Use of pharmaceutical composition in preparation of medicament for treating gvhd

Abstract

Provided is a pharmaceutical composition for treating GVHD, the pharmaceutical composition comprising a recombinant protein as shown below: 1) an scFV of an anti-CD3 antibody; and 2) diphtheria toxin. The recombinant protein of the present invention can specifically bind to CD3 on the surface of T cells, and after binding, the diphtheria toxin functional group in the recombinant protein enters the T cells under the translocation effect of CD3, leading to cell apoptosis, thereby eliminating the T cells. Therefore, the recombinant protein of the present invention can be used for treating GVHD.

Description

Application of a pharmaceutical composition for preparing a drug for treating GVHD Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular, relates to an application of a pharmaceutical composition, and more particularly, relates to an application of a pharmaceutical composition for treating GVHD. Background Art

[0002] Allogeneic hematopoietic cell transplantation (allo-HSCT) is an effective immunotherapy for a variety of blood diseases, including leukemia. Improved survival rates after allo-HSCT have led to an increase in its use, but the main cause of non-relapse mortality (NRM) remains graft-versus-host disease (GVHD). Severe infection and systemic immune impairment are the main causes of death from GVHD. Graft-versus-host disease (GVHD) is caused by a series of "cytokine storms" initiated by the recipient in the allogeneic donor's graft after transplantation, which greatly enhances its immune response to the recipient's antigens and launches a cytotoxic attack against the recipient's target cells. This can affect multiple organs, with the skin, liver, and intestines being the main targets, and the eyes, kidneys, and lungs also potentially affected.

[0003] The first-line treatment for GVHD is corticosteroids, but some people develop resistance to them. There's currently no consensus on second- and third-line treatments for those with steroid resistance, and the options available remain limited. Therefore, there's a need for a product that can treat GVHD, providing more options for clinical use and delivering superior efficacy.

[0004] Summary of the Invention

[0005] In view of this, in a first aspect, the present invention provides a use of a pharmaceutical composition for preparing a drug for treating GVHD, wherein the pharmaceutical composition comprises a recombinant protein as shown below:

[0006] 1) scFV of anti-CD3 antibody; and

[0007] 2) Diphtheria toxin.

[0008] Furthermore, the recombinant protein heavy chain is sequentially 1) and 2) from N-terminus to C-terminus, or 2) and 1) in sequence.

[0009] Furthermore, the recombinant protein heavy chain from N-terminus to C-terminus is 2) and 1).

[0010] Furthermore, the recombinant protein also includes linkers of different types and lengths.

[0011] In some specific embodiments, the linker can be PW, (G4S) n,AADP,(EAAAK) n , KESGSSVSSEQLAQFRSLD, EGKSSGSGSESKST, etc.

[0012] Furthermore, the scFV of the anti-CD3 antibody has the light chain CDR sequences shown in SEQ ID NOs. 1 to 3.

[0013] Furthermore, the scFV of the anti-CD3 antibody has the heavy chain CDR sequences shown in SEQ ID NOs. 4 to 6.

[0014] Furthermore, the scFV of the anti-CD3 antibody has a light chain variable region sequence as shown in SEQ ID NO.7.

[0015] Furthermore, the scFV of the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO.8.

[0016] Furthermore, the scFV of the anti-CD3 antibody has an amino acid sequence as shown in SEQ ID NO.9.

[0017] The recombinant protein of the present invention can specifically bind to CD3 on the surface of T cells. After binding, the diphtheria toxin functional group in the recombinant protein enters the T cells under the translocation effect of CD3, causing cell apoptosis, thereby eliminating T cells, making the recombinant protein of the present invention capable of being used to treat GVHD.

[0018] In a specific embodiment, the diphtheria toxin has the amino acid sequence shown in SEQ ID NO.10.

[0019] In some specific embodiments, the structure of the recombinant protein is shown in FIG1 , and its sequence is shown in SEQ ID NO.11.

[0020] The use of the above-mentioned recombinant proteins has the best effect in eliminating T cells and can treat GVHD to the greatest extent.

[0021] Furthermore, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0022] Furthermore, the pharmaceutical composition also includes another pharmaceutically active agent for combined use.

[0023] Furthermore, the dosage of the pharmaceutical composition used is no more than 25 μg / kg / person / day.

[0024] Furthermore, the dosage of the pharmaceutical composition used is no more than 15 μg / kg / person / day.

[0025] Furthermore, the dosage of the pharmaceutical composition used is no more than 10 μg / kg / person / day.

[0026] Furthermore, the dosage of the pharmaceutical composition used is no more than 5 μg / kg / person / day.

[0027] Furthermore, the dosage of the pharmaceutical composition used is no more than 4 μg / kg / person / day.

[0028] Furthermore, the dosage of the pharmaceutical composition used is no more than 2.5 μg / kg / person / day.

[0029] Furthermore, the dosage of the pharmaceutical composition used is no more than 1.25 μg / kg / person / day.

[0030] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a recombinant protein of the present invention. In certain preferred embodiments, the isolated nucleic acid molecule encodes a recombinant protein of the present invention.

[0031] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present invention.

[0032] In certain preferred embodiments, the vector comprises a nucleotide sequence encoding the recombinant protein of the present invention.

[0033] In certain preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a virus, etc. In certain preferred embodiments, the vector is capable of expressing the recombinant protein of the present invention in a subject (eg, a mammal, such as a human).

[0034] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention.

[0035] Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, e.g., mouse cells, human cells, etc.).

[0036] In certain preferred embodiments, the host cell of the present invention is a mammalian cell, such as CHO (eg, CHO-K1, CHO-S, CHO DG44) or HEK293.

[0037] In another aspect, the present invention provides a method for preparing the recombinant protein of the present invention, comprising culturing the host cell of the present invention under conditions allowing expression of the recombinant protein, and recovering the recombinant protein from the cultured host cell.

[0038] In certain preferred embodiments, the method comprises:

[0039] 1) constructing an expression vector comprising a nucleotide sequence encoding the recombinant protein of the present invention;

[0040] 2) transforming the expression vector described in step 1) into a host cell;

[0041] 3) culturing the host cell described in step 2) under conditions that allow expression of the recombinant protein of the present invention; and

[0042] 4) Recovering the recombinant protein from the cultured host cell.

[0043] In another aspect, the present invention provides a pharmaceutical composition comprising the recombinant protein of the present invention and a pharmaceutically acceptable carrier and / or excipient.

[0044] The recombinant protein or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.

[0045] The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: in an appropriate solvent, the recombinant protein of the present invention is mixed with the necessary dose, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0046] In addition, the recombinant protein of the present invention can be present in a pharmaceutical composition in a unit dosage form, so that it can be used. In certain embodiments, the unit dosage is at least 0.01 mg, at least 0.1 mg, at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg. In the case where the pharmaceutical composition is a liquid (e.g., injection) formulation, it can include a concentration of at least 0.1 mg / ml, such as at least 0.25 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml of the recombinant protein of the present invention.

[0047] Recombinant protein of the present invention or pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In a preferred embodiment, recombinant protein of the present invention or pharmaceutical composition are given by intravenous infusion or injection.

[0048] In the present invention, the subject may be a mammal, such as a human.

[0049] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the recombinant protein of the present invention. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the recombinant protein of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0050] In another aspect, the present invention provides a method of treatment comprising:

[0051] The above-mentioned pharmaceutical composition of the present invention is administered to a subject.

[0052] Furthermore, the subject is preferably a human.

[0053] Furthermore, the dosage of the pharmaceutical composition used is no more than 25 μg / kg / person / day.

[0054] Furthermore, the dosage of the pharmaceutical composition used is no more than 15 μg / kg / person / day.

[0055] Furthermore, the dosage of the pharmaceutical composition used is no more than 10 μg / kg / person / day.

[0056] Furthermore, the dosage of the pharmaceutical composition used is no more than 5 μg / kg / person / day.

[0057] Furthermore, the dosage of the pharmaceutical composition used is no more than 4 μg / kg / person / day.

[0058] Furthermore, the dosage of the pharmaceutical composition used is no more than 2.5 μg / kg / person / day.

[0059] Furthermore, the dosage of the pharmaceutical composition used is no more than 1.25 μg / kg / person / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG1 is a schematic diagram of the structure of the recombinant protein of the present invention;

[0061] FIG2 is a survival curve of a mouse GVHD model of the recombinant protein of the present invention;

[0062] FIG3 shows the weight changes of the mouse GVHD model induced by the recombinant protein of the present invention;

[0063] FIG4 shows the clinical scores of the mouse GVHD model of the recombinant protein of the present invention. DETAILED DESCRIPTION

[0064] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0065] Example 1: Recombinant protein used in the present invention

[0066] The sequences used in the present invention are shown in Table 1 below.

[0067] Table 1

[0068] The constructed fusion protein is transformed into the working library strain VG712 through conventional strain transformation and preservation, which is the recombinant protein VG712 of the present application.

[0069] Example 2: Expression and purification of the recombinant protein of the present invention

[0070] 1. Fermentation culture

[0071] 1). Materials

[0072] Culture medium: YSG culture medium (Thermo fisher Bacto TM Yeast extract 10g / L, Thermo fisher Difco TM Soytone 20g / L, BD anhydrous glycerin 10g / L)

[0073] Strain: VG712 working library strain

[0074] 2). Operation steps

[0075] A. Add 60 ml of YSG medium to a 250 ml shake flask. Thaw a working bacterial strain at room temperature and inoculate it into the medium. Transfer the inoculated bacterial solution to a shaker and culture for 48 hours.

[0076] B. Add 200 ml of YSG medium to 1 L of shake flask seed, inoculate 20 ml of the superior seed into the medium, and transfer the inoculated bacterial liquid to a shaker for 24 hours.

[0077] C. Remove the secondary culture that has been incubated for 24 ± 1 h, transfer the desired strain to the inoculation bottle, and transfer the strain to the fermenter. Turn on temperature, DO, and pH control.

[0078] D. When the DO peak appears, add 75% glycerol solution.

[0079] E. After a period of cultivation, start feeding methanol and 10% yeast extract and continue feeding until the end of the cultivation.

[0080] F. Use hollow fiber columns to collect the culture supernatant and transfer it to downstream for subsequent protein purification.

[0081] 2. Purification process

[0082] 1)Butyl 650M HIC

[0083] A material

[0084] Buffer: 0.5 mol / L sodium hydroxide solution, TE buffer, 4*BHIC binding buffer, BHIC binding buffer

[0085] TEG buffer, HS packing buffer, LS packing buffer, 20% ethanol solution

[0086] Filler: TOYOPEARL Buty1-650M

[0087] B. Process Description

[0088] Sample dilution: Adjust the conductivity of the clarified harvest solution to 34-42 mS / cm with 4*BHIC banding buffer;

[0089] Chromatography process: Equilibrate the column with BHIC banding buffer and then load the sample. After loading, re-equilibrate with BHIC banding buffer, elute the target protein with TEG buffer, regenerate the column with water for injection, CIP the column with 0.5 mol / L sodium hydroxide solution, and preserve the column with 20% ethanol solution.

[0090] 2)Borate Poros 50HQ AEC

[0091] A material

[0092] Buffer: 0.5mol / L sodium hydroxide solution, TE buffer, HS packing buffer, LS packing buffer, 20% ethanol solution, 0.05mol / L borate in TEG buffer, 0.075mol / L borate in TEG buffer, 0.1mol / L borate in TEG buffer

[0093] Filler: Poros 50HQ

[0094] B. Process Description

[0095] Sample dilution: Dilute the hydrophobic chromatography collection solution 8 times with TE buffer.

[0096] Chromatography process: Equilibrate the column with TE buffer before loading the sample. Re-equilibrate with TE buffer after loading. Elute the target protein with 0.05 mol / L borate in TEG buffer (elution 1), 0.075 mol / L borate in TEG buffer (elution 2), and 0.1 mol / L borate in TEG buffer (elution 3). Regenerate the column with HS packing buffer. CIP the column with 0.5 mol / L sodium hydroxide solution and store it in 20% ethanol. Combine the elution 2 and elution 3 fractions to form the anion exchange chromatography fraction B.

[0097] 3) Poros 50HQ AEC

[0098] A material

[0099] Buffer: 0.5mol / L sodium hydroxide solution, TE buffer, HS packing buffer, LS packing buffer, 20% ethanol solution, Low TEG buffer, 0.05mol / L NaCl in low TEG buffer, 0.15mol / L NaCl in low TEG buffer

[0100] Filler: Poros 50HQ

[0101] B. Process Description

[0102] Sample dilution: Dilute the collected anion exchange chromatography solution of B 4-fold with TE buffer.

[0103] Chromatography process: Equilibrate the chromatography column with TE buffer and then load the sample. Re-equilibrate with Low TEG buffer after loading. Elute the target protein with 0.05 mol / L NaCl in low TEG buffer (elution 1) and 0.15 mol / L NaCl in low TEG buffer (elution 2). Regenerate the chromatography column with HS packing buffer. CIP the chromatography column with 0.5 mol / L sodium hydroxide solution and preserve the chromatography column with 20% ethanol solution. Check the purity of the collected solutions of elution 1 and elution 2 (purity ≥ 98%, combine the collected solutions and proceed to the next step).

[0104] 4) Conductivity adjustment and dilution

[0105] A material

[0106] Buffer: 0.5mol / L NaCl in low TEG buffer, 0.15mol / L NaCl in low TEG buffer

[0107] B. Process Description

[0108] The conductivity of the anion exchange chromatography collected solution was adjusted with 0.5 mol / L NaCl in low TEG buffer, and the protein concentration was diluted to ≥ 0.8 mg / ml with 0.15 mol / L NaCl in low TEG buffer (stock buffer).

[0109] 5) Stock solution filtration

[0110] A material

[0111] Consumables: 0.22μm sterilizing filter

[0112] The stock solution (filter) sample was filtered through a 0.22 μm filter to obtain the VG712 stock solution.

[0113] Example 3: Construction of GVHD mouse model

[0114] Ten female NCG mice aged 6-8 weeks were pretreated with X-ray (1Gγ) and then injected with 5*10 6 Humanized PBMCs were injected into the mouse model of GVHD. The day of human PBMC injection was defined as Day 1. Mice were randomly divided into three groups based on their body weight on the day of PBMC injection: a model group (4 mice), a low-dose group (3 mice), and a high-dose group (3 mice).

[0115] Example 4: Treatment of GVHD with the recombinant protein of the present invention

[0116] Dosing began on day 3 after modeling. Model group mice were given solvent buffer, low-dose group mice were given 7.5 μg / kg of the recombinant protein of the present invention, and high-dose group mice were given 30 μg / kg of the recombinant protein of the present invention. Dosing frequency was twice daily, 6 hours apart, for 4 consecutive days, for a total of 8 doses, and the administration route was intraperitoneal injection. After modeling, the experimental animals were evaluated for clinical GVHD scores by observing changes in five indicators: body weight, body position, activity, coat texture, and skin integrity every day. Observation was continued until day 56.

[0117] The therapeutic effect of the recombinant protein of the present invention on GVHD was evaluated based on three indicators: mouse survival rate, body weight change, and GVHD score after modeling and administration.

[0118] After the mice were pretreated with X-rays (1Gγ) and injected with human PBMCs through the tail vein, all the mice in the model group died of acute GVHD on the 14th day. Except for one mouse in the high-dose group that died on the 14th day, all the other mice in the treatment group survived (see Figure 2).

[0119] After pretreatment with X-rays (1Gγ) and tail vein injection of human PBMCs, mice in all groups experienced transient weight loss due to irradiation. Subsequently, mice in the model group experienced rapid weight loss and death due to acute GVHD. Mice in the treatment group experienced transient weight loss due to the drug administration, but their weight gradually recovered after the drug administration ended (see Figure 3).

[0120] After the mice were pretreated with X-rays (1Gγ) and injected with human PBMCs via the tail vein, the model group mice developed GVHD and the clinical scores continued to rise. The clinical score on the 13th day was 5.5±0.5, and the clinical score on the 14th day was 7.0 (1 mouse remaining). The clinical scores of the mice in the treatment group remained at a low level (see Figure 4), indicating that the high-dose and low-dose groups had excellent therapeutic effects on GVHD.

Claims

1. A pharmaceutical composition for use in preparing a drug for treating GVHD, the pharmaceutical composition comprising a recombinant protein as shown below: 1) scFV of anti-CD3 antibody; and 2) Diphtheria toxin.

2. The use according to claim 1, wherein: The recombinant protein heavy chain from N-terminus to C-terminus is 2) and 1).

3. The use according to claim 1, wherein: The scFV of the anti-CD3 antibody has the light chain CDR sequences shown in SEQ ID NOs. 1 to 3 and the heavy chain CDR sequences shown in SEQ ID NOs. 4 to 6.

4. The use according to claim 1, wherein: The scFV of the anti-CD3 antibody has a light chain variable region sequence as shown in SEQ ID NO.7 and a heavy chain variable region sequence as shown in SEQ ID NO.

8.

5. The use according to claim 1, wherein: The scFV of the anti-CD3 antibody has the amino acid sequence shown in SEQ ID NO.

9.

6. The use according to claim 1, wherein: The diphtheria toxin has an amino acid sequence as shown in SEQ ID NO.

10.

7. The use according to claim 1, wherein: The sequence of the recombinant protein is shown in SEQ ID NO.

11.

8. The use according to claim 1, wherein: The dosage of the pharmaceutical composition used is not more than 25 μg / kg / person / day.

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