PSMA Antibody and Use Thereof

Abstract

The present disclosure relates to an antibody fragment specifically binding to PSMA, particularly to a single-chain Fv (scfv), a bispecific antibody containing the scFv, and a chimeric antigen receptor (CAR) and the preparation and the use thereof.

Claims

1. An antibody fragment specifically binding to human PSMA, comprising: A) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; and B) a light chain variable region with an amino acid sequence selected from the followings: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 52.

2. The antibody fragment according to claim 1, wherein the antibody fragment is a single-chain Fv.

3. The antibody fragment according to claim 2, wherein the single-chain Fv has an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 54 or SEQ ID NO: 56.

4. A bispecific antibody, the bispecific antibody comprising: 1) a first antigen binding domain specifically binding to PSMA, which is the antibody fragment specifically binding to human PSMA according to claim 1, comprising: A) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; and B) a light chain variable region with an amino acid sequence selected from the followings: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 52, and 2) a second antigen binding domain.

5. The bispecific antibody according to claim 4, wherein the first antigen binding domain is a single-chain Fv, preferably, the single-chain Fv has an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 54 or SEQ ID NO: 56.

6. The bispecific antibody according to claim 4, wherein the second antigen binding domain binds to a specific acceptor molecule on T cells, and wherein the specific acceptor molecule on T cells is preferably CD3.

7. The bispecific antibody according to claim 6, wherein the second antigen binding domain specifically binding to CD3 has a heavy chain variable region as shown in SEQ ID NO: 40, and a light chain variable region as shown in SEQ ID NO: 42.

8. The bispecific antibody according to claim 4, comprising a group consisting of two polypeptide chains bindable to CD3 and PMSA: SEQ ID NO: 44 and SEQ ID NO: 46; SEQ ID NO: 58 and SEQ ID NO: 60; SEQ ID NO: 62 and SEQ ID NO: 64; SEQ ID NO: 58 and SEQ ID NO: 66; and SEQ ID NO: 68 and SEQ ID NO: 64.

9. A chimeric antigen receptor, comprising a single-chain Fv, specifically binding to PSMA, a membrane spanning domain and an intracellular domain, wherein the single-chain Fv, is the antibody fragment specifically binding to human PSMA according to claim 1, comprising: A) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; and B) a light chain variable region with an amino acid sequence selected from the following: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 52.

10. The chimeric antigen receptor according to claim 9, having an amino acid sequence as shown in SEQ ID NO: 50.

11. A polynucleotide, encoding the antibody fragment according to claim 1.

12. The polynucleotide according to claim 11, having a nucleotide sequence as shown in SEQ ID NOS: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 43, 45, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67.

13. A vector, comprising the polynucleotide according to claim 11.

14. A cell, comprising the polynucleotide according to claim 11.

15. The cell according to claim 14, wherein the cell is a T cell.

16. A composition, comprising the antibody fragment according to claim 1.

17. The composition of claim 16, further comprising a pharmaceutically acceptable carrier.

18. The composition of claim 16, used for chimeric antigen receptor T-cell immunotherapy.

19. A method for treating a subject suffering from a disease associated with PSMA expression, comprising administering to the subject an effective amount of the composition according to claim 16.

20. A method for diagnosing a disease associated with PSMA expression in a mammal, wherein the method comprises: using the antibody fragment according to claim 1 to detect the binding to human PSMA in a tissue sample separated from the mammal, and thus the specific binding of the antibody fragment to the human PSMA in the tissue sample is indicative of a disease associated with PSMA expression in the mammal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting the present application are used to provide a further understanding to the prevent disclosure. The exemplary embodiments and descriptions of the prevent disclosure are intended to explain the prevent disclosure, but not be construed as improper limitation to this disclosure. In the drawings:

[0029] FIG. 1 shows the ELISA result of aPSMA LH RM and RM2 monoclonal phage, wherein RM denotes the first round of panning, and RM2 denotes the second round of panning.

[0030] FIG. 2 shows the ELISA result of aPSMA HL monoclonal phage.

[0031] FIG. 3A shows the ELISA binding of an aPSMA scfv antibody with amino acid mutation at position 42 of VL to PSMA-his; and FIG. 3B shows the binding of a PSMA-targeted scfv-Fc antibody (on the 42nd amino acid mutation of a VL) to LnCap cells as detected by flow cytometry.

[0032] FIG. 4 shows the binding of bispecific antibodies to Jurkat T cells as detected by flow cytometry.

[0033] FIG. 5 shows the binding of bispecific antibodies to LnCap cells as detected by flow cytometry.

[0034] FIG. 6 shows the cytotoxicity of the bispecific antibody on LnCap cells by recruiting human T cells.

[0035] FIG. 7 shows the PK curve of the bispecific antibody in mice.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Examples

[0036] It should be noted that, in the case of no conflict, the embodiments of the present application are merely examples for illustration, and are not intended to limit the present disclosure in any way.

EXAMPLES

Example 1 Phage Library Screening

[0037] aPSMA HL (SEQ ID NO: 37) and aPSMA LH (SEQ ID NO: 39) were synthesized, and respectively cloned into the phagemid vector, subjected to random mutation with GeneMorph II random mutagenesis (Agilent), and transformed into XL1-blue competent cells. When OD600=0.6-0.8, the transformed XL1-blue cells were infected with an helper phage M13K07. After 12 hours, the phage was collected and the phage titer (phage library capacity) was measured.

[0038] A microwell plate was coated with Fc-PSMA (300 ng/well) and blocked by BSA, the preceding two phage libraries were subjected to 2-3 rounds of panning and affinity detection, and single clones were picked randomly from the panned plates and sent for sequencing. Sequencing result analysis was performed. The single clones with sequence enrichment or mutations in the CDR regions were subjected to phage packaging and affinity detection. The detection result was shown in FIG. 1.

[0039] FIG. 1 shows the affinity detection result of the aPSMA LH RM and aPSMA LH RM2 mutant libraries. It can be seen from the figure that except for aPSMA LH RM-R13 and aPSMA LH RM2-R15, the affinity of other clones is much higher than that of the original sequence; FIG. 2 shows the affinity detection result of the aPSMA HL RM mutant library, and only aPSMA HL RM-R1/R10/R15 has a higher affinity.

[0040] After analyzing the above-mentioned sequences with higher affinity, it is found that in the aPSMA LH RM, aPSMA LH RM2 and aPSMA HL RM mutant libraries, the clones with higher affinity have a same amino acid mutation at the 42nd position of VL.

TABLE-US-00001 TABLE 1 Sequence Listing Nucleotide Amino acid sequence sequence Description SEQ ID NO: SEQ ID NO: aPSMA VH VH 1 2 aPSMA VL VL 3 4 aPSMA VL (L42) VL 5 6 aPSMA VL (H42) VL 7 8 aPSMA VL (T42) VL 9 10 aPSMA VL (S42) VL 11 12 aPSMA VL (Q42) VL 13 14 aPSMA HL scfv 15 16 aPSMA LH scfv 17 18 aPSMA HL (L42) scfv 19 20 aPSMA HL (H42) scfv 21 22 aPSMA HL (T42) scfv 23 24 aPSMA HL (S42) scfv 25 26 aPSMA HL (Q42) scfv 27 28 aPSMA LH (Q42) Scfv 29 30 aPSMA LH (S42) Scfv 31 32 aPSMA LH (T42) Scfv 33 34 aPSMA LH (H42) Scfv 35 36 aPSMA LH (L42) Scfv 37 38 aCD3 VH VH 39 40 aCD3 VL VL 41 42 aCD3-aPSMA Chain-1 43 44 Chain-2 45 46 aCD3 Chain-1 43 44 Chain-2 47 48 aPSMA CAR PSMA-targeted 49 50 chimeric antigen receptor (CAR) aPSMA VL (K42) VL 51 52 aPSMA HL (K42) scfv 53 54 aPSMA LH (K42) scfv 55 56 aCD3-aPSMA.2 Chain-1 / 57 Chain-2 / 58 aCD3-aPSMA.3 Chain-1 / 59 Chain-2 / 60 aCD3-aPSMA.4 Chain-1 / 57 Chain-2 / 61 aCD3-aPSMA.5 Chain-1 / 62 Chain-2 / 60

Example 2 Detection of the Binding Affinity of PSMA-Targeted Antibody

[0041] 2.1 Cloning and Expression of PSMA-Targeted Scfv-Fc Antibody

[0042] aPSMA HL (SEQ ID NO: 15), aPSMA LH (SEQ ID NO: 17), aPSMA HL (L42) (SEQ ID NO: 19), aPSMA HL (H42) (SEQ ID NO: 21), aPSMA HL (T42) (SEQ ID NO: 23), aPSMA HL (S42) (SEQ ID NO: 25), aPSMA HL (Q42) (SEQ ID NO: 27) and aPSMA LH (Q42) (SEQ ID NO: 29) were cloned into the N-terminal of pFuse-hIgG1-Fc2 by traditional enzyme digestion and ligation methods, and subjected to sequencing for verification.

[0043] The constructed eukaryotic expression vector was transiently transfected into FreeStyle HEK293 cells (ThermoFisher), respectively: 28 ml FreeStyle HEK293 (3×10.sup.7 cell/ml) were seeded into a 125 ml cell culture flask, plasmids were diluted with 1 ml Opti-MEM (Invitrogen) and then added to 1 ml Opti-MEM containing 60 μl 293 Fectin (Invitrogen, Inc). After incubating at room temperature for 30 min, the plasmid-293fectin mixture was added to the cell culture, and then incubated at 125 rpm, 37° C., 5% CO2. Cell culture supernatant was collected at 48 h and 96 h after transfection, respectively, and purified by Protein A Resin (Genscript) according to the manufacturer's instruction. The purified proteins were analyzed by SDS-PAGE.

[0044] 2.2 Affinity Detection of PSMA-Targeted Scfv-Fc Antibody Against PSMA-his

[0045] PSMA-His(Acro) (100 ng/well) was coated in a 96-well plate and incubated at 4° C. overnight. After blocked by PBST (0.5% Tween-20 in PBS) containing 2% skim milk powder for 1 h at room temperature, gradiently diluted scfv-Fc antibodies were added and incubated for 2 h at room temperature. After washed by PBST containing 2% skim milk powder for 4-5 times, anti-human Fc-HRP secondary antibody was added for incubation for 1 h at room temperature. After washed by PBST containing 2% skim milk powder for 4-5 times, TMB reagent (BioLegend, Cat. 421101) was added for color development, and readings at 650 nm (not termination) or 450 nm (termination) were recorded. Data was analyzed by nonlinear regression with specific binding model by Prizm Graphpad software.

[0046] As shown in FIG. 3A, the aPSMA LH-Fc or aPSMA LH-Fc with wildtype VL has weak affinity agonist PSMA-his, while aPMSA HL (Q42)-Fc or aPSMA LH (Q42)-Fc with 42.sup.nd-position amino acid mutation in VL has enhanced affinity to PSMA-his.

[0047] 2.3 Binding Analysis of the PSMA-Targeted Scfv-Fc Antibody to LnCap Cells by Flow Cytometry

[0048] LnCap cells were cultured in RPMI 1640 medium containing 10% FBS in a 5% CO2 incubator. 2*10.sup.5 cells were washed for 3 times by pre-cooled PBS. After blocked by 2% FBS diluted by PBS, gradiently diluted antibodies (100 nM, gradiently diluted for 3 folds successively) were added and incubated for 1 hour at 4° C. Unbonded antibodies were washed away by 2% FBS. Mouse anti-human IgG Fc-APC (southern biotech) was added for 1 hour incubation at 4° C., and then subjected to flow cytometry after washed by 2% FBS. As shown in FIG. 3, the PSMA-targeted scfv-Fc with 42.sup.nd-position amino acid mutation in VL has higher affinity against PSMA on the surface of LnCap cells, which is 190-220 times higher than that of PSMA-targeted scfv-Fc with wild type VL.

Example 3 Bispecific Antibody Targeting PSMA and CD3

[0049] 3.1 Construction and Expression of Bispecific Antibodies Targeting PSMA and CD3

[0050] Standard molecular biological methods were used to construct chain-1 and chain-2 containing different aCD3-aPMSA bispecific antibody as shown in Table 1 Sequence Listing, and subjected to sequencing for verification.

[0051] Eukaryotic expression vectors, containing the above-mentioned two chains, were verified by sequencing and transiently co-transfected into FreeStyle HEK293 cells (ThermoFisher) respectively: 28 ml FreeStyle HEK293 (3×10.sup.7 cell/ml) were seeded in a 125 ml cell culture flask, plasmids were diluted by 1 ml Opti-MEM (Invitrogen), and then added to 1 ml Opti-MEM containing 60 μl 293 Fectin (Invitrogen, Inc). After incubating at room temperature for 30 min, and the plasmid-293fectin mixture was added to the cell culture, and incubated at 125 rpm, 372, 5% CO2. Cell culture supernatant was collected at 48 hours and 96 hours after transfection, respectively, and purified by Protein A Resin (Genscript) according to the manufacturer's instruction, The purified proteins were analyzed by SDS-PAGE detection, and the yield was calculated. The results showed that the yield of aCD3-aPSMA bispecific antibody (15-20 mg/L) with 42.sup.nd-position mutation in VL is significantly higher than that of aCD3-aPSMA bispecific antibody (2.5-5 mg/L) with a wild type VL.

[0052] 3.2 In Vitro Activity Evaluation of Bispecific Antibody Targeting PSMA and CD3

[0053] 3.2.1 Flow Cytometry Analysis on Binding to Jurkat (CD3+) Cells

[0054] Jurkat cells were cultured in RPMI 1640 medium containing 10% FBS in a 5% CO2 incubator. 2×10.sup.5 cells were washed for 3 times with pre-cooled PBS. After blocked by PBS containing 2% FBS, bispecific antibody at concentrations of 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM or 0.4 nM was added and incubated for 1 hour at 4° C. Unbonded antibodies were washed by PBS containing 2% FBS, and mouse anti-human IgG Fc-APC (southern biotech) secondary antibody was added for 1 hour incubation at 4° C. After washing 3 times by PBS containing 2% FBS, flow cytometry was performed. As shown in FIG. 4, the bispecific antibody could effectively bind to Jurkat (CD3+) cells.

[0055] 3.2.2 Flow Cytometry Analysis on Binding to LnCap (PSMA+) Cells

[0056] LnCap cells were cultured in RPMI 1640 medium containing 10% FBS in a 5% CO2 incubator. 2*10.sup.5 cells were washed for 3 times by pre-cooled PBS. After blocked by 2% FBS diluted by PBS, bispecific antibody at concentrations of 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM or 0.4 nM was added and incubated for 1 hour at 4° C. Unbonded antibodies were washed by 2% FBS, and mouse anti-human IgG Fc-APC (southern biotech) was added for incubation for 1 hour at 4° C. After washing by 2% FBS, flow cytometry was performed. As shown in FIG. 5, the bispecific antibody could effectively bind to LnCap cells.

TABLE-US-00002 TABLE 2 EC.sub.50 of the bispecific antibody binding to Jurkat and LnCap cells EC.sub.50 (nM) aCD3-aPSMA aCD3 aPSMA Jurkat 0.51 0.51 NA LnCap 0.05 NA 0.01

[0057] 3.2.3 Detection of Human T Cell-Dependent Cytotoxicity

[0058] Peripheral blood was collected from healthy volunteer donor and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (GE Healthcare) gradient centrifugation. The isolated PBMC was washed and incubated in RPMI medium containing 10% (vol/vol) FBS for 1 h to remove adherent cells. After activation by anti-CD3 and anti-CD28, cells were expanded in IL-2 for 10 days.

[0059] 1×10.sup.4 LnCap cells (a RPMI medium containing 5% FBS) were mixed with activated PBMCs at a ratio of 1:5, and then incubated with bispecific antibody at different dilutions for 16 h at 37° C. The content of lactic dehydrogenase (LDH) in supernatant was detected by Cytotox-96 nonradioactive cytotoxicity assay kit (Promega). The value obtained from wells only containing target cells treated by the lysate was the Target Cell Maximum LDH Release Control; the value obtained from wells containing both effector cells and target cells treated by PBS (vehicle) was Spontaneous LDH Release Control. Color development was performed as the manufacturer's instruction, and absorbance at 490 nm was recorded in BMG LAB TECH CLARIOstar microplate reader (Bio-Gene Technology Ltd.). cytotoxicity was calculated as follows: % cytotoxicity=(absorbance experimental−absorbance spontaneous average)/(absorbance maximum killing average−absorbance spontaneous average).

[0060] As shown in FIG. 6, aCD3-aPSMA bispecific antibody could effectively recruit T cells to exert killing effects on target cells LnCap when compared with the control aPSMA.

TABLE-US-00003 TABLE 3 Killing of the bispecific antibody on the target cell LnCap via calling on PBMC aCD3-aPSMA aCD3 aPSMA LDH Killing EC.sub.50 (pM) 184.4 712.3 NA LDH max killing (pM) 79.79 42.76 NA

[0061] 3.3 PK Study on the PSMA and CD3-Targeted Bispecific Antibody

[0062] The bispecific antibody was intraperitoneally injected (I.P.) into C57 female mice (3 pieces/group, at a dose of 10 mg/kg). Whole blood was collected 30 min, 1 h, 2 h, 4 h, 10 h, 24 h, 3 d, 5 d, 7 d, and 14 d after injection. After centrifugation, plasma was collected and kept at −80° C. for further use. The bispecific antibody in the plasma was detected with reference to the Example 2.2. As shown in FIG. 7, the half-life of the bispecific antibody in mice is about 5 d.