Improved anti-ige antibody (options) and method for improving polypeptides
The invention relates to biotechnology, in particular to the immunoglobulin E (IgE), IgE antagonists, anti-IgE antibodies capable of binding to human IgE, and to a method of improving polypeptides, including anti-IgE antibodies. This invention allows to obtain improved polypeptides, including antibodies, which not only show “deaktivirovana” when isomerization of aspartyl, but also have an affinity for the molecule to the target, such as antigen. 11 N. and 5 C.p. f-crystals, 15 ill., 19 table.
Background of the invention
This invention relates to immunoglobulin E (IgE), IgE antagonists, anti-IgE antibodies capable of binding to human IgE, and to a method of improving polypeptides, including anti-IgE antibodies.
IgE is a member of the family of immunoglobulin, which transfers allergic reactions such as asthma, food allergies, hypersensitivity and widespread normal sinus inflammation. IgE is secreted by b-cells and b-lymphocytes and is expressed on their surface. IgE binds to b-cells (and monocytes, eosinophils and platelets) through its Fc region with IgE receptor low affinity, known as FcRII. Exposing the La antigen, “activated” and are found in IgE secreted by plasma cells (plasma cells). The resulting allergen-specific IgE then circulates in the blood stream and binds to the surface of mast cells in tissues and basophils in the blood by receptor high affinity (affinity), also known as FcRI. Thus, mast cells and basophils become sensitive to the allergen. Subsequent exposure to the allergen causes cross-linking basophilic and mast cell FcRI, which leads to the release of histamine, leukotrienes and factors of platelet activation, eosinophilic and neutrophilic hemotoxicity factors and cytokines IL-3, IL-4, IL-5 and GM - CSF, which are responsible for clinical hypersensitivity and anaphylaxis.
A pathological condition of hypersensitivity is characterized by excessive immune reaction to the allergen(s), leading to changes in fat tissue, if the allergen is present in relatively large quantities or if the humoral and cellular immune status is at a higher level.
Physiological changes in anaphylactic hypersensitivity could the expansion of capillaries. Predisposition to this condition, apparently, is the result of interaction between genetic factors and factors related to the environment. Common allergens related to the environment, which induce anaphylactic hypersensitivity, was found in the pollen, food, mites, household dust, animal dander, fungal disputes and poisons insects. Atopic Allergy is associated with anaphylactic hypersensitivity and includes disorders such as asthma, allergic rhinitis and conjunctivitis (hay fever), eczema, urticaria and food allergies. However, anaphylactic shock, a life-threatening condition anaphylaxis, usually triggered by insect bites or source of drug therapy.
Recently was continued treatment strategy hypersensitivity type 1 or anaphylactic hypersensitivity, which is trying to block IgE from binding to the receptor (FcRI) is a high affinity (affinity), located on basophils and mast cells, and thereby inhibit the release of histamine and other allergic factors leading to pathological conditions.
WO 93/04173, published on March 4, 1993 spruce related applications USSN 08/405617 describe the purified anti-IgE antibodies where the antibody is specific to mice or rats, directed against IgE (Mae) a person who was used to ensure the CDR regions, which were replaced by IgGI structure of immunoglobulin (rhuMaE25). The method of refining described in Reichman, L. et al., Nature 332:323 (1988) and Jones. P. T. et al., Nature 321:522 (1986).
Found that although humanizing antibodies mice or rats receiving anti-IgE molecules, which provide a similar affinity to IgE, as Mae mice or rats, no immunogenic reaction, revealed last (Shields et al., Int. Arch. Allergy Immunol. 107: 308-213 (1995)), it is still not led to the creation of anti-IgE with an affinity for IgE, which is certainly better than Me or anti-IgE mice or rats.
Recombinant monoclonal antibodies are subjected to degradation reactions, which affect all polypeptides or proteins, such as the isomerization of aspartic acid or asparagine residues. As shown below, the remains of aspartate (I) in-Asp-Gly-sequences can be isomerized in isoaspartate (III) through the cyclic emenee derivative (II) (Geiger and Clarke. J. il, Chem. 262:785-794 (1987)). Side chain carboxylic acids (aspartic acid (I) reacts with the amide nitrogen of the adjacent glycine with the formation of cyclic derivative asparaginase and pH dependence of this reaction was studied on model peptides allocated using liquid chromatography high-resolution reversed-phase (Oliyai and Borchardt, Pharm. Res. 10:95-102 (1993)). Believe that the tendency to undergo isomerization also depends on the local stiffness of the molecule containing-Asp-Gly sequence (Geiger and Clarke, supra).
An example of known antibodies, which undergoes isomerization of aspartic acid, is a strong anti-IgE antibody, known as rhuMabE-25 (E-25). This transformation can occur spontaneously, but can the induction to go through, if the E-25 incubated at 37°C for 21 days. The end result is the introduction of an additional methyl group in the polypeptide skeleton antibodies, which may lead to conformational changes - decrease in binding affinity. Study E-25 using-c-Asp-Gly - and ISO-Asp-Gly variants in position VL 32-33 showed that although isomerization transformation can be minimized by substitution of alanine or glutamic acid residue VL32, the transformation leads to a three-fold reduction in binding Cacia et al., supra.
Thus, there is an urgent need to establish improved polypeptides, including antibodies, which do not t the Le target (for example, antigen), is equal to or greater than the affinity of unimproved polypeptide.
This invention relates to a method of improving polypeptide, with an affinity (affinity) to the molecule target, by combining stages, including: (1) determination of residues aspartyl, which are prone to isomerization; (2) the substitution of alternative residues and verification of the obtained mutants with regard to affinity (affinity) to the molecule target. In a preferred embodiment, the method of substitution of the residues is an “affinity maturation” with rahovym display (AMPD). In another preferred embodiment, the polypeptide is an antibody molecule and the target is an antigen. In another preferred embodiment, the antibody is an anti-IgE molecule and a target - IgE.
In a more preferred embodiment, the invention relates to a method for improving the affinity of anti-IgE antibodies of E-25 by substitution of residue Asp 32 VL CDR-L1 on Glu, along with the modification of residues 27 Gln, Ser 28 and 31 Tight VL CDR-L1 on Lys, Pro, and Gly, respectively. In another preferred embodiment, the E-25 anti-IgE antibody has an additional modification of residues in VH CDR2: 53 Thr to Lys, 55Asp on Ser, 57 Ser to Glu and 59 Asn to Lys.
In another embodiment, izobreteniya antibody includes the remains of the heavy and light chain, containing fragments of the sequence designated “E27” and “e” in Fig.2. On the contrary, anti-IgE antibody includes a full length sequence heavy and light chain marked “E27” and “E” in Fig.12.
This invention also relates to compositions of anti-IgE improved affinity or functional fragments that have pharmaceutical applications. This invention relates also to the production product containing anti-IgE antibody with improved affinity.
In another embodiment, this invention relates to a method of reducing or inhibiting IgE-mediated production of histamine.
In another embodiment, the invention relates to a method for treatment of IgE-mediated disorders by introducing antibodies described in the invention or their functional fragments.
Other aspects of the invention will be clear from the following detailed description and the claims.
Brief description of drawings
In Fig.1 compare the VH and VL region MAEII antibodies mouse or rat, “the consensus sequence of subgroup III (hum III) the heavy chain and of subgroups I (humk1) light chain and a fragment F(ab)-2 belonging to the human race; the modified fragment and the or rat.
In Fig.2 compare the differences between the sequences of the CDR regions of the light chain and heavy chain for rhuMabe 25, e and sequence e and E27. The residue numbering is sequential, as opposed to Cabot with TCS. Also note that these sequences are only excerpts and does not represent the actual residues of the full length heavy and light chain.
Fig.3 is a graph showing FACS analysis showing the capacity of the investigated antibodies to inhibit PTS-conjugated IgE binding of the a-chain of the high affinity FcRI receptor expressed on Cho 3D10 cells. Shows the percentage of inhibition of mAb MaEII mice or rats (a), negative control treated mAb4D5 (), F(ab)-2 (o), F(ab)-9 (), F(ab)-11 (), F(ab)-12 (). Data are average of three experiments, except mAb4D5, which are the only experimental value. The results indicate that MaEII and analyzed F(ab)s block FITC - IgE binding to Cho 3D10 cells expressing FcRIchain.th-subunit of high-affinity FcRI receptor expressed on Cho 3D10 cells. Linking in percent with mAb MaEII mouse or rat (a), the improved version 12 (), positive control mAb MaEI mouse or rat (), negative control MORS mouse or rat () and negative control treated mAb4D5 (). On arithmetic linear scale averages of flow fluorescence at 0.1 µg/ml was for MARS - 7,3, MaEI - 32,1, MaEII - 6,4 hu4D5 - 4.7 and huMaEII - 4,6. All three mAbs mice or rats were lgG1 isotype mouse or rat, and both purified mAbs were lgG1 isotype person. Data represent the average of three experiments. The results indicate that MaEII and F(ab)-12 is not associated with IgE-loaded Cho 3D10 cells expressing FcRIchain.
Fig.5 represents curves molar ratio of anti-IgE from inhibiting the excretion of histamine induced pollen allergies (in percent). See E-25 (and E-26 (Oh). The results indicate the maximum inhibition at a molar ratio of 44:1 (anti-IgE: RsIgEI).
Fig.6 is a graphical representation of the increase in the affinity after different cycles of selection by affinity, as described in section II of example 4. Shows the relationship linking enrichment (increase) for each pool to that of wild-type (Emut/Ewt). The results indicate that VL library (marked “a” and “b”), consistently showing improved relative enrichment of up to ~10-fold improvement compared to the wild type after 5-6 cycles of enrichment. In addition, VH library (“C” and “d”), showing ~3-fold improvement after -3 cycles. It should be borne in mind that “a” refers to the Fab-ragovoy library, subjected to mutations in the residues 27, 28, 30 and 31 CDR-1 VL, while “b” refers to mutations in 30, 31, 32, and 34, and “C” and “d” are independent F(ab) libraries with mutations at residues 101, 102, 103, 105 and 107.
Fig.7 represents the dependence of the observed optical density on the concentration of competitive IgE antibodies in phage ELISA competitive learning target variation from combinations of VL CDR1 mutations in e with VH with CDR2 mutations in clones 235-5,1; 235-5,2; 235-5,3 and 235-5,4, respectively renamed E27, e, e and e and described in section V of example 4.
Fig.8 represents the dependence abscisic cups, described in section VI of example 4.
Fig.9 shows the F(ab) apparent binding affinity (affinity), E25, e and E27 measured using BIAcore TM-2000 resonant system of the surface plasmon. 1,5-Serial dilution of F(ab) fragments of antibodies was injected through IgE chip in PBS/Tween buffer (0.05% of Tween-20 in phosphate buffered saline) at 25°C, using a flow rate of 20 ál/min give the equilibrium dissociation constants (Kd) is calculated from the ratio of the observed kon/kofffor each Fab option.
Fig.10 shows the sequence of plasmid R, which was used as template (matrix) to create a library specific “stop” template in example 4.
Fig.11A. Diagrams insertion plasmids pDH188 containing DNA which encodes the light and heavy chain (variable and constant region
1) Fab purified antibodies directed to the HER2 receptor. VL and VH are the variable regions of the light and heavy chains, respectively. Ck-constant region Kappa” () the light chain of the human CH1G1the first constant region of the gamma () chain I person. Both coding region begins with bacteriana, described in 11A. After transformation of the plasmids in E. coli SR101 cells and adding the phage helper plasmid is packaged into phage particles. Some of these particles (corpuscles) find Fab-p III merger (where the pIII is a protein encoded M13 gene III DNA).
Fig.12 depicts the overall length of the residues of the heavy and light chain of an anti-IgE antibody E25, E and E27.
Fig.13 is F(ab) fragments of anti-IgE antibodies a and E27.
Fig.14 represents sFV fragments of anti-IgE antibodies a and E27.
Fig.15 is a F(ab)'2 fragments of anti-IgE antibodies a and E27.
SEQ ID No.1 represents a sequence expressing plasmids e used in the invention, also shown in Fig.10.
SEQ ID No.2 represents the sequence of the variable region of the heavy chain Mae shown in Fig.1.
SEQ ID No.3 represents the sequence of the variable region of the heavy chain F(ab)-2 shown in Fig.1.
SEQ ID No.4 represents the sequence of the variable region of the heavy chain hum11 shown in Fig.1.
SEQ ID No.5 represents a sequence of variable region of the light chain Mae shown in Fig.1.
SEQ ID No.6 represents the sequence of the variable region of the light chain F(ab)-2 shown in Fig.1.
SEQ Ecstasy sequence of the variable region of the light chain a and E27, shown in Fig.2.
SEQ ID No.9 represents the sequence of the variable region of the light chain e shown in Fig.2.
SEQ ID No.10 represents the sequence of the variable light chain E25, shown in Fig.2.
SEQ ID No.11 is a sequence of variable regions of the heavy chain E27, shown in Fig.2.
SEQ ID No.12 is a sequence variable regions of the heavy chain E25, e and e shown in Fig.2.
SEQ ID No.13 represents the sequence of the full length of the variable region of the light chain E25, as shown in Fig.12.
SEQ ID No.14 represents the sequence of the full length variable regions of the heavy chain E25, as shown in Fig.12.
SEQ ID No.15 represents the sequence of the full length of the variable region of the light chain a, as shown in Fig.12.
SEQ ID No.16 is the sequence of the full length variable regions of the heavy chain a, as shown in Fig.12.
SEQ ID No.17 represents the sequence of the full length of the variable region of the light chain E27, as shown in Fig.12.
SEQ ID No.18 represents the sequence of the full length variable regions of the heavy chain E27, as shown in Fig.12.
SEQ ID No.19 represents a variable region light chain is th circuit e, as shown in Fig.13.
SEQ ID No.21 is a variable region of the Fab fragment heavy chain E27, as shown in Fig.13.
SEQ ID No.22 represents sFv fragment e, as shown in Fig.14.
SEQ ID No.23 is a sFv fragment E27, as shown in Fig.14.
SEQ ID No.24 represents a variable region light chain F(ab)'2 fragments for e and E27, as shown in Fig.15.
SEQ ID No.25 represents a variable region of the heavy chain F(ab)'2 fragments for e, as shown in Fig.15.
SEQ ID No.26 represents a variable region of the heavy chain F(ab)'2 fragments for E27, as shown in Fig.15.
A detailed description of the preferred options
Referred to in the invention, the separate references, patent applications and patents should be considered as the references given in the text description.
Terms used in this application should be interpreted in the usual and common sense usually used in this field. However, the applicants wish the following terms have been defined below.
The terms “protein” or “polypeptide” will be used interchangeably. They belong to a chain of two (2) or more amino acids that are linked together by peptide or amide appolagies, in particular to antibodies covered by this definition.
The polypeptides of this invention may include more than one subunit, each subunit encoded by separate DNA sequence.
The phrase “almost identical”, which refers to the polypeptide sequences of the antibodies to be construed as an antibody exhibiting at least 70%, preferably 80%, more preferably 90% and most preferably 95% sequence identity relative to the polypeptide sequence. The term in relation to the nucleic acid sequence should be interpreted as a nucleotide sequence exhibiting at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity relative to the nucleic acid sequence. For polypeptides, the length of the sequence comparison is usually at least 25 amino acids. For nucleic acids, the length should normally be at least 75 nucleotides.
The concept of “identity” or “gomologichnosti” should be interpreted as meaning the percentage of amino acid residues in Kandidat, after comparing the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the complete sequence, and not considering any conservative substitutions as part of the identity sequence. Either N - or C-terminal extension or insertion segments should not be interpreted as reducing identity or gomologichnosti. Methods and computer programs for comparison are well known. The sequence identity can be determined using software for sequence analysis (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software is suitable for such sequences by determining the degree of gomologichnosti to various substitutions, deletions (eliminirovali) and other modifications.
The term “antibody” is used in its broadest sense and specifically covers monoclonal antibodies (including monoclonal body full length), polyclonal, multispecific (for example, bespecifically antibodies) and antibody fragments (such as Fab, F(ab')2and Fv), so long as they exhibit the desired biological activity. Antibodies (Abs as antibodies exhibit binding specificity to a specific antigen, immunoglobulin and include antibodies and other antibody molecules that do not possess antigenic specificity. The polypeptides of the latter type, for example, are produced with a low concentration of the lymphatic system and with the increased concentration of myeloma.
Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins with a molecular weight of about 150,000 daltons, composed of two identical light (L) and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced disulfide bridges within the chain. Each heavy chain has at one end of the variable region (VH) followed by a number of constant domains. Each light chain has a variable region at one end (VL) and a constant region at its other end. Constant region light chain compared with the first constant region of the heavy chain and the variable region of the light chain compared with the variable region of the heavy chain. I believe that opredeleniya et al., J. Mol. il. 186, 651-660 (1985); Novothny, Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596 (1985)).
“Isolated” antibody is an antibody that has been identified and separated and/or isolated from the environmental medium in which it was produced. Contaminante (impurity) components in its production are substances which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other reminiscent of protein and protein is not reminiscent of such dissolved substances. In preferred embodiments, the antibody will be clear, measuring the purity of using at least three different methods: 1) to more than 95 wt.% antibodies, as determined by the method of Lowry, and most preferably more than 99 wt.%;
2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a rapidly rotating Cup sequencing machine; or 3) to homogeneity by SDS-PAGE in reducing or non conditions, applying Kumasi blue or preferably the dye of silver. Isolated antibody includes the antibody in situ within recombinant cells, until will prisutstvovat to get with, at least one stage of cleaning.
“Videosanime antibody, for example, the antibody of a mammal against human IgE is an antibody that has a stronger binding affinity for the antigen from the first species of mammal than it has for the homology of this antigen from the second species of mammal. Usually videosanime antibody “binds specific to the antigen, human (i.e., has a value binding affinity (affinity) (Kd) no more than ~1×10-7M, preferably no more than 1×10-8M and most preferably no more than ~1×10-9M) but has a binding affinity to the homology of the antigen from a second species of mammal, but not human, which is at least 50-fold, or at least 500 times, or at least 1000 times weaker than the binding affinity to the antigen of human rights. Videosanime antibody may be any of various types of antibodies, as described above, but preferably is purified antibody or a human antibody.
The term “mutant antibody” refers to amino acid sequence variants of antibodies, in which the modified one or more amino acid residues. Such mutants have the th, at least 75% amino acid sequence identity or similarity with the amino acid sequence or variable regions of the heavy or light chain of an antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95%. Since the method of the invention is applicable to polypeptides, antibodies and their fragments, these terms are sometimes used interchangeably.
The term “variable” in the context of the variable region of an antibody refers to the fact that some (specific) part of the variable regions differ widely among antibody sequence and used in the binding and specificity of each particular antibody for its particular antigen. However, diversity is not distributed evenly along the variable regions of antibodies. It is concentrated in three segments called hypervariable sites (complementarity determining regions, CDRs) also known as hypervariable sites in the light and heavy chain variable regions. There are at least two methods for determining CDRs:
(1) an approach based on the diversity of the sequence PE is on crystallographic studies of complexes of antigen-antibody (Chothia, S. et al., Nature 342:877 (1989)). With regard to antibodies of the applicant, anti-IgE, some CDRs were determined by combining approaches Kabat et al., and Chothia et al. More highly preserved parts of the variable regions are called spanning region (framework, FR). Variable regions of native heavy and light chains comprise four FR region, largely host-folded configuration associated with three CDRs, which form loops, and in some cases forming part of-folded configuration. The CDRs in each chain are kept together in the immediate vicinity of the FR regions and, with the CDRs from the other chain, contribute to the formation of antigennegative center of antibodies (see Kabat et al.). Constant region not directly involved in the binding of an antibody to an antigen, but exhibit various effector function, such as participation of the antibody in antibody-dependent cellular toxicity.
The term “antibody fragment” refers to a piece of full length antibodies, usually antigennegative or variable regions. Examples of fragments of antibodies include Fab, Fab', F(ab')2and Fv fragments. Papirovy hydrolyzate of antibodies produces two identical antigenspecific fragmentaion, called so for its ability to easily crystallize. Treatment with pepsin yields F(ab')2the fragment that has two antigenspecific fragment, which can cross-link the antigen and other residual fragment (called pFc'). Additional fragments may include diately, linear antibodies, antibody molecules with one chain and multispecific antibodies formed from fragments of antibodies. “Functional fragment” as antibodies, refers to Fv, F(ab) and F(ab')2the fragments.
“Fv” fragment is the minimum antibody fragment which contains a complete (completed) site of antigen recognition and binding site. This area contains a dimer of one heavy and one light chain variable regions in a stable non-covalent Association (VH-VLdimer). It is in this configuration that the three CDRs of each variable regions interact to determine antigennegative center on the surface of the VH-VLdimer. Together, the six CDRs give antigennegative the specificity of the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the able is also designated as F(ab)] also contains a constant region light chain and the primary constant region (SN) the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl end of the heavy chain SN region, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation of this invention Fb'-fragment, in which residue(s) cysteine constant regions has a free Tilney group. F(AB')-fragment produced by cleavage of disulfide bonds in cysteine the hinge region of the F(ab')2product pepsinogen of the hydrolyzate. Experts know more chemical compounds fragments of antibodies.
Light chains of antibodies (immunoglobulins) from any vertebrate species can be attributed to one of the two certainly different types, called Kappa () and lambda (depending on the amino acid sequences of their constant region.
Depending on the amino acid sequences of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG-1, lgG-2, IgG-3 and IgG-4; DSA,,,andrespectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known. Preferred immunoglobulin for use in this invention is an immunoglobulin E.
The term “monoclonal antibody” as used in this invention refers to an antibody obtained from a population, essentially homogeneous antibodies, i.e., the individual antibodies containing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies of vysokospetsifichnymi, and directed to a separate area of the determinants. In addition, in contrast to conventional drugs (polyclonal) antibodies, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous because they are synthesized by the hybridoma culture, uncontaminated by other immunol the Eski homogeneous population of antibodies, and should not be construed as requiring production of the antibody in a certain way. For example, the monoclonal antibodies used in this invention, it is possible to obtain the hybridoma method first described by Kohler and Milstein, Nature 256:495 (1975) or can be obtained recombinant methods, for example as described in U. S. Pat. No. 4816567. Monoclonal antibodies used in this invention, can also be separated from phage libraries of antibodies, using the methodology described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991).
Monoclonal antibodies that specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a separate class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies, but also fragments of these antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4816567); Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855 (1984).
“Refined” form of the antibody, the e immunoglobulins, chain of the immunoglobulin or its fragments such as Fv, Fab, Fab', F(ab')2or other antigennegative sequences of antibodies) that contain minimal sequence derived from not belonging to the human race immunoglobulin. Mostly purified antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable segment (CDR) of the recipient are replaced by residues from a CDR, not belonging to the human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some examples, the remains of Fv spanning region of human immunoglobulin replaced by corresponding non-human race remains. In addition, the purified antibody can include residues which are not found in the recipient antibody nor in the imported CDR sequence or a sequence spanning the region. Data modifications made for additional purification and optimization of characteristics of antibodies. Usually purified antibody must contain substantially all of at least one or usually two variable regions, in which all or substantially all of the CDR region soo is blasti are “consensus” (generalizing typical) sequence of a human immunoglobulin. Optimally, if purified antibody will also include at least part of a constant region of immunoglobulin (Fc), typically a region of human immunoglobulin. For more information, see: Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).
Fragments of “single chain Fv” or “sFv” antibody comprise the VH and VL region of an antibody, where the data field present in a single polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and VL regions, which enables the sFv to form the desired structure for antigen binding. A review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994).
The term “ditelo” refers to small fragments of antibodies with two antihistamine centers, which fragments comprise variable region heavy chain (VH) associated with the variable region of the light chain (VL) in the same polypeptide chain (VH-VL). When using a linker that is too short for that to happen the pairing between the two domains on the same chain, the field is forced to mate with complementary areas of another chain and create two antigenspecific center. Diately described in more detail the log antibodies represents a connection, having a qualitative biological activity as the full length antibody. For example, a functional fragment or analog of an anti-lgE antibody is an antibody which can bind with IgE immunoglobulin in such a way as to prevent or substantially reduce the ability of such molecules to contact the high-affinity receptor FcRI.
The term “amino acid” and “amino acids” refer to all naturally occurring L--amino acids. Identify amino acids, as described in section A. Obtaining antibodies: (IV) the Generation of mutant antibodies. The term “variant amino acid” refers to molecules with some differences in their amino acid sequences compared to the native amino acid sequence.
“Replaced by” options are options, of which at least one amino acid residue in the native sequence removed and this place was in the same position entered another amino acid. Substitution can be single, when replaced by only one amino acid in the molecule or they can be multiple, when in the same molecule are substituted with two amino acids, directly adjacent to the amino acid at a certain position in the native sequence. Directly adjacent to the amino acid means or associated with-carboxyl or-amino group of amino acids. “Eliminated” (deletezone) options are options with the remote one or more amino acids in the native amino acid sequence. Typically eliminated options should have one or two amino acids deleted in a particular part of a molecule.
The terms “cell”, “cell line” and “cell culture” are used revezamento, and all such designations include progeny. It is also clear that all offspring should not be precisely identical in DNA due to deliberate or inadvertent mutations. Included mutant progeny that have the same function or biological property, as tested for normal and transformed cells.
“Cell bosses” used in this invention are typically prokaryotic or eukaryotic hosts. Examples of appropriate host cells described in section C. the Vectors, cells of the host and recombinant months, however, to DNA replicated or as an extrachromosomal element or by using chromosomal integration.
“Transfection” refers to the absorption of expressing the vector in the host-cell, regardless of whether expressed in reality any coding sequences.
The term “transfectional a host cell” and “transformed” refers to the introduction of DNA into the cell. The cell is called a host cell can be prokaryotic or eukaryotic. Typical prokaryotic cell hosts include various strains of e.coli.
A typical eukaryotic cell hosts include mammals, such as Chinese hamster ovary or cells of human origin. The introduced DNA sequence may be of a similar cell-host species or from other than the host cell species or it may be a sequence of hybrid DNA containing some alien and some homologous DNA.
The term “replication competent expressing vector and expressing the vector” refers to a piece of DNA, usually double-stranded, which can be part of the alien DNA. Foreign DNA is defined as the heterologous DNA, not vstrechayushchiyesya cell host. If a cell host, the vector may replicate independently of the chromosomal DNA of the host, you can generate multiple copies of the vector and introduced (alien) DNA.
The term “vector” means a construct DNA containing a DNA sequence, which is directly connected with the corresponding control sequence capable of expression of the DNA in an appropriate host. Such control sequences include a promoter for transcription and arbitrary operator sequence to control transcription; a sequence encoding a corresponding ribosome-mRNA binding sites, and sequences which control the termination (termination) transcription and translation. The vector may be a plasmid, fagboy corpuscle or simply potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the genome of the host or may in some cases be integrated into the genome. In this definition, “plasmid” and “vector” are sometimes used interchangeably as the plasmid is currently the most frequently used f the th function, those already known or will be known in this field. Typical expressing vectors for the expression of cell culture mammals based on, for example, pRK5 (EP 307247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen).
“Liposome” is a small vesicle composed of various lipids species, phospholipids and/or surfactant which is suitable for delivery of drugs mammals (e.g., mutant antibodies described in this invention, and, perhaps, a chemotherapeutic agent). Components of liposomes are usually located in the double layer like lipid unit of biological membranes.
The expression “control sequences” refers to DNA sequences necessary for the expression of operable linked coding sequence in an individual host organism. The control sequences that are suitable for prokaryotes include, for example, the promoter may operator sequence and ribasushi website. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.
Molecule “isolated” nucleic acid molecule is a nucleic acid the swarm it is usually associated in the natural source of the nucleic acid antibodies. An isolated nucleic acid molecule differs from the form or the environment in which it is found in nature. Therefore, molecules isolated nucleic acid different from the molecules of nucleic acid, which exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that normally Express the antibody, where, for example, the nucleic acid molecule is in a chromosomal location different from the location in natural cells.
Nucleic acid directly (resectable) linked” when it is placed in a functional relationship with the sequence of another nucleic acid. This may be a gene and a regulatory sequence(s) that are connected in such a way as to permit gene expression when the appropriate molecules (e.g., protein activator of transcription) are linked with the regulatory sequence(s). For example, DNA predpolagavshegosja or secretory leader directly binds to DNA polypeptide, if he expresses as preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is linked directly to xit directly associated with the coding sequence, if it affects the transcription of the sequence; or ribosomally site directly associated with the coding sequence if it is positioned so as to facilitate translation. Usually directly (resectable) linked” means that the DNA sequences being linked are contiguous (touching) and in the case of a secretory leader, contiguous (touching) in the phase of reading. However, enhancers need not be contiguous (touching). The connection is achieved by legirovaniem (crosslinking) in a convenient restriction sites. If such sites do not exist, use synthetic oligonucleotide adaptors or liqueurs in accordance with accepted practice.
“Treatment” refers to both therapeutic treatment and preventive and protective measures. Those who need treatment include those who already have the disorder, and those with the disorder should be avoided.
“Disorder” is any condition which is improved by treatment with the polypeptide. This includes chronic and acute disorders or diseases including such pathological conditions that predisposed lecanii for applied therapy, refers to substances that act to suppress or mask the immune system of the host, in which the graft is transplanted. It should include substances that inhibit the production of cytokines that regulate the type of negative communication or inhibit the expression of autoantigen or masking of MHC antigens. Examples of such agents include 2-amino-5-aryl-5-substituted pyrimidines (see U. S. Pat. No. 4665077); azathioprine or cyclophosphamide in the event of adverse reactions to azathioprine); bromkriptin; glutaric aldehyde (which masks the MHC antigens, as described in U. S. Pat. No. 4120649); antiidiotypic antibodies for MHC antigens and NHC fragments; cyclosporin a; steroids such as glucocorticosteroid, such as prednisone, methylprednisone and dexamethasone; cytokine or receptor antagonists of cytokines, including anti-interferon-, -orantibodies, antibody anticancer Nekrasovo factor-; antitumor antibodies Nekrasovo factor-; antibodies, anti-interleukin-2 antibodies, anti-IL-2 receptor; anti-L34 antibodies; heterologous anti-lymphocyte chap who asiausa region (WO 90/08187 published on July 26, 1990); streptokinase; TGF-; streptodornase; RNA or DNA from the host; FK506; RS-61443; desoxypeganine; rapamycin; T-cell receptor (U. S. Pat. No. 5114721); fragments of the T-cell receptor (Offner et al., Science 251: 430 to 432 (1991); WO 90/11294 and WO 91/01133); and antibodies T-cell receptor (ER 340109), for example TV. These agents are injected at the same time or at separate times with D11 antibody and used in the same or lower doses than previously known. Preferred additional immunosuppressive agent should depend on many factors, including the type of treat disorders, including type implemented transplantation, as well as the medical history of the patient, but the General preference is given to the agent, which is selected from cyclosporin a, glucocorticosteroid (most preferred prednisone or methylprednisolone), OKT-3 monoclonal antibody, azathioprine, bromocriptine, heterologous anti-lymphocyte globulin or mixtures thereof.
The terms “cancer” and “cancerous” refer to the physiological state or describe him in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, leukemia the th cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, carcinoma of the salivary gland, kidney cancer, renal cancer, prostate cancer, cancer of the external female organs, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
“Mlekopitayushchie” from the perspective of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, primates and animals zoo, sports or pet animals, such as dogs, horses, cats, cows, etc.
The term “epitope tagged” when used in this invention means a polypeptide that is fused with “epitope tag”. Polypeptide epitope tag has enough residues to provide the epitope against which you can obtain antibody, yet it is short enough so that it does not affect the activity of the polypeptide. It is preferable that the epitope tag is also fairly unique so that the antibody did not cross-react with other epitopes. The corresponding labeled polypeptide 9-30 residues). Examples include flu ON the labeled polypeptide and its antibody SA (Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)); c-myc tagged and the 8F9, SS, E, G4, B7 and E antibody (Evan et al., Mol. Cell. Biol. 5 (12): 3610-3616 (1985)); and glycoprotein D (g.D) labeled herpes simplex virus and its antibody (Paborsky et al., Protein Engineering 3(6): 547-553 (1990)). In some embodiments, epitope tag is “saved receptorligand epitope”.
Used in this invention, the term “saved receptorligand epitope” refers to an epitope of the Fc region of IgG molecules (e.g., IgG1, IgG2, IgG3or IgG4), which is responsible for increasing the in vivo half-life in the blood line of IgG molecules.
The term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes their destruction. The term is intended to include radioactive isotopes (e.g., I131I125, Y90and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
“Chemotherapeutic agent” is a chemical compound suitable for the treatment of cancer. Examples of chemotherapeutic agents vklyuchitel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Karminomitsin, Aminopterin, Dactinomycin, Mitomycin, Spiramycin (see U. S. Pat. No. 4675187), Melphalan and other related nitrogen-containing preparations of mustard.
The term “precursor drug” refers to a precursor or derivative form of the pharmaceutically active substance that is less toxic to tumor cells compared to the original drug and are capable of enzyme to be activated or to become more active in its original form. See, for example, Wilman "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, 14, pp. 375-382, 615 Meeting Belfast (1986) and Stella et al., (ed.), "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.) pp.247-267, Human Press (1985). Predecessors of medicines described in this invention, include, but are not limited to phosphate-containing, thiophosphoramide, sulfadimidine, peptidoergicheskih, modified D-amino acid; a glycosylated,-lastmodifiedtime may zalesennymi phenoxyacetaldehyde or perhaps replaced what Estonica medicines, can transform into the more active cytotoxic drugs. Examples of cytotoxic drugs that can derivateservlet in the form of precursor drugs include, but are not limited to, chemotherapeutics, described above.
The word “label” refers to detectivemisa the compound or composition, which conjugates directly or indirectly with the antibody. The label can be detected itself (e.g., radioisotope labels or fluorescent labels) or, in the case of fermentative label, may catalyze chemical alteration of the substrate substance or composition that detects.
In this invention, “solid phase” means anhydrous matrix, which may stick antibody. Example of solid phases include phase partially or completely derived from glass (e.g., glass, controlled pore size), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In some embodiments, depending on the context, the solid phase can include just analytical tablet; in others this column for purification (for example, column affinity chromatography). This term also includes presribes antibody which is connected with IgE person in such a way that it inhibits or reduces the binding of IgE to the high-affinity receptor, FcRI. Preferably this anti-IgE antibody is the E-25.
The term “disorder associated with IgE” means a condition or disease that is characterized by overproduction and/or hypersensitivity to the immunoglobulin IgE. Especially it is necessary to include conditions associated with anaphylactic hypersensitivity and atopic allergies, including asthma, allergic rhinitis and conjunctivitis (hay fever), eczema, urticaria and food allergies. However, a serious physiological condition of anaphylactic shock, usually caused by a bee or snake bites or source of drug therapy, is also included in the scope of this term.
“Affinity maturation using phage display (AMPD) refers to the process described in Lowman et al., Biochemistry 30(45): 10832-10838 (1991), see also Hawkins et al, J. Mol. Biol. 254: 889-896 (1992). This process, although not strictly limited to the following description can be briefly described as follows: some sites hypervariable region (e.g., 6-7 sites) mutate to genererated in monovalent form of filamentous phage particles as mergers with product M13 gene III, Packed inside each corpuscle. Phage expressing different mutants, can make loops on circles linked selection, followed by isolation and sequencing data mutants which exhibit high affinity. The method is also described in WO 92/09690, published 11 June 1992. A modified technique which involves a combined indication of the affinity described in Cunningham, B. C. et al., EMBO J. 13(11): 2508-2515 (1994).
The method proposes a method for the identification of new binding polypeptides, comprising: (a) create replicates of expression vector comprising a first gene that encodes a polypeptide, a second gene encoding at least part of the natural or wild-type protein with ragovoy shell, where the first and second genes are acid, and a regulatory element, the transcription is directly connected with the first and second genes, thereby forming a fusion gene encoding a protein; b) mutating the vector at one or more selected positions within the first gene, thereby defining (creating) a family of related plasmids; C) transforming appropriate host cells with the plasmids; (d) infecting the transformed host cells phage helper with the gene encoding the prot for the formation of recombinant pagemenu particles, containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a small number pagemenu corpuscles play more than one copy of the fused protein on the surface of the particles; g) contacting pagemenu particles with the molecules of the target so that at least part pagemenu particles were contacted with a molecule-target; and C) the Department pagemenu particles that bind from those that do not bind. Preferably, if the method further includes transforming appropriate host cells with recombinant pagaminami corpuscles (particles) that are associated with the molecule-target, and the repetition of stages d) to h) one or more times.
On the contrary, the method includes polypeptides that are composed of more than one subunit, where replicate an expression vector comprising a regulatory element transcription directly linked to a DNA coding interest subunit, connected with protein with ragovoy oblucki.
Used in this invention, the term “phage library antibody” refers to ragovoy the library used in the process of maturing affin which contains a hypervariable region (for example, 6-7 sites), which generates all the possible amino acid substitutions. Thus the generated mutants antibodies are reproduced in monovalent form of filamentous phage particles as merging with the M13 gene product III, Packed in each corpuscle and espressivo on the outer side of the phage.
Used in this invention is a “room” or “ambient temperature” will be 23-25°C.
“Binding polypeptide” refers to any polypeptide that binds to a molecule selected target affinity. Preferably, when the polypeptide will represent the protein, which is most preferably contains more than about 100 amino acid residues. Typically, the polypeptide will be a hormone, an antibody or its fragment.
“High affinity” means the value of the affinity constant Kd<10-5M and preferably Kd<10-7M under physiological conditions.
“Molecule-target” means a molecule, not necessarily the protein from which it is desirable to produce the antibody or ligand. However, preferably, if the target is a protein, and most preferably, if the antigen. However, it is particularly important to include in the scope of this term receptors, for example R is the situation peptides specific parts of IgE, mutant IgE molecules and chimeric (hybrid) IgE molecules that are referred to in this invention is carried out in accordance with the nomenclature of Cabot to the amino acid residues of the immunoglobulin (Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, MD 1987)).
Ways of carrying out the invention
I. Method for improving the affinity of a target molecule A. Identification capable of isomerization residues of aspartyl
Identification capable of isomerization residues of aspartyl can be carried out by any technique known to the specialists. For example, Cacia et al., Biochemistry 35:1897-1903 (1996), describe how, where anti-IgE antibody E-25 (which includes-Asp-Gly-residues) were incubated at 37°C for 21 days. Identification of isomerized-Asp-Gly - was carried out by chromatographic and mass spectrometric analysis of raw and treated with protease fragments. As it was reported that the isomerization occurs with the remnants of asparaginyl (T. Geiger, S. Clarke. J. il. Chem. 262(2): 785-794 (1987)), the invention preferably also can be used to systematically assess and improve the quality of polypeptides containing residues of asparagine.
C. Selection of alternative mod is t to optimize the affinity of the receptor. Usually, all of these methods include substitution of different amino acid residues in the interest of the site, followed by screening (testing) receptor affinity of the mutant polypeptide. The method, which is preferred for use in this invention is affinity maturation using phage display (Hawkins et al., J. Mol. Bil. 254: 889-896 (1992); Lowman et al., Biochemisry 30(45): 10832-10838 (1991)). Briefly several sites hypervariable region (e.g., 6-7 sites) multiroute to generate all possible amino acid substitutions at each site. Thus the generated mutants antibodies are reproduced in monovalent form of filamentous phage particles as m M13 gene product III, Packed in each corpuscle. Phage expressing the various mutants, can make loops through the circles of selection, binding, followed by isolation and sequencing of these mutants, which show high affinity.
The method of selection of new binding polypeptides preferably uses a library of structurally related polypeptides. A library of structurally related polypeptides, fused with protein ragovoy shell, produced through mutagenesis, and the second particles, containing DNA encoding the polypeptide. These pagemedia corpuscles further contact with the molecule-target, and those particles having the greatest affinity for the target are separated from the particles with lower affinity. High-affinity binder further amplified by infecting a bacterial host and the stage of competitive binding repeat. The process is repeated as many times until you get the desired polypeptides affinity.
On the contrary, multivalent phage (McCafferty et al., Nature 348: 552-554 (1990); Clackson et al., Nature 352: 624-628) (1991)) can also be used for the expression of random point mutations (generated by the application is prone to erroneous action of DNA polymerase) to generate a library of phage fragments of antibodies, which then can be tested for affinity to the antigen (Hawkins et al., J. Mol Biol. 254: 889-896 (1992).
Preferably, when during the process of affinity maturation” replicate the expression vector is under the steady control of the regulatory element, the transcription and the culturing conditions are regulated so that the quantity or number pagemenu particles, reproducing more than one copy of the fused protein on the surface of the particles was less than ~1%. Also predpochtitel is it 10% of the number of pagemenu particles, reproducing only a copy of the fused protein. Most preferably, when the amount is less than 20%.
Usually in the way described in this invention, expressing the vector must also contain a secretory signal sequence, fused to the DNA encoding each subunit polypeptide, and a regulatory element, the transcription must be promotor system. The preferred promoter system selected from LacZ,PL,TC, T7 polymerase, tryptophan and alkaline phosphatase promoters and mixtures thereof.
Also usually the primary gene should encode a protein of a mammal, preferably a protein must be anti-IgE antibody. Additional antibodies are given in section II.A. Obtaining antibody, (VI) multispecificity antibodies (I want to note, however, that antibodies should not be multispecificity). Additional polypeptides include human growth hormone (human growth hormone, hGH), N-methionyl-human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, A-chain, insulin b-chain of insulin, proinsulin, the a-chain of relaxin, In-circuit relaxin, prolactin, glycoprotein hormones such as follicle-stimulating mountains is, eceptor glycoprotein hormone, calcitonin, glucagon, factor VIII, lung surfactant, urokinase, streptokinase, tissue plasminogen activator human (human tissue-type plasminogen activator (t-PA), bombezin, factor IX, thrombogenicity growth factor,factor andfactor that causes necrosis of tumor cells, enkephalinase, serum albumin human, Müller inhibitory substance, gonadotropin-binding peptide mouse, microbial protein, such as-lactamase, protein tissue factor, inhibin, activin, vascular endodermally growth factor, receptors for hormones and growth factors, integrin, thrombopoietin, protein a or D, rheumatoid factors, nerve growth factors such as NGF-, platelet-derived growth factor, transforming growth factors (transforming growth factors, TGF) such as TGF-and TGF-, insulin-like growth factor-I and-II binding proteins insulin-like growth factor, CD-4, Tnkase, latent associated peptide, erythropoietin, osteoinductive factors, interferons such as-,Preferably, if the first gene will encode one or more polypeptide subunits, which contain more than 100 amino acid residues and will bend (fold), forming a multitude of rigid secondary structures, reproducing many amino acids capable of interacting with the target. Preferably, if the first gene will mutate in the codons that are specific to amino acids capable of interacting with a target in order to retain the integrity of the rigid secondary structures.
Usually the method described in this invention, must apply the phage helper, which is selected from MG, M13R408, M13-VCS, and Phi X 174. Preferred phage helper is MC and preferred protein shell is M13 phage gene II protein shell. The preferred host is E. coli and protease deficient strains of E. coli. Were detected new hGH variants selected using sposobny termination codon, functionally located between the nucleic acids encoding the polypeptide and protein ragovoy shell.
1. The selection of polypeptides to play (display) on the surface of phage
Repeated cycles of selection “polypeptide” is used for selection of greater and greater binding affinity using pagemenu selection of multiple amino acid changes, which are selected via multiple selection cycles. After the first round pagemenu selection, including primary area breeding of amino acids in the polypeptide ligand or antibody, conduct additional circles pagemenu breeding in other areas or the amino acid ligand. Cycles pagemenu breeding repeat up until achieved affine properties. To illustrate this process in example 4, phage display was performed cycles. Group affinity, the combination of mutations from different CDRs and so on
From the above it is clear that amino acid residues that form the binding region of the polypeptide will not be consistently communicate and can reside on different polypeptide subunits, which means tracks linking the region with a specific secondary structure in the binding is islote in a particular secondary structure in the sites, directed from the inner side of the polypeptide, so that they had the opportunity to interact with the target.
However, there is no necessary conditions so that the polypeptide is selected as the ligand or antibody to a target molecule, normally associated with this target. Thus, glycoprotein hormone, such as TSH, may be selected as a ligand for FSH receptor and a library of mutant TSH molecules used in the method described in this invention to produce new variants of medicines.
Thus, the present invention is considered any polypeptide that binds to the molecule target, especially with antibodies. Preferred polypeptides are polypeptides that have pharmaceutical applications. Examples of antibodies are listed in section II.A. Obtaining antibodies, (IV) multispecificity antibodies (note that antibodies should not be multispecificity). Preferred polypeptides include growth hormone, including human growth hormone; des-N-methionyl-human growth hormone and bovine growth hormone; parathyroid hormone; thyrostimulin hormone; thyroxine, A-chain, insulin b-chain of insulin; prolactin, gonadotropin-SW growth; inhibin; activin; vascular endothelial growth factor; receptors for hormones or growth factors; integrin; thrombopoietin; protein a or D; rheumatoid factors, nerve growth factor such as NGF-; platelet-derived growth factor; fibroblastic growth factor such as aFGF and bFGF; epidermal growth factor; transforming growth factor, such as TGF-and TGF-; insulin-like growth factor-I and-II; binding proteins insulin-like growth factor; CD-4; DNA-ASE, latent associated peptide; erythropoietin; osteoinductive factors, such as part of the HIV-shell; immunoglobulins and fragments of any of the above polypeptides. In addition, one or more predetermined amino acid residues in the polypeptide may be substituted, inserted or eliminirovat, for example, to produce products with improved biological properties. In addition, include fragments of these polypeptides, particularly biologically active fragments. More preferred polypeptides are factor human growth and atrial natriuretic peptides a, b and C, endotoxin, subtilisin, trypsin and other Surinov the amino acid sequence, was produced in a primary cell, which is specific binds to a receptor on the same cell type (autocrine hormones) or on the secondary cell type (neutogena) and evoked physiological response characteristic of cells secreting the hormone. Such polypeptide hormones include cytokines, lymphokines, neurotrophic hormones and adenohypophysis polypeptide hormones such as growth hormone, prolactin, placental lactogenic, luteinizing hormone, follicle-stimulating hormone-lipotropin,-lipotropin and endorphins; hypothalamus realizin-inhibitory hormone, such as corticotropin-releasing hormones, growth hormone realizin-inhibitory hormone, growth hormone-releasing factor; and other polypeptide hormones such as atrial natriuretic peptides a, b or C.
2. The first gene (gene I) encoding the desired polypeptide
The gene encoding the desired polypeptide (e.g. antibody), can be obtained by known methods (see Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York, (1989)). If you know the sequence of a gene, DNA encoding the gene can be synthesized chemically (Merrifield. J. Am. Chem. Soc. 85:s cDNA library made from RNA, obtained from the corresponding tissue in which the desired gene is expressed) or from the corresponding genomic DNA library. Further gene allocate, using the appropriate probe. For cDNA libraries corresponding probes include monoclonal or polyclonal antibodies (provided that the cDNA library is a library of expressed sequences), oligonucleotides and complementary or homologous cDNA-you or fragments thereof. Probes that can be used to isolate the gene of interest from a library of genomic DNA includes cDNA-you or fragments thereof that encode the above-mentioned or similar gene, which is homologous to genomic DNA or DNA enzymes and oligonucleotides. Testing screening cDNA or genomic libraries selected probe is carried out using standard techniques described in chapters 10-12 of the monograph Sambrook et al., supra.
An alternative technique to isolate the gene encoding the desired polypeptide (e.g. antibody), is the application of the methodology polymerase reaction synthesis circuit (polymerase chain reaction, PCR), as described in section 14 of the monograph Sambrook et al., supra. This method requires the use of oligonucleotides that should hybridisierung with nugnes, to generate oligonucleotides.
After isolation of the gene for amplification, it can be inserted into an appropriate vector (preferably a plasmid), as generally described in Sambrook et al., supra.
3. Creating (designing) replicating expression vectors
While available and can be applied in practice, several types of vectors, the preferred plasmid vectors because they can be created relatively easily and can easily amplify. Plasmid vectors usually contain a number of components, including the promoter, signal sequence, genes phenotypic selection, the source replication site, and other necessary components, known to specialists.
Promoters most commonly used in prokaryotic vectors include the lac Z promoter, pho And the alkaline phosphatase promoter, the APL promoter of bacteriophage (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and T7 promoter of the bacteriophage. Relative to the General descriptions of promoters, see section 17 of monograph Sambrook et al., supra. Although there are the most commonly used promoters can also be used on other is about to be regulated, so what can be controlled expression of the fused gene. If the expression of uncontrolled, leading to multiple copies of the fused protein on the surface of the plasmid can occur multipoint fixation plasmids with target. I believe that this multipoint fixation, also called “avidity” or “chelate effect” that leads to the selection (selection) erroneous polypeptides “high affinity” caused by multiple copies of the fused protein, played on pagemenu the corpuscle in close proximity to each other, to helatirovat” target (complete with target). If there is multipoint fixation, the effective or apparent Kd can be as high as Kds individual product for each copy reproduced fused protein.
Using sustainable regulation of the expression of the fused protein, so only a small amount, i.e. less than 1% pagemedia particles contain numerous copies of the fused protein to overcome the “chelate effect” that allows the appropriate selection of polypeptides high affinity. Thus, depending on the promoter, culturing conditions of the host to regulate the maximum number pagemenu is cool, containing numerous copies of the fused protein.
A preferred promoter used in this invention are the lac Z promoter and pho And the promoter. lac Z promoter is regulated by the lac repressor protein lac i, and thus, the merged transcription of a gene can be controlled by manipulation of the level of lac repressing protein. To illustrate phagemid containing the lac Z promoter, grown in cell strain, which contains a copy of the lac i repressing gene, repressor to the lac Z promoter. Typical cell strains containing the lac i gene, include JM 101 and XL-1 blue. In another case, a host cell can cotransfection with a plasmid containing the repressor l i and lac Z promoter. Sometimes both methods are used simultaneously, i.e. pagemedia corpuscles containing the lac Z promoter, grown in cell strains containing the lac i gene, and cell strains cotransfection with a plasmid containing the lac Z and lac i genes. Usually, when one wants to Express the gene, to transfectional above the owner should be added to the inductor, such as isopropylthioxanthone (isopropylthiogalactoside, IPTG). However, in this invention, this stage is omitted, in order to minimize the expression m is the ora, for example, IPTG, even at low concentrations. Usually, if the inductor is not added, the number of fused proteins on pagemenu corpuscle is above 0.1 (amounts fused proteins in volume number pagemenu corpuscles). The most preferred promoter is pho A. I Believe that this promoter should be regulated by levels of inorganic phosphate in the cell, where the phosphate acts to regulate the type of negative communication activity of the promoter. Thus, when the depletion in cells phosphate activity of the promoter can be increased. The desired result is achieved by growing cells in the enriched phosphate environment, for example 2YT or LB, thereby controlling the expression of the fusion gene III.
Other suitable component of the vectors used in this invention, is a signal sequence. This sequence is usually located directly in 5'-terminal region to the gene, codereuse fused protein, and, thus, should read aminobenzo fused protein. However, in some cases it has been demonstrated that the signal sequence is located at the position different from the 5' to gene, codereuse secretory protein. This polnoy cells. DNA encoding the signal sequence can be obtained as a fragment restriction endonuclease from any gene encoding a protein that includes a signal sequence. Appropriate prokaryotic signal sequence can be obtained from genes encoding, for example, LamB or OmpF (Wong et al., Gene 68:193 (1983)), MalE, Pho and other genes. Preferred prokaryotic signal sequences used in this invention is the signal sequence of E. coli thermostable enterotoxin II(STII), as described in Chang et al., Gene 55:189 (1987).
Other suitable component vectors are genes phenotypic selection. Typical genes phenotypic selection genes are genes encoding proteins that give resistance to antibiotics host cells. As an illustration for this purpose are widely used resistant to ampicillin (amp) and tetracycline (tet) gene.
Construction of appropriate vectors, including the aforementioned components, as well as the gene encoding the described polypeptide (gene I), is obtained by applying standard techniques of recombinant DNA as described by Sambrook et al., supra. Isolated DNA fragments that need SoE generate the desired vector.
DNA digested using the appropriate restriction enzyme or enzymes in a suitable buffer. Usually use about 0.2-1 μg of plasmid or DNA fragments with ~1-2 restriction enzyme in about 20 μl of buffer solution. Appropriate buffer solutions, DNA concentration, the time and temperature of incubation is determined by the manufacturers of restriction enzymes. Typically, the incubation times of about one or two hours at 37°C are acceptable, although some enzymes require higher temperatures. After incubation, the enzymes and other contaminants are removed by the extraction of hydrolyzed solution with a mixture of phenol and chloroform and the DNA is recovered from aqueous fractions by precipitation with ethanol.
To ligitamate DNA fragments together to form a functional of the vector, the ends of the DNA fragments must be compatible with one another. In some cases, the ends must be compatible directly after hydrolysis by the endonuclease. However, it may be necessary to first turn sticky ends, usually preziraemy endonuclease hydrolysis, dephosphorylating the ends to make them compatible for ligation. For dephosphorylation of DNA ends are treated in the appropriate bofiii four triphosphates-deoxynucleotide. Then the DNA purified using phenol-chloroform extraction and precipitation with ethanol.
Hydrolyzed DNA fragments can be separated according to size and select using DNA gel electrophoresis. DNA can be subjected to electrophoresis using a matrix of agarose or polyacrylamide. The choice of the matrix will depend on the size of the partial DNA fragments. After electrophoresis the DNA extracted from the matrix by Electrosila or, if the matrix is used discopleurus the agarose, melting agarose and extraction of her DNA, as described in sections 6.30-6.33 in the monograph by Sambrook et al., supra.
DNA fragments that need to be ligitamate together (pre-hydrolyzed relevant enzymes, so that the ends of each ligiriemale fragment compatible), are placed in solution in approximately equimolar amounts. The solution should also contain ATP, buffer and ligase, such as T4 DNA ligase at ~10 units per 0.5 μg DNA. If the DNA fragment should be ligitamate in the vector, the first vector linearized using cutting (cutting) with an appropriate endonuclease(s) restriction. Next, the linearized vector is treated with alkaline phosphatase or intestinal phosphatase calf. Processing FOSFA who are what introduced alien genome transform in the corresponding cell of the host. Preferred cells of the host are prokaryotes. Appropriate prokaryotic cell hosts include E. coli strain M101, E. coli K12 strain 294 (ATSS room 31446), E. coli strain W3110 (ATSS room 27325), E. coli X1776 (ATSS number 31537), E. coli XL-1 Blue (Stratagene) and E. coli B; however, you can also use many other strains of E. coli, for example NV, NM522, NM538, NM539, and many other species and varieties of prokaryotes. In addition to the E. coli strains listed above, bacilli such as Bacillus subtilis, other enterobacteria such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species; they can all be used as hosts.
Transformation of prokaryotic cells is easily done when using calcicludine way as described in section 1.82 of the monograph Sambrook et al., supra. On the contrary, to transform these cells can be used electroporation (Neumann et al., EMBO J. 1: 841 (1982)). Transformed cells are selected by growth on antibiotics, usually tetracycline (tet) or ampicillin (amp), to which they lend stability due to the presence of tet and /or AMR resistant genes in the vector.
After selection of transformed cells, these cells can be grown in culture and then isolate plasmid DNA (or another vector with the introduced foreign gene). Plasmas the scrap quantity and the production of DNA on a large scale, as described in sections 1.25-1.33 monograph Sambrook et al., supra. Isolated DNA can be cleaned with known methods, for example, which are described in section 1.40 in Sambrook et al., supra. Next, the purified plasmid DNA analyzed by restriction mapping and/or DNA sequencing. DNA sequencing is usually carried out or by the method of Messing et al. Nucleic Acids Res. 9:309 (1981) or by the method of Maxam et al., Meth. Enzymol 65:499 (1980).
4. The fusion gene
Stage ragovoy affinity involves the fusion gene comprising the desired polypeptide (gene 1), with a second gene (gene 2), so that the fused gene is generated during transcription. Gene 2 is usually the gene of the protein shell of the phage, and it is preferable that the M13 phage gene III protein shell or its fragment. Fusion genes 1 and 2 can be performed by inserting a gene 2 at a specific site on the plasmid that contains the gene 1, or by inserting a gene 1 in a specific site on the plasmid that contains the gene 2.
Inserting a gene into a plasmid requires that the plasmid was cut in a specific location, so gene could be inserted. Thus, there should be a site restriction endonuclease in this place (preferably a unique website, so that the plasmid is cut only in one place while g is then inserted into this linearized plasmid when legirovanii two DNA together. Ligation can be performed, if the ends of the plasmid is compatible with the ends of the gene insert. If cuts and the plasmid and isolating the inserted gene using the same restriction enzymes, DNA-you can ligitamate directly together using a ligase, for example bacteriophagous T4 DNA ligase and inquira the mixture at 16°C for 1-4 hours in the presence of ATP and ligase buffer as described in section 1.68 of the monograph Samrook et al., supra. If the ends are not compatible, they can first be dephosphorylate using fragment maple DNA polymerase I or the bacteriophage T4 DNA polymerase, both of which require four triphosphates of deoxyribonucleotide to fill speakers (“sticky”) single-stranded ends of the hydrolyzed DNA. On the contrary, the ends can dephosphorylates using nucleases, such as nuclease S1 or bean nuclease, both of which operate by cutting the speakers (“sticky”) single-stranded DNA. Next, DNA is classified using ligase as described above. In some cases it may be impossible dephosphorylation of the ends of the inserted gene, because of a change of reading frame coding region. To overcome this problem, you can ispolzovat linkers can be obtained synthetically in the form of a double-stranded or single-stranded DNA using standard methods. The linkers have one end that is compatible with the ends of the inserted gene; linkers are ligated first with this gene using the above-described methods of ligation. Assume that the other end of the linkers compatible with the plasmid for ligation. When designing linkers measures should be taken not to destroy the reading frame of the inserted gene or reading frame of the gene contained in the plasmid. In some cases it may be necessary to create linkers to encode part of the amino acids or that they encode one or more amino acids.
Between gene 1 and gene 2 can be inserted DNA encoding a termination codon, such codons are UAG(amber), UAA(ocher) and UGA (opel), Microbiology, Davis et al., Harper and Row, New York, 1980, pp.237, 245-47 and 274). The termination codon expressed in the cell-master of the wild type, leading to the synthesis of gene 1 protein product without attaching protein 2 gene. However, the increase in suppressor cell host leads to the synthesis of significant amounts of the fused protein. Such suppressor cells-owners contain tRNA modified to insert an amino acid at the position of the termination codon of the mRNA, thereby leading to the production of significant quantities merged hnologies 5: 376-379 (1987). Can be any acceptable methods for placing such a termination codon in an mRNA encoding a fused polypeptide.
Capable of suppressing the codon can be inserted between the first gene coding for the polypeptide and a second gene encoding at least part of the protein ragovoy shell. On the contrary, capable of suppressing the termination codon can be inserted next to the site of the confluence with the substitution of the last triplet of amino acids in polypeptide or the first amino acid in the protein ragovoy shell. If phagemid, capable of containing the codon suppression, grown in suppressor cell host, this leads to significant production of the polypeptide of the fusion containing the polypeptide and the protein shell. If phagemid grown in supressornah cell host, the polypeptide synthesized without-fusion protein ragovoy shell due to the termination of the inserted suppressor the triplet encoding a UAG, UAA or UGA. In supressornah cell synthesize and secrete the polypeptide from the host cell due to the absence of the fused protein ragovoy shell, which otherwise anchored her in the cell host.
5. The change (mutation) of the gene 1 in selected positions
Gene 1 encoding capable of isomerization residue of aspartyl, should be replaced. Change is defined as substitution, elimination (deletion or addition of one or more codons in the gene encoding the polypeptide, which leads to changes in the amino acid sequence of the polypeptide compared to an unmodified or native sequence of the same polypeptide. Preferably, when the changes will be the replacement of at least one amino acid with any other amino acid in one or more regions of the molecule. Changes can be produced using a number of known ways. These methods include, but are not limited to, siteprovides by mutagenesis using oligonucleotides and cassette mutagenesis.
A. Siteprovides mutagenesis using oligonucleotides
Siteprovides mutagenesis using oligonucleotides is the preferred method of obtaining substituted, eliminated and the inserted gene variants 1. This technique is well known, as described by Zoller et al., Nucleic Acids Res. 10: 6487-6504 (1987). Briefly, gene 1 change the hybridization of the oligonucleotide encoding the desired mutation, DNA template, where template is a single-stranded form of a plasmid, which contains for the synthesis of full secondary complementary chain of the template, which, therefore, will include oligonucleotide primer and encode the selected change in gene 1.
Usually use oligonucleotides containing at least 25 nucleotides. Optimal oligonucleotide will contain from 12 to 15 nucleotides that is fully complementary to the template on either side of the nucleotide(s), coding mutations. This ensures that the oligonucleotide will hybridisierung properly with single-stranded DNA template molecule. The oligonucleotides are readily synthesized using known methods described, for example, Shea et al., Proc. Natl. Acad. Sci. USA 75: 5765 (1978).
DNA template can only be generated by vectors that are obtained or bacteriophobic M13 vectors (usually suitable MMR and MMR vectors) or vectors that contain a single-stranded phage origin or replication as described by Viera et al., Meth. Enzymol. 153:3 (1987). Thus, the DNA that you want to mutate, must be inserted in one of these vectors in order to generate the single-stranded template. The production of single-stranded template is described in sections 4.21-4.41 monograph Sambrook et al., supra.
To change the sequence of amplified DNA, oligonucleotide hybridizing ONT maple DNA polymerase I, then they add for the synthesis of a complementary chain of the template using the oligonucleotide as a primer for synthesis. So get heteroduplex molecule, so that one strand of DNA encodes the mutated form of the gene 1 and the other strand (the original template) encodes the native, unaltered sequence of gene 1. This heteroduplex molecule is then transformed into the corresponding cell of the host, usually a prokaryote, such as E. coli JM 101. After growing the cells, they are placed on agar plates and perform the screening using oligonucleotide primer is labeled with32phosphate, to identify the bacterial colonies that contain the mutated DNA.
The method described just above can be modified in order to create homoduplex molecule, where both strands of the plasmid containing the mutation(s). The modification is as follows: single-stranded oligonucleotide is annealed to the single-stranded template, as described above. A mixture of three deoxyribonucleotides; desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (sttp-based) combined with a modified thio-dezoksiribozimov called dCTP-(aS) (Amersham). This mixture is added to template-oligonuclear the exception of mutant bases. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from hydrolysis with restriction endonucleases. After the template double-stranded chain of heteroduplex cut the appropriate restriction enzyme, template chain can hydrolyze by ExoIII nuclease or other suitable nuclease in addition to the field that contains subjected to mutagenesis site(s). Next, the reaction is stopped for separating molecules, which is only partially single-stranded. Next, there is formed a complete double-stranded DNA homoduplex using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates of ATP and DNA ligase. This homoduplex molecule can be transformed into the appropriate cell host, such as E. coli JM101, as described above.
Mutants with more than one substituted amino acid can be generated in one of the following ways. If amino acids are located close in the polypeptide chain, they can metrovacesa simultaneously using one oligonucleotide that encodes all of the required substitution of amino acids. If, however, amino acids are located at some distance from each other (separated boltenia. Instead, you can use one or two alternative methods.
In the first method, a separate oligonucleotide generate for each of the substituted amino acids. Next, the annealed oligonucleotides into single-stranded template DNA simultaneously, and the second strand of DNA, which is synthesized from a template, will encode all the necessary substitution of amino acids. An alternative method involves two or more circles mutagenesis to produce the desired mutant. The first round is as described for the single mutants, is that wild type DNA is used for a template and oligonucleotide encoding the first desired substitution(I) amino acids, annealed to the template and then generate heteroduplex DNA molecule. The second round of mutagenesis uses mutated DNA produced in the first round of mutagenesis in the form of a template. Thus, this template already contains one or more mutations. Oligonucleotide encoding an additional substitution(I) the necessary amino acids, then annealed to this template, and the obtained DNA chain now encodes the mutation from both the first and second circles mutagenesis. The resulting DNA can be used as a template in the third round of mutagenesis and so on
B. Cassette mutagenic variants of the gene. The method is based on described by Wells et al., Gene 34: 315 (1985) method. The starting material is the plasmid (or other vector) comprising the gene 1, the gene that you want to mutate. Identify the codon(s) in multiroom 1 gene. Must be a unique restriction site for the endonuclease on each side of the identified area(s) of mutation. If no such restriction sites exist, they can be generated using the above method siteprovides mutagenesis using oligonucleotides to introduce them at appropriate locations in the gene 1. After the restriction sites introduced into a plasmid, the plasmid cut at these sites for linearization. Double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites, but containing the desired mutation(s) is synthesized using standard techniques. Two chains are synthesized separately and then hybridize together using standard methods. This double-stranded oligonucleotide is called a cartridge. The cartridge is constructed so that there is a 3' 5' ends that are compatible with the ends of the linearized plasmid, so that the cartridge was directly luigirules with the plasmid. This plasmid now ternative embodiment, the invention provides the production of variants of the desired protein, containing one or more subunits. Each subunit is usually encoded by separate genes. Each gene, encoding each subunit can be obtained using known methods (see, for example, section II). In some cases it is necessary to obtain the gene encoding the different subunits, using separate methods selected from any of the methods described in section II.
If construct (create) replicate the expression vector, where the protein contains more than one subunit, all subunits can be regulated by the same promoter, usually located in the 5' to DNA that encodes a subunit, or each may be regulated by the same promoter, usually located in the 5' to DNA that encodes a subunit, or each may be regulated by a separate promoter, appropriately oriented in the vector so that each promoter is operable associated with DNA intended for regulation. The selection of promoters were carried out as described above in section III.
When designing replicate the expression vector containing DNA encoding the desired protein with multiple subunits, the reader refer to Fig.11, where for the purpose of illustration vector depicted schematics is of the subunits of the desired protein will merge with protein ragovoy shell, for example, M13 gene III. This fused gene will typically contain its own signal sequence. A single gene encodes another subunit or subunit and, obviously, each subunit usually has its own signal sequence. Fig.11 also shows that a single promoter may regulate the expression of both subunits. On the contrary, each subunit can be independently regulated by a different promoter. Fused design “subunit of the desired protein - protein ragovoy shell” can be obtained as described above in section IV.
If you are creating a family of variants need multispherical protein, DNA encoding each subunit in the vector can metrovacesa in one or more positions in each subunit. If you create options multispherical antibodies, the preferred sites of mutagenesis are codons coding for amino acid residues located in the hypervariable sites (CDRs) of the light chain, heavy chain or both chains. CDRs are usually referred to as hypervariable regions. Methods mutagenesis of the DNA encoding each subunit of the desired protein, mainly described above in section V.
7. Receiving target molecules and is the returns using known recombinant methods. To illustrate glycoproteinoses hormone receptors can be obtained using the method described by McFarland et al., Science 245: 494-499 (1989), deglycosylated form expressed in E. Li, described in the article Fuh et al., J il. Chem. 265: 3111-3115 (1990). Other receptors can be obtained using standard methods.
The purified protein targets can join the appropriate matrix, such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic the copolymers, hydroxyalkyl-methacrylate gels, polacrilin and polymethacrylimide the copolymers, nylon, neutral and ionic carriers, and so on Accession of the protein target to the matrix can be accomplished using the methods described in Methods in Enzymol. 44 (1976), or by other known methods.
After the accession of the protein target to the matrix immobilized target is associated with a library pagemenu particles under conditions suitable for binding at least part pagemenu particles with the immobilized target. Usually conditions, including pH, ionic strength, temperature, etc. should be close to physiological conditions.
Related pagemedia particles (binder, binders), which has high affinity to immobile) care of the patient. The binder can be separated from the immobilized matrix using a number of ways. These methods include competitive separation from the immobilized target with the help of a number of ways. These methods include competitive Department using the wild-type ligand, a change in pH and/or ionic strength, and other known methods.
Appropriate cell hosts infect the binder and phage-helper cells and the hosts are cultivated under conditions suitable for amplification pagemenu particles. Next pagemedia particles are collected and the selection process repeated one or more times until, until you have selected the binder desired affinity for a target molecule.
It is possible that the library pagemenu particles can consistently be in contact with more than one immobilized target to improve the selectivity of individual targets. For example, the fact that the ligand, such as hGH contains more than one natural receptor. In the case of hGH and hormone receptor growth, and prolactinemia receptor bind hGH ligand. It is desirable to improve the selectivity of hGH for hormone receptor growth compared to Prolactinum receptor. This can be achieved by initial binding library pagemenu particles is of the first kind) prolactinomas receptor and further linking prolactinemia “binder” or “binder” low affinity or with immobilized receptor growth hormone and selecting the binder of the high affinity receptor for growth hormone. In this case hGH mutant with a lower affinity prolactinoma receptor must have a therapeutic application, even if the affinity of the receptor for growth hormone is somewhat lower than the affinity of wild-type hGH. This same strategy can be used to improve the selectivity of a particular hormone or protein for receptor primary functions compared to the "clearance"receptor.
In another embodiment of the present invention can be obtained an improved substrate amino acid sequence. This can be useful for obtaining a better “cut” sites of liquor to a protein or to the best of substrate/protease inhibitors. In this embodiment, the immobilized molecule, for example hGH receptor, Biotin-aydinbey receptor or a receptor capable of covalently contact matrix), subjected to fusion with a gene III through a linker. The linker will preferably comprise from 3 to 10 amino acids, and will act as a substrate for the protease. Phagemid will be created, as described above, where the DNA encoding the linker region, arbitrarily motirola to obtain a randomized library pagemenu particles with different amino acid sequences in the binding site. Biblioteca, with the preferred or best substrate amino acid sequences in the linear region of the desired protease will buyouts, initially producing enriched fraction pagemenu particles encoding preferred linkers. Data pagemedia particles do next cycles several times for producing enriched fractions of particles encoding the consensus sequence(s).
II. The generation of antibodies
The original antibody can be obtained using known methods or by generating antibodies. Typical methods of generating antibodies are described in more detail in the following sections.
The antibody directed against the desired antigen. Preferably, when the antigen is a biologically important polypeptide and the introduction of antibodies mammal suffering from a disease or disorder, can lead to a therapeutic effect for a given mammal. However, also discusses antibodies directed against polipeptidnyh antigens (such as tumor-specific glycolipid antigens; see U. S. Pat. No. 5091178).
If the antigen is a polypeptide, it may be a transmembrane molecule (e.g., receptor or ligand, for example factor the third growth hormone; factor secretion of growth hormone; parathyroid hormone, glucagon; factors that prevent blood clotting, such as protein C; atrial naturethese factor; lung surfactant; a plasminogen activator, such as urokinase, or urinary or tissue plasminogen activator (tPA), bombezin; thrombin; hemopoietic growth factor;andfactor that causes necrosis of tumor cells; enkephalinase; RANTES (regulated activation normally T-cell expressed and secreted), which regulates the activation of expression and secretion of normal T-cells); human phagocytic inflammatory protein (MIP-1-); serum albumin such as serum albumin person; Müller inhibiting substance; And-chain relaxin; In-circuit relaxin; prolactin; mouse gonadotropin-binding peptide; a microbial protein, such as-lactamase; DNA-ASE; IgE; a cytotoxic T-lymphocytopenia antigen (cytotoxic T-lymphocyte associated antigen CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (vascular endothelial growth factors, VEGF); receptors for hormones or growth factors; protein a and D; rheumatoid factors; a neurotrophic factor such as NT-6), or nerve growth factor (NGF-B; platelet-derived growth factor (platelet-derived growth factor, PDGF), fibroblast growth factors (fibroblast growth factor, FGF), such as aFGF and bFGF; epidermal growth factor (epidermal growth factor, EGF); transforming growth factor (transforming growth factor, TGF) such as TGF-and TGF-including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-like growth factor-I and-II (insulin-like growth factor binding protein; CD proteins such as CD-3, CD-4, CD-8 CD1-9 and CD-20; erythropoietin; osteoinductive factors; immunotoxins; a protein involved in osteogenesis (bone morphogenetic protein, BMP); an interferon such as-,and-interferon; colony stimulating factors (ln stimulating factors, CSFs) such as M-CSF, GM-CSF and G-CSF; interleukins (IIs), such as IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; homing” receptors, adressin; regulatory proteins; integrins; such as CD11a, CD11b, CD11c, CD18, and ICAM, VLA-4 and VCAM; a tumor-specific antigen such as HER2, HER3, or HER4 receptor; and fragments of any of the above CD proteins, for example, CD3, CD4, CD8, CD 19, CD20 and CD34; members of the rb receptor family such as the EGF receptor, HER2, HER3, or HER4 receptor; cell adhesion molecules such as LFA-1, Mac 12, P150,95, VLA-4, ICAM-1, VCAM andv/3 integrin, which includes, orand-subunit (e.g., anti-CDIIa, awH-CD18 or anti-CDIIb antibodies); growth factors such as VEGF; IgE; antigens of blood groups; flk2/flk3 receptor; receptor obesity (obesity receptor, OB); mpl receptor; CTLA-4; protein C, etc. are Particularly preferred target is IgE.
Antibody grow against an antigen derived from a first species of mammal. The first species of mammal, preferably a human being is. However, considering other species of mammal, such as agricultural, domestic Pets or zoo animals, for example, if the antibody intend to use for the treatment of mammals. The antigen from the first mammalian can be isolated from its natural source for the purpose of generating antibodies against him. However, as described below, cells, including antigen, can be used as immunogens to generate antibodies. In other embodiments, the antigen producerin to have a sufficiently strong binding affinity for the antigen. For example, the antibody can bind the antigen from the first species of mammal with a magnitude of binding affinity (d) not more than ~1×10-7M, preferably no more than 1×10-8M and most preferably no more than 1×10-9M. the Affinity of the antibody can, for example, be determined by the saturated binding; enzyme-linked immunosorbent assay (ELISA) and competitive analysis (for example, RIAs).
The antibody may be subjected to other tests of biological activity, for example, to evaluate its effectiveness as a therapeutic agent. This check is known and depends on the target antigen and involves the use of antibodies. Examples include checking adhesieve monolayer of keratinocyte and analysis of a mixed reaction of lymphocytes to CDIIa (each described below in the example); the test of inhibition of tumor growth (as described, for example, in WO 89/06692); analyses of antibody-dependent cell-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) and complement-dependent cytotoxicity (complement-mediated cytotoxicity, CDS) (U. S. Pat. No. 5500362) and analysis of agonistic activity or hematopoesis (see WO 95/27062).
For selection of antibodies that are associated with a single epitope on representing in the data block, for example that described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Ed. Harbow and David Lane (1988). On the contrary, the mapping of the epitope, for example, as described by Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), can be performed to determine links if desired antibody epitope.
Next, determine the interdependence of antibodies. Binding affinity of antibodies for the homology of the antigen used to generate antibodies (where homology consists of “second” type of mammal), determined using the methods described above. In a preferred embodiment, the second type of mammal is a mammal that does not belong to the human race, to which the antibody is administered in preclinical studies. Thus, the second type of mammal may be a Primate, non-human kind, for example RH, peacock, baboon, chimpanzee and macaque. In other embodiments, the second species of mammal can be, for example, rodents, cat or dog. Videosanime antibody should normally possess binding affinity to the antigen from a second species of mammal that are not relevant for the human race, which, at least 50-fold, or at least 500 times, or at least 1000 times weaker than swasti such to videosanime antibody cannot be used effectively for preclinical studies in a second species of mammal.
While the preferred method for determining videosalvesti (and for the evaluation of mutant antibodies with improved properties; see below) is to quantify the binding affinity of antibodies, in other embodiments of the invention evaluate one or more of the biological properties fedosovskogo antibodies and mutant antibodies in addition to or instead of the definitions of binding affinity. Such typical biological test described above. Such tests are particularly useful when given evidence in relation to therapeutic efficacy of antibodies. Usually, though not necessarily, antibodies which show improved properties such inspections will also have an improved affinity. Thus, in one embodiment of the invention, where the analysis is to validate the biological activity, and not checking the binding affinity, videosanime antibody usually has to have a “biological activity” using “material” (e.g., antigen, cell, tissue, organ or whole animal) from the second species of mammal, which is extremely aktivnosti in the respective test, using reagents from a first species of mammal.
Videosanime antibody further change in order to generate the mutant antibody, which has a stronger binding affinity to the antigen from a second species of mammal than videosanime antibody. The mutant antibody preferably has a binding affinity to the antigen from the mammal, not belonging to the human race, which is at least 10 times, preferably at least 20 times stronger, more preferably at least 500 times stronger and sometimes, at least 1000 or 2000 times stronger than the binding affinity fedosovskogo antibodies to the antigen. The increase is needed or desired binding affinity will depend on the initial binding affinity fedosovskogo antibodies. However the test is a test of biological activity; mutant antibodies preferably possess biological activity in the test of choice, which at least has 10 times better, preferably at least has 20 times better, more preferably at least 50 times better and sometimes, at least 100 or 200 times better than the biological activity VI the pilot changes (for example, substitution is introduced into one or more changes (e.g., substitutions) residues spanning region) can be entered in videosanime antibody, if the result is an improvement in the binding affinity of the mutant antibodies to the antigen from a second species of mammal. Examples of modified residues spanning the area include the remains, which ecovalence bind antigen directly (Amit et al., Science 233: 747-753 (1986)); interact with / affect the conformation of CDR (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)); and/or participate in the VL-VH interface (ER V). In some embodiments, the modification of one or more residues spanning region leads to an increase in binding affinity of antibodies to the antigen from a second species of mammal. For example, from about one to five residues spanning region can be changed in this embodiment of the invention. Sometimes it may be sufficient to obtain mutant antibodies suitable for use in preclinically research, even if you have not changed the remnants of the hypervariable region. Typically, however, the mutant antibodies should include additional changes variable regions.
The remains of the variable regions that change, can be changed arbitrarily, the OS is nourishing, is such that such randomly produced mutants antibodies can easily be subjected to screening (selection, validation).
Methods of producing antibodies, which may be interdependent and therefore require modification according to the methods developed in this invention are the following:
A. Obtaining antibodies
(I) Obtaining antigen
Soluble antigens or fragments thereof, may conjugated with other molecules, can be used as immunogens to generate antibodies. Transmembrane molecules, such as receptors and fragments thereof (e.g., the extracellular region of the receptor) can be used as an immunogen. Conversely, cells expressing transmembrane molecule, can be used as an immunogen. Such cells can be obtained from a natural source (for example, lines of cancer cells) or can be cells that are transformed with recombinant methods for the expression of transmembrane molecules. It is clear that other antigens and their form suitable for the production of antibodies.
(II) polyclonal antibodies
Polyclonal antibodies usually occur in mammals, non-human kind, when numerous is Olesno for conjugation of the corresponding antigen with protein, which is immunogenic in the immunized species, such as hemocyanin lymph snails, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivateservlet agent, for example an ether maleimidomethyl-sulfosuccinimidyl (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaric aldehyde, succinic acid anhydride, thionyl chloride or R' - N=C=CR, where R and R' represent different alkyl groups.
Animals subjected to immunization with the antigen, immunogenic conjugates, or derivatives by mixing, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with three volumes complete adjuvant's adjuvant and the introduction of a solution through the skin in many places. A month later the animals revaccinate from 1/5 to 1/10 part of the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. After 7-14 days the animals took the blood and the serum was analyzed for titer antibodies. Animals revaccinate up until the titer did not cease to change. Preferably animals revaccinate conjugate of the same antigen, but conjugating is o be catching in recombinant cell culture by merging the protein. Also the agents that cause aggregation, such as alum, is successfully used to enhance the immune response.
The selected antibody of a mammal usually should have a sufficiently strong binding affinity to the antigen. For example, the antibody can bind anti-IgE antigen person with the magnitude of the binding affinity (Kd) of not more than 1×10-7M, preferably no more than 1×10-8M and most preferably no more than 1×10-9M. the affinity of antibodies can be determined using saturating binding; enzyme-linked immunosorbent assay (ELISA) and competitive analysis (e.g., radioimmunoassay).
For selection of anti-IgE antibodies person can carry out routine analysis of cross-linking, which is described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed. Harlow and David Lane (1988). On the contrary, the mapping of the epitope, for example, as described by Champe et al., J. il. Chem. 270: 1388-1394 (1995), can be performed to determine the binding.
While the preferred method of determining the effectiveness of the polypeptide or antibody is to quantify the binding of affinity antibodies; other options evaluate one or more biological properties of the antibodies, or in addition to the, what that relates to therapeutic efficacy of antibodies. Usually, though not necessarily, antibodies which show improved properties such checks should also have improved binding affinity.
(III) Monoclonal antibodies
Monoclonal antibodies are antibodies that recognize a separate area of the determinants. Their constant specificity makes monoclonal antibodies are more suitable than polyclonal antibodies, which usually contain antibodies that recognize a number of different areas of the determinants.
Monoclonal antibodies can be obtained using the hybrid method, first described by Kohler et al., Nature 256: 495 (1975), or can be obtained by methods rekombinantnoi DNA (U. S. Pat. No. 4816567).
In the method a hybrid mouse or other suitable animal host, such as a hamster or macaco, subjected to immunization, as described above, to identify lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. On the contrary, the lymphocytes can undergo immunization in vitro. The lymphocytes are then fused with myeloma cells using a relevant factor that causes a confluence of the cells, how)).
Thus, the obtained hybrid cells are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or natural selection nalivshihsya parental myeloma cells. For example, if the parental myeloma cells lack the enzyme-hypoxanthineguanine (HGPRT or HPRT), then culture medium for the hybridomas typically must contain gipoksantin, aminopterin and thymidine (GAT-Wednesday), substances that prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that are easy to blend, support stable high-level production of antibodies selected antibody-producing cells, and are sensitive to the environment, as, for example, to GAT environment. Among them, preferred myeloma cell lines are related to mice or rats myeloma lines, such as, for example, obtained from the MORSE-21 and MPC-11 tumors of mice available from the center Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American culture collections (American Type Culture Collection, Rockville, Maryland USA). For production of monoclonal antibodies person is. mmunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker. Inc., New York, (1987)).
The cultural environment in which cultured cells are hybridomas were tested for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybrid cells, determined using the thus or reaction binding in vitro, such as radioimmunoassay (radioimmunoassay, RIA) or enzyme-linked immunosorbent assay (enzyme-linked immunoabsorbent assay, ELISA).
After cell hybridoma that produce antibodies of the desired specificity, affinity and/or activity are identified, the clones can be subclinical using technology limited dilution and to cultivate by standard methods (Goding, Monoclonal Antibodies: Principals and Practice, pp. 59-103, Academic Press, 1986). A suitable culture medium for this purpose is, for example, D-MEM or RPMI-1640 medium. In addition, cell hybridomas can be grown in standard in vivo in the form of ascitic tumors in animals.
Monoclonal antibodies are excreted (secreted) by the subclones can easily be separated from the culture medium, ascites fluid, or serum by conventional means of purification of immunoglobulins,matography.
DNA encoding the monoclonal antibodies is easily detected, and sequeiros using conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chain of monoclonal antibodies). The hybrid cells are a preferred source of such DNA. Once the selected DNA may be placed in expressing vectors, which are then transfections in the cells of the host, for example, cells of E. Li., cells COS monkey cells of the ovary of the Chinese hamster (Chinese hamster ovary, CHO) or myeloma cells that otherwise do not produce the protein of the immunoglobulin by applying the synthesis of monoclonal antibodies in the recombinant cell host. Recombinant production of antibodies will be described in more detail below.
In another embodiment, antibodies or antibody fragments can be isolated from phage libraries of antibodies generated using the techniques described in McCafferty et at., Nature, 348: 552-554 (1990). In the works of Clackson et al. Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) described the selection of antibodies specific to mice or rats and humans, respectively, using phage libraries. Subsequent publications describe Produzione, 0: 779-783 (1992)), as well as combinatorial infection and recombination in vivo as a strategy for creating very large phage libraries (Water-house et al., Nuc. Acids. Res., 21: 2265-2266 (1993)). Thus, these methods are viable options compared to traditional methods hybrid monoclonal antibodies for the separation of monoclonal antibodies.
DNA also can be modified, for example, by substituting the coding sequence for the constant region of the heavy and light chains of a human instead of a homologous sequences related to mice or rats (U. S. Patent No. 4816567; Morrison, et al., Proc. Natl. Acad. Sci., USA, 81: 6851 (1984)), or covalent joining to the immunoglobulin polypeptide.
Typically, such nimmanahaeminda polypepide replaced in the constant region of the antibody, or they are substituted for variable regions of one antigennegative centre of antibody to create a chimeric bivalent antibody comprising one antigennegative centre antibody, having specificity for the antigen, and the other antigennegative centre antibody, having specificity for a different antigen.
(IV) the Generation of mutant antibodies
If videosanime antibody identified and selected, it is often poleznoe or more hypervariable regions of the antibodies of the mammal. Conversely, or in addition, one or more changes (e.g., substitutions) residues spanning the area can enter in the antibody of a mammal, where it leads to the improvement of the binding affinity of the mutant antibodies to human IgE. Examples of residues spanning the field for modification include residues that directly ecovalence associated antigen (Amit et al., Science 233: 747-753 (1986)); interact with / affect the conformation of CDR (Chothia et al., J. Mol Biol. 196: 901-917 (1987)); and/or participate in the VH-VL interface (EP 239400 B1). In some embodiments, the modification of one or more residues spanning region leads to an increase in binding affinity of the antibody for antigen person. For example, from about one to five residues spanning region can be changed in this embodiment of the invention. Sometimes it may be sufficient to obtain mutant antibodies suitable for use in preclinical studies, even if you have not changed the remnants of the hypervariable region. Typically, however, the mutant antibodies should include additional changes variable regions.
Mutable residues hypervariable region can be changed arbitrarily, especially if the initial binding affinity viatica selection (validation).
One suitable to generate the mutant antibody technique known as “alanine scanning mutagenesis” (Cunningham, B. C., J. A. Wells Science, 244: 1081-1085 (1989); B. C. Cunningham, J. A. Wells, Proc. Natl. Acad. Sci. USA 84, 6434-6437 (1991)). In this case, to influence the interaction of amino acids with antigen from the second mammalian one or more residues of the hypervariable region is replaced by the residue(s) alanine or polyalanine. These remnants of the hypervariable region, demonstrating functional sensitivity to the substitutions then are cleaned by the introduction of additional or other mutations at the sites of substitution and for the sites of substitution. Thus, while the site for introducing changes in the amino acid sequence determined in advance, the nature of the mutation, in essence, should not be pre-defined. Ala-mutants produced in this way are subjected to selection based on their biological activity described in this invention. Such substitution may be taken with other amino acids, depending on the desired properties generated by scanning the remains.
The invention also relates to a synthetic method of identifying amino acid residues that modify. According to this method, the ID manually the type of mammal, and residues from a hypervariable region involved in the binding homology of this antigen from the second species of mammal. To achieve this, you can make ladirovannye scanning residues of the hypervariable region fedosovskogo antibodies, each ala mutant is screened for binding to the first and second species of mammal. Thereby identify the remains of the hypervariable region involved in the binding of the antigen from the first mammalian (e.g. human), and residues involved in binding the homology of the antigen from a second species of mammal (e.g., non-human kind). Preferably, the residue(s), largely involved(s) in the binding of the antigen from a second species of mammal (e.g., mammalian, non-human kind), and not the antigen from the first mammalian (e.g. human) were chosen as candidates for modification. In another embodiment for modifying selected these balances are largely involved in binding the antigen from the first and second species of mammal. Another, but less preferred variant of these residues, which are involved in binding to the antigen of the requirements. Such modification may include a deletion (elimination) of residue, or insertion of one or more residues in the vicinity interest balance. Usually, however, the modification includes the substitution of residue to another amino acid.
Usually you can start with conservative substitutions, for example, shown in table a below in the heading of “preferred substitutions”. If such substitutions result in a change in biological activity (e.g. binding affinity), then impose more substantial changes, called “typical substitutions” in table.And, or, as further described with respect to classes of amino acids, and the products subjected to the test.
Even more substantial modifications in the biological properties of the antibodies make selecting substitutions that differ significantly in their effect on maintaining: (a) structure of the polypeptide backbone in the area of the substitution, for example, folded or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (C) the volume of the side chain. Naturally occurring residues are divided into groups, depending on the General properties of the side chain:
(1) hydrophobic: norleucine (norleucine), met a, ys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will cause data representative of one of these classes for another class.
The molecule is a nucleic acid that encodes the amino acid sequence mutants, obtained using a number of known ways. These methods include, but are not limited to siteprovides by mutagenesis using oligonucleotides by PCR mutagenesis and cassette mutagenesis previously obtained mutant or nematanthus version fedosovskogo antibodies. The preferred method of obtaining mutants is siteprovides mutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)).
In some embodiments, the mutant antibodies will have only replaced the remainder of the hypervariable region, for example, from 2 to 15 possible substitutions hypervariable region.
Typically, the mutant antibodies with improved biological properties must have an amino acid sequence that is at least 75% identical or similar amino acid sequences of variable regions of the heavy or light chain of IgE antibodies against mammalian, more preferably, is more preferable, at least 95%. Identity or similarity in terms of consistency is defined as the percentage of amino acid residues in the sequence of the candidate, which is identical (i.e. the same residue) or similar (i.e., amino acid residue from the same group based on common properties of the side chain, supra) residues fedosovskogo antibodies, after conducting a comparative analysis of the sequences and introducing two filament breaks, if necessary, to achieve the maximum percent sequence identity.
On the contrary, mutant antibodies can be generated using systematic mutation CDR regions of the heavy and light chains of anti-IgE antibodies. The preferred technique for generating these mutants antibodies includes the use of affinity maturation using phage image (Hawkins et al., J. Mol. Biol. 254: 889-896 (1992) and Lowman et al., Biochemistry 30(54): 10832-10838 (1991)). It is known that merge the bacteriophage membranes with protein (Smith, Science 228: 1315 (1985); Scott, Smith, Science 249: 386 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 8: 309 (1990); Devlin et al., Science 249: 404 (1990); in the above review Wells, Lowman, Curr. Opin. Struct. Biol. 2: 597 (1992); U. S. No. Pat. 5223409) suitable for binding phenotype displayed proteins or peptides with genotype bacteriophobic., Proc. Natl. Acad. Sci. USA 88: 7978 (1991); Garrard et al., Biotechnol. 9: 1373 (1991)).
Monovalent phage display contains a mapping of the set of variants of the protein in the form of mergers with bacteriophage protein shell in such a way as to limit the playback options to only one copy for a few phage particles (Bass et al., Proteins 8: 309 (1990)). “Affinity maturation” or better equilibrium binding affinely different proteins has previously been achieved through consistent application of mutagenesis, monovalent phage display of functional analysis and the addition of favorable mutations, as it is an example in the case of human growth hormone (Lowman, Wells, J. Mol. Biol. 234: 564-578 (1993); U. S. Pat. No. 5534617), and F(ab) region of the antibody (Barbas et al., Proc. Natl. Acad. Sci. USA 91: 3809 (1994); Yang et al., J. Mol. Biol. 254: 392 (1995)).
Library of many (106) protein variants, which differ in certain provisions of their sequence, you can create bacteriophobic particles, each of which contains DNA encoding a protein. After cycles of affinity purification using immobilized antigen individual clones of bacteriophages isolated and the amino acid sequence of the reproduced sposobem obtain chimeric antibodies, where significantly smaller part undamaged variable regions specific for the human race, was substituted by the corresponding sequence from species not belonging to the human race. Purified antibody has one or more amino acid residues introduced into it from a source that does not belong to the human race. The amino-acid residues that do not belong to the human race, often referred to as “alien” residues, which are usually taken from the “alien” variable regions. Improvement can be done, mainly following the method of winter with TCS. (Winter) (Jones et al., Nature 321: 522-525 (1986); Riechman et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)), by the replacement of hypervariable sites CDRs or CDR sequences of rodents on the corresponding sequences of human antibodies. Accordingly, such “refined” antibodies are chimeric antibodies (U.S. Pat. No. 4816567), in which a much smaller proportion of intact variable regions of human substituted by the corresponding sequence from a species not belonging to the human race. In practice, purified IgE antibodies have some CDR residues and possibly some FR Stadnyk areas of the person, both light and heavy, to be used to obtain purified antibodies is very important to reduce antigenicity. According to the so-called method of “best match”, conduct the screening sequence variable regions of the antibodies of the rodent relatively full (all) library of known sequences of variable regions of human rights. The sequence belonging to the human race, which is closest to the sequence of the rodent, then accepted as spanning region (FR) of a person for purified antibody (Sims et al., J. Immunol., 151: 2296 (1993), Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular spanning region obtained from consensuses” sequence of all human antibodies of a particular subgroup of light or heavy chains. The same spanning region can be used for some other purified antibodies (Carter et al., Proc. Natl. Acad. Sci., USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).
In addition, it is important that antibodies are refined with retention of high affinity for the antigen and other important biological properties. In accordance with the preferred option to achieve this goal purified antibodies produced by the method of analysis shahmatnye model parent and enhanced sequences. Model specific regions of the antibodies, such as VHand VLareas, creating separate from the “consensus” sequences, based on the F(ab) structures that have similar sequences. Three-dimensional models of immunoglobulin commonly available and known in the art. There are computer programs which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Analysis of these maps allows to make a conclusion about the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., to conclude that residues that influence the ability of the candidate immunoglobulin to bind antigen. For example, in the simulation of fragment F(ab)-12 in example 2 was used MAE mice or rats as a template to promote CDR residues and spanning areas for modification (modification) together with molecular modeling to obtain the mutant sequence.
As another example, we can mention the control antibody Mab4d5. The model was created based on several Fab structures from the protein data Bank of Brookhaven (Brookhaven) (inputs 1FB4, 2RHE, MSR, 3FAB, 1 FBg, 2H whom she and additional structures were then superimposed on this structure, using the coordinates of their main chain atoms (INSIGHT, Biosym Technologies). These programs and techniques used for modeling the desired antibodies.
A typical analysis using molecular modeling, can be performed as follows. The distance from templateto the same Sa in each of the superimposed structures calculated for each position of this residue. Usually, if all (or almost all) With- distances for this residue1then this position include the “consensus” structure. In some cases the remains offolded conformation spanning region will meet these criteria, while the CDR loop fragments may not meet. For each of these selected residues calculate the average coordinates for individual N, WithWith, and Withatoms and then adjusting for deviations obtained from the geometry of a non-standard connection using 50 cycles of minimization of energy, using commercially available software, such as progradiate fix. Side chains are highly preserved residues, for example a disulfide bridge cysteine residues, then include in the resulting “consensus” structure. Then include sequence specific VL and VH regions of antibodies, since the CDR residues and using tabular data CDR conformation of publication Chothia et al. (Chothia C. et al., Nature 342: 877-883 (1989)) as a guide. The conformation of the side chain is chosen on the basis of Fab crystal structures, rotameric library (Ponder, J. W., Richards F. M. J. Mol. Biol. 193: 775-791 (1987)) and considerations of package. As VH-CDR3 may not meet the above criteria, models can be created in the study of loop fragments of similar size, using the INSIGHT gained when using the considerations packaging and exposure to solvent, or can be created using a different path and commercially available methods. The model should preferably be subjected to 5000 cycles of minimization of energy.
Thus, residues spanning region can be selected and separated from the recipient and alien sequences in order to achieve the desired characteristics of the antibodies, such as increased affinity for the antigen(s) target. Typically, the CDR residues are directly and primarily include to provide VLE CDR residues rats or mice together with molecular modeling to create a fragment purified anti-IgE antibodies of mice or rats.
On the contrary, at the present time it is possible to produce transgenic animals (e.g. mice) that when immunization is able to produce a full range of human antibodies in the absence of producing endogenous immunoglobulin. For example, it has been described that the homozygous deletion of the heavy chain region of the connection (JH) gene antibodies in chimeric and line-embryo mutant mice results in complete inhibition of the production of endogenous antibodies. The transfer of the ranked list immunoglobulin gene lines of a human embryo in this line-embryo mutant mice will result in the production of human antibodies with the antigenic stimulus (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993)); Jacobovits et al., Nature 362: 255-258 (1993); Bruggermann et al., Year in Immunol. 7: 33 (1993); and Duchosal et al., Nature 355: 258 (1992)). Human antibodies can also be obtained from libraries of phage-display (Hoogenboom et al., J. Mol. Biol. 227: 381 (1991); Marks et al, J. Mol. Biol., 222: 581-597 (1991); Vaughan et al., Nature Biotech. 14: 309 (1996)).
(b) Additional modifications
After production of the mutant antibodies determine the biological activity of this molecule over fedosovskogo antibodies. As noted above, this may include determining binding affinity and/or other biological activities and the activity to the antigen from a second species of mammal. One or more mutant antibodies selected using this initial screening, is subjected to one or more further tests of biological activity, to confirm that the mutant(s) of an antibody with enhanced binding affinity is really suitable for preclinical studies. In preferred embodiments, the mutant antibody retains the ability to bind the antigen from the first species of mammal with a binding affinity that is similar to fedosovskogo antibody. This can be achieved by canceling the change in stocks hypervariable region included in the binding to the antigen of antibodies against human rights. In other embodiments, the mutant antibodies can be considerably modified binding affinity from the first species of mammal (e.g., binding affinity to the antigen better, but it could be worse than videosanime antibody).
The mutant(s) of the antibodies, thus selected, can be subjected to further modifications, depending on the intended use of the antibody. Such modifications may involve further changes in amino acid sequence, fusion with heterologous(them) polypeptide(s) and/or covalent modification, the AI carefully developed below. For example, any remnants cysteines not involved in maintaining the appropriate conformation of the mutant antibodies can also usually replaced with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-stitching. On the contrary, (a), cysteine bond(s) can be added to the antibody to improve its stability (particularly if the antibody is a fragment of the antigen, such as an Fv fragment). Another type of amino acid mutant has altered the structure of glycosylation. This can be achieved through deletions (elimination) of one or more carbohydrate residues present in the antibody, and/or adding one or more sites (centers) glycosylation sites that are not.present in the antibody. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate residue to the side chain of an asparagine residue. Sequence Tripeptide asparagine-X-serine and aspargine-X-threonine, where X is any amino acid except Proline, are the recognition sequences of enzymatic attaching a carbohydrate residue to the side chain of asparagine. Thus, the presence of any of this is related glycosylation refers to the attachment of sugar through the ether oxygen; for example, N-atsetilgalaktozamin, galactose, fucose or xylose associated with hydroxyquinolinato, more commonly serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine. Addition of glycosylation sites to the antibody is convenient to do by modifying amino acid sequences that contain one or more of the above Tripeptide sequences (for N-linked glycosylation sites). Change can also be made by adding or substituting one or more residues of serine or threonine in the sequence of the original antibody (for O-linked glycosylation sites).
(V) Fragments of antibodies
Developed various ways of producing fragments of antibodies. Traditionally, these fragments get via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Meth. 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced directly by recombinant cell host. For example, fragments of antibodies can be isolated from phage antibody libraries discussed above. On the contrary, F(ab')2-SH fragments can be directly distinguished from E. SMU approach F(ab') fragments can be isolated directly from recombinant cell culture host. Other methods of producing fragments of antibodies obvious for specialists. In other embodiments, the selected antibody is a Fv fragment single chain (scFv) (PCT Patent Application WO 93/16185).
(VI) Multispecificity antibodies
Multispecificity antibodies have binding specificnosti at least two different antigens. While such molecules usually have to link only two antigen (i.e., bespecifically antibodies, BsAbs), antibodies with additional specificnosti, for example thespecification antibodies included in the invention under the same name. Examples of BsAbs include bespecifically antibodies with one arm directed against the antigen of tumor cells, and the other shoulder against stimulated cytotoxic molecules, such as anti-FyRI/anti-D15, anti-p185HER2/FcyRIII (CD16), anti-D3/cancer b-cell (1D10), anti-D3/anti-RHER2anti-D3/anti-n, anti-D3/anti-renal cell carcinoma, anti-D3/anti-VR-3, anti-D3/L-D1 (anti-colonic carcinoma), anti-D3/anti analogue melanocyte stimulating hormone, anti-EGF receptor/anti-D3, anti-D3/anti-ITSELF and the folate-binding protein (folate binding protein, PBP)/anti-D3, anti-“Peng” carcinoma antigen binding (AMOS-31)/anti-D3; BsAbs with one arm that specifically binds to a tumor antigen and the other arm that binds to a toxin such as anti-saponin/anti-Id-1, anti-D22/anti-saporin, anti-BD7/anti-saporin, anti-D38/anti-saporin, anti-CEA/anti-a chain of ricin, anti-D22/anti-saporin, anti-BD7/anti-saporin, anti-D38/anti-saporin, anti-CEA/anti-a chain of ricin, anti--interferon (IFN-)/anti-idiotype of hybridoma, anti-CEA/anti-Vinca alkaloid; BsAbs for converting enzyme activated precursors of drugs, such as anti-D30/anti-alkaline phosphatase (which catalyzes conversion of the precursor drug of mitomycin phosphate in mitomycin alcohol); BsAbs which can be used as fibrinolytic agents such as anti-fibrin/anti-tissue plasminogen activator (tissue, plasminogen activator, tPA), anti-fibrin/anti-Urogynecology plasminogen activator (urokinase-type plasminogen activator, uPA), BsAbs for conjugation of immune complexes to cell surface receptors, such as antiapoptotic low density (low density lipoprotein, LDL)/anti-Fc receptor (e.g., F use in the treatment of infectious diseases, for example, anti-SW-anti-herpes simplex virus (herpes simplex virus, HSV), anti-T-cell receptor. CD3 complex/anti-influenza, anti-RSR/anti-HIV, BsAbs for detection of tumors, in vitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA, anti-RHER2/anti-hapten; BsAbs as adjuvants virus; and BsAbs as diagnostic tools such as anti-rabbit IgG/anti-ferritin, anti-peroxidase from horseradish (horseradish buffer, R)/anti-hormone, anti-somatostatin/anti-substance P, anti-R/anti-FI, anti-CEA/anti--galactosidase. Examples respecifies antibodies include anti-D3/anti-CD4/anti-D37, anti-D3/anti-D5/anti-D37 and anti-D3/anti-CD8/anti-D37. Bespecifically antibodies can be obtained in the form of full length antibodies or fragments of antibodies (e.g., F(ab')2bespecifically antibodies).
Methods of obtaining bespecifically antibodies known. Traditional production bespecifically antibodies full length based on the co-expression of two pairs of heavy chain - light chain immunoglobulin, where the two chains have different specificnosti (Millstein et al., Nature 305: 537-539 (1983)). Due to an arbitrary set of heavy and light chains of the immunoglobulin data hybridoma (quadroma) spetsificheskoi structure. Clearing the “right” molecules which are usually performed using affinity chromatography, is a rather cumbersome procedure, and the product yield is low. Such methods are described for the first time in WO 93/08829 and in Traunecker et at., EMBO J. 10: 3655-3659 (1991).
According to various approaches, the variable regions of the antibodies with the desired binding specificnosti (antibody-antigen binding sites) were merged with the sequences of the constant region of the immunoglobulin. Fusion occurs with a constant region of a heavy chain immunoglobulin comprising at least part of the hinge, CH2 and CH3 regions. Preferably, the first constant region of the heavy chain (SN) containing the center (site) required for binding to the light chain, was attended by at least one merge. DNA-are you encoding the fusion heavy chain immunoglobulin and, if required, the light chain of the immunoglobulin, are inserted in certain expressing vectors and cotransfected in a suitable host organism. This ensures a high rigidity in the regulation of the mutual proportions of the three polypeptide fragments in the case when unequal ratios of the three polypeptide chains used in the structure, the second polypeptide in one expressing vector, when the expression of at least two polypeptide chains, taken in equal ratios results in high outputs or when the ratios are of no particular value.
In a preferred embodiment, one shoulder especifismo antibodies composed of the heavy chain of the hybrid immunoglobulin with the primary binding specificity and a second shoulder of a pair of heavy-light chain hybrid immunoglobulin (providing secondary binding specificity). It was found that this asymmetric structure facilitates the separation of the corresponding especifismo connection from unnecessary combinations of chains of immunoglobulin, because the presence of the light chain of immunoglobulin only in one part of bespecifically molecules provides a convenient way of separating. This approach is proposed in WO 94/04690. For a more detailed description of obtaining bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).
According to another approach described in WO 96/27011, the boundary surface between a pair of antibody molecules can be created, thus ensuring maximum percentage of heterodimers, which is recovered from recombinant cell culture. The preferred surface includes at least part of blastermaster section of the first molecule antibodies replace (replace) large side chains (for example, tyrosine or tryptophan). Balancing “cavities” of identical or similar size to the large side chain(s) created on the surface section of the second molecule antibodies by replacing large amino acid side chains with smaller (e.g., alanine or threonine). This provides a mechanism to increase the output of heterodimer compared to other unwanted end-products such as homodimers.
Bespecifically antibodies include cross-linking or “heteroconjugate” antibodies. For example, one of the antibodies in heteroconjugate can connect with Avidya, and the other with Biotin. Such antibodies, for example, been proposed to target cells of the immune system to unnecessary (unnecessary) cells (US Patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be obtained using any convenient cross-linking methods. Suitable cross-linking agents, along with some cross-linking techniques are well known and described in US Patent No. 4676980.
Methods of obtaining bespecifically antibodies, fragments of antibodies have also been described in the literature. For example, bespecifically antibodies can be obtained by using chemical Shiva the I with the formation of F(ab')2the fragments. These fragments regenerate in the presence of dicylcopentadiene agent sodium arsenite, stabilizing benzenamine the dithioles and preventing the formation of intramolecular disulfide bonds. Generated F(ab)' fragments of the next turn in dinitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives transform in Fb'-thiol recovery using mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative with education especifismo antibodies. Produced bespecifically antibodies can be used as agents for the selective immobilization of enzymes.
Recent progress contributed to the direct allocation of Fab'-SH fragments from E. Li, which can chemically bind with the formation of bespecifically antibodies. Shalaby et al., J. Exp. Med 175; 217-225 (1992) describe the production of molecules completely “sanitized” especifismo antibody F(ab')2. Each Fab' fragment separately secretively from E. coli and subjected to direct chemical binding in vitro with education especifismo antibodies. The thus obtained bespecifically antibody was able to contact the cells, overproducers rb2 receptor, is the ne against targets tumor human breast.
Were also described different ways of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, bespecifically antibodies were obtained using binding-leucine (Kostelny et al., J. Immunol. 148(5); 1547-1553 (1992). Latinlatin peptides from the Fos and Jun proteins were attached to Fab' portions of two different antibodies at the confluence of the gene. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used for the production of homodimeric antibodies. Technology “diately” described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for obtaining fragments especifismo antibodies. The fragments comprise variable region heavy chain (VH) associated with the variable region of the light chain (VL), by using a linker that is too small to allow the connection of two regions on the same circuit. Thus, VHand VLthe scope of a single fragment of force combined with complementary (hypervariable) VLand VHareas of another fragment, thereby the image of the antibodies with using dimers single chain Fv(sFv) (see Gruger et al., J. Immunol., 152: 5368 (1994)).
Consider more than bivalent antibodies. For example, you can get thespecification antibodies (Tutt et al., J. Immunol. 147: 60 (1991)).
(VII) Engineering effector functions
It is desirable to modify the antibody for its effector functions in order to increase the efficiency of antibody binding, for example, with IgE. For example, the residue of cysteine may be introduced in the Fc region, thereby contributing to the formation of disulfide bonds between the chains in this area. Thus, the obtained homodimeric antibody may have improved the ability of internalization and/or increased complement-mediated lysis and increased antibody-dependent cellular cytotoxicity (antibody-dependent cellular cytotoxicity, ADCC), see Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes Century, J. Immunol. 148: 2918-2922 (1993). On the contrary, can be created with an antibody that has dual Fc region and thus may have increased complementry lysis and ADCC capabilities. Cm. Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).
The invention also relates to immunoconjugates containing antibody conjugated with a cytotoxic agent, such as, for example, chemotherapeutic agent, toxin (e.g., enzymatic active the main isotope (i.e., radioconjugates).
Chemotherapeutic agents suitable for the formation of such immunoconjugates described above. Enzymatically active toxins and fragments thereof that can be used include the A-chain of diphtheria, neisvaziuosiu active enzymes of diphtheria toxin And exotoxin a chain of (from Pseudomonas aeruginosa), A-chain of ricin, A-circuit abrina And-chain Medicina,-sarcin, proteins, Aleurites fordii proteins of diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, Kurin, krotin, inhibitor sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. A number of radionuclides are available for producing radioconjugates antibodies. Examples include212Bi131I131In90Y and186Re.
Conjugates of the antibody and cytotoxic agent receive, using a variety of bifunctional protein-coupling agents such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional prosodie of imidapril (for example, dimethylacetamide HCL), active esters (for example, disuccinimidyl), aldehydes (for example, dimethylacetamide HCL), active esters (for example, disuccinimidyl), aldehydes (such as glutaric aldehyde), bystriansky)Ethylenediamine), the diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained, as described in Vitetta et al., Science 238: 1098 (1987). With14-labeled 1-isothiocyanatobenzene-3-metallitron)triaminotoluene acid (MX-DTPA) is a typical complexes agent for conjugation of radionucleotide with the antibody. Cm. WO 94/11026.
In another embodiment, the antibody may konjugierte with the “receptor” (such as streptavidin) for use in przekonywanie tumors, in which the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then enter a “ligand” (e.g. avidin) which conjugates with cytotoxic agent (e.g., radionucleotide).
Described in this invention mutants antibodies can also be viewed as immunoliposome. Liposomes containing the antibody, get known ways, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980) and U.S. Pat. Nos. 4485045 and 4544545. Liposomes with enhanced circulation time of first described in U. S. Pat. No. 5013556. the second composition, containing phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-PE). Liposomes extruded through filters with defined pore size with the formation of liposomes of the desired Demetra. Fab' fragments of antibodies can konjugierte with liposomes, as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982), through a disulfide exchange reaction. The chemotherapeutic agent (such as doxorubicin) may be found in the liposome (See. Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989)).
(X) antibody-dependent enzyme therapy precursor drugs (ADEPT)
The antibody described in this invention can also be used in ADEPT when the conjugation of antibodies with the enzyme that activates predecessor medicines, which turns predecessor (for example, peptidyl-chemotherapeutic agent, see WO 81/01145) in the active antitumor agent (See, for example, W88/07378 and U. S. Pat. No. 4975278).
The enzyme component immunoconjugate suitable for ADEPT, includes any enzyme capable of affecting the precursor of a drug in such a way as to transform it into the more active cytotoxic form.
Enzymes, which are suitable for described in this invention --- the members of medicines into free drugs; arylsulfatase suitable for turning sulfidogenic predecessors medicines available drugs; citizendiumissue suitable for converting non-toxic 5-fertilizin in the anticancer drug 5-fluorouracil; proteases, such as protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins b and L), which is suitable for turning petitcodiac predecessors, drug free drugs; D-alanintranspherases suitable for the conversion of precursors of drugs that contain D-amino acid substituents; uglevodorodami enzymes, such as-galactosidase and neuraminidase, are suitable for converting glycosylated precursors of drugs into free drugs;-lactamases suitable for making medicines, derivatizing-lactams into free drugs; and penicillinases, such as penicillin-V imidazol or penicillin-G imidazol suitable for the conversion of the precursors is lname groups, respectively, into free drugs. On the contrary, antibodies with enzymatic activity, also known as the “abzyme”, you can apply for the conversion of precursors of drugs into free active drugs (Massey, Nature 328: 457-458 (1987)). Antibody-abinya conjugates can be prepared for delivery of abzyme to a population of tumor cells.
Enzymes are covalently to contact the mutant antibodies by well known methods, for example by applying heterobifunctional cross-linking reagents reported above. On the contrary, fused proteins, including at least antigennegative area antibodies associated, at least a functionally active portion (part) of the enzyme, can be created using well known methods of recombinant DNA (Neuberger et al., Nature 312: 604-608 (1984)).
(XI) the Fusion antibody is saved receptornegative epitope
In some embodiments, it is possible to apply the fragment of the antibody, but not all of the antibody to increase, for example, penetration of the tumor. In this case, it is possible to modify the fragment antibodies in order to increase the half-life of serum. This can be achieved, for example, ulucesti in fragments of antibodies, or the inclusion of the epitope into a peptide tag, which then merge with the fragment of the antibody at the end or in the middle, for example, using DNA or peptide synthesis).
Saved receptornegative epitope preferably represents an area in which one or more amino acid residues from one or two loops Fc region is transferred to a similar position of a fragment of the antibody. More preferably, when transferred three or more residues from one or two loops Fc region. Even more preferably, when the epitope is taken from the CH2 domain of the Fc region (e.g., IgE) and transferred to CN, CH3, or VH region, or more than one such region of the antibody. On the contrary, the epitope is taken from the CH2 domain of the Fc region and transferred to CLregion, or VLregion, or both regions of the antibody fragment.
(XII) Other covalent update antibodies
Covalent modifications of the antibody included in the scope of the invention. You can get them, if necessary, by chemical synthesis or by enzymatic or chemical hydrolysis of the antibody. Other types of covalent modifications of the antibody is introduced into the molecule by reacting amino acid residues of the antibody with the organic derivatizing agent, which is capable of reacting with selected N - or C-terminal OCTA is a tatami (and corresponding amines), for example Chloroacetic acid or chloroacetamide, obtaining carboxymethyl - or carboxylatomethyl-derivatives. Cysteinyl-debris derivatized by reaction with BROMOTRIFLUOROMETHANE,-bromo--(5-imidazolyl)propionic acid, chloroacetylation, N-alkylamide, 3-nitro-2-pyridyldithio, methyl-2-pyridyldithio, n-chloromercuribenzoate, 2-chloromercuri-4-NITROPHENOL, or chloro-2-nitrobenzo-2-oxa-2,3-diazelam.
His-tag residues derivatized by reaction with diethylpyrocarbonate at pH of 5.5 to 7.0 because this agent is relatively specific for the histidine side chain. n-Brompheniramine also suitable; the reaction is preferably carried out in 0.1 M solution of cacodylate sodium at pH 6.0.
Lysine and aminobenzene residues react with anhydrides of succinic or other carboxylic acids. The derivatization of these agents has an impact on the change of charge of the lysine residues. Other suitable reagents for derivatization-aminecontaining residues include imidiately, such as methylphenidate, pyridoxal phosphate, pyridoxal, Harborside, trinitrobenzenesulfonic, O-methylisoleucine reaction with one or more standard reagents, among them, phenylglyoxal, 2,3-butandiol, 1,2-cyclohexandione and ninhydrin. The derivatization arginine residues requires that the reaction was carried out in alkaline conditions because of the high values of the PKandguanidino functional groups. In addition, the reagents can react with the groups of lysine as well as with-amino group of arginine.
Specific modification of tyrosine residues can be done especially when introducing spectral labels into the tyrosine residues by reaction with aromatic diazonium compounds or tetranitromethane. Often use N-acetylimidazole, tetranitromethane for the formation of O-acetyltyrosine-derivatives and 3-nitro-derivatives respectively. The tyrosine residues coderoute125I or131I get labeled proteins for use in radioimmunoassay.
Carboxyl-containing group (aspartyl or glutamyl) are selectively modified by using carbodiimides (R-N=C=C-R'), where R and R' represent different accelgroup, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.
In addition, aspartyl and glutaradehyde residues into asparaginyl and glutaminergic in the corresponding glutamyl and asperdistria residues, respectively. These residues in neutral or alkaline conditions. Deliciously form of these residues falls out of the scope of this invention.
Other modifications include hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups was seryl or travelstories residues, methylationthe amino groups of the side chains of lysine, arginine and histidine (I.e., Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman and Co., San Francisco, pp. 79-86 (1983)), acetylation of N-terminal amino group and amidation of any C-terminal carboxyl group.
Another type of covalent modification includes chemically or enzymatically linked glycosides with the antibody. These techniques are advantageous in that they do not require the production of antibodies in the cell host, which has glycosylamine abilities for N - or O-linked glycosylation. Depending on which method to use binding, sugar(s) may accede to: (a) arginine and histidine; (b) free carboxyl groups; (C) free sulfhydryl groups such as cysteine; (d) free hydroxyl groups such as serine, threonine or hydroxyprolin; (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; or (e) Lenoir. 259-306 (1981).
Removal of any carbohydrate residues present on the antibody may be carried out chemically or enzymatically. Chemical deglycosylation requires addressing antibody connection - triftoratsetata or equivalent connection. This treatment leads to the hydrolysis of most or all sugars except the linking carbohydrate (N-acetylglucosamine or N-atsetilgalaktozamin), leaving the intact antibody. Chemical glycosylation described by Hakimuddin et al., Arch. Biochem. Biophys. 259: 52 (1987) and Egde et al., Anal. Biochem. 118: 131 (1981). Enzymatic hydrolysis of the carbohydrate residues on the antibody can be achieved by the use of a range of endo - and ectoparasites, as described in Thotakura et al., Meth. Enzymol. 138: 350 (1987).
Another type of covalent modification of the antibody comprises linking the antibody to one of a range of not reminiscent of proteinaprima, for example polyethylene glycol, polypropyleneglycol or polyoxyalkylene, by the method described in U. S. Pat. No. 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337.
Century Vectors, cell host and recombinant methods
The invention also relates to an isolated nucleic acid that encodes a mutant antibodies, as described in this invention, vectors and cells khozyaistvyennogo producing mutant antibody nucleic acid, coding his isolated and inserted into replicating vector for further cloning (amplifire DNA) or for expression. DNA encoding the mutant monoclonal antibodies, easily isolated and is sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specific contact with genes encoding the heavy and light chain mutant antibodies). Are available a lot of vectors. Components of vectors typically include, but are not limited to, one or more signal sequences, the source of replication, one or more marker genes, an enhancer element, a promoter and a transcription end sequence.
(I) Component signal sequence
The mutant antibodies can produce recombinante not only directly, but also in the form of a fused polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific website hydrolysis of the N-end of the Mature protein or polypeptide. Selected heterologous ashnola sequence is preferably a sequence that recognize the, which do not recognize and ProcessInput signal sequence to a native antibody signal sequence replace the prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penitsillinazy, RR or leader sequence of thermostable enterotoxin II. When the yeast secretion the native signal sequence may be substituted, for example, a leader sequence of yeast invertase, a leader sequencefactor (including the leader sequencefactors Saccharomyces and Kluyveromyces) or leader sequence of acid phosphatase, a leader sequence of C. Albicans glucoamylase or signal sequence described in WO 90/13646. In the expression of a mammalian cell available signal sequence mammals, as well as viral secretory leader sequence, such as gD signal sequence of the herpes simplex virus.
The DNA for such precursor region are ligated in reading frame to DNA encoding the mutant antibodies.
(II) the Source component replication
And expressing and cloning vectors contain placentas the early cells of the host. Generally, in cloning vectors this sequence is a sequence that enables the vector to replicate independently of the host chromosomal DNA, and includes sources of replication or autonomously replicating sequences. Such sequences are known for a wide range of bacteria, yeast and viruses. The source of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the source 2plasmids suitable for yeast, and various viral sources (SV40, polyoma, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells. Typically, the source component replication is not required for expressing vectors mammals (SV40 source usually can be used only because it contains the early promoter).
(III) Component gene selection
Expressing and cloning vectors may contain a gene selection, also known breeding marker. Typical genes breeding encode proteins that (a) possess a resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic failure (deficits) or (racemase for Bacilli.
One example of a selection scheme uses a drug to delay the growth of the host cell. Data cells that are successfully transformed heterologous gene produce a protein with drug resistance, and, thus, maintain (retain) mode selection. Examples of such dominant selection use the drugs - neomycin, mikofenolna acid and hygromycin.
Another example of appropriate breeding markers for mammalian cells are markers that facilitate the identification of the component cells for making nucleic acid antibodies, such as DHFR, thymidine kinase, metallothionein-I and-II, preferably metallothionein genes of primacy, adelaideans, ornithine-decarboxylase and so on
For example, cells transformed DHFR breeding gene, originally identified in the cultivation of all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. Corresponding to a host cell when using wild-type DHFR is a cell line of Chinese hamster ovary (Cho), devoid of DHFR activity.
On the contrary, the cells of the host (preferably hosts dinasty, encoding the antibody, DHFR protein wild-type and other breeding markers, such as aminoglycoside-3'-phosphotransferase (ARN) can be selected by cell growth in medium containing the agent (SPF) breeding for breeding marker such as aminoglycoside antibiotic kanamycin, neomycin, or G418 (U. S. Pat. No. 4965199).
Suitable gene selection for use in yeast is the trp1 gene present in the yeast plasmid Yrp7 (Stinchcomb et al., Nature 282: 39 (1979)). Gene trp1 breeding is a marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATSS No. 44076 or RER-1. Jones, Genetics 85: 12 (1977). The presence of the trp1 damage in the yeast genome of the host cell then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATSS 20622 or 38626) are complemented by known plasmids producing Leu2 gene.
In addition, vectors derived from the 1.6m circular plasmids (pKD1), can be used for transformation of Kluyveromyces yeasts. On the contrary, expressing system for large-scale production of recombinant calf chymosin together is ecchi Mature recombinante serum albumin human industrial strains of Kluyveromyces. Fleer et al., Bio/Technology, 9: 968-975(1991).
(IV) promoter Component
Expressing and cloning vectors usually contain a promoter that is recognized by the host-cell and directly (resectable) is associated with a nucleic acid antibodies. Promoters suitable for use with prokaryotic hosts include the phoA promoter,-lakhamandal and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. However, other suitable known bacterial promoters. The promoters used in bacterial systems also must contain a Shine-Dalgarno (S. D.) sequence directly related to DNA that encodes the antibody.
Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an at-rich region located approximately 25 to 30 bases in 3'-5' direction from site (centre), where transcription is initiated. Another sequence found from 70-80 bases in 3'-5'-direction from the start of transcription of many genes is CNCAAT area, where N can be any nucleotide. 3' the end of most eukaryotic genes have a sequence. All these sequences are appropriately inserted into eukaryotic expressing vectors.
Examples of relevant promoter sequences used with yeast hosts include the promoters for 3-phosphoglycerate or other glycolytic enzymes, such as enolase glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, pyruvate kinase, triosephosphate, phospho-glucose and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter region of the alcohol dehydrogenase 2; sociogram; acid phosphatase; causing enzymes; associated with nitrogen metabolism; metallothionein, glyceraldehyde-3-phosphate dehydrogenase; and the enzymes responsible for the utilization of maltose and galactose. Appropriate vectors and promoters for use in yeast expression, additionally described in EP 73657. Yeast enhancers can also be useful with yeast promoters.
Transcription antibodies of Viktorovich as virus polyoma, the smallpox virus of birds, adenovirus (such as adenovirus 2), bovine human virus, avian sarcoma, cytomegalovirus, a retrovirus, hepatitis b virus and most preferably simian vacuolating virus 40 (SV40); from heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter, from “hitchcover” promoters, provided such promoters are compatible with the system host cell.
Early and late promoters of SV40 virus are usually obtained in the form of an SV40 restriction fragment which also contains the SV40 viral replication source. The closest early promoter of human cytomegalovirus are usually obtained in the form of HindIIIE restriction fragment. System for expression of DNA in mammalian hosts using bovine human virus as a vector, is described in U. S. Pat. No. 4419446. Modification of this system is described in U. S. Pat. No. 4601978. On the contrary, cDNAinterferon-man was expressed in mouse cells under the control of timeintensive promoter from herpes simplex virus. On the contrary, the virus rous ' long terminal repeat can be used as a promoter.
(V) Component enhancer
Transcription of DNA, actor. Currently, there are many enhancer sequences from mammalian genes (globin, elastase, albumin,-fetoprotein and insulin). You can usually use the enhancer of virus in eukaryotic cells.
Examples include the SV40 enhancer on the last section of the origin of replication (b 100-270), the cytomegalovirus early promoter enhancer, Poliany enhancer on the last section of the origin of replication, adenovirus enhancers. Cm. also Yaniv, Nature 297: 17-18 (1982) on the reinforcing elements for activation of eukaryotic promoters. The enhancer may rasiwasia in the vector at position 5' or 3' to antitelomerase sequence, but preferably is located in position 5' from the promoter.
(VI) The termination of transcription
Expressing the vectors used in eukaryotic cells-the masters (yeast, fungi, insects, plants, animals, human or nucleated cell from other mammalian organisms) must also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA t or controlled part of the mRNA, encoding the antibody. One suitable component of the termination of transcription is bovine growth hormone polyadenylation area. Cm. WO 94/11026 and described expressing vector.
(VII) Selection and transformation of host cells
Appropriate cell hosts for cloning or expression of the DNA in the vectors are the prokaryote, yeast, or higher eukaryote cells described above. Appropriate prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteria, such as Esherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, for example Serratia marcescans, and Shigella; as well as Bacilli such as B. Subtilis and B. Licheniformis (e.g., B. Licheniformis 41 P described in DD 266710, published April 12, 1981); Pseudomonas such as P. aeruginosa, and Streptomyces. Preferred E. coli cloning host is E. coli 294 (ATSS 31446), although suitable and other strains, such as E. coli B, E. coli X ATSS 31537), and E. coli W3110 (ATSS 27325). These examples are illustrative and not restrictive.
In addition, the prokaryotes, eukaryotic microbes such as hyphomycetes or yeast are suitable cloning or expressing the hosts for antitelomerase obychnye Baker's yeast. Usually accessible and usable a number of other species, varieties and strains, such as Schizosaccharomyces pombe'; Kluyveromyces hosts such as K. lactis, K. fragilis (ATSS 12424), K. bulgaricus (ATSS 16045), K. wickeramii (ATSS 24178), K. waltii (ATSS 56500), K. drosophilarum (ATSS 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and hyphomycetes, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. Nidulans and A. niger.
Appropriate cell hosts for the expression of glycosylated antibodies derived from multicellular organisms. Basically, any higher eukaryotic cell culture is real or vertebral or invertebrate culture. Examples of invertebrate cells include cells of plants and insects, Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., Genetic Engineering, Setlow et al. eds. Vol. 8, pp.277-279 (Plenam publishing 1986); Mseda et al., Nature 315: 592-594 (1985). Identified numerous baculovirus strains and variants and corresponding recommended cell-hosts of insects from hosts such as Spodoptera frugiperda (caterpillar), Aedas aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (butterfly) and Bombyx mori. A special cell line of interest is the sf9 cell line insects. Publicly available a number of viral strains for transfection, particularly for transfection of cells Spodoptera frugiperda. In addition, as the host can be used by plant cell cultures of cotton, corn, potato, soybean, Petunia, tomato, and tobacco.
However, the biggest interest are cells of vertebrates, and the cultivation of vertebrate cells in culture (tissue culture) has become a routine procedure, see Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973). Examples of suitable cell lines of mammalian hosts are CVI line of monkey kidney transformed by SV40 (COS-7, ATCC CRL 1651); the line of embryonic human kidney (293 or 293 cells subcloned for growth in suspended culture, Graham et al., J. Gen. Virol, 36: 59 (1977)); kidney cells baby hamster (KSS, ATSS CCL10); DHFR cells of the Chinese hamster ovary (Cho, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77,: 4216 (1980)); the Sertoli cells of the mouse (TM, Mather, il. Reprod. 23: 243-251 (1980); cells monkey kidney (CVI, ATSS CCL70); kidney cells of the African green monkey (VERO-76, ATSS CRL-1587); cell cervical carcinoma human (HELA, ATSS CCL 2); cells, dog kidney (MDCK, ATSS CCL 34); liver cells Bovalino rats (BRL 3A, ATSS CRL 1442); human lung cells (W138, ATSS CCL 75); the cells of the human liver (ner G2, HB 8065); tumor of the mammary gland of the mouse (MMT 060562, ADS CCL51); TRI cells (Mather et al., Annals N. J. Acad. Sci. Nemi expressing or cloning vectors for the production of antibodies and cultured in normal (standard) nutrient medium, modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
(VIII) Culturing host cells
Cell owners used for the production of mutant antibodies, can be cultivated in a wide range of environments. For culturing host cells suitable commercially available medium such as Ham's F10 (Sigma), minimal maintenance medium (Minimal Essential Medium, MEM, Sigma), RPMI-1640 (Sigma) and modified by way of Dulbecco Wednesday Needle (Dulbecco's Modified Eagle's Medium, DMEM, Sigma). In addition, any medium described in the reviews, papers, patents, and applications (Ham et al., Meth. Enzymol. 58: 44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat No. 4767704; 4657866; 4927762; 4560655 or 5122469; WO 87/00195 or U. S. Pat. No. Re. 30985, can be used as a culture medium for the cells of the host. Any of these media may be supplemented if necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (e.g., X-chlorides, where X is sodium, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (e.g., drug GENTAMYCIN), microlam omolara level), and glucose or an equivalent energy source. Any other necessary conglutinin can also be included at appropriate concentrations that are known to experts. Culturing conditions, such as temperature, pH and other conditions are used for cell-hosana selected for expression, and will be known to specialists.
(IX) Purification of antibodies
When using recombinant methods mutant antibody can be produced intracellularly, in periplasmatic space or to secrete directly into the environment. If the mutant antibody is produced intracellularly, in the first stage of the debris particles, or cells of the owners, or lysed (broken) fragments are removed, for example, by centrifugation or ultrafiltration. Carter (Carter et al., Bio/Technology 10: 163-167 (1992)) have described a method of isolating antibodies that secrete in periplasmatic space of E. coli. Briefly, cell paste is thawed (soften) in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl (phenylmethylsulfonylfluoride, PMSF) for ~30 minutes. Cell debris can be removed by centrifugation. If the mutant secretes antibodies on Wednesday, supernatant from expressing such systems generally first concentrated using the industry is in. The protease inhibitors, such as PMSF, may engage in any of the previous stages for the inhibition of proteolysis and may include antibiotics to prevent the growth of accidental contamination.
The composition of the antibody obtained from the cell can be cleaned, for example, chromatography on hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography as a purification method is preferable. The suitability of protein And as an affinity ligand depends on the type and isotype of any Fc region of an immunoglobulin that is present in the mutant antibodies. Protein And can be used for purification of antibodies, which are based on heavy chains1,2 or4 belonging to the human race (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all isotypes of mice and3 person (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which is attached an affine ligand, is often a agarose, but fit and other matrices. A mechanically stable matrices such as glass with a defined pore size or poly(Stradivari)benzene, allow for greater skotel contains the CH3 region, for cleaning suitable polymer Bakerbond AHTM (J. T. Baker, Phillipsburg, NJ). Depending on allocated mutant antibody is also suitable for other methods of protein purification such as fractionation (separation) on ion-exchange column, ethanol precipitation, HPLC with reversed phase chromatography on silica, chromatography on heparin-SEPHAROSE, chromatography on an anion-exchange or cation-exchange polymer (for example, on a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.
After any preliminary stage(s) of the cleaning mixture containing the desired mutant antibodies and contaminants may be subjected to hydrophobic interaction chromatography with a low pH using a buffer for elution with a pH of 2.5 to 4.5, preferably performed at low salt concentration (e.g., 0-0,25 M salt).
Century Pharmaceutical composition
Therapeutic compositions of the polypeptide or antibody receive in the form of lyophilised compositions or aqueous solutions by mixing the polypeptide desired degree of purity with possible "pharmaceutically acceptable" carriers, excipients and stabilizers commonly used in this area (all of which are called atomicdust, non-ionic detergents, antioxidants and other additives mixed (see Remington's Pharmaceutical Sciences, 16thedition, A. Osol, Ed. (1980)). These additives must be non-toxic to recipients at the dosages and concentrations.
Buffering agents are used to maintain pH in the range, which almost correspond to physiological conditions. They are preferably present at concentrations ranging from 2 mM to 50 mm. Suitable for use buffering agents include organic and inorganic acids and their salts, such as citrate buffers (e.g., a mixture of one-deputizing sodium citrate-disubstituted sodium citrate, a mixture of citric acid - triple-substituted sodium citrate, a mixture of citric acid - monosubstituted sodium citrate, and so on), succinate buffers (for example, a mixture of succinic acid - one-deputizing sodium succinate mixture, succinic acid - sodium hydroxide mixture, succinic acid - disubstituted sodium succinate), tartrate buffers (for example, a mixture of tartaric acid - sodium tartrate, a mixture of tartaric acid - potassium tartrate, a mixture of tartaric acid - sodium hydroxide, and so on), fumaric buffers (for example, a mixture of fumaric acid - monosubstituted fumarate sodium, a mixture of fumaric acid - disubstituted fumarate sodium, ü gluconic acid gluconate sodium, a mixture of gluconic acid - sodium hydroxide, the mixture of gluconic acid, a gluconate, potassium and so on), oxalate buffer (for example, a mixture of oxalic acid - sodium oxalate mixture of oxalic acid - sodium hydroxide, the mixture of oxalic acid - potassium oxalate, and so on), lactate buffers (e.g., a mixture of lactic acid - sodium lactate, a mixture of lactic acid - sodium hydroxide, a mixture of lactic acid - potassium lactate and so on) and acetate buffers (e.g., a mixture of acetic acid - sodium acetate mixture, acetic acid - sodium hydroxide, and so on). In addition, mention should be made of phosphate buffers, his-tag buffers and salts of trimethylamine, for example, Tris (Tris) buffers.
Preservatives are added to retard microbial growth in amounts from 0.2% to 1% (wt./vol.). Suitable preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyltrimethylammonium chloride, benzalconi the halides (e.g. chloride, bromide, iodide), hexamethonium chloride, alkylarene, for example methyl - or propylparaben, catechin, resorcinol, cyclohexanol and 3-pentanol.
Isotonicity, sometimes referred to as “stabilizers” are present to ensure isotonicity of liquid compositions is glycerin, aritra, Arabic, xylitol, sorbitol and mannitol. Polynuclear alcohols may be present in amounts of from 0.1% to 25% (weight), preferably from 1% to 5%, taking into account the relevant quantities of the other ingredients.
Stabilizers belong to the broad category of fillers that can vary in purpose from fillers to Supplement that solubilities therapeutic agent or helps to prevent denaturation or adhesion to the wall of the vial. Typical stabilizers may be polynuclear alcohols carbohydrates (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and so on, organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, monoset of galactin, glycerin and the like, including cility, such as Inositol, polyethylene glycol, polymers of amino acids; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, digluconate, diglycerin,-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., less than 10 residues); proteins such as serum albumin man, the don, monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides, such as lactose, maltose, sucrose and trisaccharide, such as raffinose; polysaccharides, such as dextran. The stabilizers are present in amounts of from 0.1 to 10,000 weight parts per weight part of the active protein.
Non-ionic surfactants or detergents (also known as “moisturizing agents”) are to assist in the dissolution of therapeutic agent, as well as to protect therapeutic protein from aggregation-induced mixing (shaking), which also allows the composition to be subjected to the action of surface shear stress without denaturation of the protein. The corresponding non-ionic surfactants include Polysorbate (20, 80, and so on), poloxamer (184, 188, and so on), pluronic (Pluronic®), polyole associated with polyoxyethylene, monetary sorbitan (Tween®-20, (Tween®-80, and so on). Nonionic surfactants are present in amounts of ~0.05 mg/ml to ~1 mg/ml, preferably from ~0.07 mg/ml to ~0.2 mg/ml
An additional variety of fillers include fillers, such as starch, complexing agents (such as EDTA), antioxidants (for example, as the active compound, when it is necessary to treat the individual readings, preferably active compounds with complementary activities that do not adversely affect each other. For example, in the future it may be desirable to introduce immunosuppressive agent. Such molecules are present, respectively, together in an amount that is effective for their intended purpose.
The active ingredients can also be included in the microcapsule obtained, for example, ways koatservatsii or polymerization on the surface of separation of the phases, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal systems drug delivery (for example, liposomes, albumin microspheres, microemulsions, microparticles, and microcapsules) or microemulsion. These methods are discussed in Remington's Pharmaceutical Sciences, 16thedition A. Osal. Ed. (1980).
The composition used for administration in vivo, must be sterile. This can be easily achieved, for example, by filtration through sterile filtration membranes.
It is possible to obtain compositions with prolonged action (continuous release). Examples of extended release compositions include p in the form, for example, films, or microcapsules. Examples of extended-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or poly(vinyl) alcohol, polyactide (U. S. Pat. No. 3773919), copolymers of L-glutamic acid and ethyl-L-glutamate, are not subject to the degradation of the ethylene vinyl acetate exposed to degradation of copolymers of lactic acid-glycolic acid such as LUPRON DEPOT(introduced by injection of microspheres consisting of a copolymer of lactic acid - glycolic acid and leuprolide) and poly-D-(-)-3-hydroxipropionic acid. While polymers such as ethylene vinyl acetate and lactic acid - glycolic acid enable you to select molecules for 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denaturing or aggregate as a result of moisture at 37°C, leading to loss of biological activity and possible changes in immunogenicity. You can offer a rational strategy for stabilization depending on the mechanism under consideration. For example, if you discovered that the mechanism of aggregation before the folder by modifying sulfhydryl residues, liofilizirovannam from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer compositions of the matrix.
The amount of therapeutic polypeptide, antibody or its fragment, which will be effective in the treatment of individual disorder or condition will depend on the nature of the disorder or condition and may be determined by standard clinical techniques. If possible, it is desirable to define the curve dose-response relationships and to test pharmaceutical composition for the people in the beginning to test in vitro, and then in animal model systems. However, based on the General concepts of a pharmaceutical composition effective in promoting the survival of sensory neurons may provide a local concentration of therapeutic agent, equal to from 5 to 20 ng/ml, preferably between 10 and 20 ng/ml In an additional specific embodiment, a pharmaceutical composition effective in promoting growth and survival of retinal neurons may provide a local concentration of therapeutic agent from 10 ng/ml to 100 ng/ml.
In a preferred embodiment, an aqueous solution of a therapeutic polypeptide, antibody or its fragment in the a or more preferably from about 3 μg to about 30 μg per kilogram of body weight.
Dose scheme for subcutaneous administration may vary from once a week to daily injection depending on a number of clinical factors, including the type of disease, severity of disease and the sensitivity of the subject to therapeutic agent.
G. non-therapeutic use of mutant antibodies
The mutant antibodies can be used as agents for purification by affinity chromatography. In this way antibodies immobilized on the solid phase, for example the resin (polymer) Sephadex (Sephadex) or filter paper, using known methods. Immobilized mutant antibodies are contacted with the sample containing the purified antigen, and then the carrier is washed with a suitable solvent, which should remove essentially all substances in the sample, with the exception of the purified antigen, which binds to the immobilized mutant antibodies. Finally, the carrier is washed with another suitable solvent, such as glycine buffer with pH 5, which will separate the antigen from mutant antibodies.
Mutant antibodies can also be useful in diagnostic assays, for example, to detect expression of the desired antigen-specific cells, tissues, or serum.
For mnogochislennye label, which can be grouped into the following categories:
(a) Radioisotopes, such as36S14C,125I3H and131I. the Mutant antibodies can be marked with the radioisotope using the techniques described, for example, in Current Protocols in Immunology, Vol. 1 and 2, Coligen et al., Ed., Wiley. Interscience, New York, Pubs. (1991), and radioactivity can be measured using scintillation counters.
(b) Available fluorescent label, for example, chelate compounds of rare earth metals (chelation abronia) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lisanin (Lissamine), phycoerythrin and Texas Red. Fluorescent labels can konjugierte with mutant antibodies using, for example, the methods described in the Currrent Protocols in Immunology, supra. Fluorescence can be quantified using fluorimetry.
(C) various enzyme-substrate labels, and U. S. Pat. No. 4275149 provides an overview of some of them. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured in various ways. For example, the enzyme may catalyze a color change of the substrate, which can be measured spectrophotometrically. On the contrary, the enzyme may alter the fluorescence or chemiluminescence substrate. Sposoby becomes electronically excited, and then can emit light, which can be measured (for example, chemiluminometer), or to transfer energy fluorescencia the acceptor. Examples of enzymatic labels include luciferase (e.g., luciferase from fireflies and bacterial luciferase; U.S. Pat. No. 4737456); luciferin, 2,3-dihydropteridine, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (horseradish buffer, HRPO), alkaline phosphatase,-galactosidase, glucoamylase, secrete lysozyme, SharedAccess (for example, glucoseoxidase, galactosidase and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides and similar enzymes. Methods of conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (Ed. J. Langone, H. Van Vunaksi), Academic press, New York, 73: 147-166 (1981).
Examples of enzyme-substrate combinations include for example:
(I) horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate, where the hydrogen peroxide oxidizes a dye precursor substances (for example, o-phenylenediamine, (orthophenylene diamine, OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB);
(II) alkaline phosphatase (alkaline phosphatase, AP) n--tp://img.russianpatents.com/chr/946.gif" border="0">-D-Gal) with a chromogenic substrate (e.g., n-nitrophenyl--D-galactoside or fluorogenic substrate 4-methylumbelliferyl-D-galactoside.
Specialists are available and numerous other enzyme-substrate combinations. For example, their review, see U. S. Pat. No. 4275149, 4318980.
Sometimes the label is indirectly conjugates with mutant antibodies. Experts know a variety of ways to achieve this. For example, mutant antibodies can konjugierte with Biotin and any of the three main categories mentioned above labels may konjugierte with Avidya or vice versa. Biotin is selectively associated with Avidya, and thus, the label can konjugierte with mutant antibodies indirectly. On the contrary, to achieve indirect conjugation of the label with the mutant antibodies, mutant antibody conjugates with a small hapten (e.g., digoxinum) and one of the types mentioned above label conjugates with mutant antiJapanese antibodies (for example, antidigoxin antibody). Thus, it is possible to achieve indirect conjugation of the label with the mutant antibodies.
In another embodiment, not requiring labels mutant antibody and its presence can be used to detect the yubom known method of analysis, for example, the analysis of competitive binding, direct and indirect sandwich assays and immuno-precipitation analyses. Zola, Monoclonal antibodies: A Manual of Technologies, pp. 147-158 (CRS Press, Inc., 1987).
Validation of competitive binding based on the ability of a labeled standard to compete with the test sample for binding to a limited number of mutant antibodies. The amount of antigen in the sample is inversely proportional to the number of standard binds to antibodies. To facilitate determining the amount of standard that binds antibodies is usually translated into an insoluble form before or after competition. As a result, the standard and the test sample that are associated with antibodies, can as usual be separated from the standard and the sample, which remains unbound.
“Sandwich” assays include the use of two antibodies, each capable of binding to a different immunogenic region or epitope, or protein, found. In a sandwich analysis analyzed the sample binds to the primary antibody, which is immobilized on a solid carrier and then the secondary antibody binds to a test specimen, forming, thus, not the dummy fragment (direct sandwich assays) or may be determined using antiimmunoglobulin antibodies which is observed detektivami fragment (indirect sandwich assay). For example, one type of sandwich ELISA analysis is the analysis, in which detektivami balance is an enzyme.
For immunohistochemistry of tumor sample, for example, can be fresh or frozen or may be included in paraffin and bound with a preservative, such as formalin.
Antibodies can also be used in diagnostic assays in vivo. Typically, the mutant antibodies have been labelled with a radioisotope (for example,111In99TC14C,131I3H,32P,35S) so that the tumor can be localized using immunoscintigraphy.
D. Diagnostic kits (kits)
For convenience, the polypeptide or antibody described in this invention can be included in the set, i.e., in the packaged kit of reagents in predetermined amounts with instructions for performing a diagnostic test (analysis). If the mutant antibodies Machen enzyme, the kit should include substrates and cofactors required for the enzyme (e.g., the predecessor of the substrate, which provides the detected chromophore or fluorophore). In addition, may include other additives, for example stabilizers, BU can vary within wide limits, to ensure that the concentration of reagents in the solution, which substantially optimize the sensitivity analysis. In particular, the reagents may be in the form of powders, usually lyophilized, including excipients, which upon dissolution will provide for a reagent solution of appropriate concentration.
That is, the Use of the polypeptide or antibody in vivo
It is assumed that the polypeptide or antibody may be used to treat mammals. In one embodiment, the antibody is administered, for example, mammals, do not belong to the human race, for the purpose of obtaining preclinical data. Typical mammals that do not belong to the human race, which treat include primates, dogs, cats, rodents and other mammals, for which conduct preclinical studies. Such mammals you can create animal models for the disease, which is treated with an antibody, or can be used to study the toxicity of the desired antibodies. Each of these options research to increase the dose can be carried out on mammals.
The antibody or polypeptide is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intra-lungs and intranasal introduction, interline infusion include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the mutant antibody is suitably administered using infusion momentum, particularly with declining doses of the mutant antibodies. Preferably, when the dosage is in the form of injections, most preferably in the form of intravenous or subcutaneous injection, in part, depending on whether the introduction of short-lived or permanent.
For the prevention or treatment of disease, the appropriate dosage of the antibody or polypeptide should depend on the type of disease that is being treated, the severity and course of the disease, is entered whether the mutant antibodies for prophylactic or therapeutic purposes, depend on previous therapy, the patient's clinical presentation and response to mutant antibodies, and prudence visited doctors. Antibody against human IgE appropriately administered to the patient once or during the treatment cycles.
Depending on the type and severity of the disease from ~1 μg/kg to 150 mg/kg (for example, 0.1 to 20 mg/kg of antibody or polypeptide) is an initial candidate dosage for administration to the patient, or, for example, when one or more separate injections or by continuous infusion. Oba is tori. With repeated introductions in a few days or longer, depending on the condition, treatment continues until, until a desired suppression of disease symptoms. However, you can use other values doses. The progress of this therapy is easily monitored by conventional techniques and assays. Typical dosage of anti-LFA-1 or anti-SAM-1 antibodies are described in WO 94/04188.
The composition of the mutant antibodies must be created, dosed and administered in a manner consistent with contemporary medical practice. Factors for consideration in this context include certain disorders that are treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the violation, place of delivery of the agent, route of administration, schedule of administration and other factors known to medical professionals. Appointed by “therapeutically effective amount” of the mutant antibody will depend on such considerations, and is the minimum amount necessary for the prevention, delay or treatment of a disease or disorder. The mutant antibody is not necessary, but is included in the composition with one or more agents currently used for the prevention of the present antibodies against human IgE in the composition, type of illness or treatment, and other of the factors discussed above. They are usually used in the same doses and routes of administration as described above, or from 1 to 99% from before the applied doses.
J. Industrial products
In another embodiment of the invention offer industrial products containing materials suitable for the treatment of the above disorders. Industrial product includes a container and a label. Suitable containers include, for example, a bottle, a vial, syringes, and test tubes. Containers can be made of various materials, such as glass or plastic. The container holds a composition which is effective for treatment and may have a sterile inlet (for example, a container can be a container for intravenous solution or vial with a stopper capable of with a needle for subcutaneous injection). The active agent of the composition is a mutant antibodies. The label on the container or associated with it indicates that the composition is used to treat the selected state. Industrial product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, ringer's solution and the solution dexte user, including other buffer solutions, solvents, filters, needles, syringes and packaging loose sheets with application instructions.
The following examples are provided to illustrate and not to limit.
Obtaining monoclonal bodies for IgE
Received eight monoclonal antibodies (MAE-MAE) that can block the binding of IgE to FcRI. Monoclonal antibodies to IgE was obtained from supernatants U266B1 cells (ATSC TIB 196) using affinity chromatography on a stand-alone anti-IgE antibody (Genentech ME). For ME five BALB/c female mice six weeks of age were immunized in their paws, 10 μg of purified IgE in Freund, Ribi. Subsequent injections were performed in the same way one and three weeks after the initial immunization. Three days after the last injection poovae and popliteal lymph thickening was removed and collected, the suspension of individual cells was prepared by passing the fabric through the steel mesh. The cells were discharged at a ratio of 4:1 with myeloma mouse RJ-Ad,653 (ATSS CRL 1580) in a highly concentrated glucose (DMEM), containing 50% wt./about. polyethylene glycol 4000. For ME, MAE and MAE immunization was performed similarly except ME, for the booster. For ME and MAE injections were performed subcutaneously in two doses of 100 μg and final booster injection of 50 μg and to merge the used cells of the spleen.
Fused cells were then placed on a plate at a density of 2×105the hole in the plate tissue culture with 96 wells. After 24 hours was added a HAT selective medium (gipoksantin/aminopterin/thymidine, Sigma, #H0262). From 1440 placed holes 365 contained cell growth after HAT selection.
15 days after the merger supernatant tested for the presence of antibodies specific for human IgE using enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA). ELISA was carried out as follows (with all incubations performed at room temperature). The investigated plates (Nunc Immunoplate) were coated for 2 hours with antibody rats against mouse IgG (Boehringer Mannheim, #605-500) at 1 µg/ml in 50 mM sodium carbonate buffer, pH 9,6, then tied with 0.5% bovine serum albumin in phosphate buffered saline (PBS) for 30 minutes, then washed four times with PBS containing 0.05% tween (Tween 20, PBST). Added study supernatant and incubated for 2 hours with shaking and then washed four times with PBST. IgE human (purified from which the washed four times with PBST. Peroxidase from horseradish, conjugate with the antibody goat against human IgE (Kirkegard &Perry Labs, #14-10-04, 0.5 mg/ml), was added at a 1:2500 dilution and incubated for one hour, then washed 4 times with PBST. Plates were processed by the addition of 100 μl/well of a solution containing 10 mg of o-phenylenediamine dihydrochloride (Sigma, #R) and 10 μl of 30% hydrogen peroxide solution in 25 ml of phosphate-citrate buffer, pH 5.0 and incubation for 15 minutes. The reaction was stopped by adding 100 μl/well of 2.5 M sulfuric acid. Data were obtained by reading the plates in an automated ELISA spectrophotometer to read the tablets when the absorbance at 490 nm. For ME, investigated 365 supernatant and 100 were specific to human IgE. A similar frequency of IgE specificity was received, when conducting a screening for other antibodies. All monoclonal antibodies described in this invention were of the IgG1 isotype; except ME, which was IgG2b and except ME, which was IgG2a.
Each of IgE specific antibodies additionally investigated in cell-based and records analysis for the selection of antibodies that bound IgE thus, in order to inhibit IgE binding to FcRI, and ://img.russianpatents.com/img_data/86/868690-s.gif" border="0">
1. Based on FACS method validation for the analysis of monoclonal antibodies in rats and mice against human IgE
Screening of monoclonal antibodies in rats or mice against human IgE mice or rats, associated with IgE on Cho 3D10 (FcRI a+).
(a) Cho 3D10 cells (FcRI stable transfectant-circuit, Hakimi et al., J. Biol. Chem. 25: 22079) at 5x105cells in the sample incubated with U266 IgE standard (series N13068-46) at 10 μg/ml in 100 μl FACS buffer (0.1% of BSA, 10 mM of sodium azide in PBS, pH 7.4) for 30 minutes at 4°C, followed by one washing FACS buffer. The amount of bound IgE determine when the incubation of an aliquot of IgE-loaded cells with polyclonal FITC conjugated antibody rabbit against human IgG (Accurate Chem. Co., AXL-475F, series No. 16) at 50 ál/ml for 30 minutes at 4°C, followed by three washing FACS buffer.
b) IgE-loaded cells are incubated with 100 μl of the supernatant of hybridoma mouse or rat IgE against human IgG concentration rats or mice ranged from 1 to 20 μg/ml) for 30 minutes at 4°C, followed by one washing FACS buffer. Genentech-monoclonal anticlinal antibody MAD6P, which does not recognize IgE, used at 10 μl/ml as a negative control.
C) Binding of monoclonal antibodies to human IgE in Cho cells was detected by incubating cells with 20 μg/ml FITC-conjugated purified using affinity chromatography F(ab')2antibodies goat against mouse IgG (Organon Teknica, #10711-0081) for 30 minutes at 4°C, followed by three washing FACS buffer. Cells are added to 400 μl of buffer containing 2 µl/ml of iodide of propecia (Sigma, #P4170) for staining cells undergoing.
d) Cells analyzed on a Becton Dickinson FACSCAN flow cytometer. Install shutters direct light scatter and 90° side scatter analysis of a homogeneous population of cells. Experiencing the cells that stained propedy iodide, exclude from the analysis. Supernatant hybrid, which do not bind IgE on Cho 3D10 cells, were considered candidates for further screening.
2. Histamine release from basophils in peripheral blood
Heparinized blood was obtained from normal donors was diluted 1:4 in modified Tyrodes buffer (25 mM Tris, 150 mM NaCl, 10 mM CaCl2, MgCl2, 0.3 mg/ml HSA, pH 7,35), then incubated with 1 nm human IgE (ND) at 4°C for 60 mi the polyclonal antibodies against human antisera as positive control and were incubated at 37°C for 30 minutes. Cells were besieged by centrifugation, histamine in supernatant has azetilirovanie and content of histamine was determined using a RIA kit (AMAC, Inc. Wesbrook, Main). The total number of histamine was determined from cells subjected to multiple rounds of freeze-thawing.
3. The block Fits conjugated IgE binding to FcRI chain
The influence of antibodies on IgE binding was investigated by pre-incubation Fits labeled with different IgE antibodies May at 37°C for 30 minutes in PBS containing 0.1% BSA and 10 mM of sodium azide with a pH of 7.4, hereinafter incubare complex with 5×1053D10 cells at 4°C for 30 minutes. The cells were then washed three times and measured the average flow fluorescence at 475 nm. As control was used mAb (Mae) rat or mouse against human IgE, which does not block IgE binding to FcRI-a chain.
4. Analysis of antibody binding sites of the rat or mouse against human IgE person with positive cells U266 IgE membrane
(a) U266 B1 cells (membrane IgE+) were cultured in an alkaline environment, dopolnenie 15% serum, fetal calf, V / V heat inactivated (Hyclone, #A-1111-L), penicillin, with the holding monoclonal antibodies rat or mouse against human IgE at 10; 5; 1; 0.5 and 0.1 ág/ml for 30 minutes on ice in 96-well titration the microplate with round bottom, followed by two washing FACS buffer. As a positive control, use Genentech monoclonal ME.
in Cells incubated in 100 μl FACS buffer containing 50 µg/ml (1:20 biomass) or FITC-conjugated F(ab')2antibodies goat, purified by affinity chromatography against mouse IgE (Organon Teknika, #1711-0084) for 30 minutes on ice, followed by three washing FACS buffer. Cells are added to 400 μl FACS buffer containing propedy iodide at 2 µg/ml for staining cells undergoing.
5. Based on FACS analysis of binding to FCRI (CD23)+B cell M
a) M IN myeloma cells of a person (ATSS CCL 159, Ann. N. Y. Acad. Sci. 190: 221-234 (1972)) was supported in gif alkaline medium with 10% bovine fetal serum inactivated by heating, penicillin, streptomycin (100 u/ml) and L-glutamine (2 mM).
b) Cells (5×105/aliquot) were incubated in 100 μl FACS buffer containing U266 IgE standard with 2 μl/ml for 30 minutes at 4°C in 96-well titration the microplate, followed by two washing FACS buffer. As a control cells were incubated in the same buffer or in the morning is Linyi antibodies rat or mouse against human IgE at 0.1 to 10 μg/ml for 30 minutes on ice. As a positive control was used Genentech monoclonal ME.
g) Next, cells were incubated in 100 μl FACS buffer containing FITC conjugated F(ab')2goat against mouse IgG at 50 µg/ml (Organon Teknika, # 1711-0084) for 30 minutes at 4°C, followed by 3 washing FACS buffer.
d) Then the cells were added to 400 μl of a buffer containing propedy iodide at 2 µg/ml for staining cells undergoing.
(e) Cells were analyzed on a Becton Dickenson FACSCAN flow cytometer. Installed shutters direct light scatter and 90° lateral scattering for the analysis of a homogeneous population of cells and experiencing the cells, which were stained with propedy iodide, were excluded from the analysis. FITC positive cells (IgE binding) were analyzed relative to cells stained one FITC antibody rabbit against human IgE.
W) as a positive control to determine the level of CD23 on the surface of IM9 cells in each experiment, aliquots of cells were stained with Becton Dickinson monoclonal antibody Leu 20 rat or mouse (anti-CD23) at 10 µg/ml for 30 minutes at 4°C, followed by two rinses. Next, cells were incubated with FITC conjugated F(ab')2 goat against rat IgG or mouse, purified affine x and
The binding of 40 nm FITC labeled IgE and IgE receptor low affinity (CD33 or FcRI), expressed In lymphoblastoid cells IM-9, were analyzed by flow cytometry on a FACSCAN flow cytometer. The influence of antibodies on FITS IgE binding was studied by pre-incubation FITS with IgE antibodies rat or mouse against human IgE at 0.1-10 μg/ml chimeras at 37°C for 30 minutes in PBS containing 0.1% BSA and 10 mM of sodium azide with pH 7.4, hereinafter incubare complex with 5×105cells at 4°C for 30 minutes. Cells were then washed three times and measured the average flow fluorescence at 475 nm.
7. Procedure (scheme) analysis of IgE in vitro
(a) Mononuclear cells peripheral blood was separated from normal donors.
b) the Cells were washed with PBS to remove the maximum number of platelets.
C) Mononuclear cells were counted and resuspendable in an environment with 1×106cells/ml Medium was a mixture of DMEM with penicillin and streptomycin, 15% horse serum, IL-2 (25 u/ml) and IL-4 (20 ng/ml).
g) Antibodies were added at appropriate concentrations at day 0, 5 and 8.
d) Cultures were incubated in 24-hole Falcon cultural tablet tissue within 14 days.
e) On 14 detente in the affinity (Kd) mouse mAb or rat compared to human IgE was determined by analysis of equilibrium binding (Scatchard)
(a) IgE (ND and PS allotype was codiroli with chloramine T and was separated from free125I use speakers with D10 Sephadex G25 (Pharmacia, #17-0851-01) in RIA buffer: PBS, 0.05% of bovine serum albumin (Sigma, #A-7888), 0.5% Tween (Tween 20, Sigma, #P-1379), 0.01% thimerosal (Sigma, #T-5125), pH 7.4. Approximately 78-95% politologicznych counts (counts) besieged 50% trichloroacetic acid and the specific activity of iodine IgE preparations ranged from 1.6 to 13Ci/μg, assuming 70% efficiency account.
b) a Certain concentration of125I (approximately 5×104the number of pulses/min) was added to varying concentrations of unlabeled IgE (1 to 200 nm) in a final volume of 0.1 ml of RIA buffer 12×75 mm polypropylene test tubes. mAB's mouse or rat IgE against the person (20 mM final concentration) in 0.1 ml of RIA buffer was then added to a final sample volume of 0.2 ml.
C) the Samples were incubated 16-18 hours at 25°C under continuous stirring.
g) bound and free125I IgE were separated by addition of 0.3 ml of the mixture purified by affinity chromatography antibodies goat against mouse IgG (Boehringer Mannheim #605-208) associated with CNBr-activated separate 4B (Sepharose 4B, Pharmacia, #17-0430-10) and a carrier protein And sepharose (Repligen, #IPA 300)) and the tubes were centrifuged for 5 minutes at HD. The samples were then read to determine the total number of pulses. Supernatant was removed with finely calibrated Pasteur pipettes, specimen re-read and to calculate the associated pulses (counts) compared to free.
d) Scatchard analysis was carried out using the Fortran program (Scanplot), based on the program Ligand, written P. Munson at the NIH. Scanplot uses the equation of the effective masses of the corresponding limit as a function of full use Rodbard type of regression analysis.
Obtaining refined Maen
The following example describes the various ways of obtaining refined Mae, in which residues modified by siteprovides mutagenesis to achieve 12 anti-IgE Mae options [F(ab)1-12]. The remains of the F(ab)12 was used as template to create rhuMaE25 or E25, vysokovalentnogo anti-IgE antibodies, as described in the application PCT/US92/06860, filed August 14, 1992.
mAb MaE11 rat or mouse against human IgE, shown in Fig.1, modified with siteprovides mutagenesis (Kunkel, T. A. OEWG.Natl. Acad. Sci. USA 82: 488 (1985)) from desoxyribonuclease a template that includes a light chain-subgroups I, belonging to the human who et al., The OEWG.Natl. Acad. Sci. USA 89: 4285 (1992)) to obtain the option F(ab)-1. F(ab)-2 created from F(ab)-1 template and all other purified F(ab) variants created from template F(ab)-2. Plasmids were transformed into E. coli strain JM101 to obtain double-stranded or single-stranded DNA (Messing J. Recomb. DNA Tech. Bull. 2:43 (1979); Ausuble et al., Current Protocols in Molecular Biology, Unit 1 (1997)). The remains of the light and heavy chain fully sequenced using dideoxynucleotide way. DNA encoding light and heavy chains, then was subcloned into a derivative of E. coli F(ab) expressing plasmid rack (Carter et al., Biotechology 10: 163 (1992)). Derived lacked cysteine in the hinge region, which form disulfide bonds between heavy chains in F(ab)'2 fragments. F(ab) fragments in contrast to IgG antibodies full length facilitated the analysis of moderately large number of options, because you can use E. coli expression, and not the methods of cultivation of mammalian cells. These individual (private) options described in the application WO 93/04173, published on March 4, 1993, If it was determined the best option, it was subsequently subcloned into a plasmid encoding human IgG1 full length (see below).
Expressing plasmids were transformed into E. coli strain MM (Meselon M., R. Yuan, Nature 217: at 37°C. Next, the culture (5 ml) was added to 100 ml OR environment - carbenicillin (100 μg/ml) and gave the opportunity for culturing for 16 hours in a 500 ml shaker at 37°C. the Culture was centrifuged at HD and removing the supernatant. After freezing for 1 hour the precipitate after centrifugation resuspendable in 5 ml of cold 10 mM Tris buffer, 1 mm EDTA and 50 μl of 0.1 M of benzamidine (Sigma, St. Louis), the last of which was added to inhibit proteolysis. After careful shaking on ice for 1 hour, the sample was centrifuged at 10000xg for 15 minutes. The supernatant was applied to a column of protein a-sepharose CL-4B (Pharmacia) (0.5 ml net volume of the matter), then washed with 10 ml of 3M potassium chloride / 100 ml Tris-buffer, pH 8.0, followed by elution with 100 mM acetic acid (2.5 ml), pH 2.8 in 1 M Tris buffer, pH 8.0 (0.5 ml).
F(ab), the buffer was then replaced with PBS using Method-30 (Amicon) and concentrated to a final volume of 0.5 ml of SDS-PAGE gels of each F(ab) fragments was performed in order to evaluate the purity. F(ab) concentrations were determined using a 0.1%280equal to 1.0. The coefficient of estingly was determined using the concentration of the protein from amino acid analysis cleaned the th / mass spectrometry to confirm the molecular weight. Samples were injected in a liquid chromatographic system with a filled capillary column (Henzel, W. J. et al., Anal. Biochem. 187: 228 (1990)) and directly analyzed using a Sciex API 3 mass spectrometer. For calibration used the higher the charged state of the human growth hormone (M. C. 22256,2), using the same instrumental parameters, as the parameters used for the samples.
To generate refined versions Mae IgG1 human full length heavy and light chains were subclinically separately in the previously described pRK plasmids (German et al., DNA Protein Eng. Tech. 2:3 (1990)). The corresponding heavy and light chain plasmids (depending on the desired change(s) sequence) was cotransfection in adenovirus-transformed cell line of embryonic human kidney, known as 293 (Graham et al., J. Gen. Virol 36: 59 (1977)), using a highly effective method (Graham et al., supra and Gorman, Science 221:551). The medium was replaced with desterrado serum medium was collected daily for 5 days. Antibodies were purified from the collected supernatants using protein a-sepharose CL-4B (Pharmacia). Replace the buffer in buyowner antibody in PBS using a G25 gel filtration, was concentrated by ultrafiltration using Centriprep-30 or Method-100 (Millipore) and Granny immunoassay receptor
Analytical plate with 96 wells (Nunc) were coated 0.05 ml FcRI-chain IgG chimeric receptor in the buffer for sensitization of the surfaces (50 mM carbonate, bicarbonate, pH 9,0) for 12 hours at 4-8°C. the Wells were removed and added to 250 μl of blocking buffer (PBS, 1% BSA, pH of 7.2), and incubated for one hour at 4°C. In a separate analytical tablet samples and control Mae rat or mouse was titrated from 200 up to 0.001 μg/ml at 1:4 dilutions analyzed buffer (0,5% BSA and 0.05% tween-20, PBS, pH of 7.2) and added equal volume of 10 ng/ml biotinylated IgE (O. Shannessy D. J. et al., Immunol. Let. 8: 273 (1984)), followed by incubation tablet for 2-3 hours at 25°C. Covered FcRI wells were washed three times with PBS and 0.05% tween-20 (Sigma) and 50 μl of the sample from the wells is transferred and incubated with shaking for 30 minutes at 25°C. a Solution of Streptavidin-HRP (500 μg/ml, Sigma), diluted to 1:5000 in the sample buffer, was added at 50 μl/well, followed by incubation tablet for 15 minutes with shaking and washing, as described above. 50 μl/well Microwell Substrate Buffer (peroxidase substrate, Kirkgaard and Perry Laboratories) was added, and the color appeared in tealy at 50% inhibition was calculated by constructing a curve of percentage inhibition on the concentration of the blocking antibodies using non-linear four parameter curve selection, using data analysis Kalelidagraph (Synergy Software). The results are presented in table.3.
Analysis (review) binding, based on FACS
The ability of antibodies to inhibit FI-conjugated (Goding J. W. J. lmmunol. Methods, 13: 215 (1976)) IgE binding-chain of the high-affinity FcRI receptor, expressed on Cho 3D10 cells (J. Hakimi et al., J. Biol. Chem., 265: 22079 (1990)), were determined using flow cytometry. FITC-Konyushenny IgE (40 nm) was plaincourault with antibody(0,3-1,0×10-6M) at 37°C for 30 minutes in FACS buffer (PBS with 0.1% BSA and 10 mM sodium azide, pH 7.4). The complex is then incubated with 5×105SNO D10 cells at 4°C for 30 minutes. Cells were washed three times in FACS buffer and measured average flow fluorescence at 475 nm on a FACScan flow cytometer (Becton Dickinson). MaE1, mAb rat or mouse against human IgE, which does not block IgE binding to FcRI-chain was used as positive control and MARS (Cappel), monoclonal antibody rat or mouse, which does not recognize IgE was used as a negative control. The results are shown in Fig.3.-Fc subunitRI, expressed on Cho 3D10 cells with 10 μg/ml human IgE for 30 minutes at 4°C. the Cells were washed three times, followed by 30 minute incubation with varying concentrations of mAbs or MaE1 or Mae rat or mouse against human IgE or purified mAb variant 12[F(ab)12]. MOPC21 (IgG1 mouse or rat) was used as a control for mAbs rats or mice, whereas purified 4D5 mAb (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)) human IgG1 was used as a control for F(ab)12. Binding of mAbs rats or mice were detected with FI-conjugated F(ab')2 goat against mouse IgE (10 μg/ml). The binding of purified mAb were detected (detected) using FI-conjugated F(ab')2 goat against human IgG (50 μg/ml), which is purified using affinity chromatography on IgE column to minimize cross-interaction with IgE. The results are presented in Fig.4.
Computer-graphics model related to rats or mice and purified F(ab' s
Sequences of VL and VH regions F(ab) Fig.1 used to create a computer-graphics model of the VL and VH regions Mae related to rats or mice. Derogene antibody which leads to the creation of F(ab)-2. Also created a model refined variants to confirm the correctness of the choice of residues spanning the area related to rats or mice. The creation of the models was carried out as described in Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992), C. Eigenbrot et al., J. Mol. Biol. 229: 969 (1993)).
Design refined Mae antibodies
This study refined antibodies used “consensus” sequence of human rights. This is in contradiction with other ways of refining that use sequences belonging to the person closest to Ig rat or mouse. Sherman et al., J. Immunol. 147: 4366 (1991); Kettleborough et al., Protein Eng. 4: 773 (1991); Tempest et al., Biotechnology 9: 266 (1991); Co et al., Proc. Natl. Acad. Sci. USA 88: 2869 (1991); Routledge Eur. J. Immunol. 21: 2717 (1991). This consensus sequence of a person, consisting of spanning region, based on the subgroup III VH and subgroup I VLk person, as defined in monopatia of Cabot (Kabat et al., Sequences of Proteins of Immunological Interest, 5ed., National Institute of Health, Bethesda, MD (1991)). F(ab)-1 created transplantation six CDR's, as defined by Cabot (Kabat, supra) spanning region of human IgG1. All residues spanning the field were preserved such as the human Ig. This option is a better description of what was reported about the impossibility of such “CDR sharing” options to bind their antigens. (Carter et al., supra; Tempest et al., supra). It should be noted that the exact sequence F(ab)-1 is not described in the table.3, however, this sequence can be deduced by replacing Cabot CDR residues Mae rat or mouse (given in parentheses) to the corresponding residues of the fragment of human antibodies. Fig.1 shows CDRs of Cabot in brackets to the left and to the right, while the CDRs Hatia (Chothia) is underlined.
In order to help interpret and to reduce confusion, the remains relating to the fragments of antibodies man, wrote a normal font, while the remains relating to rats or mice, are presented in italics. If show substitution of residues, the first residue is substituted by the residue, and the second is inserted residue, and use the numbering Cabot to refer to native sequence.
F(ab)-2 option based on molecular modeling. In this molecule, several residues spanning region of rats or mice included in the spanning region of the person. In F(ab)-2 used to define CDR's proposed by Kabat et al., supra (which is based on diversity (variability) interspecific sequence), with the exception of CDR-H1 and CDR-H2.
Definition of CDR-H1, based on diversity (variability) follower complexes antigen-antibody (Chothia et al., Nature 342: 877 (1989)) (Fig.1). Therefore, CDR-H1 re-defined to include both definitions, i.e., residues 26-35 of the person.
The definition of CDR-H2, based on the diversity of sequences (Kabat et al.), contains more residues than a definition based on the crystal structures of antibody-antigen (Chothia et al.) [see Fig.1 CDR's Cabot denoted by brackets, Hatia - underlined]. Because not found crystal structure, which would indicate communication antibody-antigen for residues 60-65 human antibodies, CDR-H2 modified to include a hybrid of the two definitions, i.e., residues 50-58 human antibodies. As a result, in F(ab)-2 used a shorter variant CDR-H2 compared with the F(ab)-1.
In the F(ab)-2 was created with the minimum number of changes remnants of human antibodies on the remains of antibodies in rats or mice that believed, required to preserve the tie. Created 10 additional options in order to investigate the influence of depth residues on CDR conformation, and to assess the predicted impact of molecular modeling significant residues spanning region and explore other residues of interest.
To study the influence of depth residues in CDR conformant antibodies person. As shown in the table.4 (using F(ab)-3 and F(ab)-4), side chains in the VL4 and VL33 had a minimal effect on the binding and, presumably, the conformation of CDR-L1 in improved antibody.
The modeling assumed that the remainder VH24 spanning region may influence CDR-L1 conformation and VH37 could affect the surface section VL-VH. However, substitution of the residue in the antibody man in VH24 [F(ab)-5] or VH37[F(ab)-7] showed a minimal decrease in the binding. On the contrary, the substitution of the Phe residue in spanning region of rats or mice in position VH78 on Leu [F(ab)-6] man caused a significant decrease in the binding. Models suggest that this residue affects the conformation of CDR-H1 and/or CDR-H2.
F(ab)-9-F(ab)-12 investigated the substitution of residues of human antibodies on rat or mouse. All four of these variants showed a significant improvement in binding compared with the F(ab)-2 (see tab.3, 4 and Fig.3). In F(ab)-9, which showed a 5-fold greater binding compared with the F(ab)-2, two of the residues in CDR-H2 (as defined by Kabat et al., supra) was replaced with the remains of antibodies rat or mouse: Ala VH 60 Asn Asp H61 Pro. Replacement Pro should change CDR-H2 conformation and/or stiffness and suggest that Asn H60 buried in the surface of section VL-VH, possibly interacting with Asp VL1.which all depth residues (defined as residues with the available surface area, less than 5% of the amount of free amino acids) in the VL and VH regions were Mae rat or mouse. Basically, F(ab)-10 can be thought of as Mae rat or mouse, in which the only available is not related to CDR residues in the VL and VH regions were replaced by residues belonging to the person.
To determine whether it was improved binding F(ab)-10 consequence of one or more residues have created variants of the F(ab)-11 and F(ab)-12. Instead of F(ab)-2, F(ab)-9 was used as the main template, which enables these options, because they showed a 5-fold better binding. The modeling assumed that the side chain at VH63 and VH67 may affect the conformation of CDR-H2. I believe that VH63 part of chr-H2, as determined by Cabot with TCS. (Kabat et al., supra), and not as defined by Chothia et al., supra. I believe that V67-residue core region in both definitions. In F(ab)-11, VH63 and VH67 were remnants Leu and ll rats or mice, respectively. In F(ab)-12 only VH67 replaced ll rat or mouse.
In restorelocation and cellular assays (table.4) and cell analysis (table.4, Fig.3) both F(ab)-11 and F(ab)-12 showed binding to at least the same as in F(ab)-10, and better than the F(ab)-9. This suggests that the enhanced binding of F(ab)-10 not , what about it was due to the influence of only a single residue, i.e., VH 67.
F(ab)-8 created by substitution VL55 residue Glu person on Gly rat or mouse, and similar substitutions in VL57 Gly to Glu. F(ab)-2 used the remains belonging to the man, while F(ab)-8 replaced these provisions remains related to rats or mice. As you can see from the table.3, the substitution of these residues does not affect receptor binding.
Found F(ab) variants with binding close to Mae rats or mice, namely F(ab)-2, F(ab)-9, F(ab)-10 and F(ab)-12, used to generate IgG1 molecules full length. Binding of these molecules in relation to option F(ab)-2 or Mae was comparable to the binding, show F(ab) fragments. These results are presented in table.4.
Linking Me with IgE-loaded FcRI
Mae rat or mouse prevents the binding of free IgE FcRI on mast cells, but does not cause histamine release upon binding with IgE-loaded FcRI. As shown in Fig.4, and Mae rats or mice and refined version 12 (IgG1-12), and negative control isotype antibody /img.russianpatents.com/chr/949.gif" border="0">RI on Cho 3D10 cells. On the contrary, Mae antibody rat or mouse, which binds to IgE, but does not prevent IgE binding to FcRI was associated with IgE-loaded FcRI. In contrast to IgG1 control person (purified 4D5) isotype IgG1 rats or mice (as shown MORS) shows nonspecific background binding on these cells, accounting for 10%. Mae does not staining over control MORS, and refined version 12 does not staining than control treated 4D5 (Fig.4).
Partial scan (scan) CDR residues of Aponina important in IgE binding
Sequence Mae CDR's point to the predominance of charged residues (Fig.1). CDR-L1 contain three Asp residue, whereas CDR-L3 has His, Glu and Asp. CDR-H3 has three His balance. Model Mae rats or mice and treated Me illustrated the spatial proximity of all these charged residues (not shown). On the contrary, single Asp45 in CDR-H2 spatially separated from other charged residues. Alanine was substituted using site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)) on each of these charged residues to generate variants. In CDR-L1 change one of the three Asp stately effect [F(ab)-14; F(ab)-15]. Simultaneous substitution of Glu VL93 and Asp VL94 by alanine in CDR-L3 [F(ab)-17; PL.5] also reduced the binding, although not to the same extent as in the substitution VL32b. Individual substitution of the three His residues in CDR-H3 to Ala resulted in a small improvement in binding [F(ab)-21] or three-fold reduction in binding [F(ab)-20, F(ab)-22]. However, the simultaneous change of all three His residues were eliminated binding of [F(ab)-19]. Although it is not easy to determine whether the charged residues in direct binding to IgE or provide some conformational stability to their respective CDR's variants, F(ab)-13 to F(ab)-22 show that CDR-L1 and CDR-H3 are important determinants of IgE binding.
Summary and conclusion
Creating functional, refined anti-IgE antibodies rat or mouse comprises a replacement of several residues spanning the area related to rats or mice, spanning an area owned by the person. In addition, the mapping charged CDR residues indicated that some of them are important in antibody-IgE interaction.
According to previous studies (Carter et al., supra; Shearman et al., J. Immunol. 147: 4366 (1991); Kettleborough et al., Protein Eng. 4: 773 (1991); Tempest Biotechnology 9: 266 (1991)), options from F(ab)-1 and F(ab)-12 wk is but given the value when considering F(ab)-1, which is a direct CDR exchange, in which only six CDR's related to rats or mice transplanted (transplanted) in residues spanning the areas related to the person. A potential explanation for this includes CDR-H2. Advanced hydrophobic residues in position VH63 and VH67 may affect the conformation of CDR-H2. Created variants containing four combinations provisions VH63 and N, i.e., Leu and ll related to rats or mice, respectively [Mae and F(ab)-11], VaI and Phe [F(ab)-2] Leu and Phe [F(ab)-1] and VaI and lle [F(ab)-12]. A clear conclusion from the data linking these four options indicates that important balance is VH67, which must be ll rat or mouse in order to ensure the affinity associated with Mae rat or mouse. In F(ab)-1 this residue represented Phe person.
Of the 12 residues in F(ab)-1, held as belonging to the person [in comparison with F(ab)-2], 8 separately replaced related to rats or mice in other embodiments. Three changes had no effect on binding: VL4 [F(ab)-4]; VL55 and VL57 [F(ab)-8]. Substitution of two residues: VH60 and VH61 [F(ab)-9], improved binding, whereas three were reduced binding: VH24 [F(ab)-5]; VH37 [F(ab)-7] and VH78 [F(ab)-6].
Option F(ab)-10 was created with the help of the hypothesis proposed PE is th when replacing only available residues spanning region. In this embodiment the hydrophobic inner region of the VL and VH regions, in other words, the variant represented Mae rat or mouse, in which the only available residues spanning the field in the VL and VH were replaced with a sequence that belongs to the person. Although F(ab)-10 showed binding close to the binding Mae rat or mouse, changes in individual amino acid region, VH67 person on rats or mice, would have the same impact on improving binding [F(ab)-12, IgG1-12].
Refined version with binding comparable to Mae rat or mouse, which also required the least change was a F(ab)-12. This option was replaced only 5 residues spanning the field of human antibodies rat or mouse (VL4, VH24, VH37, VH67 and VH78). Four of these residues was determined by molecular modeling. Fifth, VH67, and CDR-H2 residues VH60 and VH61 included using molecular models to attempt to improve the binding source variation F(ab)-2.
Verify the selection of histamine
It is the analysis of histamine mast cells of the rat (the rat mast cell histamine assay, RMCHA), which quantitatively measures the biological activity of recombinant purified monoclonal and RBL 48 cells. In addition, this definition is carried out in physiological conditions, analogous to the human body conditions. RBL 48 cells received from the parent line of the fat cells of the rat RBL 2H3, which were subsequently transfectional-subunit of high-affinity IgE receptor human (FcRI) (Gilfillan, A. M. J. Immunol. 149 (7): 2445-2451 (1992)).
RBL 48 cells (Gilfillan et al., supra) were cultured in sIMDM, environment, Dulbecco modified according to the method Iscove, supplemented with 10% serum, fetal calf, 2 mm glutamine, and 500 μg/ml of active geneticin (geneticin, Gibco, #860-1811) in T mattress tissue culture (Falcon #3028) at 37°C in a humidified 5% CO2-incubator (Fischer, model #610). Cells were collected during curing 4 ml PBS/0.05% trypsin/ of 0.53 mM EDTA for 2 minutes at 37°C, followed by centrifugation (HD, 10 min) and re-suspension in fresh sIMDM. Cells in suspension were counted using hemocytometer (Reichert-Jung) and the density was brought to 0.4×106cells/ml was Then produced by seeding cells at 100 μl/well (40,000 cells per well) in the inner 60 wells of 96-hole, U-shaped tablet (Linbro) tissue culture and were cultured for 24 hours at 37°C in humidified and 30 minutes at 90 μl/well of a solution of diluent (sIMDM, 3 u/ml Na-heparin) with specific pollen Allergy IgE (RSIgE, 10 ng/ml, 23,48 ng/ml total IgE, 1,43% specific for pollen Allergy human plasma, North American Biological, lot #42-365054).
After incubation 10 μl/well of anti-IgE antibodies (diluted with diluent 0,06 to 39.4 μg/ml) or diluent (control full selection of histamine, background and pollen Allergy) was added to the cells and the plate is incubated for 24 hours in 5% CO2at 37°C in the incubator. After incubation, the cells are aspirated (removed) and washed 3 times with 200 μl/well sIMDM. After washing cells were incubated with 100 μl/well of either (1) 0.5% solution of Triton (for full selection of histamine), (2) buffer for isolation of histamine (HRB, 50% D2O, 0.8% of NaCl, 1,3 mM CaCl2sIMDM or (3) antigen pollen Allergy (NIH #A-601-A-185, 0.1 ág/ml in HRB) at 37°C for 30 minutes and the reaction stopped when placed on ice (100% D2O=100%, and 0.8% NaCl, 1,3 mM CaCl2).
The tablet was centrifuged 5 minutes at HD (2460 rpm) at 4°C, supernatant was collected and diluted 1:80 in PBS (1:1000 in PBS for control full selection of histamine) for the determination of histamine using histamine enzyme immunoassay kit with affinity chromatography (Histamine Enzyme Immunoassay Kit, Immunotech #1153). Supernatual reaction with 50 μl Alliluyeva buffer (set) for 30 minutes at room temperature. Acylated histamine (50 µg/well) is then transferred to a tablet for conjugation (in the set), and incubated with 200 μg/well gistaminoliberatora conjugate (set) for 18 hours at 4°C.
After incubation the wells were subjected to blotting and washed to remove unbound conjugate with 4 times washing, 300 μg/well washing buffer (Immunotech kit, #1153). Added chromatogaphy substrate (acetylthiocholine, detenidamente, 200 μl/well, and incubated in the dark at room temperature for 30 minutes. The reaction was stopped by adding stop solution (50 μl/well, in the set) and was determined by the absorbance at 405 nm with 620 nm compared to SLT 340 ATTC spectrophotometer to read the tablets. The intensity of the absorption is inversely proportional to the concentration of histamine (expressed in nm), which is determined from the standard curve for histamine (from enzyme immunodiagnostics set AMAC). The percentage of full selection of histamine was calculated from the data of the concentration of histamine and the percentage of inhibition was calculated using a 100% full selection of histamine. The results are shown in Fig.5.
Summary and conclusion
A plot of the molar ratio of anti-IgE on antibody possesses the best properties allocation of histamine, induced pollen allergies, compared with the F(ab) form E25 antibody. E inhibits the release of histamine induced by pollen allergies, depending on the dose of a molar ratio equal to 44:1 for half-maximum inhibition (anti-IgE: RSIgE). On the contrary, E25 only inhibits the release of histamine from between 200:1 to 1550:1 anti-IgE: RSIgE). The molar ratio of the half maximum inhibition curve E25 determined from 400:1 to 500:1. Therefore, based on the data values of the molar ratios for half maximal inhibition, which is a measure of the binding affinity of molecules, a molecule associated with RSIgE about 10 times better than E25 molecule.
Example phage display
This example describes the anti-IgE antibodies with improved specific affinity generated by monovalent phage display and selection of F(ab) fragments derived from E25 purified anti-lgE antibodies (Presta et al., J. Immunol. 151: 2623 (1993)).
I. creating a monovalent F(AB)-phage libraries
Created several F(ab) libraries. As the original vector E25 variant containing the substitution D32E VL (to exclude IsoAsp isomerization), was merged with asmita, which is known as R shown in Fig.10. First, F(AB)-phage “wild-type” R used as template to create a library-specific “stop” template. The introduction of stop codons (TAA and TGA) of the original molecule becomes inactive, thereby reducing the shadow (background) effects and template-specific hybridization) offset in stages mutagenesis when creating a library (Lowman, Wells: Methods: OMRS. Methods Enzymol. 3: 205 (1991)). Data template created using the single-stranded template-directed mutagenesis (Kunkel et al., Methods Enzymol. 204: 125 (1991)) with the oligonucleotides shown in table.10.
Subsequently, these stop-template used in the second round of mutagenesis, using listed in the table.11 oligonucleotides to generate libraries in each of these CDR regions. NNS generating the codons used for all twenty amino acids in each of these CDR regions (Bases of the nucleotides denoted by single letters according to the IUPAC nomenclature; N=A, G, C or T; S=G or C). NNS generating the codons used for all twenty amino acids in each randomly selected position, using 32 different possible codon. The amber stop codon (TAG) encodes Gln used here in Spresso what actu heavy chain antibodies and Dr area on the phage allows the expression of the phage-detected fused protein only in amber-suppressor strains of E. coli, while soluble F(ab) protein of the same design can be obtained in despressing strains of E. coli (Lowman, et al., Biochemistry 30:10832 (1991); Lowman and Wells, Methods Comp. Methods Enzymol. 3: 205 (1991); Hoogenboom et al., Nucl. Acids Res. 19: 4133 (1991)). However, experts know about the other stop codons for use in other E. coli phage-expressing systems.
Products of random mutagenesis reactions were transformed into E. coli cells (Stratagene, XL-1 Blue) by electroporation and amplified by culturing overnight at 37°WITH MK phage helper (Essen and Messing, Methods Enzymol. 153: (1987)).
II. The phage-binding breeding
For selections on the affinity of phage particles that detect F(ab) variants, the phages were obtained using precipitation of sodium chloride/polyethylene glycol (NaCl/PEG) supernatants of E. coli culture. Phages suspended in PBS buffer, and then diluted in horse serum (catalog no. A-3311-D, Hyclone, Logan, UT) containing 0.05% Tween-20 (Tween-20), and neonarrative phages as a negative control. As a positive control e F(AB)-phage “wild-type” mixed with neonatorium phage and subjected to false selection.
Maxisorp 96-hole Board over night at 4°C. Next IgE solution was removed and the plates were incubated with a blocking solution of horse serum (without Tween-20) 2 hours at room temperature.
The blocking solution was removed and the phage solution was incubated on the plates for one hour at room temperature. Then, the phage solution was removed and the tablets were washed 10 times with buffer PBS/Tween-20 (0,05%). The wells were filled with PBS/Tween, and incubated for 10 minutes, after which the tablets again washed 10 times.
F(ab)-phage remaining bound on the tablet was suirable 20 mM HCl, neutralized with buffer Tris-HCl, pH 8, and cultivated with the phage helper, as described above. An aliquot of the phage serially diluted, mixed with fresh cells XL-1 Blue were placed on the appropriate antibiotic tablets and counted the number of CFUs (colony-forming units, colony forming units) F(AB)-detect (carbenicillin-resisting; CFUa) or neonarrative (chloramphenicol-resistant CFUc) aliremove phage. Improvement (enrichment) (Emut) F(ab)-detect compared to neonatorium phage in each round was determined as (CFUa/CFUc) for aliremove pool, dividing by (CFUa/CFUc) original pool. Improvement (enrichment) for control fagiano above, except that the incubation period after the first 10 washings increased each round. In order to compare the effectiveness of ragovoy selection from round to round with increasing stringing conditions, the enrichment factor in each round were normalized (arranged) in relation to the factor of enrichment control wild-type. The ratio of binding enrichment for each pool to bind to the enrichment of wild-type (Emut/Ewt) is shown in Fig.6. As at the balance a large fraction of high-affinity variant should contact IgE tablet compared to the low-affinity variant, more high-affinity variants should be removed more efficiently and therefore discover a large relative improvement (enrichment). Indeed, VL1 library consistently showed improved relative enrichment, up to about ten times greater relative enrichment than wild type after 5-6 rounds (rounds) selection. With this measurement VL1 library showed greater improvement in affinity compared with the wild type than it showed VH3 library. The disparity in results between the two series CDR libraries can reflect a large energy contribution VL1 to connect the zoom, allowing a greater relative improvement in binding interactions made VL1, through substitution of the side chain.
DNA sequencing showed that the majority of the F(AB)-phage variants of the first VL CDR1 library (randomly selected positions 27, 28, 29 and 31) kept the balance D30 wild-type and preferably mutated Y31G (PL.15, in which the clones from the third round (round) designated as 212-3.x and clones from the sixth circle (round) indicated 212-6.x). Although watched a number of substitutions in positions Q27 and S28, one clone containing Q27K and S28P, dominated the phage pool after six rounds (rounds) selection. This clone also contained remains the preferred D30 and G31, suggesting that the combination of the side chains may be optimal for IgE binding.
In the second VL CDR1 library (randomly selected positions 30, 31, 32, and 34), the majority of selectnow preserved remains of the wild type in D30 and e; only D34 wild type was observed among the sequenced clones. This library has observed a number of varieties of residues in Y31. Watched more false mutation G33S - two clones 213-6.7 and 213-6.8 (PL.15).
Analysis of the sequencing of clones from VH CDR3 library after three rounds (rounds) breeding showed, Thu is the operating NT and H107Y (PL.15).
IV Phage-ELISA tests selected F(ab) clones
Results phage-binding selection phages were transfectional in E. coli XL-1 Blue cells and cultured in suspension culture or placed on containing the antibiotic tablets. Clones were randomly collected from these tablets for analyzing sequencing and linking using competitive phage-ELISA. (Cunningham et al., EMBO J. 13: 2508 (1994); Lowman, Chapter 24, in Methods in Molecular Biology, vol. 87, S. Cabilly (ed.), Humana Press Inc., Totawa, NJ (1997)).
To evaluate the relative IgE binding affinely phage were titrated on the tablet with adsorbed IgE as described above to normalize the detected F(ab) concentrations. The phage was pre-mixed with serial solutions IgE, was then added to IgE-adsorbed to the plate and incubated for one hour at room temperature. Then the tablets were washed 10 times in PBS/Tween was added a solution of antiferromag antibodies rabbit mixed with a conjugate antibodies goat or goat against rabbit and peroxidase from horseradish. After one-hour incubation at room temperature the tablets were processed chromogenic substance-o-phenylenediamine (Sigma). The reaction was stopped by adding1 In some cases, phage pools from this circle (round) selection tested en masse in order to obtain an estimate of the average relative affinity of the population [S(weight)/ S(mutant)] for IgE. For example, VL CDRI library, residues 32, 33, 35 and 37 showed a 3.6-fold improvement in affinity against a after 5 rounds (rounds) selection, although the parent version of this library (e), apparently, had a 25-fold improved affinity. Therefore, the VL-CDR1 library these individual residues are not further considered (continued). On the other hand, VH CDR2 phage pool showed a 6.2 fold improvement in affinity compared to the parent a phage. Also created a phage library CDR regions of a VL CDR2, residues 54-57, and VL CDR3, residues 96-98, 99 and 100. However, amino acid substitutions in these provisions have not been able to generate any enrichment compared to e. Phage library was generated for VH CDR1, ottenibili when contamination e-phage. This suggests that variations greater affinity than he was not present in the original library. Phage libraries CDR regions of a VL CDR1, remains 27, 28, 30, 31, 32, 34, as well as VH CDR1, residues 101, 102, 103, 105 and 107 are shown in table.15, while the VH CDR2 is presented in table.16. In table.15 and 16 library clones that did not show a significantly higher affinity than e, are not further considered (continued) not determined factor of the improvement of the binding. V. Mixed (combined) mutations of phage screening Mutations at different sites within a protein often demonstrate an additional effect on the function of the protein (Wells, Biochemistry 29: 8509 (1990)). Therefore, several mutations from the original phage libraries described above were combined to improve the binding of IgE. In order to reduce the probability of increasing the immunogenicity of anti-IgE antibodies, it is necessary to minimize the degree of mutations from the E-25. As a result, if measured independently, used only mutation of phage variants, which exhibited the greatest improvement in the affinity. In addition, the frequency with which observed this phage clone, may be associated with the level of expression and/or proteolytic stability (Lowman and Wells, 25 times superior compared to e in phage-ELISA assays (table.17). VH CDR2 library also showed improved affinity compared to e, although this improvement, only 6.2 times greater compared with the collected phage. The affinity of the harvested phage showed improved binding affinity for at least several members of the pool, not measuring the affinity of all individual members. Using the collected phage also allows you to identify which increase the affinity was achieved after this round (round), and whether to continue the selections on the affinity (i.e., if the pool affinity peaked, subsequent rounds (rounds) are unlikely to give additional improvement (enrichment)). As such, the use of data collected affinity is a very useful screening tool (validation testing). It is obvious that mutations in the VH CDR2 region can act additively with mutations in the VL CDR1, because VH CDR2 loop fragment is at a distance from VH CDR1 loop fragment and in the crystal structure, and molecular models. However, as some combinations of these mutations may, however, be incompatible, we investigated four different mixed mutant: a savali using mutagenesis of Kunkele (Kunkel et al., Methods Enzymol. 204: 125 (1991)), using a F(AB)-phage as template with mutagenic oligonucleotides encoding VH2 mutations. Phage-ELISA tests (Lowman, Methods in Molecular Biology, vol. 87, Cabilly (ed), Humana Pres Inc., Totawa, N. J. (1997)) was used to compare the target variation of mixed VL CDR1 mutations in e with VH CDR2 mutations in clones 235-5.1; 235-5.2; 235-5.3 and 235-5.4. Soluble F(ab) proteins were also obtained and compared by analysis of Biotin-IgE-tablet presented below in table.17 and in Fig.7. VI. Biotin-tablet analysis (competitive analysis FcRI-IqG chimeras) Introduction: the purpose of this example is to compare how the various anti-IgE F(ab)s compete with immobilized IgG Chimera high-affinity IgE receptor for binding to biotinylated human IgE in the liquid phase, when anti-IgE F(ab) and Biotin-IgE was added simultaneously to the tablet with adsorbed chimeric IgE receptor. Since the concentration of anti-IgE F(ab) increases, the amount of biotinylated IgE that can bind to a receptor on the tablet decreases, leading to lower optical density, measured with a spectrophotometer. Nunc maxisorp plates (catalog no. F96) covered (adsorbing) 100100 μl concentration 1 mg/ml initial solution of antibody in 50 nm natrocarbonatite buffer (pH 9,6), during 12 to 24 hours at 4°C. The tablets were washed three times with ELISA proryvnym buffer (0.05% Polysorbate 20 (Sigma) in PBS (pH 7.4) and blocked by incubation with 200 μl of buffer for ELISA (Tris-buffered saline, pH of 4.45 0.5% RIA purity bovine serum albumin (Sigma); 0.05% Polysorbate 20 and 4 mM EDTA) for 60 minutes. After three washes proryvnym buffer three times was added 100 μl of serial 2 fold diluted anti-IgE F(ab)s in the buffer for analysis with an initial concentration of 200 nm by ELISA plate. Dilution was performed multichannel pipette Titertek®. Biotinylated IgE in the buffer for analysis (100 μl, 1:500 diluted to 0.5 mg/ml of the original solution) was added to all wells and the mixture is incubated in miniorities shaker (Bellco) for 60 minutes at 25°C. IgE was purified using affinity chromatography from the supernatant U266B1 myeloma (ATSS TIB 196) culture and biotinilated sheet bilingualised (O'Shannessy et al., Immunol. Lett. 8: 273 (1984); Pierce Chemical). The samples were washed five times with wash buffer and bound IgE was detected using 100 μl of peroxidase-conjugated streptavidin (Zymed) at a dilution of 1:3000 in 90 minutes. The sample is then again washed six times proryvnym buffer, followed by addition of 100 ál of substrate
Next, the reaction was stopped by 4.5 M H2SO4(100 μl) and the absorbance was measured at 490 nm on a spectrophotometer to read the tablets Uvmax (Molecular Devices). The absorbance at different values of F(ab) concentrations E25, e and E27 F(ab) fragments of the antibodies is shown in Fig.8. Conclusion: the Curves in Fig.8 indicate that E and E27 have a greater affinity than E25 to high-affinity receptor, and that E27 showed the greatest affinity. VII. BIAcore analysis of soluble F(ab) protein Receptornegative affinity of several F(ab) fragments was calculated (Lofas and Johnson, J. Chem. Soc. Commun. 21: 1526-1528 (1990)) of the rate constants of Association and dissociation, as measured by BIAcore TM-2000 resonant system of the surface plasmon (BIAcore, Inc.). A biosensor chip was activated for covalent binding of IgE using the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions (BIAcore). IgE was diluted to 10 nm buffer sodium acetate (pH 4.5), which was further diluted to about 30 μg/ml and was administered by injection through the chip for receiving the signal from 800 to 12400 units allergic reactions (RU) of immobilized substances. As the signal EN is proportional to the mass of the immobilized substance, this means that the range is on as a blocking agent. Regeneration was performed with 4.5 M MgCl2. For kinetic measurements of 1.5 serial dilution of F(ab) fragments of antibodies was injected through IgE chip in PBS/Tween buffer (0.05% of Tween-20 in phosphate buffered saline) at 25°C, at a flow rate of 20 μl/min (Fig.9). Data dissociation is consistent with a single-site model for koff±s.d. (the standard deviation of the measurements). The rate constant of pseudobersama order (ks) was calculated for each curve of the Association and depicted as a function of the concentration of the protein to obtain the kon±s.d.e. (standard error tolerance). The equilibrium dissociation constants for F(ab):IgE binding, kd'scalculated from SPR measurements in the form of koff/kon. In the absence of experimental artifacts, such as rebinding of dissociated F(ab), the observed koffnot depend on the concentration of F(ab). Also, since the equilibrium dissociation constant kd, is inversely proportional to koffthe assessment of the improvement in the affinity can be done, assuming that the rate of Association (kon) constant for all variants. koffalong with a calculated half-life period is shown in table.18. VIII. Expressi who was preservati in the strain E. W Li, Toothpick culture (10 ml) in 2YT medium with 50 μg/ml carbenicillin were incubated for 8 hours at 37°C and then transferred into 1 liter of modified AR-5, containing 50 μg/ml of carbenicillin, and incubated for 24 hours at 37°C. the Culture was centrifuged in 500 ml bottles at 7000 rpm for 15 minutes at 4°C. the Precipitate after centrifugation was frozen for at least 3 hours at 20°C. Every 500 ml of sediment after centrifugation suspended in 12.5 ml of a cold solution of 25% sucrose in 50 mM Tris buffer pH 8.0, containing 1 mM of benzamidine (Sigma) at 4°C. the Suspension was solubilizers under stirring at 4°C for 3 hours. The suspension was centrifuged at 18000 rpm for 15 minutes at 4°C and F(ab)sexpressed in the supernatant was purified using affinity chromatography on protein G (Pharmacia). The column was washed with a solution of 10 mM Tris (pH 7.6) and 1 mM EDTA (pH 8.0) and F(ab)s were suirable 100 mM acetic acid (pH 3.0), an amount equal to 2.5 column volumes, and immediately neutralized to a neutral pH of 0.5 volumes of 1M Tris pH 8.0. Eluate was concentrated and the buffer was replaced with PBS using method 30 microconcentrators (Amicon). The concentration of protein was determined by absorbance at 280 nm using a spectrophotometer (Beckman DU 64)946.gif" border="0">-mercaptoethanol. IX. Results and conclusion The results of the phage-ELISA competition experiments show that while a F(AB)-phage approximately 9 times improves the affinity compared to e, Raman options a, e and e 20-40 times superior compared to e-phage. Additional combinations of phage-derived mutations can give options antibodies with similarly improved appendectomy. When F(ab) soluble proteins was investigated in the Biotin-IgE tablet analysis, a F(ab) and E27 F(ab) was improved about 10 times and 30 times, respectively, compared to E25, with regard to the inhibition of IgE binding FCeRI-IgG. Koffdetermination using BIAcore analysis confirms the relative affinity of. In particular, e and E27 showed 7.7 times and 22 times less Koffthan E25. Longer half-life means that IgE busy or assists variants unable to bind with high-affinity receptor for a longer period, thus resulting in increased therapeutic efficacy of anti-IgE. Thus, the data of the equilibrium and kinetic binding supports the conclusion that a and E27 F(ab)2 bind IgE is about 10 times and 30 times stronger, respectively, than the E25. Predpolagaetsya affinity to E25 IgG. Claims 1. Antibody anti-IgE that specifically bind to the IgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, including the sequence of the light chain and the sequence of the heavy chain, which exhibit at least 70% identity with the sequences of the light chain and heavy chainaFig.13 [SEQ ID NOS:19 and 20] and which include the remains 32Glu, 27Lys and go variable light chain CDR1. 2. The antibody under item 1, includingasequence selected from the following series: F(ab) fragment [SEQ ID NOS:19-20], sFv fragment [SEQ ID NO:22], F(ab)2fragment [SEQ ID NOS:24-25]. 3. The antibody specifically binding to IgE and inhibiting or substantially reducing the binding to high-affinity receptor Fc 4. The antibody under item 3, includingE27sequence selected from the following series: F(ab) fragment [SEQ ID NOS:19 and 21], sFv fragment [SEQ ID NO:23], F(ab)2fragment [SEQ ID NOS:24 and 26]. 5. The nucleic acid molecule encoding the antibody under item 1, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody comprises the sequence of the light chain and the sequence of the heavy chain, which exhibit at least 70% identity with the sequences of the light chain and heavy chainaFig.13 [SEQ ID NOS:19 and 20] and which include the remains 32Glu, 27Lys and go variable CDR1 light chain . 6. The nucleic acid molecule encoding the antibody under item 2, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody is an antibody fragment “26b> [SEQ ID NOS:24-25]. 7. The nucleic acid molecule encoding the antibody under item 3, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody comprises the sequence of the light chain and the sequence of the heavy chain, which exhibit at least 70% identity with the sequences of the light chain and heavy chainE27Fig.13 [SEQ ID NOS:19 and 21] and which include the remains 32Glu, 27Lys and go variable CDR1 light chain. 8. The nucleic acid molecule encoding the antibody under item 4, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody is an antibody fragment “E27”, selected from the following list: F(ab') [SEQ ID NOS:19-21], sFv fragment [SEQ ID NO:23], F(ab)2[SEQ ID NOS:24 and 26]. 9. The composition is enhanced antibody anti-IgE designed to reduce or prevent IgE-mediated production of histamine in a mammal, comprising a pharmaceutically acceptable filler or fillers in smeureanu of histamine in mammals, involving the introduction of a therapeutically effective amount of the antibody molecules according to any one of paragraphs.1-4. 11. A method of treating IgE-mediated disorders, comprising introducing a therapeutically effective amount of the antibody molecules according to any one of paragraphs.1-4. 12. A method of obtaining antibodies anti-IgE according to any one of paragraphs.1-4, not deaktivirovana when isomerization of aspartyl and has affinity to the molecule target, which is remotefilename the polypeptide or greater, comprising the following stages: a) identification of residues of aspartyl, which are prone to isomerization, b) substitution of alternative residues and screening the resulting mutants in relation to the affinity to the molecule target bythe affinity maturationusing the phage display technique, which further includes the following steps: (i) substitution, a deletion (elimination) or insertion of one or more codons in the gene encoding the polypeptide, resulting in a change of amino acid sequence of the polypeptide thus obtained library of structurally related polypeptides, fused with protein ragovoy shell, (ii) display (nd DNA encoding this polypeptide, (C) the selection of modified polypeptides in which the remnant of aspartyl replaced and which have affinity to the molecule target, which is equal to remotefilename the polypeptide or higher. 13. The method according to p. 12, in which the unmodified antibody has a sequence denoted asE25in Fig.12 [SEQ ID NOS:13-14]. 14. The method according to p. 13, in which the substituted residues are residues Asp32Glu, Gln27Lys and Ser28Pro variable light chain CDR1. 15. The method according to p. 14, which is optionally substituted residues are residues Thr53Lys, Asp55Ser, Ser57Glu and Asp59Lys variable heavy chain CDR2. 16. A way to improve the inhibitory properties of antibodies against histamine induced pollen Allergy, including the implementation of the method under item 12.
In some cases, phage pools from this circle (round) selection tested en masse in order to obtain an estimate of the average relative affinity of the population [S(weight)/ S(mutant)] for IgE. For example, VL CDRI library, residues 32, 33, 35 and 37 showed a 3.6-fold improvement in affinity against a after 5 rounds (rounds) selection, although the parent version of this library (e), apparently, had a 25-fold improved affinity. Therefore, the VL-CDR1 library these individual residues are not further considered (continued). On the other hand, VH CDR2 phage pool showed a 6.2 fold improvement in affinity compared to the parent a phage.
Also created a phage library CDR regions of a VL CDR2, residues 54-57, and VL CDR3, residues 96-98, 99 and 100. However, amino acid substitutions in these provisions have not been able to generate any enrichment compared to e. Phage library was generated for VH CDR1, ottenibili when contamination e-phage. This suggests that variations greater affinity than he was not present in the original library.
Phage libraries CDR regions of a VL CDR1, remains 27, 28, 30, 31, 32, 34, as well as VH CDR1, residues 101, 102, 103, 105 and 107 are shown in table.15, while the VH CDR2 is presented in table.16. In table.15 and 16 library clones that did not show a significantly higher affinity than e, are not further considered (continued) not determined factor of the improvement of the binding.
V. Mixed (combined) mutations of phage screening
Mutations at different sites within a protein often demonstrate an additional effect on the function of the protein (Wells, Biochemistry 29: 8509 (1990)). Therefore, several mutations from the original phage libraries described above were combined to improve the binding of IgE.
In order to reduce the probability of increasing the immunogenicity of anti-IgE antibodies, it is necessary to minimize the degree of mutations from the E-25. As a result, if measured independently, used only mutation of phage variants, which exhibited the greatest improvement in the affinity. In addition, the frequency with which observed this phage clone, may be associated with the level of expression and/or proteolytic stability (Lowman and Wells, 25 times superior compared to e in phage-ELISA assays (table.17).
VH CDR2 library also showed improved affinity compared to e, although this improvement, only 6.2 times greater compared with the collected phage. The affinity of the harvested phage showed improved binding affinity for at least several members of the pool, not measuring the affinity of all individual members. Using the collected phage also allows you to identify which increase the affinity was achieved after this round (round), and whether to continue the selections on the affinity (i.e., if the pool affinity peaked, subsequent rounds (rounds) are unlikely to give additional improvement (enrichment)). As such, the use of data collected affinity is a very useful screening tool (validation testing).
It is obvious that mutations in the VH CDR2 region can act additively with mutations in the VL CDR1, because VH CDR2 loop fragment is at a distance from VH CDR1 loop fragment and in the crystal structure, and molecular models. However, as some combinations of these mutations may, however, be incompatible, we investigated four different mixed mutant: a savali using mutagenesis of Kunkele (Kunkel et al., Methods Enzymol. 204: 125 (1991)), using a F(AB)-phage as template with mutagenic oligonucleotides encoding VH2 mutations.
Phage-ELISA tests (Lowman, Methods in Molecular Biology, vol. 87, Cabilly (ed), Humana Pres Inc., Totawa, N. J. (1997)) was used to compare the target variation of mixed VL CDR1 mutations in e with VH CDR2 mutations in clones 235-5.1; 235-5.2; 235-5.3 and 235-5.4. Soluble F(ab) proteins were also obtained and compared by analysis of Biotin-IgE-tablet presented below in table.17 and in Fig.7.
VI. Biotin-tablet analysis (competitive analysis FcRI-IqG chimeras)
Introduction: the purpose of this example is to compare how the various anti-IgE F(ab)s compete with immobilized IgG Chimera high-affinity IgE receptor for binding to biotinylated human IgE in the liquid phase, when anti-IgE F(ab) and Biotin-IgE was added simultaneously to the tablet with adsorbed chimeric IgE receptor. Since the concentration of anti-IgE F(ab) increases, the amount of biotinylated IgE that can bind to a receptor on the tablet decreases, leading to lower optical density, measured with a spectrophotometer.
Nunc maxisorp plates (catalog no. F96) covered (adsorbing) 100100 μl concentration 1 mg/ml initial solution of antibody in 50 nm natrocarbonatite buffer (pH 9,6), during 12 to 24 hours at 4°C. The tablets were washed three times with ELISA proryvnym buffer (0.05% Polysorbate 20 (Sigma) in PBS (pH 7.4) and blocked by incubation with 200 μl of buffer for ELISA (Tris-buffered saline, pH of 4.45 0.5% RIA purity bovine serum albumin (Sigma); 0.05% Polysorbate 20 and 4 mM EDTA) for 60 minutes. After three washes proryvnym buffer three times was added 100 μl of serial 2 fold diluted anti-IgE F(ab)s in the buffer for analysis with an initial concentration of 200 nm by ELISA plate. Dilution was performed multichannel pipette Titertek®. Biotinylated IgE in the buffer for analysis (100 μl, 1:500 diluted to 0.5 mg/ml of the original solution) was added to all wells and the mixture is incubated in miniorities shaker (Bellco) for 60 minutes at 25°C. IgE was purified using affinity chromatography from the supernatant U266B1 myeloma (ATSS TIB 196) culture and biotinilated sheet bilingualised (O'Shannessy et al., Immunol. Lett. 8: 273 (1984); Pierce Chemical). The samples were washed five times with wash buffer and bound IgE was detected using 100 μl of peroxidase-conjugated streptavidin (Zymed) at a dilution of 1:3000 in 90 minutes. The sample is then again washed six times proryvnym buffer, followed by addition of 100 ál of substrate Next, the reaction was stopped by 4.5 M H2SO4(100 μl) and the absorbance was measured at 490 nm on a spectrophotometer to read the tablets Uvmax (Molecular Devices). The absorbance at different values of F(ab) concentrations E25, e and E27 F(ab) fragments of the antibodies is shown in Fig.8.
Conclusion: the Curves in Fig.8 indicate that E and E27 have a greater affinity than E25 to high-affinity receptor, and that E27 showed the greatest affinity.
VII. BIAcore analysis of soluble F(ab) protein
Receptornegative affinity of several F(ab) fragments was calculated (Lofas and Johnson, J. Chem. Soc. Commun. 21: 1526-1528 (1990)) of the rate constants of Association and dissociation, as measured by BIAcore TM-2000 resonant system of the surface plasmon (BIAcore, Inc.). A biosensor chip was activated for covalent binding of IgE using the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions (BIAcore). IgE was diluted to 10 nm buffer sodium acetate (pH 4.5), which was further diluted to about 30 μg/ml and was administered by injection through the chip for receiving the signal from 800 to 12400 units allergic reactions (RU) of immobilized substances. As the signal EN is proportional to the mass of the immobilized substance, this means that the range is on as a blocking agent. Regeneration was performed with 4.5 M MgCl2.
For kinetic measurements of 1.5 serial dilution of F(ab) fragments of antibodies was injected through IgE chip in PBS/Tween buffer (0.05% of Tween-20 in phosphate buffered saline) at 25°C, at a flow rate of 20 μl/min (Fig.9).
Data dissociation is consistent with a single-site model for koff±s.d. (the standard deviation of the measurements). The rate constant of pseudobersama order (ks) was calculated for each curve of the Association and depicted as a function of the concentration of the protein to obtain the kon±s.d.e. (standard error tolerance). The equilibrium dissociation constants for F(ab):IgE binding, kd'scalculated from SPR measurements in the form of koff/kon. In the absence of experimental artifacts, such as rebinding of dissociated F(ab), the observed koffnot depend on the concentration of F(ab). Also, since the equilibrium dissociation constant kd, is inversely proportional to koffthe assessment of the improvement in the affinity can be done, assuming that the rate of Association (kon) constant for all variants. koffalong with a calculated half-life period is shown in table.18.
VIII. Expressi who was preservati in the strain E. W Li, Toothpick culture (10 ml) in 2YT medium with 50 μg/ml carbenicillin were incubated for 8 hours at 37°C and then transferred into 1 liter of modified AR-5, containing 50 μg/ml of carbenicillin, and incubated for 24 hours at 37°C. the Culture was centrifuged in 500 ml bottles at 7000 rpm for 15 minutes at 4°C. the Precipitate after centrifugation was frozen for at least 3 hours at 20°C. Every 500 ml of sediment after centrifugation suspended in 12.5 ml of a cold solution of 25% sucrose in 50 mM Tris buffer pH 8.0, containing 1 mM of benzamidine (Sigma) at 4°C. the Suspension was solubilizers under stirring at 4°C for 3 hours. The suspension was centrifuged at 18000 rpm for 15 minutes at 4°C and F(ab)sexpressed in the supernatant was purified using affinity chromatography on protein G (Pharmacia). The column was washed with a solution of 10 mM Tris (pH 7.6) and 1 mM EDTA (pH 8.0) and F(ab)s were suirable 100 mM acetic acid (pH 3.0), an amount equal to 2.5 column volumes, and immediately neutralized to a neutral pH of 0.5 volumes of 1M Tris pH 8.0. Eluate was concentrated and the buffer was replaced with PBS using method 30 microconcentrators (Amicon). The concentration of protein was determined by absorbance at 280 nm using a spectrophotometer (Beckman DU 64)946.gif" border="0">-mercaptoethanol.
IX. Results and conclusion
The results of the phage-ELISA competition experiments show that while a F(AB)-phage approximately 9 times improves the affinity compared to e, Raman options a, e and e 20-40 times superior compared to e-phage. Additional combinations of phage-derived mutations can give options antibodies with similarly improved appendectomy.
When F(ab) soluble proteins was investigated in the Biotin-IgE tablet analysis, a F(ab) and E27 F(ab) was improved about 10 times and 30 times, respectively, compared to E25, with regard to the inhibition of IgE binding FCeRI-IgG. Koffdetermination using BIAcore analysis confirms the relative affinity of. In particular, e and E27 showed 7.7 times and 22 times less Koffthan E25. Longer half-life means that IgE busy or assists variants unable to bind with high-affinity receptor for a longer period, thus resulting in increased therapeutic efficacy of anti-IgE.
Thus, the data of the equilibrium and kinetic binding supports the conclusion that a and E27 F(ab)2 bind IgE is about 10 times and 30 times stronger, respectively, than the E25. Predpolagaetsya affinity to E25 IgG.
1. Antibody anti-IgE that specifically bind to the IgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, including the sequence of the light chain and the sequence of the heavy chain, which exhibit at least 70% identity with the sequences of the light chain and heavy chainaFig.13 [SEQ ID NOS:19 and 20] and which include the remains 32Glu, 27Lys and go variable light chain CDR1.
2. The antibody under item 1, includingasequence selected from the following series: F(ab) fragment [SEQ ID NOS:19-20], sFv fragment [SEQ ID NO:22], F(ab)2fragment [SEQ ID NOS:24-25].
3. The antibody specifically binding to IgE and inhibiting or substantially reducing the binding to high-affinity receptor Fc
4. The antibody under item 3, includingE27sequence selected from the following series: F(ab) fragment [SEQ ID NOS:19 and 21], sFv fragment [SEQ ID NO:23], F(ab)2fragment [SEQ ID NOS:24 and 26].
5. The nucleic acid molecule encoding the antibody under item 1, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody comprises the sequence of the light chain and the sequence of the heavy chain, which exhibit at least 70% identity with the sequences of the light chain and heavy chainaFig.13 [SEQ ID NOS:19 and 20] and which include the remains 32Glu, 27Lys and go variable CDR1 light chain .
6. The nucleic acid molecule encoding the antibody under item 2, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody is an antibody fragment “26b> [SEQ ID NOS:24-25].
7. The nucleic acid molecule encoding the antibody under item 3, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody comprises the sequence of the light chain and the sequence of the heavy chain, which exhibit at least 70% identity with the sequences of the light chain and heavy chainE27Fig.13 [SEQ ID NOS:19 and 21] and which include the remains 32Glu, 27Lys and go variable CDR1 light chain.
8. The nucleic acid molecule encoding the antibody under item 4, specifically communicating with lgE and inhibiting or substantially reducing the binding to high-affinity receptor FcRI, the antibody is an antibody fragment “E27”, selected from the following list: F(ab') [SEQ ID NOS:19-21], sFv fragment [SEQ ID NO:23], F(ab)2[SEQ ID NOS:24 and 26].
9. The composition is enhanced antibody anti-IgE designed to reduce or prevent IgE-mediated production of histamine in a mammal, comprising a pharmaceutically acceptable filler or fillers in smeureanu of histamine in mammals, involving the introduction of a therapeutically effective amount of the antibody molecules according to any one of paragraphs.1-4.
11. A method of treating IgE-mediated disorders, comprising introducing a therapeutically effective amount of the antibody molecules according to any one of paragraphs.1-4.
12. A method of obtaining antibodies anti-IgE according to any one of paragraphs.1-4, not deaktivirovana when isomerization of aspartyl and has affinity to the molecule target, which is remotefilename the polypeptide or greater, comprising the following stages:
a) identification of residues of aspartyl, which are prone to isomerization,
b) substitution of alternative residues and screening the resulting mutants in relation to the affinity to the molecule target bythe affinity maturationusing the phage display technique, which further includes the following steps:
(i) substitution, a deletion (elimination) or insertion of one or more codons in the gene encoding the polypeptide, resulting in a change of amino acid sequence of the polypeptide thus obtained library of structurally related polypeptides, fused with protein ragovoy shell,
(ii) display (nd DNA encoding this polypeptide,
(C) the selection of modified polypeptides in which the remnant of aspartyl replaced and which have affinity to the molecule target, which is equal to remotefilename the polypeptide or higher.
13. The method according to p. 12, in which the unmodified antibody has a sequence denoted asE25in Fig.12 [SEQ ID NOS:13-14].
14. The method according to p. 13, in which the substituted residues are residues Asp32Glu, Gln27Lys and Ser28Pro variable light chain CDR1.
15. The method according to p. 14, which is optionally substituted residues are residues Thr53Lys, Asp55Ser, Ser57Glu and Asp59Lys variable heavy chain CDR2.
16. A way to improve the inhibitory properties of antibodies against histamine induced pollen Allergy, including the implementation of the method under item 12.