations can occur due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but also falls under the definition of "a host cell" in the context of the application.

Various aspects of the invention are described in detail in the following subsections.

I. Human antibodies that bind TNFα man

The invention is selected human antibody or antigen-binding fragments that bind human TNFα with high affinity, low dissociation rate and high neutralizing capacity. Preferably human antibodies, corresponding to the invention are neutralizing recombinant human antibodies against hTNFα. The most preferred recombinant, neutralizing antibody according to the invention, is D2E7, which has sequences of VL and VH, as shown in Figures 1A, 1B and Figures 2A, 2B, respectively (amino acid sequence plot VL D1 E7 also represented as SEQ ID No 1; amino acid sequence plot VH D1E7 also represented as SEQ ID No 2). Binding properties of D2E7, as compared with murine mAB MAK 195 against hTNFαthat possess high affinity and low dissociation kinetics, and other human antibodies against hTNFα 2SD4 close D2E7 sequence are summarized below in table is:

AntibodyKoffwith-1konM-1with-1TodMStoichiometry
D2E7 lgG18,81×10-51,91×1056,09×10-101,2
2SD4 lgG48,4×10-34,20×1052,00×10-80,8
MAC 195 F(ab')28,70×10-51,90×1054,60×10-101,4

Antibody D2E7 and close antibodies also have a high ability to neutralize the activity of hTNFαthat was identified in several in vitro and in vivo (see Example 4). For example, these antibodies neutralize hTNFαinduced cytotoxicity of L929 cells with values IC50in the range of from about 10-7M to about 10-10M D2E7 when the expression in the form of antibody lgG1 full length neutralizes hTNFαinduced cytotoxicity of L929 cells with IC50approximately 1.25 x 10-10M moreover, the neutralizing activity of D2E7 supported when the antibody is expressed as Fab, F(ab')2or scFv fragment. D2E7 also inhibits hTNFα-induced activation of the cells is then measured by hTNFα induced expression of ELAM-1 on HUVEC (IC50= approximately 1,85×10-10M) and binding hTNFα receptors hTNFα on the cells U-937 (IC50=approximately 1,56×10-10M). Concerning the last case, D2E7 inhibits the binding of hTNFα both receptors hTNFα - P55 and P75. Moreover, the antibody inhibits hTNFα-induced death in vivo in mice (ED50=1-2,5 μg/mouse).

As for the specificity of binding of D2E7, this antibody binds to human TNFα in various forms, including soluble hTNFα, transmembrane hTNFα and hTNFαassociated with cellular receptors. D2E7 is not associated specifically with other cytokines such as lymphokines (hTNFβ), IL(interleukin)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IFNγ and TGFβ. However, D2E7 really has cross-reactivity with factors tumor necrosis from other species. For example, the antibody neutralizes the activity of TNFα at least five primates (chimpanzee, baboon, marmoset, cynomolgus and RH) with approximately the same value IC50as for the neutralization activity of murine TNFαalthough approximately 1000 times weaker than human TNFα (see Example 4, subsection E). D2E7 also binds canine and porcine hTNFα.

In one aspect, the invention relates to antibodies and fragments of antibodies, D2E7, antibodies and fragm the tov antibodies close D2E7, and other human antibodies and fragments of antibodies with equivalent D2E7 properties, such as high affinity binding hTNFβ with low dissociation kinetics and high neutralizing activity. In one embodiment, the invention is selected human antibody or antigen-binding fragment that dissociate from human TNFβ cd1×10-8M or less, and the rate constant for the dissociation of Koff1×10-3with-1M or less, measured using surface plasma resonance, and neutralizes the cytotoxicity of human TNFβ in the standard analysis using L929 in vitro with IC501×10-7M or less. More preferably, if the selected human antibody or antigen-binding fragment dissociate from human TNFβ Coff5×10-4with-1M or less, or even more preferably Tooff1×10-4with-1M or less. More preferably, if the selected human antibody or antigen-binding fragment neutralize the cytotoxicity of human TNFβ in the standard analysis using L929 in vitro with IC501×10-8M or less, even more preferably with IC501×10-9M or less and even more predpochtitel what about - IC505×10-10M or less. In a preferred embodiment, the antibody is selected recombinant human antibody or antigen-binding fragment. In another preferred embodiment, the antibody also neutralizes hTNFα-induced activation of cells that evaluate the standard in vitro assays hTNFα-induced expression of ELAM-1 on endothelial cells of the umbilical vein of a person (HUVEC).

Analysis using surface plasma resonance measurement Todand Koffcan be performed as described in Example 1. Standard in vitro assays using L929 for determining the values of the IC50described in Example 4, subsection A. the Standard in vitro assays hTNFα-induced expression of ELAM-1 on endothelial cells of the umbilical vein of a person (HUVEC) described in Example 4, subsection A. Examples of recombinant human antibodies that meet or assumed to correspond to the above criteria kinetics and neutralization include antibodies having the following [VH/VL] pair, the sequence of which is presented in Figures 1A, 1B, 2A and 2B (see also the analysis of the kinetics and neutralization in Examples 2, 3 and 4): [D2E7 VH/D2E7 VL]; [HD2E7*.A1/D2E7 VL]; [HD2E7*A2/D2E7 VL]; [HD2E7*A3/D2E7 VL]; [HD2E7*.A4/D2E7 VL]; [HD2E7*.A5/D2E7 VL]; [HD2E7*A6/D2E7 VL]; [HD2E7*.A7/D2E7 VL]; [HD2E7*.A8/D2E7 VL]; [HD2E7*.A9/D2E7 VL]; [D2E7 VH/LD2E7*.A1]; [D2E7 V/LD2E7*.A4]; [D2E7 VH/LD2E7*.A5]; [D2E7 VH/LD2E7*.A7]; [D2E7 VH/LD2E7*.A8]; [HD2E7*A9/LD2E7*.A1]; [VH1-D2/LOE7]; [VH1-D2.N/LOE7.T]; [VH1-D2.Y/LOE7.A]; [VH1-D2.N/LOE7.A]; [VH1-D2/EP B12] and [3C-H2/LOE7].

In the prior art it is well known that the CDR3 domains of the heavy and light chains of antibodies play an important role in the specificity/affinity in the binding of an antibody to the antigen. Accordingly, in another aspect, the invention relates to human antibodies, which have a low dissociation kinetics for Association with DTNFα and who have the CDR3 domains of light and heavy chains, which are structurally identical or similar to those of D2E7. As shown in Example 3, position 9 D2E7 VL CDR3 can be occupied by Ala or Thr without significant effect on the Koff. Accordingly, a consensus site D2E7 VL CDR3 contains the amino acid sequence Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID No 3). In addition, the position 12 D2E7 VH CDR3 can be occupied by Tyr or Asn no significant effect on Koff. Accordingly, a consensus site D2E7 VH With DR3 contains the amino acid sequence of V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID No 4). Moreover, as demonstrated in Example 2, in the domain CDR3 of the heavy and light chains D2E7 easy to replace by a single alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 in VH CDR3) without significant effect on the Koff. Moreover, the expert will appreciate that with the possibility of easy replacement by alanine in the domains D2E7 VL and VH CDR3 possible substitute is s other amino acids in the CDR3 domains while maintaining low values of the rate constant for dissociation of an antibody, in particular conservative replacement amino acids. Under the "conservative replacement amino acids in the context of the application refers to the replacement, in which the amino acid residue is substituted by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the prior art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Preferably in the domains D2E7 VL and/or VH CDR3 do nothing more than from one to five conservative substitutions of amino acids. More preferably in the domains D2E7 VL and/or VH CDR3 do nothing more than from one to three conservative substitutions of amino acids. In addition, conservative replacement amino acid should not be in positions of amino acids that are critical for binding hTNFα. As shown in Example 3, position 2 and 5 of the D2E7 VL CDR3 and positions 1 and 7 of the D2E7 VH CDR3 was critical for EOI is to interact with hTNFα and thus, it is preferable not to make conservative substitutions of amino acids in these positions (although the replacement of the alanine in position 5 of the D2E7 VL CDR3 is acceptable, as described above).

Accordingly, in another embodiment, the invention is selected human antibody or antigen-binding fragment with the following characteristics:

a) dissociates from the Association (complex) with hTNFα with a rate constant koff1×10-3with-1or less than that determined using surface plasma resonance;

b) has domain CDR3 light chain containing the amino acid sequence of SEQ ID No 3 or SEQ ID No 3, modified by replacing the single alanine at position 1, 4, 5, 7, or 8, or replacement of one to five conservative amino acid positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a domain CDR3 of the heavy chain containing the amino acid sequence of SEQ ID No 4 or SEQ ID No 4, modified by replacing the single alanine in position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or changing from one to five conservative amino acid positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

More preferably, if the antibody or antigen-binding fragment dissociate from human TNFα Coff5×10-4with-1or less. Even more preferably, if the antibody or antigen-St is binding fragment dissociate from human TNFα Tooff1×10-4with-1or less.

In yet another embodiment, the invention is selected human antibody or antigen-binding fragment with variable area light chain (LCVR)having a CDR3 domain containing the amino acid sequence of SEQ ID No 3 or SEQ ID No 3, modified by replacing the single alanine at position 1, 4, 5, 7, or 8, or variable plot heavy chain (HCVR)having a CDR3 domain containing the amino acid sequence of SEQ ID No 4 or SEQ ID No 4, modified by replacing the single alanine in position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, if the LCVR further has a CDR2 domain containing the amino acid sequence of SEQ ID No 5 (i.e. D2E7 VL CDR2), and the HCVR further has a CDR2 domain containing the amino acid sequence of SEQ ID No 6 (i.e. D2E7 VH CDR2). Even more preferably, if the LCVR further has CDR1 domain containing the amino acid sequence of SEQ ID No 7 (i.e. the D2E7 VL CDR1) and HCVR further has CDR1 domain containing the amino acid sequence of SEQ ID No 8 (i.e. the D2E7 VH CDR1). The preferred skeletal sites VL family of germline human VkI, more preferred from gene Vk germline human A20 and most preferred from skeletal sequences D2E7 VL shown in Figures 1A and 1B. The preferred skeletal parts of VH with whom the family germline human V H3, more preferred from gene VH germline human DP-31, and most preferred from skeletal sequences D2E7 VH shown in Figures 2A and 2B.

In yet another embodiment, the invention is selected human antibody or antigen-binding fragment with variable area light chain (LCVR), containing the amino acid sequence of SEQ ID No : 1 (i.e D2E7 VL) and variable plot heavy chain (HCVR), containing the amino acid sequence of SEQ ID No 2 (i.e. D2E7 VH). In some embodiments, the implementation of the antibody contains a constant plot heavy chain, such as constant plot IgG1, IgG2, IgGS, IgG4, IgA, IgE, IgM, IgD. Preferably constant plot heavy chain is a constant plot heavy chain IgG1 or constant plot IgG4 heavy chain. Moreover, the antibody can contain a constant-area light chain or Kappa constant area light chain or lambda constant area light chain. Preferably the antibody contains Kappa is a constant site of the light chain. On the other hand, the fragment of the antibody can be, for example, the fragment Fab or Fv fragment of the same chain.

In some other embodiments, implementation of the invention is selected human antibody or antigen-binding fragments having domains VL CDR3 is VH, close D2E7, for example, antibodies or their antigen-binding fragments with variable area light chain (LCVR)having a CDR3 domain containing the amino acid sequence selected from the group consisting of SEQ ID No 3, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21, SEQ ID No 22, SEQ ID No 23, SEQ ID No 24, SEQ ID No 25, SEQ ID No 26 or variable plot heavy chain (HCVR)having a CDR3 domain containing the amino acid sequence selected from the group consisting of SEQ ID No 4, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 34, SEQ ID No 35.

In yet another embodiment, the invention is a recombinant human antibody or antigen-binding fragment that neutralizes the activity of human TNFαbut not a human TNFβ. Preferably, if the antibody or antigen-binding fragment neutralizes the activity of TNFα chimpanzees and at least one additional TNFα primates selected from the group consisting of TNFα baboon, TNFα marmoset, TNFα cynomolgus and TNFα rhesus. Preferably, if the antibody or antigen-binding fragment neutralizes TNFα human, chimpanzee and/or additional of the primacy in standard in vitro assays using L929 with IC501×10-8M or less, more predpochtitel is about 1× 10-9M, even more preferably 5×10-10M or less. In one subvariant implementation of the antibody neutralizes the activity of canine TNFα, preferably in a standard in vitro assays using L929 with IC501×10-7M or less, more preferably 1×10-8M or less and even more preferably 5×10-9M or less. In another subvariant implementation of the antibody neutralizes the activity of porcine TNFαpreferably with IC501×10-5M or less, more preferably 1×10-6M or less and even more preferably 5×10-7M or less. In another subvariant implementation of the antibody neutralizes the activity of mouse TNFα preferably with IC501×10-4M or less, more preferably 1×10-5M or less and even more preferably 5×10-6M or less.

The antibody or antibody fragment, corresponding to the invention, can form derivatives or be associated with another active molecule (e.g., another peptide or protein). Accordingly, antibodies or fragments of antibodies, corresponding to the invention, include derivatives or otherwise modified forms of human antibodies against hTNFαdescribed here, including molecules immunoadhesin. N the example, the antibody or antibody fragment, corresponding to the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent Association or otherwise) to one or more molecular structures, such as another antibody (e.g., bespecifically antibody or diatel), a detectable agent (label), a cytotoxic agent, a pharmaceutical agent and/or a protein or peptide that may affect the Association of the antibody or antibody fragment with another molecule (such as an area of core streptavidin or polyhistidine label).

One type is derived antibodies receive cross-linking two or more antibodies (of the same type or different types, for example, to create bispecific antibodies). Suitable cross-linking agents include agents that are heterobifunctional having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidomethyl-N-hydroxysuccinimidyl ether) or homobifunctional (for example, disuccinimidyl suberate). Such linkers are available Pierce Chemical Company, Rockford, IL.

Suitable detectable agents with which can be formed derivatives of the antibodies or fragments of antibodies, corresponding to the invention include fluorescent compounds. Examples of fluorescent who's labels include fluorescein, isothiocyanate fluorescein, rhodamine, chloride, 5-dimethylamine-1-naphthalenesulfonyl, phycoerythrin etc. Antibody can also be derivative detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, etc. When the antibody forms derived with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses with the formation of a detectable reaction product. For example, when there is detectable agent is horseradish peroxidase, adding hydrogen peroxide and diaminobenzidine leads to the formation of colored reaction product, which is defined. The antibody can also be derived form with Biotin and can be detected through indirect measurement of the binding avidin or streptavidin.

II. Expression of antibodies

The antibody or antibody fragment, corresponding to the invention can be obtained by recombinant expression of the gene for the light and heavy chains of immunoglobulin in the cell host. For recombinant expression of the antibody to the cell-master transferout one or more recombinant expression vectors carrying DNA fragments encoding the light and heavy chain immunoglobulin antibody such that the light and heavy chains are expressed in the cell the owner and, preferably, are highlighted in the food, in which the cultured host cell, this environment can be selected antibodies. Standard methods with the use of recombinant DNA are used to obtain genes of the heavy and light chains, incorporation of these genes into recombinant expression vectors and introduce the vectors into host cells as described in Sambrook, Fritsch and Maniatis (eds) Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989; Ausubel F.M. et al. (eds.) Current Protocols in Molecular Biology, Green Publishing Associates, (1989) and U.S. Patent No 4816397 Boss et al.

For the expression of D2E7 or related D2E7 antibodies are first obtained DNA fragments encoding the light and heavy chain variable regions. These DNA can be obtained by the amplification and modification of light and heavy chains of the germline of the polymerase chain reaction (PCR). The DNA sequence of a germ-line gene variable regions of the heavy and light chains of a man known from the prior art (see, for example, "Vbase" - a database of sequences of the germline of the person; see also E.A. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson I.M. et al. (1992) "The Repertoire of Human Germline VHSequences Reveals about Fifty Groups of VHSegments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; Cox J.P.L et al. (1994) "A Directory of Human Germ-line VkSegments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836; the contents of each of which were specially introduced here by reference). To obtain a DNA fragment encoding the variable plot D2E7 heavy chain or close D2E7 antibody, a member of the family of VH3 genes VH germline human amplified using standard PCR. Most preferred is the amplification sequence germline VH DP-31. To obtain a DNA fragment encoding the variable plot D2E7 light chain or close D2E7 antibody member of a family of VkI VL genes germline human amplified with standard PCR. Most preferred is the amplification sequence germline VL A20. PCR primers suitable for use in amplification of sequences of the germline VH DP-31 and germline VL A20, can be determined on the basis of the nucleotide sequences described in the references cited supra, using standard methods.

After receiving the fragments of the VH and VL germline of these sequences can be subjected to mutagenesis to encode described herein amino acid sequence of the D2E7 or close D2E7. Amino acid sequence encoded by the DNA sequences of germ line VH and VL, are first compared with the amino acid sequences of VH and VL D2E7 or close D2E7 for the identification of amino acid residues in serial is a major D2E7 or close D2E7, which differ from the germline. Then the corresponding nucleotide sequences of the DNA of germ line is subjected to mutagenesis in such a way that the mutant germline sequence encodes the amino acid sequence of D2E7 or close D2E7, using the genetic code in order to identify nucleotide changes should be made. Mutagenesis of germline sequences carried out by standard methods, such as PCR-induced mutagenesis (in which the mutated nucleotides are introduced in the PCR primers such that the PCR product contains a mutation or site-directed mutagenesis.

Moreover, it should be noted that, if the sequence "germ line", the obtained PCR amplification, encode amino acid differences between skeletal sites on the correct configuration of the germ line (i.e. differences in the amplified sequence is compared with the correct sequence of the germline, for example, as a result of somatic mutation), it may be desirable to modify these differences of amino acids in the opposite direction - to the correct sequences of the germ line (i.e. "reverse mutation "skeletal remains in the configuration of the germ line).

After receiving the DNA fragments encoding VH segments and D2E7 VL or Liska D2E7 (by amplification and mutagenesis of genes VH and VL germline, as described above, can be carried out further manipulation of these DNA fragments by standard methods using recombinant DNA, for example the transformation of the genes of the variable segment genes in chain antibodies full-length genes of fragment Fab or scFv gene. In these manipulations, a DNA fragment that encodes a VL or VH, operatively linked to another DNA fragment, encoding another protein, such as constant plot antibody or a flexible linker. The term "operatively linked", as used in this context, is intended to denote that two fragments of DNA are linked so that the amino acid sequence encoded by the two DNA fragments remain in the frame are read.

The selected DNA encoding a plot VH, can be turned into a gene of the heavy chain of the full length through operational linking the VH-encoding DNA to another DNA molecule that encodes a constant sections of the heavy chain (CN, CH2 and CH3). The sequences of genes const plots the heavy chain of human known from the prior art (see, for example, Kabat E.A. et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments containing these plots can be obtained by standard PCR amplification. Constant plot heavy chain may be constant plot IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, IgD, but most is impactfully is a constant phase lgG1 or lgG4. For gene fragment Fab heavy chain VH-encoding DNA can be operatively linked to another DNA molecule encoding only a constant plot SN heavy chain.

The selected DNA encoding a plot of VL, can be transformed into gene light chain of the full length (as well as gene light chain Fab) via the operational link VL-code of soup DNA to another DNA molecule that encodes a constant area light chain CL. The sequences of genes const plots the light chain of the human known from the prior art (see, for example, Kabat E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments containing these plots can be obtained by standard PCR amplification. Constant area light chain can be a constant area of Kappa - or lambda, but most preferably is a constant plot Kappa.

To obtain the gene of scFv VH - and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3so that sequences of VL and VH can be expressed in the form of adjacent protein from a single thread, in which areas VL and VH are connected by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

For expression of the antibodies or fragments of antibodies, corresponding to the invention, encoding the light and heavy chains of the full length or fragments of DNA obtained as described above, is introduced into expression vectors such that the genes become operatively associated with sequences that control transcription and translation. In this context, the term "operatively linked" is intended to indicate that the antibody gene are ligated in the vector so that the sequence control transcription and translation in the vector serve the function that they regulate transcription and translation of the antibody gene. Expression vector and a sequence controlling the expression chosen so that they are compatible we used expressing the host-cell. Gene light chain antibody gene and the heavy chain of the antibody can be introduced into separate vectors or, more typically, both the gene is introduced into the same expression vector. Gene antibodies injected into the expression vector by standard methods (for example, legirovaniem complementary restriction sites on the fragment of the antibody gene and the vector or legirovaniem blunt ends, if restriction sites are absent). Before the introduction of sequences of light and heavy chains D2E7 or close D2E7 expression vector can carry the sequence to nstantly plots antibodies. For example, one approach to converting sequences of VH and VL D2E7 or close D2E7 in the genes of antibodies full-length is their insertion into the expression vector already encoding the constant plot heavy chain and the constant-area light chain, respectively, such that the VH segment is operatively associated with the segment(s) SN in the vector and the VL segment is operatively linked to the segment of the CL in the vector. Additionally or alternatively the recombinant expression vector can encode a signal peptide that facilitates secretion of chain antibody from the host cell. Gene chain antibodies can be cloned into a vector such that the signal peptide is linked in reading frame with the gene of aminocore chain antibodies. The signal peptide may be a signal peptide immunoglobulin or a heterologous signal peptide (i.e. the signal peptide of the protein of nimmanapalli).

In addition to genes chain antibodies recombinant expressing the vectors corresponding to the invention carry regulatory sequences that control gene expression chain antibodies in the cell host. The term "regulatory sequence" includes the incorporation of promoters, enhancers and other control elements of expression (e.g., polyadenylation signals)that control the transcription or tra is slalu genes chain antibodies. Such regulatory sequences are described, for example, see Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Any expert will appreciate that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of host cell for transformation, the desired level of expression of the protein, etc. of the Preferred regulatory sequences for expression in the cell host mammals include viral elements that direct the expression of the protein in mammalian cells at a high level, such as promoters and/or enhancers selected from cytomegalovirus (CMV) (such as the promoter/enhancer CMV), Simian virus 40 (SV40) (such as the promoter/enhancer of SV40), adenovirus (such as, adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and their sequences, see, for example, U.S. Patent No 5168062 Stinski; U.S. Patent No 4510245 Bell et al. and U.S. Patent No 4968615 Schaffner et al.

In addition to genes chain antibodies and regulatory sequences, the recombinant expressing the vectors corresponding to the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., originy replication genes and breeding is Askerov. Gene breeding marker facilitates the selection of host cells into which has been introduced vector (see, for example, U.S. Patents NoNo 4399216, 4634665 and 5179017 all Axel et al.). For example, in a typical case, the gene of breeding marker causes the resistance to medicinal substances, such as G418, hygromycin or methotrexate, host cell, which was Introduzione vector. Preferred genes of breeding markers include gene digidrofolatreduktazy (DHFR) (for use host cells dhfr-with the selection/amplification on background methotrexate) and the neo gene (for selection on the background of G418).

For the expression of light and heavy chain expression vector(s)encoding the heavy and light chains, transferout cell-host by standard methods. Different interpretations of the term "transfection" pregnancey to include a wide variety of ways, usually used for introduction of exogenous DNA into a prokaryotic or eukaryotic cell host, for example, electroporation, precipitation with calcium phosphate, transfection using DEAE-dextran, etc. Although theoretically it is possible to Express the antibodies, corresponding to the invention, either prokaryotic or eukaryotic cells masters, expression of antibodies in eukaryotic cells, and most preferably, in the cells of the host mammal is most prepost the tion, because such eukaryotic cells, and especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly educated and immunologically active antibody. Prokaryotic expression of antibody genes, as described, is ineffective to obtain active antibodies with high yield (Boss M.A. and Wood C.R. (1985) Immunology Today 6:12-13).

Preferred cells of mammalian hosts for expression of recombinant antibodies, corresponding to the invention include cells Chinese hamster ovary cells Cho (including cells of the Cho dhfr-described by Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a selective marker DHFR, for example, as described R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When expressing recombinant vectors encoding antibody genes, being introduced to the cells of the host mammal, antibodies are formed during the cultivation of the host cells for a period of time sufficient for expression of the antibody in the cell-the owner, or (more preferably) the secretion of antibodies into the culture medium in which to grow the host cells. Antibodies can be isolated from the culture medium using standard methods of protein purification.

Cell host can also be used to obtain fragments INTA is the shaft antibodies such as Fab fragments and scFv molecules. It should be understood that variations of the above procedure are included in the scope of the invention. For example, it may be desirable to transliterate cell host DNA encoding either the light chain or the heavy chain (but not both) antibodies, corresponding to the invention. The method using recombinant DNA can also be used to remove some or all of the DNA encoding separately or both light and heavy chains that is not necessary for binding hTNFα. The molecules expressed from such truncated DNA molecules are also covered by the antibodies corresponding to the invention. In addition, cross-linking antibodies, corresponding to the invention, with the second antibody using standard chemical methods, cross-stitching can also be obtained bifunctional antibodies, in which one heavy and one light chain are an antibody corresponding to the invention and the other heavy and the other light chain are specific for an antigen other than hTNFα.

In the preferred system for recombinant expression of the antibodies or antigen-binding fragment corresponding to the invention, a recombinant expression vector encoding both the heavy chain of the antibody and the light chain of the antibody being introduced into cells Cho dhfr-by Tran the infection using calcium phosphate. In recombinant expression vector of each of the genes of the heavy and light chains of the antibody operatively linked to regulatory elements of the enhancer/promoter (e.g., isolated from SV40, CMV, adenovirus and the like, such as a regulatory element of the CMV enhancer/AdMLP promoter or a regulatory element of the SV40 enhancer/AdMLP promoter) to direct the transcription of genes at a high level. The recombinant expression vector also carries the gene for DHFR, which provides a selection of cells SNO, which were transliterowany vector using the selection/amplification on a background of methotrexate. Selected cells-owners-transformants are culture, ensuring the expression of the heavy and light chains of the antibody, and the intact antibody isolated from the culture medium. To obtain recombinant expression vector, transfection of host cells, selection of transformants, culturing host cells and selection of antibodies from the culture medium using standard molecular biological methods.

In light of the above another aspect of the invention relates to compositions of nucleic acids, vectors and host cells that can be used for recombinant expression of the antibodies and fragments of antibodies, corresponding to the invention. The nucleotide sequence encoding variabel the hydrated area D2E7 light chain, presented in Figure 7 and SEQ ID No 36. Domain LCVR CDR1 comprises the nucleotide 70-102, CDR2 domain encompasses nucleotides 148-168 and CDR3 domain encompasses nucleotides 265-291. The nucleotide sequence encoding the variable plot D2E7 heavy chain, presented on Figure 8 and SEQ ID No 37. Domain HCVR CDR1 comprises the nucleotide 91-105, CDR2 domain encompasses nucleotides 148-198 and CDR3 domain encompasses nucleotides 295-330. The expert will appreciate that the nucleotide sequence encoding the antibody, close D2E7, or fragments thereof (e.g., domain, CDR, such as the domain CDR3) may be selected from the nucleotide sequences encoding the LCVR and HCVR D2E7 using the genetic code and standard molecular biological methods.

In one embodiment, the invention is selected nucleic acid encoding a domain of the light chain CDR3 with the amino acid sequence SEQ ID No 3 (i.e. the D2E7 VL CDR3) or SEQ ID No 3, modified by replacing one alanine residue in position 1, 4, 5, 7, or 8, or by replacement of one to five conservative amino acid positions 1, 3, 4, 6, 7, 8 and/or 9. This nucleic acid can encode only the plot CDR3 or, more preferably, encodes a variable area light chain whole antibodies (LCVR). For example, nucleic acid can encode LCVR having a CDR2 domain containing linakis is now the sequence of SEQ ID No 5 (i.e. D2E7 VL CDR2, and CDR1 domain containing the amino acid sequence of SEQ ID No 7 (i.e. the D2E7 VL CDR1).

In yet another embodiment, the invention is selected nucleic acid encoding a CDR3 domain of the heavy chain with the amino acid sequence SEQ ID No 4 (i.e. the D2E7 VH CDR3), or SEQ ID No 4, modified by substitution of one residue alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by replacement of one to five conservative amino acid positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12. This nucleic acid can encode only the plot CDR3 or, more preferably, encodes variable plot heavy chain whole antibodies (HCVR). For example, nucleic acid can encode HCVR having a CDR2 domain containing the amino acid sequence of SEQ ID No 6 (i.e. D2E7 VH CDR2 and CDR1 domain containing the amino acid sequence of SEQ ID No 8 (i.e. the D2E7 VH CDR1).

In some other embodiments, the invention is selected nucleic acid encoding the CDR3 domain, close D2E7, for example, contains the amino acid sequence selected from the group consisting of SEQ ID No 3, SEQ ID No 4, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21, SEQ ID No 22, SEQ ID No 23, SEQ ID No 24, SEQ ID No 25, SEQ ID No 26, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 34, SEQ ID No 35.

In yet another embodiment, Khujand the exercise of the invention is selected nucleic acid, coding variable area light chain antibodies containing the amino acid sequence of SEQ ID No : 1 (i.e LCVR D2E7). Preferably, this nucleic acid contains a nucleotide sequence of SEQ ID No 36, although the expert will appreciate that due to degeneration of the genetic code, other nucleotide sequences can encode the amino acid sequence of SEQ ID No 1. The nucleic acid can encode only LCVR or can also encode a constant area light chain antibodies, operatively associated with the LCVR. In one embodiment, this nucleic acid is a recombinant expression vector.

In yet another embodiment, the invention is selected nucleic acid encoding a variable plot heavy chain antibodies containing the amino acid sequence of SEQ ID No 2 (i.e. HCVR D2E7). Preferably, this nucleic acid contains a nucleotide sequence of SEQ ID No 37, although the expert will appreciate that due to degeneration of the genetic code, other nucleotide sequences can encode the amino acid sequence of SEQ ID No 2. The nucleic acid can encode only HCVR or can also encode a constant plot heavy chain antibodies, operatively associated with the HCVR. For example, a nucleic acid which may contain a constant plot IgG1 or IgG4. In one embodiment, this nucleic acid is a recombinant expressing the vector.

The invention is also expressing recombinant vectors encoding both the heavy chain of the antibody and the light chain antibodies. For example, in one embodiment, the invention is a recombinant expression vector encoding:

a) light chain antibody having a variable area, with amino acid sequence SEQ ID No : 1 (i.e LCVR D2E7), and

b) a heavy chain antibody having a variable area, with amino acid sequence SEQ ID No 2 (i.e. HCVR D2E7).

The invention also provides cell-owners, which was introduced one or more recombinantly expression vector, corresponding to the invention. Preferably, a host cell is a cell of the host mammal, more preferably a host cell is a cell Cho, NSO cell or a COS cell.

Further, the invention provides a method for the synthesis of recombinant human antibodies, corresponding to the invention by culturing a host cell corresponding to the invention in a suitable medium cultivation until such time as not synthesized recombinant human antibody, corresponding to the invention. The method may further provide you the bookmark recombinant human antibodies from the culture medium.

III. Selection of recombinant human antibodies

Recombinant human antibodies, corresponding to the invention, in addition to described here antibody D2E7 or close D2E7 can be selected by viewing recombinant combinatorial libraries of antibodies, preferably a library of scFv-displaying phages obtained using cDNA of human VL and VH obtained from mRNA isolated from human lymphocytes. The methodology for obtaining and screening such libraries are known in the prior art. In addition to commercially available kits for obtaining libraries with demonstration of phage (e.g., the Pharmacia Recombinant Phage Antibody System, catalog No 27-9400-01; set demonstration of phage Stratagene SurfZAP™, catalogue No 240612), examples of methods and reagents particularly suitable for use when creating and screening libraries that display antibodies can be detected, for example, Ladner et al. U.S. patent No 5223409; Kang et al. The PCT publication No WO 92/18619; Dower et al. The PCT publication No WO 91/17271; Winter et al. The PCT publication No WO 92/20791; Markland et al. The PCT publication No WO 92/15679; Breitling et al. The PCT publication No WO 93/01288; McCafferty et al. The PCT publication No WO 92/01047; Garrard et al. The PCT publication No WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; CIackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-33580; Garra et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. ((1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982.

In the preferred embodiment, for the selection of human antibodies with high affinity and a low rate constant of dissociation in relation to hTNFα mouse antibodies against hTNFαhaving a high affinity and a low rate constant of dissociation in relation to hTNFα (for example, MAK 195, hybridoma for which has the number of the depositor ESAS 87 050801), first used for the selection of the sequences of human heavy and light chains having similar binding activity against hTNFαapplying the epitope imprinting or directed selection, the methods described in Hoogenboom et al., The PCT publication No WO 93/06213. Libraries of antibodies used in this method are preferably of the library of scFv obtained and analyzed as described in McCafferty et al.; The PCT publication No WO 92/01047; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734. Screening libraries of scFv antibodies preferably carried out using as antigen recombinant human hTNFα.

After you select the source segments of human VL and VH experiment "mixing and determination of conformity", which perform the screening of different pairs of the initially selected segments of the VL and VH in relation linking hTNFα order of selection is repectfully combinations of pairs of VL/VH. In addition, to further improve the affinity and/or reduce the rate constants of dissociation binding hTNFαsegments VL and VH preferred pair (s) VL/VH can be subjected to an undirected mutagenesis, preferably within the CDR3 region of VH and/or VL, in a manner analogous to the method of obtaining a somatic mutation in vivo, contributing to the affinity maturation of antibodies during a natural immune response. This affinity maturation in vitro can be carried out by amplification plots VH and VL using PCR primers complementary VH CDR3 or VL CDR3, respectively, these primers "drive" with a random mixture of the four nucleotide bases at certain positions, so that the resulting PCR products encode the segments of VH and VL, in which random mutations have been introduced in areas CDR3 VH and/or VL. These segments of the VH and VL with random mutations can be again subjected to screening for binding hTNFαand can be selected sequences that exhibit high affinity and a low rate constant of dissociation in relation to hTNFα.

Amino acid sequence selected heavy and light chains of the antibodies can be compared with the amino acid sequences of the heavy and light chain germline. In cases where certain skeletal remains of Ottobrunn the x chain VL and/or VH differ from the germline configuration (for example, the result of somatic mutation of immunoglobulin genes used to produce ragovoy library), it may be desirable to reverse the mutation altered the skeletal remains of the selected antibodies to return to the configuration of the germ line (i.e. changing skeletal amino acid sequence selected antibodies, so that they become the same as skeletal amino acid sequence of the germ line). This "reverse mutation" (or "bringing to the germ line") the skeletal remains can be performed by standard molecular biological methods for the introduction of specific mutations (e.g., site-directed mutagenesis, PCR-based mutagenesis and others).

After screening and selection of antibodies against hTNFαcorresponding to the invention, from the library of recombinant immunoglobulins, nucleic acid encoding the selected antibody can be selected from display package (e.g., from the phage genome) and subcloned into other expressing vectors by standard methods using recombinant DNA. If desirable, further manipulation of the nucleic acid to create other forms of antibodies, corresponding to the invention (for example, binding to a nucleic acid that encodes an additional immunoglobulin is Omani, such as additional constant plots). For expression of recombinant human antibodies are selected by screening a combinatorial library, the DNA encoding the antibody, clone in recombinant expression vector and being introduced to the cells of the host mammal, as described in detail above in Section II.

IV. Pharmaceutical compositions and pharmaceutical application

Antibodies and antibody fragments, corresponding to the invention can be introduced into pharmaceutical compositions suitable for administration to a subject. In a typical case, the pharmaceutical composition comprises the antibody or antibody fragment, corresponding to the invention, and a pharmaceutically acceptable carrier. Used herein, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, shell, antibacterial and antifungal agents, isotonic and delaying absorption agents, etc. that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following media: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases it will be preferable to include in the composition isotonic agents, for example sugars, m is agatone alcohols, such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further contain minor amounts of additional substances, such as moisturizing agents or emulsifying agents, preservatives or buffers, which enhance the retention time or the effectiveness of the antibody or antibody fragment.

Composition, corresponding to this invention may be in various forms. They include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., solutions for injection and infusion), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended route of administration and therapeutic application. A typical preferred composition is represented by the form of solutions for injection or infusion, such as compositions similar to the composition used for passive immunization of humans with other antibodies. The preferred method of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions in typical is the second case must be sterile and stable under conditions of manufacture and storage. The composition can be made in the form of solutions, microemulsions, dispersions, liposomes or other designated structures suitable for high concentration of the medicinal substance. Sterile injectable solutions can be prepared by introduction of active compounds (i.e. antibody or fragment of antibody) in the required amount in the appropriate solvent with one or a combination of the above ingredients as required, followed by sterilization by filtration. Basically dispersion is prepared by introduction of the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, with which to produce a powder of the active ingredient with any additional desired ingredient from its pre-sterilized filtered solution. The proper fluidity of the solution can be maintained, for example, by use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be shortcuts is bent by the inclusion in the composition of the agent, which delays absorption, for example, monostearate salts and gelatin.

Antibodies and antibody fragments, corresponding to the invention can be administered in a variety of ways known in the prior art, though in many ways therapeutic applications, the preferred method/by injection is intravenous injection or infusion. As will be appreciated by the specialist, the method and/or route of administration will vary depending on the desired results. In some embodiments, the active compound can be prepared with a carrier that will protect the compound against rapid release of the drug with controlled access, including implants, transdermal patches and microencapsulation delivery system. Can be used biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. Many methods of preparation of such drugs are patented and mostly known in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems. J.R.Robinson ed. Marcel Dekker, Inc., New York, 1978.

In some embodiments, the implementation of the antibody or antibody fragment, corresponding to the invention can be administered orally, for example, with an inert diluent or an assimilable edible carrier. With the unity (and other ingredients if desirable) may also be enclosed in a gelatin capsule with a hard or soft shell, compressed into tablets, or entered directly into the food of the subject. For oral therapeutic administration connections can be entered with excipients and used in the form of tablets for oral administration, cheek tablets, pellets, capsules, elixirs, suspensions, syrups, wafers and the like, For introducing connections, corresponding to the invention, in a manner different from parenteral, it may be necessary floor connection sheath material, preventing its inactivation, or joint introduction connection with such material.

Supplementary active compounds can also be introduced into the composition. In some embodiments, the implementation of the antibody or antibody fragment, corresponding to the invention are contained in the product and/or are administered together with one or more therapeutic agents are used to treat disorders in which the activity of hTNFα is harmful. For example, the antibody or fragment antibodies against hTNFαcorresponding to the invention, can enter into a drug and/or be administered in conjunction with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or associated surface of the mole is uly cells), with one or more cytokines, soluble receptor of TNFα (see, for example, PCT Publication No WO 94/06476) and/or one or more chemical agent that inhibit the production or activity of hTNFα (such as derivatives of cyclohexylidene, as described in PCT Publication No WO 93/19751. Moreover, one or more antibodies, corresponding to the invention can be used in combination with one or more of the foregoing therapeutic agents. In such methods of combination therapy can successfully be used a low dose of an administered therapeutic agents, thus avoiding possible toxicities or complications associated with different ways monotherapy.

Non-limiting scope of the invention examples of therapeutic agents from rheumatoid arthritis, which can be combined antibody or antibody fragment, corresponding to the invention include the following: non-steroidal anti-inflammatory drug substance(s) (NSAIDs); cytokine-suppressive anti-inflammatory drug substance(s) (CSAIDs); CDP-571/BAY-10-3356 (humanitariannet antibody against hTNFα; Celltech/Bayer); cA2 (chimeric antibody against hTNFα; Centocor); TNFR-IgG molecular weight of 75 KD (protein TNF receptor-IgG mol. weight of 75 KD; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol.37, 295; J. Invest. Med. (1996) Vol.44, A); TNFR-IgG molecular weight 55 is D (protein TNF receptor-IgG mol. mass of 55 KD; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (sustainable primaryservername antibody against CD4; IDEC/SmithKline; see, e.g., Arthritis & Rheumatism (1995) Vol. 38, 185; DAB 486-IL-2 and/or DAB 389-IL-2 (slit proteins IL-2; Seragen; see, e.g., Arthritis & Rheumatism (1993) Vol. 36, 1223; Anti-Tac (humanitariannet antibody against IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, an anti-inflammatory cytokine; DNAX/Schering); IL-4; agonists of IL-10 and/or IL-4 (e.g., agonist antibodies); IL-1RA (receptor antagonist IL-1; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF binding protein; see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 284; Amer. J. Physiol. - Heart and Circulatory discrimination (1995) Vol.268, pp.37-42); R973401 (inhibitor of phosphodiesterase type IV; see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 282); MK-966 (inhibitor SOH-2, see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 81); iloprost (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 82); methotrexate; thalidomide (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 282) and close to the thalidomide drug substance (e.g., Celgen); Leflunomide (anti-inflammatory and inhibitor of cytokines, see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 131; Inflammation Research (1996) Vol 45, pp.103-107); tranexamic acid (inhibitor of plasminogen activation; see, e.g., Arthritis & Rheumatism (1996) Vol.39, No 9 (optional), 284); T-614 (inhibitorzetia (see, for example, Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 282); prostaglandin E1 (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 282); tenidap (non-steroidal anti-inflammatory drug; see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 280); naproxen (non-steroidal anti-inflammatory drug; see, e.g., Neuro Report (1996) Vol.7, pp.1209-1213); meloxicam (non-steroidal anti-inflammatory drug); ibuprofen (non-steroidal anti-inflammatory drug); piroxicam (non-steroidal anti-inflammatory drug); diclofenac (non-steroidal anti-inflammatory drug); indomethacin (non-steroidal anti-inflammatory drug); sulfasalazin (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 281); azathioprine (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 281); ICE inhibitor (inhibitor of the enzyme interleukin - 1β-converting enzyme); inhibitor of zap-70 and/or Ick (inhibitor of the tyrosine kinase zap-70 or Ick); VEGF inhibitor and/or an inhibitor of VEGF-R inhibitors of growth factor of vascular endothelium cells or growth factor receptor of vascular endothelium cells; inhibitors of angiogenesis); corticosteroid anti-inflammatory drug substances (for example, S203580); inhibitors of TNF-is invertase; antibodies against IL-12; interleukin-11 (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 296); interleukin-13 (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 308); inhibitors of interleukin-17 (see, e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (optional), 120); gold, penicillamine, chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine; total lymphoid irradiation: globulin against thymocytes; antibodies against CD4; toxins CD5; peptides and collagen for oral administration; disodium lobenzarit; Cytokine-regulating agents (CRA) HP228 and NR (Houghten Pharmaceuticals, Inc.); antisense phosphorothioate oligodeoxynucleotide ICAM-1 (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TR; T Cell Sciences, Inc.); prednisone; orgotein; fractions polysulphate; minocycline; antibodies against IL2R; marine and Botanical lipids (fatty acids of marine animals and plants; see, e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic acid; floranova acid: immune globulin intravenous; zileuton; mycofenolate acid (RS-61443); tacrolimus (FK-506), sirolimus (rapamycin); amiprilose (terpentin); cladribine (2-chloromethoxypropyl) and azaribine.

Non-limiting scope of the invention examples of therapeutic agents from inflammatory bowel disease with which to monoaminergic antibodies or fragments of antibodies, corresponding to the invention, include budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazin; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; inhibitors of thromboxane; antagonists of the receptor for IL-1; monoclonal antibodies against IL-1β; monoclonal antibodies against IL-6; growth factors; elastase inhibitors; compounds pyridinyl-imidazole; CDP-571/BAY-10-3356 (humanitariannet antibody against hTNFα; Celltech/Bayer); cA2 (chimeric antibody against hTNFα; Centocor); TNFR-IgG molecular weight of 75 KD (protein TNF receptor-IgG mol. weight of 75 KD; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol.37, 295; J. Invest. Med. (1996) Vol.44, 235A); TNFR-IgG molecular weight of 55 KD (protein TNF receptor-IgG mol. mass of 55 KD; Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plough); IL-4; agonists of IL-10 and/or IL-4 (e.g., agonist antibodies); Il-11; glucuronide - or dextran-conjugated proletarienne form of prednisolone, dexamethasone or budezonida; antisense phosphorothioate oligodeoxynucleotide ICAM-1 (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TR; T Cell Sciences, Inc.); slow-release mesalazine; methotrexate; antagonists of platelet activating factor (PAF); ciprofloxacin, lignocain.

A non-limiting amount of the Britania examples of therapeutic agents from multiple sclerosis, you can combine antibodies or fragments of antibodies, corresponding to the invention, include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1A (Avonex™; Biogen); interferon-β1b (Betaseron™; Chiron/Berlex); Copolymer 1 (COP-1; Copaxone™; Teva Pharmaceutical Industries Inc.); hyperbaric oxygen; intravenous immunoglobulin; claritin; CDP-571/BAY-10-3356 (humanitariannet antibody against hTNFα; Celltech/Bayer); cA2 (chimeric antibody against hTNFα; Centocor); TNFR-IgG molecular weight of 75 KD (protein TNF receptor-IgG mol. weight of 75 KD; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol.37, 295; J. Invest. Med. (1996) Vol.44, 235A); TNFR-IgG molecular weight of 55 KD (protein TNF receptor-IgG mol. mass of 55 KD; Hoffmann-LaRoche); IL-10; IL-4; agonists of IL-10 and/or IL-4 (e.g., agonistic antibodies).

Non-limiting examples of therapeutic agents from sepsis, which can be combined antibodies or fragments of antibodies, corresponding to the invention, include the following: hypertonic saline solutions; antibiotics; γ-globulin; continuous hemofiltration; carbapenems (e.g. Meropenem); antagonists of cytokines such as TNFα, IL-1β, IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanitariannet antibody against hTNFα; Celltech/Bayer); cA2 (chimeric antibody against hTNFα; Centoco); TNFR-IgG molecular weight of 75 KD (protein TNF receptor-IgG mol. weight of 75 KD; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol.37, 295; J. Invest. Med. (1996) Vol.44, 235A); TNFR-IgG molecular weight of 55 KD (protein TNF receptor-IgG mol. mass of 55 KD; Hoffmann-LaRoche); cytokine-regulating agents (CRA) HP228 INR (Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular weight peptide; SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (Picower Institute); inhibitor of the biosynthesis pathway of tissue factor (TFPI; Chiron); PHP (chemically modified hemoglobin; APEX Bioscience); chelators and chelates of iron, including complex diethylenetriaminepentaacetic acid - iron (III) (DTPA iron (III); Molichem Medicines); lisofylline (synthetic small molecule of methylxanthines; Cell Therapeutics, Inc.); PGG-glucan (soluble β-1,3-glucan; Alpha-Beta Technology); apolipoprotein a-1, the reconstructed lipids; chiral hydroxamic acid (synthetic antibacterial agents which inhibit the biosynthesis of lipid A), antibody against endotoxin; E (synthetic antagonist of lipid A; Eisai America, Inc.); rBPI21(recombinant N-terminal fragment of human bactericidal/increasing permeability of the protein and synthetic peptides against endotoxin (SAEP; BiosYnth Reserch Laboratories).

Non-limiting examples of therapeutic agents for respiratory distress syndrome in adults (ARDS), which can be combined antibodies or fragments is s antibodies corresponding to the invention include antibodies against IL-8; replacement therapy surfactants; CDP-571/BAY-10-3356 (humanitariannet antibody against hTNFα; Celltech/Bayer); cA2 (chimeric antibody against hTNFα; Centocor); TNFR-IgG molecular weight of 75 KD (protein TNF receptor-IgG mol. weight of 75 KD; Immunex; see, e.g., Arthritis & Rheumatism (1994) Vol. 37, 295; J. Invest. Med. (1996) Vol.44, 235A); TNFR-IgG mol. mass of 55 KD (protein TNF receptor-IgG mol. mass of 55 KD; Hoffmann-LaRoche).

The use of antibodies or fragments of antibodies, corresponding to the invention, in combination with other therapeutic agents discussed below in section V.

Pharmaceutical compositions corresponding to the invention, may include a "therapeutically effective amount" or "prophylactically effective amount" of the antibody or antibody fragment, corresponding to the invention. "Therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody fragment may vary in accordance with factors such as the disease state, age, sex and weight of the subject and the ability of the antibody or antibody fragment to cause the desired response in the subject. Therapeutically effective amount I have is also a number, in which any toxic or detrimental effects of the antibody or fragment antibodies were outweighed by therapeutically favorable effects. Prophylactically effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. In a typical case, since a prophylactic dose is used in subjects prior to or at the early stage of the disease, the prophylactically effective amount will be less than therapeutically effective amount.

Schema dosage can be increased to obtain the optimum desired response (e.g., therapeutic or prophylactic response). For example, there may be one dose, there may be several individual doses over the period of time or the dose may be proportionally reduced or increased, depending on the needs of therapeutic situation. A special advantage is the preparation of parenteral compositions in a standard dosage form for ease of application and the same dosage. Standard dosage form, as used here, refers to physically discrete units suitable as individual doses for subjects-mammal being treated; each unit contains a certain amount of active travel is, calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The definition of standard dosage forms, corresponding to the invention, is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect, which plan to get, and (b) the limitations inherent in the method of obtaining such an active compound for the treatment of sensitivity in the regions.

Exemplary non-limiting interval for therapeutically or prophylactically effective amount of the antibody or antibody fragment, corresponding to the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg it Should be noted that the values of the dose may vary depending on the type and severity of the facilitated state. In addition, it should be understood that for any particular subject should be over a period of time selected specific schema dosage in accordance with the needs of the subject and the professionalism of the specialist who introduces or leads to the introduction of the compositions, and that the intervals doses are only exemplary and are not intended to limit the scope or application of the claimed composition.

V. Application of antibodies, corresponding to the invention

Considering their is sposobnosti link hTNFα antibodies against hTNFα or fragments thereof, corresponding to the invention can be used for detection hTNFα (e.g., in a biological sample, such as serum or plasma), using a known method immunoassay such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or immunohistochemistry tissue. The invention provides a method of detecting hTNFα in a biological sample, involving contacting a biological sample with the antibody or antibody fragment corresponding to the invention and detecting either the antibody (or antibody fragment)associated with hTNFαor unbound antibody (or antibody fragment) to identify so hTNFα in a biological sample. The antibody directly or indirectly have been labelled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable complexes prosthetic groups include streptavidin/Biotin and avidin/Biotin; umbelliferone, fluorescein, isothiocyanate fluorescein, rhodamine, DeHart ezineminicourse, chloride of dansili or phycoerythrin; an example of a luminescent material includes luminal and examples of suitable radioactive material include125I131I35S or3H.

Alternative to labeling the antibody hTNFα can be determined in biological samples is a competitive immunoassay utilizing standards rhTNFαmarked-defined substance, and unlabeled antibodies against hTNFα. In this analysis, mix the biological sample, the labeled standards rhTNFα and antibody against hTNFα and determine the number of standard labeled rhTNFαassociated with the unlabeled antibody. The number of hTNFα in the biological sample is inversely proportional to the number of standard labeled rhTNFαassociated with antibody against hTNFα.

The antibody D2E7, corresponding to the invention can also be used for detection of TNFα from species other than human, in particular TNFα primates (e.g., chimpanzee, baboon, marmoset, cynomolgus and rhesus), pig and mouse, as D2E7 can associate each of these TNFα (to be discussed later in Example 4, subsection E).

Antibodies or fragments of antibodies, corresponding to the invention, capable of neutralizing the activity of hTNFα in vitro and in vivo (see Example 4). Moreover, at least some of the antibodies corresponding to the invention, such as D2E7, can n teralithe activity of TNFα isolated from other species. Accordingly, the antibodies or fragments of antibodies, corresponding to the invention can be used for inhibiting the activity of TNFαfor example, in a cell culture containing hTNFα, y of man or other mammals with TNFαthat antibodies corresponding to the invention, the cross-reactive (e.g., chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig, and mouse). In one embodiment, the invention provides a method of inhibiting the activity of TNFαproviding for the engagement of TNFα with the antibody or antibody fragment corresponding to the invention, the activity TMFα inhibited. Preferably TNFα is human TNFα. For example, the antibody or antibody fragment, corresponding to the invention, can be added to a cell culture containing, or suspected of containing TNFαwith the purpose of inhibiting the activity of TNFα in culture.

In another embodiment, the invention provides a method of inhibiting the activity of TNFα y subject suffering from disorders in which the activity of TNFα is harmful. TNFα participated in the pathophysiology of a wide range of disorders (see, for example, Moeller, A., et al. (1990), Cytokine 2:162-169; U.S. Patent No 5231024 Moeller et al.; European Patent Publication No. 260610 B1 A. Moeller). The invention is the means, concerning the activity of TNFα the subject with such violations, this method provides an introduction to the subject the antibody or antibody fragment, corresponding to the invention, while inhibited the activity of TNFα the subject. Preferably TNFα is human TNFα and the subject is human. Alternative a subject can be a mammal expressing TNFαwith which the antibodies corresponding to the invention, the cross-reactive. Further, the subject may be a mammal, which was introduced hTNFα (for example, through the introduction of hTNFα or expression of the transgene hTNFα). The antibody corresponding to the invention can be administered to the person for therapeutic purposes (further discussion below). Moreover, the antibody corresponding to the invention can be administered to the mammal animal expressing TNFαwith which the antibody cross-reactive (e.g., a Primate, pig or mouse) for veterinary purpose or as into a useful animal model of human disease. Regarding the latter, such animal models can be used to evaluate therapeutic efficacy of antibodies, corresponding to the invention (e.g., testing of dosages and time courses of the application).

As used here, the term "a disorder in which the activity of TNFα is harmful," p is educative the inclusion of diseases and other disorders when the presence of hTNFα for a subject suffering from violations, as was shown or believed or causes pathophysiology of violation, or is a factor that exacerbates the violation. Accordingly, a disorder in which the activity of TNFα is harmful, is a disorder in which inhibition of the activity of TNFαas expected, reduces symptoms and/or development disorders. Such violations can be detected, for example, by increasing the concentration of TNFα in a biological fluid of a subject suffering from disorders (for example, increasing the concentration of TNFα in serum, plasma, synovial fluid, etc. of the subject), which can be determined, for example, using antibodies against TNFαas described above. There are many examples of disorders in which the activity of TNFα is harmful. The use of the antibodies and fragments of antibodies, corresponding to the violation for the treatment of specific disorders will be discussed further below:

A. Sepsis

The tumor necrosis factor plays an established role in the pathophysiology of sepsis with biological effects, including hypotension, weakness infarction, syndrome leaking vessels, necrosis of organs, stimulation of release of toxic secondary mediators and activation of the cascade of blood coagulation (see, for example, Moeller, A., et al. (199), Cytokine 2:162-169; U.S. Patent No 5231024 Moeller et al.; European Patent No 260610 B1 Moeller, A.; Tracey K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503; Russel D. and Thompson, R.C. (1993) Curr. Opin. Biotech. 4:714-721). Accordingly, human antibodies and antibody fragments, corresponding to the invention, can be used to treat sepsis in any of its clinical manifestations, including septic shock, endotoxic shock, sepsis caused by gram-negative bacteria, and toxic shock syndrome.

Moreover, for the treatment of sepsis antibodies against hTNFα and fragments of antibodies, corresponding to the invention, can be introduced together with one or more therapeutic agent, which may further weaken sepsis, such as an inhibitor of interleukin-1 (described in PCT Publications No WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see, for example, PCT Publication No WO 93/11793) or antagonist of platelet activating factor (see, for example, European Patent application No EP 374510). Other methods of combination therapy for the treatment of sepsis are discussed below in section III.

In addition, in the preferred embodiment, the antibody against hTNFα and fragments of antibodies, corresponding to the invention, is administered to a person in a subgroup of patients with sepsis, with the concentration of IL-6 in serum or plasma from more than 500 PG/ml and more preferably 1000 PG/ml during treatment (see Publications the PCT No WO 95/20978 L. Daum et al.).

C. Autoimmune diseases

The tumor necrosis factor has been found to play a role in the pathophysiology of a number of autoimmune diseases. For example, TNFα involved in the activation of inflammation and caused destruction of the joints in rheumatoid arthritis (see, for example, Moeller, A., et al. (1990), Cytokine 2:162-169; U.S. Patent No 5231024 Moeller et al.; European Patent No 260610 B1 Moeller, A.; Tracey K.J. and Cerami, A., supra; Arena W.P. and Dayer J-M. (1995) Arth. Rheum. 38:151-160; R. A. Fava et al. (1993) Clin. Exp. Immunol. 94:261-266). TNFα also involved in stimulating the destruction of the islets cells and in the formation of insulin resistance in diabetes (see, e.g., Tracey and Cerami, supra; PCT Publication No WO 94/08609). TNFα also participated in the appearance of cytotoxicity against oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see, e.g., Tracey and Cerami, supra). Chimeric and humanized mouse antibodies against hTNFα have undergone clinical testing for treatment of rheumatoid arthritis (see, for example, Elliot M.J. et al. (1994); Lancet 344:1125-1127; M.J. Elliot et al. (1994) Lancet 344:1105-1110; Rankin E.C. et al. (1995) Br. J. Rheumatol. 34:334-342).

Human antibodies and antibody fragments, corresponding to the invention, can be used to treat autoimmune diseases, in particular, associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, Allergy, multiple MS who is, autoimmune diabetes, autoimmune uveitis and kidney syndrome. In a typical case, the antibody or fragment antibodies are applied systematically, although some violations can be successful local application of the antibody or antibody fragment of inflammation (for example, local injection into joints, rheumatoid arthritis or external use in diabetic ulcers, alone or in combination with derivatives of cyclohexylidene, as described in PCT Publication No WO 93/19751). Antibody and antibody fragment corresponding to the invention can also be used with one or more additional therapeutic agents used in the treatment of autoimmune diseases, as described later in section III.

C. Infectious diseases

The tumor necrosis factor involved in the occurrence of biological effects observed in a number of infectious diseases. For example, hTNFα participated in the development of inflammation of the brain and thrombosis of capillaries and heart with malaria. TNFα also participated in the development of inflammation of the brain, inducing destruction of the blood-brain barrier, the development of the syndrome of septic shock and activation of venous infarction with meningitis. TNFα was also involved in the induction of cachexia, stimulating the proliferation of viruses and education of damage to the Central nervous system when the syndrome is e acquired immunodeficiency syndrome (AIDS). Accordingly, the antibodies and fragments of antibodies, corresponding to the invention can be used for the treatment of infectious diseases, including bacterial meningitis (see, for example, European Patent application No EP 585705), cerebral malaria, AIDS, and AIDS-associated complex (ARC) (see, for example, European Patent application No EP 230574)and secondary cytomegalovirus infection after transplantation (see, for example, Fietze, E., et al. (1994) Transplantation 58:675-680). Antibodies and antibody fragments, corresponding to the invention, can be used to relieve symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza), and secondary cachexia during infection (e.g., secondary with AIDS or ARC).

D. Transplantation

The tumor necrosis factor considered as a key mediator in graft rejection and disease graft-versus-host (GVHD) and in the formation of the adverse reactions that were observed when rat antibody ACTS directed against a complex of T-cell receptor - CDR, used for the inhibition of rejection of kidney transplants (see, for example, Eason J.D. et al. (1995) Transplantation 59:300-305; Suthanthiran M. and Strom T.V. (1994) New Engl. J. Med. 331:365-375). Accordingly, the antibodies and fragments of antibodies, corresponding to the invention, can be used to inhibit the tworzenia transplant, including the rejection of allografts and xenografts, and for inhibition of GVHD. Although the antibody or antibody fragment can be used separately, preferably using them in combination with one or more agents that inhibit the immune response against the allograft or inhibit GVHD. For example, in one embodiment, the antibody or antibody fragment, corresponding to the invention, used in combination with one or more antibodies directed at other targets involved in regulating immune responses, such as surface molecules CD25 cells (receptor-β interleukin-2), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment, the antibody or antibody fragment, corresponding to the invention, used in combination with one or more of the essential IMMUNOSUPRESSIVE agents, such as cyclosporin a or FK506.

E. Malignant neoplasms

The tumor necrosis factor involved in the induction of cachexia, stimulating tumor growth, increased metastatic potential and the appearance of cytotoxicity in malignant tumors. Accordingly, the antibodies or fragments of antibodies, corresponding to the invention, used in the treatment of malignant tumors to inhibition of tumor growth or metastasis and/or to about what lahcene secondary cachexia in malignant tumors. The antibody or antibody fragment may be administered systemically or locally into the tumor.

F. Pulmonary disorders

The tumor necrosis factor involved in the pathophysiology of respiratory distress syndrome in adults (ARDS), including the stimulation of activated endothelial cells, the orientation of cytotoxicity on the pneumocytes and induction syndrome leaking vessels. Accordingly, the antibodies or fragments of antibodies, corresponding to the invention can be used in the treatment of various lung diseases, including respiratory distress syndrome in adults (see, for example, PCT Publication No WO 91/04054), pulmonary shock, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The antibody or antibody fragment, corresponding to the invention, can be administered with one or more additional therapeutic agent used in the treatment of pulmonary disorders, as described below in section III.

G. Intestinal disorders

The tumor necrosis factor involved in the pathophysiology of inflammatory intestinal disorders (see, for example, Tracy K.J., et al. (1986) Science 234:470-474; Sun X-m, et al. (1988) J. Clin. Invest. 81:1328-1331; MacDonald T.T., et al. (1990) Clin. Exp. Immunol. 81: 301 to 305). Chimeric mouse antibodies against hTNFα have undergone clinical testing for treatment of Crohn's disease (van Dullemen H.M. et al. (1995) Gastroenterology 109:129-135). Human antibody or fra is ment antibodies corresponding to the invention can also be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndrome - Crohn's disease and ulcerative colitis. The antibody or antibody fragment, corresponding to the invention may also be administered with one or more additional therapeutic agent used in the treatment of intestinal disorders, as described below in section III.

N. Heart disorders

Antibodies or fragments of antibodies, corresponding to the invention can also be used to treat various cardiac disorders, including ischemia of the heart (see, for example, European Patent application No EP 453898) and heart failure (weak heart muscle) (see, for example, PCT Publication No WO 94/20139).

I. Other

Antibodies or fragments of antibodies, corresponding to the invention can also be used to treat a variety of disorders in which the activity of TNFα is harmful. Examples of other diseases and disorders in which the activity of TNFα participates in the pathophysiology, and thus can be treated using the antibodies or fragments of antibodies, corresponding to the invention include inflammatory bone disorders, and diseases associated with bone resorption (see, for example, Bertolini / Marian Fisher D.R., et al. (196) Nature 319:516-518; Konig, A., et al. (1988) J. Bone Miner. Res. 3:621-627; Lerner Uriah Heep show; and Ohlin, A. (1993) J. Bone Miner. Res. 8:147-155 and Shankar G. and Stern, P.H. (1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (see, e.g., McClain C.J. and Cohen D.A. (1989) Hepatology 9:349-351; Felver M.E. et al. (1990) Alcohol Clin. Exp. Res. 14:255-259, and J. Hansen et al. (1994) Hepatology 20:461-474), viral hepatitis (Sheron N. et al. (1991) J. Hepatol. 12:241-245 and Hussain M.J. et al. (1994) J. Clin. Pathol. 47:1112-1115) and fulminant hepatitis; clotting disorders (see, for example, van der Poll T. et al. (1990) N. Engl. J. Med. 322:1622-1627; van der Poll T. et al. (1991) Prog. Clin. Biol. Res. 367:55-60; burns (see, for example, Giroir V.R. et al. (1994) Am. J. Physiol. 267:H118-124; Liu X.S. et al. (1994) Burns 20:40-44), reperfusion injury (see, for example, Scales W.E. et al. (1994) Am. J. Physiol. 26:1122-1127; Serrick S. et al. (1994) Transplantation 58:1158-1162; Y.M. Yao et al. (1995) Resuscitation 29:157-168), the formation of keloid zone (see, for example, McCauley R.L. et al. (1992) J. Clin. Immunol. 12:300-308), scar-tissue formation, pyrexia, periodontal disease, obesity and toxicity of radiation.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited in this application, introduced here by reference.

Example 1: Kinetic analysis of binding of human antibodies with hTNFα

What is happening in the moment of interaction between the binding ligand (biotinylated recombinant human hTNFα (rhTNFα)immobilized on mA is the ricks biosensor) and analyte (antibodies in solution) is measured by using surface plasma resonance (SPR), using the BIAcore system (Pharmacia Biosensor, Piscataway, NJ). The system uses the optical properties of SPR for detection of changes in the concentrations of protein in dextranomer matrix biosensor. Proteins covalently linked to dextranomer matrix with known concentrations. Antibodies Inuktitut in dextranase metrics, and specific binding of the injected antibodies and immobilized ligands leads to increased concentration of proteins in the matrix and, as a result, the change of the SPR signal. These changes signal SPR register as units resonance (RU) and expressed relative to the time on the Y-axis sensogram.

To facilitate immobilization of the biotinylated rhTNFα on the matrix biosensor streptavidin covalently linked through free amine groups with dextranomer matrix with carboxyl groups on the matrix, pre-activated with 100 mm N-hydroxysuccinimide (NHS) and 400 mm hydrochloride N-ethyl-N'-(3-diethylaminopropyl)carbodiimide (EDC). Then streptavidin is introduced into the activated matrix. Thirty-five microlitres streptavidin (25 μg/ml)diluted in sodium acetate, pH 4.5, is introduced into the activated biosensor and free amines on the protein directly bind the activated carboxyl group. Unreacted EDC-esters matrix inactivate injection of 1 M ethanolamine. Chips connected the CSOs with streptavidin biosensor are also on sale (Pharmacia BR-1000-16, Pharmacia Biosensor, Piscataway, NJ).

To obtain biotinylated rhTNFα first dissolve 5.0 mg of Biotin (N-hydroxysuccinimidyl ester D-biotinyl-∈-aminocaproic acid; Boehringer Mannheim, Catalogue No 1008 960) and 500 ál of DMSO to obtain a solution of 10 mg/ml Ten microlitres Biotin is added to 1 ml of rhTNFα (2.65 mg/ml) to obtain the molar ratio of Biotin to rhTNFα 2:1. The reaction mixture was gently mixed and incubated for two hours at room temperature in the dark. Column PD-10, Sephadex G-25M (Pharmacia, catalog number 17-0851-01) balance 25 ml of cold PBS (phosphate buffered saline) and put 2 ml of rhTNFα-Biotin on the column. Column elute 10×1 ml of cold PBS. Fractions are collected and determined at OD280 (0,1 OD=1.25 mg/ml). The appropriate fractions are combined and stored at -80°s to use. Biotinylated rhTNFα also commercially available (R & D Systems, Catalog No FTA00, Minneapolis, MN).

Biotinylated rhTNFα for immobilization on the matrix using streptavidin diluted in buffer PBS (Gibco Cat. No 14190-144, Gibco BRL, Grand Island, NY) with the addition of 0.05% (BIAcore) surfactant P20 (Pharmacia BR-1000-54, Pharmacia Biosensor, Piscataway, NJ). To determine the ability of rhTNFα-specific antibodies to bind immobilized rhTNFα analysis of binding performed as follows. The aliquot is biotinylated rhTNFα (25 nm; aliquots of 10 µl) is injected into the streptavidin-linked dextranase matrix a flow rate of 5 μl/min Before injection of the protein and immediately after it pure PBS buffer wash each flow-through chamber. In order to represent the magnitude of the binding of this sample (approximately 500 RU) take the difference between the basic signal and the signal obtained after approximately 30 s after injection of the antibody. Measure direct binding of rhTNFα-specific antibodies with immobilized biotinylated rhTNFα. Antibodies (20 μg/ml) diluted in PBS-buffer and injected aliquots of 25 μl in the matrices with immobilized protein a flow rate of 5 μl/min Before injection of the protein and immediately after it pure PBS buffer wash each flow-through chamber. In order to represent the magnitude of the binding of this sample, we take the difference between the basic signal and the signal obtained after injection of the antibody. The matrices biosensor regenerate using 100 mm HCL before injection of the next sample. To determine the rate constants (Koff), the speed of the link (Kon), the rate of Association (Kaand the rate of dissociation (Kd) use the software for evaluation of the kinetics BIAcore (version 2.1).

In case they are obtained the results of binding of D2E7 (lgG4 antibodies full-length) with biotinylated rhTNα compared with the murine mAb MAK 195 (fragment F[ab')2) are presented below in Table 1.

Table 1

Linking lgG4 D2E7 or MAC 195 with biotinylated rhTNFα
Antibody[Ab], nmrhTNFαassociated RUsAb associated RUsrhTNFα/AbKoffwith-1, (Avg)
D2E726737312151,148,45×10-5
13342015691,305,42×10-5
6743416331,314,75×10-5
3345015321,194,46×10-5
1746012960,983,47×10-5
84869360,672,63×10-5
44895360,38 2,17×10-5
24702440,183,68×10-5
(of 4.38×10-5)
MAC 1954003758811,205,38×10-5
20040010801,384,54×10-5
10041911411,393,54×10-5
5042711061,323,67×10-5
254469571,09to 4.41×10-5
134647080,783,66×10-5
64744330,477,37×10-5
34512310,26 6,95×10-5
(4,94×10-5)

In the second series of experiments the molecular-kinetic interaction between lgG1 form of D2E7 full length and biotinylated rhTNFα analyze quantitatively using the method BIAcore as described above, and the resulting constants of the kinetics of speed are presented below in Tables 2, 3 and 4.

Table 2

Experimentally obtained rate constants of dissociation of the interaction of D2E7 with biotinylated rhTNFα
ExperimentKd(with-1)
19,58×10-5
29,26×10-5
37,60×10-5
Average8,81±1,06×10-5
Table 3

Experimentally obtained rate constants of Association of the interaction of D2E7 with biotinylated rhTNFα
ExperimentKa(M-1with-1)
11,33×105
21,05×105
33,36×105
Average1,91±1,26×105

Table 4

Experimentally obtained constants of the kinetics of the rate and affinity of D2E7 and biotinylated rhTNFα
ExperimentToa(M-1with-1)Kd(with-1)Tod(M)
11,33×1059,58×10-57,20×10-10
21,05×1059,26×10-58,82×10-10
33,36×1057,60×10-5of 2.26×10-10
Average1,91±1,26×1058,81±1,06×10-56,09±3,42×10-10

The rate constants of dissociation and Association calculated by analyzing the plots of dissociation and Association sensogram using software analysis BIA. For the interaction between D2E7 and biotinylated molecules rhTNFα accept conditional values of the kinetics of chemical reactions: the zero order kinetics of dissociation and the first order kinetics of the Association. When choosing molecular models for the analysis of kinetic data is the analysis subjected to the interaction of only one shoulder bivalent antibody D2E7 and one unit of three-dimensional biotinylated rhTNFα . Spend three independent experiments and analyze the results separately. Average received rate constant of dissociation (kdinteraction between D2E7 and biotinylated rhTNFα is 8,81±1,06×10-5with-1and the average received rate constant of Association (ka) is 1,91±1,26×105M-1with-1. The resulting constant internal dissociation (Kd) is then calculated according to the formula: Kd=kd/ka. Thus, the value of Kdantibody D2E7 for rhTNFα, derived from the kinetic parameters, is 6,09±3,42×10-10M. Minor differences in the kinetic values for the form lgG1 antibody D2E7 (presented in Tables 2, 3 and 4) and forms lgG4 D2E7 (presented in Table 1 and Examples 2 and 3) cannot be considered reliable differences due to the presence of constant sites or lgG1 or lgG4, and are viewed as associated with more accurate measurements of the concentrations of the antibodies used in the kinetic analysis lgG1. Accordingly presented here the kinetic values for the form lgG1 D2E7 are considered to be the most accurate kinetic parameters for antibody D2E7.

Example 2. Scanning alanine mutagenesis in the CDR3 domains D2E7.

A series of mutations at the same residue alanine being introduced by standard methods ove the position of the CDR3 domain plots D2E7 VL and VH D2E7. Mutations in the light chain is illustrated by Figure 1B (LD2E7*.A1, LD2E7*.A3, LD2E7*.A4, LD2E7*.A5, LD2E7*.A7 and LD2E7*.A8 with alanine mutation at position 1, 3, 4, 5, 7, or 8, respectively, in the domain CDR3 D2E7 VL). Mutations in the heavy chain is illustrated by Figure 2B (HD2E7*.A1, HD2E7*.A2, HD2E7*.A3, HD2E7*.A4, HD2E7*.A5, HD2E7*.A6, HD2E7*.A7, HD2E7*.A8 and HD2E7*.A9 with alanine mutation at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, respectively, in the domain CDR3 VH D2E7). The kinetics of the interaction of rhTNFα with the antibody containing the VL and VH D2E7 wild-type, compared with the same kinetics of antibody containing 1) D2E7 VL wild-type associated with the alanine-substituted D2E7 VH; 2) D2E7 VH wild type associated with the alanine-substituted D2E7 VL, or 3) the alanine-substituted D2E7 VL associated with the alanine-substituted D2E7 VH. All antibodies tested as molecules lgG4 full length.

The kinetics of the interaction of antibodies with rhTNFα determined using surface plasma resonance, as described in Example 1. Rate constants Kofffor different pairs of VH/VL below in Table 5:

D2E7 VL
Table 5

The binding of alanine-scanbutton D2E7 with biotinylated rhTNFα
VHVLKoff(with-1)
D2E7 VHD2E7 VL9,65×10-5
HD2E7*.A11,4×10-4
HD2E7*.A2D2E7 VL4,6×10-4
HD2E7*.A3D2E7 VL8,15×10-4
HD2E7*.A4D2E7 VL1,8×10-4
HD2E7*.A5D2E7 VL2,35×10-4
HD2E7*.A6D2E7 VL2,9×10-4
HD2E7*.A7D2E7 VL1,0×10-4
HD2E7*.A8D2E7 VL3,1×10-4
HD2E7*.A9D2E7 VL8,1×10-4
D2E7 VHLD2E7*.A16,6×10-5
D2E7 VHLD2E7*.A3not defined
D2E7 VHLD2E7*.A41,75×10-4
D2E7 VHLD2E7*.A51,8×10-4
D2E7 VHLD2E7*.A71,4×10-4
D2E7 VHLD2E7*.A83,65×10-4
HD2E7*.A9LD2E7*.A11,05×10-4

These results demonstrate the, most of the provisions of the CDR3 domains plot VL and plot D2E7 VH suitable for replacement by a single alanine residue. Replacement of one alanine at position 1, 4, 5, or 7 in the domain CDR3 VL D2E7 or in position 2, 5, 6, 8, 9 or 10 in the CDR3 domain D2E7 VH does not have a significant influence on the rate of dissociation binding hTNFα compared to the original antibody D2E7 wild type. Replacement of the alanine in position 8 VL CDR3 D2E7 or in position 3 VH CDR3 D2E7 increases Tooff4 times, and has been replaced by alanine at position 4 or 11 CDR3 D2E7 VH increases Tooff8 times, indicating that these provisions are more critical for binding hTNFα. However, the replacement of one alanine at position 1, 4, 5, 7, or 8 in the domain CDR3 VL D2E7 or in position 2, 3, 4, 5, 6, 8, 9, 10 or 11 in the CDR3 domain D2E7 VH leads to the fact that the antibody against hTNFα hasoff1×10-3with-1or less.

Example 3: Analysis of antibody binding, close D2E7

A series of antibodies with a sequence of close D2E7, analyze binding with rhTNFα compared to D2E7 using surface plasma resonance, as described in Example 1. Amino acid sequences tested plots VL lead on the Figures I 1B. Amino acid sequences tested areas VH lead in Figures 2A and 2B. Rate constants Kofffor different pairs of VH/VL (in the specified format, Lieb is in the form of antibody lgG1 or lgG4 full length, or scFv) are presented below in Table 6:

Table 6

The linking loved ones D2E7 antibody with biotinylated rhTNFα
VHVLFormatKoff(with-1)
D2E7 VHD2E7 VLlgQ1/lgG49,65×10-5
VH1-D2LOE7lgG1/lgG47,7×10-5
VH1-D2LOE7scFv4,6×10-4
VH1-D2.NLOE7.lgG42,1×10-5
VH1-D2.YLOE7.AlgG42,7×10-5
VH1-D2.NLOE7.AigG43,2×10-5
VH1-D2EP B12scFv8,0×10-4
VH1-D22SD4 VLscFv1,94×10-3
CS-H2LOE7scFv1,5×10-3
2SD4 VHLOE7scFv6,07×10-3
2SD4 VH2SD4 VLscFv1,37×10-2
VH1A112SD4 VL scFv1,34×10-2
VH1B122SD4 VLscFv1,01×10-2
VH1B112SD4 VLscFv9,8×10-3
VH1E42SD4 VLscFv1,59×10-2
VH1F62SD4 VLscFv2,29×10-2
VH1D82SD4 VLscFva 9.5×10-3
VH1G12SD4 VLscFv2,14×10-2
2SD4 VHEP B12scFv6,7×10-3
2SD4 VHVL10E4scFv9,6×10-3
2SD4 VHVL100A9scFv1,33×10-2

VHVLFormatKoff(with-1)
2SD4 VHVL100D2scFv1,41×10-2
2SD4 VHVL10F4scFv1,11×10-2
2SD4 VHVLLOE5scFv1,16×10-2
2SD4 VHVLLOF9scFv6,09&x000D7; 10-3
2SD4 VHVLLOF10scFv1,34×10-2
2SD4 VHVLLOG7scFv1,56×10-2
2SD4 VHVLLOG9scFv1,46×10-2
2SD4 VHVLLOH1scFv1,17×10-2
2SD4 VHVLLOH10scFv1,12×10-2
2SD4 VHVL1B7scFv1,3×10-2
2SD4 VHVL1C1scFv1,36×10-2
2SD4 VHVL1C7scFv2,0×10-2
2SD4 VHVL0.1F4scFv1,76×10-2
2SD4 VHVL0.1H8scFv1,14×10-2

Low decomposition rate (i.e. Tooff≤1×10-4with-1for full length antibodies (for example, forms IgG)having a VL selected from the D2E7, LOE7, LOE7.T and LOE7. that contain either threonine or alanine at position 9, indicate that the position 9 CDR3 D2E7 VL may be taken by any of these two residues without significant effect on the Koff. Accordingly, a consensus site CDR3 D2E7 VL has the amino acid on sledovatelnot Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID No 3). Moreover, the low rate of dissociation (i.e. Tooff≤1×10-4with-1for antibody having a VH selected from the D2E7, VH1-D2.N and VH1-D2.Y, which have either tyrosine or asparagine at position 12, indicate that the position 12 CDR3 D2E7 VH may be taken by any of these two residues without significant effect on the Koff. Accordingly, a consensus site for CDR3 D2E7 VH has the amino acid sequence of V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID No 4).

The results are shown in Table 6, demonstrate that the format of scFv antibodies containing the plot CDR3 VL or VH 2SD4 have a higher Koff(ieoff≥1×10-3with-1compared with antibodies containing the plot CDR3 VL or VH D2E7. In CDR3 2SD4 VL differs from the D2E7 in positions 2, 5 and 9. However, as mentioned above, position 9 may be employed Ala (as in 2SD4) or Thr (as D2E7) without significant effect on the Koff. Thus, when comparing 2SD4 and D2E7 position 2 and 5 VL CDR3 D2E7 (both residues) can be identified as critical for the Association of the antibody with hTNFα. These residues may be directly involved in the binding of the antibody, as the contacting residues, or can play a critical role in maintaining the structure of the folding of the molecule antibodies in this area. As regards the importance of position 2 note that replacement of Arg (LOE7, which has the same CDR3 VL, as D2E) to Lys (EP B12) increases the dissociation rate by a factor of two. As regards the importance of position 5, note that replacement of Arg (D2E7) to Ala (LD2E7*.A5), as described in Example 5, also increases the dissociation rate twice. Moreover, without any of Arg at positions 2 and 5 (2SD4) the dissociation rate increases five times. However, it should be noted that, although the position 5 is important for improved binding hTNFαand the change in this position may be negative in respect of changes in other provisions, as can be seen from VLLOE4, VLLOH1 or VLO.1H8.

Among CDR3 2SD4 VH differs from the D2E7 in positions 1, 7 and 12. However, as discussed above, the position 12 may be occupied by Asn (as in 2SD4) or Tyr (as D2E7) without significant effect on the rate constant Koff. Thus, when comparing 2SD4 and D2E7 positions 1 and 7 CDR3 D2E7 VH can be identified as critical for binding with hTNFα. As described above, the data remains can be directly placed in the binding site of the antibody, as the contacting residues, or can play a critical role in maintaining the structure of the folding of the molecule antibodies in this area. Both provisions are important for binding hTNFαbecause, when using the VH CDR3 3C-H2 (which contains a substitution of valine by alanine at position 1 relative to the VH CDR3 D2E7), the scFv has a dissociation rate that is three times higher than when using a VH CDR3 D2E7, but this rate is still four the Aza less than using a VH CDR3 D2E7 (which has changes in both positions 1 and 7 relative to the VH CDR3 D2E7).

Example 4: Functional activity of D2E7

To study the functional activity of this antibody is D2E7 used in several analyses, which determine the ability of antibodies to inhibit the activity of hTNFα either in vitro or in vivo.

A. Neutralization of TNFα-induced cytotoxicity in L929 cells

Human recombinant TNFα (rhTNFα) causes cellular cytotoxicity of murine L929 cells after incubation for 18-24 hours Human antibodies against hTNFα evaluate the analyses using L929 when the joint incubation of antibodies with rhTNFα and cells as follows. In 96-hole tablet for micrometrology containing 100 ál of Ab against hTNFαmake duplicate serial dilution 1/3 the depth of the tablet, using RPMI medium containing 10% fetal calf serum (FBS). Add 50 microliters rhTNFα to a final concentration of 500 PG/ml in each sample well. Then the tablets incubated for 30 min at room temperature. Next, add 50 ál of TNFαand-sensitive murine L929 fibroblasts to a final concentration of 5×104cells per well, including 1 μg/ml of actinomycin D. the Control contains a medium with cells and rhTNFα with cells. These control experience is s and a standard curve of TNFα in the range from 2 ng/ml to 8.2 PG/ml are used to determine the quality of the analysis and obtain a "window" of neutralization. Then the wells incubated overnight (18-24 h) at 37°With 5% CO2.

From each well is removed hundred microlitres environment and add 50 ál of bromide 3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazole (MMT; commercially available from Sigma Chemical Co., St. Louis, MO) in PBS with a concentration of 5 mg/ml. Then tablets incubated for 4 hours at 37°C. Then each well was added 50 μl of a 20%aqueous solution of sodium dodecyl sulfate (SDS) and the plate incubated at 37°With during the night. Measure the optical density at 570/630 nm, build curves for each sample and determine the IC50standard methods.

Typical results for human antibodies having different pairs of VL and VH in comparison with the murine mAb MAK 195 shown below in Figure 3 and in Table 7.

Table 7

Neutralization of TNFα-induced cytotoxicity of L929
VHVLStructureIC50M
D2E7D2E7scFv1,1×10-10
D2E7D2E7lgG44,7×10-11
2SD42SD4scFv/lgG1/lgG4 3,0×10-7
2SD4LOE7scFv4,3×10-8
VH1-D22SD4scFv1,0×10-8
VH1-D2LOE7scFv/lgG1/lgG43,4×10-10
VH1.D2.YLOE7.lgG48,1×10-11
VH1-D2.NLOE7.lgG41,3×10-10
VH1-D2.YLOE7.AIgG42,8×10-11
VH1-D2.NLOE7.AIgG46,2×10-11
MAC 195MAC 195scFv1,9×10-8
MAC 195MAC 195F(ab')26,2×10-11

The results presented on Figure 3 and in Table 7, demonstrate that the human antibody D2E7 against hTNFα and various close D2E7 antibodies neutralize TNFαinduced cytotoxicity of L929 activity approximately equal to the activity of murine mAb MAK 195 against hTNFα.

In another series of experiments examined the capacity forms lgG1 D2E7 to neutralize TNFαinduced cytotoxicity of L929, as described above. The results of three independent experimental now and their average value is presented below in Table 8:

Table 8

Neutralization of TNFα-induced cytotoxicity of L929 through lgG1 D2E7
ExperimentIC50[M]
11,26×10-10
21,33×10-10
31,15×10-10
Average1,25±0,01×10-10

This series of experiments confirms that D2E7 in the form of lgG1 full length neutralizes TNFαinduced cytotoxicity of L929 with a mean IC50[M] 1,25±0,01×10-10.

C. Inhibition of binding TNFα TNF receptorsα on the cells U-937

The ability of human antibodies against TNFα to inhibit the binding of hTNFα with receptors hTNFα on the surface of cells examined using the cell line U-937 (ATCC No CRL 1593), a line of histiocytes person, which expresses the receptor hTNFα. Cells U-937 grown in RPMI medium with addition of 10% fetal calf serum (Hyclone A-1111, Hyclone Laboratories, Logan, UT), L-glutamine (4 nm), HEPES buffer solution (10 mm), penicillin (100 μg/ml) and streptomycin (100 µg/ml). To study the activity of IgG antibodies full-length cells U-937 pre-incubated with PBS with the addition of 1 mg/ml human IgG (Sigma I4506, Sigma Chemical Co., St. Louis, MO) for 45 min on ice and then the cells three times washed with binding buffer. To analyze the binding of the receptor cells U-937 (5×106cells/well) are incubated in binding buffer (PBS with addition of 0.2% bovine serum albumin) in 96-well tablets for micrometrology (Costar Corp., Cambridge, MA) together with labeled125I rhTNFα (3×10-10M; 25 mccoury/ml, obtained from NEN Research Products, Wilmington, DE) in the presence or without antibodies against hTNFα in a total volume of 0.2 ml Tablets incubated on ice for 1.5 hours Then 75 μl of each sample is transferred into test tubes with a capacity of 1.0 ml (Sarstedt 72.700, Sarstedt Corp., Princeton, NJ)containing dibutyl phthalate (Sigma-D-2270, Sigma Chemical Co., St. Louis, MO) and dinonylphenol (ICN 210733, ICN, Irvine, CA). The test tubes containing 300 μl of a mixture of dibutyl phthalate and dinnerplate in volumetric ratio of 2:1, respectively. Free (i.e. unbound) labeled125I rhTNFα remove with microcentrifuge within five minutes. Then the end of each of the test tubes containing sediment cells cut by scissors for microtubes (Bel-Art 210180001, Bel-Art Products, Pequannock, NJ). Sediment cells contains labeled125I rhTNFαassociated with the receptor of TNFα p60 or P80, whereas the aqueous phase above the oil mixture contains an excess of free labeled125I rhTNFα. All precipitation cells collect in the tube defined by the I (Falcon 2052, Becton Dickinson Labware, Lincoln Park, NJ) and shall count in scintillation counter.

Illustrative results are shown in Figure 4. The value of the IC50for inhibition through binding of D2E7 hTNFα with receptors hTNFα on the cells U-937 is in these experiments is approximately 3×10-10M. These results indicate that the human antibody D2E7 against hTNFα inhibits binding of rhTNFα with receptors hTNFα on the cells U-937 at concentrations approximately equal to the concentrations of mAb MAK 195 against hTNFα.

In another series of experiments, the ability to form lgG1 D2E7 to inhibit the binding of rhTNFα with receptors hTNFα on the cells U-937 examined as described above. The results of three independent experiments and their average are shown in Table 9.

Table 9

Inhibition of binding of the receptor TNFα on the cells U-937

through lgG1 D2E7
ExperimentIC50[M]
11,70×10-10
21,49×10-10
31,50×10-10
Average1,56±0,12×10-10

This series of experiments confirms that D2E7 in the form of lgG1 full length inhibits SV is the statements of the TNF receptor on the cells U-937 with an average IC 50[M] 1,56±0,12×10-10.

To study inhibitory activity of D2E7 when linking125I-rhTNFαassociated with individual receptors P55 and P75, conduct solid-phase radioimmunoassay. To determine the values IC50D2E7 in relation to selected receptors TNF various concentrations of the antibody is incubated with a concentration of 3×10-10125I-rhTNFα. The mixture is then tested on a separate cups containing TNF receptors P55 or P75 dose-dependent way. The results are presented below in Table 10:

Inhibition of binding125l-rhTNFα TNF receptors P55 and P75 in the cells U-937 with D2E7 is accompanied by a simple sigmoid curve, showing similar values IC50for each receptor. In experiments using solid-phase radioimmunoassay (RIA) with recombinant TNF receptors values IC50for inhibition of binding125l-rhTNFα TNF receptors P55 and P75 with D2E7 calculated as 1,47×10-9and 1.26×10-9M, respectively. Lower values IC50in the solid phase, possibly due to the high density of receptors in the form of RIA, since unlabeled rhTNFα also inhibited with similar values IC50. The values of the IC50for inhibition of binding125l-rhTNFα with receptors P55 and pee rhTNFα are 2,31×10-9and 2.70×10-9m, respectively.

C. Inhibition of the expression of ELAM-1 on HUVEC

You can induce the expression of human endothelial cells of the umbilical vein (HUVEC) adhesion molecule 1 and endothelial leukocyte (ELAM-1) on the cell surface processing rhTNFαthat can be determined by reaction of rhTNFα-treated HUVEC with mouse antibody against ELAM-1 person. The ability of human antibodies against hTNFα to inhibit this TSRα-induced expression of ELAM-1 on HUVEC examined as follows: HUVEC (ATCC N0 CRL 1730) placed in 96-well plates (5×104cells/well) and incubated overnight at 37°C. the next day, prepare a serial dilution of human antibodies against hTNFα (1:10) in microlaterolog Cup, starting with 20-100 μg/ml antibody. The mother liquor rhTNFα prepared with a concentration of 4.5 ng/ml, aliquots rhTNFα add each containing the antibody well and well mix the contents. Control experiments include only the environment, environment plus antibody against hTNFα and Wednesday plus rhTNFα. Stop incubation tablets with HUVEC at 37°and the environment is carefully removed by suction from each hole. Two hundred microlitres mixture of antibody-rhTNFα transferred to each well of tablets with HUVEC. Then the tablets with HUVEC further incubated at 37°C for 4 hours is. Then the mother liquor mouse antibodies against ELAM-1 was diluted in the ratio 1:1000 in RPMI. The environment from each of the wells with HUVEC gently sucked off, add 50 ál per well of a solution of antibodies against ELAM-1 and tablets with HUVEC incubated for 60 minutes at room temperature. Solution125I-labeled antibody against mouse Ig prepared in RPMI (approximately 50,000 pulses/min 50 ál). The medium from each well of tablets with HUVEC carefully removed by aspiration, and the wells washed twice RPMI and to each well add 50 ál of solution125I-labeled Ig against mouse. Tablets incubated for one hour at room temperature, and then each well three times washed with RPMI. One hundred and eighty microlitres 5% SDS (sodium dodecyl sulfate) is added to each well for lizirovania cells. Cell lysate from each well is then transferred into a test tube and counted in the scintillator.

The obtained results are shown in Figure 5. The value of the IC50for inhibition using D2E7 hTNFα-induced expression of ELAM-1 on HUVEC is in these experiments is approximately 6×10-11M. these results demonstrate that the human antibody against hTNFα D2E7 inhibits hTNFα-induced expression of ELAM-1 on HUVEC at concentrations approximately equal concentrations of murine mAb MAK 195 against hTNFα.

In another series of experts who cops investigate the ability of the form lgG1 D2E7 to inhibit hTNFα -induced expression of ELAM-1 on HUVEC as described above. The results of three independent experiments and the average value of them is presented below in Table 11:

Table 11

Inhibition of TNFα-induced expression of ELAM-1 receptor lgG1 D2E7
ExperimentIC50[M]
11,95×10-10
21,69×10-10
31,90×10-10
Average1,85±0,14×10-10

This series of experiments confirms that D2E7 in the form of lgG1 full length inhibits TNFα-induced expression of ELAM-1 on HUVEC with an average IC50[M] 1,85±0,14×10-10.

Neutralizing activity lgG1 D2E7 examined in relation to rhTNFα-induced expression of two other adhesion molecules ICAM-1 and VCAM-1. Because titration curve rhTNFα for the expression of ICAM-1 after 16 hours very close to the curve of the expression of ELAM-1, using the same concentration of rhTNFα in experiments on neutralization with antibodies. HUVEC incubated with rhTNFα in the presence of various concentrations of D2E7 at 37°C incubator with CO2for 16 hours, and measuring the expression of ICAM-1 with mouse antibodies against ICAM - and then 125I-labeled sheep antibodies against mouse. Spend two independent experiment and calculate values of IC50. Unrelated human lgG1 antibody did not inhibit the expression of ICAM-1.

Experimental testing method of inhibiting the expression of VCAM-1 is the same as the way of expression of ELAM-1, except that instead of Mab against ELAM-1 using Mab against VCAM-1. Spend three independent experiments and calculate values of IC50. Unrelated human lgG1 antibody did not inhibit the expression of VCAM-1. The results are summarize in the following Table 12:

Table 12

Inhibition of the expression of ICAM-1 and VCAM-1 through lgG1 D2E7
Inhibition of ICAM-1IC50[M]
ExperimentIC50[M]ExperimentIC50[M]
11,84×10-1011,03×10-10
22,49×10-1029,26×10-11
31,06×10-10
Average2,17±0,46×10-10Average1,01±0,01×10-10

These experiments demonstrate that treatment of primary endothelial cells of the umbilical vein of a person rhTNFα leads to optimal expression of adhesion molecules: ELAM-1 and VCAM-1 after 4 hours and the maximum regulated expression of ICAM-1 after 16 hours. D2E7 is able to inhibit the expression of three adhesion molecules in a dose-dependent manner. The value of the IC50for inhibition of ELAM-1, ICAM-1 and VCAM-1 amount to 1.85×10-10, 2,17×10-10and 1.01×10-10M, respectively. These values are very close, which indicates that similar requirements to the dose of the activation signal rhTNFαrequired for the induction of expression of ELAM-1, ICAM-1 and VCAM-1. Interestingly, D2E7 has similar effectiveness when analyzing longer inhibiting the expression of ICAM-1. Analysis of the inhibition of ICAM-1 requires a 16-hour joint incubation rhTNFα and D2E7 with HUVEC in contrast to the 4 hours required for analysis of inhibition of ELAM-1 and VCAM-1. As D2E7 has a low decay rate in respect of rhTNFαit is clear that during the 16-hour period joint incubation no significant competition for TNF receptors on HUVEC.

D. Neutralization of rhTNFα in vivo

Three different systems used for in vivo demonstration that the D2E7 is effective in the inhibition of the activity of rhTNFα in vivo.

I. Inhibition of TNF-indutsirovanno the death of the D-galactosamine-sensitized mice

Injection of recombinant human TNFα (rhTNFα) D-galactosamine-sensitized mice causes death within 24-hour period of time. TNFαneutralizing agents are shown to prevent death in this model. To study the ability of human antibodies against hTNFα to neutralize rhTNFα in vivo in this model, mice C57B1/6 intraperitoneally (I.P. Pavlova.) injected with different concentrations D2T7-lgG1 or control protein in PBS. After 30 minutes, mice injected with 1 mg of rhTNFα and 20 mg D-galactosamine in PBS I.P. Pavlova. and observe after 24 hours. This number rhTNFα and D-galactosamine, as previously set, causes 80-90% mortality in these mice. The results, presented as a graph in the form of columns survival in % relative to the concentration of antibodies is shown in Figure 6. Black bars indicate D2E7, while the shaded bars indicate the POPPY 196. Injection of 2.5-25 µg of the antibody D2E7 each mouse protects animals from TNFα-induced death. The value of the ED50approximately 1-2,5 μg per mouse. Antibodies positive control MAC 195 close on its protecting ability. Injection of D2E7 in the absence of rhTNFα does not cause any harmful effect on mice. Injection of non-specific human antibody lgG1 not give any protection from TNFα-induced death.

In the second experiment, forty-nine mice divided into 7 equal groups. Each group receives a different dose of D2E7 for thirty minutes to obtain a dose LD80a mixture of rhTNFα/D-galactosamine (10 µg rhTNFα and 20 mg D-galactosamine on the mouse). The control unit 7 receives a normal human Kappa-lgG1 antibody at a dose of 25 μg per mouse. Mice examined after 24 hours. The survival rate for each group are presented below in Table 13.

Table 13

Survival at 24 h after treatment D2E7
GroupSurvival (live/total)Survival, %
1 (without antibody)0/70
2 (1 µg)1/714
3 (2.6 mg)5/771
4 (5,2 μg)6/786
5 (26 g)6/786
6 (26 µg; without rhTNF)7/7100
7 (25 µg Hu lgG1)1/714

II. Inhibition of TNF-induced hyperthermia in rabbits

Examine the effectiveness of D2E7 in the inhibition of rhTNFα-induced hyperthermia (fever) in rabbits. Groups of three female NZW rabbits with mass priblizitel is but 2.5 kg each injected intravenously D2E7, rhTNFα and immune complexes D2E7 and rhTNFα. Rectal temperature measured by the temperature probes on thermograph Caue every minute for about 4 hours. Recombinant human TNF in saline solution, injected at a dose of 5 mg/kg, causes a temperature rise of more than 0.4°approximately 45 minutes after injection. The drug antibody in saline solution at a dose of 138 mg/kg does not cause a temperature rise in rabbits even after 140 minutes after injection. In all further experiments D2E7 or control reagents (human IgGq or saline vehicle) is injected to rabbits intravenously 15 minutes after intravenous injection of rhTNFα in saline solution at a dose of 5 mcg/kg Representative results of some experiments are presented below in Table 14:

Tablica

Inhibition of rhTNFα-induced hyperthermia in rabbits using D2E7
Dose of D2E7 (ug/kg)The temperature increase*, °% inhibition**The molar ratioPeak temperature
rhTNFrhTNF + D2E7D2E7: rhTNFminutes after rhTNF
140,53 0,2553160
240,430,13701,640
480,530,03943,350
1370,530,001009,560
7920,800,001005560
* peak temperature

** - % inhibition - (1-{temperature increase during rhTNF & D2E7/temperature increase during the same rhTNF})·100.

Intravenous pretreatment D2E7 at a dose of 14 mg/kg partially inhibits the pyrogenic response in comparison with rabbits, which previously administered only saline. D2E7, introduced at a dose of 137 mg/kg, completely suppresses the pyrogenic reaction rhTNFα in this experiment. In the second experiment, D2E7, introduced at a dose of 24 µg/kg, also partially suppresses the pyrogenic response in comparison with rabbits, which previously administered only saline. The molar ratio of D2E7 to rhTNFα is in this experiment 1/6:1. In the third experiment, D2E7, injectable intravenously at a dose of 48 mg/kg (molar ratio of D2E7: rhTNFα=3.3:1)completely suppresses the pyro is military response in comparison with rabbits, which pre-enter human lgG1 (control) in saline at a dose of 30 µg/kg In the last experiment in rabbits, which pre-enter D2E7 (792 mg/kg) at a very high molar ratio with rhTNFα (55:1), there is no temperature rise within 4 hours of observation. Treatment of rabbit immune complexes obtained from a mixture of D2E7 and rhTNFα, which is incubated at 37°C for 1 hour with a molar ratio of 55:1, without the subsequent introduction rhTNFα also does not lead to any increase of temperature in this experiment.

III. Prevention of arthritis in transgenic mice Tg197

The effect of D2E7 on the development of the disease investigated in models of arthritis in transgenic mice. Get Tg197 transgenic mice that Express human wild-type TNF (modified at 3'-site after the coding sequence), and these transgenic mice develop chronic polyarthritis with 100% probability at the age of 4-7 weeks (see EMBO J. (1991) 10:4025-4031 for further description of the model of polyarthritis in Tg197).

Transgenic mice identified by PCR in three days of age. Progeny transgenic mice are divided into six groups. Transgenic mice verify hybridization analysis type shotblasting at the age of 15 days. Provide the following treatment protocols for six g is UPP: Group 1 = no treatment; Group 2 = saline (media); Group 3 = D2E7, 1.5 mcg/g; Group 4 = D2E7, 15 µg/g; Group 5 = D2E7, 30 ág/g and Group 6 = control lgG1 isotype, 30 μg/g Seed with necroshine mice also include in the study as control (Group 7 - necroshine, without treatment). Each group will be given three intraperitoneal injections per week in accordance with prescribed treatment. Injections continue for 10 weeks. Every week register macroscopic changes in the morphology of the joints for each animal. At 10 weeks all animals killed and tissues of mice kept in formalin. Conduct a microscopic examination of tissue.

The weight of the animal in grams is determined for each mouse at the beginning of each week. At the same time determine the size of the joint (in mm) as an indicator of the severity of the disease. The size of the joint is defined as the average of three measurements rear right ankle using a micrometer. Indicators of arthritis register on a weekly basis as follows: 0 = no arthritis (normal appearance and flexion); + = weak arthritis (deformation of the joint); ++ = moderate arthritis (swelling, deformation of the joint), and +++ = heavy arthritis (ankylosis, as defined by flexion and highly complicated movement). Histopathological indicators on the basis of staining of sections of the joints hematoxylin/eosin indicate as follows: 0 = UTS is DTIE defined disease; 1 = the proliferation of the synovial membrane; 2 = significant synovial seal; 3 = destruction of cartilage and bone erosion.

The effect of treatment with D2E7 on the average size of the joint transgenic mice with arthritis Tg197 shown in the graph of Figure 9. Histopathological indicators and indicators of arthritis in transgenic mice TD at the age of 11 weeks summarised below in Table 15:

Table 15

The effect of D2E7 on the histopathology and the rate of arthritis in mice Tg 197
GroupThe treatmentHistopathologic indicatorFigure arthritis
1without treatment3 (7/7)+++ (7/7)
2saline3(8/8)+++ (8/8)
6control IgG13(9/9)+++ (7/9)
3D2E7, 1.5 mcg/g0(6/8)0 (8/8)
4D2E7, 15 µg/g0(7/8)0 (8/8)
5D2E7, 30 µg/g0(8/8)0 (8/8)

This experiment demonstrates that the antibody D2E7 has a favorable effect in relation to transgenic mice expressing human TNF (g197) wild-type, expressed in the absence of arthritis after treatment period.

E. Neutralization of rhTNFα other species with D2E7

The specificity of binding of D2E7 examined by determining their ability to neutralize factors tumor necrosis other species of primates and mouse, using the analysis of the cytotoxicity of L929 (as described above in Example 4, subsection (A). The results are presented below in Table 16:

Table 16

The ability of D2E7 to neutralize TNF different species in the analysis with L929
TNFα*SourceIC50For neutralization of D2E7 (M)**
PeopleRecombinant7,8×10-11
ChimpanzeesLPS-stimulated RVMS5,5×10-11
BaboonRecombinant6,0×10-11
IgrankaLPS-stimulated RVMS4,0×10-10
CynomolgusLPS-stimulated RVMS8,0×10-11
RHLPS-stimulated RVMS3,0×10-11
DogLPS-stimulated WBC2,2×10-1
PigRecombinant1,0×10-7
MouseRecombinant>1,0×10-7

The results in Table 16 show that D2E7 can neutralize the activity of TNFα five primates, approximately equivalent to human TNFα and, moreover, can neutralize the activity of the canine TNFα (approximately 10 times weaker than human TNFα), and porcine and murine TNFα (approximately ˜1000 times weaker than human TNFα). Moreover, the binding of D2E7 with soluble phase rhTNFα not inhibited by other cytokines, such as lymphokine (TNFβ), IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IFNγ and TGFβ, indicating that the D2E7 is very specific in relation to its ligand TNFα.

F. No release of cytokines in whole blood of man, inkubiruemykh with D2E7

In this example, investigate the ability of D2E7 to induce the secretion of cytokines or highlight cell surface molecules in normal human blood cells. D2E7 incubated with diluted whole blood from three different normal donors at various concentrations for 24 h LPS-positive control is carried out in the same time using predetermined concentrations, stimulating the secretion of cytokines immunocompetent blood cells. Supernatant collect and test panel kits ELISA out of ten of soluble cytokines, receptors and adhesion molecules: IL-1α, IL-1β, receptor antagonist IL-1, IL-6, IL-8, TNFβsoluble receptor I, TNF soluble receptor II TNF, soluble ICAM-1 and soluble E-selectin. The co-incubation of the antibody D2E7 at concentrations up to 343 mg/ml not determined any significant quantities of cytokines or secreted cell surface molecules. Control cultures without added antibodies also do not produce cytokines in defined quantities, whereas the control joint culture LPS gives higher values in the interval from many picograms to a few nanograms. These results indicate that D2E7, does not induce the secretion of cytokines or allocation of cell surface proteins in excess of the normal levels in cultures ex vivo.

Part of this description is the attached List of Sequences, the contents of which are presented in the table below:

Table 17
SEQ ID No:Chain antibodiesPlotSequence type
1D2E7VLamino acid
2D2E7 VHamino acid
3D2E7VL CDR3amino acid
4D2E7VH CDR3amino acid
5D2E7VL CDR2amino acid
6D2E7VH CDR2amino acid
7D2E7VL CDR1amino acid
8D2E7VH CDR1amino acid
92SD4VLamino acid
102SD4VHamino acid
112SD4VL CDR3amino acid
12EP B12VL CDR3amino acid
13VL10E4VL CDR3amino acid

SEQ ID No:Chain antibodiesPlotSequence type
14VL100A9VL CDR3amino acid
15VLL100D2VL CDR3amino acid
16 VLLOF4VL CDR3amino acid
17LOE5VL CDR3amino acid
18VLLOG7VL CDR3amino acid
19VLLOG9VL CDR3amino acid
20VLLOH1VL CDR3amino acid
21VLLOH10VL CDR3amino acid
22VL1B7VL CDR3amino acid
23VL1C1VL CDR3amino acid
24VLO.1F4VL CDR3amino acid
25VLO.1H8VL CDR3amino acid
26LOE7.AVL CDR3amino acid
272SD4VH CDR3amino acid
28VH1B11VH CDR3amino acid
29VH1D8VH CDR3amino acid
30VH1A11VH CDR3amino acid
31VH1B12VH CDR3amino acid
32VH1E4VH CDR3amino acid
33VH1F6VH CDR3amino acid

SEQ ID No:Chain antibodiesPlotSequence type
34CS-H2VH CDR3Amino acid
35VH1-D2.NVH CDR3Amino acid
36D2E7VLnucleic acid
37D2E7VHnucleic acid

Equivalent solutions

Specialists recognize or be able to ascertain using no more than routine investigations, that there are many solutions, equivalent to a specific implementation options described here. It is envisaged that such equivalent solutions are covered by the following claims.

1. The selected human antibody or antigen-binding fragment having a variable area light chain (LCVR), containing the amino acid sequence of SEQ ID No 1, and variable plot heavy chain (HCVR), containing the amino acid sequence of SEQ ID No 2, and binding of human TNFα.

2. The selected human antibody according to claim 1, characterized in that it is an Fv fragment of the same chain.

3. The selected human antibody according to claim 2, characterized in that it is a recombinant antibody or antigen-binding fragment.

4. The selected human antibody according to any one of claims 1 to 3, characterized in that it is designed for inhibiting the activity of human TNFα the person suffering the surrounding abuse when the activity of TNFα is harmful.

5. The selected human antibody according to any one of claims 1 to 3, characterized in that it is intended for the production of a medicinal product for treating disorders in which the activity of TNFα is harmful.

6. The selected human antibody according to any one of claims 1 to 3, characterized in that it is intended for use in therapy.

7. The selected human antibody according to any one of claims 1 to 3, characterized in that it is intended for use in therapy in combination with at least one additional therapeutic agent for treating disorders in which the activity of TNFα is harmful.

8. The selected nucleic acid containing the nucleotide sequence of SEQ ID No 36 encoding a variable area light chain antibodies containing the amino acid sequence of SEQ ID No 1.

9. The selected nucleic acid of claim 8, characterized in that it is included in a recombinant expression vector.

10. The selected nucleic acid containing the nucleotide sequence of SEQ ID No 37, encoding a variable plot heavy chain antibodies containing the amino acid sequence of SEQ ID No 2.

11. The selected nucleic acid of claim 10, characterized in that it is included in a recombinant expression vector.

12. Ryoko is pinentry expression vector, comprising the nucleic acid represented in SEQ ID 36 and 37, and expressing the variable region of the heavy and light chains of the antibody according to claim 1.

13. The use of cell lines of Chinese hamster ovary CHOdhfr-as host cells in which the introduced recombinant expression vector according to item 12, expressing the antibody according to claim 1.

14. The method of synthesis of human antibodies, as described in claim 1, which binds to human TNFαand, wherein the cultured host cell according to item 13 in the medium for cultivation as long as human antibody that binds human TNFα, is synthesized by the cell.

15. Pharmaceutical composition for treating disorders in which the activity of TNFα is harmful, containing the antibody or antigen-binding fragment according to claim 1, and optionally at least one therapeutic agent and a pharmaceutically acceptable carrier.

16. A method of inhibiting the activity of human TNFα, characterized in that exercise contacting human TNFα with the antibody or antigen-binding fragment according to claim 1 and inhibit the activity of human TNFα.

17. The method according to item 16, wherein the person suffering from disorders in which the activity of TNFα is harmful, enter the antibody or the anti-Christ. Yong-binding fragment according to claim 1 and inhibit the activity of human TNFα .



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: invention is directed to using microbiological methods in petrochemical field and can be used to simultaneously prepare vitamin-rich biomass and dewaxed component of motor fuel. Method of invention consists in that yeast, preparing Candida maltoza, is grown under aeration conditions nutritive medium composed of, mg/L: phosphorus 120.0, potassium 20.0, magnesium 33.0, iron 20.0, zinc 6.0, manganese 2.9, residual nitrogen 190.0, and petroleum distillates, as carbon source wherein ratio of n-alkanes to aromatic hydrocarbons ids 1:(1-3). Cultivation is carried out in flow mode at flux velocity 0.12-0.3 h-1.

EFFECT: increased yield of biomass and protein content therein at no loss in quality of motor fuel.

1 tbl, 2 ex

FIELD: biotechnology, in particular mammalian cell cultivation and protein production from mammalian cells.

SUBSTANCE: claimed medium represents synthetic medium containing soy hydrolyzate in amount of 0.1-100 g/l, wherein at least 40 % of said hydrolyzate has molecular weight 500 D or less. Optionally medium contains buffer, antioxidant, etc. Cell cultivation in claimed medium provides both increased yield of recombinant cells and productivity thereof (increased protein yield).

EFFECT: universal medium for method selection for mammalian cell cultivation.

14 cl, 5 dwg, 7 tbl, 9 ex

FIELD: biotechnology, virology.

SUBSTANCE: invention relates to preparing a new strain of hybrid cells of Mus musculus L., NIIMB-280 (9E2), as a producer of monoclonal antibodies to the West Nile virus (WNV) protein E. West Nile virus (strain WNV/LEIV-VIg99-27889) is isolated in Volgograd district in 1999 year from a patient. Producing monoclonal antibodies can be used effectively for detection of the strain WNV/LEIV-VIg99-27880 of WNV that causes human diseases in Russia territory. New hybrid strain of cells is obtained by fusion of murine myeloma cells p3-X63/Ag8.653 (NS0/1) with murine splenocytes BALB/c immunized with the purified and inactivated preparation WNV (strain WNV/LEIV-VIg99-27889). The strain of hybrid cells Mus musculus L., NIIMB-280 (9E2), is deposited in Collection of cellular cultures of NII cellular cultures GNTS VB "VEKTOR" at number № NIIMB-280. Author's name of hybridoma cellular strain is 9E2. Using hybridoma allows preparing specific monoclonal antibodies raised to the West Nile virus protein E that, in turn, gives a possibility for identification of WNV and to standardize the content of protein E in immunodiagnostics.

EFFECT: valuable properties of strain.

1 dwg, 3 ex

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: biotechnology, medicine, oncology, peptides.

SUBSTANCE: invention relates to a method based on phage display for preparing peptides interacting specifically with mammary Ehrlich tumor and can be used in therapy and diagnosis of malignant neoplasm. Peptides are prepared by affinity selection from phage peptide libraries comprising ten millions of different peptides of size 15 amino acid residues, the group of nine peptides wherein each peptide shows ability for accumulation in Ehrlich tumor. For practice using mimetic-peptides selected by such manner can be prepared by chemical synthesis and to use for preparing conjugates on their basis with the known cytotoxic preparations, radioactive isotopes and they can be incorporated in the composition of liposomal preparations for visualization of tumor neoplasm also.

EFFECT: valuable medicinal properties of peptides.

2 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: heterologous protein is obtained by cultivation of strain Corynebacterium glutamicus AJ 12036, which does not produce cell surface protein and contains gene expressing construct wherein nucleic acid sequence encoding signal peptide of cell surface protein of Corynebacterium glutamicus or C. ammoniagenes is bound in direct direction to promoter sequence, and nucleic acid sequence encoding heterologous protein is bound in direct direction to abovementioned nucleic acid sequence encoding signal peptide. Further heterologous protein secreted from cells is isolated.

EFFECT: high effective method for heterologous protein production.

6 cl, 8 tbl, 10 ex

FIELD: medicine, allergology, toxins, pharmacy.

SUBSTANCE: invention relates to recombinant allergens of insect venom and to specific methods for their preparing, in particular, antigen 5 of wasp venom allergen. Recombinant antigen 5 is prepared in bacterial cells as insoluble aggregates followed by their denaturation and transfer to a soluble monomeric allergen. Transfer is carried out by dialysis using acid buffer solution (pH = 3.5-6.5) that can comprise guanidine hydrochloride, or by using a cysteine-containing solvent. Based on describes methods the practically pure recombinant antigen 5 of wasp venom allergen is isolated and used in pharmaceutical composition for hyposensibilization of body to wasp venom allergen. Invention provides preparing protein with reduced reaction capability JgE owing to it can be used in immunotherapy in treatment of allergy.

EFFECT: improved preparing method, valuable properties of allergen.

8 cl, 1 dwg

FIELD: immunology; treatment of mediated diseases IL-1 and failures.

SUBSTANCE: bonding molecule IL-1β which is antibody to human IL-1β and especially human antibody to human IL-1β where hypervariable sections CDRs of heavy and light chains have definite amino acid sequences. Antibody may be used for treatment of mediated disease IL-1, for example osteoarthritis, osteoporosis and other inflammatory processes of bones of rheumatism or podagra nature. Constructions of deoxyribonucleic acid are described which code heavy and light chains or their fragments and expressive vectors which may be replicated in cells including deoxyribonucleic acid constructions. Method of obtaining bonding molecule IL-1β by means of cell transformed by vector is described. Proposed antibody may be used both in prophylactic and treatment of diseases.

EFFECT: enhanced efficiency.

15 cl, 3 dwg, 5 ex

FIELD: medicine, immunobiology, pharmacy.

SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.

EFFECT: valuable medicinal properties of antibody.

75 cl, 78 dwg, 14 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

FIELD: biotechnology, in particular biosensors.

SUBSTANCE: claimed method includes production of sensitive cells producing in exited state signals being detectable by peripheral device. In one embodiment cells cultivated in cell cultures prepared form animal receptor cells are used as sensitive cells. In another embodiment as sensitive cells receptor cells which are functionally analogous to animal receptor cells cultivated in cell cultures prepared form animal stem cells are used. As peripheral device electrical signal receiver is used, wherein said electrical signal is generated by cell in exited state. Claimed invention is useful both in investigations and in industry.

EFFECT: biosensors with increased sensitivity, accuracy and integrity.

14 cl, 1 dwg, 4 ex

FIELD: biology, genetic engineering, biotechnology, medicine.

SUBSTANCE: invention relates to preparing glycosylated polypeptide (glycoprotein) as a component of human erythropoietin by using the technology of recombinant DNAs. This polypeptide shows ability to increase production of reticulocytes and erythrocytes, to enhance the level of hemoglobin synthesis and consumption of iron by marrow cells and characterized by the higher molecular mass as compared erythropoietin isolated from human urine. Invention describes variants DNA sequences encoding this polypeptide that comprise vector constructions with these sequences, a method for preparing transformed mammalian cell lines producing the recombinant human erythropoietin, and a method for its preparing and purification. Also, invention proposes pharmaceutical compositions comprising glycosylated polypeptide (glycoprotein) of erythropoietin as an active component. Applying this invention provides scaling the process for preparing active human erythropoietin useful for its using in medicine.

EFFECT: improved preparing method, valuable properties of polypeptide.

10 cl, 4 dwg, 21 tbl, 12 ex

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

The invention relates to biotechnology and can be used to obtain Malinov human IL-4 with an activating T-cell activity and reduced activating endothelial cells activity

The invention relates to biotechnology, in particular to the creation of transgenic plants with insecticidal properties

The invention relates to the field of genetic engineering and can be used in the biomedical industry

FIELD: medicine.

SUBSTANCE: method involves increasing receptor activity, that is activated with proliferating agent of peroxis, by introducing NADP+-dependent isocitrate dehydrogenase (IDPc), IDPc gene or NADPH, selecting antisense molecule inhibiting fat deposit or nitroglycerides and cholesterol production using IDPc gene, and treating metabolism disorder diseases like adiposity, hyperlipiemia or hepatic fat infiltration by introducing antisense molecule of IDPc gene or IDPc gene inhibitor like oxalomalic acid or methyl isocitric acid.

EFFECT: enhanced effectiveness of treatment; reduced NADP level in cells.

5 cl, 10 dwg

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