JPH06505396A - Soluble CD28 protein and therapeutic methods using the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Soluble CD28 protein and therapeutic method using the same 1-! The present invention relates to the CD28 protein, which is associated with T-cell immune responses. Even more special If so, the present invention provides soluble CD2B protein fragments and the use of such fragments. Regarding treatment methods.

CD28 (also known as Tp44: fefss, A,; Mana Ger.

B, and fboden, J, , J, [mmunol, , 137.81 9-825.1986; Aruffo, A., and 5eed, B. Pro c, Natl, Acad, Set, USA.

84, 8573-8577.1987) The protein is a transmembrane protein. is a membrane-bound T-cell differentiation antigen containing in. It has a molecular weight of 44 kDa A large subset of peripheral blood T-lymphocytes is a homodimeric membrane protein with Expressed on the host. It has been demonstrated that it is involved in T-cell activation by antigen. (Weiss, A, Manager, B, and Embode n, J, J, Immunol, 137, 819-825.1986 >. The complete gene sequence encoding CD211 is published by Aruffo, A., and 5 eed, B, Proc, Natl, Acad, Sci, USA, 84, 8573-11577.1987.

Recent studies have shown that the CD28 antigen expressed in Chinese hamster ovary cells in human lymphoblastic and leukemic B-cell lines and activated mediate specific cell-cell adhesion with primary mouse B cells. It was shown that B-cell activation antigen 87 by CD28 (also known as BB-1) specific members of the immunoglobulin superfamily (known) are members of the immunoglobulin superfamily. The interaction represents a heterophilic interaction between T-cell cells at the site of B-cell activation. May play a role in regulating itokine levels. The results of that study showed that CD2B-mediated adhesion but in maintaining or amplifying the immune response rather than initiating it. This led the researchers to conclude that it could play a role. (Lfnsley, P. , S, , C1ark, E, A, , Ledbetter, J, A, , P roc, Natl, Ac Hiroe 1. , USA, 87.5031-5035 ．． 1990).

In order for T-cells to increase the immune response against a given antigen, T-cells and anti-antigens must Two types of interaction with the original presenting cell are required. The first interaction is , T-cell antigen receptor of T-cells and antigen-histocompatibility molecule complex of antigen-presenting cells. occurs between the body. A second interaction is also required, ■ - the cell's CD28 molecule and B-7 molecules on antigen-presenting cells have been shown to provide the necessary second signal. Instigated. However, soluble human proteins can be used to suppress this specific pathway. There are no publications demonstrating that it can be used. (Freeman, G. J. et al. , J. Immunol L. 143.2714-2722.1989).

22! standing Therefore, it blocks antigen-presenting cells, thereby inhibiting specific T-cell activation, i.e. That is, to provide a molecule that inhibits the interaction between biomolecule B7 and CD28. is the object of the present invention.

The 87 molecule is sometimes referred to in the art as the BB-1 molecule. Applicant: B-7 It has been discovered that there are soluble compounds that bind to T-cells and inhibit T-cell activation. good Preferred compounds include soluble fragments of CD2g or soluble analogs or derivatives thereof. and the most preferred compound is a soluble fragment of CD2B or its It is a dimer of soluble analogs or derivatives of

Another object of Applicant's invention is to ensure a high yield of soluble CD28 dimers. This is a method for producing soluble CD2g in order to

As used herein, the term "fragment," "derivative," or "analog" refers to a compound This refers to the modification of the sequence of an amino acid, without essentially impairing the properties of the original sequence. Residues may be deleted, inserted, substituted, and such properties may affect solubility and B-7 It encompasses the ability to combine. As used herein, the term "tissue" refers to whole organs. include. Soluble fragments of CD28 that bind to B-7 molecules are sometimes referred to herein as ``Soluble CD28J.'' Furthermore, the term ``animal'' in its broadest sense used and includes mammals, including humans.

1 plate of Ranga Yu! five dishes The figures illustrated and described herein further explain the invention and which Nothing in the law is intended to limit the invention.

['', COMB cDNA sequence.

This cDNA sequence was derived from Aruffo, A., and 5eed, B., Proc. , Natl.

Acad, Set, USA, 84.8573-8577, 1987 Come on. Soleha, for the CD28 gene, i.e. nucleotide number 100~ It includes the entire protein coding region for 762 proteins. ATG (number 100-10 2) and TGA (numbers 750-762>) The lines above indicate that they are associated with the CD2B gene. act as start and stop codons, respectively, for the translation of shows. The two underlined clusters of sequences are marked A and B and are MIT and CIT of the two 3° end primers cited in the Examples section, respectively. We will respond accordingly.

ILj = DNA sequences of T7, MIT and CIT primers (sequence numbers, respectively) No. = 7 and 8).

The T7 primer is 20 nucleotides long and its sequence is Is it a PC3N vector purchased from GA, Madison, WI, USA? It originates from The T7 primer is a truncated primer that directs the production of soluble CD2g protein. In the PCB reaction to obtain the morphomorphic CD28 gene, any 3' end primer can be used. – used as a 5° end primer, e.g. to pair with MIT and CIT be done. MIT and CIT are both 21 nucleotides long; From Figure 1 marked as clusters A and B, respectively.

MIT ranges from nucleotide number 556 to 570 and is the nucleotide of clT. Ocide ranges from numbers 538 to 552. 5° end of MIT and CIT There are six additional nucleotides added to the end, ATCATC, which act as a termination signal for translation. Play the role of Don.

Plate 3. Construction of CD28 expression vector.

A, pG3N-CD28 vector and Xbal-Pstl CD28 cDNA Fragment (Aruffo, A, and 5eed, B, Proc, Natl, Aead, Sci, USA, 84.8573-8577, 1987) POEM-3fz plasmid (herein sometimes % pG3N; Pro mega, Madison, 11) at the Xbal-Pst1 site. Constructed by combining. Arrows indicate genes for T7, T3 and CD28. Indicates the direction of transfer. The shaded area indicates the transmembrane content of CD28 protein after expression. display the blank area.

B, pG3N-CD2awo, (-re('shiMITOyohiCIT)DNAlFr Pc using a pair of primers T7-MIT and T7-CIT for single generation It was used as DNA #ffi in the R reaction. Next, both CD2B genes The fragment was transferred to the p9 vector (long, G, G, et al., 5science 228. 810-815°1985) into the EcoRIN position. obtained expression vector, p9-MIT, p9-CIT, soluble cD28 protein To transfect CO5-7 cells and GHQ for quality production. used.

Otori” Yu C02g protein sequence 0 CD2a cDNA encodes a protein that is 220 amino acids (aa) long (Sequence number: Engineering). It contains a signal peptide of amino acid numbers 1-18 and is cleaved during maturation of the CD2g protein. of mature CD2g protein The N-terminus is ASN of amino acid number 19 (ASN of nucleotide number 153-155). (marked with a thick line above AC). Amino acid numbers 153-179 The underlined sequence is the transmembrane region of the CD2B protein. Soluble C Production of soluble CD2g protein from D28 protein, e.g. p9-CIT Because of this, the transmembrane region was removed. The dashed line above the DNA sequence is A and B, indicating the positions of the MIT and CIT primers, respectively. vinegar. Therefore, as discussed herein in the examples provided, P 9-MIT-derived CD28 protein is not soluble CD28, but LN139 has the length of amino acids (amino acid numbers 19-157) (SEQ ID NO: 9), and The CD2B protein of p9-CIT contains 133 amino acids, which is soluble CD28. amino acid (amino acid number 19-151) (SEQ ID NO: 2).

K” Yu Constructs of the entire CD2g gene and its truncated forms C3T, C2T and CIT, and whether the truncated form of CD28 is soluble, dimer or not. Figure 2 shows a chart identifying forces that induce T-cell anergy.

Figure-”- A, DNA sequences of primers Pl, Pl, P3 and P4 used in PCR. , and the phrasing of each of them.

B, DNA sequence for the ligation between CIT and PAP.

K12 Construction of CLPAP expression vector is shown.

The CD211 gene segment CIT uses PL and Pl as 1. Nuru PCH4ko thus generated and then gene segments CI Tit, Xhol and Xho 1 and then subcloned into the expression vector pBJ-neo.

The resulting vector CIP contains a gene segment at the 3' end of the CIT gene segment. The resulting vector (LPAP expression vector Constructed of.

Otori” CIT and Il used independently in PCH [can start with PAP Production of soluble dimeric CD2g and pBJ-ne to form CIPAP o Their ligation into expression vectors is shown. CIPAP is then applied to C 0 cells. transfected, amplified, subcloned, cell sorted, and It is then scraped off from the cell membrane, yielding a soluble dimer of CD28.

& bean yu 1 dish craft and clothes Applicants have discovered that during antigen presentation by antigen-presenting cells to T-cells, CD28 and B-7 found that blocking the interaction between (See Examples cited from pages 6 to 19 herein). against antigen Such inactivation of T-cells may have certain clinical uses in treating diseases. It's a benefit. Furthermore, Applicants have determined that the soluble compound that binds to B-7 is We have discovered that it is possible to generate sex.

Although the scope of the invention is not intended to be limited in any way by theoretical evidence, There are two theories that may explain the effect of soluble CD28 on cell activation.

③ Soluble CD28 or its derivatives or analogs thereof bind to B-7 cells. , thereby transmitting the initiation signal from B-7 cells to the CD28 protein receptor. permanently inactivates the ability of T-cells to recognize foreign antigens. It is possible. Thereby, soluble CD28 or derivatives or analogs thereof are T-cell responses can be permanently inhibited.

Il, in the case of autoimmune diseases in which T-cells recognize foreign antigens, T-cell activity It is believed that it is possible to attenuate T-cell responses to prevent higher levels of is being given. This blocks B-7 cells from CD28 protein signaling. Soluble CD28 was used to maintain T-cell attenuation by This includes chronic treatment.

Therefore, the soluble peptides or proteins of the invention may be useful for specific clinical responses. may be useful for Applicants hereby disclose possible clinical findings for their invention. Provide examples of practical use. However, their use is indicated for illustrative purposes and is of the invention. It is not intended to limit applications. For example, treatment of the following diseases: autoimmune diseases disease, immunosuppressive disease, T-cell immunosuppression, rejection of transplanted cells and tissues , and specific prevention or mitigation of graft-versus-host disease in bone marrow transplants.

According to one aspect of the invention, soluble compounds capable of binding to B-7 are provided.

In a preferred embodiment of the invention, soluble CD2g or derivatives or analogs thereof are provided. Such soluble CD28 or To obtain a truncated CD28 gene that directs the production of derivatives or analogs thereof. The polymerase chain reaction (P CR) utilizes the 3' end primer.

Therefore, the 3° end primer in the range of nucleotide numbers 538-552 is Sequences for preferred embodiments of soluble CD28 are derived. Its soluble CD28 is an amino acid of human CD2g protein or soluble derivative or its analogue. It has an amino acid sequence of acid residue numbers 19-151 (CIT) (SEQ ID NO: 2).

The above sequence CIT is the human CD28 molecule, and the transmembrane region (amino Acid residue numbers 153-179) were deleted from the full-length CD28 molecule. obtained The obtained peptide or protein contains amino acid residue numbers 19 to 151, and It is a soluble CD28 molecule. The origin of this sequence can be found in the examples provided herein. Described in detail.

In yet another embodiment of the invention, in the range of nucleotide numbers 409-425 The 3′ end primer directs the sequence for soluble CD28, which sequence Amino acid residues of CD28 protein or soluble derivative or analog thereof It has an amino acid sequence number 19-108 (C2T) (SEQ ID NO: 4).

In yet another embodiment of the invention, in the range of nucleotide numbers 502-529 The 3′ end primer directs the sequence for soluble CD28, which sequence Amino acid residues of CD28 protein or soluble derivative or analog thereof It has an amino acid sequence number 19-140 (C3T) (SEQ ID NO: 3).

According to yet another aspect of the invention, encoding a soluble compound capable of binding to B-7. DNA is provided. Preferably, the soluble compound is as described herein above: human CD28 protein or a soluble derivative thereof or any sequence thereof; It is an analog and has an amino acid sequence of amino acid residue numbers 19-151. soluble The DNA encoding sexual CD28 herein is a fragment of the CD28 amino acid sequence described above. Deletions, insertions or deletions of nucleotides to encode fragments, derivatives or analogs These fragments, derivatives or analogs may be modified by It has the same properties as the CD28 amino acid sequence, which has the same properties as the CD28 amino acid sequence, and this property has the ability to bind B-7. It should be understood that it encompasses power.

The DNA encoding the soluble compound described herein above can be obtained by techniques known to those skilled in the art. Such techniques include recombinant DNA techniques or can be chemically synthesized. ClTm products produced by these methods consist of monomers and Both soluble and dimerized CD28 can occur naturally. It is a T-cell apoptotic It is a dimer of soluble CD28 that is responsible for inducing energy (90% mono See Example 3 which yields 10% dimer body. ).

According to yet another aspect of the invention, D encoding a soluble compound as herein above described. Expression vectors containing NA are provided.

In one embodiment, the expression vector can be a eukaryotic expression vector. .

The following are examples of expression vectors that can be expressed as eukaryotic expression vectors: pMSG is the promoter from murine breast cancer virus long terminal repeat (MMTV) use. A suitable host for pMSG is Chinese hamster ovary cells. , He1a cells and mouse Lkt-negative cells (Lee, F., et al., Na ture 294.228-232.1981).

psvt, uses the SV40 late promoter and its suitable host cells have high levels of CO3 for the transient expression of BEL (Sprague, J9 et al., J. Virol, 45.773-781.1983; Gempleton, D , and Eckhart, W., Mo1. Ce1l Biol, 4.8 17-821.1984).

pRsV uses the Rous sarcoma virus promoter and its suitable host is the mouse. fibroblasts, lymphoblastocytes and CO3 cells (Gorman, C. , Padmanabhan, R., and Hovard, B.H.

, 5science 221.551-553.19H).

pBPV is a DNA viral vector derived from bovine papillomavirus. , it is suitable for stable expression in mouse breast cancer cells, C127 (Zin, , , DiMaio, D., and Maniatis, T. , Ce1134, 8 65-879. 1983; 5arver, N. et al., Mo1. Ce1l Btol, 1゜486-496.1981: 5arver, N,, Byr ne, J.C., and Howley, P.

M, , Proc, Natl, Acad, Set, ll5A, 79 ．． 7147-7151, 1982; Lam, M.F., Byrne, J. , C., and Howley, P.M., Mo1. Ce1l Bi.

1, 3.2110-2115.1983).

Baquinillovirus expression vectors are suitable for stable expression in inserted cells, sf9. (Luckow, ”/, A, and Summers, M, D,.

Bio, Technolo 6.47-55.19H; Miller, L , , , Ann Rev ■ Anal Erosion 42.177-199.1988)

In yet another embodiment, the expression vector can be a prokaryotic vector.

The following expression vectors can be expressed as prokaryotic expression vectors: The pOX expression system is E, Co11 system. a) using the oxygen-dependent promoter of the hemoglobin gene (Khosla , G, et al., Bio, Technolo 8. 554-558. 1990 ).

The pPL expression system uses the strong PL promoter of the λ phage (Reed, R,R,, Ce1l 25.713-719.1981; Simatake , H., Z., and Rosenberg, M., Nature 292.　 128-132. 1981; Mott, J.

D, et al., Proc, Natl, Acad, Sci, USA, 82. 88-9 2. 1985).

pKK223-3 contains the tryptophan operon and lactophane operon of E, Ca1f origin. Hybrid promoter derived from fusion between promoters of the tos operon (Brosius, J., and H.

ty, A, , Proc, Natl, Acad, Set, , USA, 81.6929-6933.1984).

Expression vectors can be used to transform cells and produce soluble compounds. obtain. An example of a cell that can be transformed with an expression vector is a Chinese hamster egg. nest cells, lymphoblastoid cells, (M, Okamoto et al., Bio, Techn. ol, 8.550-553.1990) and C127 mouse breast cancer cells (J , Virol, 26.291-298.1978; -375.1980>. Transformed cells expressing soluble compounds are It can be used in an in vitro expression system to produce soluble CD28 molecules.

As mentioned above, it is possible that the dimerization of soluble CD211 is responsible for T-cell inactivation. Since it is integral, it is therefore inherently difficult to prepare CIT by known techniques. This is advantageous because it produces a larger amount of dimerite than the dimerite produced in .

Therefore, another embodiment of the present invention provides for more efficient dimerization of soluble CD28. A method for efficiently producing and isolating the present invention is provided. The method is (i) CIT ( PCR and primers P1 and P2 to generate soluble CD28 truncated form) CCD28-1 used) 3N vector treatment, and (ii) PAP (49 using PCR and primers P3 and P4 to generate Both independent treatments of human placental alkaline phosphatase (PAP) treatment includes. CIT and PAP are then combined into an expression vector and CIPA To form P, the CIT sequence (with only 18 amino acids left) Connected to the P array. The last of the 18 amino acids in the PAP portion of CIPAP is luginine, and arginine is a phosphatidylinositol glycan ( pl-G). pr-a retains CIPAP in the cell membrane. It acts as an anchor for holding. Once on the cell membrane, the membrane As a protein, CIT is expressed as dimeric recombinant soluble CD28.

Dimeric proteins are then recovered from the membrane after treatment with phospholipase C. can be done. After removal from the cell membrane, soluble CD28 remains in its dimer form.

The ability of soluble CD28 to form dimers is thought to occur via Cys1. and cysteine residues (Figure 5) form dimeric CD2a via the formation of Cyst. including crosslinking, such as the formation of disulfide bonds within. Soluble CD28 dimer The production of CD2g is increased by fixation of CIT (soluble CD2g) to the cell membrane. It is also conceivable that they will be rejected.

The presence of Cys2 and Cys3, i.e., cysteine residues (Figure 5), - believed to be responsible for the biological activity of soluble CD211 compounds. However, Other amino acids can be added to Cys2 and/or C while maintaining bioreactivity of the compound. What can be replaced with ys3 is:

Claims (21)

[Claims] 1. A soluble compound that binds to B-7 protein, the compound binding to soluble CD 28, and soluble derivatives and analogs thereof. Compound. 2. The soluble CD28 or its soluble derivatives and analogs thereof are dimers. 2. The compound according to claim 1, which is a compound of claim 1. 3. The compound may contain the CD28 protein (SEQ ID NO: 2) or the protein The amino acid sequence from amino acid residue position 19 to amino acid residue position 151 of a derivative or analogue of 3. A compound according to claim 2, having a column. 4. 3. A method of treating T-cell immunosuppression in an animal, comprising: A method comprising administering to an animal an effective amount of a compound. 5. The effective amount further comprises about 2 μg to about 2 mg per kg of body weight of the animal. 4. The method of claim 3, comprising intravenous injection of the compound. 6. A method for preventing rejection of transplanted cells or tissues within a host, the method comprising: A method comprising administering to a host an effective amount of a compound according to claim 2. 7. The effective amount further comprises about 2 μg to about 2 mg of the above compound per kg body weight of the host. 7. The method of claim 6, comprising intravenous injection of the compound. 8. A method of treating autoimmune diseases in an animal, comprising a compound according to claim 2. A method comprising administering to an animal an effective amount of. 9. The effective amount further comprises about 2 μg to about 2 mg per kg of body weight of the animal. 9. The method of claim 8, comprising chronic intravenous injection of the compound. 10. A method of preventing graft-versus-host disease in a bone marrow transplant, comprising: About 100 cc to about 1000 cc of bone marrow cells from a bone marrow graft donor are treated with appropriate pharmaceutical agents. at least 1 cc of the compound according to claim 2 in the medium for no more than 24 hours. perfusing, mixing, or treating ex vivo during combining donor bone marrow cells and recipient bone marrow cells, and A method comprising the step of reintroducing said combination to a recipient. 11. Bone marrow cells from said donor are treated with about 1 cc to about 50 cc of said compound. , perfused, mixed, or treated for a period of about 10 minutes to about 4 hours. 10. 12. T-cells containing the CD28 receptor of cells containing the B7 protein 3. A method of preventing binding to: a cell containing said B7 protein; A method comprising the step of contacting an effective amount of a compound as described. 13. T-cells containing CDZ8 receptors containing B-7 protein A composition that prevents binding to: B-7 protein holes and an acceptable pharmaceutical agent. comprising soluble CD28 or a soluble derivative or analog thereof bound to a carrier; A composition with. 14. The above-mentioned soluble CD28 or its soluble derivative or analogue is dimeric. 14. The composition according to claim 13, which is a 15. Soluble CD28 or its soluble derivatives capable of binding to B7 protein DNA encoding a compound selected from the group consisting of 16. The above-mentioned soluble CD28 or its soluble derivatives and analogs are dimers. 16. A DMA encoding a compound according to claim 15, which is a compound according to claim 15. 17. The encoded compound may be the CD28 protein (SEQ ID NO: 2) or is the amino acid residue number 19 to 151 of its soluble derivative or analogue 17. The DNA according to claim 16, having the amino acid sequence. 18. An expression vector containing the DNA according to claim 16. 19. A cell transformed with the expression vector according to claim 20. 20. The expression vector is expressed as a eukaryotic or prokaryotic vector. The expression vector according to claim 18. 21. 20. The cell according to claim 19, wherein the cell is a mammalian or bacterial cell. .