Antibody targeting B cell maturation antigen and related products thereof and medical applications

Abstract

The present disclosure belongs to the fields of biotechnology and biological medicine, and specifically relates to an antibody targeting a B cell maturation antigen (BCMA) and related products thereof and medical applications. Specifically, the antibody contains a variable light chain and a variable heavy chain. The variable light chain contains a light chain complementarity-determining region (CDR) 1, a light chain CDR2, and a light chain CDR3. The light chain CDR1 has a sequence as shown in SEQ ID NO. 1, the light chain CDR2 has an amino acid sequence as shown in SEQ ID NO. 2, and the light chain CDR3 is as shown in SEQ ID NO. 3 or SEQ ID NO. 4.

Claims

1. An antibody or an antigen binding fragment thereof targeting a B cell maturation antigen (BCMA), the antibody containing a variable light chain and a variable heavy chain, the variable light chain containing a light chain complementarity-determining region (CDR) 1, a light chain CDR2, and a light chain CDR3, the light chain CDR1 having the sequence as shown in SEQ ID NO. 1, the light chain CDR2 having the amino acid sequence as shown in SEQ ID NO. 2, and the light chain CDR3 being as shown in SEQ ID NO. 3 or SEQ ID NO. 4; and the variable heavy chain containing a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, the heavy chain CDR1 having the sequence as shown in SEQ NO. 5, the heavy chain CDR2 having the amino acid sequence as shown in SEQ ID NO. 6, and the heavy chain CDR3 being as shown in SEQ ID NO. 7 or SEQ ID NO. 8.

2. The antibody or an antigen binding fragment thereof according to claim 1, wherein the antibody is linked as the variable light chaina connexonthe variable heavy chain.

3. The antibody or an antigen binding fragment thereof according to claim 1, wherein the antibody is humanized.

4. The antibody or an antigen binding fragment thereof according to claim 1, wherein the antibody has the sequence as shown in any one of SEQ ID NO. 28-35.

5. The antibody or an antigen binding fragment thereof according to claim 1, wherein the antibody contains a heavy chain constant region and a light chain constant region.

6. An antibody complex, formed by ligating the antibody or an antigen binding fragment thereof according to claim 1 to a cluster of differentiation (CD) 8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory structural domain, and an intracellular signal transduction structural domain, the intracellular signal transduction structural domain having the amino acid sequence as shown in SEQ ID NO. 48.

7. The antibody complex according to claim 6, wherein the antibody complex further comprises a signal peptide.

8. The antibody complex according to claim 7, wherein the signal peptide is a CD8 signal peptide.

9. An antibody conjugate, containing the antibody or an antigen binding fragment thereof according to claim 1, and further containing a conjugated portion selected from the following group: a detectable marker, a drug, a toxin, a cytokine or an enzyme.

10. A drug composition for treating BCMA-related diseases, containing the antibody complex according to claim 6 and a diluent, the BCMA-related disease is multiple myeloma (MM), Burkitt's lymphoma, and plasma cell leukemia.

11. A drug composition for treating BCMA-related diseases, containing the antibody complex according to claim 6 and a diluent, the BCMA-related disease is myeloma.

12. A method for detection of BCMA, comprising a step of contacting the antibody or an antigen binding fragment thereof according to claim 1 with a sample, the detection being for non-diagnostic purposes, and detecting binding between a) BCMA in the sample and b) the antibody or antigen-binding fragment thereof, wherein binding indicates the presence of BCMA in the sample.

13. The method according to claim 12, wherein the antibody or an antigen biding fragment thereof is an antibody having a detectable marker.

14. The method according to claim 13, wherein the detectable marker is selected from an enzyme, a fluorescent material, a luminescent material, a radioactive material, a positron emission metal, and a non-radioactive paramagnetic metal ion.

15. The method according to claim 12, wherein the sample comprises a physiological body fluid, the physiological body fluid being selected from blood, a serum, plasma, saliva, an eye secretion, a cerebrospinal fluid, pus, an exudate, breast milk, sweat, a tear, an ear effluent, a sputum, lymph, urine or feces; and/or the sample comprises a tissue, the tissue being from a lung, a spleen or a kidney.

16. The method according to claim 12, wherein the sample to be detected contains B cells.

17. A method of preparing a drug for treating diseases, the disease being multiple myeloma, Burkitt's lymphoma, or plasma cell leukemia, comprising providing the antibody complex according to claim 6, and combining the antibody complex with a pharmaceutically acceptable excipient.

18. A method of preparing a drug for treating diseases, the disease being myeloma comprising providing the antibody complex according to claim 6, and combining the antibody complex with a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is a graph showing the affinity of a mouse-derived antibody to BCMA in an H929 blank group;

(2) FIG. 1B is a graph showing the affinity of the mouse-derived antibody to BCMA in an H929+PE fluorescence group;

(3) FIG. 1C is a graph showing the affinity of the mouse-derived antibody to BCMA in a negative control group;

(4) FIG. 1D is a graph showing the affinity of the mouse-derived antibody to BCMA in a positive control group; and

(5) FIG. 1E is a graph showing the affinity of the mouse-derived antibody to BCMA.

(6) FIG. 2A is a graph showing the expression of BCMA-CAR in NK cells in a negative control group;

(7) FIG. 2B is a graph showing the expression of mouse-derived BCMA-CAR in NK cells;

(8) FIG. 2C is a graph showing the expression of humanized BCMA-CAR-V0 in NK cells;

(9) FIG. 2D is a graph showing the expression of humanized BCMA-CAR-V1 in NK cells;

(10) FIG. 2E is a graph showing the expression of humanized BCMA-CAR-V2 in NK cells;

(11) FIG. 2F is a graph showing the expression of humanized BCMA-CAR-V3 in NK cells;

(12) FIG. 2G is a graph showing the expression of humanized BCMA-CAR-V4 in NK cells;

(13) FIG. 2H is a graph showing the expression of humanized BCMA-CAR-V6 in NK cells;

(14) FIG. 2I is a graph showing the expression of humanized BCMA-CAR-V7 in NK cells; and

(15) FIG. 2J is a graph showing the expression of humanized BCMA-CAR-V8 in NK cells.

(16) FIG. 3A is a graph showing the killing ability of BCMA-CAR in a negative control group;

(17) FIG. 3B is a graph showing the killing ability of mouse-derived BCMA-CAR;

(18) FIG. 3C is a graph showing the killing ability of humanized BCMA-CAR-V0;

(19) FIG. 3D is a graph showing the killing ability of humanized BCMA-CAR-V1;

(20) FIG. 3E is a graph showing the killing ability of humanized BCMA-CAR-V2;

(21) FIG. 3F is a graph showing the killing ability of humanized BCMA-CAR-V3;

(22) FIG. 3G is a graph showing the killing ability of humanized BCMA-CAR-V4;

(23) FIG. 3H is a graph showing the killing ability of humanized BCMA-CAR-V6;

(24) FIG. 3I is a graph showing the killing ability of humanized BCMA-CAR-V7; and

(25) FIG. 3J is a graph showing the killing ability of humanized BCMA-CAR-V8.

DETAILED DESCRIPTION

(26) The present disclosure is further described below by reference to the embodiments, and what described in the following is merely the preferred embodiments of the present disclosure, rather than a limitation to the present disclosure in any other form. The above disclosed technical contents can be altered to equivalent embodiments by any skilled familiar with the field, and without departing from the content of the solutions of the present disclosure, any simple modifications or equivalent changes to the following embodiments based on the technical substance of the present disclosure fall within the scope of protection of the present disclosure.

Embodiment 1, Determination of the Affinity of Mouse-Derived Antibody to Myeloma Cells

(27) A mouse-derived antibody was obtained and the affinity of the antibody to myeloma cells was detected. The mouse-derived antibody had an amino acid sequence as shown in SEQ ID NO. 27 and a nucleotide sequence as shown in SEQ ID NO. 36.

Determination Method

(28) 1. H929 myeloma cells were cultured, and then subjected to cell counting. 510.sup.5 cells were taken and placed in a 1.5 mL Eppendorf (EP) tube for centrifuging at 1000 rpm for 5 min, and supernatant was removed.

(29) 2. 500 L of phosphate buffered saline (PBS) was added to resuspend the cells, and the resuspended cells were centrifuged at 1000 rpm for 5 min, and supernatant was removed.

(30) 3. Supernatant from blank cell culture and supernatant produced from antibody were separately used to resuspend cells to be tested. The resuspended cells were protected from light for reacting at 4 C. for 30 min, and then centrifuged at 1000 rpm for 5 min, and supernatant was removed.

(31) 4. 500 L of PBS was added to resuspend the cells, and the resuspended cells were centrifuged at 1000 rpm for 5 min, and supernatant was removed.

(32) 5. 100 L of secondary antibody (PE anti-human IgG Fc Antibody, diluted by 1:600 with PBS) was added to resuspend the cells. The resuspended cells were protected from light for reacting at 4 C. for 30 min, and then centrifuged at 1000 rpm for 5 min, and supernatant was removed.

(33) 6. 500 L of PBS was added to resuspend the cells, and the resuspended cells were centrifuged at 1000 rpm for 5 min, and supernatant was removed.

(34) 7. 400 L of PBS was added to resuspend the cells, and the resuspended cells were detected by a machine.

(35) The results prove that the mouse-derived BCMA antibody provided in the present disclosure has good affinity to H929 cells. The positive rate of PE-fluorescence labeled H929 cells is shown in Table 1, and the detection results are shown in FIG. 1.

(36) TABLE-US-00001 TABLE 1 PE positive rate statistics Allophycocyanin (APC) Group fluorescent positive rate H929 blank 0.05% H929 + PE 0.01% Negative control 0.05% Positive control 98.09% BCMA-scFv candidate 73.05%

Embodiment 2, Detection of Transduction Rate of CAR Constructed by Humanized Antibody to Cells

(37) Acquisition of Humanized Antibody

(38) Mouse-derived antibodies were humanized. Specifically, a sequence of a light chain CDR1 of each of the humanized antibodies was as shown in SEQ ID NO. 1, an amino acid sequence of a light chain CDR2 was as shown in SEQ ID NO. 2, and a light chain CDR3 was as shown in SEQ ID NO. 3 or SEQ ID NO. 4. A sequence of a heavy chain CDR1 of each of the humanized antibodies was as shown in SEQ ID NO. 5, an amino acid sequence of a heavy chain CDR2 was as shown in SEQ ID NO. 6, and a heavy chain CDR3 was as shown in SEQ ID NO. 7 or SEQ ID NO. 8.

(39) In the case that the above CDR (also known as hypervariable region) remains unchanged, different FRs were also tested, proving that the antibodies formed by different FRs were functional in the case that the above CDR remains unchanged. Specifically, a light chain FR1 could be shown as any one of SEQ ID NO. 9-10, a light chain FR2 could be shown as any one of SEQ ID NO. 11-12, a light chain FR3 could be shown as any one of SEQ ID NO. 13-14, and a light chain FR4 could be shown as any one of SEQ ID NO. 15-16. A heavy chain FR1 could be shown as any one of SEQ ID NO. 17-18, a heavy chain FR2 could be shown as any one of SEQ ID NO. 19-21, a heavy chain FR3 could be shown as any one of SEQ ID NO. 22-24, and a heavy chain FR4 could be shown as any one of SEQ ID NO. 25-26.

(40) Humanized antibodies V0, V1, V2, V3, V4, V6, V7, and V8 were further prepared for testing. Specifically, amino acid sequences of the humanized antibodies V0, V1, V2, V3, V4, V6, V7, and V8 were sequentially shown as SEQ ID NO. 28-35, and coding nucleic acid sequences were sequentially shown as SEQ ID NO. 37-44.

(41) Construction of CAR

(42) A CD8 signal peptide (shown as SEQ ID NO. 45) was ligated in front of the above mouse-derived and humanized antibodies, and a CD8 hinge region+a transmembrane domain (shown as SEQ ID NO. 46), a 4-1BB co-stimulatory structural domain (shown as SEQ ID NO. 47), and an intracellular signal transduction structural domain (shown as SEQ ID NO. 48) were ligated after the mouse-derived antibody, and BCMA-CAR-V0/V1/V2/V3/V4/V6/V7/V8 was obtained for transduction.

(43) That is, the function of CAR was verified in the present disclosure through the following structure: CD8 signal peptide-antibody-CD8 hinge region-CD8 transmembrane domain-4-1BB co-stimulatory structural domain-intracellular signal transduction structural domain.

(44) Preparation of CAR-NK Cells

(45) A third-generation lentiviral packaging system and a four-plasmid system were employed to package three plasmids: pRSV-Rev, pMDLg/pRRE, and pMD2.G, and shuttle pLVX-puro, and HEK293T cells served as virus production cells, at a mixing ratio of pLenti-puro:pRSV-Rev:pMDLg/pRRE:pMD2.G of 2:1:1:1. A lipidosome mirusTransIT-293 (mir2700) acted as a transfection reagent, with a lipidosome-to-DNA ratio of 3:1. After the transfection, supernatant was collected in three days and centrifuged at a high speed of 40000 g for 6 h.

(46) Viruses at an infection concentration of 10 MOI were directly added to a culture medium of NK cells, and at the same time, 0.8 g/mL of polybrene was added, followed by incubating for 6 h. An NK medium containing viruses was removed, and a fresh NK medium was added for continuous culture. The CAR positive rate was determined by a flow cytometry at different time points.

(47) Results Analysis

(48) TABLE-US-00002 TABLE 2 Flow cytometry results of BCMA-CAR expression and stability Numerical order Group Day 2 Day 4 Day 6 Day 8 1 Negative control 0.47% 0.31% 0.27% 0.38% 2 Mouse-derived 81.93% 30.50% 11.29% 10.08% BCMA-CAR 3 Humanized BCMA- 89.83% 19.99% 18.35% 16.38% CAR-V0 4 Humanized BCMA- 72.01% 14.20% 12.83% 10.68% CAR-V1 5 Humanized BCMA- 87.10% 15.45% 14.50% 12.81% CAR-V2 6 Humanized BCMA- 78.23% 24.43% 7.67% 7.16% CAR-V3 7 Humanized BCMA- 74.15% 29.51% 14.30% 13.19% CAR-V4 8 Humanized BCMA- 75.54% 17.01% 14.47% 13.53% CAR-V6 9 Humanized BCMA- 76.82% 27.09% 11.16% 8.85% CAR-V7 10 Humanized BCMA- 76.30% 30.95% 13.87% 11.89% CAR-V8

(49) The flow cytometry results of humanized BCMA-CAR expression and stability are shown in Table 2, showing that BCMA-CAR-V0/V1/V2/V3/V4/V6/V7/V8 can transduce NK cells well, and the transduction rate of each group can reach between 70%-90% on Day 2.

(50) From Day 4, the BCMA-CAR expression tends to be stable, and the difference in the stability of expression between groups is shown. The BCMA-CAR expression in the mouse-derived group decreases significantly to 30.5%, with inconspicuous clustering. The stability of expression in each humanized group is different. In the four groups of BCMA-CAR-V0/V1/V2/V6, the stability of expression is better, with conspicuous cell clustering, and the proportions of CAR-NK cells are 19.99%, 14.2%, 15.45% and 17.01%, respectively. The histogram shows an obvious BCMA-CAR-NK cell cluster forming on the right side.

(51) On Day 6-Day 8, the BCMA-CAR expression in the mouse-derived group further decreases to 11.29%, with inconspicuous CAR-NK cell clustering, while the stability of expression in the five humanized groups of BCMA-CAR-V0/V1/V2/V4/V6 is better, with conspicuous cell clustering and CAR-NK cells accounting for 18.35%, 12.83%, 14.5%, 14.3%, and 14.47%, respectively.

(52) Compared with mouse-derived BCMA-CAR, the humanized BCMA-CAR has a more stable and uniform expression on an NK cell membrane, and can form a clearer group of NK cell clusters (V0/V1/V2/V4/V6) expressing CAR. In the histogram, BCMA-CAR-NK cells are shown as a small cell peak on the right side, and the more conspicuous of this peak is, the more uniform and stable of the CAR expression is.

Embodiment 3, Cell Killing Experiment

(53) BCMA-CAR-NK cells in each group were expanded, cultured, and counted.

(54) A target cell line with green fluorescence: H929-GFP cell was cultured, and counted.

(55) The BCMA-CAR-NK cells and the H929-GFP cells were co-cultured at an effector-target ratio of 1:2.

(56) The proportion of H929-GFP cells in a co-culture system was detected by a flow cytometry at different time points (24 h, and 48 h).

(57) Results Analysis

(58) TABLE-US-00003 TABLE 3 Detection results of cell killing at effector-target ratio of 1:2 24 h 48 h Numerical killing killing order Group 0 h 24 h 48 h ratio ratio 1 Negative control without virus 63.51% 46.93% 29.19% 26.11% 54.04% 2 Mouse-derived BCMA-CAR 67.82% 45.16% 22.78% 33.41% 66.41% 3 Humanized BCMA-CAR-V0 66.02% 30.51% 3.45% 53.79% 94.77% 4 Humanized BCMA-CAR-V1 63.85% 34.48% 5.82% 46.00% 90.88% 5 Humanized BCMA-CAR-V2 66.73% 36.92% 6.37% 44.67% 90.45% 6 Humanized BCMA-CAR-V3 64.90% 36.85% 12.52% 43.22% 80.71% 7 Humanized BCMA-CAR-V4 63.98% 32.17% 4.30% 49.72% 93.28% 8 Humanized BCMA-CAR-V6 69.65% 41.36% 11.01% 40.62% 84.19% 9 Humanized BCMA-CAR-V7 63.57% 30.68% 4.17% 51.74% 93.44% 10 Humanized BCMA-CAR-V8 62.79% 30.40% 4.66% 51.58% 92.58%

(59) The detection results of cell killing at effector-target ratio of 1:2 are shown in Table 3. At 24 h, compared with the proportion of 45.16% of the H929-GFP cells accounting in the mouse-derived BCMA-CAR-NK group, the H929-GFP cells account less in each humanized BCMA-CAR-NK group. The H929-GFP cells in the four groups of BCMA-CAR-V0/V4/V7/V8 account for 30.5%, 32.17%, 30.68%, 30.4%, and 30.4%, respectively.

(60) At 48 h, compared with the proportion of 22.78% of the H929-GFP cells in the mouse-derived BCMA-CAR-NK group, the proportion of the H929-GFP cells in each humanized BCMA-CAR-NK group is significantly decreased. The H929-GFP cells in the four groups of BCMA-CAR-V0/V4/V7/V8 account for 3.45%, 4.3%, 4.17%, and 4.66%, respectively. The histogram shows that, the lower or even disappearing of the small cell peak on the right side, the higher the killing ability.

(61) Compared with the mouse-derived BCMA-CAR-NK cells, the humanized BCMA-CAR-NK cells have a better killing ability to basically remove the tumor cells at 48 h, and show different killing ability in the presence of different humanized modifications. On the whole, the humanized BCMA-CAR-NK cells have a better killing ability than that of the mouse-derived BCMA-CAR-NK cells.