CN115581765A - Recombinant humanized anti-BCMA/CD 3 bispecific antibody injection - Google Patents

Abstract

The invention provides a stable recombinant humanized anti-BCMA/CD 3 bispecific antibody injection, which is prepared by adding other pharmaceutically acceptable auxiliary materials into sulfobutyl betacyclodextrin sodium and propyl gallate, has an ideal glass transition temperature, can effectively prevent the antibody components in the preparation from being broken or degraded by adding the sulfobutyl betacyclodextrin sodium and the propyl gallate, and ensures the stability of the product in storage, transportation or use.

Description

Recombinant humanized anti-BCMA/CD 3 bispecific antibody injection

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a recombinant humanized anti-BCMA/CD 3 bispecific antibody injection.

Background

Multiple myeloma is the second most common hematologic malignancy in which uncontrolled proliferation of monoclonal plasma cells in the bone marrow results in overproduction of monoclonal immunoglobulins and immunosuppression, as well as osteolysis and end-organ damage. Two monoclonal antibodies are currently being used clinically, and multiple myeloma treatment regimens have significantly improved patient survival over the past decade. Nevertheless, existing treatment regimens do not meet the current therapeutic needs, particularly for relapsed/refractory patients who are resistant to current therapies.

B Cell Maturation Antigen (BCMA) is a highly plasma cell specific antigen that plays an important role in regulating B cell maturation and differentiation into plasma cells by participating in proliferation-inducing ligands (APRIL). BCMA expression is restricted to the B cell lineage and is found primarily on plasma and plasmablasts, and to some extent on memory B cells, but is virtually absent on peripheral and naive B cells, nor is it seen in other normal tissue cells. BCMA is also expressed on multiple myeloma cells and is involved in leukemias and lymphomas. Along with its family members, TACI (transmembrane activator and cyclophilin receptor ligand interactor) and BAFF-R (B cell activator receptor), BCMA regulates different aspects of humoral immunity, B cell development and homeostasis. BCMA expression occurs late in B-cell differentiation and contributes to the long-term survival of plasmablasts and plasma cells in the bone marrow. Targeted deletion of the BCMA gene in mice resulted in a significant reduction in the number of long-lived plasma cells in the bone marrow, indicating that BCMA is important for its survival. BCMA overexpression or stimulation of APRIL by BCMA in multiple myeloma cells may directly upregulate key immune checkpoint molecules, which may contribute to immunosuppression of the bone marrow microenvironment.

In the cellular immune process, T lymphocytes play an important role. The cellular immunity mediated by T cells is mainly to specifically recognize antigen peptides presented by Major Histocompatibility Complex (MHC) on the cell surface through a T Cell Receptor (TCR), and further activate signals in the T cells to specifically kill the target cells. The method plays an important role in timely clearing diseased cells in vivo and preventing tumor. Tumors develop because the expression of MHC on the surface of most cancer cells is down-regulated or even absent, allowing tumor cells to escape immune killing.

T cell-engaging bispecific antibodies (TCBs) represent a very effective way to redirect activated cytotoxic T cells to tumors. CD3, which is part of the T cell receptor, is expressed on mature T cells and is capable of transducing the activation signal generated by TCR recognition of the antigen. TCBs can bind both surface tumor antigens and the CD3 epsilon subunit of the T cell receptor, providing a physical link between T cells and tumor cells, thereby effectively activating quiescent T cells to kill tumor cells, and achieving a tumor-treating effect (Smits nc, sentman C L, journal of Clinical Oncology, 2016. Because of the co-stimulatory requirement of T cell bispecific alternative TCR antigen recognition and T cell activation, they eliminate the need for tumor specific immunity and overcome many of the obstacles faced by T cells in the tumor microenvironment.

In recent years, scientists have designed and developed bispecific antibodies of various structures in order to solve the problem of properly assembling two different half antibodies. There are two broad categories to which the population can be attributed, one class of bispecific antibodies is free of Fc regions, including BiTE, DART, trandAbs, bi-Nanobody, and the like. The structure double antibody has the advantages of small molecular weight, capability of being expressed in prokaryotic cells and no need of considering the problem of correct assembly; the disadvantages are that the half-life period is short due to the lack of Fc segment of the antibody and low molecular weight, and the double antibody in the form is extremely easy to polymerize, has poor stability and low expression level, so the clinical application is limited. Another class of bispecific antibodies retains the Fc domain, e.g., triomabs, kih IgG, cross-mab, orthoFab IgG, DVD IgG, igG scFv, scFv2-Fc and like configurations. Such double antibodies form IgG-like structures, are large in molecular structure, and have a longer half-life due to the process of endocytosis and recycling mediated by FcRn; while retaining some or all of the effector functions mediated by Fc, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). However, such double antibodies cannot completely prevent the generation of mismatched products, and any residual fraction of mismatched molecules is difficult to separate from the products, and this method requires a great deal of genetic engineering modification such as mutation on two antibody sequences, and cannot achieve the purpose of simplicity and universality.

Therefore, research has focused on developing a BCMA bispecific molecule with improved properties in terms of product half-life, stability, safety and producibility, such as a BCMA bispecific antibody molecule reported in patent CN 111138542A. However, few reports on the preparation research of the BMCA bispecific antibody molecule exist at present, and the proper preparation product has important influence on the development of the clinical application value of the novel bispecific antibody molecule.

Disclosure of Invention

The invention aims to provide a stable recombinant humanized anti-BCMA/CD 3 bispecific antibody injection which is prepared by adding other pharmaceutically acceptable auxiliary materials into sulfobutylbetacyclodextrin sodium and propyl citrinate and has an ideal glass transition temperature.

The technical scheme of the invention is as follows:

a recombinant humanized anti-BCMA/CD 3 bispecific antibody injection contains a recombinant humanized anti-BCMA/CD 3 bispecific antibody, sulfobutylbetacyclodextrin sodium, propyl gallate and other pharmaceutically acceptable auxiliary materials.

Preferably, the concentration of the recombinant humanized anti-BCMA/CD 3 bispecific antibody is 30-70 mg/ml.

Preferably, the injection contains 30 to 100 mass ratio of sulfobutylbetacyclodextrin sodium, propyl citrinate and the recombinant humanized anti-BCMA/CD 3 bispecific antibody: 1 to 10:30 to 70.

Further preferably, the injection comprises 50 to 100 mass ratio of sulfobutylbetacyclodextrin sodium, propyl gallate and the recombinant humanized anti-BCMA/CD 3 bispecific antibody: 5 to 10:30 to 70 percent.

Preferably, the other pharmaceutically acceptable excipients include excipients, solubilizers, and buffers.

Further preferably, the excipient is one or more selected from trehalose, sucrose, mannitol or NaCl.

Further preferably, the content of the excipient in the injection is 100-250 mM; preferably 180 to 250mM.

Further preferably, the solubilizer is polysorbate 20 or polysorbate 80; more preferably polysorbate 80.

Further preferably, the content of the solubilizer in the injection is 0.1-0.5 mg/ml.

Further preferably, the buffering agent is a sodium citrate/citric acid buffering system.

Further preferably, the content of the buffer in the injection is 10-50 mM; preferably 10 to 20mM; the pH range is preferably 5.5 to 6.5.

Preferably, the recombinant humanized anti-BCMA/CD 3 bispecific antibody injection comprises the following components:

preferably, the recombinant humanized anti-BCMA/CD 3 bispecific antibody injection comprises the following components:

a preparation method of the recombinant humanized anti-BCMA/CD 3 bispecific antibody injection comprises the following specific steps:

preparing sodium citrate/citric acid buffer solution with the concentration and pH value of a prescription, adding the recombinant humanized anti-BCMA/CD 3 bispecific antibody ultrafiltration exchange solution into the sodium citrate/citric acid buffer solution, and regulating the protein concentration; accurately weighing sulfobutylbetacyclodextrin sodium, propyl citrinate, excipient and solubilizer according to the prescription amount, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Compared with the prior art, the invention has the following technical effects:

the invention provides a stable recombinant humanized anti-BCMA/CD 3 bispecific antibody injection which has the characteristics of good stability and high safety, and further reduces the potential safety hazard of clinical medication. The injection provided by the invention has the advantages of simple preparation process, stable and controllable quality, and convenience in storage and transportation, and is suitable for amplification. The use of cyclodextrin and propyl gallate can reduce the dosage of excipient and solubilizer, reduce production cost, and reduce the potential safety hazard of drug administration.

Detailed Description

The invention is further illustrated by the following examples, which should be properly understood: the examples of the present invention are intended to be illustrative only and not to be limiting, and therefore, the present invention is intended to be simply modified within the scope of the present invention as claimed.

Example 1

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 2

(1) Prescription

(2) Preparation method

Preparing sodium citrate/citric acid buffer solution with the concentration and pH value of a prescription, ultrafiltering the recombinant humanized anti-BCMA/CD 3 bispecific antibody to change the solution into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, naCl and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 3

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 50mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 4

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the sulfobutyl-beta-cyclodextrin sodium, the propyl citrinate, the trehalose and the polysorbate 20 according to the prescription amount, dissolving and uniformly mixing, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 5

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 70mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, mannitol and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 6

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 7

(1) Prescription

(2) Preparation method

Preparing sodium citrate/citric acid buffer solution with the concentration and pH value of a prescription, ultrafiltering the recombinant humanized anti-BCMA/CD 3 bispecific antibody to change the solution into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Example 8

(1) Prescription

(2) Preparation method

Preparing sodium citrate/citric acid buffer solution with the concentration and pH value of a prescription, ultrafiltering the recombinant humanized anti-BCMA/CD 3 bispecific antibody to change the solution into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Comparative example 1

(1) Prescription

(2) Preparation method

Preparing sodium citrate/citric acid buffer solution with the concentration and pH value of a prescription, ultrafiltering the recombinant humanized anti-BCMA/CD 3 bispecific antibody to change the solution into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing sucrose and polysorbate 80 according to the prescription amount, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Comparative example 2

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing sulfobutyl-beta-cyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80 according to the prescription amount, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the sodium sulfobutyl-beta-cyclodextrin sodium citrate.

Comparative example 3

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Comparative example 4

(1) Prescription

(2) Preparation method

Preparing a sodium citrate/citric acid buffer solution with the concentration and the pH value of a prescription, carrying out ultrafiltration exchange on a recombinant humanized anti-BCMA/CD 3 bispecific antibody into the sodium citrate/citric acid buffer solution, and adjusting the protein concentration to 30mg/ml; accurately weighing the formula amounts of sulfobutylbetacyclodextrin sodium, propyl citrinate, sucrose and polysorbate 80, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.

Verification examples

1. Differential calorimetric scanning (DSC)

The solutions of the recombinant humanized anti-BCMA/CD 3 bispecific antibodies of examples 1 to 8 and comparative examples 1 to 4 were measured for their melting temperature Tm values by gradually increasing the temperature. The results are shown in Table 1.

The method comprises the following steps: the Tm of the protein in solution was determined using a MicroCalTM VP-Capillary DSC instrument at the instrument program controlled temperature. The samples were diluted to 1mg/mL with the corresponding buffer solutions. And (3) adding 350 mu L of sample into a sample hole of a 96-well plate, adding 350 mu L of corresponding buffer solution into a buffer hole, setting the scanning temperature to be 20-90 ℃, and setting the scanning speed to be 60 ℃/hr. Data analysis MicroCal VP-Capillary DSC automatic analysis software was used.

TABLE 1 DSC results

Sample(s)	TmOnset/℃	Tm1/℃	Tm2/℃
				Example 1	48.74	63.46	80.20
Example 2	47.53	62.75	78.34
				Example 3	47.34	62.33	77.69
Example 4	48.69	63.12	79.66
				Example 5	45.18	60.86	76.34
Example 6	45.53	61.39	77.55
				Example 7	43.74	56.38	76.43
Example 8	42.36	59.75	77.14
				Comparative example 1	42.12	53.38	75.04
Comparative example 2	42.93	55.36	75.09
				Comparative example 3	42.36	54.69	78.69
Comparative example 4	44.32	57.63	76.05

The result shows that Tmonset and Tm1 of the preparation product added with sulfobutylbetacyclodextrin and propyl citrinate are obviously improved, and the obtained medicine has better thermal stability.

2. Stability test

Samples were prepared according to examples 1 to 8 and comparative examples 1 to 4, respectively, and storage stability was examined using accelerated stability test and long term test.

The accelerated test was carried out at 25 ℃. + -. 2 ℃ for 6 months. The used equipment can control the temperature to +/-2 ℃ and monitor the actual temperature. Samples were taken at the end of 0, 3, and 6 months during the test period and examined according to stability focus examination. The long-term test is carried out under the condition of 2-8 ℃, and the test is carried out according to the stability key investigation items at the end of 0 month, 6 months, 12 months and 24 months respectively; purity check was determined according to general rule 0514 molecular grouping chromatography and general rule 0542 capillary electrophoresis, activity was determined according to bioluminescence-based reporter gene method, experimental cells: CHOK1-BCMA-Luc cells, the results are shown in tables 2 and 3.

TABLE 2 Long-term stability test results at 8 deg.C

TABLE 3 accelerated stability test results at 25 ℃

Claims (10)

1. The injection is characterized by comprising the recombinant humanized anti-BCMA/CD 3 bispecific antibody, sulfobutylbetacyclodextrin sodium, propyl gallate and other pharmaceutically acceptable auxiliary materials.

2. The injection of claim 1, wherein the mass ratio of sulfobutylbetacyclodextrin sodium, propyl gallate to the recombinant humanized anti-BCMA/CD 3 bispecific antibody is 30 to 100:1 to 10:30 to 70 percent; preferably 50 to 100:5 to 10:30 to 70.

3. The injection of claim 1, wherein said other pharmaceutically acceptable excipients comprise excipients, solubilizers, and buffers.

4. The injection of claim 1, wherein the excipient is one or more selected from the group consisting of trehalose, sucrose, mannitol, and NaCl.

5. The injection of claim 1, wherein the solubilizer is polysorbate 20 or polysorbate 80.

6. The injection of claim 1, wherein said buffering agent is a sodium citrate/citric acid buffer system.

7. The injection of claim 1, wherein the buffer is present in an amount of 10 to 50mM.

8. The injection of claim 1, wherein said injection of a recombinant humanized anti-BCMA/CD 3 bispecific antibody comprises the following components:

9. the injection of claim 1, wherein said injection of a recombinant humanized anti-BCMA/CD 3 bispecific antibody comprises the following components:

10. a method for preparing an injection according to any one of claims 1 to 9, comprising the steps of:

preparing sodium citrate/citric acid buffer solution with the concentration and pH value of a prescription, adding the recombinant humanized anti-BCMA/CD 3 bispecific antibody ultrafiltration exchange solution into the sodium citrate/citric acid buffer solution, and regulating the protein concentration; accurately weighing sulfobutylbetacyclodextrin sodium, propyl citrinate, excipient and solubilizer in a prescribed amount, dissolving and mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the product.