CN1272345C - Silensed anti-CD 28 antibodies and use thereof - Google Patents

Abstract

The present invention provides anti-CD28 antibodies which are defective in mitogenic activity (silenced anti-CD28 antibodies), methods of producing, compositions containing the antibody and methods of immunosuppression, inducing T-cell tolerance and treating organ and/or tissue transplant rejections.

Description

Silent anti-CD-28 antibody and its application

field of invention

The present invention relates to anti-CD-28 antibodies lacking mitogenic activity and uses thereof.

Background of the invention

The immune response, especially the rejection of organ transplantation, is mainly caused by the activation of T lymphocytes. This activation of T cells is induced by a signal from antigen presenting cells (APCs). This signal from APCs involves a first signal through the T cell receptor (TCR) and a second signal (co-stimulatory signal) through co-stimulatory molecules. When APCs present T cell antigens via the TCR, the first signal comes from the major histocompatibility (MHC) complex of peptide antigens. The second signal is mediated by several co-stimulatory molecules; examples of such co-stimulatory molecules include: B7 called ligands (B7-1(CD80) and B7-2(CD86)) on the APC side and B7-2(CD86) on the APC side CD28, CTLA-4, etc. as receptors on the T cell side. Ligand B7 is a glycoprotein belonging to the immunoglobulin superfamily, and is expressed in B cells and the like belonging to the antigen-presenting cell group. Both CD28 and CTLA-4, which recognize B7 as a common ligand, are transmembrane glycoproteins belonging to the immunoglobulin superfamily. Thus, activation of T cells is regulated by simultaneous transduction of a first signal via the TCR and a second signal from eg B7 and CD28/CTLA-4. It is known that signaling from B7 to CD28 promotes T cell activation, whereas signaling from B7 to CTLA-4 inhibits T cell activation [Waterhouse et al., Science, 270:985-988 (1995)].

Until now, in order to induce immunosuppression or tolerance, attempts have been made to block B7-CD28 signaling by administering CTLA-4Ig, anti-B7-1 antibody/anti-B7-2 antibody, anti-CD28 antibody, and the like. For example, CTLA-4Ig binds B7, thereby interfering with the reaction between B7 and CD28, and thus blocks the signal from CD28, exhibiting immunosuppressive activity. However, since the reaction between B7 and CTLA-4 is also inhibited at the same time, CTLA-4 signaling, which negatively regulates T cell activation, is also inhibited, so that the required tolerance cannot be induced (Kirk et al., Proc. Natl USA, 94:8789-8794 (1997)). In addition, anti-B7 antibodies were produced, which were reported to inhibit T cell activation only in the presence of CTLA-4Ig, which also inhibited CTLA-4 signaling. In in vitro experiments, it was found that anti-CD28 antibody had a mitogenic effect on T cells, and combined stimulation with this antibody and anti-CD3 antibody promoted the growth and activation of T cells, and increased the production of cytokines [WO 90/05541, Eur. J. Immunology, 16, 1289-1296 (1986) etc.]. Furthermore, mitogenic stimulation of T cell CD28 receptors by eliciting anti-CD28 antibodies in vivo results in the generation of T cell activation signals similar to the second signal from B7 to CD28 [Yin et al., J. Immunology, 163:4328- 4334 (1999)]. These T cell activation functions imply that anti-CD28 antibodies could be used as immunopotentiators in the treatment of cancer and AIDS (WO 90/05541).

Invention Summary

Anti-CD28 antibodies prepared by conventional techniques have mitogenic effects on T cells. Although the reason for this mitogenic activity is not fully understood, it is believed that binding of the Fc region of anti-CD28 to Fc receptors on antigen-presenting cells is the likely cause (Cole et al., J. Immunology, 36:159 (1997)). Therefore, by using genetic engineering techniques, we introduced mutations into the binding site of the Fc receptor of the anti-CD28 antibody to modify the antibody so that it no longer has mitogenic activity. The present inventors have produced one such antibody, TN228 IgG2M3, in which IgG2M3 has two amino acid substitutions in the IgG gene. Furthermore, we demonstrated that the generated silent anti-CD28 antibody has no mitogenic activity, which is very useful for inducing T cell tolerance.

Therefore, the present invention provides an anti-CD28 antibody having no mitogenic activity (hereinafter referred to as a silent anti-CD28 antibody), and provides a method for suppressing an immune response (especially transplant rejection) and inducing immune tolerance by using the antibody .

One object of the present invention is a silent anti-CD28 antibody, wherein said anti-CD28 antibody may be a chimeric antibody and/or a humanized antibody. The variable region of the anti-CD28 antibody may include the amino acid sequences shown in SEQ ID NO: 2, 4, 7 and 9 and polynucleotides encoding such amino acid sequences. For example, such polynucleotides include SEQ ID NO: 1, 3, 6 and 8.

Another object of the present invention is: a vector and a cell host comprising the polynucleotide encoding the anti-CD28 antibody.

Yet another object of the present invention is to produce a silent anti-CD28 antibody by culturing a cell host comprising a polynucleotide encoding an anti-CD28 antibody under conditions that allow expression of said polynucleotide and collecting the gene product produced Methods.

Yet another object of the present invention is a pharmaceutical composition comprising one or more silencing anti-CD28 antibodies, preferably in admixture with one or more pharmaceutically acceptable ingredients.

The silent anti-CD28 antibody can be used to induce T cell tolerance, immunosuppression, and can be used as a preventive/therapeutic drug for organ or tissue transplant rejection. Accordingly, the present invention provides methods for inducing T cell tolerance, immunosuppression by administering one or more silent anti-CD28 antibodies to a mammal, and during organ or tissue transplant rejection by administering one or more A method for preventive or therapeutic treatment using a silent anti-CD28 antibody. Such silent anti-CD28 antibodies are preferably administered in the form of pharmaceutical compositions described herein, and may contain suitable additional drugs/drugs.

Brief description of the attached drawings

Figure 1: Plasmid constructs for expression of ChTN228 antibody. The VL and VH of murine TN228 were constructed as small exons adjacent to the Xbal site. The VL sequence was inserted into the expression vector pVk, and the VH sequence was inserted into the expression vector pVg2M3.

Figure 2: Nucleotide sequence and deduced amino acid sequence of ChTN228 light chain in the small exon. The signal peptide sequence is in italics. CDRs are underlined. The mature light chain begins with an aspartic acid residue (bold letters). Untranslated sequences and intronic sequences are indicated in lower case letters. (SEQ ID NO: 1 and 2).

Figure 3: Nucleotide sequence and deduced amino acid sequence of the ChTN228 heavy chain variable region in the small exon. The signal peptide sequence is in italics. CDRs are underlined. The mature heavy chain begins with a glutamine residue (bold letters). Untranslated sequences and intronic sequences are indicated in lower case letters. (SEQ ID NO: 3 and 4).

Figure 4: Competition experiment. P815/CD28 ⁺ cells were incubated with 25ng MuTN228-FITC and 2-fold serial dilutions of either ChTN228, or MuTN228 as described ^. Likewise, P815/CD28' cells were incubated with MuTN228-FITC alone without any competitor added. The average amount of channel fluorescence for each sample was plotted against the concentration of competitor.

Figure 5: Inhibition of human primary MLR(1) by TN228-IgG2m3. Percent inhibition of primary MLR from four individuals is shown separately.

Figure 6: Inhibitory effect of TN228-IgG2m3 on human primary MLR (2). Percent inhibition of primary MLR from four individuals is shown separately.

Figure 7: Effect of TN228-IgG2m3 on secondary MLR. Data from two volunteers are shown separately. [3H]-thymidine uptake in the secondary MLR was expressed as a percentage of dpm of Raji stimulation alone in the primary MLR as 100. TN228-IgG2m3: 0.1 mg/ml.

Figure 8: Plasmid constructs for expression of HuTN228 antibody. The VL and VH of humanized TN228 were constructed as small exons adjacent to the Xbal site. The VL sequence was inserted into the expression vector pVk, and the VH sequence was inserted into the expression vector pVg2M3.

Figure 9: Nucleotide sequence and deduced amino acid sequence of the HuTN228 heavy chain variable region in the small exon. The signal peptide sequence is in italics. CDRs are underlined. The mature heavy chain begins with a glutamine residue (bold letters). (SEQ ID NOS: 6 and 7).

Figure 10: Nucleotide sequence and deduced amino acid sequence of the HuTN228 light chain variable region in the small exon. The signal peptide sequence is in italics. CDRs are underlined. The mature light chain begins with an aspartic acid residue (bold letters). (SEQ ID NO: 8 and 9).

Figure 11: FACS competition experiment. Binding of FITC-labeled MuTN228 to P815/CD28' cells in the presence of different amounts of competitor MuTN228 antibody or HuTN228 antibody was analyzed using the flow cytometric competition assay described in the Examples.

Figure 12: ELISA competition experiment. The binding of biotinylated MuTN228 to sCD28-Fc in the presence of different amounts of competitor MuTN228 antibody or HuTN228 antibody was analyzed using the ELISA competition assay described in the Examples.

Figure 13: I-125 competition experiment. Binding ^of125I -labeled MuTN228 to P815/CD28 ⁺ cells in the presence of different amounts of competitor MuTN228 antibody or HuTN228 antibody was analyzed using ^the125I -labeled antibody competition assay described in the Examples.

Figure 14: Plasmid constructs for expression of PV1-IgG3 antibodies. The _VL and _VH of PV1 were constructed as small exons adjacent to the Xbal site. The _VL sequence was inserted into the expression vector pMVk.rg.dE, and the _VH sequence was inserted into the expression vector pMVg3.D.Tt. The two plasmids were then recombined to generate a single plasmid co-expressing the PV1-IgG3 heavy chain and the PV1-IgG3 light chain.

Figure 15A: cDNA sequences and deduced amino acid sequences of the light and heavy chains described in the small exon. CDRs are underlined. The mature light chain begins with an aspartic acid residue at position 20 (double underlined). (SEQ ID NO: 10 and 11).

Figure 15B: cDNA sequence and deduced amino acid sequence of the PV1 variable region in the small exon. CDRs are underlined. The mature heavy chain begins with a glutamine at position 20 (double underlined). (SEQ ID NOS: 12 and 13).

Figure 16: Analysis of PV-1-IgG3 by size exclusion chromatography using HPLC as described in the Methods. Proteins were monitored by their absorbance at 280 nM.

Figure 17: SDS-PAGE analysis of mouse IgG3 isotype control (lane 1), PV1 (lane 2) and PV1-IgG3 (lane 3). Proteins in plate A were electrophoresed under non-reducing conditions, and proteins in plate B were electrophoresed under reducing conditions. MW means molecular weight marker. The numbers listed are MW standards in kD.

Figure 18: EL4 cells stained with PV1 (A), 37.51 (B) or PV1-IgG3 (C) and analyzed by flow cytometry. Secondary antibodies used were: FITC-conjugated donkey anti-Armenian hamster IgG (H+L) for PV1, FITC-conjugated donkey anti-golden hamster IgG for 37.51, and FITC-conjugated FITC for PV1-IgG3 goat anti-mouse κ. Profiles with solid lines represent cells stained only by the secondary antibody. The dashed profile represents cells stained by both the primary and secondary antibodies described in Methods. Mouse IgG3 isotype control did not stain EL4 cells (data not shown).

Figure 19: (A) Excess PV1 or PV1-IgG3 competes with R-PE-conjugated PV1 for binding to EL4 cells. Thin solid lines (black) in the flow cytometry frequency histogram represent cells that were not stained in any color, thick solid lines (dark blue) represent cells stained only by R-PE-PV1, and thin dashed lines (magenta) represent Cells stained by R-PE-PV1 and excess unconjugated PV1, while thin double dashed lines (light blue) indicate cells stained by R-PE-PV1 and excess unconjugated PV1-IgG3. Excess mouse IgG3 isotype control had no effect on the binding of R-PE-PV1 to EL4 cells (data not shown). (B) Excess 145.2C11 or 145.2C11-IgG3 competes with R-PE-conjugated 145.2C11 for binding to EL4 cells. Thin solid line (black) indicates cells without any staining, thick solid line (dark blue) indicates cells stained only by R-PE-145.2C11, thin dashed line (magenta) indicates cells stained by R-PE-145.2C11 and excess unstained Conjugated 145.2C11 stained cells, while thin double dashed lines (light blue) indicate cells stained with R-PE-145.2C11 and excess unconjugated 145.2C11-IgG3. (C) Excess PV1 competes with PV1-IgG3 for binding to EL4 cells. EL4 cells were stained with PV1-IgG3 and excess PV1, or with PV1-IgG3 in the absence of excess PV1. Cells were washed and stained with mouse IgG3-specific, FITC-conjugated donkey anti-mouse IgG (H+L). The thin solid line (black) indicates cells stained by the secondary antibody only, the thick solid line (dark blue) indicates cells stained by both PV1-IgG3 and the secondary antibody, and the thin dashed line (magenta) indicates cells stained by PV1-IgG3 and excess Cells were stained with PV1 as well as secondary antibodies.

Figure 20: Murine splenocytes stained with PV1-IgG3 and 145.2C11. Cells were stained with mouse IgG3 isotype control (A) or PV1-IgG3 (B), counterstained with R-PE-conjugated goat anti-mouse IgG and FITC-conjugated 145.2C11, and analyzed with Materials and methods. Cells were analyzed by two-color flow cytometry as described. Only cells in the lymphocyte gate were analyzed. PV1-IgG3 positive cells are in the upper "quadrant", while CD-3 positive cells are in the right "quadrant". The numbers in each "quadrant" indicate the percentage of cells in that particular "quadrant".

                    Invention Details

Within the scope of the present invention, the term "silent anti-CD28 antibody" means any anti-CD28 antibody that lacks mitogenic activity. More precisely, it is an antibody that specifically binds to the antigen CD28 receptor on the surface of T cells and does not promote T cell growth or activation through co-stimulation with anti-CD3 antibodies.

Silent anti-CD28 antibodies can be constructed on the basis of anti-CD28 antibodies or on the basis of hybridomas that produce anti-CD28 antibodies, by using genetic engineering techniques or by chemical modification to mutate or modify agonistic anti-CD28 antibodies. CD28 antibody. Taking the application of genetic engineering technology as an example, by introducing mutations into the amino acid sequence of the Fc region of the antibody, the binding affinity of the anti-CD28 antibody to the Fc receptor can be reduced or eliminated. For example, by isolating cDNA from hybridoma cells capable of producing anti-CD28 monoclonal antibodies, and introducing mutations into the sequence region corresponding to the Fc region that plays an important role in binding to Fc receptors, the silent type can be obtained. Anti-CD28 antibody (WO 88/07089). There is no particular limitation on the site to be mutated, because all can inhibit the binding to Fc receptors. Thus, in the case of antibodies of the IgG class, for example H-chain amino acid residues 234, 235, 236, 237, 318, 320 and 322 are preferred, and by substituting at least one of these amino acids with a different amino acid And construct silent anti-CD28 antibody.

The source of such a silent anti-CD28 antibody can be carefully selected according to the target animal to which the antibody is applied. For example, non-human monoclonal antibodies contain amino acid sequences that exhibit antigenicity in humans over a fairly wide range. Many studies have shown that after the antibody is injected, the patient's immune response to the foreign antibody is very strong; and only giving the antibody may put the patient into a dangerous state or make the antibody lose its therapeutic effect. Therefore, it is recommended to replace the Fc region so that the antibody is relatively more homologous to the treatment target animal, to replace the framework part of the variable region, or to use the antibody obtained from a transgenic animal into which the antibody gene has been introduced . For example, when the antibody is to be administered to humans, the following antibodies can be used: chimeric antibodies obtained by substituting the Fc region (EP125023), humanized antibodies with the Human antibodies obtained from transgenic animals carrying the human antibody genes (EP546073, WO 97/07671). The mitogenic activity of the antibodies can be reduced or eliminated by introducing mutations in these antibodies using genetic engineering techniques such as those described above, or introducing mutations in these antibodies by chemical modification.

As specific examples of the anti-CD28 antibody having a silent Fc region, not only the antibodies described in the Examples section below but also the combination of the constant region gene of the target animal to be treated and the expression based on SEQ ID NO: 2 and 4 or An antibody prepared synthetically from the variable region polynucleotides of the variable region amino acid sequences shown in SEQ ID NO: 7 and 9. Examples of such polynucleotides are SEQ ID NO: 1, 3, 6 and 8.

More specific examples of the present invention are HuTN228 and MuTN228 and their Fab fragments, their F(ab)'2 fragments, their derivatives and the like.

As expected by those skilled in the art, due to the degeneracy of the third base, almost every amino acid can be represented by more than one triplet codon in the nucleotide coding sequence. Furthermore, minor base pair changes may result in variations in the encoded amino acid sequence (conservative substitutions); but are not expected to significantly alter the biological activity of the gene product. Therefore, in sequence, the nucleic acid sequence encoding the protein or peptide disclosed herein can be slightly modified (for example, replacing a nucleotide in a triplet codon), but the sequence still encodes its corresponding gene product of the same amino acid sequence .

The term "expression vector" refers to a polynucleotide that encodes a peptide of the invention and provides the sequences necessary for expression of the peptide in a host cell of choice. Expression vectors will usually contain a transcriptional promoter and terminator, or will allow for the incorporation of an adjacent endogenous promoter. An expression vector will usually be a plasmid further comprising an origin of replication and one or more selectable markers. Alternatively, however, the expression vector may be a viral recombinant designed to infect the host, or an integrating vector designed for integration at a preferred site within the host genome. Examples of expression vectors are disclosed in Molecular Cloning: A Laboratory Manual, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989.

Suitable host cells for expressing silencing anti-CD28 antibodies include: prokaryotes, yeast, archae and other eukaryotic cells. Suitable cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are well known in the art, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York (1985). The cells are preferably mammalian cells. The vector may be a plasmid vector, a single- or double-stranded phage vector, or a single- or double-stranded RNA or DNA viral vector. Such vectors can be introduced into cells in the form of polynucleotides, preferably DNA, using well known techniques for introducing DNA and RNA into cells. In the case of bacteriophage and viral vectors, the vectors can also and are preferably introduced into cells in the form of packaged or enveloped viruses using well known infection and transduction techniques. Viral vectors can be replication competent or replication defective. In the latter case, viral multiplication will normally only occur in complementary host cells. Cell-free translation systems can also be used to produce the proteins using RNA derived from the DNA constructs of the invention.

The silencing anti-CD28 antibody/protein can be purified according to protein isolation/purification methods generally known in the field of protein chemistry. In more detail, there may be mentioned, for example: extraction, recrystallization, salting out with ammonium sulfate, sodium sulfate, etc., centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography , reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, etc., and combinations of these methods.

According to the present invention, purified antibodies can be produced using the recombinant expression systems described above. The method comprises: cultivating a host cell transformed with an expression vector containing a DNA sequence encoding the protein under conditions sufficient to promote the expression of the protein. The protein is then recovered from the culture medium or cell extracts, depending on the expression system used. As is known to the skilled artisan, the procedure for purifying the recombinant protein will vary depending on factors such as the type of host cell used and whether the recombinant protein is secreted into the culture medium.

If the silencing anti-CD28 antibody is formulated as a pharmaceutical composition, it can be used for: (a) transplant rejection after organ or tissue transplantation, such as heart, kidney, liver, bone marrow, skin, Cornea, lung, pancreas, small intestine, muscle, nerve, etc.; (b) graft-versus-host reaction in bone marrow transplantation; (c) such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Autoimmune diseases such as type I diabetes; and (d) immune diseases such as asthma, atopic dermatitis and the like.

Although silencing anti-CD28 antibodies can be expected to suppress immune responses and graft rejection and induce immune tolerance by themselves, they can also be used in combination with other drugs. Among such other drugs that can be used in combination with silent anti-CD28 antibodies are various immunosuppressive drugs such as rapamycin, deoxyspergualin, anti-CD40 antibodies, anti-CD40L antibodies, Kefu, cyclosporin A, anti-IL-2 antibody, anti-IL-2 receptor antibody, and MMF. Rapamycin, in particular, inhibits the transduction of signals from the IL-2 receptor involved in T cell growth, but not the transduction of apoptosis-related signals; it is therefore expected to interact with specific inhibitors of CD28 signaling. A combination would be useful.

The silent anti-CD28 antibody of the present invention can be administered orally or parenterally, preferably intravenously, intramuscularly or subcutaneously.

The silent anti-CD28 antibody of the present invention can be prepared in the form of a solution or lyophilized powder; and if necessary, various pharmaceutically acceptable additives such as excipients, diluents, stabilizers, isotonic agents and buffers can be used agent to prepare the silent anti-CD28 antibody of the present invention. Preferred additives include: sugars such as maltose, surfactants such as polysorbate, amino acids such as glycine, proteins such as human serum albumin, and salts such as sodium chloride.

Also, dosage forms such as injections (solutions, suspensions, emulsions, solids dissolved at the time of use, etc.), tablets, capsules, granules, powders, liquids, etc. can be appropriately selected depending on the mode of administration. formulations, liposome inclusions, ointments, gels, topical powders, sprays, inhalation powders, eye drops, eye ointments, suppositories, vaginal suppositories, etc.; and the peptides of the present invention can be formulated accordingly. Formulations are fully described in Comprehensive Medicinal Chemistry, Vol. 5, Hansch et al. eds., Pergamon Press 1990, Chapter 25.2.

The dose of the pharmaceutical composition of the invention depends inter alia on the particular composition, the type of disease targeted for treatment or prevention, the mode of administration, the age and health of the patient and the duration of the treatment. However, in the case of intravenous administration, intramuscular administration or subcutaneous administration, 0.01-100 mg/kg, preferably 0.1-10 mg/kg can be administered per adult per day.

When using the silent anti-CD28 antibody of the present invention to suppress transplant rejection or induce immune tolerance, after organ or tissue transplantation, it can be injected intravenously, intramuscularly or subcutaneously at a dose of about 1 mg/kg/day at The composition is administered immediately before transplantation, immediately after transplantation, and at 3 days, 7 days, 12 days, 18 days, 25 days, 35 days, 45 days and 60 days after transplantation. The frequency and dose of administration can be increased or decreased judiciously while monitoring the course of rejection following transplantation.

Although the interval of administration depends inter alia on the mode of administration used and the condition of the patient; not only continuous but also intermittent administration is possible. Thus, since the silencing anti-CD28 antibody of the present invention is an antibody, it provides a sustained effect so that intermittent administration can achieve the desired efficacy. As for the treatment period, once a state of tolerance has been established, this tolerance can be maintained even if the use of the silencing anti-CD28 antibody is discontinued. In this regard, this silent anti-CD28 antibody is undoubtedly superior to other immunosuppressive drugs whose immunosuppressive effect decreases after discontinuation.

Example

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and not limitation unless otherwise indicated. Unless otherwise described in detail, the following examples were performed using standard techniques well known and conventional to those skilled in the art.

Example 1 Determination of the amino acid sequence of the mouse anti-human CD28 antibody

A hybridoma (clone: TN228, mouse IgG1κ) producing anti-human CD28 antibody was generously provided by Dr. Yagita (Juntendo University School of Medicine, Japan). Allow approximately 0.2 mg of purified anti-human CD28 antibody (TN228) to be reduced in 0.64M guanidine hydrochloride, 0.28M Tris-HCl, pH 8.5, 0.055M DTT at 60°C (in argon) for 90 minutes, at room temperature ( In the dark) carboxymethylation was carried out by adding iodoacetic acid to 0.13M for 45 minutes, followed by the addition of DTT to 0.32M (to terminate the carboxymethylation reaction), and immediately on a PD-10 column (cat. no. 17-0851-01, Amersham Pharmacia Biotech, Uppsala, Sweeden) to carry out buffer exchange in 0.002M EDTA of 0.1M sodium phosphate, pH 8.0. The eluate was adjusted to 0.005 MDTT, 0.02% glycerol; and a third of this solution (approximately 0.35 ml) was transferred to a separate tube for deblocking of the heavy chain N-terminus. The sample was digested with 1800 µU of pyroglutamate aminopeptidase (Cat. No. 7334, Takara Shuzo Co., Ltd., Tokyo, Japan) at 45°C for 24 hours. The N-terminal sequences of the light and heavy chains from deblocked samples were determined by 20 cycles of automated Edman degradation and PTH analysis with a Model 241 Protein Sequencer (Hewlett Packard, Palo Alto, CA). PTH derivatives were analyzed using a Hypersil ODS C18 column. The sequencer and HPLC were operated according to the manufacturer's instructions with reagents, solvents and columns from Hewlett Packard.

The results of N-terminal sequencing of TN228 deblocked with pyroglutamate aminopeptidase are as follows: residue number amino acid residue number amino acid residue number amino acid residue number amino acid 1 D, Q 6 Q,E 11 L 16 G, Q 2 l,v 7 the s 12 A, V 17 Q, S 3 V, Q 8 P, G 13 V, A 18 R, L 4 L 9 A, P 14 S, P 19 A, S 5 T, K 10 S, G 15 L, S 20 T, I

Example 2 Cloning of variable region cDNA

Using the anchored polymerase chain reaction (PCR) method described by Co et al. (Co, MS, NMAvadalovic, PCCaron, MVAvadalovic, DAScheinberg and C.Queen. 1992. Chimeric and humanized antibodies specific for the CD33 antigen . J. Immunol. 148: 1149-1154), the light chain and heavy chain V region cDNAs of TN228 were cloned from the hybridoma cells. Amplification was performed on the cDNA using 3' primers annealing to the mouse kappa chain C region and gamma chain C region respectively and a 5' primer annealing to the G tail of the added cDNA. For VLPCR, the 3' primer has the following sequence (SEQ ID NO: 14): 5' TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC 3' which hybridizes residues 17-46 to the mouse _CK region. For VH PCR, the 3' primers had the following degenerate sequences (SEQ ID NOs: 15, 16 and 17):

A A G G T

5′TATAGAGCTCAAGCTTCCAGTGGATAGACCGATGGGGCTGTCGTTTTGGC 3′

T

Its residues 17-50 hybridize to mouse IgG _CH1 . Non-hybridizing sequences in both primer sets contain restriction enzyme sites for cloning. The VL cDNA and VH cDNA were subcloned into TOPOII Blunt vector (Invitrogen, Inc., Carlsbad, CA) for sequence determination.

Several light and heavy chain clones were sequenced from two independent polymerase chain reactions. As for the light chain, two unique sequences homologous to the mouse light chain variable region were identified. One VL sequence was non-functional due to a frameshift mutation and was identified as a non-productive allele. Another VL sequence is typical of a functional mouse kappa chain variable region. For the heavy chain, a unique sequence homologous to the typical mouse heavy chain variable region was identified. In Figure 2 and Figure 3, the nucleotide sequence of its variable region and its deduced amino acid sequence are depicted.

Example 3 Construction and expression of chimeric TN228-IgG2M3

(method)

E-selectin and P- Humanization and Pharmacokinetics of Monoclonal Antibodies with Selectin Specificity. J. Immunol.160: 1029-1035) described, by PCR, the V _L and V _H of TN228 were transformed into adjacent XbaI positions The small exon segment of the dot; and subcloned into the light chain and heavy chain expression plasmids (Figure 1). Each small exon contains a signal peptide sequence, a mature variable region sequence and a splice donor sequence derived from the most homologous mouse J chain gene. Using such a splice donor sequence, the V region exons are spliced onto the constant region of a human antibody. After cloning the mini-exons into expression vectors, each mini-exon was sequenced to ensure the correct sequence and no PCR errors. Likewise, the constant region exons of the light and heavy chain expression plasmids were confirmed by sequencing.

In this specification, ChTN228 refers to a chimeric antibody comprising mouse TN228 VL and VH variable regions, human IgG2M3 heavy chain constant region, and human light chain kappa constant region. This heavy chain constant region from a human germline 2 genomic fragment is modified (Cole, MS, C. Anasetti and JYTso. 1997. Chimeric anti-CD33 human IgG2 variants are not mitogenic to T cells. J. Immunol 159:3613-3621), and the light chain is derived from a human germline K genome fragment. Both the heavy and light chain genes are driven by the major immediate early promoter and enhancer of human cytomegalovirus. The heavy chain gene is followed by a transcription terminator derived from the human complement gene C2 (Ashfield, R., P. Enriquez-Harris and NJ Proudfoot. 1991. Transcription termination between the closely linked human complement gene C2 and factor B: C2 and c-myc co-termination factor? EMBO J.10:4197-4207). Light chain selection marker gpt gene (Mulligan, RC and P.Berg.1981. Selection of animal cells expressing the xanthine-guanine phosphoribosyltransferase encoding gene of Escherichia coli.Proc.Natl.Acad.Sci.USA78:2072- 2076) and the heavy chain selectable marker dhfr gene (Simonsen, CC and AD Levinson.1983. Isolation and expression of a modified mouse dihydrofolate reductase cDNA. Proc. Natl. Acad. Sci. USA 80: 2495-2499) Both are driven by the SV40 early promoter. For expression of chimeric TN228, transient transfection into COS-7 cells (monkey kidney cell line) was accomplished with lipofectamine (cat. no. 10964-013, GIBCO BRL). Spent media from transient transfectants were analyzed for human IgG2M3 antibody production by ELISA using a goat anti-human IgG gamma chain-specific antibody as capture reagent and an HRP-conjugated goat anti-human kappa chain antibody as chromogenic reagent . In addition, the spent medium was tested for the ability of ChTN228 to bind to P815/CD28 ⁺ cells (stably transfected cells obtained by transfecting P815 (mouse mastocytoma) with CD28) by indirect immunofluorescent staining; and analyzed by flow cytometry. As for the production of stable cell lines, the chimeric expression plasmid was transfected into the mouse myeloma cell line Sp2/0 by electroporation; and the transfectants were selected according to the expression of gpt. For transient transfections, the spent medium of stable transfectants was analyzed by ELISA.

result

The cloned VL and _VH genes were converted to small exons by PCR (Figures 2 and 3) and subcloned into the light and heavy chain expression vectors _described above and shown in Figure 1 .

Transient transfection of COS-7 cells: The chimeric expression vector was transiently transfected into the monkey kidney cell line COS-7 to produce chimeric TN228 ⁺ antibody. The production ^of chimeric IgG2M3 antibodies in the medium used by the transfected cells was checked by ELISA ^; The state of cell binding. Spent medium was positive in both assays. The yield of chimeric antibody from transient transfection was approximately 0.9 μg/ml. ChTN228 antibodies from transient supernatants bound to P815/CD28 ⁺ cells in a concentration-dependent manner (data not shown).

Stable transfection of Sp2/0 cells: To generate a stable cell line, the chimeric expression vector was transfected into Sp2/0 cells. Several transfectants were tested for production of TN228 chimeric antibody in spent media; and as for the transient transfectants, binding to P815/CD28 ⁺ cells was tested. Most transfectants were positive for both assays. One transfectant was selected for its high antibody production rate and was culture-expanded to grow in 5 liters of serum-free medium. ChTN228 was purified from 5 liters of spent medium by affinity chromatography. The purified antibody yield was approximately 25 mg.

Example 4 Protein purification of chimeric antibody ChTN228

One of the high-expressing ChTN228 transfectants from the stable transfection (clone 7H) was grown in 5 liters of GIBCO hybridoma serum-free medium (Catalog No. 12045-076, GIBCOBRL). When the cell viability reached 10% or less, the spent culture supernatant was harvested; it was concentrated to 500ml, and it was loaded into a cell using a Pharmacia P1 pump (2-3ml/min). 5ml protein-A Sepharose column. The column was washed with PBS and the antibody was then eluted with 0.1M Glycine, 0.1M NaCl pH 2.7. The eluted protein was dialyzed against 2 liters of PBS with 3 changes of PBS; it was then desalted by applying it to a PD-10 column equilibrated with PBS containing additional 0.1M NaCl. The desalted protein solution was filtered through a 0.2 mm filter paper before storage at 4°C.

Example 5 Purity Determination by Size Exclusion HPLC and SDS-PAGE

Use a Perkin consisting of a PE ISS 200 Advanced LC Sample Processor (sample processor), a PE Series 410 Bio LC Pump (pump), a PE 235C Diode ArrayDetector (diode array detector) and PE Nelson 600 Series LINK Elmer HPLC system for size exclusion HPLC. Perkin Elmer Turbochrom Navigator Version 4.1 software was used to control autosamplers, pumps and detectors; and to acquire, store and process data using this software. Separation was accomplished with two TosoHaas TSK-GEL G3000SWXL size exclusion HPLC columns connected in series; data for said columns: 7.8 mm x 300 mm, particle size 5 mm, pore size 250 Å (Cat. No. 08541, TosoHaas, Montgomeryville, MD). The mobile phase was 200 mM potassium phosphate/150 mM potassium chloride pH 6.9 with a flow rate of 1.00 ml/min. The column eluate was monitored with a spectrophotometer at a wavelength of 220 nm and a wavelength of 280 nm. The injection volume of ChTN228 samples was 50 μl (50 μg).

SDS-PAGE was performed using a 4-20% gradient gel (Cat# EC6025, Novex, San Diego, CA) following standard procedures.

The purity of the isolated ChTN228 was analyzed by size exclusion HPLC and SDS-PAGE. According to this analysis, the protein is 96.5% monomeric and has a mobility equivalent to that of a protein with a molecular weight of approximately 160 kD. SDS-PAGE analysis of MuTN228, isotype control MuFd79 (mouse IgGl), ChTN228, and isotype control HuEP5C7 (human IgG2M3) under non-reducing conditions also showed that all four antibodies had molecular weights of approximately 150-160 kD. Analysis of the same four proteins under reducing conditions showed that all four antibodies consisted of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD.

Embodiment 6 competition experiment

method

Titration experiments were performed using two-fold serial dilutions of MuTN228-FITC antibody starting at 250 ng/assay. P815/CD28 ⁺ cells (5×10 ⁵ cells/assay) were incubated with FITC-labeled antibody for 1 hour on ice, then washed with PBS and analyzed by flow cytometry. For competition experiments, 25 ng of MuTN228-FITC and two-fold serial dilutions of competing ChTN228 or MuTN228 antibody starting at 800 ng/assay were added to P815/CD28 ⁺ cells (5×10 ⁵ cells/assay). As a control, 25 ng of MuTN228-FITC alone was incubated with P815/CD28 ⁺ cells (5×10 ⁵ cells/assay) (ie without any competitor). Likewise, HuEP5C7 isotype control antibody (800 ng/test) and MuFd79 isotype control antibody (800 ng/test) were tested as competitors. Cells were incubated with the antibody mixture for 1 hour on ice (in the dark) in a final volume of 150 ml, then washed and analyzed by flow cytometry.

result

The binding specificity of the MuTN228 antibody and the ChTN228 antibody were compared using flow cytometry competition experiments as described in Methods. Different amounts of unlabeled MuTN228 or ChTN228 were mixed with 25ng FITC-labeled MuTN228 antibody and incubated with P815/CD28' cells. Both MuTN228 and ChTN228 competed with MuTN228-FITC in a concentration-dependent manner, suggesting that the binding of these two antibodies to the CD28 antigen was specific (Fig. 4). Isotype control antibodies MuFd79 and HuEP5C7 did not compete with MuTN228-FITC, indicating that MuTN228 antibody and ChTN228 antibody recognize CD28 antigen through the specific interaction of the V region.

Example 7 Chimeric anti-human CD28 antibody with reduced affinity to human FR inhibits primary mixing

Combined Lymphocyte Response

cell preparation

Human peripheral blood mononuclear cells (PBMC) from healthy normal volunteers were prepared by density gradient centrifugation with Ficoll-Paque plus (Amersham Pharmacia Biotech, Tokyo, Japan). Human blood was diluted with an equal volume of RPMI1640, and spread on top of Ficoll-Paque plus. After centrifugation at room temperature for 30 minutes, peripheral blood mononuclear cells were collected and washed with RPMI1640. Thereafter, peripheral blood cell mononuclear cells were suspended with medium (RPMI1640 containing 2.5% of human AB serum, 2-mercaptoethanol, and antibiotics) and applied to a nylon fiber column (Wako junyaku, Osaka, Japan) . After incubation for 1 h at 37 °C in 5% _CO2 , T cells were eluted with warmed medium.

In mixed lymphocyte reactions, human B cell lines (Raji and JY) were used as stimulator cells. The cells were X-ray irradiated (2000R) before use.

primary mixed lymphocyte reaction (primary MLR)

Purified human T cells (1×10 ⁵ cells/well) and irradiated Raji (1×10 ⁵ cells/well) were seeded into 96-well flat-bottomed miniplates. Antibiotics were added to the medium, and the cells were cultured for 7 days. All cultures were labeled with 10 kBq/well of [ ³ H]thymidine (AmershamPharmacia biotech) during the final 6 hours of incubation. Cells were harvested and incorporated radioactivity was measured using a liquid scintillation counter.

The effect of TN228-IgG2m3 (ChTN228) on primary MLR is shown in FIGS. 5 and 6 . The original anti-human CD28 antibody TN228 (MuTN228) did not inhibit primary MLR, however, chimeric antibody TN228-IgG2m3 inhibited primary MLR in a dose-dependent manner. Therefore, converting the Fc region of an anti-human CD28 antibody to an Fc region with reduced affinity for human FcR renders the antibody antagonistic to T cell proliferation.

An anti-human CD28 chimeric antibody with reduced affinity for human FcR attenuates T cell hyporesponsiveness in a secondary mixed lymphocyte reaction.

Secondary mixed lymphocyte reaction (secondary MLR)

Purified human T cells (1×10 ⁵ cells/well) and irradiated Raji cells (1×10 ⁵ cells/well) were seeded into 96-well flat-bottomed small plates. Add antibiotics to the medium and incubate the cells. After 5 days, cells were harvested and washed with fresh medium. The cells were suspended with fresh medium and cultured for 8 days. The cells were restimulated with irradiated Raji cells or JY cells. After an additional 7 days of culture, 10 kBq/well of [ ³ H]thymidine was incubated with the cells for 6 hours. Cells were harvested and radioactivity was measured with a liquid scintillation counter.

TN228-IgG2m3 inhibited primary MLR (Figure 5 and Figure 6). Next, we analyzed the effect of this antibody on the secondary MLR. The antibody is added to the primary MLR culture, and then the antibody is removed from the culture supernatant. After culturing cells in antibody-free medium, cells were restimulated with the same stimulator (Raji) or a third-party stimulator (JY). The proliferation of cells treated with TN228-IgG2m3 by primary MLR was reduced compared to the proliferation of untreated cells. However, with a third-party stimulator (JY), both cells proliferated to almost the same extent (Fig. 7). This result indicates that an anti-human CD28 antibody with reduced affinity for human FcR can induce T cell energy by stimulation with an allogeneic (alo-) antigen.

Example 8 Design of the variable region of humanized TN228

The V region sequence of MuTN228 was analyzed by computer modeling. Based on the sequence identity of the Kabat antibody sequence database (8. Johnson, G. and T.T.Wu. 2000. The Kabat database and its application: 30 years after the first variability map. Nucleic Acids Res. 28:214-218) Origin search, pick out IC4 (Manheimmer-Lory, A., J.B.Katz, M.Pillinger, C.Ghossein, A.Smith, B.Diamond.1991. Molecular characterization of antibodies with anti-DNA-associated idiotypes. J .Exp.Med.174:1639-1652), to provide the framework for both the heavy and light chain variable regions of humanized TN228. Among the 85 framework residues in the humanized TN228 heavy chain variable region, 65 residues are identical to those of the mouse TN228 heavy chain framework, that is, there is a sequence identity of 76%. Among the 80 framework residues in the humanized TN228 light chain variable region, 56 residues are identical to those of the mouse TN228 light chain framework, that is, there is 70% sequence identity.

Use the computer program ABMOD and ENCAD (Levitt, M.1983. Molecular Dynamics of Natural Proteins. I. Computer Simulation of Orbitals. J.Mol.Biol.168:595-620), to construct the molecular model of the variable region of TN228; This model was used to determine the positions of amino acids in the framework of mouse TN228 that are close enough to the CDRs to potentially interact with them. In order to design the heavy and light chain variable regions of humanized TN228, the CDRs from the mouse TN228 heavy chain were grafted into the human IC4 heavy chain framework region, and the CDRs from the mouse TN228 light chain were grafted into In the framework region of the human IC4 light chain. Amino acids from the mouse antibody were substituted for those of the original human framework at framework positions where the computer model suggested notable contacts with the CDRs. For humanized TN228, such substitutions were made at residues 27, 29, 30, 48, 67, 71 and 78 of the heavy chain. For the light chain, no substitutions were made (ie MuTN228 CDRs were grafted directly into the IC4 framework regions). In addition, as for framework residues that are only rare at the corresponding positions in the human antibody database, human consensus sequence amino acids at those residue positions were substituted. For humanized TN228, such substitutions were made at residues 23, 40, 73, 83 and 85 of the heavy chain and at residues 69 and 77 of the light chain. Figure 9 and Figure 10 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized TN228 antibody.

Example 9 Construction and expression of humanized TN228-IgG2M3

method

Once the amino acid sequences of the humanized variable regions were designed as described above, genes were constructed to encode them, including signal peptides, splice donor signals, and appropriate restriction enzyme sites (Figure 8). A heavy chain variable region gene and a light chain variable region gene were constructed and amplified with eight overlapping synthetic oligonucleotides approximately 65-80 bases in length (He, X.Y., Z. Xu, J. Melrose , A.Mullowney, M.Vasquez, C.Queen, V.Vexler, C.Klingbeil, M.S.Co and E.L.Berg.1998. Human origin of monoclonal antibodies specific for both E-selectin and P-selectin Chemistry and Pharmacokinetics. J. Immunol. 160: 1029-1035). The oligonucleotides were annealed in pairs and extended with the Klenow fragment of DNA polymerase I to generate four double-stranded fragments. The resulting fragments were denatured, annealed in pairs, and extended with Klenow fragments to generate two fragments. These fragments are denatured, annealed in pairs, and extended again to generate the full-length gene. The resulting product was amplified by polymerase chain reaction (PCR) with Taq polymerase, gel purified, and digested with XbaI; gel purified again, then subcloned into pVg2M3 for heavy chain expression and for In the XbaI site of pVk expressing the light chain. The pVg2M3 vector for human gamma 2 heavy chain expression has been described previously (Cole, M.S., C. Anasetti and J.Y. Tso. 1997. Chimeric anti-CD33 human IgG2 variant is not mitogenic to T cells. J. Immunol. 159 : 3613-3621) and pVk vectors for human kappa light chain expression (Co, M.S., N.M.Avadalovic, P.C.Caron, M.V.Avadalovic, D.A.Scheinberg and C.Queen.1992. Chimeric antibodies specific for CD33 antigen and Humanized Antibodies. J. Immunol. 148:1149-1154).

The sequences of the V region and constant region exons of the final heavy chain plasmid and light chain plasmid were verified by nucleotide sequence determination. The entire structure of the final plasmid was verified by restriction enzyme mapping. All manipulations of DNA were performed according to standard methods.

In this specification, HuTN228 refers to a humanized antibody comprising humanized TN228 V _H and V _L variable regions, a human IgG2M3 heavy chain constant region, and a human light chain kappa constant region. This heavy chain constant region from a human germline 2 genomic fragment is modified (Cole, MS, C. Anasetti and JYTso. 1997. Chimeric anti-CD33 human IgG2 variants are not mitogenic to T cells. J. Immunol 159:3613-3621), and the light chain is derived from a human germline K genome fragment. The major immediate early promoter and enhancer of human cytomegalovirus drive both the heavy and light chain genes. The heavy chain gene is followed by a transcription terminator derived from the human complement gene C2 (Ashfield, R., P. Enriquez-Harris and NJ Proudfoot. 1991. Transcription termination between the closely linked human complement gene C2 and factor B: C2 and c-myc co-termination factor? EMBO J.10:4197-4207). Light chain selection marker gpt gene (Mulligan, RC and P.Berg.1981. Selection of animal cells expressing the xanthine-guanine phosphoribosyltransferase encoding gene of Escherichia coli.Proc.Natl.Acad.Sci.USA78:2072- 2076) and the heavy chain selectable marker dhfr gene (Simonsen, CC and AD Levinson.1983. Isolation and expression of a modified mouse dihydrofolate reductase cDNA. Proc. Natl. Acad. Sci. USA 80: 2495-2499) Both are driven by the SV40 early promoter.

For expression of HuTN228, transient transfection into COS-7 cells (monkey kidney cell line) was accomplished with Lipofectamine 2000 (Cat# 11668-027, Life Technologies). Human IgG2M3 antibody production on spent media of transient transfectants by ELISA using goat anti-human IgG gamma chain-specific antibody as capture reagent and HRP-conjugated goat anti-human kappa chain antibody as chromogenic reagent aspect analysis. In addition, the spent medium was tested for the ability of HuTN228 to bind to P815/CD28 ⁺ cells by indirect immunofluorescence staining; and the spent medium was analyzed by flow cytometry (data not shown). For stable cell line generation, the humanized expression plasmid was transfected into the murine myeloma cell line Sp2/0 by electroporation; and transfectants were selected for gpt expression. For transient transfections, the spent medium from stable transfectants was analyzed by ELISA.

result

Based on the design of the amino acid sequence of the humanized V region, the V region gene of the heavy chain and the V region gene of the light chain were constructed as described in the Methods (Fig. 9 and Fig. 10). As shown in Fig. 8, the V region gene of the heavy chain and the V region gene of the light chain were cloned into pVg2M3 vector and pVk vector, respectively. Several clones were analyzed by nucleotide sequence determination, and the correct clones of both the heavy and light chain expression vectors were used for transfection. Also, the constant regions of both the heavy chain expression vector and the light chain expression vector were further confirmed by sequencing.

Example 10 Expression of HuTN228

Transient transfection of COS-7 cells: The expression vector was transiently transfected into the monkey kidney cell line COS-7 to produce HuTN228 antibody. The culture medium of the transfected cells was used to test the production of humanized IgG2M3 antibody by ELISA and the binding to P815/CD28 ⁺ cells by flow cytometry (data not shown). Spent medium was positive in both assays. The yield of humanized antibody from transient transfection was about 3.7g/ml. HuTN228 antibody from transient supernatants bound to P815/CD28 ⁺ cells in a concentration-dependent manner (data not shown).

Stable transfection of Sp2/0 cells: To generate a stable cell line, the humanized expression vector was transfected into Sp2/0 cells. As with the transient transfectants, aliquots of media used for several transfectants were tested for HuTN228 antibody production. One transfectant (clone 4) was selected for higher antibody production and expanded in GIBCO hybridoma serum-free medium. HuTN228 antibody was purified from 570 ml of spent medium by affinity chromatography. The purified antibody yield was approximately 7 mg.

Example 11 Purification of protein

One of the high HuTN228 expressing transfectants from the stable transfection (clone 4) was grown in 570 ml of GIBCO Hybridoma Serum Free Medium (Catalog No. 12045076, Life Technologies). Harvest the spent culture supernatant when cell viability reaches 10% or less; load it onto a 2 ml column of Protein-A Sepharose. The column was washed with PBS and the antibody was eluted with 0.1M Glycine, 0.1M NaCl pH 2.5. The eluted protein was dialyzed against 2 liters of PBS with 3 changes of PBS; it was then desalted on a PD-10 column equilibrated with PBS containing additional 0.1M NaCl. The desalted protein solution was filtered through a 0.2 mm filter paper before storage at 4°C.

Example 11 Purity Determination by Size Exclusion HPLC and SDS-PAGE

method

Use a Perkin Elmer HPLC consisting of a PE ISS 200Advanced LC Sample Processor (sample processor), a PE Series 410Bio LC Pump (pump), a PE 235C Diode ArrayDetector (diode array detector) and PE Nelson 600 Series LINK system for size exclusion HPLC. Perkin Elmer Turbochrom Navigator Version 4.1 software was used to control autosamplers, pumps and detectors; and to acquire, store and process data using this software. Separation was accomplished using two TosoHaas TSK-GEL G3000SWXL size exclusion HPLC columns (7.8 mm x 300 mm, 5 mm particle size, 250 Å pore size, Cat. No. 08541, TosoHaas, Montgomeryville, MD) connected in series. The mobile phase was 200 mM potassium phosphate/150 mM potassium chloride pH 6.9 with a flow rate of 1.00 ml/min. The column eluate was monitored with a spectrophotometer at a wavelength of 220 nm and a wavelength of 280 nm. The injection volume of the HuTN228 sample was 60 1 (60 g).

SDS-PAGE was performed on a 4-20% gradient gel (Cat# EC6025, Novex, San Diego, CA) following standard procedures.

The isotype of the purified antibody was confirmed using the Human IgG Subclass Profile ELISA Kit (Cat. No. 99-1000, Zymed Laboratories, South San Francisco, CA) following the manufacturer's recommendations .

result

The purity of the isolated HuTN228 antibody was analyzed by size exclusion HPLC and SDS-PAGE. The HPLC elution profile of HuTN228 is not shown. According to this analysis, the protein is approximately 98% monomeric and has a mobility equivalent to that of a protein with a molecular weight of approximately 160 kD.

SDS-PAGE analysis of MuTN228, isotype control MuFd79 (mouse IgGl), HuTN228, and isotype control HuEP5C7 (human IgG2M3) under non-reducing conditions also showed that all four antibodies had a molecular weight of approximately 150-160 kD. Analysis of the same four proteins under reducing conditions showed that all four antibodies consisted of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD.

Isotype assays showed that the isotype of the HuTN228 antibody was consistent with the expected IgG2 isotype (data not shown).

Embodiment 12 FACS competition experiment

method

Titration experiments were performed using two-fold serial dilutions of MuTN228-FITC antibody starting at 250 ng/assay. FITC-labeled antibody was mixed with P815/CD28 ⁺ cells (3× ₁₀ ⁵ cells/ Assay) were incubated together for 1 hour, then washed with 2 ml of FBS, and analyzed by flow cytometry (data not shown).

For competition experiments, MuTN228-FITC (50ng/assay) in 251 FSB was mixed with competing HuTN228 antibody in 251 FSB or three-fold serial dilutions of MuTN228 antibody (starting with 200 g/ml antibody) and added to 501 FSB in P815/CD28 ⁺ cells (3×10 ⁵ cells/test). As a control, P815/CD28 ⁺ cells were incubated with MuTN228-FITC alone (50 ng/assay in 50 1 of FSB). Likewise, HuEP5C7 (human IgG2M3) isotype control antibody (200 g/ml) and MuFd79 (mouse IgG1 ) isotype control antibody (200 g/ml) in 25 1 FSB were tested as non-specific competitors. The antibody mixture was incubated with the cells in a final volume of 100 1 for 1 hour on ice (in the dark), then washed with 2 ml FSB and analyzed by flow cytometry. Repeat this experiment three times.

result

Binding specificity of MuTN228 antibody and HuTN228 antibody to the CD28 molecule on P815/CD28 ⁺ cells was compared using a flow cytometry competition experiment as described in Methods. Representative results are shown in Figure 5. Both MuTN228 and HuTN228 competed with MuTN228-FITC in a concentration-dependent manner, suggesting that the binding of these two antibodies to the CD28 antigen was specific. The relative binding of HuTN228 was a fraction of that of MuTN228. Isotype control antibodies MuFd79 and HuEP5C7 did not compete with MuTN228-FITC, indicating that MuTN228 antibody and HuTN228 antibody recognize CD28 antigen through the specific interaction of the V region.

Embodiment 13 ELISA competition experiment

method

Coat with 100 l/well of sCD28-Fc (0.5 g/ml in PBS) (sCD28-Fc is a fusion protein in which the extracellular domain of CD28 binds to the CH2 and CH3 domains of IgG1) at 4°C for 96 Well ELISA plates (Nunc-Immuno plates, Cat# 439454, NalgeNunc, Naperville, IL) overnight. Block the plate for 30 minutes with 300 l/well of SuperblockBlocking Buffer (blocking buffer) in TBS (Catalogue No. 37535, Pierce, Rockford, IL) and with 300 l/well of ELISA ELISA Wash Buffer (wash buffer) ( EWB=PBS, 0.1% Tween-20) to wash the plate. According to the final volume of 200l/hole, MuTN228-biotin (0.5g/ml) in 100l ELISA Buffer (ELISA buffer) (EB=PBS, 1%BSA, 0.1%Tween-20) was added in triplicate with competition A mixture of three-fold serial dilutions (starting at 100 g/ml) of HuTN228 antibody or MuTN228 antibody in 100 lEB. Isotype control antibodies HuEP5C7 and MuFd79 (100 g/ml) were also tested in 1001EB as non-specific competitors. 100 l of EB was added to 100 l of MuTN228-biotin (0.5 g/ml) as a 'no competitor' control. 200 1 of EB was added to the remaining wells (without MuTN228-biotin) as a blank (control). The plate was incubated with shaking at room temperature for 1.5 hours. After washing the wells 4 times with 300 l/well of EWB, 100 l/well of streptavidin-HRP (1 g/ml, cat. no. 21124, Pierce) was added to all wells. The plate was incubated with shaking at room temperature for 1 hour. After washing the wells as described above, 100 1/well of ABTS substrate (cat. no. 507602 and 506502, KPL, Gaithersburg, MD) was added to all wells. The plate was incubated at room temperature for 5 to 7 minutes before reading the optical density at 415 nm. Repeat this experiment three times.

result

Binding specificity of HuTN228 antibody and MuTN228 antibody to sCD28-Fc were compared using an ELISA competition assay as described in Methods. Representative results are shown in Figure 12. Both MuTN228 and HuTN228 competed with MuTN228-biotin in a concentration-dependent manner. Isotype control antibodies MuFd79 and HuEP5C7 did not compete with MuTN228-biotin, indicating that MuTN228 antibody and HuTN228 antibody recognize CD28 antigen through the specific interaction of the V region. In Table 2 the _IC50 values for MuTN228 and HuTN228 for all three experiments are shown. The relative binding of HuTN228 was on average 2.6 times that of MuTN228.

Table 2 Summary of ELISA competition

_IC50 (g/ml) competitor Experiment 1 Experiment 2 Experiment 3 average value standard deviation MuTN228 0.21 0.20 0.15 0.19 0.03 HuTN228 0.37 0.64 0.48 0.50 0.14

Example 14 Competition experiment of ¹²⁵ I-labeled antibody

method

According to Queen et al. (Queen, C., WPSchneider, HE Selick, PWPayne, NF Landolfi, JFDuncan, NMAvdalovic, M.Levitt, RP Junghans, TAWaldmann.1989. A humanized antibody that binds to the interleukin 2 receptor. Proc. Natl .Acad.Sci.86:10029-10033), the relative binding affinities of MuTN228 antibody and HuTN228 antibody were determined. Briefly, approximately 10 ng ¹²⁵ I-labeled MuTN228 in 50 l of Binding Buffer (BB=PBS, 2% FBS, 1 g/ml mouse IgG, 0.1% NaN ₃ ) was mixed with competitor MuTN228 antibody or HuTN228 Three-fold serial dilutions of the antibody in 501BB (starting at 400 g/ml) were mixed in triplicate; then, added to incubation tubes (Skatron Macrowell Tube Strips, cat. no. 15773, Molecular Devices, Sunnyvale , CA) in 1001 P815/CD28 ⁺ cells (2.5×10 ⁵ cells/assay); and incubated at 4°C for 90 minutes with gentle shaking. Isotype control antibodies HuEP5C7 and MuFd79 (400 g/ml) in 501BB were also tested as non-specific competitors. After incubation, the cell-antibody mixture was transferred to a centrifuge tube (Sarstedt MicroTubes, Cat. No. 72.702, Sarstedt, Newton, NC) containing 0.1 ml of 80% dibutyl phthalate-20% olive oil, and was washed with 50 l of BB washed the incubation tube once and centrifuged to separate bound and free counts by the method described (Kuziel, WA, SJ Morgan, TC Dawson, S. Griffin, O. Smithies, K. Ley, N. Maeda. 1997 . Severe reduction of leukocyte adhesion and monocyte extravasation in CC chemokine receptor 2 deficient mice. Proc. Natl. Acad. Sci. 94: 12053-12058). Repeat this experiment three times.

result

The relative binding affinities of the MuTN228 antibody and the HuTN228 antibody were compared using125I ^- labeled antibody competition experiments as described in Methods. Representative results are shown in FIG. 13 . Both MuTN228 and HuTN228 competed ^with125I -labeled MuTN228 in a concentration-dependent manner. The isotype control antibody MuFd79 showed weak and reproducible competition at high concentrations, but the isotype control antibody HuEP5C7 did not compete with ¹²⁵ I-labeled MuTN228; this suggests that the HuTN228 antibody recognizes CD28 through specific interactions of the V region antigen. In Table 3 the _IC50 values for MuTN228 and HuTN228 for all three experiments are shown. The apparent binding affinity of HuTN228 was approximately 2.4 times that of the MuTN228 antibody.

Table 3 Summary of ¹²⁵ I competition

IC ₅₀ (nM) competitor Experiment 1 Experiment 2 Experiment 3 average standard deviation MuTN228 0.93 1.05 1.02 1.00 0.06 HuTN228 2.65 2.43 2.13 2.40 0.26

Example 15 Determination of the amino acid sequence of the hamster anti-mouse CD28 antibody

method

Hybridomas and antibodies: Armenian hamster anti-mouse CD28 hybridoma PV1 was obtained from ATCC (ATCC HB-12352). Purified PV1, R-phycoerythrin (R-PE)-conjugated PV1 was purchased from Southern Biotechnology (Birmingham, AL). Syrian hamster anti-CD28 antibody 37.51 was from PharMingen (San Diego, CA). Secondary antibodies Fluorescein (FITC)-conjugated donkey anti-Armenian hamster IgG (H+L), FITC-conjugated donkey anti-Syrian hamster IgG (H+L), FITC-conjugated donkey anti-mouse IgG (H+L) ), R-PE-F(ab') ₂ donkey anti-mouse IgG (H+L) from Jackson ImmunoResearch (West Grove, PA); and FITC-conjugated goat anti-mouse κ, conjugated R-PE Goat anti-mouse IgG3 and goat anti-mouse Kappa conjugated to horseradish peroxidase (HRP) were from Southern Biotechnology. Goat anti-mouse IgG3 and mouse IgG3 isotype control FLOPC 22 were from Sigma Chemicals (St. Louis, MO). In our laboratory, Armenian hamster anti-mouse CD3 antibody 145.2C11 and its hamster/mouse chimera 145.2C11-IgG3 were produced. FITC-conjugated 145.2C11 was from Boehringer Mannheim (Indianapolis, IN).

Cloning of variable region cDNA: anchored polymerase chain reaction (PCR) method described by Co et al. (Co, MS, NMAvadalovic, PC Caron, MVAvadalovic, DAScheinberg and C.Queen. 1154), cloning the cDNAs of the light and heavy chain V regions of PV1 from said hybridoma cells. Amplification was performed on the cDNA using 3' primers that annealed to the C region of the hamster kappa chain and the C region of the gamma chain, respectively, and a 5' primer that annealed to the G tail of the added cDNA. For _VL PCR, the 3' primer has the sequence 5'TATAGAGCTCCACTTCCAGTGCCC 3' (SEQ ID NO: 21 ), which hybridizes to the hamster _CK region from residues 11-24. For _VH PCR, the 3' primers had the following degenerate sequences (SEQ ID NOs: 18, 19 and 20):

A A G T

5′ TATAGAGCTCAAGCTTCCAGTGGATAGACCGATGGGGCTGTCGTTTTGGC,

T

Its residues 19-50 hybridize to most rodent IgG _CH1 . Non-hybridizing sequences in both primer sets contain restriction enzyme sites for cloning. The cDNAs of VL and VH were subcloned into pUC19 vector for sequence determination. In order to avoid errors caused by PCR, the sequences of 5 independent clones were determined for each cDNA; and only clones whose sequences were consistent with the consensus sequences were selected to express chimeric PV1.

result

Cloning of PV1 V region cDNA: The PV1 light chain variable region cDNA and heavy chain variable region cDNA were cloned from hybridoma cells as described in Methods. For VLPCR, only the 3' primer corresponding to the hamster C _gamma region was able to generate a V _L cDNA product from PV1. On the other hand, a 3' primer from the hamster C _gamma region did not produce any PCR products. These results indicate that the light chain of hybridoma PV1 uses kappa. Several clones of the light and heavy chains were sequenced and found to contain identical _VL and _VH , respectively. Limited sequence data for _CH1 and _Cγ indicated that the cloned heavy and light chains were not of murine origin.

Example 16 Construction and expression of chimeric PV1-IgG3

method

According to the method already described (He, XY, Z. Xu, J. Melrose, A. Mullowney, M. Vasquez, C. Queen, V. Vexler, C. Klingbeil, MSCo and EL Berg. 1998. J. Immunol. 160: 1029-1035), by PCR, the V _L and V _H of PV1 were prepared into a small exon segment adjacent to the XbaI site; and they were respectively introduced into the light chain expression plasmid and the heavy chain expression plasmid (Figure 14) . Each small exon contains a signal peptide sequence, a mature variable region sequence and a 5' splice donor sequence derived from the most homologous mouse J chain gene. Using such a splice donor, the variable region exons are spliced onto the constant region of the mouse antibody. After cloning the mini-exon into the expression vector, the sequence of each mini-exon was determined again to ensure that the correct splicing signal was introduced and no PCR errors occurred.

A vector was constructed to express both the heavy and light chain genes of chimeric PV1-IgG3 from a single plasmid. In this report, PV1-IgG3 denotes a chimeric antibody comprising the VL and _VH variable regions of hamster PV1, the heavy chain constant region of mouse IgG3, and the mouse light chain kappa constant region. By a two-step cloning method similar to that described by Cole et al. (Cole, MS, C.Anasetti and JYTso.1997.J.Immunol.159:3613-3621), the expression vector pV1.g3.rg.dE (Fig. 14 ). The heavy chain constant region is derived from the mouse γ3 genome fragment, and the light chain is derived from the κ fragment. Both the heavy and light chain genes are driven by the major immediate early promoter and enhancer of human cytomegalovirus, and they are terminated by a transcription terminator derived from human complement gene C2 (Ashfield, R., P. Enriquez-Harris and NJ Proudfoot. 1991. EMBO J. 10: 4197-4207). The selectable marker gpt gene (Mulligan, RC and P. Berg. 1981. Proc. Natl. Acad. Sci. USA 78:2072-2076) is driven by a modified SV40 early promoter. To express chimeric PV1-IgG3, the single plasmid vector was transfected into the murine myeloma cell line NS0; and transfectants were selected based on the expression of gpt. Spent media from transfectants were analyzed for mouse IgG3 antibody production by ELISA using goat anti-mouse IgG3 as capture reagent and goat anti-mouse kappa chain conjugated to HRP as chromogenic reagent. This assay is specific for mouse IgG3, in which other mouse IgG isotypes were negative.

result

Expression of Chimeric PV1-IgG3: Cloned _VL and _VH were prepared as mini-exons (Figure 15) and incorporated into an expression vector as described in Materials and Methods and Figure 15. Then, this expression vector was transfected into the murine myeloma cell line NSO to generate chimeric PV1-IgG3. Spent media from several transfectants were analyzed for production of mouse IgG3 antibodies by ELISA and for binding to EL4 cells by FACScan. In both assays, most transfectants were positive. One transfectant, selected for high antibody production, was expanded in 1 liter of serum-free medium. PV1-IgG3 was purified from 1 liter of spent medium by affinity chromatography. Yield > 10 mg/l.

Example 17 Characterization of purified chimeric PV1-IgG3 by HPLC and SDS-PAGE

method

Purification of protein: One of the transfectants with high expression of IgG3 (clone No. 1) was cultured in 1 liter of Gibco serum-free hybridoma medium. Harvest the spent culture supernatant when cell viability reaches 30% or less; concentrate it to 200ml and pipette it into a 5ml tube with a PharmaciaP1 pump (2-3ml/min) Protein-A Sepharose column. Next, the column was washed with PBS containing an additional 0.1M NaCl (final concentration of NaCl was 0.25M), and the antibody was then eluted with 3.5M MgCl. Next, the eluted protein was desalted on a PD-10 column equilibrated with PBS containing additional 0.1M NaCl. The desalted protein solution was filtered through a 0.2 mm filter paper before storage at 4°C. Like all mouse IgG3, PV1-IgG3 at high concentrations (>1 mg/ml) precipitates at low temperatures and dissolves back into solution by warming at 37°C. At room temperature, the antibody remains in solution. Repeated cycles of cryoprecipitation did not appear to affect the antigen-binding activity of the antibody.

Purity determination by size exclusion HPLC and SDS-PAGE: with a PEISS 200 Advanced LC Sample Processor (sample processor), a PE Series410Bio LC Pump (pump), a PE 235C Diode Array Detector (diode array detection Device) and a Perkin Elmer HPLC system composed of PE Nelson 600Series LINK for size exclusion HPLC. Perkin Elmer Turbochrom NavigatorVersion 4.1 software was used to control the autosampler, pumps and detectors; and the software was used to acquire, store and process data. Separation was accomplished using two TosoHaas TSK-GELG3000SWXL size exclusion HPLC columns (TosoHaas, cat. no. 08541, 7.8 mm x 300 mm, 5 mm particle size, 250 Å pore size) connected in series. The mobile phase was 200 mM potassium phosphate/150 mM potassium chloride pH 6.9 with a flow rate of 1.00 ml/min. The column eluate was monitored with a spectrophotometer at a wavelength of 220 nm and a wavelength of 280 nm. The injection volume of undiluted PV1-IgG3 samples was 50 μl (63.5 μg). Following standard methods, SDS-PAGE was performed.

result

The purity of the isolated PV1-IgG3 was analyzed by size exclusion HPLC and SDS-PAGE. The HPLC elution profile of PV1-IgG3 is shown in FIG. 16 . According to this analysis, the protein is 99% monomeric and has a mobility corresponding to a molecular weight of 150 kD. SDS-PAGE analysis of PV1, PV1-IgG3 and isotype control under non-reducing conditions also showed that all three antibodies had a molecular weight of approximately 150 kD (Fig. 17A). The weaker band seen in Figure 17A is an artifact of the sample being boiled in SDS without reduction. They reflect the number of outstanding interchain disulfide bonds in the antibody. But analysis of the same three proteins under reducing conditions (Fig. 17B) revealed that PV1, but not PV1-IgG3 or the isotype control, had a heavy chain with a molecular weight slightly higher than the 50 kD normally seen for IgG. Therefore, either the hamster antibody PV1 is glycosylated on the heavy chain at Asn ₂₉₇ in _CH3 , or it has an additional glycosylation site elsewhere in the heavy chain. As discussed later, this rare glycosylation pattern may contribute to the nonspecific binding of PV1 to EL4 cells, perhaps through lectin/carbohydrate interactions.

Example 18

method

Flow cytometry: Murine T cell line EL4 cells (2.5 x ¹⁰⁵ cells/0.2ml) were stained with 1 μg/ml of PV1, 37.51 or PV1-IgG3 for 30 min at 4°C, and then washed with 2 ml of cold PBS, And it was stained with 20 μl of a specific secondary antibody (10 μg/ml) conjugated to a fluorescent dye. After incubation for 20 minutes at 4°C in the dark, the cells were washed with PBS and analyzed with a FACScan (Becton Dickenson, Milpitas, CA).

In the competition experiment, EL4 cells (2.5×10 ⁵ cells/ 0.2 ml) for 30 minutes; the cells were washed with PBS and analyzed by FACScan. Similar competition experiments were also performed with different forms of 145.2C11. In the reversible competition experiment, EL4 cells (2.5×10 ⁵ cells/0.2ml) were stained with 1 μg/ml PV1-IgG3 and 25 μg/ml PV1 for 30 minutes at 4°C; They were stained with FITC-conjugated donkey anti-mouse IgG (H+L), washed, and analyzed by FACScan. To control for non-specific binding of the secondary antibody to PV1, EL4 cells were stained with excess PV1 in the absence of PV1-IgG3 and analyzed.

For mouse T cell staining, splenocytes (2.5 x ¹⁰⁵ cells/0.2ml) from BALB/c mice were treated with 1 μg/ml mouse IgG3 isotype control (FLOPC 21) or PV1-IgG3 at 4°C. Stained for 30 minutes; then washed with 2 ml cold PBS and stained with 20 μl FITC-conjugated 145.2C11 (10 μg/ml) and 20 μl R-PE-conjugated goat anti-mouse IgG3 (10 μg/ml). After incubation for 20 minutes at 4°C in the dark, the cells were washed with PBS and analyzed with FACScan.

result

Characterization of PV1 and PV1-IgG3 by flow cytometry: The CD28 positive T cell line EL4 was stained with PV1 and analyzed with FACScan. The pattern of staining indicated that PV1 bound EL4 cells at two distinct sites (Fig. 17A). In addition, PV1, as well as several Armenian hamster anti-mouse T-cell antibodies (145.2C11, anti-CD3; H57-597, anti-TCR; and UC10-4F10-11, anti-CTLA4), also interacted with the CD28-negative myeloma cell line NS0 Binds non-specifically (data not shown). In contrast, Syrian hamster anti-CD28 antibody 37.51 bound specifically to only one site on EL4 cells (Fig. 17B). It appears that PV1 also binds non-specifically to other sites besides CD28; possibly through carbohydrate/lectin-type interactions. As shown in Figure 17C, chimeric PV1-IgG3 did not contain this non-specific binding activity. This antibody bound to EL4 cells in a manner similar to that of 37.51, and it did not bind to CD28 negative NSO cells (data not shown). Thus, the nonspecific binding properties of PV1 depend on the heavy chain constant region of this particular antibody and are abolished by chimerization.

To confirm that PV1-IgG3 contains CD28-specific binding activity, we employed FACScan competition analysis. In these experiments, R-PE-conjugated PV1 was mixed with an excess (25-fold) of unlabeled PV1, PV1-IgG3, or mouse IgG3 control, and this mixture was used to stain EL4 cells. As shown in Figure 18A, both PV1 and PV1-IgG3, but not the isotype control, prevented the binding of R-PE-conjugated PV1 to EL4 cells. Inhibition by PV1-IgG3 was less than that by PV1, we interpret these data as PV1-IgG3 competes with R-PE-conjugated PV1 for CD28 sites, rather than for non-specific sites. Similarly, both 145.2C11 (Armenian hamster anti-mouse CD3) and chimeric 145.2C11-IgG3 prevented binding of R-PE-conjugated 145.2C11 to EL4 cells (Fig. 18B); R-PE-145.2C11 binds non-specifically to cells, so the antibody is less efficient.

We also used excess (25-fold) PV1 to compete with PV1-IgG3 for binding to EL4 cells, and performed reversible competition experiments. Although PV-1-IgG3 was not labeled in this case, it was specifically recognized with a FITC-conjugated donkey anti-mouse antibody. The results in FIG. 18C show that the inhibition of PV1-IgG3 binding to EL4 cells caused by excess PV1 is almost complete; thus confirming that PV1 and PV1-IgG3 bind to the same epitope.

Finally, mouse splenocytes were stained with PV1-IgG3. Splenocytes coated with PV1-IgG3 were specifically recognized by the secondary antibody goat anti-mouse IgG3 conjugated to R-PE. At the same time, FITC-conjugated 145.2C11 was also added to splenocytes to label CD3-positive cells. PV1-IgG3 specifically stained CD3-positive cells but not CD3-negative cells in dual-color flow cytometry analysis (Fig. 19B). In contrast, the mouse IgG3 isotype control did not stain CD3 positive cells (Figure 19A). Thus, chimeric PV1-IgG3 recognized an antigen expressed on murine T cells, an antigen-binding activity consistent with anti-CD28 antibodies.

Example 19 Induction of collagen-induced arthritis

method

Mice were immunized in the basal dermis of mouse tails with 125 μg of bovine CII (Collagen Gijutsu Kenkyukai, Japan) emulsified with an equal volume of CFA (Wako, Japan). On day 21, mice were boosted with 125 μg of bovine CII in CFA by intradermal injection. After the primary immunization, mice were treated with continuous infusion of anti-CD28 antibody (PV1-IgG3) at a dose of 1 mg/kg/day by means of an isotonic pump for 7 days. On day 11 after the second immunization, four paws were examined for the development of arthritis; and as previously described (Tada, Y., A.Ho, D.-R.Koh, T.W. 1996. J. Immunol. 156: 4520, . Tada, Y., A. Ho, T. Matsuyama, T. W. Mak. 1997. J. Exp. Med. 185: 231), the inflammation of the four paws on a scale of 0 to 3 Rating level. Each paw is graded and the four scores are added; thus the maximum score for each mouse is 12. Arthritis index was calculated by dividing the total score of experimental mice by the total number of mice.

result

Mice were immunized with bovine CII, and the occurrence of arthritis in mice was observed. On day 11 after the second immunization, the arthritis index (0.63±0.50) (P<0.01) was significantly decreased in the mice treated with anti-CD28 antibody compared to the control (7.50±0.66).

Example 20

method

mouse: animal

Female BALB/c mice and C3H mice were obtained from Charles River Japan, Inc. (Yokohama, Japan). Animals were all housed in small isolation cages with filtered air in a pathogen-free facility with free access to food and water. All mice were 6 to 8 weeks old when experiments were started.

Antibody:

Silent anti-mouse CD28 (PV1-IgG3) has the same specificity as the PV-1 clone, but it does not have strong agonistic activity in vitro (Fc→IgG3). Anti-mouse CD154 (TRAP1, IgG1) was purchased from BD PharMingen (San Diego, CA). CTLA4-Ig (CTLA-4/Fc chimera) was purchased from Genzyme (Cambridge, MA).

Tail skin transplant:

A full-thickness skin graft (0.5 cm2) from the tail of a donor mouse (BALB/c: H-2d) was grafted onto the dorsal chest of a recipient mouse (C3H: H-2b) and secured with a first aid bandage 7 days. Graft survival was then followed by daily visual observations. Rejection was defined as loss of >80% viable epidermal graft tissue. Statistical analysis was performed with Dunnett's multiple comparison test. Values of p<0.05 were considered significant.

Treatment programs:

Recipients of skin grafts were treated with 10 μg, 50 μg, 250 μg of silencing anti-mouse CD28. 250 μg anti-mouse CD154 and 100 μg CTLA4-Ig were administered intraperitoneally on the day of transplantation (day 0) and on days 3 and 6 after handing.

result:

Simultaneous blockade of the CD40 and CD28 T cell co-stimulatory pathway by administration of silencing anti-mouse CD28 and anti-mouse CD154 effectively promoted skin allograft survival in C3H mice. Control animals rejected their grafts on day 9. Anti-CD40L mAb alone moderately prolongs allograft survival (MST of 10 days), but when combined with CD28, a significant improvement in survival is observed, extending the median survival time (MST) to more than 10 days. 33 days. This strategy was significantly less effective when administered with CTLA4-Ig and anti-mouse CD40L monoclonal antibody, with a MST of 12 days.

Example 21 Preparation of Fab and F(ab')2 fragments of anti-CD28 antibody

Preparation of Fab Fragment of Anti-CD28 Antibody

Anti-human CD28 antibody (HuTN228) was digested with immobilized ficin (Pierce, USA). The immobilized ficin was activated with 50 mM Tris-HCl pH 6.8 buffer containing 5 mM EDTA and 11.5 mM cysteine hydrochloride; and packed into a column. The antibody solution was added to the column and incubated at 37°C for 2 or 3 days. The column was washed with PBS and the digest was concentrated by ultrafiltration. The concentrated digest was applied to a gel filtration column (TSKgel-3000SWxl, Tosoh, Japan), and appropriate fractions were collected and concentrated by ultrafiltration. Protein concentrations were determined from absorbance at 280 nm (Abs280 = 1.4 for 1 mg/ml); and fragment sizes were confirmed by SDS-PAGE.

Preparation of F(ab')2 fragment of anti-CD28 antibody

An anti-human CD28 antibody was prepared in the same manner as the preparation of the Fab fragment (except for the concentration of cysteine (1.15 mM) and incubation time (once overnight)).

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Sequence Listing

<110> VASQUEZ. Maximiliano

HINTON. Paul

TAMURA, Kouiehi

HIGASHI, Yauyuki

SEKI. Nobuo

UEDA, Hirotsugu

TSO, J. Yun

<120> Silent anti-CD28 antibody and its application

<130>200071USOPROV

<150>US 60/255,155

<151>2000-12-14

<160>21

<170>PatentIn version 3.1

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         Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu

1 5 10

tgg gtt cca ggc tcc act ggt gac att gtg ctc acc caa tct cca gct 98

Trp Val Pro Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala

15 20 25

tct ttg gct gtg tct ctg ggg cag aga gcc acc atc tcc tgc aga gcc 146

Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala

30 35 40 45

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Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gln Trp Tyr Gln

50 55 60

cag aaa cca gga cag cca ccc aaa ctc ctc atc tat gct gca tcc aac 242

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

65 70 75

gta gat tct ggg gtc cct gcc agg ttt agt ggc agt ggg tct ggg aca 290

Val Asp Ser Gly Val Pro Ala Arg PheSer Gly Ser Gly Ser Gly Thr

80 85 90

gac ttc agc ctc aac atc cat cct gtg gag gag gat gat att gca atg 338

Asp Phe Ser Leu Asa Ile His Pro Val Glu Glu Asp Asp Ile Ala Met

95 100 105

tat ttc tgt cag caa agt agg aag gtt cca ttc acg ttc ggc tcg ggg 386

Tyr Phe Cys Gln Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Ser Gly

110 115 120 125

aca aag ttg gaa ata aaa cgtaagtaga cttttgctct aga 427

Thr Lys Leu Glu Ile Lys

130

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<212>PRT

<213> heterozygous

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Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

35 40 45

Val Glu Tyr Tyr Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu IIe Tyr Ala Ala Ser Ash Val Asp Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

85 90 95

Leu Asn Ile His Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys

100 105 110

Gln Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

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<400>3

tctagaccac c atg gag tca gac aca ctc ctg cta tgg gtg ctg ctg ctc 50

         Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu

1 5 10

tgg gtt cca ggc tcc act ggt gac att gtg ctc acc caa tct cca gct 98

Trp Val Pro Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala

15 20 25

tct ttg gct gtg tct ctg ggg cag aga gcc acc atc tcc tgc aga gcc 146

Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala

30 35 40 45

agt gaa agt gtt gaa tat tat gtc aca agt tta atg cag tgg tac caa 194

Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gln Trp Tyr Gln

50 55 60

cag aaa cca gga cag cca ccc aaa ctc ctc atc tat gct gca tcc aac 242

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

65 70 75

gta gat tct ggg gtc cct gcc agg ttt agt ggc agt ggg tct ggg aca 290

Val Asp Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

80 85 90

gac ttc agc ctc aac atc cat cct gtg gag gag gat gat att gca atg 338

Asp Phe Ser Leu Asn Ile His Pro Val Glu Glu Asp Asp Ile Ala Met

95 100 105

tat ttc tgt cag caa agt agg aag gtt cca ttc acg ttc ggc tcg ggg 386

Tyr Phe Cys Gln Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Ser Gly

110 115 120 125

aca aag ttg gaa ata aaa cgt aag tag act ttt gct cta gat cta 431

Thr Lys Leu Glu Ile Lys Arg Lys Thr Thr Phe Ala Leu Asp Leu

130 135

131

gaccaccatg gctgtcctgg tgctgttcct ctgcctggtt gcatttccaa gctgtgtcct 491

gtcccaggtg cagctgaagg agtcaggacc tggcctggtg gcgccctcac agagcctgtc 551

catcacttgc actgtctctg gattttcatt aaccagctat ggtgtacact gggttcgcca 611

gcctccagga aagggtctgg aatggctggg agtcatatgg cctggtggag gcacaaattt 671

taattcggct ctcatgtcca gactgagcat cagcgaagac aactccaaga gccaagtttt 731

cttaaaaatg aacactctgc aaactgatga cacagccata tattattgtg ccagagatcg 791

ggcgtatggt aactacctct atgccatgga ctactggggt caaggaacct cagtcaccgt 851

ctcctcaggt aagaatggcc tctaga 877

<210>4

<211>133

<212>PRT

<213> heterozygous

<400>4

Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

35 40 45

Val Glu Tyr Tyr Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Asp Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

85 90 95

Leu Asn Ile His Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys

100 105 110

Gln Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Lys

130

<210>5

<211>6

<212>PRT

<213> heterozygous

<400>5

Thr Phe Ala Leu Asp Leu

1 5

<210>6

<211>450

<212>DNA

<213> heterozygous

<220>

<221> CDS

<222>(12)..(431)

<223>

<400>6

tctagaccac c atg gct gtc ctg gtg ctg ttc ctc tgc ctg gtt gca ttt 50

        Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe

1 5 10

cca agc tgt gtc ctg tcc cag gtg cag ctg cag gag tca gga cct ggc 98

Pro Ser Cys Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly

15 20 25

ctg gtg aag ccc tca gag acc ctg tcc ctc act tgc gct gtc tct gga 146

Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly

30 35 40 45

ttt tca tta acc agc tat ggt gta cac tgg att cgc cag cct cca gga 194

Phe Ser Leu Thr Ser Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly

50 55 60

aag ggt ctg gaa tgg ctg gga gtc ata tgg cct ggt gga ggc aca aat 242

Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Pro Gly Gly Gly Thr Asn

65 70 75

ttt aat tcg gct ctc atg tcc aga ctg acc atc agc gaa gac acc tcc 290

Phe Asn Ser Ala Leu Met Ser Arg Leu Thr Ile Ser Glu Asp Thr Ser

80 85 90

aag aac caa gtt tcc tta aaa ttg agc tct gtg aca gct gct gac aca 338

Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr

95 100 105

gcc gta tat tat tgt gcc aga gat cgg gcg tat ggt aac tac ctc tat 386

Ala Val Tyr Tyr Cys Ala Arg Asp Arg Ala Tyr Gly Asn Tyr Leu Tyr

110 115 120 125

gcg atg gac tac tgg ggt caa gga acc tta gtc acc gtc tcc tca 431

Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

ggtaagaatg gcctctaga 450

<210>7

<211>140

<212>PRT

<213> heterozygous

<400>7

Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys

1 5 10 15

Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu

35 40 45

Thr Ser Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Pro Gly Gly Gly Thr Asn Phe Asn Ser

65 70 75 80

Ala Leu Met Ser Arg Leu Thr Ile Ser Glu Asp Thr Ser Lys Asn Gln

85 90 95

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Asp Arg Ala Tyr Gly Asn Tyr Leu Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

<210>8

<211>427

<212>DNA

<213> heterozygous

<220>

<221> CDS

<222>(12)..(404)

<223>

<400>8

tctagaccac c atg gag tca gac aca ctc ctg cta tgg gtg ctg ctg ctc 50

         Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu

1 5 10

tgg gtt cca ggc tcc act ggt gac att cag atg acc caa tct cca tct 98

Trp Val Pro Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser

15 20 25

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Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

30 35 40 45

agt gaa agt gtt gaa tat tat gtc aca agt tta atg cag tgg tac caa 194

Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gln Trp Tyr Gln

50 55 60

cag aaa cca gga aag gca ccc aaa ctc ctc atc tat gct gca tcc aac 242

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

65 70 75

gta gat tct ggg gtc cct tcc agg ttt agt ggc agt ggg tct ggg aca 290

Val Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

80 85 90

gac ttc acc ctc acc atc tct tct ctg cag ccg gag gat att gca acg 338

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr

95 100 105

tat tac tgt cag caa agt agg aag gtt cca ttc acg ttc ggc ggg ggg 386

Tyr Tyr Cys Gln Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Gly Gly

110 115 120 125

aca aag gtg gaa ata aaa cgtaagtaga cttttgctct aga 427

Thr Lys Val Glu Ile Lys

130

<210>9

<211>131

<212>PRT

<213> heterozygous

<400>9

Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser

35 40 45

Val Glu Tyr Tyr Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Asp Ser

65 70 75 80

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Ser Arg Lys Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys

130

<210>10

<211>445

<212>DNA

<213> heterozygous

<220>

<221> CDS

<222>(21)..(419)

<223>

<400>10

tctagacagt gggaacaat atg gat tca cag atc cag gtc ctc atg tcc ctg 53

                                    Met Asp Ser Gln Ile Gln Val Leu Met Ser Leu

1 5 10

ctc ctc tgg atg tct ggt gcc tgt gga gat att gtg atg acc cag tct 101

Leu Leu Trp Met Ser Gly Ala Cys Gly Asp Ile Val Met Thr Gln Ser

15 20 25

cca tat tcc ctg gct gtg tca gca gga gag aag gtc acc atg agt tgc 149

Pro Tyr Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys

30 35 40

agg tcc agt cag agc ctc tat tac agt gga atc aaa aag aac ctc ttg 197

Arg Ser Ser Gln Ser Leu Tyr Tyr Ser Gly Ile Lys Lys Asn Leu Leu

45 50 55

gcc tgg tac cag cag aaa cca ggc cag tct ccg aaa ctg ctg atc tac 245

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

60 65 70 75

ttt aca tct act cgg tta cct ggg gta ccg gat cgc ttc aca ggc agt 293

Phe Thr Ser Thr Arg Leu Pro Gly Val Pro Asp Arg Phe Thr Gly Ser

80 85 90

gga tct ggg aca gat tac act ctc acc atc acc agt gtc cag gct gaa 341

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Ala Glu

95 100 105

gac atg ggg cat tat ttc tgt cag cag ggt ata agc act ccg ctc acg 389

Asp Met Gly His Tyr Phe Cys Gln Gln Gly Ile Ser Thr Pro Leu Thr

110 115 120

ttc ggt gat ggc acc aag ctg gag aga cgtaagtaga atccaaagtc 439

Phe Gly Asp Gly Thr Lys Leu Glu Ile Arg

125 130

tctaga 445

<210>11

<211>133

<212>PRT

<213> heterozygous

<400>11

Met Asp Ser Gln Ile Gln Val Leu Met Ser Leu Leu Leu Trp Met Ser

1 5 10 15

Gly Ala Cys Gly Asp Ile Val Met Thr Gln Ser Pro Tyr Ser Leu Ala

20 25 30

Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Arg Ser Ser Gln Ser

35 40 45

Leu Tyr Tyr Ser Gly Ile Lys Lys Asn Leu Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Phe Thr Ser Thr Arg

65 70 75 80

Leu Pro Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Tyr Thr Leu Thr Ile Thr Ser Val Gln Ala Glu Asp Met Gly His Tyr

100 105 110

Phe Cys Gln Gln Gly Ile Ser Thr Pro Leu Thr Phe Gly Asp Gly Thr

115 120 125

Lys Leu Glu Ile Arg

130

<210>12

<211>467

<212>DNA

<213> heterozygous

<220>

<221> CDS

<222>(16)..(444)

<223>

<400>12

tctagagtct tcacc atg gta tgg ggc ttg atc atc atc ttc ctg gtc aca 51

           Met Val Trp Gly Leu Ile Ile Ile Phe Leu Val Thr

1 5 10

gca gct aca ggt gtc cac tcc cag gtc cag ttg aag cag tct ggg gct 99

Ala Ala Thr Gly Val His Ser Gln Val Gln Leu Lys Gln Ser Gly Ala

15 20 25

gag ctt gtg aag cct gga gcc tca gtg aag ata tcc tgc aaa act tca 147

Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser

30 35 40

ggc tat acc ttc act gat ggc tac atg aac tgg gtt gag cag aag cct 195

Gly Tyr Thr Phe Thr Asp Gly Tyr Met Asn Trp Val Glu Gln Lys Pro

45 50 55 60

ggg cag ggc ctt gag tgg att gga aga att gat cct gat agt ggt aat 243

Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Asp Ser Gly Asn

65 70 75

act cgg tac aat cag aaa ttc cag ggc aag gcc aca ctg act aga gac 291

Thr Arg Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Arg Asp

80 85 90

aaa tcc tcc agc aca gtc tac atg gac ctc agg agc ctg aca tct gag 339

Lys Ser Ser Ser Thr Val Tyr Met Asp Leu Arg Ser Leu Thr Ser Glu

95 100 105

gac tct gct gtc tat tac tgt gcg aga gat ggg acc ttc tac ggt acc 387

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Gly Thr Phe Tyr Gly Thr

110 115 120

tac ggc tac tgg tac ttc gat ttc tgg ggc cag ggg acc cag gtc acc 435

Tyr Gly Tyr Trp Tyr Phe Asp Phe Trp Gly Gln Gly Thr Gln Val Thr

125 130 135 140

gtc tcc tca ggtgagtcct taaaacctct aga 467

Val Ser Ser

<210>13

<211>143

<212>PRT

<213> heterozygous

<400>13

Met Val Trp Gly Leu Ile Ile Ile Phe Leu Val Thr Ala Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Asp Gly Tyr Met Asn Trp Val Glu Gln Lys Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Arg Ile Asp Pro Asp Ser Gly Asn Thr Arg Tyr Asn

65 70 75 80

Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Arg Asp Lys Ser Ser Ser Ser

85 90 95

Thr Val Tyr Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Gly Thr Phe Tyr Gly Thr Tyr Gly Tyr Trp

115 120 125

Tyr Phe Asp Phe Trp Gly Gly Gln Gly Thr Gln Val Thr Val Ser Ser

130 135 140

<210>14

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>14

tatagagctc aagcttggat ggtgggaaga tggatacagt tggtgc 46

<210>15

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>15

tatagagctc aagcttccag tggatagacc gatggggctg tcgttttggc 50

<210>16

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>16

tatagagctc aagcttccag tggatagaca gatgggggtg ttgttttggc 50

<210>17

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>17

tatagagctc aagcttccag tggatagacc gttggggctg tcgttttggc 50

<210>18

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>18

tatagagctc aagcttccag tggatagacc gatggggctg tcgttttggc 50

<210>19

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>19

tatagagctc aagcttccag tggatagacc gatgggggtg ttgttttggc 50

<210>20

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>20

tatagagctc aagcttccag tggatagtcc gatggggctg tcgttttggc 50

<210>21

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA

<400>21

tatagagctc cacttccagt gccc 24

Claims (13)

1. anti-CD28 antibody of reticent type, it has: have the variable region of heavy chain and the variable region of light chain with aminoacid sequence among the SEQ ID NO:2 of aminoacid sequence among the SEQ ID NO:4, perhaps have the variable region of heavy chain and the variable region of light chain with aminoacid sequence among the SEQID NO:9 of aminoacid sequence among the SEQ ID NO:7; And has human IgG2 M3 CH.

2. the polynucleotide of the antibody of the claim 1 of encoding.

3. expression vector that comprises the polynucleotide of claim 2.

4. host cell that comprises the polynucleotide of claim 2.

5 one kinds of host cells that comprise the expression vector of claim 3.

6. method of producing the anti-CD28 antibody of reticent type, described method comprises:

Be suitable under the condition of described antibody expression, cultivating the host cell of claim 4; And from described culture, reclaim expressed antibody.

7. method of producing the anti-CD28 antibody of reticent type, described method comprises:

Be suitable under the condition of described antibody expression, cultivating the host cell of claim 5; And from described culture, reclaim expressed antibody.

8. method of producing the anti-CD28 antibody of reticent type, described method comprises:

The polynucleotide of claim 2 are incorporated in the host cell;

Be suitable for cultivating described host cell under the condition of described antibody expression; And from described culture, reclaim expressed antibody.

9. method of producing the anti-CD28 antibody of reticent type, described method comprises:

The expression vector of claim 3 is incorporated in the host cell;

Be suitable for cultivating described host cell under the condition of described antibody expression; And from described culture, reclaim expressed antibody.

10. medicinal compositions, described composition comprises anti-CD28 antibody of the reticent type of claim 1 and pharmaceutically acceptable composition.

11. the antibody of claim 1 is used for the treatment of application in the medicine that patient's intracorporeal organ or tissue transplantation repel in preparation.

12. the application of claim 11, wherein said medicine and another kind of immunosuppressive drug coupling.

13. antibody HuTN228, it has: have the variable region of heavy chain of aminoacid sequence among the SEQ ID NO:7, the variable region of light chain with aminoacid sequence among the SEQ ID NO:9 and human IgG2 M3 CH.

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