JP2014124186A - Multivalent antibody fragment and trimer complex thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a multivalent Fab fragment.SOLUTION: The method for producing a trimer Fab fragment by coexpression of a gene construct comprising an inframe fusion collagen-like peptide and the heavy chain part of a Fab fragment, and a gene construct consisting of the light chain part of IgG, in mammalian cells. And also the use of a molecule produced by the method of this invention is disclosed.

Description

  The present invention relates to a multivalent antibody fragment and a method for producing the same. The multivalent antibody fragment comprises an amino acid sequence comprising the heavy chain portion of an Fab fragment and an in-frame fused collagen-like peptide, a light chain variable region of human IgG and a kappa light chain constant domain ( and an amino acid sequence including kappa light chain constant domain).

  Functional affinity (avidity) is a measure of the combined binding strength of an antigen having multiple antigenic determinants. Polymerization of the antigen binding partner significantly increases their effectiveness (or valence) to bind to a group of specific same ligands in close proximity to the target cells, resulting in stronger target binding strength, lower dissociation rate, And provide a cross-linking effect that can prolong the modulation of the ligand and promote biological effects.

Single chain variable region fragments (scFv) are immunoglobulin heavy chain (V _H ) and light chain (V _L ) variable region fusion proteins linked by a short linker peptide. The main drawback of scFv lies in the monovalency of the product that prevents increased avidity due to multivalent binding compared to the corresponding divalent immunoglobulin G (IgG). To increase avidity, several strategies have been developed for scFv multimerization.

  C-propeptide of procollagen, coiled-coil neck domain of collectin family protein, C-terminal part of FasL and bacteriophage T4 fibrin foldon domain (Non-patent Document 1) The production of a recombinant trivalent single-chain antibody fragment (scFv) fusion protein using a trimerization domain including Non-Patent Document 2 and Non-Patent Document 3) has been reported. A short alpha-helical collagen-like peptide with the ability of self-trimerization and growth of heterologous fusion proteins from the C- or N-terminal direction is also reported in US Pat. . The heterologous fusion domain used in Patent Document 1 was shown as an scFv antibody fragment. However, scFv has drawbacks in that it is multivalent for therapeutic applications.

  Easy purification by affinity chromatography using protein A or G conjugated resin by binding of IgG to Fc fragment results in a homogeneity of product of 98% or more in the first step of the purification scheme Unlike immunoglobulin G (IgG) molecules, purification of multimeric scFv fusions for therapeutic applications is a difficult task because there is no commercially available affinity column available. Multivalent scFvs have significantly different stability depending on the specific variable domain in which they are constructed (Non-Patent Document 4, Non-Patent Document 5). Multimerization of the chimeric anti-CD20 single chain Fv-Fc fusion protein is performed through variable domain exchange, which has been reported to cause mutation of heterologous antibodies (Non-Patent Literature). 6).

  CFY196 consists of a humanized Fab fragment of mAb 1A616 fused with a linker derived from the hinge region of human immunoglobulin D, and a tetramerization domain derived from a coiled-coil sequence of human transcription factor ATFα (Non-patent Document 7). However, ATFα is not a plasma-derived protein and may be associated with the risk of an immune response that can greatly limit the potential for therapeutic applications.

  Patent Document 2 discloses a trimeric antibody comprising an anti-CD3 scFv N-terminal three-chain peptide fused with a self-trimerization collagen-like scaffold containing a GPP triplet. Proven ability to form fragments. However, downstream purification of the trimer scFv of these collagen-like scaffold fusions has been cumbersome because there is no affinity resin to efficiently purify the trimer scFv. In addition, the low protein expression level and thermal instability of the trimeric scFv were not suitable for biotherapy applications. Therefore, it is necessary to design a new type of trimeric collagen scaffold antibody.

European Patent No. 1798240 US Patent Application Publication No. 2008/0176247 US Pat. No. 5,641,870 US patent application Ser. No. 13 / 588,752 US Pat. No. 5,208,020 US Pat. No. 5,475,092 US Pat. No. 5,585,499 US Pat. No. 5,846,545 US Pat. No. 5,908,626 US Pat. No. 5,223,409 International Publication No. 92/18619 International Publication No. 91/17271 International Publication No. 92/20791 International Publication No. 92/15679 International Publication No. 93/01288 International Publication No. 92/01047 International Publication No. 92/09690 International Publication No. 90/02809 International Patent Application No. 91/00906 International Publication No. 91/10741 International Patent Application No. 92/03918 International Patent Application No. 92/03917 International PCT / US86 / 02269 European Patent Application No. 184,187 European Patent Application No. 171,496 European Patent Application No. 173,494 International Patent Application No. 86/01533 US Pat. No. 4,816,567 European Patent Application No. 125,023

Hoppe, H.M. J. et al. , P.M. N. Barlow, et al. (1994). “A parallel three-stranded alpha-helical bundle at the nucleation site of collagen triple-helix formation.” FEBS Lett 344 (2-3): 191-195; Frank, Kammerer et al. 2001 "Stabilization of short collagen-like triple helices by protein engineering." J Mol Biol 308 (5): 1081-1089. Holler, N .; A. Tardivel, et al. (2003). “Two adadient trimeric Fas ligands are required for Fas signaling and formation of a death signaling complex.” Mol Cell Biol 23- (14) 14 (14). Jung, S.M. A. Honegger, et al. (1999). “Selection for improved protein stability by phase display.” J Mol Biol 294 (1): 163-180. Worn, A.M. and A. Pluckthun (2001). “Stability engineering of anti-body single-chain Fv fragments.” Mol Biol 305 (5): 989-1010 Wu, A .; M.M. , G. J. et al. Tan, et al. (2001). "Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange." Protein Eng 14 (12): 1035-103. Charles, Luo et al. (2003). “Prevention of human rhinovirus effect by multiple Fab molecules directed against ICAM-1.” Antimicrob Agents Chemother 47 (5): 1503-1508. Zapata et al. , Protein Eng. 8 (10): 1057-1062 [1995]. Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994) Fan, et al. (2008) “Production of multiple protein binders using a self-trimerizing collagen-like peptide scaffold.” FASEB J 22 (11): 3795-3804. Frank et al. (2001) "Stabilization of short collagen-like triple helices by protein engineering." Mol. Biol. 308 (5): 1081-1089 Persikov et al. (2000) Biochemistry 39, 14960-14967. Persikov et al. (2004) Protein Sci. 13: 893-902 Mohs et al. , (2007) J. Am. Biol. Chem. 282: 29757-29765 Nucl. Acids Res. 12: 387, 1984 J. et al. Mol. Biol. 48: 443, 1970 Smith and Waterman (Adv. Appl. Math 2: 482, 1981) Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986 Schwartz and Dayhoff, eds. , Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979 Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987) Richards et al (2003) “Engineered fibrinctin type III domain with a RGDWXE sequence bindings with the enhanced affinity and specificity. Mol. Biol. 326 (5): 1475-1488 Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Gluzman (1981) Cell 23: 175-182 Bird et al. (1988) Science 242: 423-426. Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883 Fuchs et al. (1991) Bio / Technology 9: 1370-1372. Hay et al. (1992) Hum Antibody Hybridomas 3: 81-85 Huse et al. (1989) Science 246: 1275-1281. Griffths et al. (1993) EMBO J. et al. 12: 725-734 Hawkins et al. (1992) J Mol Biol 226: 889-896 Clackson et al. (1991) Nature 352: 624-628. Gram et al. (1992) Proc Natl Acad Sci USA 89: 3576-3580 Garrad et al. (1991) Bio / Technology 9: 1373-1377 Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137 Barbas et al. (1991) Proc Natl Acad Sci USA 88: 7978-7982. Lonberg, et al. (1994) Nature 368: 856-859 Green, L.M. L. et al. (1994) Nature Genet. 7: 13-21 Morrison et al. (1994) Proc. Natl. Acad. Sci. USA 81: 6851-6855 Bruggeman et al. (1993) Year Immunol 7: 33-40 Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724 Bruggeman et al. (1991) Eur J Immunol 21: 1323-1326. Better et al. (1988) Science 240: 1041-1043. Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443 Liu et al. (1987) J Immunol. 139: 3521-3526 Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218 Nishimura et al. , (1987) Canc. Res. 47: 999-1005 Wood et al. (1985) Nature 314: 446-449. Shaw et al. (1988) J. Am. Natl Cancer Inst. 80: 1553-1559) Li, J. et al. , J .; Davis, et al. (2006). "Modulation of antigen-specific T cell response by a non-mitogenic anti-CD3 antibody." Int Immunopharmacol 6 (6): 880-891. Cole, M.C. S. , C.I. Anasetti, et al. (1997). “Human IgG2 variants of chimeric anti-CD3 arenon-mitogenic to T cells.” J Immunol 159 (7): 3613-3621. Ochi et al. , (2006) "Oral CD3-specific anti-suppresses autoimmune encephalomyelitis by inducing CD4 + CD25-LAP + T cells." Nat Med 12 (6): 627-635.

  Multivalent scFvs can vary significantly in stability depending on the particular variable domain in which they are constructed, and multimerization of chimeric anti-CD20 single chain Fv-Fc fusion proteins can result in variable domain exchange. exchange), which has been reported to result in heterologous antibody mutations. Immunoglobulin G, which can be easily purified by affinity chromatography using protein A or G conjugated resin by binding IgG to Fc fragment, resulting in a product homogeneity of 98% or more in the first step of the purification scheme Unlike (IgG) molecules, purification of multimeric scFv fusions for therapeutic applications is a difficult task because there is no commercially available affinity column available.

  Furthermore, downstream purification of trimer scFv of these collagen-like scaffold fusions was complicated because there was no affinity resin for efficiently purifying the trimer scFv. In addition, the low protein expression level and thermal instability of the trimeric scFv was not suitable for biotherapy applications.

  Therefore, it is necessary to design a new type of trimeric collagen scaffold antibody.

  The present invention provides a method for producing novel multivalent Fab fragments in eukaryotic cells since it is necessary to design a new type of trimeric collagen scaffold antibody.

  The method comprises a gene construct encoding an amino acid sequence comprising a heavy chain portion of a Fab fragment and an in-frame fusion collagen-like peptide, and a gene encoding an amino acid sequence comprising a light chain variable region and a kappa light chain constant domain of human IgG Co-expressing the construct with a host cell (eg, a eukaryotic cell).

Embodiments of the present invention
(1) a gene construct encoding an amino acid sequence comprising a heavy chain portion of a Fab fragment and an in-frame fusion collagen-like peptide;
(2) a gene construct encoding an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
A method of making a multivalent Fab fragment in a eukaryotic cell, comprising co-expressing in a host cell (eg, a eukaryotic cell).

  In one embodiment, the gene construct of (1) further comprises a sequence encoding a hinge region derived from human IgG.

In one embodiment, the human IgG is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .

  In one embodiment, the sequence encoding the hinge region is located between the Fab fragment and the collagen-like peptide in the genetic construct.

  In one embodiment, the gene construct of (1) further includes a sequence encoding a single chain Fv.

In one embodiment, the gene construct of the (2) comprises a kappa light chain of human IgG _1.

  Embodiments of the present invention further comprise purifying the multivalent Fab fragment with a ligand that binds to the kappa light chain of human IgG.

  Embodiments of the invention include multivalent Fab fragments produced by the above method.

  Another embodiment comprises a trimeric multivalent Fab fragment comprising three multivalent Fab fragments joined by at least the collagen-like domain of the multivalent Fab fragment.

Further embodiments are:
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Including multivalent antibody fragments.

  In one embodiment, the amino acid sequence of (1) includes an amino acid sequence of a hinge region derived from human IgG.

  In one embodiment, the hinge region is located between the Fab fragment and the collagen-like peptide in the amino acid sequence.

  In one embodiment, the amino acid sequence of (1) further comprises a single-chain Fv sequence.

In one embodiment, the amino acid sequence of the (2) comprises a kappa light chain sequences of human IgG _1.

  In one embodiment, the multivalent antibody fragment is bispecific.

  The ligand of the multivalent antibody fragment of the embodiments described above may be human CD3, or human CD3 and human epidermal growth factor receptor.

  Another embodiment is a nucleic acid encoding a protein embodiment, an expression vector expressing the protein embodiment, or a host cell comprising the expression vector and / or nucleic acid.

A further embodiment of the invention is a method of modulating the biological activity of a ligand comprising:
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Culturing a multivalent antibody fragment comprising

A further embodiment of the invention is a method of treating an autoimmune disease in a subject comprising
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Administering an effective amount of a multivalent antibody fragment comprising: to a subject in need thereof.

A further embodiment of the present invention is a method for detecting a ligand comprising:
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain; (3) a label;
Culturing a trimeric multivalent antibody fragment complex comprising a ligand with a ligand, and detecting binding of the trimeric multivalent antibody fragment complex,
including.

  The present invention provides a method for treating, preventing or ameliorating the symptoms of T cell mediated immune diseases, particularly autoimmune diseases, by use of anti-CD3 antibody fragments. Specifically, the methods of the invention provide for the administration of an antibody that specifically binds to an epsilon subunit within the human CD3 complex. Such antibodies modulate T cell receptor / alloantigen interactions and thus control T cell mediated cytotoxicity associated with autoimmune diseases. Furthermore, the present invention provides a modification of an anti-CD3 antibody wherein the modified anti-CD3 antibody exhibits reduced or eliminated effector function and T cell activation compared to an unmodified anti-CD3 antibody. Cytokine release syndrome manifests, for example, headache, nausea, vomiting, fever, myalgia, arthralgia and tremors, and is caused by elevated serum levels of IL-2, IL-6, IL-10, TNFα and IFNγ, for example. Can be.

  According to the present invention, a multivalent antibody fragment that can solve all the above-mentioned conventional problems is provided.

FIG. 1 shows a schematic representation of each different form of antibody fragment molecule according to an embodiment of the present invention. Format A: Fab CSA, a trimeric antibody fragment consisting of a Fab fragment in which the heavy chain fragment is fused with a hinge region and a collagen-like peptide capable of self-trimerization. Format B: FabCSA-scFv, a trimeric bispecific antibody fragment consisting of a Fab fragment and a single chain antibody located respectively at the N- and C-termini of a collagen-like peptide capable of self-trimerization . Format C: Fab CSA-sdAb, a trimeric bispecific antibody fragment consisting of a Fab fragment and a single domain antibody (sdAb) located at the N- and C-termini, respectively, of a collagen-like peptide capable of self-trimerization It is. FIG. 2 shows the structural characterization of h145 FabCSA molecules purified from media by continuous chromatography on KappaSelect and Superdex 200 columns, according to an embodiment of the present invention. (A) Separation of h145 FabCSA species by gel filtration. The sample eluted from the KappaSelect column was concentrated and loaded onto a Superdex 200 gel filtration column equilibrated with PBS (pH 7.4). (B) Different peak fractions (numbered in peaks 1 to 3 in A) and sample loading, non-reduced (lanes 2, 4, 6, and 8), and sample 75% at DTT 50 mM. Analysis by SDS-PAGE under reducing conditions (lanes 3, 5, 7, and 9) treated at 10 ° C. (C) Schematic representation of the structure corresponding to the protein band indicated by the arrow displayed in B. The dotted line indicates the putative disulfide bond. All samples containing the same amount of protein were electrophoresed on a 4-12% SDS / Bis-Tris polyacrylamide gel using MES as the running buffer. Gels were stained with InstantBlue protein stain. FIG. 3 shows the structural characterization of hOKT3 FabCSA molecules purified from the medium by continuous chromatography on KappaSelect and Superdex 200 columns, according to an embodiment of the present invention. The sample eluted from the KappaSelect column was concentrated and loaded onto a Superdex 200 gel filtration column equilibrated with PBS (pH 7.4). Upper right panel: different peak fractions separated by Superdex 200 (numbered peaks 1 to 3) were analyzed by SDS-PAGE under non-reducing conditions. The protein bands corresponding to hOKT3 FabCSA trimer and monomer are indicated by arrows, respectively. All samples were electrophoresed on 4-12% SDS / Bis-Tris polyacrylamide gels using MES as the running buffer. Gels were stained with InstantBlue protein stain. FIG. 4 shows trimeric hOKT3 FabCSA separated by SDS-PAGE under non-reducing (lanes 2, 3 and 4) and reducing (lanes 5, 6 and 7) conditions where samples were treated with DTT 50 mM for 10 min at 75 ° C. (FIG. 3). Shows the purity analysis of peak 2), low-Fc-conjugated anti-human CD3 antibody-hOKT3IgG-AA (purified on protein A column), and muromonab-CD3 (OKT3). All samples containing the same amount of protein were electrophoresed on a 4-12% SDS / Bis-Tris polyacrylamide gel using MES as the running buffer. M, BenchMark® molecular weight standard. FIG. 5 shows a trimeric hOKT3 Fab CSA (separated from peak 2 in FIG. 3), a low-Fc binding anti-human CD3 antibody-hOKT3 IgG-AA (dimer), and a monomer, according to an embodiment of the present invention. FIG. 6 shows the binding affinity of hOKT3 Fab CSA (separated from peak 3 in FIG. 3) for purified human T lymphocytes. FIG. 6 shows the binding affinity of anti-mouse CD3 antibody-145-2C11 and trimer h145 FabCSA (separated from peak 2 in FIG. 2A) to purified mouse spleen T lymphocytes according to an embodiment of the present invention. Yes. FIG. 7 shows two independent competitive displacement binding assays, according to one embodiment of the present invention. Jurkat cells expressing the CD3 (+) T cell receptor were cultured for 1 hour at 4 ° C. with serial dilutions of anti-CD3 antibody-purified hOKT3 FabCSA trimer (black circle) and OKT3 (white circle), respectively. A saturating amount of FITC-conjugated OKT3 was added and further cultured for 1 hour. Cells were washed and bound FITC conjugate OKT3 was quantified by flow cytometry. Values are expressed as percent inhibition of maximum fluorescence intensity, which is defined as the mean fluorescence intensity obtained by adding FITC-conjugated OKT3 without pre-blocking of anti-CD3 antibody. FIG. 8 shows T cell proliferation in response to purified OKT3, low Fc binding anti-human CD3 antibody-hOKT3IgG-AA and hOKT3FabCSA trimer. Human PBMCs were collected from 3 healthy donors, individually cultured for 66 hours with serial log dilutions of OKT3 (black circles), hOKT3IgG-AA (white circles) or hOKT3 FabCSA (white triangles), and further pulsed with BrdU 10 μM for 6 hours. Cell proliferation was measured by BrdU-ELISA using a chemiluminescence immunoassay, and BrdU incorporation during DNA synthesis was quantified. Each point represents the mean ± standard deviation of 3 donors. FIG. 9 shows cytokine release induced by purified OKT3, hOKT3IgG-AA and hOKT3FabCSA trimer. Human PBMCs were collected from 3 healthy donors and cultured individually with serial log dilutions of OKT3 (black circles), hOKT3IgG-AA (white circles) or hOKT3 FabCSA (white triangles). The levels of IL-2 and the indicated remaining cytokines in the culture supernatant were measured by ELISA at 24 and 72 hours. Each point represents the mean ± standard deviation of 3 donors. FIG. 10 shows inhibition of mixed lymphocyte reaction by purified OKT3, hOKT3IgG-AA and hOKT3FabCSA trimer. A mixture of responder PBMC and mitomycin C treated stimulator PBMC in the presence of different concentrations of OKT3 (black circles), hOKT3IgG-AA (white squares) or hOKT3FabCSA (white triangles). For 5 days and pulsed with BrdU for another 16 hours. Cell proliferation was measured by BrdU-ELISA. A mixture of responder PBMC and stimulator PBMC treated with mitomycin C in the absence of antibody is indicated by black squares and black triangles, respectively. Cell growth of untreated stimulator PBMC in the absence of antibody is indicated by open circles. FIG. 11 shows the modulation / coating quantification of T cell receptor (TCR) -CD3 by purified hOKT3FabCSA (trimer), hOKT3IgG-AA (dimer) and hOKT3FabCSA (monomer). The CD3 modulation data represents the percentage of TCR-CD3 complex on the surface of treated CD5 positive T cells as a percentage of TCR-CD3 complex on the surface of untreated CD5 positive T cells. CD3 coating is shown as the percentage of TCR-CD3 complex that was not detected by FITC conjugate OKT3. FIG. 12 shows the blood clearance of purified h145 FabCSA trimer in mice according to the present invention. Male BALB / c mice were injected intravenously with 25 μg of h145 FabCSA trimer. Blood samples were taken at different times. Antibody levels remaining in plasma were measured by ELISA plates coated with anti-human Fd using horseradish peroxidase conjugated anti-human kappa light chain as the detection antibody. Results are the average of 3 animals at each time point. FIG. 13 shows the biological effects of different anti-mouse CD3 antibodies on an experimental autoimmune encephalomyelitis (EAE) mouse disease model based on embodiments of the present invention. (A) Efficacy of different biologics against EAE mice. For the group of vehicle (hamster control IgG), 145-2C11 (hamster IgG) and purified trimeric h145 FabCSA, dose levels of 1.0, 0 once daily for 5 consecutive days (indicated by arrows), respectively. Mice were injected intravenously at .1 and 1.0 μg / mouse. For the interferon-β1a (positive control) group, mice were injected intraperitoneally at a dose level of 10000 units / mouse once a day for the entire treatment period. Results are shown as the mean ± standard deviation of each group of representative experiments (n = 10) from two independent experiments. (B) Flow cytometry of Treg cell populations for h145-2C-11 IgG and trimeric h145 FabCSA treatment to EAE mice. One day after the last intravenous injection, spleen lymphocytes from 3 mice from each of the vehicle (hamster control IgG), 145-2C11 IgG and the trimeric h145 FabCSA group were individually prepared and then subjected to LAP for flow cytometry. -Stained with APC and CD4-FITC. Measurement of the Treg population was gated on LAP + cells. Data are from 1 representative mouse in 3 experiments. 14A and B show the therapeutic effect of 145-2C11 IgG and h145 FabCSA trimer on the SLE mouse model. FIG. 15 shows a time course study of serum cytokine levels in mice after intravenous injection of different anti-mouse CD3 antibodies as a single dose of 50 μg. Mice were grouped and purified 145-2C11 IgG (light gray squares), low Fc-conjugated anti-mouse CD3 antibody-145 IgG-AA (dark gray squares), or 50 μg h145 FabCSA trimer (white squares) intravenously. Administered. Blood (100 μl / mouse) was collected at 0 (pre-bleed), 0.5, 24, and 144 hours. Cytokine levels were measured using ELISA. Each point represents the mean ± standard deviation of 3 wells. PBS (black square) was used as a control. FIG. 16 shows the structural characterization of hOKT3FabCSA763scFv molecules from non-single (A) or single stable clones (B). Each medium was purified by continuous chromatography on a KappaSelect and Superdex 200 column. (A) Separation of non-clone-derived hOKT3FabCSA763scFv species by gel filtration. Upper right panel: different peak fractions (peaks 1 to 3 are numbered) were analyzed by SDS-PAGE under non-reducing conditions. The positions of bands corresponding to the structures of disulfide bond trimer (T), non-disulfide bond trimer (Mt), and monomer (Mm) are indicated by arrows. (B) Separation of stable clone-derived hOKT3FabCSA763scFv species by gel filtration. Peak fractions were analyzed by SDS-PAGE under non-reducing conditions. The positions of the bands corresponding to the structures of disulfide bond trimer (T) and non-disulfide bond trimer (Mt) are indicated by arrows. All samples were electrophoresed on 4-12% SDS / Bis-Tris polyacrylamide gels using MES as the running buffer. Gels were stained with InstantBlue protein stain. FIG. 17 shows a trimeric bispecific anti-CD3 × EGFR antibody, hOKT3FabCSA763scFv, obtained from the trimeric peak fraction containing both the non-disulfide and disulfide-bonded trimers shown in FIG. 16B. FIG. 6 shows flow cytometric analysis of binding to EGFR (+) cells-A431, WiDr, HCT116, and CD3 (+) Jurkat T cells. FIG. 18 shows the cytotoxicity of a trimeric bispecific anti-CD3 × EGFR antibody, hOKT3FabCSA763scFv, against EGFR-expressing tumor cells by human PBMC. Stimulated human PBMCs were cultured with different A / T ratios at different E / T ratios (A) or WiDr or HCT116 cells (B) at a constant ratio of 10: 1 under various trimeric hOKT3 Fab CSA 763 scFv concentrations. FIG. 19 shows time-lapse photographs of redirected lysis of EGFR-overexpressing tumor cells with trimeric bispecific hOKT3FabCSA763scFv. Time points (hours: minutes) indicated in the absence (upper figure) or presence (lower figure) of A431 cells in the absence (upper figure) or presence (lower figure) of human T lymphocytes prestained with PKH26 and trimeric hOKT3FabCSA763scFv 1 μg / ml ), And images were recorded with a time-lapse microscope. Note the completion of apoptosis of A431 cells by T cells after 13 hours of co-culture. FIG. 20 shows the in vivo efficacy of the trimeric hOKT3FabCSA763scFv in the HCT116 colon cancer NOD / SCID mouse model. On day 0, two groups—763 IgG and PBS controls were inoculated subcutaneously with 5 × 10 ⁶ HCT116 cells in the absence of human PBMC. The remaining three groups were inoculated subcutaneously on day 0 with a mixture of 5 × 10 ⁶ HCT116 cells and 5 × 10 ⁶ healthy donor-derived non-stimulated human PBMC, followed by PBS solvent control (100 μl) on day 1. 50 μg and 15 μg of hOKT3FabCSA763scFv were injected into the tail vein for 10 consecutive days. Tumor growth curves obtained from each group consisting of the indicated n number of animals are shown. Statistically significant difference (P <0.001) between administration of hOKT3 Fab CSA 763 scFv group and unstimulated human PBMC control group is shown.

  In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, it will be apparent that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.

The disclosure provides a method of making a multivalent antibody fragment in a host cell (eg, a eukaryotic cell) comprising:
(1) a gene construct encoding an amino acid sequence comprising a heavy chain portion of an antibody fragment and an in-frame fusion collagen-like peptide;
(2) a gene construct encoding an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Including the step of expressing.

  The gene construct of (1) may further include an IgG hinge region and / or scFv.

  The multivalent antibody fragment described in the present method described above may then be associated with a trimeric construct.

  The present disclosure includes a nucleic acid encoding a multivalent antibody fragment and an expression vector that expresses the multivalent antibody fragment when introduced into a host cell. The present disclosure also encompasses host cells comprising an expression vector that expresses the multivalent antibody fragment.

  The present disclosure encompasses methods and kits for modulating (ie, suppressing or increasing) the biological activity of a ligand comprising culturing a trimer comprising three multivalent antibody fragments with the ligand.

  The present disclosure also provides methods for treating, preventing or ameliorating symptoms of T cell mediated immune diseases, particularly autoimmune diseases, by administration of multivalent anti-CD3 antibody fragments.

Definition 1. Antibody Fragments The multivalent antibody fragments of the present disclosure include one or more “antibody regions”, “antibody domains” or “antigen binding domains”, also referred to as “antibody binding domains”. Antibody fragment ". “Antibody fragments” as referred to herein comprise a portion of an intact antibody, eg, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ and Fv fragments, diabodies, linear antibodies (US Pat. No. 5,641,870). 3), Example 2; see Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995] (non-patent document 8), single chain antibody molecules, and multispecific antibodies. .

The papain degradation of the antibody results in two identical antigen-binding fragments called “Fab” fragments and the remaining “Fc” fragments, which are designated for their ability to crystallize easily. The Fab fragment may contain all the light chains and the variable region domain of the heavy chain (V _H ) and the first constant domain of one heavy chain (C _H1 ). The constant region of the light chain may be either lambda or kappa. Each Fab fragment is monovalent for antigen binding, ie it has a single antigen binding site.

An Fd or Fd fragment is an antibody heavy chain fragment consisting of V _H and C _H1 domains.

Pepsin treatment of the antibody yields a single large F (ab ′) ₂ fragment that corresponds approximately to a Fab fragment having two disulfide bonds with divalent antigen binding activity and also has the ability to crosslink antigen. Fab ′ fragments differ from Fab fragments in that they additionally have several residues at the carboxy terminus of the C _H1 domain, such as one or more cysteines in the antibody hinge region.

  The multivalent antibody fragment of the invention can comprise a portion of a heavy chain, a light chain (or a portion thereof), or both a heavy chain portion and a light chain or a portion thereof.

An antibody fragment can include a single chain Fv. “Single-chain Fv”, also abbreviated as “sFv” or “scFv”, is an antibody fragment comprising V _H and V _L antibody domains linked to form a single polypeptide chain. In one embodiment, the scFv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that allows the scFv to form a preferred structure for antigen binding. For a description of scFv, see Plugthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994) (9); Borrebaeck 1995 (see below).

  A “single domain” antibody (sdAb) is an antibody whose complementarity determining region is part of a single domain polypeptide. Examples include heavy chain antibodies, antibodies in which the light chain is naturally deleted, single domain antibodies derived from conventional 4-chain antibodies, modified antibodies and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any of those in the art, or other single domain antibody. Single domain antibodies can be derived from any species including, but not limited to, mouse, human, camel, llama, shark, goat, rabbit and cow.

  A “bispecific antibody” is an antibody that has binding specificities for at least two different antigens.

Fd-CS in an embodiment of the present invention, an antibody Fd fragment of the present invention, it is subsequently made of the hinge region, and collagen-like peptide-human IgG ₁ with a fusion polypeptide chain.

  The term “monomer” can be used herein to describe an embodiment of a multivalent antibody fragment having a single collagen-like peptide. In one embodiment, “monomer” can be used to refer to a multivalent antibody fragment in which heavy and light chains are associated. The “monomer” is distinguished from the “trimerization” structure of the multivalent antibody fragment.

  In one embodiment, the multivalent antibody fragment is bispecific. In one embodiment, the monomer structure of the present invention may have not only antibody fragments but also two or more antigen binding domains and can bind two or more antigens. For example, the single monomer structure of the multivalent antibody fragment of the present invention may have a Fab region at the N-terminus of the monomer structure and an scFv region at the C-terminus of the monomer structure.

2. Hinge Region The protein of embodiments of the present invention optionally includes a “hinge region”. In one embodiment, the hinge region is a sequence of approximately 4-15 amino acids in length. The hinge region can be a human IgG hinge region or a glycine linker. In one embodiment, the human IgG hinge region is a human IgG ₁ , IgG ₂ , IgG ₃ or human IgG ₄ hinge region.

Fan, et al. (2008) “Production of multiple protein binders using a self-triaging collagen-like peptide scaffold.” Collagen as already demonstrated by FASEB J 22 (11): 3795-3804 (non-patent document 10). (GPP) ₁₀ alone can promote the formation of non-covalently linked trimer fusion proteins. Thus, the “hinge region” is optional and, if present, has no trimerization effect on the fusion peptide.

3. Collagen-like peptide Collagen is the most abundant protein in mammals. Collagen is an extracellular matrix protein containing one or more triple helix regions (collagen domains or collagen “scaffolds”) having a repetitive triplet sequence of Gly-Xaa-Yaa, where Xaa and Yaa are any amino acid residues. Proline (amino acid code, P or Pro) is the most frequently incorporated residue. At the Yaa position, Pro is usually enzymatically modified to 4-hydroxyproline (amino acid code, O or Hyp), whereby Gly-Pro-Hyp is the most abundant in collagen and at the same time the most stabilizing triplet . The presence of such a triplet allows the three collagen polypeptide chains (α chains) to be folded to form a triple helical structure. A description of collagen-like peptides can be found in the description of collagen-like domains in US patent application Ser. No. 13 / 588,752, which is hereby incorporated by reference in its entirety. Included explicitly. The collagen-like polypeptide of the present invention can trimerize the heavy chain portion of the Fab fragment alone to form a trivalent structure without requiring a separate trimerization domain. The collagen-like polypeptide of the present invention comprises at least one and at least one consecutive repeating sequence of at least 10 Gly-Pro-Pro or Gly-Pro-Hyp triplets. The collagen-like peptide of the present invention may contain a Gly-Pro-Pro or Gly-Pro-Hyp motif and / or other Gly-Xaa-Yaa motif, where Xaa and Yaa are any amino acid residue. The collagen-like peptide of the present invention may contain a complete repetitive Gly-Xaa-Yaa triplet, but the residue at the first position of Gly or the third position of Yaa is deleted, resulting in a short defect (short may be interrupted by impact, which is found in many naturally occurring proteins and proteins that contain collagen-like domains.

  The stability of the collagen multimeric structure can be determined by measuring the melting temperature of the trimer. Many studies have verified the melting temperature / stability of GP-X1 repeats. Frank et al. (2001) "Stabilization of short collagen-like triple helices by protein engineering." Mol. Biol. 308 (5): 1081-1089 (Non-Patent Document 11); Persikov et al. (2000) Biochemistry 39, 14960-14967 (Non-patent Document 12), Persikov et al. (2004) Protein Sci. 13: 893-902 (Non-Patent Document 13), and Mohs et al. , (2007) J. Am. Biol. Chem. 282: 29757-29765 (Non-Patent Document 14). Based on these studies, the stability of various repeating structures can be predicted.

4). Linker A linker is a short peptide sequence that can optionally be placed between an antibody fragment and a collagen-like peptide region, or between a binding domain and a collagen-like peptide region. The scFv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that allows the scFv to form the desired structure for antigen binding. In some embodiments, in any case, the linker is between 4 and 10 amino acids in length.

The multivalent antibody fragment of the present invention can bind a ligand in the binding domain. A ligand is a biomolecule that forms a complex with a binding domain of an embodiment of the invention. The ligand can bind to the binding domain by intermolecular forces with a certain functional affinity. In one embodiment, the functional affinity of the multivalent antibody fragment for the ligand is greater than 10 ⁻⁶ M. In one embodiment, the functional affinity of the multivalent antibody fragment for the ligand is greater than 10 ⁻⁸ M. In one embodiment, the functional affinity of the multivalent antibody fragment for the ligand is greater than 10 ⁻¹⁰ M. In certain embodiments, the functional affinity (or avidity) for a ligand of a soluble trimeric or hexameric fusion protein is between 10 ⁻⁷ M and 10 ⁻¹² M, 10 ⁻⁸ M to 10 ⁻¹¹ M. Between 10 ⁻⁷ M and 10 ⁻¹⁰ M, between 10 ⁻⁸ M and 10 ⁻¹⁰ M, and between 10 ⁻⁹ M and 10 ⁻¹⁰ M.

  In one embodiment, the trimeric or hexameric protein composed of multivalent antibody fragments is a soluble protein. A soluble protein is a protein that is readily soluble under physiological conditions. In one embodiment, the soluble trimer or hexamer construct of the multivalent antibody fragment is a secreted protein. A secreted fusion protein is a protein secreted from cells. Protein secretion can be targeted by having a signal sequence or signal peptide on a polypeptide comprising an antibody domain.

  The signal sequence can include the following sequences:

  MetGluThrAspThrLeuLeuLeuTrpValLeuLeuLeuLeuTrpValProGlySerThrGly (SEQ ID NO: 11)

  The signal peptide can be cleaved during the expression, association, and / or secretion process. Mouse myeloma NS0 cells are an expression system suitable for production of recombinant collagen or collagen-like protein and expression of the fusion protein of the invention. In addition, CHO and CHO-S cells can be used for the production of recombinant collagen or collagen-like proteins and the expression of the fusion proteins of the invention.

  The associated trimer of embodiments of the present invention comprises three monomers, a first, second and third multivalent antibody fragment (forms a multivalent antibody fragment complex). In one embodiment, the first, second and third multivalent antibody fragments are substantially the same and are at least 75% of each other (eg, any number between 75% and 100%, for example, 75%, 76% ... 95%, 96%, 97%, 98%, or 99%). The complex formed by three identical multivalent antibody fragments is a homotrimer. The three multivalent antibody fragments can be functionally equivalent. “Functional equivalent” refers to a polypeptide derivative of a common polypeptide, eg, a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, forming a triple helical coil And a protein that substantially retains the activity of a heterologous domain, such as binding to a ligand. In one embodiment, three copies of a first monomeric multivalent antibody fragment structure and three copies of a second multivalent antibody fragment structure. In one embodiment, there may be two copies of the first multivalent antibody fragment structure, two copies of the second multivalent antibody fragment structure, and two copies of the third polypeptide structure.

  The percent identity is described, for example, by Devereux et al. (Nucl. Acids Res. 12: 387, 1984 (Non-Patent Document 15)) and comparison of sequence information using the GAP computer program version 6.0 available from the University of Wisconsin Genetics Computer Group (UWGCG). Can be determined. The GAP program was modified by Smith and Waterman (Adv. Appl. Math 2: 482, 1981 (Non-Patent Document 17)), Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970 (Non-Patent Document 16)). ) Alignment method is used. The default parameters of the GAP program include (1) a unary comparison matrix for nucleotides (including values of 1 for identical and 0 for nonidentical), and Schwartz and Dayhoff eds. , Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979 (Non-patent Document 19), Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap, and (3) The last gap includes no penalty.

  As used herein, the term “consensus sequence” refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (eg, Winnaker, From Genes to Clones Verlagsgesellschaft, Weinheim, Germany 1987 (Non-patent Document 20), and Richard et al., (2003) “Engineered fibrinectine type 3 bioin the um. 3 rd affiliation in the world. RGDWSE sequence binds with the enc. ): 1475-1488 (see Non-Patent Document 21) Figure 9). In a family of proteins, each position of the consensus sequence is occupied by the amino acid that appears most frequently at that position in the family. Both may be included in the consensus sequence if the two amino acids appear with equal frequency.

  A heterologous polypeptide, nucleic acid, or gene is a polypeptide, nucleic acid, or gene that associates with another polypeptide, nucleic acid, or gene that is not naturally associated. Two fusion domains or sequences are heterologous to each other if they are not in close proximity to each other in a naturally occurring protein or nucleic acid.

  An “isolated” polypeptide (or multivalent antibody fragment) or protein complex refers to a polypeptide or protein complex that is substantially free of naturally associated molecules, that is, by dry weight. It is at least 75% pure (ie, any number between 75% and 100%). Purity can be measured by any appropriate standard technique, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Isolated polypeptide or protein complexes of embodiments of the invention can be purified from natural sources and made by recombinant DNA techniques.

  The three monomeric antibody fragments that are trimerized to form a trimeric multivalent antibody fragment may not be contiguous (non-contiguous). In another embodiment, three monomeric antibody fragments that are trimerized to form a trimeric multivalent antibody fragment are contiguous, ie, translated as a single translation product.

  On the other hand, when two or more of the six binding domains are the same as each other, the protein complex has one to three binding domains specific for one binding partner (eg, antigen) compared to conventional antibodies or receptors. Traditional antibodies or receptors have only one or two such domains. In other words, unlike conventional antibodies or receptors that are monovalent or only bivalent to the antigen, the protein complex can be bivalent, trivalent, tetravalent, pentavalent, or hexavalent. As a result, it can be made to have a higher affinity than conventional antibodies or receptors. Because of the higher affinity, a smaller amount of protein complex and shorter incubation time is required to achieve the desired goal, eg, therapeutic effect, compared to conventional antibodies, thereby reducing therapeutic costs and side effects ( For example, undesirable immune reactions) will be minimized.

  On the other hand, when 2 or more of the 6 domains are different from each other, the protein complex of the invention may have 2 to 6 binding domains specific for 2 to 6 different binding partners. Combining multiple binding partner sites with different specificities into one unit provides the ability to consolidate multiple binding partners, thus assembling therapeutic, tissue reconstruction, and active protein mechanisms (eg, multi-subunit enzymes) Can be desirably used at the nanometer level.

  The protein complex of embodiments of the present invention can be conjugated to a therapeutic moiety such as a cytotoxin, therapeutic agent, or radioactive ion. A cytotoxin or cytotoxic agent includes any substance that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, amitomycin Mycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol (US Pat. No. 5,208, No. 020 (Patent Document 5)), CC-1065 (US Pat. No. 5, 75,092 (Patent Document 6), US Pat. No. 5,585,499 (Patent Document 7), and US Pat. No. 5,846,545 (Patent Document 8)) and its Analogs or homologs are included. The therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thioepachlorbucil) ), CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamineplatinum (II) (DDP) (cisplatin) ), Anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin, (formerly actino) Leucine), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, vincristine, vinblastine, taxol and maytansinoids) is.

Radioactive ions contemplated in embodiments of the present invention include, but are not limited to, ¹¹¹ indium, ¹¹³ indium, ⁹⁹ rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ⁹⁹ M technetium, ¹²¹ M tellurium, ¹²² M tellurium, ¹²⁵ M tellurium, ¹⁶⁵ Thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodo, ¹²⁵ iodo, ¹²⁶ iodo, ¹³¹ iodo, ¹³³ iodo, ⁸¹ krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium, ⁶⁸ gallium, ³⁵ sulfur, and ¹⁴ carbon.

  Conjugates can be used to modify a given biological response by administering the conjugate to a host. The drug component should not be construed as limited to classic chemotherapeutic drugs. For example, the drug component may be a protein or polypeptide with the desired biological activity. Examples of such proteins include abrin, ricin A, toxins such as Pseudomonas aeruginosa exotoxin or diphtheria toxin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminol A protein such as a gene activator or, for example, lymphokine, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocytes Biological response modifiers such as macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or other growth factors may be included.

  In further embodiments, the multivalent antibody (or trimeric complex thereof) can be conjugated or conjugated to a labeling agent (ie, a “marking agent”) such as a fluorescent agent or a radioactive agent. Marker proteins include, but are not limited to, luciferase, green fluorescent protein, and sensitive green fluorescent protein. The multivalent antibody of an embodiment of the invention comprising a marker protein can be used for diagnosis and molecular imaging. In an embodiment of the invention, a multivalent antibody fragment comprising a marker protein or radioactive ion or other fusion component is packaged in a kit containing the multivalent antibody fragment and other reagents necessary for imaging a particular molecule. Also good. These reagents can include, but are not limited to, reagents used to make biological samples and reagents used to visualize marker proteins.

  In a further embodiment of the invention, the multivalent antibody fragment (or its trimer complex) can be conjugated to a polymer. Such polymers include, but are not limited to, polyethylene glycol, polypropylene glycol, and polyoxyethylated polyols.

  Embodiments of the invention also include isolated nucleic acids that contain a sequence encoding a multivalent antibody fragment as described above, or the complement of the sequence. Nucleic acid refers to a DNA molecule (eg, cDNA or genomic DNA), an RNA molecule (eg, mRNA), or a DNA or RNA analog. DNA or RNA analogs may be synthesized from nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but in one embodiment is double-stranded DNA. An “isolated nucleic acid” is a nucleic acid whose structure is not identical to the structure of any naturally occurring nucleic acid or the structure of any naturally occurring fragment of a nucleic acid. Thus, this term includes, for example, (a) DNA having a sequence of a portion of a naturally occurring genomic DNA molecule, but without two coding sequences flanking that portion of the molecule in the genome of a naturally occurring organism, (B) a nucleic acid that is incorporated into a prokaryotic or eukaryotic vector or genomic DNA in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA, (c) eg a cDNA, genomic fragment , Isolated molecules such as fragments generated by polymerase chain reaction (PCR) or restriction fragments, and (d) recombinant genes, ie recombinant nucleotide sequences that are part of a gene encoding a fusion protein. The nucleic acids described above can be used to express the polypeptides of the present invention. For this purpose, an expression vector can be generated by operably linking the nucleic acid to suitable regulatory sequences.

  A vector refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. The vector can be self-replicating or incorporated into the host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. The vectors of the present invention comprise a nucleic acid in a format suitable for expression of the nucleic acid in a host cell. The vector includes one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. In one embodiment, the expression vector is pSecTag2 / Hygro (Invitrogen).

  “Regulatory sequences” include promoters, enhancers, or other expression control elements (eg, polyadenylation signals). Regulatory sequences include those that induce constitutive expression of nucleotide sequences as well as tissue-specific regulatory and / or inducible sequences. The design of the expression vector will depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. Expression vectors can be introduced into host cells to produce the polypeptides of the present invention. Host cells containing the nucleic acids described above are also included within the scope of embodiments of the present invention. Examples include E.I. E. coli cells, insect cells (eg, using Drosophila S2 cells or insect cells infected with baculovirus), yeast cells, or mammalian cells (eg, mouse myeloma NS0 cells). See, for example, Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif (22).

  The sequence encoding the monomer structure of the present invention may include nucleotide or protein sequences that allow identification and purification. Such sequences can include restriction enzyme recognition sites, labels, spacers, and other means for purifying or identifying nucleotide or protein sequences. Often, such sequences are contained in nucleotides and encode short amino acid sequences of 4-6 amino acids in length. They often appear as artifacts between domains of multivalent antibody fragments, but do not substantially affect the basic and novel features of the present invention.

  To produce a multivalent antibody fragment, host cells can be cultured in a medium under conditions that allow expression of the polypeptide encoded by the nucleic acid, and the polypeptide can be purified from the cultured cells or cell culture medium. . Peptides that contain collagen-like peptides can be difficult to purify in the absence of affinity tags. In the multivalent antibody fragment of the present invention, the Fab region is useful for purification. Alternatively, the 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 embodiment of the present invention, a host cell containing the first, second and third nucleic acids encoding the first, second and third fusion polypeptides, respectively, described above is used. Culturing in culture medium under conditions that allow expression of a polypeptide encoded by two nucleic acids and formation of a triple helical coil between the expressed polypeptides, and purifying the protein complex from the cultured cell or cell culture medium can do. A host cell is a eukaryotic cell that contains an enzymatic activity that hydroxylates a proline residue.

  The host cell can be any prokaryotic or eukaryotic cell. Proteins of embodiments of the present invention may be bacterial cells (eg, E. coli), insect cells, yeast, or mammalian cells (eg, Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell 23: 175-182 (non-patent document 23)) Other suitable host cells are known to those skilled in the art.

  Vector DNA can be introduced into host cells by conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (eg, DNA) into a host cell. This includes coprecipitation of calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, or electroporation.

  For in vivo use in humans, the multivalent antibody fragments of the embodiments of the invention are of human origin. For example, a multivalent antibody fragment can comprise a sequence fused in-frame to a human-like collagen-like domain. Since many collagen-like proteins having a collagen domain are extremely stable in blood, the multivalent antibody fragment should also maintain structural integrity in blood. Furthermore, the hinge region and Fc domain can be obtained from human IgG or humanized antibodies.

  Embodiments of the present invention also provide methods for treating, preventing or ameliorating symptoms of T cell mediated immune diseases, particularly autoimmune diseases, through the use of multivalent anti-CD3 antibody fragments. Specifically, the methods of the invention provide for the administration of a multivalent antibody fragment or antibody that specifically binds to an epsilon subunit within a human CD3 complex. Such antibodies and antibody fragments modulate T cell receptor / alloantigen interactions and thus control T cell mediated cytotoxicity associated with autoimmune diseases. Furthermore, the present invention provides modifications of anti-CD3 antibodies or anti-CD3 antibody fragments in which the modified anti-CD3 antibody exhibits reduced or eliminated effector function and T cell activation compared to an unmodified anti-CD3 antibody.

  Autoimmune disease refers to a disease in which the immune system mistakenly attacks and destroys healthy living tissue, thereby causing tissue damage. Autoimmune diseases include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, mixed connective tissue disease, progressive Systemic scleroderma, antiphospholipid syndrome, psoriasis, scleroderma, glomerulonephritis, dermatomyositis, Hashimoto's thyroiditis and Graves' disease.

  Effector function is due to phagocytosis and disruption of antibody-coated particles through complement-dependent cytotoxicity (CDC), cross-linking of antibody Fc fragments to Fcγ receptors of activated effector cells such as natural killer cells. Lysis of antibody-coated target cells through antibody-dependent cell-mediated cytotoxicity (ADCC), cell membrane disruption, release of inflammatory mediators including IL-1α, IL-1β, IL-6, and TNFα, and immunoglobulin production Can be indicated by the controls.

  T cell activation can be demonstrated by measuring T cell proliferation upon stimulation of T cells with an antigen or agonist antibody to the T cell receptor (TCR). TCR activation is the induction of specific protein tyrosine kinases (PTK), degradation of phosphatidylinositol-4,5-diphosphate (PIP2), activation of protein kinase C (PKC), and increased intracellular calcium ion concentration Can lead to the initiation of signal transduction pathways. These early events are transmitted to the nucleus, resulting in clonal proliferation of T cells, up-regulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion such as IL-2, Induction of apoptosis occurs.

  Cytokine release syndrome manifests, for example, headache, nausea, vomiting, fever, myalgia, arthralgia and tremor, such as IL-1α, IL-1β, IL-2, IL-6, IL-10, TNFα and IFNγ. May be caused by elevated levels in serum.

  The protein complex described above can be used to treat various diseases including autoimmune disease, inflammatory disease, metabolic disease, fibrosis disease, cancer, and cardiovascular disease depending on the specificity of the heterologous binding domain. Can do. Specific symptoms of these diseases include headache, nausea, vomiting, fever, myalgia, arthralgia and tremor, such as serum levels of IL-2, IL-6, IL-10, TNFα and IFNγ. May be caused by a rise. Thus, the invention features methods of treating such diseases, for example, by administering an effective amount of a protein complex of the invention to a subject in need thereof to treat the disease. A subject to be treated can be considered to have or be at risk of becoming characterized by the disease. The method can be performed alone or in combination with other drugs or treatments.

  One embodiment is used to treat diseases caused or exacerbated by T cell receptor / alloantigen interactions and thus control T cell mediated cytotoxicity associated with autoimmune diseases. In another embodiment, the invention is used to modulate the biological activity of CD3, the level of CD3 signaling, or the T cell receptor / alloantigen interaction in a patient in need thereof. In one embodiment, the present invention reduces the level of unbound CD3 or CD3 signaling.

  Due to the multispecific nature of the protein complex of the present invention, it can be used to crosslink molecules or cells that do not normally associate with each other. This feature is particularly useful for cell-based therapies. In one example, a multivalent Fab antibody fragment of the invention can bind CD3 to one domain while another domain binds to EGFR.

  Activation of cytotoxic T cells can occur by binding CD3 antigen as an effector antigen on the surface of cytotoxic T cells by the protein complex of the present invention. 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 leads to activation of effector cells such as monocytes, neutrophil granulocytes, and dendritic cells. These activated cells then have a cytotoxic or apoptotic effect on the target cells.

  The term “treating” is used to cure, alleviate, reduce, or cure a disease, a symptom of the disease, a medical condition that follows the disease, a disease that is exacerbated by a ligand of the protein of the present invention, or a predisposition to the disease. , Defined as administration to a subject for the purpose of preventing or ameliorating. An “effective amount” is an amount of a composition that can produce a medically desirable result, such as described above, in a subject to be treated.

  In one in vivo approach, a therapeutic composition (eg, a composition comprising a protein complex of the invention) is administered to a subject. Usually, the complex is suspended in a pharmaceutically acceptable carrier (eg, saline) and administered orally or by intravenous injection, or subcutaneous, muscle, intrathecal, intraperitoneal, intrarectal. Injected or implanted into the vagina, intranasal, stomach, trachea or lung.

  The required dosage will depend on the choice of route of administration, the nature of the dosage form, the nature of the subject's illness, the subject's size, weight, surface area, age and gender, other drugs being administered, and the judgment of the attending physician. Determined. A suitable dose is in the range of 0.01 to 100.0 mg / kg. A suitable dose is in the range of 0.01 to 100.0 mg / kg. More specifically, 0.1 to 100, 0.1 to 75, 0.1 to 50, 0.1 to 25, 0.1 to 10, 0.5 to 100, 0.5 to 75, 0.5 to The range is 50, 0.5 to 25, 0.5 to 10, 1 to 100, 1 to 75, 1 to 50, or 1 to 25 mg / kg. Dosages include 1-10, 10-100, 10-75, 10-50, 10-25, 25-50, 50-75, 25-100, 25-50, 50-100, or 75-100 mg / kg may be included. Alternatively, the dosage may be 1-2, 3-4, 5-6, 7-8, or 9-10 mg / kg.

  The therapeutic compositions of embodiments of the present invention may be used daily for about 1 to 10 weeks, such as 2 to 8 weeks, or about 3 to 7 weeks, or even about 4, 5, or 6 weeks daily. It can be administered once, twice, three times or more once a week. Considering the different types of compositions that can be used and the different efficiencies of the various routes of administration, the required dosage variation is expected. For example, oral administration is expected to require a higher dose than administration by intravenous injection. Variations in these dosage levels can be adjusted by standard experiments for optimization, as is well understood in the art. Encapsulation of the composition in a suitable delivery medium (eg, polymeric microparticles or implantable device) can improve delivery efficiency, particularly in oral delivery.

  Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents. Specifically, these substances include saline, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, anti-corrosive agents such as ascorbic acid or sodium bisulfite. Oxidants, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate or phosphate, and substances used for tonicity adjustment such as sodium chloride or dextrose may be included. The pH of the pharmaceutical composition can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.

  Pharmaceutical compositions containing a pharmaceutically acceptable carrier and an effective amount of a protein complex of embodiments of the present invention are also within the scope of embodiments of the present invention. The pharmaceutical composition can be used to treat the diseases listed above. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents. The pharmaceutical composition can be formulated into dosage forms suitable for different administration routes using conventional methods.

  The effectiveness of the compositions of embodiments of the present invention can be evaluated both in vitro and in vivo. For in vivo testing, the composition can be injected into an animal (eg, a mouse model) and its therapeutic effect is evaluated. Based on the results, an appropriate dosage range and administration route can be determined.

As used herein, the terms “directed against” and “specifically bind to” mean that the fusion protein of the invention comprises an antibody domain and the antibody or fragment of an antibody thereof It means having a functional affinity of at least 10 ⁻⁶ M for the ligand.

  The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

II. Configuration of Embodiment A. First Embodiment Antibody Fragments A monomer of a multivalent antibody fragment of the invention according to one embodiment can have a single antibody fragment (including a binding domain / region or antigen binding fragment) or more than one antibody fragment. Examples of monomeric antibody fragments representing multivalent antibody fragments include, but are not limited to: (i) a Fab fragment that is a monovalent fragment consisting of V _L , V _H , C _L and C _H1 domains; (ii) hinge region An F (ab ′) ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at, (iii) an Fd fragment consisting of V _H and C _H1 domains, (iv) one arm of the antibody Fv fragment consisting of V _L and V _H domains of (v) dAb fragment consisting of V _H domain (Ward et al., (1989) Nature 341: 544-546), (vi) isolated complementarity determining region (CDR), as well as (vii) _VL or _VH domains are included.

  In one embodiment, the antibody fragment is a Fab fragment.

In addition, the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, but using recombinant methods they are paired with the V _L and V _H regions to form a monovalent molecule 1 Can be linked by a synthetic linker that can be linked to a single protein chain (known as single chain Fv (scFv). For example, Bird et al., (1988) Science 242: 423-426 (Non-Patent Document 24). And Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883 (Non-patent Document 25)). Such single chain antibodies (scFv) are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are selected in the same manner as intact antibodies so that they can be put to practical use.

  In one embodiment, the antibody fragment is an scFv.

  In one embodiment, the multivalent antibody comprises two antibody fragments, Fab and scFv.

An Fd or Fd fragment is an antibody heavy chain fragment consisting of V _H and C _H1 domains.

  The antibody may be a monoclonal antibody. In one embodiment, antibodies can be generated by recombinant techniques, such as phage display or combinatorial techniques. Phage display and combinatorial techniques for generating antibodies are known in the art (eg, Ladner et al. US Pat. No. 5,223,409); Kang et al. No. 92/18619 (Patent Document 11); Dower et al., International Publication No. 91/17271 (Patent Document 12); Winter et al., WO 92/20791 (Patent Document 13); International Publication No. 92/15679 (Patent Document 14); Breitling et al., International Publication No. 93/01288 (Patent Document 15), McCafferty et al., International Publication No. 92/01047. Garrard et al., WO 92/09690 (patent document 17); Ladner et al., WO 90/02809 (patent document 18); Fuchs et al. (1991). Bio / Technology 9: 1370-1372 (Non-patent Document 26); Hay et al. (1992) Hum Antibody Hybridomas 3: 81-85 (Non-patent Document 27); Huse et al. (1989) Science 246: 1275-1281 (Non-patent Document 28); Griffiths et al. (1993) EMBO J 12: 725-734 (Non-patent Document 29); Hawkins et al. (1992) J Mol Biol 226: 8. 9-896 (Non-Patent Document 30); Clackson et al. (1991) Nature 352: 624-628 (Non-Patent Document 31); Gram et al. (1992) PNAS 89: 3576-3580 (Non-Patent Document 32); Garrad et al. (1991) Bio / Technology 9: 1373-1377 (Non-patent Document 33); Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137 (Non-patent Document 34), and Barbas et al. 1991) Proc Natl Acad Sci USA 88: 7978-7982 (all of which are hereby incorporated by reference). In one embodiment, the antibody is a fully human antibody (eg, an antibody made in a mouse that has been genetically engineered to produce antibodies derived from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or Rat), goat, primate (eg, monkey), or camel antibody. In one embodiment, the non-human antibody is a rodent (mouse or rat antibody). Methods for making rodent antibodies are known in the art.

  Human monoclonal antibodies can be generated using transgenic mice with human immunoglobulin genes rather than mouse systems. Splenocytes from these transgenic mice immunized with the antigen of interest are used to generate hybridomas that secrete human mAbs with specific affinity for human protein-derived epitopes (eg, Wood et al. International Patent Application No. 91/00906 (Patent Document 19); Kucherlapati et al., International Patent Application No. 91/10741 (Patent Document 20), Lonberg et al., International Patent Application No. 92/03918 (Patent Document 21). Kay et al., International Patent Application No. 92/03917 (Patent Document 22); Lonberg et al. 1994 Nature 368: 856-859 (Non-Patent Document 36), Green, LL et al. (1994) Nature. Genet.7: 13-2 Morrison et al. (1994) Proc. Natl. Acad. Sci. USA 81: 6851-6855 (Non-patent document 38); Bruggeman et al. (1993) Year Immunol 7: 33-40. Non-Patent Document 39; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724 (Non-Patent Document 40); Bruggeman et al. 1991 Eur J Immuno 21: 1323-1326 (Non-Patent Document 39). (See Patent Document 41)).

  One example of a human monoclonal antibody is panitumumab (VECTIBIX®), formerly ABX-EGF, which is a fully human monoclonal antibody specific for epidermal growth factor receptor (EGFR). Panitumumab is used to treat patients suffering from metastatic cancer of the colon or rectum that express EGFR.

  An antibody may be one in which a variable region, or a portion thereof, eg, a CDR, is produced in a non-human organism, eg, a rat or mouse. Chimeras, CDR-grafts, and humanized antibodies can be used. Antibodies made in non-human organisms, such as rats or mice, and then modified, eg, with variable frameworks or constant regions, to reduce antigenicity in humans are within the scope of the invention.

  Chimeric antibodies can be made by recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding mouse Fc, and the equivalent of the gene encoding the human Fc constant region Substituting with a moiety (Robinson et al., International Publication No. PCT / US86 / 02269 (Patent Document 23); Akira et al., European Patent Application No. 184,187 (Patent Document 24); Taniguchi, M, European Patent Application No. 171,496 (Patent Document 25); Morrison et al., European Patent Application No. 173,494 (Patent Document 26); Neuberger et al., International Patent Application No. 86/01533. (Patent Document 27); Cabilly et al., National Patent No. 4,816,567 (Patent Document 28); Cabilly et al., European Patent Application No. 125,023 (Patent Document 29); Better et al. (1988) Science 240: 1041- Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443 (non-patent document 43); Liu et al., (1987) J Immunol.139: 3521. Sun et al. (1987) Proc.Natl.Acad.Sci.USA 84: 214-218 (Non-patent document 45); Nishimura et al., (1987) Canc. : 999-1005 (Non-Patent Document 4) Wood et al. (1985) Nature 314: 446-449 (Non-Patent Document 47); and Shaw et al., (1988) J. Natl Cancer Inst. 80: 1553-1559 (Non-Patent Document 48). ).

  A humanized or CDR-grafted antibody will have at least one or two but usually all three recipient CDRs (of heavy and / or light immunoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. The number of CDRs required for binding of the humanized antibody or fragment thereof may be replaced. The donor may be a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or a human consensus framework. In general, an immunoglobulin that provides a CDR is called a “donor” and an immunoglobulin that provides a framework is called an “acceptor”. In one embodiment, the donor immunoglobulin is non-human (eg, a rodent). The acceptor framework is a naturally occurring (eg, human) or consensus framework, or a sequence that is about 85% or more, or 90%, 95%, 99% or more identical thereto.

  In one embodiment, the ligand or antigen is CD3. In one embodiment, the ligand or antigen is EGFR. In one embodiment, the bispecific structure has both an anti-CD3 region and an anti-EGFR region.

  In one embodiment, the Fab is anti-CD3 and may have one or more of the following structures:

Binding region included in h145 Fab CSA The binding region of the antibody fragment may be derived from an anti-CD3 antibody or may include both heavy and light chains. For example, the heavy chain variable region derived from a hamster anti-mouse CD3 antibody (145-2C11) is as follows.

  AspGluValGlnLeuGlnGluSerGlyGlyGlyLeuValGlnProGlyLysSerLeuLysLeuSerCysGluAlaSerGlyPheThrPheSerGlyTyrGlyMetHisTrpValArgGlnAlaProGlyArgGlyLeuGluSerValAlaTyrIleThrSerSerSerIleAsnIleLysTyrAlaAspAlaValLysGlyArgPheThrValSerArgAspAsnAlaLysAsnLeuLeuPheLeuGlnMetAsnIleLeuLysSerGluAspThrAlaMetTyrTyrCysAlaArgPheAspTrpAspLysAsnTyrTrpGlyGlnGlyThr etValThrValSerSer (SEQ ID NO: 12)

  The light chain variable regions derived from the hamster anti-mouse CD3 antibody (145-2C11) are as follows.

  AspIleGlnMetThrGlnSerProSerSerLeuProAlaSerLeuGlyAspArgValThrIleAsnCysGlnAlaSerGlnAspIleSerAsnTyrLeuAsnTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrTyrThrAsnLysLeuAlaAspGlyValProSerArgPheSerGlySerGlySerGlyArgAspSerSerPheThrIleSerSerLeuGluSerGluAspIleGlySerTyrTyrCysGlnGlnTyrTyrAsnTyrProTrpThrPheGlyProGlyThrLysValGluIleLys (SEQ ID NO: 13)

Binding region included in hOKT3FabCSA The heavy chain variable region of humanized muromonab-CD3 (orthoclone OKT3 antibody) is as follows.

  AspGlnValGlnLeuValGlnSerGlyGlyGlyValValGlnProGlyArgSerLeuArgLeuSerCysLysAlaSerGlyTyrThrPheThrArgTyrThrMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpIleGlyTyrIleAsnProSerArgGlyTyrThrAsnTyrAsnGlnLysValLysAspArgPheThrIleSerArgAspAsnSerLysAsnThrAlaPheLeuGlnMetAspSerLeuArgProGluAspThrGlyValTyrPheCysAlaArgTyrTyrAspAspHisTyrCysLeuAspTyrTrpGly lnGlyThrProValThrValSerSer (SEQ ID NO: 14)

  The light chain variable region of humanized muromonab-CD3 (orthoclone OKT3) is as follows.

  AspAspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThrIleThrCysSerAlaSerSerSerValSerTyrMetAsnTrpTyrGlnGlnThrProGlyLysAlaProLysArgTrpIleTyrAspThrSerLysLeuAlaSerGlyValProSerArgPheSerGlySerGlySerGlyThrAspTyrThrPheThrIleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysGlnGlnTrpSerSerAsnProPheThrPheGlyGlnGlyThrLysLeuGlnIleThr (SEQ ID NO: 15)

In Fab, heavy chain can include a _{C H1} domain and hinge region of human IgG _1. An example of this arrangement is as follows:

  AlaSerThrLysGlyProSerValPheProLeuAlaProSerSerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPheProGluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPheProAlaValLeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValProSerSerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLysValAspLysLysValGluProLysSerCysAspLysThrHisThrCysProPro ysPro (SEQ ID NO: 16)

In Fab, kappa light chain constant domain of the IgG _1, may be added to the C-terminus of the light chain variable region. An example of a kappa light chain constant domain is as follows.

ArgThrValAlaAlaProSerValPheIlePheProProSerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsnPheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsnSerGlnGluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThrHisGlnGlyLeuSerSerProValThrLysSerPheAsnArgGlyGlu
Cys (SEQ ID NO: 17)

  In one embodiment, the multivalent antibody fragment comprises a scFv. One such scFv sequence is for EGFR shown below (763 scFv).

  AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThrIleThrCysGlnAlaSerGlnAspIleSerAsnTyrLeuAsnTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAspAlaSerAsnLeuGluThrGlyValProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThrIleSerSerLeuGlnProGluAspIleAlaThrTyrPheCysGlnHisPheAspHisLeuProLeuAlaPheGlyGlyGlyThrLysValGluIleLysGlyGlyGlyGly erGlyGlyGlyGlySerGlyGlyGlyGlySerGlnValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGluThrLeuSerLeuThrCysThrValSerGlyGlySerValSerSerGlyAspTyrTyrTrpThrTrpIleArgGlnSerProGlyLysGlyLeuGluTrpIleGlyHisIleTyrTyrSerGlyAsnThrAsnTyrAsnProSerLeuLysSerArgLeuThrIleSerIleAspThrSerLysThrGlnPheSerLeuLysLeuSerSerValThrAlaAlaAspThrAlaIleTyrTyrCysValArgAsp rgValThrGlyAlaPheAspIleTrpGlyGlnGlyThrMetValThrValSerSer (SEQ ID NO: 9)

B. Hinge region The fusion protein of the present invention may comprise a “hinge region”. In one embodiment, the hinge region is a sequence of approximately 4-15 amino acids in length. The hinge region can be a human IgG hinge region or a glycine linker. In one embodiment, the hinge region of human IgG is the hinge region of human IgG ₁ or IgG _2.

  In one embodiment, the “hinge region” has one of the following sequences:

  GluProLysSerGlyAspLysThrHisThrCysProProCysPro (SEQ ID NO: 18), or

  GluProLysSerCysAspLysThrHisThrCysProProCysPro (SEQ ID NO: 19) or one of the following.

  In one embodiment, the “hinge region” includes a glycine linker.

  Examples of glycine linkers (G-linkers) may include:

(GGGGS) ₃ (SEQ ID NO: 24). The most commonly used scFv linker comprises a 15 amino acid combination of glycine and serine residues.

  US Pat. No. 5,908,626 (Patent Document 9): GGSGGSGGGGSGGGGS (SEQ ID NO: 25) shown in a hybrid having interferon-β and immunoglobulin Fc linked by a peptide linker.

  RGRGRGRGRGRGGS obtained from scFv-RG3 (SEQ ID NO: 26).

  Linkers can also be used in other parts of the multivalent antibody fragment of the invention.

C. Collagen-Like Peptides A description of collagen-like peptides can be found in the description of collagen-like domains in US patent application Ser. No. 13 / 588,752, which is hereby incorporated by reference in its entirety. Explicitly incorporated into the document. Collagen-like peptides of the invention can include GPP or GPO motifs and / or trimerization motifs or other structures. For example, one such collagen-like peptide may have the following sequence:

  GlyProProGlyProProGlyProProGlyProProGlyProProGlyProProGlyProProGlyProProGlyProProGlyProPro (SEQ ID NO: 27)

D. Linker A linker is a short peptide sequence that can optionally be placed between an antibody fragment and a collagen-like peptide region, or between a binding domain and a collagen-like peptide region. The scFv polypeptide also includes a polypeptide linker between the _VH and _VL domains that allows the scFv to form the desired structure for antigen binding. In some embodiments, in any case, the linker is between 4 and 10 amino acids in length and can have the following sequence:

  Alaalaalaglyclyglyser (SEQ ID NO: 28) or glylyglyclyser (SEQ ID NO: 29)

Glycine linker (G-linker): (GGGGS) ₃ (SEQ ID NO: 24). The most commonly used scFv linker comprises 15 combinations of glycine and serine residues.

  U.S. Pat. No. 5,908,626, which is expressly incorporated herein by reference, "Hybrid with interferon-beta and an immunoglobulin Fc joined by a peptide linker" GGSGGSGGGGGGGGS (SEQ ID NO: 25) "found in" Interferon-beta and hybrid with immunoglobulin Fc ").

  Glycine-alanine linker: GGAGAGAG (SEQ ID NO: 30)

  Glycine-arginine linker: RGRGRGRGRGRGGGS (SEQ ID NO: 26)

  Particular embodiments include Forms AC as trimers shown in FIG. Format A: Fab CSA, a trimeric antibody fragment having a Fab fragment at the N-terminus of a collagen-like peptide capable of self-trimerization. Format B: FabCSA-scFv, a trimeric bispecific antibody fragment having a Fab fragment and a single chain antibody fragment at the N- and C-termini, respectively, of a collagen-like peptide capable of self-trimerization. Format C: FabCSA-sdAb, a trimeric bispecific antibody fragment having a Fab fragment and a single domain antibody at the N- and C-termini of a collagen-like peptide capable of self-trimerization, respectively. In particular, formats AC include three monomers, each monomer having a heavy and light chain in the Fab region.

  The following specific examples are illustrative only and are not to be construed as limiting in any manner other than this disclosure. Without being detailed, it is believed that those skilled in the art can make maximum use of the present invention based on the description herein. All publications cited herein are hereby incorporated by reference in their entirety.

Example 1
Construction of h145FabCSA, hOKT3FabCSA and hOKT3FabCSA763scFv Described below is the polypeptide sequence of the heavy chain of h145FabCSA (SEQ ID NO: 1) and the cDNA sequence encoding it (SEQ ID NO: 2). The coding region of the heavy chain of h145 FabCSA is from the N-to C-terminus, the signal peptide (underlined) (SEQ ID NO: 11), the heavy chain variable region derived from the hamster anti-mouse CD3 antibody (145-2C11) (bold) (SEQ ID NO: 12), _{C H1} domain and hinge region of human IgG ₁ (italics) (SEQ ID NO: 16), and _{(GPP) 10} collagen-like domain (double underline) (SEQ ID NO: 31) was followed one. This synthetic sequence (SEQ ID NO: 2) was prepared by overlapping PCR.

Described below is the light chain polypeptide sequence of h145 FabCSA (SEQ ID NO: 3) and the cDNA sequence encoding it (SEQ ID NO: 4). The coding region of the light chain of h145 FabCSA is the light chain variable region (bold) derived from the signal peptide (underlined) (SEQ ID NO: 11), hamster anti-mouse CD3 antibody (145-2C11) from N- to C-terminal (SEQ ID NO: includes a 13), and kappa light chain constant domain of human IgG ₁ (italics) (SEQ ID NO: 17) was followed one. This synthetic sequence (SEQ ID NO: 4) was prepared by overlapping PCR.

The h145 FabCSA heavy and light chain inserts were then cloned into a dual expression vector derived from pSecTag2 / Hygro (Invitrogen) for antibody expression in mammalian cells. Described below is the polypeptide sequence (SEQ ID NO: 5) of the heavy chain of hOKT3 FabCSA and the cDNA sequence encoding it (SEQ ID NO: 6). The coding region of the heavy chain of hOKT3FabCSA is from N- to C-terminal, signal peptide (underlined) (SEQ ID NO: 11), heavy chain variable region of humanized muromonab-CD3 (orthoclone OKT3) (bold) (SEQ ID NO: 14 ), includes a _{C H1} domain and hinge region of human IgG ₁ (italics) (SEQ ID NO: 16), and _{(GPP) 10} collagen-like domain (double underline) (SEQ ID NO: 31) was followed one. This synthetic sequence (SEQ ID NO: 6) was prepared by overlapping PCR.

Described below is the polypeptide sequence of the light chain of hOKT3FabCSA (SEQ ID NO: 7) and the cDNA sequence encoding it (SEQ ID NO: 8). The coding region of the light chain of hOKT3FabCSA is N- to C-terminal, signal peptide (underlined) (SEQ ID NO: 11), light chain variable region of humanized muromonab-CD3 (orthoclone OKT3) (bold) (SEQ ID NO: 15 ) a, and kappa light chain constant domain of human IgG ₁ (italics) (SEQ ID NO: 17) was followed one. This synthetic sequence (SEQ ID NO: 8) was prepared by overlapping PCR.

Subsequently, the above heavy and light chain inserts of hOKT3FabCSA were cloned into a dual expression vector derived from pSecTag2 / Hygro (Invitrogen) for antibody expression in mammalian cells. Described below are the polypeptide sequence of 763 single chain Fv, 763scFv (SEQ ID NO: 9) and the cDNA sequence encoding it (SEQ ID NO: 10). Based on the published nucleotide sequence (US Pat. No. 6,235,883), the cDNA encoding 763scFv _VL and _VH was obtained from the anti-EGFR monoclonal antibody panitumumab (VECTIBIX®, Amgen, Inc.) with a primer set from the corresponding cDNA. The 763 scFv PCR fusion was generated by ligating the _VL and V _H chains with the glycine linker (GGGGS) ₃ (SEQ ID NO: 24) underlined in SEQ ID NO: 9.

An anti-CD3 × EGFR bispecific antibody, hOKT3FabCSA763scFv, was generated as follows. The cDNA sequence encoding the 763ScFv, and cloned in frame to the C- terminus of the heavy chain of hOKT3FabCSA in AgeI and BamHI sites, a heavy chain construct consisting of the heavy chain of HOKT3Fab, the human IgG ₁ hinge region, (GPP) _Ten collagen-like domains (SEQ ID NO: 31) were created, followed by 763 scFv. The heavy chain construct described above and the hOKT3 FabCSA light chain construct (SEQ ID NO: 8) were then cloned into a pSecTag2 / Hygro (Invitrogen) derived double expression vector for antibody expression in mammalian cells.

To construct a low Fc-binding anti-human CD3 antibody-hOKT3IgG-AA with structural features similar to teplizumab (also referred to as MGA031 and hOKT3-γ1 (Ala-Ala)), the heavy and light chains of hOKT3 the variable regions pSecTag2 / Hygro (Invitrogen) and cloned into the human-derived IgG ₁ expression vector, wild-type leucine residue at amino acid 234 and 235 of the heavy chain constant region in which has been substituted by two alanine residues. In order to construct a low Fc binding anti-mouse CD3 antibody-145 IgG-AA, the heavy and light chain variable regions of the hamster anti-mouse CD3 antibody (145-2C11) were cloned into a mouse IgG _2a expression vector (InvivoGen) In which the wild type leucine residues at amino acids 234 and 235 of the heavy chain constant region were replaced with two alanine residues. In order to construct anti-human EGFR antibody-763 IgG with structural features similar to panitumumab (Vectibix®), the heavy and light chain variable regions of panitumumab were transferred into a human IgG ₂ expression vector derived from pSecTag2 / Hygro (Invitrogen) Cloned into.

Example 2
Expression and purification of h145FabCSA, 145IgG-AA and hOKT3IgG-AA using expression construct, using the expression construct of h145FabCSA, 145IgG-AA and hOKT3IgG-AA Mouse myeloma NS0 cells (European Collection of Animal Cell Cultures, Wiltshire, UK). After selection with hygromycin B (400 μg / ml), stable clones are placed in shake flasks to an initial seeding density of 5 × 10 ⁵ cells / ml and HyQCDM4NS0, a chemically-defined medium. (Hyclone). The culture was maintained at 130 rpm at 37 ° C. for 5 days. CHO-S cells (Life with the expression constructs of hOK3FabCSA, hOKT3FabCSA763scFv, and 763IgG by electroporation using an Amax Nucleofector device (Amaxa, Inc., Gaithersburg, Md.) According to the manufacturer's instructions. Technologies Corporation) respectively. After selection with hygromycin B (400 μg / ml), stable clones are placed in shake flasks to an initial seeding density of 3 × 10 ⁵ cells / ml and CD OptiCHO®, a medium with a well-defined chemical composition. Incubated with (Life Technology). The culture was maintained at 130 rpm at 37 ° C. for 12 days. Glucose was controlled at 2 mg / L by adding 30 mg / ml glucose solution.

  To purify h145 FabCSA, hOKT3 Fab CSA, and hOKT3 Fab CSA 763 scFv, about 1 L of each filtered medium was added at a flow rate of 60 ml / h, phosphate buffered saline (PBS), pH 7.4 (0.01 M phosphate buffer, 0.0027 M It was applied to a KappaSelect column (5 ml in bed volume, GE Healthcare) equilibrated with KCl, 0.14 M NaCl). After washing with the same buffer, the recombinant antibodies were eluted with sodium phosphate buffer 50 mM, pH 2.5. The UV absorbance was monitored at 280 nm and the peak fractions were collected and neutralized with 1.0M sodium bicarbonate to pH 7.5. The neutralized sample was then concentrated by ultrafiltration using an Amicon Ultra-15 Centrifugal Filter Unit (EMD Millipore Corporation, Billerica, Mass.) Equipped with an Ultracel-30 membrane. 5 milliliters of the concentrate was applied to a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with phosphate buffered saline (PBS), pH 7.4, at a linear flow rate of 1.5 ml / min.

  To purify h145IgG-AA, hOKT3IgG-AA, and 763 IgG, approximately 1 L each of filtered media was added at a flow rate of 60 ml / h, phosphate buffered saline (PBS), pH 7.4 (0.01 M phosphate). It was applied to a HiTrap Protein A HP column (5 ml in bed volume, GE Healthcare) equilibrated with buffer, 0.0027 M KCl, 0.14 M NaCl). After washing with the same buffer, the recombinant antibodies were eluted with sodium phosphate buffer 50 mM, pH 2.5. The UV absorbance was monitored at 280 nm and the peak fractions were collected and neutralized with 1.0M sodium bicarbonate to pH 7.5. The neutralized sample was dialyzed against phosphate buffered saline (PBS), pH 7.4.

  SDS-PAGE was performed using MES as a running buffer (Invitrogen, San Diego, Calif.) And 4-12% of any NuPAGE bis-Tris polyacrylamide gel. Proteins were stained with InstantBlue (Expedon, Cambridgeshire, UK). Bench Mark (Invitrogen, San Diego, CA) was used as the molecular weight standard.

Results: Chromatographic and structural characterization of h145 FabCSA FIG. 2 shows the structural characterization of h145 FabCSA molecules purified from the medium by continuous chromatography on KappaSelect and Superdex 200 columns, according to an embodiment of the present invention. . (A) Separation of h145 FabCSA species by gel filtration. The sample eluted from the KappaSelect column was concentrated and loaded onto a Superdex 200 gel filtration column equilibrated with PBS (pH 7.4). (B) Different peak fractions (numbered in peaks 1 to 3 in A) and sample loading, non-reducing (lanes 2, 4, 6, and 8), and samples at 75 ° C. with DTT 50 mM Analysis by SDS-PAGE under reduction (lanes 3, 5, 7, and 9) conditions treated for 10 minutes. (C) Schematic representation of the structure corresponding to the species separated by SDS-PAGE shown in B.

  These results demonstrate that the collagen-like peptide of the present invention has the ability to trimerize the anti-mouse CD3Fd fragment and that the complete Fab fragment can associate in eukaryotic cells and be secreted as a stable trimer. did.

Results: Chromatography and structural characterization of hOKT3 Fab CSA FIG. 3 shows the structural characterization of hOKT 3 Fab CSA molecules isolated by Superdex chromatography, according to an embodiment of the invention. The sample eluted from the KappaSelect column was concentrated and loaded onto a Superdex 200 gel filtration column equilibrated with PBS (pH 7.4). Upper right panel: different peak fractions (peaks 1 to 3 are numbered) were analyzed by SDS-PAGE under non-reducing conditions. Peak 2 is the main fraction containing hOKT3 Fab CSA trimer, while the fraction of peak 3 contains monomer. The major upper band shown in peak 1 appears to be a trimeric interchain disulfide bonded dimer.

  Again, these results indicate that the collagen-like peptide of the present invention has the ability to trimerize anti-human CD3Fd fragments, and that the complete Fab fragment associates in eukaryotic cells to form a stable trimer. Proven to be secreted.

Results: Purity analysis of hOKT3FabCSA trimer, hOKT3IgG-AA, and OKT3 by SDS-PAGE. FIG. 4 shows non-reduction (lanes 2, 3 and 4) and reduction of samples treated with DTT 50 mM at 75 ° C. for 10 minutes (lane 5). , 6 and 7) shows the purity analysis by SDS-PAGE of hOKT3FabCSA, low Fc binding anti-human CD3 antibody-hOKT3IgG-AA, and OKT3 antibody from the peak 2 fraction in FIG. All samples containing equal amounts of protein were electrophoresed on a 4-12% SDS / Bis-Tris polyacrylamide gel using MES as the running buffer. Gels were stained with InstantBlue Protein Stain solution. M, BenchMark® molecular weight standard.

Under non-reducing conditions, the major band of hOKT3 Fab CSA appeared as a disulfide-bonded Fab trimer (lane 2), while under non-reducing conditions, non-disulfide-bonded Fd-Cs and light chain were separated (lane 5). hOKT3IgG-AA and OKT3 antibodies are in the normal IgG ₁ format and contain two heavy chains and two light chains (lanes 3 and 4 under non-reducing conditions, lanes 6 and 6 under reducing conditions) 7).

Example 3
Determination of CD3 binding activity Human T cells (1 × 10 ⁶ / ml) were isolated from PBMCs according to the manufacturer's instructions using the Pan T cell isolation Kit (Miltenyi Biotec, CA) for negative selection. The cells were treated with Fc blocker (2 μg / ml, eBioscience, CA) for 30 minutes at 4 ° C. and then serial dilutions of purified low Fc-conjugated anti-human CD3 antibody-hOKT3IgG-AA, hOKT3FabCSA trimer and monomer Incubated with the solution at 4 ° C. for 30 minutes. After washing, the cells were stained with goat anti-human IgG (H + L) -Alexa Fluor 647 (Invitrogen) for 30 minutes at 4 ° C., and binding of the antibody to T cells was detected by flow cytometry, and mean fluorescence intensity (MFI) As shown. As shown in FIG. 5, trimer HOKT3FabCSA (calculated _K D = 0.02 nM), the divalent counterpart (counterpart), than hOKT3IgG-AA (calculated _K D = 0.15 nM) It showed a binding strength to human T lymphocytes 7.5 times larger. The monovalent form of hOKT3 Fab CSA was weak and showed insignificant binding affinity to CD3 + human T cells.

This experiment was also performed to determine the binding affinity of purified h145 Fab CSA trimer to mouse spleen T lymphocytes compared to 145-2C11 hamster IgG (FIG. 6). Again, the trimer 145 FabCSA (calculated K _D = 4 nM) is 3.75 times larger mouse T lymphocytes than the parent hamster IgG, 145-2C11 (calculated K _D = 15 nM). The bond strength to was shown.

Example 4
Competitive displacement binding assay Since OKT3 and hOKT3FabCSA recognize the same epitope on the CD3ε chain, hOKT3FabCSA can competitively inhibit the binding of OKT3 to T cells. The activity of hOKT3 FabCSA and OKT3 for binding to CD3 molecules on the cell surface of human T cells was compared by flow cytometry analysis using an antibody displacement assay with a saturating concentration of fluorescein conjugated OKT3 as a competitor. All the following procedures were performed at 4 ° C. CD3 (+) Jurkat T cells, clone E6-1 (ATCC No. TIB-152), with a total number of cells of 1 × 10 ⁶ , 0.1 ml staining buffer (2% fetal calf serum and 0.1% azide) It was suspended in phosphate buffered saline containing sodium). The cells were cultured for 1 hour with serial dilutions of hOKT3 Fab CSA or OKT 3 IgG. A fixed, saturating amount (0.25 μg / ml, determined by flow cytometry) of FITC-conjugated OKT3 (eBioscience, San Diego, Calif.) Was added directly. After 1 hour of culture, the cells were washed with staining buffer and analyzed for immunofluorescence by flow cytometry on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) system. The data is shown as percent inhibition of maximum fluorescence intensity, which is defined as the mean fluorescence intensity obtained by staining T cells with OKT3-FITC in the absence of blocking mAb. The concentration of each mAb required to inhibit half of the maximum fluorescence intensity (IC ₅₀ ) was calculated.

Comparison of IC ₅₀ values showed that OKT3 required approximately 2-fold higher concentrations than hOKT3 FabCSA to achieve the same inhibitory effect using Jurkat T cells (FIG. 7, two independent experiments). It is shown). These results indicated that due to the avidity effect, humanized trivalent hOKT3 Fab CSA has stronger binding strength than its parent mouse IgG form, OKT3.

Example 5
T cell proliferation Tests comparing OKT3, hOKT3IgG-AA and hOKT3FabCSA in T cell activation were performed by a cell proliferation assay. Human peripheral blood mononuclear cells (PBMC) collected from 3 healthy donors were placed in a black 96 well flat bottom tissue culture plate at 2 × 10 ⁵ cells / well in 100 μl of RPMI-1640 medium containing 10% FBS. eBioscience, Inc.), hOKT3IgG-AA and hOKT3FabCSA in the presence of 10-fold serial dilutions and placed at 37 ° C. for 66 hours. Subsequently, the cells were pulsed with 10 μM BrdU for an additional 6 hours. After removing the medium, the cells were fixed, and the DNA was denatured in one step using FixDenat (Roche Applied Science, Indianapolis IN). The 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). Each point shown in FIG. 8 represents the mean ± standard deviation of 3 donors.

  The results in FIG. 8 showed that OKT3 significantly induced T cell proliferation even at very low concentrations. Low Fc-conjugated hOKT3IgG-AA was also weakly inducing, but induced T cell proliferation at relatively high antibody concentrations. These results are based on the work previously published by Li et al. (Li, J., J. Davis, et al., (2006). “Modulation of antigen-specific T cell response by a non-mitogenic anti-CD3. antibody. "Int Immunopharmacol 6 (6): 880-891. (non-patent document 49)). On the other hand, there was no detectable T cell proliferation induced by hOKT3 Fab CSA. Thus, non-Fc hOKT3 Fab CSA trimer is the weakest inducer of T cell proliferation among different anti-CD3 antibody formats.

Example 6
Cytokine measurements Human PBMC from 3 healthy donors were placed in 2 × 10 ⁵ cells in 0.1 ml of RPMI-1640 medium containing 10% FBS in the presence of 10-fold serial dilutions of OKT3, hOKT3IgG-AA and hOKT3FabCSA. Seeded at 37 ° C./well. The levels of IL-2 and the remaining cytokines (IL-1β, IL-6, IL-10, and IFN-γ) in the culture supernatant were determined using a human cytokine immunoassay kit (eBioscience, Inc.) 24 and Measurements were taken at 72 hours.

  The mitogenic activity of murine OKT3 is caused by extensive T cell receptor (TCR) -CD3 cross-linking through binding to FcR-positive cells. Therefore, in recent years, an attempt has been made to develop a non-mitogenic form that does not promote cell division of anti-CD3 by changing the binding to the Fc receptor. The ability of OKT3, hOKT3IgG-AA and trimeric hOKT3 FabCSA to induce cytokines (IL-1β, IL-2, IL-6, IL-10, and IFN-γ) was measured. As expected, OKT3 IgG significantly induced cytokine production at very low doses. Low Fc-binding anti-human CD3 antibody-hOKT3IgG-AA, which has structural features similar to teplizumab (also referred to as MGA031 and hOKT3-γ1 (Ala-Ala)), is also a relatively high antibody, although weakly acting Cytokine production was induced at the concentration. These results are consistent with the work previously published by Li (Li, J., J. Davis et al., (2006) (non-patent document 49)). On the other hand, trimeric hOKT3 Fab CSA does not induce detectable IL-2 and IFN-γ at concentrations up to 100 μg / ml. Compared to hOKT3IgG-AA, the induction levels of IL-1β, IL-6, and IL-10 at concentrations of 10 μg / ml or higher of hOKT3FabCSA were lower. These results indicated that hOKT3 Fab CSA was the weakest mitogen among different anti-CD3 antibody formats (FIG. 9).

Example 7
Mixed lymphocyte reaction (MLR)
Immunosuppression in the unidirectional mixed lymphocyte reaction was evaluated as follows. Human PBMC were obtained from two healthy donors (stimulator and responder). 25 μg of mitomycin C (Sigma-Aldrich) in complete medium (RPMI 1640 supplemented with 10% human AB serum, 2 mM glutamine, 50 nM 2-mercaptoethanol, and penicillin and streptomycin 100 units / ml each). Treated at 37 ° C. for 30 minutes in humidified air containing 5% CO ₂ , followed by 3 washes in RPMI 1640 medium. Responder cells were mixed alone or with a mitomycin C-treated stimulator or mitomycin C responder cells at a 1: 1 ratio and cultured in 200 μl of complete medium at 2 × 10 ⁵ cells / well. Immediately after seeding the responder cells, purified hOKT3FabCSA trimer, hOKT3IgG-AA, or OKT3 was added to the cultures at different concentrations. After 5 days, cultured cells were pulsed with 10 μM BrdU and harvested 24 hours later. A 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed. After removing the medium, the cells were fixed, and DNA was denatured in one step using FixDenat. The 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).

  Whether trimeric hOKT3FabCSA can exhibit greater immunosuppressive activity than parental OKT antibody and low Fc-conjugated anti-human CD3 antibody-hOKT3IgG-AA due to increased binding avidity to CD3 (+) T cells To determine, antibodies were tested for T cell division activation in a one-way mixed lymphocyte reaction (MLR). In a mixed PBMC culture (stimulator + responder treated with mitomycin C) cultured for 5 days without antibody treatment, a mixed lymphocyte reaction (MLR) occurred as a result of allogeneic stimulation of T cell activation ( FIG. 10, black square). Treatment of mixed PBMC cultures with OKT stimulated T cell proliferation at low concentration levels from 0.1 to 1.0 ng / ml (FIG. 10, black circles). When the antibody concentration is higher than 10 ng / ml, the immunosuppression of OKT3 becomes significant. Low Fc-conjugated anti-human CD3 antibody-hOKT3IgG-AA did not show significant suppression of T cell activation (FIG. 10, open squares). In contrast, the potency of hOKT3 FabCSA trimer in MLR was significant, reaching background levels at a concentration of 0.1 ng / ml (Figure 10, white triangles). These results indicated that trimeric hOKT3FabCSA is a potent immunosuppressant of T cell proliferation while showing reduced mitogenicity in vitro.

Example 8
T cell receptor (TCR) modulation PBMCs from healthy donors were seeded at 2 × 10 ⁶ cells / well in 24-well plates (Nunc) and various amounts of hOKT3 FabCSA (trivalent) in RPMI 1640 with 10% FCS Body), hOKT3IgG-AA (dimer), and hOKT3FabCSA (monomer). After culturing for 24 hours, cells were harvested and stained with FITC-conjugated OKT3 or anti-TCRα / β mAb IP26 (eBioscience). Stained cells were counterstained with phycoerythrin-conjugated anti-CD5 mAb UCHT2 (eBioscience) on T cells and analyzed by flow cytometry. CD3 modulation and coating calculations were performed as previously described (Cole, MS, C. Anasetti et al., (1997). “Human IgG ₂ variants of chimeric anti-CD3 arenon-mitogenic to T cells. "J Immunol 159 (7): 3613-3621 (non-patent document 50)).

Here, F represents the average fluorescence of the stained cells.

  In FIG. 11, the combination of TCR-CD3 complex modulation and coating achieved by the trivalent hOKT3 FabCSA is from low Fc-conjugated anti-human CD3 antibody-hOKT3IgG-AA at antibody concentrations from 0.1 ng / ml to 10 μg / ml. Is also big. Due to the low cross-linking activity against TCR, monovalent hOKT3 Fab CSA showed much lower TCR coating and modulation at each corresponding concentration. These results demonstrated that improved binding strength of the trimeric hOKT3 FabCSA resulted in an increased degree of TCR regulation.

Example 9
Pharmacokinetic assays
To assess h145 FabCSA blood clearance, 15 male BALB / c mice (randomly divided into 5 groups) were given a single dose level of 25 μg / mouse, a dose volume of 0. Purified h145 Fab CSA trimer was administered intravenously at 1 ml / mouse. Blood (100 μl / mouse) was collected from mice sequentially at 0, 2, 5, 15, 30 minutes, 1, 2, 4, 6, 8, 24, 48, 72, and 96 hours and coated with EDTA. Transfer to tube. Using ELISA, the remaining h145 FabCSA in the plasma samples at each time point was quantified by interpolation into a standard curve of serial dilutions of h145 Fab CSA standards. Anti-human Fd and horseradish peroxidase conjugated anti-human kappa light chain were used as capture and detection antibodies, respectively. 3,3 ′, 5,5 ′,-tetramethylbenzidine was used as the peroxidase substrate. The pharmacokinetic profile of h145 Fab CSA in mice is shown in FIG.

The kinetics of the non-compartment model was determined. The immunoreactive concentration in plasma decreased biphasically, with a terminal elimination phase half-life (t _1/2 ) of 8.85 hours.

Example 10
Biodistribution Purified h145 FabCSA trimer was radiolabeled with iodo-131 and the biodistribution of ¹³¹ I-h145 FabCSA in 30 male BALB / c mice was evaluated. A group of 5 mice was administered h145 Fab CSA intravenously (specific activity: 1.2 μCi / μg; 30 μCi / mouse). The time points for analysis of h145 FabCSA were 0.5, 1, 2, 6, 24, and 48 hours. Euthanized at these selected time points and the percent injected dose per gram of tissue (% ID / g) was determined. ^{The 131} I-h145 FabCSA trimer showed rapid blood clearance by 48 hours and was largely removed (Table 1). However, ¹³¹ I-h145 FabCSA showed localization in the spleen, reaching 33.76 ± 1.56% ID / g in 0.5 hours and a maximum of 42.09 ± 1.64% ID / g in 24 hours. .

Example 11
Experimental Autoimmune Encephalomyelitis Mouse Disease Model Experimental autoimmune encephalomyelitis (EAE) is a widely used mouse model of multiple sclerosis. In this study, myelin oligodendrocyte glycoprotein (MOG) induced mouse EAE was used to examine the therapeutic effect of h145 Fab CSA on paralysis. 6-8 week old female C57BL / 6 mice (National Laboratory Animal Center, Taiwan) in MOF (35-L) in complete Freund's adjuvant containing 500 μg of Mycobacterium tuberculosis H37RA (complete Freund's adjuvant). 55) 200 μg was immunized subcutaneously. Immediately after immunization, 500 ng of pertussis toxin was intraperitoneally administered to mice, and again administered 48 hours later. The following criteria were used to assess clinical EAE scores. 0 = asymptomatic, 0.5 = distal weak or tail tail, 1 = complete tail tail, 1.5 = tail droop and hindlimb weakness (Hindlimb weakness) (foot slips out of cage lattice), 2.0 = unilateral partial hindlimb paralysis, 2.5 = bilateral partial hindlimb parasitism , 3.0 = complete bilateral hindlimb paralysis, 3.5 = complete hindlimb and unilateral partial hindlimb paralysis terial partial forelimb paralysis), 4.0 = moribund and 5 = death. A total of 40 mice were assigned to each of 4 groups of 10 mice (solvent: hamster IgG isotype control, treatment: 145-2C11 IgG and h145 FabCSA (trimer), and positive control: interferon-β1a). Treatment began when the first clinical signs of EAE developed and the effects of different test substances on paralysis were tested. In the group of vehicle, 145-2C11 IgG and h145 FabCSA, mice were intravenously injected once daily for 5 consecutive days at dose levels of 1.0, 0.1 and 1.0 μg / mouse, respectively. In the interferon-β1a group, mice were injected intraperitoneally at a dose level of 10,000 units / mouse once a day for the entire treatment period.

  Initially, EAE mice treated with 145-2C-11 IgG at dose levels above 0.2 μg / mouse had a 50% mortality rate one day after treatment. Gross necropsy observations revealed that all dead mice had splenomegaly, indicating a serious uncontrolled systemic inflammatory response or cytokine storm. Therefore, the experimental dose level of the 145-2C-11 group was reduced to 0.1 μg per mouse. In the trimeric 145 Fab CSA group, treatment was performed at a dose level of 1.0 μg / mouse. These results indicated that trimer h145 FabCSA produced relatively low levels of paralysis at all stages of immunization (see FIG. 13A). Specifically, mice treated with h145 FabCSA showed a lower degree of paralysis compared to mice treated with 145-2C11 IgG or the control treatment group, and statistics between the h145 FabCSA treatment group and the 145-2C11 IgG treatment group Showed lower paralysis. Eventually, the untreated group showed as severe symptoms as complete bilateral hindlimbs, whereas the highest level of paralysis demonstrated by the h145 Fab CSA treatment group was unilateral hindlimb paralysis (ie clinical criteria 2) It was less heavy.

  Induction of regulatory T (Treg) cells is one of the major goals in immunotherapy of autoimmune diseases. Previous studies on orally administered CD3-specific antibodies have demonstrated that CD4 + CD25-LAP + Treg cells have been induced (Ochi et al., (2006) "Oral CD3-specific antigenic supraimmunis immunity in CD25 + CD4 LAP + T cells. "Nat Med 12 (6): 627-635 (Non-Patent Document 51)). A comparison of Treg populations in EAE mice after treatment with h145-2C-11 IgG and trimeric h145 FabCSA was performed. One day after the last intravenous injection, spleen lymphocytes from 3 mice from each of the vehicle (hamster control IgG), 145-2C11 IgG and the trimeric h145 FabCSA group were individually prepared and then subjected to LAP for flow cytometry. -Stained with APC and CD4-FITC. Splenocytes were stained with LAP-APC and CD4-FITC for flow cytometry. Treg population determination was gated on LAP + cells.

  These results showed that after h145 FabCSA treatment, the CD4 + LAP + T cell population was increased compared to the isotype IgG control and the 145-2C-11 group (see FIG. 13B). In particular, the h145 FabCSA treatment group also increased the CD4-LAP + T cell population, which could show CD8 + LAP + T cells.

Example 12
Systemic lupus erythematosus (SLE) in a mouse model
The therapeutic effect of anti-CD3 antibody of 145-2C11 IgG and h145 FabCSA trimer on NZB / W F1 mice with spontaneous erythema developed lupus was investigated. Approximately 6 weeks old female NZB / W F1 mice that developed spontaneous lupus erythematosus (spontaneous lupus) were divided into 6 courses every other week for 12 weeks for 6 days, 5 μg isotype IgG control, 145-2C11 IgG or h145 FabCSA ( Trimer). As shown in FIG. 14A, mice treated with h145 FabCSA survived longer than mice treated with isotype IgG control, and trimeric h145 FabCSA was more active in treatment of the SLE mouse model compared to 145-2C11 and isotype IgG control. It has been shown to be effective.

Example 13
Measurement of mouse serum antibodies against double stranded (dsDNA) When erythematous lupus is active, there are large amounts of serum anti-dsDNA antibodies. Thus, disease progression in the SLE mouse model was measured by an anti-dsDNA autoantibody test. For detection of anti-dsDNA autoantibodies, ELISA of mouse sera from the above treatment groups was measured as follows. A 96-well polystyrene ELISA plate was coated with 50 μl of bovine serum-derived methylated bovine albumin (Sigma) (50 μg / ml in distilled water) and cultured at 37 ° C. for 1 hour. Each well was then washed 3 times with phosphate buffered saline before 50 μl dsDNA (10 μg / ml) was added in PBS. dsDNA was prepared by treating bovine thymus DNA (Sigma) with S1 nuclease (Sigma) 1 U / mg for 30 minutes at 37 ° C. After overnight culture at 4 ° C., the plate was washed 4 times with PBS. These were treated with PBS containing 2% BSA (100 μl (Sigma)) so as to prevent non-specific binding, incubated at 37 ° C. for 1 hour, and then PBS containing 0.05% Tween-20 (PBS-T) ( Sigma) was washed 5 times. Test serum samples were diluted 800-fold with PBS-T and 50 μl aliquots were added in duplicate to the wells. After culturing at 37 ° C. for 1 hour, the plate was washed 6 times with PBS-T. For detection of total IgG antibody, 50 μl / well of HRP-conjugated rat anti-mouse antibody (BD Biosciences) diluted 5000 times was added and incubated at 37 ° C. for 1 hour. After washing 7 times with PBS-T, the reaction was initiated with 100 μl / well of citrate phosphate buffer containing 1 mg / ml of tetramethylbenzene (Sigma-Aldrich) and stopped by adding 50 μl / well of 1N HCl. It was. Absorbance at 450 nm was measured with an ELISA reader.

  As shown in FIG. 14B, the level of anti-dsDNA autoantibodies in h145 FabCSA treated mice appears to be significantly lower than that in the control isotype IgG group and even lower than most of the animals treated with 145-2C11 IgG. Met. These results indicated that trimeric h145 FabCSA was effective in the treatment of the SLE mouse model and there was no disease progression over the course of treatment.

Example 14
In Vivo Proinflammatory Cytokine Analysis Already administered anti-mouse CD3 antibody, 145-2C11 is specific to T cell proliferation and IL-2, IFNγ, TNFα, IL-1β, IL-6, IL-10 and IL17A. Associated with the induction of cytokines. A non-Fc h145 Fab CSA (trimer) is non-mitogenic in a time course study of serum pro-inflammatory cytokine levels in mice following intravenous injection of a single dose of 50 μg of different anti-CD3 antibodies It was evaluated whether it was useful for a therapeutic effect accompanied by sex (non-mitogenicity). Mice were grouped and 50 μg of purified 145-2C11 IgG (light gray squares), low Fc binding anti-mouse CD3 antibody-145 IgG-AA (dark gray squares), or h145 FabCSA trimer (white squares) intravenously It was administered internally. Blood (100 μl / mouse) was collected at 0 (prebleed), 0.5, 24, and 144 hours. Cytokine levels were measured using an ELISA. Each point represents the mean ± standard deviation of three wells. PBS (black square) was used as a control.

  The result is shown in FIG. As expected, the most cytokines were produced significantly in the 145-2C11 group. Low Fc binding 145 IgG-AA results in moderate transient induction of IL-2, TNFα, IL-6, and IL-10. Administration of non-Fc h145 Fab CSA (trimer) showed negligible levels of cytokine induction. These results indicated that after the first injection, trimeric h145 FabCSA did not induce cytokine production in vivo.

Example 15
Chromatography and structural characterization of the trivalent bispecific anti-CD3 × EGFR antibody, hOKT3FabCSA763scFv. The multivalent antibody fragment of the present invention is obtained by the fusion of two different antibody fragments with a collagen scaffold at either end. It is particularly suitable for making bispecific antibodies. A trivalent bispecific anti-CD3xEGFR antibody, hOKT3FabCSA763scFv, targets both CD3 and EGFR and was created to demonstrate the use of such modalities. FIG. 16 shows the structural characterization of hOKT3FabCSA763scFv molecules from non-single (A) or single stable clones (B). Each medium was purified by continuous chromatography on a KappaSelect and Superdex 200 column. (A) Separation of non-clone-derived hOKT3FabCSA763scFv species by gel filtration. Upper right panel: Different peak fractions (peaks 1 to 3 are numbered) were analyzed by SDS-PAGE under non-reducing conditions. The structures of disulfide bond trimer (T), non-disulfide bond trimer (Mt), and monomer (Mm) are shown. (B) Separation of stable clone-derived hOKT3FabCSA763scFv species by gel filtration. Peak fractions were analyzed by SDS-PAGE under non-reducing conditions. The structures of disulfide bond trimer (T) and non-disulfide bond trimer (Mt) are shown.

  These results demonstrated that the collagen-like peptide of the present invention has the ability to simultaneously trimerize the N-terminal Fd fragment and the C-terminal scFv fragment in eukaryotic expression systems. In addition, the light chain can associate with the Fd portion of the trimerized polypeptide chain to form a complete Fab trimer having the structural format shown in FIG. 1B.

Example 16
Binding Specificity of Trimeric Bispecific Antibody-hOKT3FabCSA763scFv to EGFR (+) and CD3 (+) Cells Binding of hOKT3FabCSA763scFv to each antigen was confirmed by flow cytometry. Strong reactivity was observed with A431, WiDr, and HCT116 cells (EGFR positive) and Jurkat T cells (CD3 positive, FIG. 17).

Example 17
Cytotoxicity assay Fluorescence-based Eu TDA non-radioactive cytotoxicity assay (Perkin Elmer, Boston, Mass.) Was used to induce trimeric hOKT3FabCSA763scFv against tumor cell lines with different human EGFR (EGFR-bearing) as an effector. Cytotoxicity was compared. Human PBMCs from one healthy donor were transferred to 72 by proliferating cells in plates coated with OKT3 (2 μg / ml) in RPMI + 10% FBS containing 50 units / ml (or 0.2 ng / ml) IL-2. Stimulated for hours. Approximately 1 × 10 ⁶ target cells were labeled with DELFIA BATDA Reagent according to the manufacturer's instructions. The cells were washed 3 times with PBS before Eu ³⁺ labeled target cells were resuspended in complete medium (CM) at a concentration of 5 × 10 ⁴ cells / ml. A 100 μl aliquot of target cells (5 × 10 ⁴ cells) was added to the wells of a 96 well V bottom sterile microtiter plate. An equal volume of effector PBMC was added to each well to give an effector / target (E / T) ratio of 50: 1 to 2.5: 1 for A431 cells and a constant 10: 1 ratio for WiDr and HCT116 cells, respectively. The microplate was incubated at 37 ° C. for 2 hours in a humidified hood with 5% CO ₂ . All assays were performed in triplicate. After incubation, the plate was centrifuged again and the supernatant was collected for measurement of released Eu ³⁺ . To detect released Eu ³⁺ , a 20 μl aliquot of the supernatant was transferred to a well of a flat bottom 96 well microplate and a 200 μl aliquot of enhancement solution was added to each well.

  After mixing for 15 minutes at room temperature on a rotary shaker, fluorescence was measured with a time-resolved fluorometer (Hidex, CHAMELION detection platform, Finland). The percentage of specific cytotoxicity was calculated as (experimental release-spontaneous release) / (maximum release-spontaneous release) x 100. Spontaneous release was determined by culturing target cells with 100 μl CM instead of effector cells, and maximal release was determined by culturing the target with 100 μl lysis buffer (0.5% Triton-X100).

  The ability of the trimeric bispecific hOKT3FabCSA763scFv to promote lysis of different EGFR expressing tumor cells including A431, WiDr and HCT116 cell lines was evaluated by human PBMC. FIG. 18 shows the specific cytotoxicity of various concentrations of trimeric hOKT3FabCSA763scFv against A431 cells with different E / T ratios (A), and WiDr or HCT116 cells with a constant E / T ratio of 10: 1 (B). Show. Lysis of EGFR overexpressing A431 tumor cells in the presence of stimulated human PMBC was specifically triggered by the trimeric bispecific hOKT3FabCSA763scFv in a dose and E / T ratio dependent manner (FIG. 18A). As a result, the specific cytotoxicity was maximally ˜80% at a concentration level of 0.05 μg / ml of hOKT3FabCSA763scFv and an E / T ratio of 20: 1 (black square). In the absence of hOKT3FabCSA763scFv, stimulated human PMBC alone was unable to effectively kill A431 cells. Similar results were obtained by culturing either WiDr or HCT116 cell lines with the trimeric OKT3FabCSA763scFv at a constant E / T ratio of 10: 1 (FIG. 18B). These results demonstrated that the trimeric hOKT3FabCSA763scFv can effectively induce lysis of tumor cells with EGFR by human PBMC (possibly cytotoxic T cells).

Example 18
Time-lapse microscopy The time-lapse micrograph in FIG. 19 further demonstrates the redirection of human cytotoxic T lymphocytes into tumor cell lysis by the trimeric hOKT3FabCSA763scFv. Trivalent hOKT3FabCSA763scFv can temporarily bind T cells and tumor cells by simultaneously binding CD and target antigen-EGFR, respectively. This causes T cell activation, followed by attack on the cross-linked tumor cells. Attacked tumor cells undergo programmed cell death (apoptosis). Free T cells can be newly killed by going to another tumor cell and cross-linking with another hOKT3FabCSA763scFv. In the absence of hOKT3FabCSA763scFv, human T lymphocytes did not affect the growth of tumor cells A431. In the lower diagram of FIG. 19, human T lymphocytes and A431 cells were cross-linked in the presence of the trimer hOKT3FabCSA763scFv. T lymphocytes were activated and began to attack tumor cells. In the 13 hour incubation period, the majority of tumor cells reached lysis by the apoptotic pathway. In the absence of hOKT3FabCSA763scFv, no cross-linking was observed between A431 cells and T lymphocytes even after a 24-hour co-culture time (upper figure in FIG. 19).

Example 19
Tumor xenograft mouse model In vivo studies to evaluate anti-tumor activity were performed in 8-10 week old female NOD / SCID mice (NOD. CB17-Prkdc ^scid / JNarl, National Laboratory Animal Center, Taiwan). On day 0, unstimulated human PBMC (effector) from one healthy donor was premixed with HTC116 tumor cells (target) and the mixed cells were then injected subcutaneously into NOD / SCID mice. Cell ratios as low as 1: 1 (effector to target) were used. The indicated doses of hOKT3FabCSA763scFv or PBS solvent control were administered by tail vein injection once daily for 10 consecutive days starting on day 1. Tumor progression was measured twice weekly with an external caliper meter and tumor volume was calculated using the standard hemiellipoid equation: (length x width ² ) / 2.

The result is shown in FIG. FIG. 20 shows the antitumor effect of the trimeric hOKT3FabCSA763scFv in the HCT116 colon cancer NOD / SCID mouse model. In the absence of human PBMC, two groups of NOD / SCID mice were inoculated subcutaneously with 5 × 10 ⁶ HCT116 cells (anti-EGFR 763 IgG and PBS control). The remaining three groups were injected subcutaneously with a mixture of 5 × 10 ⁶ HCT116 cells and 5 × 10 ⁶ unstimulated human PBMC from a healthy donor. After inoculating HCT116 cells on day 0, PBS solvent control (100 μl), trimer hOKT3FabCSA763scFv 50 μg and 15 μg were administered via tail vein injection for 10 consecutive days from day 1. Tumor growth curves derived from each group of the number of animals denoted n are shown.

  Statistically significant differences (P <0.001) between administration of the trimeric hOKT3 Fab CSA 763 scFv group and the unstimulated human PBMC control group are shown. These results showed that the bispecific anti-CD3xEGFR antibody of hOKT3FabCSA763scFv can effectively reduce tumor growth in the presence of human PBMC.

  It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the details and examples of the invention be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims and their equivalents.

Claims (28)

A method for producing a multivalent Fab fragment comprising:
(1) a gene construct encoding an amino acid sequence comprising a heavy chain portion of a Fab fragment and an in-frame fusion collagen-like peptide;
(2) a gene construct encoding an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Co-expressing in a host cell. The method of claim ₁ , wherein the human IgG is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .   The method according to claim 1, wherein the gene construct of (1) further comprises a sequence encoding a hinge region derived from human IgG. The method of claim 3, wherein the human IgG is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .   The method according to claim 3 or 4, wherein the sequence encoding the hinge region is located between the Fab fragment and the collagen-like peptide in the gene construct.   The method according to any one of claims 1 to 5, wherein the gene construct of (1) further comprises a sequence encoding a single chain Fv. The gene construct of (2) A method according to claim 1 comprising a kappa light chain of human IgG _1.   The method according to any one of claims 1 to 7, further comprising the step of purifying the multivalent Fab fragment with a ligand that binds to a kappa light chain of human IgG.   A multivalent Fab fragment produced by the method according to claim 1.   10. A trimeric multivalent Fab fragment comprising three multivalent Fab fragments according to claim 9 bound by at least the collagen-like domain of the multivalent Fab fragment. (1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
A multivalent antibody fragment comprising The multivalent antibody fragment according to claim 11, wherein the human IgG is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .   The multivalent antibody fragment according to claim 11, wherein the amino acid sequence of (1) comprises an amino acid sequence of a hinge region derived from human IgG. The multivalent antibody fragment according to claim 13, wherein the human IgG is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .   The multivalent antibody fragment according to claim 13 or 14, wherein the hinge region is located between the Fab fragment and the collagen-like peptide in the amino acid sequence.   The multivalent antibody fragment according to any one of claims 11 to 15, wherein the amino acid sequence (1) further comprises a single-chain Fv sequence. Multivalent antibody fragment according to any one of claims 11 to 16 amino acid sequence, including the kappa light chain sequences of human IgG ₁ of the (2).   The multivalent antibody fragment of claim 16, wherein the multivalent antibody fragment is bispecific.   The multivalent antibody fragment according to any one of claims 11 to 18, wherein the ligand of the multivalent antibody fragment is human CD3.   19. The multivalent antibody fragment according to claim 18, wherein the ligand of the multivalent antibody fragment is human CD3 and human epidermal growth factor receptor.   A trimeric multivalent antibody fragment comprising three multivalent antibody fragments according to any one of claims 11 to 20.   A nucleic acid encoding the multivalent antibody fragment according to any one of claims 11 to 20.   The expression vector which expresses the multivalent antibody fragment of any one of Claims 11-20 introduce | transduced into a host cell.   A host cell comprising the expression vector according to claim 23. A method for modulating the biological activity of a ligand comprising the steps of:
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Culturing a multivalent antibody fragment comprising: A method of treating an autoimmune disease in a subject comprising:
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain;
Administering an effective amount of a multivalent antibody fragment comprising: to a subject in need thereof.   27. The method of claim 26, wherein the ligand of the multivalent antibody fragment is human CD3. A method for detecting a ligand comprising:
(1) an amino acid sequence comprising the heavy chain portion of the Fab fragment and an in-frame fusion collagen-like peptide;
(2) an amino acid sequence comprising a light chain variable region of human IgG and a kappa light chain constant domain; (3) a label;
Culturing a trimeric multivalent antibody fragment complex comprising a ligand with a ligand, and detecting binding of the trimeric multivalent antibody fragment complex,
Including methods.