CN115197329A - A kind of 4-1BB agonist and its preparation method and application - Google Patents

Abstract

The invention discloses a 4-1BB agonist, which is a fusion protein comprising an anti-FAP antibody and 4-1BBL, wherein the anti-FAP antibody is scFv comprising VL with an amino acid sequence shown as SEQ ID NO. 1 or at least 90% of identity with the SEQ ID NO. 1, and VH with an amino acid sequence shown as SEQ ID NO. 3 or at least 90% of identity with the SEQ ID NO. 3. Also disclosed are an isolated nucleic acid encoding the 4-1BB agonist, a recombinant expression vector, a transformant or a pharmaceutical composition containing the same, and use thereof in preparing a medicament for treating tumors. The invention realizes the local immune activation of the tumor; the mediated activation caused by Fc fusion is avoided, and the molecule is safer; the monovalent 4-1BBL is also used, so that the risk of production and immunogenicity is avoided.

Description

A kind of 4-1BB agonist and its preparation method and application

technical field

The invention relates to the field of biomedicine, in particular to a tumor antigen-dependent 4-1BB agonist and a preparation method and application thereof.

Background technique

4-1BB is an inducible receptor mainly expressed on activated T cells. Using 4-1BB antibody to activate the 4-1BB pathway can promote T cell proliferation, cytokine secretion, and CD8 T cell survival. Based on this, many antibodies targeting it are in development, such as the earliest BMS-663513, but the clinical trial was terminated due to severe hepatotoxicity caused by high doses. A safe and effective 4-1BB agonist for tumor targeting. Various antibody formats and different target combinations have also appeared (M.A.Lakins, et al. Clin Cancer Res, 26(2020) 4154-4167; M.J. Hinner, et al. Clin Cancer Res, 25(2019) 5878-5889; F.C. Claus C, et al. SciTransl Med, 11(496)(2019)), mainly divided into combinations with tumor-associated antigens and immunosuppressants. Dual antibodies against tumor-associated antigens are at the forefront, such as RG7827, PRS-343, etc., as well as ALG.APV-527, which has just been incorporated into the clinic. Although RG7827 has a good effect, its expression level is very low, the production cost is expensive, and the asymmetric structure has a certain risk of mismatch. Up to now, there is still no 4-1BB antibody drug on the market. In the form of bispecific antibody fusion protein, 4-1BB aggregation can be activated only when FAP is highly expressed, avoiding off-target toxicity to a great extent, and relatively It is good to avoid the problem of low expression level of double antibody at present, and it has certain development potential. Failed to make good use of its activation effect. Therefore, it is necessary to develop tumor-targeted 4-1BB agonists that are not only effective but also selective antibodies or their fusion proteins.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a tumor antigen-dependent 4-1BB agonist in order to overcome the defect of the lack of an effective 4-1BB agonist in the prior art.

To solve the above technical problems, one of the technical solutions of the present invention is to provide a 4-1BB agonist, which is a fusion protein comprising an anti-FAP antibody and 4-1BBL, that is, an anti-FAP antibody-4-1BBL.

In a specific embodiment, the anti-FAP antibody is an scFv comprising an amino acid sequence as shown in SEQ ID NO: 1 or at least 90%, 91%, 92%, 93% identity with SEQ ID NO: 1 %, 94%, 95%, 96%, 97%, 98% or 99% of VL, and the amino acid sequence shown in SEQ ID NO:3 or at least 90%, 91% identical to SEQ ID NO:3 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% VH.

In a specific embodiment, described 4-1BBL is full-length or truncated 4-1BBL, and its amino acid sequence is shown as SEQ ID NO:5 and SEQ ID NO:6 respectively, or with SEQ ID NO:5 and SEQ ID NO:6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Preferably, the nucleotide sequences encoding VL, VH and truncated 4-1BBL of the scFv are shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 7, respectively, or the same as SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 7, respectively. ID NO:4 and SEQ ID NO:7 are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In a specific embodiment, the VL and VH of the anti-FAP antibody are linked by a linker or a chemical bond; and/or, the anti-FAP antibody and 4-1BBL are linked by a linker or a chemical bond. Preferably, the linker is (G ₄ S) ₃ or (G ₄ S) ₂ . More preferably, the VL and VH of the anti-FAP antibody are linked via (G ₄ S) ₃ and the anti-FAP antibody and 4-1BBL are linked via (G ₄ S) ₂ .

In a specific embodiment, the amino acid sequence of the 4-1BB agonist is shown in SEQ ID NO:8.

In order to solve the above technical problems, the second technical solution of the present invention is to provide an isolated nucleic acid encoding the 4-1BB agonist as described above. Preferably, the isolated nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:9.

In order to solve the above technical problems, the third technical solution of the present invention is to provide a recombinant expression vector, the recombinant expression vector comprising the isolated nucleic acid as described in the second technical solution.

In order to solve the above technical problem, the fourth technical solution of the present invention is to provide a transformant, the transformant comprising the recombinant expression vector as described in the third technical solution. Preferably, the starting cells of the transformants are prokaryotic cells or eukaryotic cells. More preferably, the eukaryotic cells are mammalian cells, such as CHOK1 cells.

In order to solve the above-mentioned technical problems, the fifth technical solution of the present invention is to provide a method for preparing a 4-1BB agonist, the method comprising the following steps: culturing the transformant as described in the fourth technical solution in a culture medium, that is, have to. Preferably, the medium is CD-CHO medium.

In order to solve the above technical problems, the sixth technical solution of the present invention is to provide a pharmaceutical composition, the pharmaceutical composition comprising the 4-1BB agonist described in one of the above technical solutions.

In order to solve the above technical problems, the seventh technical solution of the present invention is to provide a 4-1BB agonist according to the one of the technical solutions of the present invention, the isolated nucleic acid described in the second technical solution, and the third technical solution. Use of the recombinant expression vector, the transformant described in technical solution 4, or the pharmaceutical composition described in technical solution 6 of the present invention in the preparation of a medicament for treating tumors. Preferably, the tumor is one of solid tumors. More preferably, the solid tumors include: colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, lung cancer, cervical cancer and ovarian cancer.

The positive progressive effect of the present invention is:

Anti-FAP antibody was used in series with monovalent 4-1BBL to achieve local immune activation of the tumor;

No Fc fusion is used to avoid Cross-linking-mediated activation due to Fc receptors, and the molecule is safer.

Multivalent 4-1BBL was not used, avoiding its production and immunogenicity risks.

Description of drawings

Figure 1 shows the results of the preparation test for the construction of the expression vector of the present invention.

Figure 2 is the SDS-PAGE and SEC-HPLC test results of the protein expression and purification of the present invention.

Fig. 3 is the test result of the binding ability of the anti-FAP antibody of the present invention and 4-1BBL.

FIG. 4 is the test result of using BLI experiment to detect the affinity strength of human FAP (hFAP) and human 4-1BB (h4-1BB) in the present invention.

Figure 5 shows the test results of the double binding experiment.

Figure 6 is a schematic diagram of the anti-FAP antibody-4-1BBL fusion protein constructed by the present invention.

Figure 7 shows cytokine production using agonists of the present invention in the absence of FAP.

Figure 8 shows cytokine production using agonists of the present invention in the presence of FAP.

Detailed ways

The present invention will be further described below by means of embodiments, and will be described more clearly and completely in conjunction with the accompanying drawings, but the present invention is not limited to the scope of the described embodiments. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.

The following are the relevant amino acid sequences and nucleotide sequences of the present invention:

The amino acid sequence of the VL of the anti-FAP antibody (wherein, the underline indicates LCDR1, LCDR2 and LCDR3 in order) is:

EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGCGTKVEIK ( SEQ ID NO:1)

The nucleotide sequence encoding the VL of the anti-FAP antibody is:

GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCTTCTCAGAGCGTGAGCAGAAGCTACCTGGCCTGGTATCAGCAGAAGCCTGGCCAAGCCCCTAGACTGCTGATCATCGGCGCTAGCACAAGAGCCACCGGCATCCCTGACAGATTCAGCGGTAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGTCTGGAGCCTGAGGACTTCGCCGTGTATTATTGTCAGCAAGGCCAAGTGATCCCTCCTACCTTCGGCTGCGGCACGAAAGTCGAGATCAAA(SEQ ID NO:2)

The amino acid sequence of the VH of the anti-FAP antibody (wherein, underlining represents HCDR1, HCDR2 and HCDR3 in order) is:

EVQLLESGGGLVQPGGSLRLSCAASGFTFS SHAMS WVRQAPGKCLEWVS AIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS ( SEQ ID NO:3)

The nucleotide sequence encoding the VH of the anti-FAP antibody is:

GAAGTGCAGCTGTTAGAGAGCGGCGGGGGTCTCGTGCAGCCTGGTGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAGCCACGCCATGAGCTGGGTGAGACAAGCCCCTGGCAAGTGCCTGGAGTGGGTGAGCGCCATCTGGGCTAGCGGCGAGCAGTACTACGCCGACAGCGTGAAGGGCAGATTCACTATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTATTGTGCCAAAGGCTGGCTGGGCAACTTCGATTATTGGGGACAAGGCACTTTGGTGACCGTTTCGTCG(SEQ ID NO:4)

The full-length amino acid sequence of 4-1BBL is:

MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF5)

The amino acid sequence of ECD (64-254) of 4-1BBL is:

SAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:6)

The nucleotide sequence of ECD(64-254) encoding 4-1BBL is:

TCCGCTGCTAGCCCTAGACTGAGAGAGGGCCCTGAGCTGAGCCCTGACGACCCTGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGATGGTCCTCTGAGCTGGTACAGCGACCCGGGACTGGCCGGTGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTCCAACTGGAGCTTCGTCGAGTAGTGGCCGGCGAGGGCAGCGGGTCTGTTAGCCTAGCACTCCACTTGCAACCTCTGAGAAGTGCTGCCGGGGCGGCAGCCCTTGCGCTTACCGTGGACCTGCCTCCTGCTAGCAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGACAGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCTGAGATCCCGGCTGGTCTGCCTAGTCCTAGAAGCGAG(SEQ ID NO:7)

The amino acid sequence of the anti-FAP antibody-4-1BBL recombinant protein is:

EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGCGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKCLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSGGGGSGGGGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEHHHHHH(SEQID NO:8)

The nucleotide sequence encoding the anti-FAP antibody-4-1BBL recombinant protein is:

GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCTTCTCAGAGCGTGAGCAGAAGCTACCTGGCCTGGTATCAGCAGAAGCCTGGCCAAGCCCCTAGACTGCTGATCATCGGCGCTAGCACAAGAGCCACCGGCATCCCTGACAGATTCAGCGGTAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCCGTCTGGAGCCTGAGGACTTCGCCGTGTATTATTGTCAGCAAGGCCAAGTGATCCCTCCTACCTTCGGCTGCGGCACGAAAGTCGAGATCAAAGGTGGAGGCGGTTCCGGGGGAGGTGGCAGTGGGGGGGGCGGTTCTGAAGTGCAGCTGTTAGAGAGCGGCGGGGGTCTCGTGCAGCCTGGTGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAGCCACGCCATGAGCTGGGTGAGACAAGCCCCTGGCAAGTGCCTGGAGTGGGTGAGCGCCATCTGGGCTAGCGGCGAGCAGTACTACGCCGACAGCGTGAAGGGCAGATTCACTATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTATTGTGCCAAAGGCTGGCTGGGCAACTTCGATTATTGGGGACAAGGCACTTTGGTGACCGTTTCGTCGGGGGGGGGAGGGAGCGGAGGAGGCGGCTCCGCTGCTAGCCCTAGACTGAGAGAGGGCCCTGAGCTGAGCCCTGACGACCCTGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGATGGTCCTCTGAGCTGGTACAGCGACCCGGGACTGGCCGGTGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTCCAACTGGAGC TTCGTCGAGTAGTGGCCGGCGAGGGCAGCGGGTCTGTTAGCCTAGCACTCCACTTGCAACCTCTGAGAAGTGCTGCCGGGGCGGCAGCCCTTGCGCTTACCGTGGACCTGCCTCCTGCTAGCAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGACAGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCTGAGATCCCGGCTGGTCTGCCTAGTCCTAGAAGCGAGCACCACCACCACCACCAC(SEQ ID NO:9)

Example 1 Expression vector construction, protein expression and purification

The schematic diagram of the anti-FAP antibody-4-1BBL constructed by the present invention is shown in FIG. 6 , which are the VL, VH and truncated 4-1BBL of the anti-FAP antibody from the N-terminus to the C-terminus.

1.1 Expression vector construction and protein expression

Preparation: Entrust Jinweizhi to synthesize the sequence (SEQ ID NO: 9); then clone it into the pKS001 vector with EcoRI and HindIII, and the enzyme digestion identification is shown in Figure 1.

The cells were electroporated into suspended CHOK1 cells, conditions: 40μg plasmid, 2×10^7 cells, a total of 800μL in CD-CHO medium, electroporation conditions: 300V, 900μF, 4mm electroporation cup. Then, MSX was added to screen to obtain stable transfected cells, and the culture supernatant was obtained by fed-batch culture for 14 days, filtered, and purified by AKTA pure nickel column to obtain the final protein product.

1.2 Protein purification

Take the purified protein sample, the loading amount is about 1 μg, and carry out SDS-PAGE in reducing and non-reducing state, 120V, 80min, room temperature for 40min staining, after double-distilled water for multiple decolorization, take a picture as shown in Figure 2A .

Take the purified protein, use PBS as the mobile phase, and use BioCore SEC-300 (Nanochrom) on Agilent1260 HPLC to analyze the protein purity as shown in B of Figure 2. It can be seen from the purified size exclusion chromatography results that the antibody fusion protein has a main peak. Single, more than 90% pure after one-step purification.

Example 2 Analysis of the binding ability of anti-FAP antibody-4-1BBL

2.1 Anti-FAP antibody-4-1BBL binding ability

step:

1. Dilute hFAP and h4-1BB in PBS to make the final concentration 2 μg/mL, and coat 100 μL/well on the microtiter plate overnight at 4°C.

2. Discard the liquid in the plate, add 200 μL/well of washing solution, and wash three times.

3. Add 200 μL/well of blocking solution, RT for 1 h.

4. Discard the liquid in the plate, add 200 μL/well of washing solution, and wash three times.

5. Dilute the fusion protein in the diluent, take 90nM as the highest concentration, dilute 3 times, set a total of 12 concentration gradients, and add the diluted fusion protein correspondingly, 100μL/well, RT for 1h.

6. Discard the liquid in the plate, add 200 μL/well of washing solution, and wash three times.

7. Add HRP-anti-His secondary antibody (abcam ab1187) (1:20000 dilution in diluent), 100 μL/well, RT1h.

8. Discard the liquid in the plate, add 200 μL/well of washing solution, and wash three times.

9. Add 100 μL/well of TMB mixture, and react at RT in the dark for 3-6 minutes. The actual reaction time is determined by the specific observation of the color.

10. Add 50 μL/well of stop solution to stop the reaction, shake the plate slightly to mix the liquid in the well, and immediately measure the OD450 on a microplate reader.

Among them, cleaning solution: 0.1% PBST

Blocking solution: 1% BSA + 0.05% Tween-20 + 1% goat serum in PBS

Diluent: 1%BSA+0.01%Tween-20 in PBS

Stop Solution: 1M HCl

A of Figure 3 is the test result of protein binding to hFAP, and B of Figure 3 is the test result of protein binding to h4-1BB. The results show that the EC ₅₀ values of protein binding to hFAP and protein binding to h4-1BB are 0.17nM respectively. , 0.1046nM.

2.2 Detection of affinity

step:

1. Soak the anti-hFc probe in K Buffer to pre-wet. (K Buffer: 0.1% BSA+0.02% T-20 in PBS; Probe: Probe Life PL168-160003).

2. Dilute hFAP and h4-1BB in K Buffer to 10μg/mL, and add hFAP and h4-1BB respectively.

3. Take 100nM as the highest concentration, dilute the protein sample to 6.25nM, and bind for 120s.

4. Dissociate for 180s.

Among them, K Buffer: 0.1%BSA+0.02%T-20 in PBS. A in Figure 4 is the result of affinity determination with hFAP, and B in Figure 4 is the result of binding with h4-1BB. The affinities of hFAP and h4-1BB are 0.758nM and 0.306nM, respectively.

Add anti-FAP antibody-4-1BBL (100nM) for 120s, wash off the unbound part with buffer (same K Buffer, the formula is as described above) for 120s, bind hFAP (10μg/mL), and then directly bind h4-1BB (10μg /mL), each 120s, dissociation 180s. Among them, the buffer is 0.1%BSA+0.02%Tween-20 in PBS.

2.3 Detection of double binding

step:

1. Soak the anti-His probe in K Buffer to pre-wet.

2. Add anti-FAP antibody-4-1BBL for 120s, and wash off the unbound part for 120s.

3. Bind hFAP, then directly bind h4-1BB, each for 120s, and dissociate for 180s.

Figure 5 shows the results of the double binding experiment. The results show that while binding to hFAP, the binding to h4-1BB will not be affected, and the anti-FAP antibody-4-1BBL can simultaneously bind to the corresponding two targets.

Example 3FAP-dependent T cell activation assay (including HEK293-hFAP cell construction)

3.1 Construction of HEK293-hFAP cells

1. The synthetic hFAP sequence was ligated into the pcDNA3.1-hygro vector.

2. The optimal screening concentration of hygromycin (Invitrogen-10687010) for HEK293 cells (Nanjing Kebai, CBP60661) is 80 μg/mL.

3. Transfect hFAP into HEK293: add 8 μL Lipo2000 to 200 μL OptiPRO (OptiPRO ^™ SFM), 2 μg plasmid to 200 μL OptiPRO, mix well, incubate at room temperature for 10 min, add HEK293 cells, and place in a 37°C incubator.

4. After 48 hours, the cells were passed out at a ratio of 1/3, and 80 μg/mL of hygromycin was added for screening. After 14 days of screening, HEK293-hFAP cell line was obtained.

3.2 FAP-dependent T cell activation assay

HEK293 cells and HEK293-hFAP cells were plated in 96-well plates at 20,000/well (100 μL/well), and duplicate wells were set up to allow cells to adhere. Anti-FAP antibody-4-1BBL was diluted to a final concentration of 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM, 0 nM, 100 μL/well, and 10 μg/mL of OKT3 antibody (BD) was added to each well. Biosciences-566685), add hPBMC, 100,000/well, 100 μL/well, incubate at 37°C for 48 hours, then centrifuge to take the supernatant, and test samples for IFN-γ and IL-2 for 60 times respectively and 2-fold dilution, using a pre-coated ELISA kit for detection, the operation is carried out according to the instructions, and the final data processing is shown in Figure 7;

A of Figure 7 shows that in the absence of FAP, ie, wild-type HEK293, different concentrations of anti-FAP antibody-4-1BBL hardly stimulate the production of IFN-γ, while in the case of HEK293-hFAP, the release of IFN-γ Concentration-dependent with anti-FAP antibody-4-1BBL; Figure 7B shows that in the absence of FAP, that is, wild-type HEK293, different concentrations of anti-FAP antibody-4-1BBL did not stimulate the production of IL-2, while In the case of HEK293-hFAP, the release of IL-2 was concentration-dependent with the anti-FAP antibody-4-1BBL.

A of Figure 8 shows that in the presence of FAP, the release of IFN-γ is positively correlated with the concentration of anti-FAP antibody-4-1BBL, and is stronger than that of Urelumab at the same concentration, and IFN-γ is not activated in the absence of FAP. However, Urelumab activation is independent of the presence or absence of FAP, and requires a higher concentration to produce a significant activation effect; Figure 8B shows that the anti-FAP antibody-4-1BBL stimulates the production of IL-2 in a concentration-dependent manner in the case of HEK293-hFAP, while in the case of HEK293-hFAP Under HEK293 conditions, there is no activation effect. The stimulatory effect of Urelumab was independent of FAP and was weaker than that of anti-FAP antibody-4-1BBL.

sequence listing

<110> Jingyuan Biomedicine (Suzhou) Co., Ltd.

<120> A kind of 4-1BB agonist and its preparation method and application

<130> P21012096C

<160> 9

<170> PatentIn version 3.5

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Ser Ala Ile Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

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Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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cctggcaagt gcctggagtg ggtgagcgcc atctgggcta gcggcgagca gtactacgcc 180

gacagcgtga agggcagatt cactatcagc cgggacaaca gcaagaacac cctgtacctg 240

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Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro

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Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

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Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

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Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

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Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

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Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

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Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe

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Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

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Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

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Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

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Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

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Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

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Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

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Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

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Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

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Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

65 70 75 80

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

85 90 95

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

100 105 110

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

115 120 125

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

130 135 140

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

145 150 155 160

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

165 170 175

Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

180 185 190

<210> 7

<211> 573

<212> DNA

<213> Artificial Sequence

<220>

<223> ECD of 4-1BBL (64-254)

<400> 7

tccgctgcta gccctagact gagagagggc cctgagctga gccctgacga ccctgccggc 60

ctgctggacc tgagacaagg catgttcgct cagctggtgg ctcagaacgt gctgctgatc 120

gatggtcctc tgagctggta cagcgacccg ggactggccg gtgtgagcct gaccggcggc 180

ctgagctaca aggaggacac caaggagctg gtggtggcca aggccggcgt gtactacgtg 240

ttcttccaac tggagcttcg tcgagtagtg gccggcgagg gcagcgggtc tgttagccta 300

gcactccact tgcaacctct gagaagtgct gccggggcgg cagcccttgc gcttaccgtg 360

gacctgcctc ctgctagcag cgaggctaga aacagcgcct tcggcttcca aggcagactg 420

ctgcacctga gcgccggaca gagactgggc gtgcacctgc acaccgaggc tagagctaga 480

cacgcctggc agctgaccca aggcgccacc gtgctgggcc tgttcagagt gacccctgag 540

atcccggctg gtctgcctag tcctagaagc gag 573

<210> 8

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-FAP antibody-4-1BBL recombinant protein

<400> 8

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Ile Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Gln Val Ile Pro

85 90 95

Pro Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu

115 120 125

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His Ala Met Ser Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Trp Ala

165 170 175

Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Trp Leu Gly

210 215 220

Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Ala Ala Ser Pro Arg Leu Arg

245 250 255

Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu

260 265 270

Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile

275 280 285

Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser

290 295 300

Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val

305 310 315 320

Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg

325 330 335

Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu

340 345 350

Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val

355 360 365

Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe

370 375 380

Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His

385 390 395 400

Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly

405 410 415

Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly

420 425 430

Leu Pro Ser Pro Arg Ser Glu His His His His His His

435 440 445

<210> 9

<211> 1335

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-FAP antibody-4-1BBL recombinant protein

<400> 9

gagatcgtgc tgacacagag ccctggcacc ctgagcctga gccctggcga gagagccacc 60

ctgagctgca gagcttctca gagcgtgagc agaagctacc tggcctggta tcagcagaag 120

cctggccaag cccctagact gctgatcatc ggcgctagca caagagccac cggcatccct 180

gacagattca gcggtagcgg cagcggcacc gacttcaccc tgaccatcag ccgtctggag 240

cctgaggact tcgccgtgta ttattgtcag caaggccaag tgatccctcc taccttcggc 300

tgcggcacga aagtcgagat caaaggtgga ggcggttccg ggggaggtgg cagtgggggg 360

ggcggttctg aagtgcagct gttagagagc ggcgggggtc tcgtgcagcc tggtggcagt 420

ctgagactga gctgcgccgc tagcggcttc accttcagca gccacgccat gagctgggtg 480

agacaagccc ctggcaagtg cctggagtgg gtgagcgcca tctgggctag cggcgagcag 540

tactacgccg acagcgtgaa gggcagattc actatcagcc gggacaacag caagaacacc 600

ctgtacctgc agatgaacag cctgagagcc gaggacaccg ctgtgtatta ttgtgccaaa 660

ggctggctgg gcaacttcga ttattgggga caaggcactt tggtgaccgt ttcgtcgggg 720

gggggaggga gcggaggagg cggctccgct gctagcccta gactgagaga gggccctgag 780

ctgagccctg acgaccctgc cggcctgctg gacctgagac aaggcatgtt cgctcagctg 840

gtggctcaga acgtgctgct gatcgatggt cctctgagct ggtacagcga cccgggactg 900

gccggtgtga gcctgaccgg cggcctgagc tacaaggagg acaccaagga gctggtggtg 960

gccaaggccg gcgtgtacta cgtgttcttc caactggagc ttcgtcgagt agtggccggc 1020

gagggcagcg ggtctgttag cctagcactc cacttgcaac ctctgagaag tgctgccggg 1080

gcggcagccc ttgcgcttac cgtggacctg cctcctgcta gcagcgaggc tagaaacagc 1140

gccttcggct tccaaggcag actgctgcac ctgagcgccg gacagagact gggcgtgcac 1200

ctgcacaccg aggctagagc tagacacgcc tggcagctga cccaaggcgc caccgtgctg 1260

ggcctgttca gagtgacccc tgagatcccg gctggtctgc ctagtcctag aagcgagcac 1320

caccaccaccaccaccacc 1335

Claims (10)

1. The 4-1BB agonist is a fusion protein comprising an anti-FAP antibody and 4-1BBL, wherein the anti-FAP antibody is scFv comprising a VL having an amino acid sequence shown as SEQ ID NO. 1 or having at least 90%, 95% or 99% identity to SEQ ID NO. 1, and a VH having an amino acid sequence shown as SEQ ID NO. 3 or having at least 90%, 95% or 99% identity to SEQ ID NO. 3.

2. The 4-1BB agonist of claim 1, wherein the 4-1BBL is a full length or truncated 4-1BBL having an amino acid sequence as set forth in SEQ ID No. 5 and SEQ ID No. 6, or having at least 90%, 95% or 99% identity thereto; preferably, the nucleotide sequences encoding the VL, VH and truncated 4-1BBL are shown in SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 7, respectively, or have at least 90%, 95% or 99% identity to SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 7.

3. The 4-1BB agonist of claim 1, wherein the VL and VH of the anti-FAP antibody are linked by a linker or a chemical bond; and/or, the anti-FAP antibody is connected with 4-1BBL through a linker or a chemical bond;

preferably, the linker is (G) ₄ S) ₃ Or (G) ₄ S) ₂ (ii) a More preferably, the VL and VH of the anti-FAP antibody are passed through (G) ₄ S) ₃ (ii) association of the anti-FAP antibody with 4-1BBL (G) ₄ S) ₂ Are connected.

4. The 4-1BB agonist of any one of claims 1 to 3, wherein the amino acid sequence of the 4-1BB agonist is as set forth in SEQ ID NO. 8.

5. An isolated nucleic acid encoding the 4-1BB agonist of any one of claims 1-4; preferably, the isolated nucleic acid comprises the nucleotide sequence set forth as SEQ ID NO 9.

6. A recombinant expression vector comprising the isolated nucleic acid of claim 5.

7. A transformant which comprises the recombinant expression vector according to claim 6; preferably, the starting cell of the transformant is a prokaryotic cell or a eukaryotic cell; more preferably, the eukaryotic cell is a mammalian cell, such as a CHOK1 cell.

8. A method of making a 4-1BB agonist, comprising the steps of: culturing the transformant according to claim 7 in a medium; preferably, the medium is a CD-CHO medium.

9. A pharmaceutical composition comprising a 4-1BB agonist according to any one of claims 1 to 4.

10. Use of the 4-1BB agonist of any one of claims 1 to 4, the isolated nucleic acid of claim 5, the recombinant expression vector of claim 6, the transformant of claim 7, or the pharmaceutical composition of claim 9 for the preparation of a medicament for treating a tumor;

preferably, the tumor is a solid tumor; more preferably, the solid tumor comprises: colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, lung cancer, cervical cancer and ovarian cancer.

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