WO2006066463A1

WO2006066463A1 - Receptor-selective lymphotoxin deviants

Info

Publication number: WO2006066463A1
Application number: PCT/CN2004/001522
Authority: WO
Inventors: Yanjun Liu; Tong Yang; Yijun Shen; Jingsong Wu; Feng Cao; Zheng Wang; Jingwei Tan; Fang Wu; Yan Xu; Haibo Wang
Original assignee: Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd.
Priority date: 2004-12-24
Filing date: 2004-12-24
Publication date: 2006-06-29
Also published as: CN101084238A; CN101084238B

Abstract

This invention discloses some receptor-selective lymphotoxin (LT) mutants and the ratio, the ability of these lymphotoxin mutants combining with the human TNFRI to that with the human TNFRII, is more than 10. The preparation method and the use of the above said LT mutants are also disclosed. In this invention, the LT mutants bind to the TNFRI selectively and that decreases the binding to the TNFRII in a large range, thereby the toxic effect which may be caused by the TNFRII will be reduced.

Description

Bulk selective lymphotoxin derivative

Technical field

The present invention belongs to the field of protein engineering and pharmaceuticals, and in particular to a receptor-selective lymphotoxin derivative, a preparation method and use thereof. Background technique

Lymphotoxin a (LT a ), also known as TNF P, is a member of the TNF superfamily and is an important class of cytokines. Compared with TNF, LT a has similar structure and function to TNF, and has the same receptor TNFRI, TNFRI I, L'l' o inhibits tumor cell type slightly less than TNF, and kills certain tumor cells. not exactly.

LT a is produced by active lymphocytes and is a 25KD secreted glycoprotein. The LT precursor contains 205 amino acid residues, of which 34 amino acid residues encode signal peptides, and mature LT has 171 amino acid residues (i8kD). (SEQ ID NO: 3), no disulfide bond, position 62 is an N-linked glycosylation site (glycosylation is not necessary for its cytotoxic activity), LT a interacts with TNFRI, TNFRI I The points are concentrated in the two lasso areas at the bottom of the trimer. LT a homotrimer interacts with TNFRI and TNFRII to initiate signaling.

TNFRI and TNFRII differ in terms of cell distribution, molecular weight, affinity of binding ligands, and mediated biological activity. TNFRI and T 'FRII are present in all types of cells except red blood cells, and the presence of TNFRI is more prevalent. The TNFRI receptor is mainly expressed in epithelial cells. The TNFRII receptor is mainly expressed in bone marrow cells, and most of the cells express these two receptors, but the ratio is different. T! FRI consists of 455 amino acids, TNFRII 461 461 amino acids, all of which are type I transmembrane glycoproteins, which are composed of signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain. TNFRI cytoplasmic zone 326-413 is the death domain (DD). TNFRI binds to TNFR-associated proteins via DD and conducts cytotoxic signals, triggering apoptosis. There is no DD in the cytoplasmic domain of TN'FRII, and the signal transmitted by TNFRII is mainly restricted to cells in the immune system and is highly correlated with the immune response of viral infection. The intracellular portions of TNFRI and TN'FRII have very low homology, indicating that they have different signal transduction pathways and different biological functions.

The biological activities of the two receptor-mediated TNFs vary widely. Mouse TNFa binds similarly to murine TNFRI and TNFRII, whereas human TMFa binds only to murine TNFRI and does not bind murine TNFRII. In the lethal experiment, human TNFa showed a lower mortality rate than murine TNFa, suggesting that TNFRII has a large effect on systemic toxicity.

Studies have shown that TNFRII signaling has an inhibitory effect on insulin signaling, and blocking TNFRII signaling with an anti-TNFRII antibody attenuates the inhibitory effect of this signal on insulin signaling.

TNFRII signaling also plays an important role in regulating vascular permeability. The anti-TNFRII antibody inhibits the proliferation of neuroblastoma cells and has no effect on the cell killing activity of TNF. In a mouse experiment, TNFRII was found to be involved in cisplatin-mediated renal injury.

By modifying the binding region of TNF to the receptor, a TNF mutant selective for TNFRI is obtained, thereby maintaining its cell killing activity against tumor cells, and at the same time reducing the side effects caused by TNFRI I, and obtaining good results. LT and TNF have comparable anti-tumor activity in vivo and in vitro, and have less toxicity and longer half-life. In addition, LT also has obvious chemosensitization and radiosensitization. Therefore, the clinical application prospects of LT in the treatment of tumors may be better than TNF. The lymphotoxin derivative LT _a28 _ _m lacking the N-terminal sequence of LTA, 1-27, is currently in the phase II clinical phase and shows a clear anti-tumor effect. However, LT still has certain toxic side effects, and the therapeutic index is not

- 1 - Confirmation High, and the inactivation of certain tumor cells is not high, which limits the application of lymphotoxin in the field of medicine. Studies have shown that the toxic side effects of LT are related to TNFRII signaling. TNFRII signaling can activate NFKB, and NFKB is an important inflammatory factor that directly causes inflammatory reactions. It also activates multi-drug resistance genes and affects the efficacy of drugs on tumor cells. TNFRII is also able to compete with TNFRI for ligands, resulting in LT being insensitive to tumor cells with high expression of TNFRII, inhibiting the cytotoxic activity of LTa.

At present, there is an urgent need for lymphotoxin with TNFRI selectivity, which maintains the tumor cell killing activity of lymphotoxin and reduces the toxic side effects of LT. The sensitivity of LT to tumor cells with high expression of TNFRII is improved, and can be applied to more. A variety of tumor treatments: In the combination of LT and chemotherapy drugs, there will be better therapeutic effects, lower toxic side effects.

However, there is currently no satisfactory lymphotoxin with high selectivity for TNFRI. Therefore, there is an urgent need in the art to develop new lymphotoxin mutants that are highly selective for TNFRI and have very low selectivity for TNFRII. Summary of the invention

It is an object of the present invention to provide a lymphotoxin mutant having high selectivity for TNFRI and extremely low selectivity for TNFRII.

In a first aspect of the invention, there is provided a receptor-selective lymphotoxin mutant, wherein the mutant is in a 106-113 lasso structure corresponding to positions 106-113 of the natural sequence of lymphotoxin, 106, 107 , 109, 110, ΠΚ 112, or at least one amino acid at position 13 is replaced, and/or at least one amino acid is inserted in the 106-113 lasso structure,

The additional condition is that the 108-bit Y residue is unchanged,

And the ratio of the binding ability of the lymphotoxin mutant to human TNFRI to the binding ability of the lymphotoxin mutant to human TNFRII is greater than 10.

In another preferred embodiment, the ratio of the binding ability of the lymphotoxin mutant to TNFRI to the binding ability of wild-type LT to human TNFRI is greater than 0.1.

In another preferred embodiment, the lymphotoxin mutant binds to human TNFRII with wild type LT and human

The ratio of binding ability of TNFRII is less than 0.01.

In another preferred embodiment, the amino acid substitution is 106, 107, 109, 110, 111, 112 or 113 amino acids replaced by other amino acids. In another preferred embodiment, 1-3 amino acids (preferably acidic or basic amino acids) are inserted at positions 106-113.

In another preferred embodiment, the mutant has a mutation selected from the group consisting of:

Q107E; Q107D; S106E; S106D: Q107R; Q107N: Q107E/S106E; Q107E/S106D; Q107D/S106E; or Q107D/S106D.

In another preferred embodiment, the ratio of the binding ability of the lymphotoxin mutant to human TNFRI to the binding ability of the lymphotoxin mutant to human TNFRII is greater than 100, more preferably greater than 200.

In a second aspect of the invention, there is provided a pharmaceutical composition comprising a safe and effective amount of a human lymphotoxin morphamate and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises a chemotherapeutic agent selected from the group consisting of CDDP, 5-FU, ADM, VCR, or a combination thereof. In a third aspect of the invention, there is provided the use of a human lymphotoxin mutant of the invention for the preparation of a medicament for the treatment of: (a) a tumor: (b) an internal parasitic disease.

In a fourth aspect of the invention, a method for preparing a TNFRI receptor-selective human lymphotoxin mutant, wherein the residue at position 108 of the native lymphotoxin sequence is unchanged, replacing at least one amino acid at positions 106-113; Inserts 1-3 amino acids at positions 106-113.

In a fifth aspect of the invention, there is provided an isolated DNA sequence encoding a lymphotoxin mutant of the invention. Also provided is an expression vector comprising the DNA sequence. Also provided are host cells transformed with the expression vector or the -DNA sequence.

In a sixth aspect of the invention, there is also provided a method of producing a lymphotoxin mutant of the invention, the method comprising the steps of:

(a) cultivating said host cell under conditions suitable for expression to express said lymphotoxin mutant;

(b) isolating and purifying the lymphotoxin mutant.

In a seventh aspect of the invention, there is provided a method of treating a tumor or an internal parasitic disease comprising the steps of: administering to a subject in need of treatment a safe and effective amount of a lymphotoxin mutant of the invention. DRAWINGS

Figure 1 shows the in vitro killing of Jurkat (leukemia) cells by the rhLT mutant LT008.

Figure 2 shows that the rhLT mutant LT008 kills Lovo (colon cancer) cells in vitro.

Figure 3 shows that the rhLT mutant LT008 kills MCF-7 (breast cancer) cells in vitro.

Figure 4 shows that the rhLT mutant LT008 kills A549 (lung cancer) cells in vitro.

Figure 5 shows that the rhLT mutant LT008 kills A375 (melanoma) cells in vitro.

Figure 6 shows that the rhLT mutant LT008 kills Hela (cervical cancer) cells in vitro.

Figure 7 shows that the rhLT mutant LT008 kills U937 (tissue cell lymphoma) cells in vitro.

Figure 8 shows that the rhLT mutant LT008 kills HL-60 (leukemia) cells in vitro.

Figure 9 shows the sensitization of the rhLT mutant to the chemotherapeutic drug CDDP.

Figure 10 shows the sensitizing effect of the rhLT mutant on the 5-pulmonary chemotherapeutic drug.

Figure 11 shows the sensitization of the rhLT mutant to the chemotherapeutic drug ADM.

Figure 12 shows the sensitization of the rhLT mutant to the chemotherapeutic drug VCR. detailed description

The present inventors studied the effects of LT on TNFRI and TNFRII by means of molecular simulation techniques. After extensive and in-depth research, the LT sequence 106-113 lasso structure is a structural region in which LT interacts closely with its receptors TNFRI and TNFRII. By introducing mutations in the lasso structure, TNFRI selectivity can be obtained. LT mutants, receptor blocking experiments confirm that these LT mutants retain their ability to bind to TNFRI, while the ability to bind TNFRII is reduced by at least 10-fold, usually at least 20-fold, preferably at least 100, more preferably at least 1000-fold. More preferably at least 10,000 times (eg 40,000 times). Cytological experiments confirmed that this TNFRI-selective LT is superior to wild-type rhLT in killing activity of various tumor cells, and has enhanced sensitivity to tumor cells with high expression of TNFRII, and can also increase the sensitivity of tumor cells to chemotherapeutic drugs such as CDDP. As used herein, the terms "lymphoxine", "LT.", "LTc", "TNFp" are used interchangeably to mean a lymphotoxin LToc, including lymphotoxin derived from human, mouse, pig, horse or cow. Preferably, it is a human lymphotoxin. Furthermore, the lymphotoxin may be LT having a native wild-type sequence, or may be a derivative or recombinant LT having a mutated sequence (relative to the wild-type sequence). The amino acid sequence of the native wild-type human lymphotoxin TNFoc is shown in SEQ ID NO: 3.

As used herein, the amino acid numbering of lymphotoxin is based on wild-type human lymphotoxin (SEQ ID Ν 0·· 3). For example, the lasso structure at position 106-L 13 is the natural sequence of lymphotoxin, SQYPFHVP at positions 106-113 of SEQ ID NO: 3, or the region corresponding to the amino acid of native sequence 106-113.

As used herein, the term "TNFRI-selective LT" or "LT mutant of the invention" refers to an LT mutant that retains its ability to bind to TNFRI, while the ability to bind TNFRII is at least 10-fold, usually at least 20-fold. Preferably it is at least 100 times, more preferably at least 1000 times, more preferably at least 10,000 times (e.g., 40,000 times). The LT mutants of the invention may or may not contain an initial methionine.

As used herein, 'Q107E denotes 107 Gln-Glu mutations: Q107D represents 107 Gin-Asp; S106E represents 106 Ser-Glu; S106D represents 106 Ser-Asp, and so on. Q107E/S106E represents 107-bit Gin-Glu mutation and 106-position Ser→Glu, and so on.

The human lymphotoxin mutant of the present invention includes DNA, RNA or human lymphotoxin mutant protein encoding a human lymphotoxin mutant.

In a preferred embodiment, the 106-113 region structure is fine tuned by amino acid substitution. For example, the original amino acid is replaced with acidic ammonia glutamic acid or aspartic acid.

In another preferred embodiment, 1-3 amino acids are inserted by keeping the residue at position 108 unchanged in the 106-113 region. For example, an acidic or basic amino acid, particularly one of glutamic acid, aspartic acid, arginine, and asparagine, is inserted in the 106-113 region.

In another preferred embodiment, a negatively charged group can be introduced in the 106-113 region by chemical modification. The LT mutein of the present invention can be produced by synthesizing a primer according to the sequence of the disclosed human lymphotoxin, and amplifying the coding sequence of human lymphotoxin by PCR. The coding sequence of human TNFo can also be artificially synthesized. In addition, base substitutions can be made to the coding sequence to facilitate high expression (e.g., for expression in E. coli, the natural codon can be replaced with an E. coli preferred codon encoding the same amino acid). Then, genetic modification of the DNA sequence of human lymphotoxin, according to the mutant form indicated in the present invention, and genetic modification are known in the art, for example, see "Mutagenesis: a Practical Approach", MJ McPherson, Ed (IRL Press, Oxford, UK. (1991), which includes, for example, site-directed mutagenesis, cassette mutagenesis, and polymerase chain reaction (PCR) mutagenesis.

After obtaining the DNA sequence encoding the novel mutant protein of the present invention after site-directed mutagenesis, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the novel mutant protein of the present invention is obtained by separation and purification.

In the present invention, various carriers known in the art such as commercially available carriers can be used. For example, a commercially available vector is selected, and then a nucleotide sequence encoding a novel mutein of the present invention is operably linked to an expression control sequence to form a protein expression vector. As used herein, "operably linked" refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to Coding sequence: If the ribosome binding site is placed at a position where it can be translated, then it is operably linked to the coding sequence. In general, "operably linked to" means adjacent, and for secretory leader sequences means adjacent in the reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a prokaryotic cell, more preferably E. coli.

In one embodiment of the invention, a method of making an LT mutant of the invention comprises the steps of:

i. Modification of the coding sequence of LT to replace at least one of amino acids 106-113 with other amino acids while maintaining the 108 residues of the native lymphotoxin sequence; or insert 1-3 at positions 106-113 An amino acid (such as an acidic or basic amino acid, preferably glutamic acid, aspartic acid, arginine, or asparagine);

i i. cloning the above modified lymphotoxin gene into an expression plasmid;

i i i. transforming a host cell with the above expression plasmid carrying a lymphotoxin gene,

i v. Culturing transformed host cells:

v. Collect host cells and culture fluids, and isolate and purify lymphotoxin mutants.

The LT mutants of the invention are useful for treating tumors and endoparasites. Representative tumors include, but are not limited to, leukemia, histiocytic lymphoma, gastric cancer, breast cancer, lung cancer, colon cancer, cervical cancer, melanoma, bladder cancer. Representative endoparasite diseases include, but are not limited to, toxoplasmosis.

The lymphotoxin mutant of the present invention can be used alone or in combination with other drugs such as chemotherapeutic drugs to increase the sensitivity of tumor cells to chemotherapeutic drugs.

The invention also provides a pharmaceutical composition comprising an effective amount of one or more of the LT muteins of the invention, and at least one pharmaceutically acceptable carrier, diluent or excipient. In the preparation of these compositions, the active ingredient is usually mixed with excipients, or diluted with excipients, or enclosed in a vehicle in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid or liquid material as an excipient, carrier or medium for the active ingredient. Thus, the composition may be in the form of a tablet, a pill, a powder, a solution, a syrup, a sterile injectable solution or the like. Examples of suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The preparation may also include: a wetting agent, an emulsifier, a preservative (e.g., methyl and propyl hydroxybenzoate), a sweetener, and the like.

In another preferred embodiment, the pharmaceutical composition of the invention further comprises an additional chemotherapeutic agent, such as CDDP, 5-FU, ADM, VCR or a combination thereof.

The composition can be formulated in unit or multi-dose form. Each dosage form contains a predetermined amount of active material calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.

The administration mode of the LT mutant and the pharmaceutical composition of the present invention is not particularly limited and can be administered orally, topically, parenterally, for example, by muscle, intravenous, or subcutaneous injection, or by inhalation spray. A preferred mode is intravenous injection.

When the LT mutant of the present invention is orally administered in the form of a tablet or a capsule, the dose is for an average body weight of 60-70 kg. Adults can be administered parenterally in the range of about lug to lOOOO, or in the form of an injection, at a dose of about lug to 500 ug, which can be administered once or several times a day. The unit dose of the pharmaceutical composition typically comprises the active ingredient in the range of from lug to 500 ug, typically lug, 5 ug, 10 ug, 25 ug, 50 ug, 100 ug, 200 ug, 300 ug, 400 ug, 500 ug.

The amount and dosage regimen of the therapeutically active ingredients employed in treating a particular condition with a composition of the invention will depend on a variety of factors including weight, age, sex, inevitable medical condition, severity of the disease, route of administration and frequency. This can be determined by the medical staff. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually produced according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer.

General method:

(a) Evaluation of mutants

1. Enzyme-linked immunosorbent assay

TNFRI -. Fc/TNFRII: Fc was coated on the ELISA plate, and the LT mutant to be tested was diluted to the same concentration and bound to it, and then rabbit anti-LT enzyme-linked antibody was added to develop a color reading.

Comparative calculations were compared with wild-type LT to determine the percentage change, and the binding ability of the LT mutant to the receptor was predicted. The mutants with strong FcRI binding ability were further screened for further evaluation.

2. Determination of cytotoxic activity of rhLT mutant in vitro against L929 cell line

L929 cells were used as target cells, and wild type LT was used as a control to determine the in vitro killing of tumor cells by rhLT mutants. Each unit of viability refers to the amount of lymphotoxin or lymphotoxin mutant used by lymphotoxin to induce 50% of the cells inoculated with apoptosis.

3. Determination of receptor binding capacity

L929 cells were used as target cells, and lymphotoxin or lymphotoxin derivatives were added for killing. The killing effect of lymphotoxin was neutralized by TNFRI and TNFRII, respectively, and TI\'FRI: Fc blocked the killing of L929 cells by the lymphotoxin mutant to be tested. The ratio of IC50 to IC50 for blocking the killing of L929 cells by wild-type lymphotoxin was defined as the ability of LT to bind to TNFRI receptors; TNFRI I : Fc blocked the IC50 of killing of L929 cells by mutant lymphotoxin mutants and blocking wild type The IC50 ratio of lymphotoxin to L929 cell killing is defined as the ability of LT to bind to the TNFRI I receptor.

(b) Tumor killing activity of lymphotoxin mutants

The cytotoxic activity of various lymphotoxin mutants on tumor cells was examined in vitro, and the tumor cell lines used included: Jurkat (leukemia), U937 (tissue cell lymphoma), MKN-45/MGC-863 (stomach cancer), MCF- 7 (breast cancer), A549 (lung cancer), A375 (melanoma), T-24 (bladder cancer) cells, etc. Example 1 Preparation of Wild Type LT, LT ₂₄ . ₁₇ LT ₂₈ _ _m

Expression vector construction:

According to the GENBANK Chinese LT sequence (BAA00064), Shanghai Shenggong Biotechnology Service Co., Ltd. was commissioned to synthesize wild-type LT encoding the LT mature protein from position 1 to position 171, position 24 to 171, and position 28 to 171, respectively. , LT ₂ , _-17 LT ₂₈ _ _m amino acid sequence, and cloned into pET32a (+) vector (purchased from Novagen) Λ3⁄43⁄4Ι At the ΗίηΛ II site, recombinant expression plasmids LT/pET32a (+), LT _3⁄4 ., ₇₁ /pET32a (+), LT ₂₈ - l _7l /pET32a(+) were obtained.

Expression in E. coli - Recombinant expression plasmids LT/pET32a(+), LT _24-171 /pET32a (+), LT _28-17l /pET32a(+) were transformed into E. coli BL21 (DE3), and the transformation solution was coated with LB. (containing ampicillin lOOng / μ Ι) plate. Monoclones were picked from the transformation plates and inoculated into ImL LB medium, and cultured at 37 ° C, 250 rpm for 3 h, IPTG was added to a final concentration of 0.5 m, and culture was continued for 3 h. After the lOOul expression, the bacterial cell pellet was collected by centrifugation, 50 ul of SDS sample preparation solution was added, and electrophoresis was carried out in a 12.5% PAGE gel.

"Protein purification - LT/pET32a(+)/BL21(DE3), LT ₂₄ . ₁₇₁ /pET32a (+) /BL21 (DE3), LT _28- /pET32a(+)/BL21 (DE3> recombinant engineering strain 1ml Inoculated with 100 ml of LB (containing ampicillin lOOng/μ1) medium, cultured at 37 ° C, 250 rpm for 5 h, IPTG was added to a final concentration of 0.5 mM, and culture was continued for 4 h. The bacterial cell pellet with a wet weight of 5 g was collected and washed with PBS. Ultrasonic disruption, centrifugation of inclusion bodies. Solubilization of inclusion bodies with urea, renaturation with Tris buffer. Succession with Q-Sepharose FF ion exchange column chromatography, Phenyl-Sepharose FF hydrophobic chromatography and CM-Sepharose FF ion exchange column Purification by dilution. The eluate was diluted to a protein concentration of 1 mg/ml.

Wild type LT, LT ₂ .,. _17r LT _{2 m} protein 1.0, 3.5, 4.2 mg were obtained, respectively. Example 2 Preparation of mutant rhLT008

Construction of the rhLT008 mutant expression vector:

(1) PCR amplification of mutant genes:

Using LT ₂ ., _ ₁₇₁ / pET32a (+) plasmid as template, LT-P32U and Q107E- R were used as a pair of primers to obtain LT008-AB fragment, and LT-P32D and Q]07E-F- The primers were amplified to obtain a LT008-CD fragment. Then, LT008-AB and LT008-CD were mixed and used as a template, and LT-P32U and LT-P32D were used as a pair of primers to obtain a LT008 fragment containing the Q107E mutation.

LT-P32U-. 5*ACACATATGATG CAC TCT ACC CTG AAA CCG 3, (SEQ ID NO: 4)

LT-P32D: 5'TGCAAGCTTCTA CAG AGC GAA GGC TCC AAA 3' (SEQ ID NO: 5)

Q107E-F: 5'CTCTTCTCCTCCGAATACCCCTTC 3' (SEQ ID NO: 6)

Q107E-R: 5 'GAAGGGGTATTCGGAGGAG AAGAG 3' (SEQ ID NO: 7)

The PCR product was purified and ligated with pMD-18T vector, transformed into E. coli DH5a, and the 008/pMD-18T plasmid was extracted, and sequenced in forward and reverse directions.

(2) Construction of prokaryotic expression vector of rhLT mutant:

The correct clone was sequenced, and the plasmid was digested with restriction enzyme and ffindUl, and subcloned into the same site of the pET32a(+) expression vector to obtain LT008/pET32a(+) recombinant expression plasmid.

Expression of rhLT mutant in E. coli:

The recombinant expression plasmid LT008/ _P ET3a(+) was transformed into E. coli BL21 (DE3), and the transformant was plated with LB (ampicillin-containing lOOng/μ1) plate. The monoclonal antibody was picked from the transformation plate and inoculated in 1 mL of LB medium, and cultured for 3 hours at 37 ° C, 250 rpm, IPTG was added to a final concentration of 0.1 mM, and culture was continued for 3 hours. Take the lOOul expression of the bacterial solution after centrifugation The cells were precipitated, and 50 μl of SDS sample preparation solution was added, and electrophoresis was carried out in 12.5% of PAGE gel.

LT008 was purified in the same manner as in Example 1 to obtain 3. 6 mg of L.T008 protein. Example 3 Preparation of LT006

Using LT^ _{I 71} / pET32a (+) plasmid as template and LT-P32U and S 106E- Q107E-R as a pair of primers to obtain LT006- AB fragment, and LT-P32D and S106E- Q107E-F - Amplification of the primer to obtain a LT006-CD fragment. Then, LT006-AB and LT006-CD were mixed and used as a template, and LT-P32U and LT-P32D were used as a pair of primers to obtain a full-length LT006 fragment containing the S106E-Q107E mutation.

The PC primer sequence is:

S 106E-Q107E-F: 5 'CTCTTCTCCGAAGAATACCCCTTC 3 ' (SEQ ID NO : 8)

S106E-Q107E-R: 5' GAAGGGGTATTCTTCGGAGAAGAG 3' (SEQ ID NO: 9)

LT006 was cloned, expressed and purified in the same manner as in Example 2 to obtain 3. 1 mg of LT006 protein. Example 4 Preparation of LT009

Using LT ₂ , , _ _m / pET32a (+) plasmid as template, LT-P32U and Q107D- R were used as a pair of primers to obtain LT009-AB fragment, and LT-P32D and Q107D-F-pair primers were obtained. Amplification was performed to obtain a LT009-CD fragment. Then, LT009-AB and LT009-CD were mixed and used as a template, and LT-P32U and LT-P32D were used as a pair of primers to obtain a full-length LT009 fragment containing Q107D mutation.

The PCR primer sequence is - Q107D-F.- 5' CTCTTCTCCTCCGACTACCCCTTC 3' (SEQ ID NO: 10)

Q107D-R : 5' CAAGGGGTAGTCGGAGGAGAAGAG 3' (SEQ ID NO : 1 1 )

LT009 was cloned, expressed and purified in the same manner as in Example 2 to obtain LT009 protein of 4. 2 mg. Example 5 Preparation of mutant rhLT057

Using LT ₂₈ _ _m / pET32a (+) plasmid as template, LT ₂₈ - P32U and Q107E- R were used as a pair of primers to obtain LT057-AB fragment, and a pair of primers of LT-P32D and Q107E-F were used. Amplification, LT057-CD fragment was obtained. Then, LT057-AB and LT057-CD were mixed and used as a template, and LT ₂₈ -P32U and LT-P32D were used as a pair of primers to obtain a LT057 fragment containing the Q107E mutation.

LT ₂₈ -P32U : 5'ACACATATG AAA CCG GCT GCT CAC 3' (SEQ ID NO : 12)

LT-P32D: 5'TGCAAGCTTCTA CAG AGC GAA GGC TCC AAA 3' (SEQ ID NO: 5)

Q107E-F: 5'CTCTTCTCCTCCGAATACCCCTTC 3' (SEQ ID NO: 6)

Q 107E-R : 5 'GAAGGGGTATTCGGAGGAGAAGAG 3' (SEQ ID NO : 7)

LT057 was cloned, expressed and purified in the same manner as in Example 2 to obtain 3.10 mg of LT057 protein. Example 6 Preparation of LT090

Using LT^ ₁₇₁ / pET32a (+) plasmid as template and LT-P32U and Q107R-R as a pair of primers to obtain LT090-AB fragment, and LT-P32D and Q107R-F-amplification , get the LT090-CD clip. Then LT090-AB and LT090-CD are mixed to form a template, and LT-P32U and LT-P32D are used as a pair of primers. Amplification was performed to obtain a full-length LT090 fragment containing the Q107R mutation.

The PCR primer sequence is:

Q107R-F: 5' CTCTTCTCCTCCCGTTACCCCTTC 3' (SEQ ID NO: 13)

Q107R-R-. 5, GAAGGGGTAACGGGAGGAGAAGAG 3' (SEQ ID NO: 14)

LT090 was cloned, expressed and purified in the same manner as in Example 2 to obtain 3.3 mg of 1^090 protein. Example 7 Preparation of LT092

The LT ₂₄ _ / pET32a (+) plasmid was used as a template, and LT-P32U and Q107N- R were used as a pair of primers to obtain a LT092-AB fragment, and the primers were amplified by -P32D and Q107N-F-. Obtain the LT092-CD fragment. Then, LT092-AB and LT092-CD were mixed and used as a template, and LT-P32U and LT-P32D were used as a pair of primers to obtain a full-length LT092 fragment containing Q107N mutation.

The PCR primer sequence is -

Q107N-F: 5, CTCTTCTCCTCCAACTACCCCTTC 3' (SEQ ID NO: 15)

Q107 -R: 5' GAAGGGGTAGTTGGAGGAGAAGAG 3' (SEQ ID NO: 16)

LT092 was cloned, expressed and purified in the same manner as in Example 2 to obtain 4.5 mg of LTO92 protein. Example 8. Enzyme-linked immunosorbent assay for receptor binding status:

TNFRI: Fc/TNFRII: Fc was coated on the ELISA plate, and the LT mutant to be tested was diluted to the same concentration and bound to it, and then rabbit anti-LT enzyme-linked antibody was added to develop a color reading.

Comparison calculation, compared with wild type LT, to determine the percentage, speculate the binding ability of LT to the receptor

Table 1: Evaluation of binding ability to receptor

The results showed that the binding of LT006, LT008, LT009 to the receptor TNFRI was substantially maintained compared with LT, and the binding to TNFRII was greatly reduced. Example 9. Determination of cytotoxic activity of rhLT mutants on L929 cell line in vitro

The cytotoxic activity was further determined for the mutants LT006, 008, and 009 in this example.

L929 cells were inoculated into a 96-well plate at 1.5x10" cells/well, cultured at 37'C, 5% C0 _{2 for} 24 h, and 100 μL of different dilutions of the sample and actinomycin D (1 mg/L) were added to each well, and culture was continued for 24 h. Cell viability was detected by crystal violet staining. The test samples were LT international standard, and the first generation LT stock solution with 27 amino acids missing at the N-terminus. (LT^ _!71 ), N-terminal deletion of 23 amino acids of LT (LT _21-m ; ^fl LT mutants LT006, LT008, LT009. One unit refers to the amount of LT required to induce 50% of apoptosis.

The results of the cytotoxic activity of the rhLT mutant on the L929 cell line in vitro are shown in Table 2:

Example 10. TNFRI and TNFRII activity neutralization assay for binding receptor capacity of LT mutants:

(1) Cell seed plate: The L929 cells in the logarithmic growth phase were 1.5× ^{10 5} /ml, and inoculated into 96-well culture plates, each well was cultured for 24 hours.

(2) Sample dilution: On the next day of seeding, the LT mutant and the LT standard were formulated into 1 ng/ml 2-fold diluted TNFRI, and 13 dilutions were diluted from 250 ng/ml. The LT mutant and the LT standard were formulated as I ng/ml 4-fold diluted TNFRI I, diluted from 500, 000 ng/ml, 13 dilutions per well containing 1 actinomycin D1000 ng/ml. Continue to culture for 24 hours.

(3) Sample reading: The 96-well plate was taken out, the culture solution was discarded and patted as much as possible, and the survival of the cells was examined by crystal violet staining, and the colorimetric instrument was 570 nm.

(4) Analysis of results: Automated analysis using the four-parameter equation of the data processing software of PraphPad Prism 4.0: The logarithm of the concentration of the LT standard is the X-axis, and the value of the 0D490 is the Y-axis. The software will give half of the sample and the standard. Concentration (IC50), and plot the "S" curve. The receptor binding capacity of LT was defined by the ratio of the IC50 that blocks the killing of L929 cells to the lymphotoxin mutant to be tested and the IC50 that blocks the killing of L929 cells by wild-type lymphotoxin.

The results are shown in Table 3.

Table 3 Ability of LT Mutant to Bind TNFRI, TNFRII

TNFRI neutralization combined with TNFRI TNFRII neutralizing LT binding TNFRII sample decrease factor

LT's IC50 capability IC50 capability

LT1-171 20.14ng/ml 1 12.2 lng/ml 1 -

LT28-171 15.64ng/ml 1.29 10.15ng/ml 1.20 0.83

LT24-171 18.38ng/ml 1.10 12.57ng/ml 0.97 1.03

LT006 9.02ng/ml 2.23 2.86X 10 ⁵ ng/ml 4.27X10 2.34Χ 10 ⁴

LT008 8.1 lng/ml 2.48 2.91X10 ⁵ ng/ral 3.43X10" ⁵ 2.38Χ 10 ⁴

LT009 10.86ng/ml 1.86 4.98X 10 ³ ng/ml 2.45X10— ³ 4.08 X 10 ²

LT057 14.2 lng/ml 1.42 >5 10 ⁵ ng/ml <2· 44X10 〉4.10X10 ⁴

LT090 28.46ng/ml 0.71 3.12X10 ng/ml 3.91X10' ³ 2.56X10 ²

LT092 22.88ng/ml 0.88 1.80X10'ng/nil 6.78X10"' 1.47X10 ³ The results of the neutralization experiments in Table 3 indicate that the binding ability of LT006, LT008, LT009, LT057, LT090, LT092 to TNFRI is maintained, indicating that LT006, LT008. LT009 has a slightly higher binding capacity to TNFRI than LT, and the binding ability to TNFRII is decreased. More than 200-40000 times, it has high selectivity to TNFRI receptors. Example 11. In vitro anti-tumor spectrum of rhLT mutants:

rhLT mutant LT008 kills Jurkat (leukemia), U937 (tissue cell lymphoma), MKN-45/GC-863 (gastric cancer), MCF-7 (breast cancer), A549 (lung cancer), A375 (melanoma), T in vitro - 24 (bladder cancer) The ability of cells is better than LT.

(l) rhLT mutant LT008 kills Jurkat (leukemia) cells in vitro

Jurkat cells were seeded with l.OxlO ⁴ cells/well in 96-well plates, cultured at 37 ° C, 5% C0 _{2 for} 24 h, and each well was added with different dilutions of lOOul. The final concentration of actinomycin D was 25 ng/nil. After 48 h, cell viability was detected by MTS staining. The samples tested were LT, rhLT mutant LT008.

The results are shown in Figure 1. The EC50 of LT is 8.38ong/ral, and the EC50 of LT008 is 1.390ng/ml.

(2) rhLT mutant LT008 kills Lovo (colon cancer) cells in vitro

Lovo cells were seeded in 96-well plates at 2. ΟχΙΟ ⁴ cells/well, cultured for 37 hours at 37 Ό and 5% CO ₂ , and each sample was added with different dilutions of lOOul. The final concentration of actinomycin D was 2000 _n g/ml. After 24 h, cell viability was detected by crystal violet staining. The samples tested were LT and LT mutant LT008, respectively.

The results are shown in Figure 2. The EC50 of LT was 5.171 ng/ml; the EC50 of LT008 was L 845 ng/ml.

(3) rhLT mutant LT008 kills MCF-7 (breast cancer) cells in vitro

MCF-7 cells were seeded in 96-well plates with Ι.ΟχΙΟ ⁴ cells/well, cultured at 37 ° C, 5% CO _{2 for} 24 h, and each well was added with different dilutions of lOOul. The final concentration of actinomycin D was 20 n _g /ml. After 48 hours of continuous culture, cell viability was detected by MTS staining. The samples tested were LT and LT mutant LT008.

The results are shown in Figure 3. The EC50 of LT is 1.773 ng/ml _; the EC50 of LT008 is 0.889 ng/ml.

(4) rhLT mutant LT008 kills A549 (lung cancer) cells in vitro

A549 cells were seeded in 96-well plates at .0χ10 ⁴ cells/well, cultured at 37 °C, 5% CO _{2 for} 24 h, and each well was added with different dilutions of lOOul. The final concentration of actinomycin D was 20 ng/ml. After 48 h, cell viability was detected by MTS staining. The samples tested were LT and LT mutant LT008, respectively.

The results are shown in Fig. 4. The EC50 of LT is 6. OlOng/ml; the EC50 of LT008 is 3.886ng/m.

(5) rhLT mutant LT008 kills A375 (melanoma) cells in vitro

A375 cells were seeded with l.OxlO ⁴ cells/well in 96-well plates, cultured at 37 ° C, 5% CO _{2 for} 24 h, and each well was added with different dilutions of lOOul. The final concentration of actinomycin D was 10 ng/ml. After 48 h, cell viability was detected by MTS staining. The samples tested were LT and LT mutant LT008, respectively. '

Results As shown in Figure 5, the EC50 of LT was 2.128 ng/ml _; the EC50 of LT008 was 0.621 ng/ml.

(6) rhLT mutant LT008 kills He (cervical cancer) cells in vitro

HeLa cells were seeded in 96-well plates at 2.0χ10 ⁴ cells/well, 3 C, 5% (: 0 ₂ cultured 411, each well was added with 100 μl of different dilutions, and the final concentration of actinomycin D was 2000 ng/nil. After 24 h, cell viability was detected by crystal violet staining. The samples tested were LT and LT mutant LT008. Results As shown in Figure 6, EC50 of LT = 3.024 ng/ml _; EC50 of LT008 = 0.945 ngM.

(7) r h LT mutant LT008 kills U937 (tissue cell lymphoma) cells in vitro

U937 cells were seeded at 2.5x10" cells/well in 96-well plates, cultured at 37 °C, 5% CO _{2 for} 24 h, and each well was added with different dilutions of lOOul. The final concentration of actinomycin D was 20 ng/ml, and the culture was continued for 48 h. The cell survival was detected by MTS staining. The samples tested were LT and LT mutant LT008.

The results are shown in Figure 7. The EC50 of LT is 2.174 ng/ml; the EC50 of LT008 is 0.562 ng/mlo.

(8) rhLT mutant LT008 kills HL- 60 in vitro (leukemia> cells - HL-60 cells are inoculated with Ι.ΟχΙΟ ⁵ cells/well in 96-well plates, cultured at 37 °C, 5% CO _{2 for} 4 h, each well added lOOul For the diluted sample, the final concentration of actinomycin D was 300 ng/ml, and the cell survival was detected by MTS staining after 24 hours of culture. The samples were LT and rhLT mutant LT008.

The results are shown in Figure 8, EC50 - 2.263 ng / ml for LT; EC50 = 1.034 ng / ml for LT008 Example 12. Sensitization of rhLT mutants to chemotherapeutic drugs

(DrhLT mutant LT008 increases K562 (chronic myeloid leukemia), U937 (tissue cell lymphoma), A549 (lung cancer), MCF-7 (breast cancer), SW-626 (ovarian cancer) cells against alkylating agents Sensitivity of CDDP (cisplatin) and CTX (cyclophosphamide).

K562, U937, A549 MCF-7 and SW-626 cells were seeded in a 96-well plate at 1. OxlO ⁴ cells/well, and cultured at 37 ° C, 5% CO for 24 h. The dosing regimen is: (a) LT, LT mutant LT008 with 0. lng/ml;

(b) Single plus different concentrations of CDDP and CTX; (c) 0. lng/ml of LT, LT mutant LT008 were combined with different concentrations of CDDP and CTX, respectively; and (d) blank control valence plus equal culture liquid.

After 48 hours of culture, cell viability was detected by MTS staining. According to the concentration response curve, determine the IC50 of the half inhibitory concentration of LT, TNF and LT008 chemotherapeutic drugs with or without LT, and find the sensitization multiple (T) = IC50 (chemotherapy alone) / IC50 (LT, TNF and LT008 Chemotherapy)

The results are shown in Fig. 9. The IC50 of the CDDP group was 7.96 mg/ml; the IC50 of the LT+CDDP group was 0.89 mg/ml, T=8.94; and the IC50 of the LT008+CDDP group was 0.12 mg/m, T=66.33.

(2) The rhLT mutant LT008 increases the sensitivity of SW480 (colorectal cancer) and MKN-45/GC-863 (gastric cancer) cells to the metabolic chemotherapeutic drugs 5-FU (5-fluorouracil) and MTX (carbamopterin) .

SW480 and MKN 5 / MGC-863 cells Ι.ΟχΙΟ ⁴ cells / well in 96-well plates were seeded, 37 ° C, 5% C0 2 culture 2. The dosing regimen is: · (a) adding different concentrations of LT, LT mutant LT008; (b) adding different concentrations of 5-FU and MTX; (c) different concentrations of LT, LT mutant LT008 and different concentrations of 5- FU and MTX combined with drug (d) blank control valence plus equal broth. After 48 hours of culture, cell viability was detected by MTS staining.

The results are shown in Figure 10. The IC50 of the 5-FU group was 40rag/ml _; the IC50 of the LT+5-FU group was 21.5 mg/ml, and the ϊ=1.86 _: IC50 of the LT008+5-FU group was 2.3 mg/m. T=17.39 ₀

(3) rhLT mutant LT008 increases the sensitivity of Jurkat (leukemia) and HL- 60 (promyelocytic leukemia) cells against the chemotherapeutic drug ADM (doxorubicin).

Jurkat, HL-60 cells were seeded with 1. OxlO ⁴ cells/well in 96-well plates, cultured at 37 °C, 5% CO _{2 for} 24 h. Plus The drug regimen is: (a) different concentrations of LT, LT mutant LT008; (b) different concentrations of ADM; (c) different concentrations of LT, LT mutant LT008 combined with different concentrations of ADM. d) A blank control valence group plus an equal amount of broth. After 48 hours of culture, cell viability was detected by MTS staining.

The results are shown in Fig. 11. The IC50 of the ADM group was 1.07 mg/ml; the IC50 of the LT+ADM group was 0.98 mg/ml, T-l. 09; and the IC50 of the LT008+ ADM group was 0.450.013 mg/ml. T=2. 38.

(4>rhLT mutant LT008 increases the sensitivity of A375 (melanoma), HeLa (cervical cancer), and T-24 (bladder cancer) cells to the plant chemotherapeutic drug VCR (vincristine).

A375, HeLa, and T-24 cells were seeded in a 96-well plate at 1. cells/well, and cultured at 37 °C, 5% C0 _{2 for} 24 hours. The dosing regimen is: (a) adding different concentrations of LT, LT mutant LT008; (b) adding different concentrations of VCR; (c) different concentrations of LT, LT mutant LT008 combined with different concentrations of VCR: d) A blank control valence group plus an equal amount of broth. After 48 hours of culture, cell viability was detected by MTS staining.

The results are shown in Fig. 12, IC50 of the VCR group: 0. 437 mg/ml; IC50 of the LT+ VCR group = 0.1 Hlmg/ml, T=3.10; IC50 of the LT008+ VCR group = 0. 073 mg/m , T =5. 99.

The above results indicate that the rhLT mutant LT008 can significantly increase the sensitivity of tumor cells to chemotherapeutic drugs. Discussion

LT108Y is an important residue of LT and receptor. The 108Y residue is highly conserved, and any single point change at this site will result in a sharp decrease in the binding capacity of LT to TN'FR and a more than 100-fold decrease in receptor binding capacity. The present inventors conducted a large number of mutation studies on the lasso structure in which LT108Y is located, and confirmed that the lasso structure formed by LT106-1 13 is a close and different position of LT and TNFRI and TNFRI I, and mutations in this region may cause The significant change in the affinity of LT for TNFRI and TNFRI I is an ideal research area for obtaining receptor-selective mutants. In particular, the amino acid at position LT107 has a great influence on the binding of LT to the two receptors. When LT107 residue Q is mutated to E, D, N', R, it shows a high degree of TNFRI selectivity. This is demonstrated by receptor neutralization experiments. The binding of LT to TNFRI I is reduced by 200-40000 times. The binding ability to TNFRI remained basically unchanged; indicating that the amino acid properties of this region are important, the binding ability of LTS106M mutant to TNFRI is decreased, and the binding capacity to TNFRI I is maintained, and the mutation of LT109 at position 10 is also The binding of LT to the two receptors has different effects, and it is also demonstrated that this region is an important research area for obtaining receptor-selective LT. By modifying this region, LT mutants that differ in binding to both receptors can be designed.

The rhLT of the present invention has a selective binding to TNFRI as compared to native rhLT, and the ability to bind to TNFRII can be as high as 200-40000 times or more. TNFRI-selective LT reduces the toxic side effects of TNFRI I, has lower systemic toxicity; avoids the competition of TNFRI I and TNFRI for LT and the neutralization of LT by sTNFRI I, thus high expression of TNFRI I The sensitivity of tumor cells is increased, and the tumor suppressor spectrum is broader. Studies have shown that TNFRI-selective LT has enhanced killing effect on various tumor cells, and it is more effective in combination with chemotherapy drugs.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the invention.

Claims

Rights request

A receptor-selective lymphotoxin mutant, characterized in that the mutant is in a 106-113 lasso structure corresponding to positions 106-113 in the natural sequence of lymphotoxin, 106, 107, 109, 110, At least one amino acid of amino acid residue 111, 112, or 113 is replaced, and/or at least one amino acid is inserted into the 106-113 lasso structure,

The additional condition is that the 108-bit Y residue is unchanged;

2. The lymphotoxin mutant according to claim 1, wherein

The ratio of the binding ability of the lymphotoxin mutant to TNFRI to the binding ability of wild type LT to human TNFRI is greater than 0.1;

The ratio of the binding ability of the lymphotoxin mutant to human TNFRII to the binding ability of wild type LT to human TNFRII is less than 0.01.

3. The lymphotoxin mutant according to claim 2, wherein the amino acid substitution is 106, 107, 109, 110, 111, 112 or 113 amino acid replaced by another amino acid; or 106-113 Insert 1-3 amino acids.

The lymphotoxin mutant according to claim 3, wherein the mutant has a mutation selected from the group consisting of:

Q107E; Q107D; S106E; S106D; Q107R; Q107N; Q107E/S 106E; Q107E/S 106D;

Q107D/S 106E; or Q107D/S106D.

The lymphotoxin mutant according to claim 1, wherein the ratio of the binding ability of the lymphotoxin mutant to human TNFRI to the binding ability of the lymphotoxin mutant to human TNFRII is more than 100.

A pharmaceutical composition comprising a safe and effective amount of a human lymphotoxin mutant and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, further comprising a chemotherapeutic agent selected from the group consisting of CDDP, 5_FU, ADM, VCR or a combination thereof.

The use of the human lymphotoxin mutant according to claim 1, which is for use in the preparation of a medicament for treating: (a) a tumor; (b) an internal parasitic disease.

9. A method of producing a TNFRI receptor-selective human lymphotoxin mutant, characterized in that at position 108 of the native lymphotoxin sequence is unchanged, at least one of amino acids 106-113 is replaced; or at 106-113 Insert 1-3 amino acids.

An isolated DNA sequence which encodes the lymphotoxin mutant of claim 1.