CN103159857B - Complexes based on recombined triple-helix scaffolds - Google Patents

Abstract

A protein complex is disclosed comprising a fusion polypeptide chain forming a triple helical coil, each chain having a scaffold region and a heterologous region. Also disclosed are related isolated fusion polypeptides, nucleic acids, vectors, host cells, and methods of making.

Description

Complexes based on recombined triple-helix scaffolds

This application is a divisional application of the Chinese invention patent application with application number 200710104159.8 (application date: May 21, 2007; invention name: complex based on recombined triple helical scaffold).

technical field

The present invention relates to protein complexes with scaffolds, and in particular to protein complexes with triple-helix scaffolds, which can be used for anti-tumor, infectious diseases and immunomodulatory use.

Background technique

Protein-based binding reagents have a variety of uses in therapeutic or diagnostic applications. Antibody is an excellent example, and many monoclonal antibodies (mAbs) (hereinafter referred to as mAbs) have been successfully used to treat cancer, infectious diseases and inflammatory diseases (inflammatory diseases) (Adams et al., Nat.Biotechnol.2005Sep ;23(9):1147-57.).

The efficacy and safety of murine mAbs has been enhanced through recombinant DNA technology, including chimerization and humanization. However, when murine mAbs are humanized using CDR (complementarity determining region, hereinafter referred to as CDR) transplantation, the binding affinity is usually lower than that of the murine counterpart. Antibody affinity is a key factor in the success of antibodies as therapeutic agents. Antibodies with high affinity allow antibodies to compete efficiently with natural ligands for targeted receptors to reduce dosage, toxicity, and cost. Affinity also affects antibody pharmacokinetics, such as distribution and secretion within the target tissue and host circulation. A prerequisite for a monoclonal antibody to be effective in vivo is a strong affinity for the target antigen and no non-specific binding to unrelated proteins. Multimerization of antigen-binding sites has been proven to be an effective way to increase the overall strength of antibody-antigen binding, which is defined as antibody avidity (functional affinity) affinity)) (Miller et al., J Immunol170:4854-4861,2003; Rheinnecker et al., J Immunol157:2989-2997,1996; Shopes, J Immunol148:2918-2922,1992; Shuford et al., Science252:724- 727, 1991; Wolff et al., J Immunol 148:2469-2474, 1992). They have enhanced antitumor activity in vivo (Liu et al., Int Immunopharmacol 6:791-799, 2006; Wolff et al., Cancer Res 53:2560-2565, 1993). Due to the bivalent nature of the molecular structure of immunoglobulin G (IgG) (hereinafter referred to as IgG), conventional and engineered IgG cannot be used to bind more than two different antigens simultaneously. Thus there is a need for multivalent or multi-specific protein binding reagents.

In some instances, in order to reduce mitogenicity side-effects, it is necessary to avoid effector functions, such as antibody-dependent cell-mediated cytotoxicity, by engineering the Fc region. , ADCC) and complement dependent cytotoxicity (CDC). For example, murine anti-human CD3 monoclonal antibodies (Orthoclone OKT3, Muromonab-CD3) act as T-cell receptor/CD3 complexes on human T cells A potent immunosuppressive agent for the target. It has been used to prevent or treat allograft rejection (allograft rejection) for two decades (Cosimi et al., N Engl J Med305:308-314,1981; Group, N Engl J Med313:337-342,1985 ; Kung et al., Science 206:347-349, 1979). However, a major disadvantage of such treatment is the systemic release of cytokines, such as TNF-α, IL-2 and IFN-γ, which lead to a series of deleterious mitogenic effects, including flu-like symptoms (flu-like symptoms), respiratory distress (respiratory distress), neurological symptoms (neurological symptoms) and acute tubular necrosis (acute tubular necrosis) (Abramowicz et al., Transplantation47:606-608,1989; Chatenoud et al., N Engl J Med320:1420 -1421, 1989; Goldman et al., Transplantation 50:158-159, 1990; Toussaint et al., Transplantation 48:524-526, 1989). Because the mitogenic effect of OKT3 and other anti-CD3 mAbs depends on binding to Fc receptor-bearing cells (FcR-positive cells) (such as monocytes) and thus extensive TCR/CD3 cross-linking (cross-linking), so recently Many studies have sought to develop nonmitogenic forms of anti-CD3 antibodies by altering the binding to FcR. From the above description, it can be seen that there is an urgent need for a protein-binding reagent with high affinity, low mitogenic effect and high stability in vivo.

Contents of the invention

The present invention provides protein complex-based binding reagents with high affinity, low mitotic effect and high stability in vivo. Agents of the invention may also be multi-valenyt or multi-specific.

In one aspect, the invention relates to an isolated recombinant protein complex comprising a first fusion polypeptide chain comprising a first scaffold region and a first heterologous ( heterologous) region, the second fusion polypeptide chain comprising the second scaffold region, and the third fusion polypeptide chain comprising the third scaffold region. The above-mentioned first, second and third scaffold regions are aligned with each other to form a triple helical coil. Moreover, the above-mentioned first scaffold region is fused in frame with the first heterologous region and is on the same polypeptide chain.

The aforementioned heterologous regions may comprise sequences of enzymatic domains or fluorescent proteins. Examples of fluorescent proteins include GFP and dsRed and variants thereof. Examples of enzymatic domains include glutathione S-transferase (glutathione S-transferase), luciferase (luciferase), β-galactosidase (β-galactosidase) and β-lactamase (β-lactamase). lactamase).

The aforementioned heterologous region may include a binding region (eg, a ligand binding region, a ligand, a receptor, an affinity tag, or a proteoglycan) that binds to a binding partner. "Binding partner" means any molecule that has a specific, covalent or non-covalent affinity for a portion (eg, a protein) of a targeting compound of interest. Examples of binding partners include antigen/antibody pairs, protein/inhibitor pairs, receptor/ligand pairs (e.g., cell surface or nuclear receptor/ligand pairs), enzyme/substrate pairs (e.g., , kinase/substrate pair), lectin/carbohydrate pair, oligomeric or heterooligomeric protein pair, DNA-binding protein/DNA Binding site counterparts and RNA/protein counterparts. Examples of binding regions also include the sequence of an affinity tag, eg, histidine-tag, myc tag or hemagglutin tag.

The above-mentioned first heterologous region may comprise one or more CDRs of an immunoglobulin. Thus the heterologous region may comprise the antigen-binding portion of an antibody, such as the _VH region and Fab. In an embodiment, the first heterologous region comprises the sequence of an antigen-binding fragment or a single-chain antibody, for example, cluster designation 3 (CD3) (hereinafter referred to as CD3) or epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) (hereinafter referred to as EGFR) specific sequence. The above-mentioned first polypeptide chain may further comprise a second heterologous region fused in-frame with the other end of the above-mentioned first scaffold region. As with the first scaffold region described above, the second heterologous region may also comprise a binding region that binds a binding partner. The first and second heterologous regions may be the same or different from each other. They can bind to the same binding partner or to two different binding partners. For example, the first heterologous region and the second heterologous region may respectively comprise the sequences of the first single-chain antibody and the second single-chain antibody, which specifically bind to CD3 and EGFR respectively.

In the above protein complex, the second fusion polypeptide chain may comprise a third heterologous region fused in frame with one end of the second scaffold region, a fourth heterologous region fused in frame to the other end of the second scaffold region, or Two regions fused in-frame to the two ends. Likewise, the aforementioned third fusion polypeptide chain may comprise a fifth heterologous region fused in frame to one end of the third scaffold region or a sixth heterologous region fused in frame to the other end of the third scaffold region or both . As with the first and second heterologous regions described above, each of the other four heterologous regions may comprise a binding region that binds a binding partner. All six heterologous regions may be identical or different from each other. They can thus bind 1, 2, 3, 4, 5 or 6 binding partners. In other words, the protein complex can be one, two, three, four, five or hexavalent.

In order for the first, second and third scaffold regions to form triple helical coils, each of the three scaffold regions comprises one or more triple helical repeats, each repeat comprising a sequence of the following formula: (X1-X2 -X3) n, wherein X1 is a glycine (Gly) residue, X2 or X3 is any amino acid residue, preferably the imino acid proline or hydroxproline; and n is greater than or equal to 5. For example, the first, second or third scaffold region may comprise (GPP) ₁₀ or one or more triple helical repeats of human complement C1q, collagen collectin or collagen polypeptide chains.

In an embodiment, the aforementioned first, second and third fusion polypeptides are substantially identical, having at least 75% (eg, any number between, including 75% and 100%) sequence identity to each other. The complex formed by three identical fusion polypeptides is a homotrimer. The above three fusion polypeptides may be "functional equivalents". "Functional equivalent" means polypeptide derivatives of common polypeptides, for example, proteins with one or more point mutations, insertions, deletions, truncations, fusion proteins thereof, or combinations thereof, And it substantially retains the ability to form a triple helix as well as the activity of the heterologous domain, such as binding to a ligand.

Heterologous polypeptides, nucleic acids or genes may be derived from different species, or even if they are from the same species, they have been substantially modified from their original form. Two fusion regions or sequences are heterologous to each other in that they are not joined to each other even in naturally occurring proteins or nucleic acids. For example, polypeptide sequences that are not part of naturally occurring human minicollagen (type XXI), collagen lectin family proteins, or complement 1q (Clq) are heterologous to regions of the human protein.

Another feature of the invention is an isolated recombinant fusion polypeptide comprising (i) a scaffold region for forming a triple helical coil and (ii) a first heterologous region fused in-frame to one end of said scaffold region or an in-frame fusion to the second heterologous region at the other end of the scaffold region. The scaffold region may comprise one or more of the aforementioned triple helical repeat sequences, such as repeat sequences of human C1q or collagen polypeptide chains. The heterologous region may comprise one of the aforementioned binding regions and may be obtained by a number of methods known in the art such as phage display screening.

Isolated polypeptide or protein complex means substantially free of naturally associated molecules, i.e., having a purity of at least 75% (i.e., any amount between 75% and 100%, including 75% and 100%) by dry weight. Polypeptide or protein complex. Purity can be measured by any suitable standard method, for example by column chromatography, polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC) analysis. An isolated polypeptide or protein complex of the invention can be isolated from natural sources or produced using recombinant DNA techniques.

The present invention also relates to isolated nucleic acids comprising a sequence encoding a fusion polypeptide as described above or the complement of such a sequence. Nucleic acid refers to a DNA molecule (eg, cDNA or genomic DNA), an RNA molecule (eg, mRNA), or an analog of DNA or RNA. Analogs of DNA or RNA can be synthesized from nucleotide analogs. This nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

An "isolated nucleic acid" is a nucleic acid that is not structurally identical to any naturally occurring nucleic acid or to any naturally occurring fragment of a genomic nucleic acid. The term thus encompasses, for example (a) DNA, which has a partial sequence of a naturally occurring genomic DNA molecule, but which is not contiguous with coding sequences flanking it naturally in the genome of an organism; (b) nucleic acid, which begins with Introduced into a vector or into the genome of a prokaryotic or eukaryotic cell in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) isolated molecules such as cDNA, genomic fragments , a fragment or a restriction fragment (restriction fragment) produced by polymerase chain reaction (polymerase chain reaction, PCR); and (d) a recombinant nucleotide sequence, which is a hybrid gene (ie, a gene encoding a fusion protein) a part of. The nucleic acids described above can be used to express the polypeptides of the present invention. For this purpose, the nucleic acids described above can be linked to suitable regulatory sequences to generate expression vectors.

A vector refers to a nucleic acid molecule that has the ability to transfer another nucleic acid to its junction. Vectors have the ability to autonomously replicate or integrate into host DNA. Examples of vectors include plasmid (plasmid), phagemid (cosmid) or viral vectors. A vector of the invention comprises a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. "Regulatory sequences" include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Regulatory sequences include those that direct the constitutive expression and tissue-specific regulation and/or inducible sequences of a nucleotide sequence. The design of the expression vector may depend on such factors as the choice of host cell to be transfected, the degree of protein expression desired, and the like. Expression vectors can be introduced into host cells to produce polypeptides of the invention. Host cells comprising the nucleic acids described above are also within the scope of the invention. Examples include E. coli cells, insect cells (e.g. using Drosophila S2 cells or baculovirus cells), yeast cells or mammalian cells (e.g. mouse myeloma NSO cells ). See, e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA.

In order to produce the fusion polypeptide of the present invention, host cells are cultured in a medium under certain conditions to express the polypeptide encoded by the nucleic acid of the present invention, and the polypeptide is purified from the cultured cells or the culture medium of the cells. Alternatively, nucleic acids of the invention can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

In order to produce the protein complex of the present invention, host cells comprising the first, second and third nucleic acids encoding the above-mentioned first, second and third fusion polypeptides can be cultured under certain conditions to express the above-mentioned three nucleic acid-encoded and form triple helical coils between the expressed polypeptides, and purify the protein complex from cultured cells or the culture medium of the cells. Preferably, the above-mentioned host cell is a eukaryotic cell comprising an enzymatic activity to hydroxylate proline residues.

In order to make the above-mentioned and other purposes, features, and advantages of the present invention more obvious and understandable, the preferred embodiments are specifically cited below, and in conjunction with the accompanying drawings, the detailed description is as follows:

Brief description of the drawings

Figure 1 shows the protein complex with a mini-collagen triple-helical coil scaffold derived from human type XXI collagen. The six ends of the triple helical coil are fused in-frame to six Fv fragment regions with the _VL and _VH regions of a single-chain antibody (scFv), respectively.

Figure 2A shows a protein complex with a triple-helical coiled scaffold whose three N-terminals are fused in frame with three single-chain antibodies: OKT3 (anti-CD3), 528 (anti-EGFR) and erb_scFv (anti-EGFR), While Figure 2B shows a photograph of a Western blot of the protein complex. The culture medium of the stably transfected Drosophila S2 cells was subjected to SDS-PAGE electrophoresis in a non-reducing state, and then immunoblotted with a monoclonal antibody against the C-terminus of type XXI collagen (3E2). T: an interbond disulfide triplet; Mt: a monomer comprising an interbond disulfide triplet.

Figure 3 shows a protein complex with a triple helical coiled scaffold. The three N-terminals of the above-mentioned triple helical coiled scaffold were fused in-frame with three OKT3 single-chain antibodies; the three C-terminals were fused in-frame with three 528 single-chain antibodies.

Figures 4A and 4B show schematic diagrams of different formats of antibodies: Figure 4A shows a collagen scaffold antibody: scFv-Col (left panel), which contains an amino-terminal scFv, human IgG hinge (hinge) region, collagen region (GPP) ₁₀ and XXI Collagen type carboxy-terminal NCl region; NSPD-scFv (right panel), which contains surfactant protein D (surfactant protein D, SPD), collagen lectin and scFv at the carboxy-terminus. Figure 4B shows, from left to right, immunoglobulin G (IgG), chimeric (scFv-Fc) and single chain antibody (scFv), respectively, and their respective approximate molecular weights. Gray areas show _VH and _VL segments; dashed lines: interchain disulfide bonds.

Explanation of main component symbols

101～Mini Collagen

401～hinge area

Collagen region from 403 to (GPP)10

405 ~ NC1 region of collagen type XXI or other targeted binding regions

407～N-terminal region of collagen lectin

409 ~ Collagen-like domain of collagen lectin

411 ~ α-helical neck region of collagen lectin

Implementation

The present invention is based, at least in part, on the unexpected discovery that a heterologous protein binding domain fused in frame to a human triple helical coil scaffold domain retains binding activity and that the resulting fusion polypeptide forms a triple helical coil. Figure 1 shows an example of the protein complex of the present invention. As shown in the figure, the six ends of the triple-helical coiled protein complex are fused in-frame with the binding regions of six heterologous proteins, ie, the Fv fragment of a single-chain antibody (scFv).

The protein complexes of the present invention have many advantages over conventional antibodies. On the one hand, when two or more of the above six regions are identical to each other, the protein complex can have 2-6 binding regions specific for a binding partner (such as an antigen), while commonly used antibodies only have two of these regions. seed area. In other words, unlike commonly used antibodies which are only divalent to the antigen, the protein complex can be di-, tri-, tetra-, penta- or hexa-valent. Therefore, it can be manufactured to have various higher affinities than commonly used antibodies. Because of the higher affinity, it requires less protein complexes and shorter reaction times than commonly used antibodies to achieve desired goals, such as therapeutic effects, thereby reducing treatment costs And reduce side effects (eg, undesired immune responses). In another aspect, when two or more of the above six domains are different from each other, the protein complex of the present invention may have 2-6 binding domains specific for 2-6 different binding partners. Combining binding partners with different specificities into one unit has the ability to bring together multiple binding partners, so it is used in therapy, tissue reconstruction and active protein machinery at the nanoscale (eg multi-subunit enzymes) have satisfactory applications.

For use in humans, the protein complexes of the invention are preferably of human origin. For example, it may comprise a humanized single chain antibody sequence fused in frame to a helical coil scaffold of human origin, such as human C1q, a collagen lectin family protein or a collagen polypeptide chain. Because both human C1q and collagen are very stable in blood, this protein complex is more stable than typical therapeutic murine antibodies.

Collagen, the most abundant protein found in mammals, is an extracellular matrix protein comprising a repeating triplet of the sequence glycine (Gly)-X1-X2, the presence of which allows three Collagen polypeptide chains (α-chains) fold into a triple helical conformation. In the sequence glycine-X1-X2 of the triplet, the amino acid at the X2 position is often proline. In order to stabilize the triple helix structure of collagen, proline 4-hydroxylation is often performed through post-translational modification of the collagen polypeptide chain ( hydroxylated). In the absence of proline hydroxylation, the requisite triple-helix conformation of collagen is thermally unstable below physiological temperatures (Berg and Prockop, Biochem Biophys Res Commun 52:115-120, 1973; Rosenbloom et al., Arch Biochem Biophys 158:478-484, 1973). Many collagen-like proteins with a collagenous domain appear in human serum and play a role in the innate immune system in protecting against infectious organisms. These include the complement protein C1q, the collagen lectin family protein - mannose binding lectin (MBL), surfactant proteins A and D (SP-A and SP-D). The common structural feature of these collagen-like proteins is a multimeric protein subunit (unit) composed of a triple helix assembly of collagen regions, and the trimeric molecules are stacked with each other or form disulfide bond cross-links between the chains. . Thus, the functional affinity of the binding domains of these "defense collagen" molecules is greatly increased by multimerization.

The protein complexes or polypeptides of the present invention can be obtained by recombinant techniques. A nucleic acid encoding a polypeptide of this complex can be introduced into a suitable host cell and the polypeptide encoded by the aforementioned nucleic acid expressed under conditions that allow expression of the polypeptide and the formation of a triple helical coil between the polypeptides. To promote the formation of triple-helical coiled scaffolds, prolyl4-hydroxylase (P4HA), a key enzyme in collagen biosynthesis, can be co-expressed in host cells.

A domain of a heterologous protein may comprise an antibody or fragment thereof (eg, an antigen-binding fragment thereof). "Antibody" as used herein means an immunoglobulin molecule or an immunologically active portion thereof, ie, an antigen binding portion. It refers to a protein comprising at least one, preferably two heavy (H) chain variable regions (V _H ), and at least one, preferably two light (light, L) chain variable regions (V _L ). The _VH and _VL regions can be further subdivided into hypervariable regions and more conservative interspersed regions. The former is called the "complementarity determining region" (CDR) region, and the latter is called the framework region. region). The extent of complementarity determining regions and framework regions has been well defined (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al. (1987 ) J. Mol. Biol. 196:901-917). Each _VH and _VL consists of three CDRs and four FRs, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Antibodies can also comprise constant regions of the heavy and light chains, thereby forming the immunoglobulin chains of the heavy and light chains, respectively. The heavy chain constant region comprises three domains CH1, CH2 and CH3. The light chain constant region comprises domain CL. The variable regions of the heavy and light chains contain binding regions that react with antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (eg, effector cells) and CIq, the first component of the classical complement system.

"Immunoglobulin" as used herein refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Known human immunoglobulin genes include the constant region genes of kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3 and IgG4), delta and mu and a myriad of immunoglobulin variable region genes. The "light chains" of full-length immunoglobulins (about 25 KDa or 214 amino acids) are encoded by variable region genes (about 110 amino acids) at the _NH terminus and kappa or lambda constant region genes at the COOH terminus. The "heavy chain" of a full-length immunoglobulin (about 50 KDa or 446 amino acids) is similarly composed of variable region genes (about 116 amino acids), and other above-mentioned constant region genes, such as gamma (encoding about 330 amino acids). coding.

An "antigen-binding fragment" of an antibody (or "antibody portion" or "fragment") refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (eg, an EGFR or CD3 polypeptide or fragment thereof). Antigen-binding fragments of antibodies include, but are not limited to: (i) Fab fragments, monovalent fragments consisting of VL, _VH , _CL and _CH1 domains; ₍ ii) F(ab') ₂ fragments, comprising two A bivalent fragment that connects two Fab fragments by a sulfur bond at its hinge region; (iii) a Fd fragment consisting of _VH and _CH1 domains; (iv) a single arm consisting of the VL _and Fv fragments consisting of _VH domains; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546) comprising _VH domains; (vi) isolated complementarity determining regions (CDRs) and ( vii) _VL or _VH domains. Furthermore, although the two structural domains V _L and V _H of the Fv fragment are encoded by their respective genes, they can be connected using a recombination method, and they can be prepared into a single-chain protein through an artificial linker, wherein V _L and V _{The H} regions pair to form a monovalent molecule (termed a single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242:423-426; and Hustone et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879 -5883). Such single chain antibodies are also included within the scope of "antigen-binding fragments" of antibodies. These antibody fragments can be obtained using techniques common in the art, and screened for use in the same manner as intact antibodies.

Suitable antibodies may be monoclonal antibodies. In other embodiments, antibodies can be produced recombinantly, eg, by phage display or combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (see, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO92/18619; Dower et al. 17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO92/15679; Breitling et al. International Publication WO93/01288; McCafferty et al. International Publication No. WO92/01047; Garrard et al. Publication No.WO92/09690; Ladner et al.International Publication No.WO90/02809; Fuchs et al.(1991) Bio/Technology9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas3:81-85; Huse et al. al.(1989)Science246:1275-1281; Griffths et al.(1993)EMBO J12:725-734;Hawkins et al.(1992)J Mol Biol226:889-896;Clackson et al.(1991)Nature352:624- 628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982).

In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse that has been genetically engineered to make antibodies derived from human immunoglobulin sequences) or a non-human antibody, such as a rodent (mouse or rat), goat, primate (eg monkey), cameloid. The non-human antibody is preferably a rodent (mouse or rat) antibody. Methods for producing rodent antibodies are well known in the art.

Instead of the mouse system, human monoclonal antibodies can be produced by using transgenic mice carrying human immunoglobulin genes. Splenocytes from these transgenic mice immunized with an antigen of interest were used to generate hybridomas that secrete human mAbs with specific affinity for epitopes of human proteins (see, e.g., Wood et al.International Application WO91/00906, Kucherlapati et al.PCT publication WO91/10741; Lonberg et al.International Application WO92/03918;Kay et al.International Application92/03917;Lonberg, N.et al.1994Nature368:856-859 ;Green,L.L.et al.1994Nature Genet.7:13-21;Morrison et al.1994Proc.Natl.Acad.Sci.USA81:6851-6855;Bruggeman et al.1993Year Immunol7:33-40;Tuaillon et al.1993PNAS90 :3720-3724; Bruggeman et al.1991Eur J Immunol21:1323-1326).

The variable regions of antibodies or portions thereof, such as the CDRs, can be produced in non-human organisms (eg, rats or mice). Chimeric, CDR-grafted and humanized antibodies can be used. Antibodies produced in non-human organisms (eg, rats or mice) and modified (eg, in variable frameworks or constant regions) to reduce antigenicity in humans are included in the invention.

Chimeric antibodies can be produced using recombinant DNA techniques known in the technical field of the present invention. For example, the Fc constant region gene encoding the mouse (or other species) monoclonal antibody molecule is digested with restriction enzymes, the region encoding the mouse Fc is removed, and then replaced with an equivalent portion of the gene encoding the human Fc constant region (see, Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO86/01533; Cabilly et al. U.S. Patent No.4,816,567; Cabilly et al., European Patent Application 125,023; Better et al.(1988Science240:1041-1043);Liu et al.(1987)PNAS834:44339;- Liu et al., 1987, J. Immunol.139:3521-3526; Sun et al. (1987) PNAS84: 214-218; Nishimura et al., 1987, Canc.Res.47:999-1005; Wood et al et al (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

In a humanized or CDR-grafted antibody at least one or two but usually all three recipient CDRs (of the heavy or light chain of the immunoglobulin) are replaced by a donor CDR. The antibody may have at least a portion of the non-human CDRs substituted or only some of the CDRs substituted with non-human CDRs. Simply replace as many CDRs as necessary to bind the humanized antibody or humanized antibody fragment. Preferred donors are rodent antibodies, such as rat or mouse antibodies, and the recipient is a human framework or human consensus framework. Generally, the immunoglobulin that provides the CDRs is called the "donor" and the immunoglobulin that provides the framework is called the "acceptor". In one embodiment, the donor immunoglobulin is non-human (eg, rat). Acceptor frameworks are naturally occurring (eg, human) or consensus frameworks, or sequences that are about 85% or greater, preferably 90%, 95%, 99% or greater, identical. "Consensus sequence" as used herein refers to the sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987 ). In a protein family, each position in the consensus sequence is occupied by the most frequently occurring amino acid of that family at that position. If two amino acids occur with the same frequency, either of them can be included in the consensus sequence In. "Consensus framework" refers to the framework regions in the consensus immunoglobulin sequences.

Antibodies can be humanized by methods known in the art. Humanized antibodies can be produced by substituting Fv variable region sequences not directly involved in antigen binding with equivalent sequences derived from human Fv variable regions. Common methods for producing humanized antibodies are provided by Morrison, S.L., 1985, Science 229:1202-1207, Oi et al., 1986, BioTechniques 4:214, Queen et al. US5,585,089, US5,693,761 and US5,693,762, The contents of which are incorporated herein by reference. Those methods include isolating, manipulating and expressing nucleic acid sequences encoding all or a portion of an immunoglobulin Fv variable region from at least one of the heavy or light chains. Sources of such nucleic acids are well known in the art and may be obtained, for example, from hybridomas which produce antibodies against the polypeptide of interest or fragments thereof. Recombinant DNA encoding a humanized antibody or fragment thereof can be cloned into an appropriate expression vector.

Humanized antibodies, in which specific amino acids have been substituted, deleted or added, can also be fused to the scaffold. Preferred humanized antibodies have amino acid substitutions in the framework regions, eg, to improve binding to the antigen. For example, a humanized antibody has framework residues that are identical to those of the donor or amino acids other than those of the recipient. To generate such antibodies, selected minor acceptor framework residues of the humanized immunoglobulin chains are substituted with corresponding donor amino acids. Preferred substitution positions include amino acid residues adjacent to or capable of interacting with a CDR. Criteria for selecting amino acids from donors are described in US 5,585,089, the contents of which are incorporated herein by reference. Additional techniques for humanizing antibodies are described in Padlan et al. EP519596A1.

The invention also includes nucleic acids encoding fusion polypeptides that form protein complexes of the invention. Nucleic acids can be screened from phage display libraries or isolated (eg RT-PCR) from cell lines expressing the above-mentioned suitable antibodies or antibody derivatives. A nucleic acid can be functionally linked to an expression vector. Cells transformed with nucleic acids or vectors can be used to produce fusion polypeptides or protein complexes of the invention. Useful cells from which to prepare antibodies include insect cells and mammalian cells (eg, CHO or lymphatic cells).

The protein complexes of the invention can be combined with therapeutic moieties such as cytotoxins, therapeutic agents or radioactive ions. Cytotoxin or cytotoxic agent includes any agent that is harmful to cells, examples include taxol, cytochalasin B, gramicidin D, ethidium bromide , emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine ( colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D (actinomycin D), 1-dehydrotestosterone (1-dehydrotestosterone), glucocorticoids (glucocorticoids), procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol ( propranolol), puromycin, maytansinoids, for example, maytansinol (see, US Patent No. 5,208,020), CC-1065 (see US Patent Nos. 5,475,092, 5,585,499, 5,846,545) and its analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, arabinosin cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC- 1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin (streptozotocin), mitomycin C (mitomycin C) and cis-dichlorodiamine platinum (II) (DDP) cisplatin (cisplatin)), anthracyclines (such as Erythromycin (daunorubicin (formerly called daunomycin)), antibiotics (eg, dactinomycin (formerly called actinomycin), bleomycin, glaucoma Anthramycin and anthramycin (AMC) and anti-mitotic agents (eg, vincristine), vinblastine, paclitaxel, and maytansine. Radioactive ions include, but are not limited to, iodine, yttrium, and praseodymium.

Conjugates can be utilized to modify known biological responses, and drug moieties should not be limited to commonly used chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheriatoxin; proteins such as tumor necrosis factor ( tumor necrosis factor), α-interferon ( -interferon), β-interferon -interferon), nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or biological response modifier, for example, Lymphokines (lymphokines), interleukin-1 (interleukin-1, "IL-1"), interleukin-2 (interleukin-2, "IL-2"), interleukin-6 (interleukin-6, "IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"'), granulocyte colony stimulating factor ("G-CSF"), or other growth factors .

The aforementioned proteins and conjugates can be used to treat a variety of disorders according to the specificity of their heterologous binding regions, including cancer, inflammatory disease, metabolic disease, and fibrosis disease. ) and cardiovascular disease. The invention thus features treating such disorders, eg, by administering to a subject in need thereof an effective amount of a protein complex of the invention. The patient being treated can be determined to have or be at risk of a condition characteristic of the disorder. This method can be done alone or in combination with other drugs or treatments.

Because of the multispecific nature of the protein complexes of the present invention, they can be used to link molecules or cells that do not normally bind to each other. This feature is particularly useful for cell-based therapies. In one embodiment, the heterologous region in the protein complex is capable of activating a cytotoxic cell (e.g., a cytotoxic T cell) by specifically binding to an effector antigen located on the cytotoxic cell. T cell)), while other heterologous regions specifically bind to targeted antigens located on pathogen cells or malignant cells to be destroyed. In this way, protein complexes can be used to treat disorders caused by pathogens or harmful cells.

Cytotoxic cells can be activated via binding of the protein complex of the present invention to the CD3 antigen as an effector antigen on the cytotoxic cells. Other lymphoid cell-associated effector antigens include human CD16 antigen, NKG2D antigen, NKp46 antigen, CD2 antigen, CD28 antigen, CD25 antigen, CD64 antigen and CD89 antigen. Binding to these effector antigens activates effector cells such as monocytes, neutrophilic granulocytes and dendritic cells. These activated cells then exert a cytotoxic or apoptotic effect on the targeted cells.

A target antigen is an antigen that is uniquely expressed on targeted cells associated with a disease condition, but which is not expressed or expressed to a low degree or is undetectable in healthy cells. Examples of such targeted antigens associated with malignant cells include EpCAM, CCR5, CD19, HER-2neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3 , MUC4, MUC5.sub.AC, MUC5.sub.B, MUC7, .beta.hCG, Lewis-Y, CD20, CD33, CD30, ganglioside CD3 (ganglioside GD3), 9-O-acetyl-GD3, GM2 , Globo H, Fucosyl GM1, Poly SA, GD2, Carbonic Anhydrase IX (MN/CA IX)), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen, Membrane-bound IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-α precursor, STEAP, mesothelin, A33 antigen, prostate germ cell antigen (Prostate Stem Cell Antigen (PSCA)), Ly-6; desmoglein 4 (desmoglein4), E-cadherin neoepitope (E-cadherin neoepitope), fetal acetylcholine receptor (Fetal Acetylcholine Receptor), CD25, CA19 -9 marker (marker), CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen (TAG-72)) , FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS and CD63.

"Treatment" is defined as the administration of a composition to a patient for the purpose of curing, alleviating, alleviating, treating, preventing or ameliorating a disorder, a symptom of a disorder, a disease state secondary to a disorder, or a predisposition to a disease. An "effective amount" is an amount of a composition, such as described above, that produces a medically satisfactory result in a treated subject.

In vivo, a therapeutically effective composition (eg, a composition comprising a protein complex of the invention) is administered to a subject. Generally speaking, the complex is suspended in a pharmaceutically acceptable carrier (carrier) (such as physiological saline), and it is given orally, intravenously (intravenous) infusion, or subcutaneous (subcutaneous), intramuscular (intramuscular) ), intrathecal, intraperitoneal, intrarectal, intravaginal, intranasal, intragastric, intratracheal, or intrapulmonary ) administered by injection or implantation.

The required dosage will depend on the chosen route of administration; the nature of the formulation; the nature of the subject's disease; the size, weight, surface area, age and sex of the subject; other drugs administered and the decision of the attending physician. A suitable dosage range is 0.01-100.0 mg/kg. Variation in dosage requirements is expected based on the variety of compositions available and the varying efficacy of various routes of administration. For example, oral administration is expected to require higher doses than intravenous administration. Variations in these dosage levels can be adjusted to optimum levels using standard empirical routines, as is well known in the art of the invention. Efficiency of delivery, especially for oral delivery, can be enhanced by encapsulating the composition in suitable vehicles, such as polymeric microparticles or implantable devices, which are commonly used.

Also within the scope of the present invention are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a protein complex of the present invention. This pharmaceutical composition can be used to treat the disorders mentioned above. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents. The composition can be formulated into dosage forms for different modes of administration by conventional methods.

The efficacy of compositions of the invention can be assessed in vivo and in vitro. For in vivo studies, the compositions can be injected into animals (eg, mouse models), and their therapeutic efficacy recorded thereafter. From this result, an appropriate dosage range and mode of administration can be determined.

The following examples are for illustrative purposes only, and are by no means intended to limit other disclosures of the present invention in any way. Without further details, it is believed that one skilled in the art can, based on the disclosure herein, practice the present invention to its fullest extent. All publications cited are hereby incorporated by reference in their entirety.

Example 1

An M13 phage display library was screened to identify human single-chain variable fragments (scFv) that specifically bind to EGFR. Some clones were identified. After confirmation by Western blot and enzyme-linked immunoassay (Enzyme-linked immunoassay, ELISA) (hereinafter referred to as ELISA), a clone erb_scFv was selected for further experiments. The cDNA encoding erb_scFv was obtained and ligated into an expression vector by standard methods. The polypeptide sequence of erb_scFv is sequence identification number: 1 (SEQ ID NO: 1), and the nucleotide sequence encoding the sequence is sequence identification number: 2 (SEQ ID NO: 2).

SEQ ID NO: 1

MetAlaGluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGlySerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyrAlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpValSerAspIleGlyAlaSerGlySerAlaThrSerTyrAlaAspSerValLysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaLysSerThrThrThrPheAspTyrTrpGlyGlnGlyThrLeuValThrValSerSerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerThrAspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThrIleThrCysArgAlaSerGlnSerIleSerSerTyrLeuAsnTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAspAlaSerAlaLeuGlnSerGlyValProSerArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAspPheAlaThrTyrTyrCysGlnGlnTyrAlaAspTyrProThrThrPheGlyGlnGlyThrLysValGluIleLysArg

SEQ ID NO: 2

ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGATATTGGTGCTTCTGGTTCTGCTACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTACTACTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGCTGATTATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG

The expression vector was then expressed in the insect cell line Drosophila S2. The anti-EGFR erb_scFv was purified and subjected to Western blot analysis and ELISA to confirm its anti-EGFR specificity.

RT-PCR was performed to obtain cDNA encoding the variable region of the heavy chain ( _VH ) and the variable region of the light chain ( _VL ) of the anti-CD3 monoclonal antibody OKT3 from a hybridoma cell line. Afterwards, the two cDNAs were ligated to generate a fusion sequence encoding the _VH - _VL fusion protein of OKT3. The sequence of the fusion protein is sequence identification number: 3 (SEQ ID NO: 3), and the cDNA sequence encoding the sequence is sequence identification number: 4 (SEQ ID NO: 4).

SEQ ID NO: 3

ValGlnLeuGlnGlnSerGlyAlaGluLeuAlaArgProGlyAlaSerValLysMetSerCysLysAlaSerGlyTyrThrPheThrArgTyrThrMetHisTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIleGlyTyrIleAsnProSerArgGlyTyrThrAsnTyrAsnGlnLysPheLysAspLysAlaThrLeuThrThrAspLysSerSerSerThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCysAlaArgTyrTyrAspAspHisTyrCysLeuAspTyrTrpGlyGlnGlyThrThrValThrValSerSerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAspIleValLeuThrGlnSerProAlaIleMetSerAlaSerProGlyGluLysValThrMetThrCysSerAlaSerSerSerValSerTyrMetAsnTrpTyrGlnGlnLysSerGlyThrSerProLysArgTrpIleTyrAspThrSerLysLeuAlaSerGlyValProAlaHisPheArgGlySerGlySerGlyThrSerTyrSerLeuThrIleSerGlyMetGluAlaGluAspAlaAlaThrTyrTyrCysGlnGlnTrpSerSerAsnProPheThrPheGlySerGlyThrLysLeuGluLeuLysArg

SEQ ID NO: 4

GTCCAGCTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGTGCTAACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCACTTCAGGGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCGGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCATTCACGTTCGGCTCGGGGACCAAGCTGGAGCTGAAACGA

The same procedure was followed to obtain the cDNA encoding the _VH and _VL of anti-EGFR monoclonal antibody 528, and the fusion sequence encoding the _VH - _VL fusion protein of anti-EGFR antibody 528. Monoclonal antibody 528 binds to EGFR on the cell membrane, for example, on human epidermoid carcinoma A431 cells. The polypeptide sequence of the 528 single-chain antibody is sequence identification number: 5 (SEQ ID NO: 5), and the cDNA sequence encoding the sequence is sequence identification number: 6 (SEQ ID NO: 6).

SEQ ID NO: 5

ValLysLeuGlnGluSerGlySerGluMetAlaArgProGlyAlaSerValLysLeuProCysLysAlaSerGlyAspThrPheThrSerTyrTrpMetHisTrpValLysGlnArgHisGlyHisGlyProGluTrpIleGlyAsnIleTyrProGlySerGlyGlyThrAsnTyrAlaGluLysPheLysAsnLysValThrLeuThrValAspArgSerSerArgThrValTyrMetHisLeuSerArgLeuThrSerGluAspPheAlaValTyrTyrCysThrArgSerGlyGlyProTyrPhePheAspTyrTrpGlyGlnGlyThrThrValThrValSerSerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerMetThrGlnThrProLeuSerLeuProValSerLeuGlyAspGlnAlaSerIleSerCysArgSerSerGlnAsnIleValHisAsnAsnGlyIleThrTyrLeuGluTrpTyrLeuGlnArgProGlyGlnSerProLysLeuLeuIleTyrLysValSerAspArgPheSerGlyValProAspArgPheSerGlySerGlySerGlyThrAspPheThrLeuLysIleSerArgValGluAlaGluAspLeuGlyIleTyrTyrCysPheGlnGlySerHisHisProProThrPheGlyGlyGlyThrLysLeuGlu

SEQ ID NO: 6

GTCAAGCTGCAGGAGTCAGGGTCTGAGATGGCGAGGCCTGGAGCTTCAGTGAAGCTGCCCTGCAAGGCTTCTGGCGACACATTCACCAGTTACTGGATGCACTGGGTGAAGCAGAGGCATGGACATGGCCCTGAGTGGATCGGAAATATTTATCCAGGTAGTGGTGGTACTAACTACGCTGAGAAGTTCAAGAACAAGGTCACTCTGACTGTAGACAGGTCCTCCCGCACAGTCTACATGCACCTCAGCAGGCTGACATCTGAGGACTTTGCGGTCTATTATTGTACAAGATCGGGGGGTCCCTACTTCTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAATAATGGAATCACCTATTTAGAATGGTACCTGCAAAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCGACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTAGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATCATCCTCCCACGTTCGGCGGGGGGACCAAGCTGGAA

The cDNAs encoding the above-mentioned anti-EGFR scFv03, OKT3V _H -V _L and anti-EGFR antibody 528V _H -V _L were fused in-frame with human mini-collagen XXI cDNA, which contained a short hinge sequence at the 5' end and was separated at 3 ' end contains a histidine tag sequence. The polypeptide of human mini-collagen XXI and its cDNA sequence are sequence identification number: 7 (SEQ ID NO: 7) and sequence identification number: 8 (SEQ ID NO: 8).

SEQ ID NO: 7

GlyGlyArgGluProLysSerCysAspLysThrHisThrCysProProCysProArgSerIleProGlyProProGlyProIleGlyProGluGlyProArgGlyLeuProGlyLeuProGlyArgAspGlyValProGlyLeuValGlyValProGlyArgProGlyValArgGlyLeuLysGlyLeuProGlyArgAsnGlyGluLysGlySerGlnGlyPheGlyTyrProGlyGluGlnGlyProProGlyProProGlyProGluGlyProProGlyIleSerLysGluGlyProProGlyAspProGlyLeuProGlyLysAspGlyAspHisGlyLysProGlyIleGlnGlyGlnProGlyProProGlyIleCysAspProSerLeuCysPheSerValIleAlaArgArgAspProPheArgLysGlyProAsnTyrSerLeuAspAspSerSerHisHisHisHisHisHisSerSerGly

(Note: Pro=proline or hydroxyproline residue)

SEQ ID NO: 8

GGCGGCCGCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAAGATCTATTCCTGGGCCACCTGGTCCGATAGGCCCAGAGGGTCCCAGAGGATTACCTGGTTTGCCAGGAAGAGATGGTGTTCCTGGATTAGTGGGTGTCCCTGGACGTCCAGGTGTCAGAGGATTAAAAGGCCTACCAGGAAGAAATGGGGAAAAAGGGAGCCAAGGGTTTGGGTATCCTGGAGAACAAGGTCCTCCTGGTCCCCCAGGTCCAGAGGGCCCTCCTGGAATAAGCAAAGAAGGTCCTCCAGGAGACCCAGGTCTCCCTGGCAAAGATGGAGACCATGGAAAACCTGGAATCCAAGGGCAACCAGGCCCCCCAGGCATCTGCGACCCATCACTATGTTTTAGTGTAATTGCCAGAAGAGATCCGTTCAGAAAAGGACCAAACTATAGTCTAGACGACAGCAGCCATCATCACCATCACCATAGCAGCGGC

The resulting three expression vectors were co-transfected into Drosophila S2 cells. These cells were grown in the presence of blasticidin to select cells resistant to blasticidin. Cell culture supernatants were collected and antibodies with anti-EGFR and CD3 activity were screened by Western blotting and ELISA. Some cloned cells were found to stably express the triple helix complex. These triple helix complexes, such as human mini-collagen XXI, are resistant to heat and pepsin. More importantly, they specifically bind to EGFR and CD3.

Example 2

In this example, three fusion polypeptides were generated: OKT3_scFv-Col, erb_scFv-Col and erb_NSPD-scFv.

Screening of phage library

The erb phagemid (phagemid) was isolated by screening a human single fold scFv phage display library (Tomlinson I+J; kindly provided by I.M. Tomlinson and G. Winter, MRC Laboratory of Molecular Biology, Cambridge, UK) The erb phagemid contains a variable fragment (scFv) that binds to the epidermal growth factor receptor extracellular domain (EGFR-ECD). Screening was performed using immunotubes (Maxisorp; Nunc, Roskilde, Denmark) coated with 10 μg of the extracellular region of purified recombinant EGF receptors (EGFR-ECD; Research Diagnostics, Inc.) ( coated). Blocking, panning, washing, elution and reamplification of eluted phagemids were performed according to the manufacturer's instructions.

Construction of recombinant plastids

The cDNA encoding the scFv of erb was amplified by PCR from the erb phagemid. The coding sequence of murine IgG2a anti-CD3 mAb (Ortho Pharmaceutical Corporation) was obtained by reverse transcription product from OKT3 hybridoma (ATCC, CRL-8001). The cDNAs of _VL and _VH of OKT3 mAb were obtained by RT-PCR according to the published nucleotide sequence. A scFv PCR fusion of erb and OKT3 was generated by linking the _VH and _VL chains with a glycine-linker (GGGS) ₃ .

In order to generate scFv-Col, the coding region of scFv-Col comprises the scFv nucleotide sequence at the N-terminal (N-terminal) and comprises a synthetic collagen scaffold gene at the C-terminal (C-terminal), which encodes EPKSCDKTHTCPPCPRSIP( GPP) ₁₀ Peptide sequence of GICDPSLCFSVIARRDPFRKGPNY comprising the hinge region of human IgG, the collagenous domain (underlined) and the NCl region of type XXI collagen. The sequences of the synthesized collagen scaffold polypeptide and its cDNA are sequence identification number: 9 (SEQ ID NO: 9) and sequence identification number: 10 (SEQ ID NO: 10).

SEQ ID NO: 9

GluProLysSerCysAspLysThrHisThrCysProProCysProArgSerIlePro GlyProGlyProProProGlyProProProGlyProProGlyProProGlyProProProGlyProP roGlyProProGlyProProProGlyProGlyIleCysAspProSerLeuCysPheSerValIleAlaArgArgAspProPheArgLysGlyProAsnTyr

SEQ ID NO: 10

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAAGATCTATTCCTGGGCCACCTGGTCCCCCAGGTCCTCCAGGACCCCCAGGGCCCCCAGGCCCCCCCGGGCCGCCTGGACCCCCAGGGCCACCAGGCCCCCCAGGCATCTGCGACCCATCACTATGTTTTAGTGTAATTGCCAGAAGAGATCCGTTCAGAAAAGGACCAAACTAT

This synthetic sequence (SEQ ID NO: 10) was prepared by overlapping PCR and cloning of the PCR product flanked by NotI and XhoI sites into the same position in the expression vector pSecTag2/Hygro (Invitrogen). The scFv of erb and OKT3 were cloned in-frame at the AscI and NotI sites into the construct (construct) including the above-mentioned C-terminal collagen scaffold to prepare the expression constructs (expression construct) of erb_scFv-Col and OKT3_scFv-Col respectively.

The erb_NSPD-scFv was then generated. The coding region of NSPD-scFv comprises the N-terminal 254 amino acids of human surfactant protein D (SPD), a member of the collagen lectin family, and the scFv at the C-terminus. The N-terminal 254 amino acids of human surfactant protein D polypeptide and its cDNA sequence are sequence identification number: 11 (SEQ ID NO: 11) and sequence identification number: 12 (SEQ ID NO: 12)

SEQ ID NO: 11

MetLeuLeuPheLeuLeuSerAlaLeuValLeuLeuThrGlnProLeuGlyTyrLeuGluAlaGluMetLysThrTyrSerHisArgThrMetProSerAlaCysThrLeuValMetCysSerSerValGluSerGlyLeuProGlyArgAspGlyArgAspGlyArgGluGlyProArgGlyGluLysGlyAspProGlyLeuProGlyAlaAlaGlyGlnAlaGlyMetProGlyGlnAlaGlyProValGlyProLysGlyAspAsnGlySerValGlyGluProGlyProLysGlyAspThrGlyProSerGlyProProGlyProProGlyValProGlyProAlaGlyArgGluGlyProLeuGlyLysGlnGlyAsnIleGlyProGlnGlyLysProGlyProLysGlyGluAlaGlyProLysGlyGluValGlyAlaProGlyMetGlnGlySerAlaGlyAlaArgGlyLeuAlaGlyProLysGlyGluArgGlyValProGlyGluArgGlyValProGlyAsnThrGlyAlaAlaGlySerAlaGlyAlaMetGlyProGlnGlySerProGlyAlaArgGlyProProGlyLeuLysGlyAspLysGlyIleProGlyAspLysGlyAlaLysGlyGluSerGlyLeuProAspValAlaSerLeuArgGlnGlnValGluAlaLeuGlnGlyGlnValGlnHisLeuGlnAlaAlaPheSerGlnTyrLysLysValGluLeuPhe

SEQ ID NO: 12

ATGCTGCTCTTCCTCCTCTCTGCACTGGTCCTGCTCACACAGCCCCTGGGCTACCTGGAAGCAGAAATGAAGACCTACTCCCACAGAACAATGCCCAGTGCTTGCACCCTGGTCATGTGTAGCTCAGTGGAGAGTGGCCTGCCTGGTCGCGATGGACGGGATGGGAGAGAGGGCCCTCGGGGCGAGAAGGGGGACCCAGGTTTGCCAGGAGCTGCAGGGCAAGCAGGGATGCCTGGACAAGCTGGCCCAGTTGGGCCCAAAGGGGACAATGGCTCTGTTGGAGAACCTGGACCAAAGGGAGACACTGGGCCAAGTGGACCTCCAGGACCTCCCGGTGTGCCTGGTCCAGCTGGAAGAGAAGGTCCCCTGGGGAAGCAGGGGAACATAGGACCTCAGGGCAAGCCAGGCCCAAAAGGAGAAGCTGGGCCCAAAGGAGAAGTAGGTGCCCCAGGCATGCAGGGCTCGGCAGGGGCAAGAGGCCTCGCAGGCCCTAAGGGAGAGCGAGGTGTCCCTGGTGAGCGTGGAGTCCCTGGAAACACAGGGGCAGCAGGGTCTGCTGGAGCCATGGGTCCCCAGGGAAGTCCAGGTGCCAGGGGACCCCCGGGATTGAAGGGGGACAAAGGCATTCCTGGAGACAAAGGAGCAAAGGGAGAAAGTGGGCTTCCAGATGTTGCTTCTCTGAGGCAGCAGGTTGAGGCCTTACAGGGACAAGTACAGCACCTCCAGGCTGCTTTCTCTCAGTATAAGAAAGTTGAGCTCTTC

The N-terminal SPD cDNA was cloned into the expression vector pSecTag2/Hygro (Invitrogen) between the NheI and AscI sites. The scFv of erb was cloned in frame into the AscI and XhoI sites of the construct containing the above N-terminal SPD to make an expression construct of erb_NSPD-scFv.

The open reading frame (open reading frame) of each erb_scFv-Col, erb_NSPD-scFv and OKT3_scFv-Col contains the coding N-terminal leader sequence (leader sequence) and the C-terminal myc epitope/multiple for the purpose of secretion, detection and purification Sequence of polyhistidine tag. Table 1 shows various recombinant proteins/antibodies encoded by the above expression constructs.

Table 1. Summary of various antibody molecules used in this study

¹ collagen scaffold antibody

Antibody expression and purification

To generate recombinant protein complexes/antibodies, the aforementioned constructs were transfected into mouse myeloma NSO cells using Effectene (Qiagen) according to the manufacturer's instructions. After 4 weeks of selection with hygromycin (400 μg/ml), each stable clone was seeded at an initial density of 2×10 ⁵ cells/ml in shaker flasks containing 2% fetal Bovine serum was cultured in a chemically-defined medium HyQCDM4NSO (Hyclone). The culture was maintained at 37° C. at 150 rpm for 5 days. Sodium ascorbate (Sodium ascorbate) (80 μg/ml) was added to the culture medium every day to those cells carrying the expression construct encoding the protein comprising the aforementioned antibody region and the collagen scaffold region, i.e. collagen scaffold antibody ( CSA).

To purify erb_scFv, erb_scFv-Fc, erb_scFv-Col, or OKT3_scFv-Col protein or protein complexes, about 2 L of each filtered cell culture medium was loaded at a flow rate of 60 ml/hour to 50 mM Tris-HCl buffer solution (pH 8 ) equilibrated T-gel column (1.5 x 8 cm, Pierce). After washing with the same buffer solution, the recombinant protein or protein complex was eluted with 50 mM sodium acetate buffer solution (pH 4). Monitor its UV absorption at 280nm, and its eluted peak part is loaded onto the Sepharose HighTrap column (1-ml column bed volume, GE Healthcare) of zinc sulfate-charged (charged) chelation with the flow velocity of 60ml/ hour, this The column was equilibrated with 50 mM Tris-HCl buffer solution (pH 8) containing 0.5 M NaCl. After washing with 20 mM imidazole (imidazole), the bound protein or protein complex was eluted with 0.25 M imidazole in the same buffer solution. The final preparation was dialyzed against 50 mM Hepes buffer, pH 7.0.

SDS-PAGE was then performed using 10% NuPAGE bis-Tris polyacrylamide gels with MOPS or 7% SDS/Tris-acetate polyacrylamide gels with sodium acetate as running buffer (Invitrogen). Proteins were then stained with Coomassie brilliant blue R-250. Density of protein bands was quantified by densitometry using ChemiImager 5500 (Alpha Innotech, San Leandro, CA) and Alpha EaseFC (v.4.0; Alpha Innotech) software.

To verify the nature of the triple helix, purified erb_scFv-Col (1 mg/ml) was incubated in the absence or presence of DTT for 1 hour at 37°C. An aliquot from the DTT-treated sample was further reacted with 50 mM N-ethyl-maleimide (NEM) for 30 minutes at room temperature to permanently prevent free thiols from (sulfhydryl) and trimer reformation. An equal amount of protein from each sample was electrophoresed in 7% SDS/Tris-acetic acid polyacrylamide gel with sodium acetate as the electrophoresis buffer solution. The gel was stained with Coomassie blue. Purified CSA was found to be a homotrimer or an interchain disulfide bonded hexamer (heximer), which could be separated into two trimers under a slightly reducing environment.

The thermal stability of the trimeric structure of erb_scFv-Col was tested. In 50 mM Tris-HCl (pH 8) containing 2M urea (urea), the purified erb_scFv-Col was incubated at room temperature in the absence or presence of 10 mM tris (2-carboxyethyl) phosphine (tris (2-carboxyethyl) phosphine , TCEP) will be processed. The reduced samples were alkylated with 50 mM NEM at room temperature. Each sample of equal amount of protein was heated at 35, 45, 55, 65, 75 and 85°C for 10 minutes before mixing with SDS gel loading buffer. The samples were electrophoresed in 10% NuPAGE bis-Tris polyacrylamide gel and in MOPS buffer solution under non-reducing state. Gels were stained with Coomassie blue. The results show that erb_scFv-Col trimer has high thermal stability. More than 50% of trimers remained after 10 minutes of treatment at 65°C. And found that the trimer structure of erb_scFv-Col collagen domain was prolyl hydroxylated (prolyl hydroxylate).

combined research

Use BIAcore X biosensor (BIACORE, Inc., Uppsala, Sweden) to measure the change of erb antibody in running buffer (running buffer) HBS-EP (10mM HEPES, pH7.4, 150mM NaCl, 3mMEDTA, 0.005% surfactant P20). Binding kinetics of EGFR-ECD in vivo. Briefly, EGFR-ECD was immobilized on a C1 sensor chip via amine coupling (amine coupling) to reach 1700 response units (response units, RU), and different concentrations of purified antibodies were injected at a flow rate of 10 μl/min. The surface was regenerated by injecting 5 μl of 10 mM glycine-HCl (pH 3.5). A sensorgram was taken at each concentration and the sensorgram was eluted using the program BIA Evaluation 3.2. Binding data were fed into a 1:1 Langmuir binding model to calculate the affinity constant K _D , which is defined as the ratio of dissociation rate (k _diss )/association rate ( _kass ). The results are shown in Table 2.

Table 2. Binding kinetics of various forms of erb antibody binding to immobilized EGFR-ECD

As shown in Table 2, the binding affinity of erb_scFv_Col for EGFR-ECD was almost 20 and 1000 times higher than that of the bivalent (erb_scFv-Fc) and monovalent (erb_scFv) mAb counterparts, respectively.

Stability and Pharmacokinetic Analysis

For analysis of serum stability, the stability of various forms of the erb antibody of erb_scFv_Col, erb_scFv-Fc or erb_scFv was measured by incubation at 37°C with human serum. The amount of activated anti-EGFR remaining after different periods of incubation time was measured by quantitative ELISA. Use recombinant EGFR-ECD (as capture reagent (capture reagent)) and anti-c-myc mAb (9E10, Sigma Chemical Co.), and then use HRP-coupled affinity purified polyclonal goat anti-mouse IgG and Chemiluminescent substrate (Pierce Biotechnology, Inc.) was used for ELISA. For pharmacokinetic analysis, three BALB/c nude mice were used to analyze erb_scFv_Col clearance. Briefly, 25 μg (2 mg/body weight Kg) of erb_scFv_Col was injected subcutaneously (s.c.) into each mouse after prior blood collection. During the next 70 hours, periodic blood samples were collected and their erb_scFv_Col content was assessed by ELISA. The protein was found to be quite stable.

T cell proliferation (proliferation) analysis and mixed lymphocyte reaction (Mixed LymphocyteReaction)

5-bromo-2-deoxyuridine (5-bromo-2'-deoxyuridine, BrdU) cell proliferation assay was performed. Briefly, human peripheral blood mononuclear cells (peripheral blood mononuclear cells) were cultured at 2×10 ⁵ cells/well in 100 μl RPMI- Cultured in 1640 medium for 66 hours at 37°C in the presence of 10-fold serial dilutions of OKT3 (eBioscience, Inc.) or OKT3_scFv-Col. Cells were then pulsed with 10 μM BrdU for 6 hours. After removal of medium, cells were fixed and DNA was denatured in one step with FixDenat. Cells were then incubated with peroxidase-labeled anti-BrdU antibody (anti-BrdU POD, Fab fragment) for 1.5 hours at room temperature. Chemiluminescence detection and quantification were performed using a microplate-luminometer (Hidex, CHAMELEON detection platform, Finland).

T cell proliferation and immunosuppression were assessed with a one-way mixed lymphocyte reaction as follows. Human peripheral blood mononuclear cells were obtained from two healthy donors (stimulator and responder). In a humidified atmosphere containing 5% _CO2 at 37°C, 25 μg/ml mitomycin C (Sigma-Aldrich) was used in complete medium (RPMI1640, supplemented with 10% human AB serum (human AB serum), 2 mM gluten Aminoamide (glutamine), 50nM 2-mercaptoethanol (2-mercaptoethanol), and penicillin (penicillin) and streptomycin (streptomycin) each 100 units/ml) were treated with stimulator or responder cells for 30 minutes, and then cultured in RPMI1640 Wash three times in the base. Responder cells were cultured alone or mixed with mitomycin C-treated stimulators or mitomycin C-treated responder cells at a ratio of 1:1 in 200 μl of complete medium at 2 × 10 ⁵ cells/well were cultured. Immediately after placing into responder cells, purified OKT3_scFv-Col or OKT3 was added to the culture medium at different concentrations. Cultured cells were pulsed with 10 μM BrdU after 5 days and harvested after 24 hours. Cell proliferation assays were then performed as described above.

As a result, it was found that OKT3_scFv-Col was more effective for the proliferation of immunosuppressive T cells, although showing negligible mitogenic activity in stimulating T cell proliferation.

Cytokine measurement

Put human peripheral blood mononuclear cells at 2×10 ⁵ cells/well in 0.1 ml RPMI-1640 medium containing 10% FBS, in the presence of 10-fold diluted OKT3 (eBioscience, Inc.) or OKT3_scFv-Col at 37°C down to cultivate. Supernatants were collected at different time points and various cytokines were measured using Human Cytokine Immunoassay Kit (eBioscience, Inc.). The results showed that administration of OKT3_scFv-Col resulted in negligible cytokine release compared to murine OKT3 mAb.

Antibody substitution assay (replacement assay)

All following methods were performed at 4°C. Human T cells were suspended in FCM buffer solution (phosphate-buffered saline with 2% FBS and 0.1% sodium azide) at a density of 1×10 ⁶ cells/ml. Cells were treated with mouse total IgG (2 μg/ml, Jackson ImmunoResearch Laboratories) for 30 minutes and then incubated with serially diluted OKT3_scFv-Col or OKT3 antibody for 1 hour. A fixed saturating amount (determined by cytometry) of FITC-conjugated OKT3 (0.25 μg/ml, purchased from eBioscience, Inc.) was added directly. After incubation for 1 hour, cells were washed with FCM buffer solution and subjected to immunofluorescence analysis by flow cytometry on a FACScan (Becton Dickinson, San Jose, CA). Results are shown as percent inhibition of maximal fluorescence intensity, defined as the mean fluorescence intensity obtained by staining T cells with OKT3-FITC in the absence of blocking antibodies.

The results indicated that OKT3_scFv-Col binds to human CD3 ⁺ T cells more strongly than native murine OKT mAb.

Protein concentration was measured by Bradford assay (Coomassie plus reagent from Pierce Biotechnology, Inc.) using human IgG as a standard. For amino acid analysis, the purified erb_scFv-Col was dialyzed against 50 mM acetic acid, hydrolyzed in 6N HCl at Amino acid analysis is performed in-system.

These results demonstrate that collagen scaffold antibodies are ideal structures for the design of therapeutic antibodies in antitumor and immunomodulatory applications.

All features disclosed in this specification can be combined in any way. Each feature disclosed in this specification may be replaced by another feature serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed is one example only of a series of equivalent or similar features.

Although the present invention discloses the above preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope defined in the claims.

The application also provides the following technical solutions:

1. A recombinant protein complex comprising:

A first fusion polypeptide chain comprising a first scaffold region and a first heterologous region fused to one end of the first scaffold region;

a second fusion polypeptide chain comprising a second scaffold region; and

a third fusion polypeptide chain comprising a third scaffold region;

Wherein the first, second and third scaffold regions are arranged to form a triple helical coil.

2. The recombinant protein complex according to item 1, wherein the heterologous region comprises a binding region.

3. The recombinant protein complex according to item 2, wherein the binding domain comprises a ligand binding domain, a ligand, a receptor, an affinity tag or a proteoglycan.

4. The recombinant protein complex according to item 2, wherein the binding region comprises one or more complementarity determining regions of an immunoglobulin.

5. The recombinant protein complex according to item 4, wherein the binding region comprises the sequence of an antigen-binding fragment.

6. The recombinant protein complex according to item 5, wherein the antigen-binding fragment specifically binds to CD3 or EGFR.

7. The recombinant protein complex according to item 5, wherein the antigen-binding fragment comprises a sequence of a single-chain antibody.

8. The recombinant protein complex according to item 1, wherein the first fusion polypeptide chain further comprises a second heterologous region fused to the other end of the first scaffold region.

9. The recombinant protein complex according to item 8, wherein the first heterologous region comprises the sequence of a first single-chain antibody that specifically binds to CD3.

10. The recombinant protein complex according to item 8, wherein the second heterologous region comprises the sequence of a second single-chain antibody that specifically binds to EGFR.

11. The recombinant protein complex according to item 8, wherein the second fusion polypeptide chain comprises a third heterologous region fused to one end of the second scaffold region.

12. The recombinant protein complex as described in item 11, wherein the second fusion polypeptide chain further comprises a fourth heterologous region fused to the other end of the second scaffold region.

13. The recombinant protein complex according to item 12, wherein the third fusion polypeptide chain comprises a fifth heterologous region fused to one end of the third scaffold region.

14. The recombinant protein complex as described in item 13, wherein the third fusion polypeptide chain comprises a sixth heterologous region fused to the other end of the third scaffold region.

15. The recombinant protein complex of item 1, wherein the first, second or third scaffold region comprises one or more triple helical repeat sequences, each repeat sequence comprising a sequence of the following formula: (X1-X2- X3) n, wherein X1 is a Gly residue, X2 or X3 is any amino acid residue, and n is greater than or equal to 5.

16. The recombinant protein complex according to item 15, wherein the first, second or third scaffold region comprises one or more triple helix repeat sequences of C1q, collagen lectin or collagen polypeptide chain.

17. The recombinant protein complex according to item 15, wherein X3 is a proline or hydroxyproline residue.

18. The recombinant protein complex according to item 15, wherein each repeat sequence comprises the sequence of (GPP) ₁₀ .

19. The recombinant protein complex according to item 1, wherein the heterologous region comprises an enzyme region or a sequence of a fluorescent protein.

20. The recombinant protein complex according to item 1, wherein the first, second and third fusion polypeptides are substantially identical.

21. A recombinant fusion polypeptide comprising:

a scaffold region for the formation of triple helical coils, and

A first heterologous region fused to one end of the scaffold region.

22. The recombinant fusion polypeptide as described in item 21, wherein the scaffold region comprises one or more triple helical repeat sequences, each repeat sequence comprising a sequence of the following formula: (X1-X2-X3)n, wherein X1 is a Gly residue base, X2 or X3 is any amino acid residue, and n is greater than or equal to 5.

23. The recombinant fusion polypeptide according to item 22, wherein X3 is a proline or hydroxyproline residue.

24. The recombinant fusion polypeptide according to item 21, which further comprises a second heterologous region fused to the other end of the scaffold region.

25. The recombinant fusion polypeptide according to item 21, wherein the scaffold region comprises one or more triple helical repeat sequences of C1q, collagen lectin or collagen polypeptide chains.

26. The recombinant fusion polypeptide according to item 21, wherein the heterologous region comprises a ligand binding region, a ligand, a receptor or a polysaccharide.

27. The recombinant fusion polypeptide according to item 21, wherein the heterologous region is obtained by phage display screening.

28. An isolated nucleic acid comprising a sequence encoding the fusion polypeptide of item 21 or its complement.

29. A host cell comprising the nucleic acid of item 28.

30. The host cell according to item 29, wherein the cell is a mammalian or insect cell.

31. The host cell according to item 30, wherein the mammalian cell is a mouse myeloma NS0 cell.

32. An expression vector comprising the nucleic acid of item 28.

33. A method of producing a fusion polypeptide comprising:

Cultivating the host cell of item 29 under the condition of allowing the expression of a polypeptide, wherein the polypeptide is encoded by a nucleic acid, and

The polypeptide is purified from the cultured cells or the culture medium of the cells.

34. The method for producing the protein complex described in item 1, comprising:

A host cell is cultured in a medium allowing expression of polypeptides encoded by the three nucleic acids and formation of a triple helical coil between them, the host cell comprising:

a first nucleic acid encoding a first fusion polypeptide chain comprising a first scaffold region and a first heterologous region fused to one end of the first scaffold region;

A second nucleic acid encoding a second fusion polypeptide chain comprising a second scaffold region; and

A third nucleic acid encoding a third fusion polypeptide chain comprising a third scaffold region; and

The protein complex is purified from the cultured cells or the culture medium of the cells.

35. The method for producing protein complexes according to item 34, wherein the host cell is a eukaryotic cell comprising enzymatic activity to hydroxylate proline residues.

Claims (6)

1. recombinant protein mixture, it comprises:

The first fusion polypeptide chain, the first heterology district that comprises the first rack area and hold frame endomixis with this first rack area N;

The second fusion polypeptide chain, the San heterology district that comprises the second rack area and hold frame endomixis with this second rack area N; And

The 3rd fusion polypeptide chain, the Wu heterology district that comprises the 3rd rack area and hold frame endomixis with the 3rd rack area N;

The aminoacid sequence of wherein said rack area is as shown in SEQ ID NO:9, and wherein said first, second and the 3rd rack area are arranged mutually to form triple helix curling, described first, the 3rd, the aminoacid sequence in Wu heterology district be SEQ ID NO:1 or described first, the 3rd, the aminoacid sequence in Wu heterology district is SEQ ID NO:3.

2. the nucleic acid of separation, it is that sequence by coding the first fusion polypeptide chain claimed in claim 1 is formed.

3. host cell, it comprises nucleic acid claimed in claim 2, and this host cell is eukaryotic cell and comprises prolyl 4 hydroxylase.

4. host cell as claimed in claim 3, wherein this cell is the cell of Mammals or insect.

5. host cell as claimed in claim 4, wherein this mammiferous cell is mouse myeloma NS0 cell.

6. produce the method for protein complex claimed in claim 1, comprising:

In substratum, cultivate host cell curling in the situation that allow expressing by the polypeptide of three kinds of nucleic acid encodings and forming triple helix between them, this host cell is eukaryotic cell and comprises prolyl 4 hydroxylase, and this host cell comprises:

Encode the first nucleic acid of the first fusion polypeptide chain, this first fusion polypeptide chain comprise the first rack area and with the first heterology region of the N end frame endomixis of the first rack area;

Encode the second nucleic acid of the second fusion polypeptide chain, this second fusion polypeptide chain comprise the second rack area and with the San heterology district of this second rack area N end frame endomixis; With

Encode the 3rd nucleic acid of the 3rd fusion polypeptide chain, the 3rd fusion polypeptide chain comprise the 3rd rack area and with the Wu heterology district of the 3rd rack area N end frame endomixis; And

This protein complex of purifying from the substratum of cultured cells or this cell, the aminoacid sequence of wherein said rack area as shown in SEQ ID NO:9, described first, the 3rd, the aminoacid sequence in Wu heterology district be SEQ ID NO:1 or described first, the 3rd, the aminoacid sequence in Wu heterology district is SEQ ID NO:3.