Patent Application
20240247043
The present invention relates to the field of biopharmaceutics, and in particular, to an LAG-3 fusion protein mutant, a method for preparing same, and use thereof.
Lymphocyte-activation gene 3 (LAG-3, or CD223) encodes a 498-amino acid type I transmembrane protein consisting of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region has four Ig-like domains designated domain 1 to domain 4 (D1-D4). The extracellular region is similar to CD4, but has only 20% amino acid homology to CD4. The intracellular region consists of 3 parts: a serine phosphorylation site, a “KIEELE” motif, and EP repeats, wherein the “KIEELE” motif is a highly conserved sequence that is involved in intracellular signal transduction, but is not found in other proteins.
Under physiological conditions, LAG-3 is mainly expressed on the cell membrane of activated T cells, NK cells, B cells, and dendritic cells, and modulates the immune response of T cells by three predominant means: direct inhibition of T cell proliferation and activation through negative regulation; indirect inhibition of T cell responses by promoting inhibitory function of regulatory T cells (Treg); and prevention of T cell activation by modulating the function of antigen-presenting cells (APCs). LAG-3 inhibits T cell activation by transmitting inhibitory signals through the intracellular region.
LAG-3 distinguishes the conformation of pMHCII and selectively binds to stabilized pMHCII. To date, several molecules have been reported as potential ligands of LAG-3 in addition to the stabilized pMHCII. Galectin-3 and liver sinusoidal endothelial cell lectin (LSECtin) have been demonstrated to interact with the glycans on LAG-3. In 2019, Chen Lieping et al. demonstrated that FGL1 is an important functional ligand of LAG-3, and revealed that the LAG-3-FGL1 pathway is another tumor immune escape pathway independent of the PD-L1-PD-1 pathway, and the blockage of the pathway may play a synergistic role with anti-PD-1 therapies.
Current pharmaceutical development for LAG-3 includes anti-LAG-3 blocking antibodies, depleting antibodies, agonist antibodies, and fusion proteins of LAG-3. IMP321 is a soluble recombinant fusion protein consisting of the extracellular region of LAG-3 and the Fc domain of IgG, and can activate antigen-presenting cells by MHCII-mediated reverse signaling, resulting in increased IL-12 and TNF and the up-regulation of CD80 and CD86. Use of the drug for cancer treatment is currently in the clinical stage.
The present invention relates to an LAG-3 protein mutant, a fusion protein thereof, and use thereof. Specifically, the present invention relates to the following: 1. An LAG-3 protein mutant, comprising mutations at one or more of the following positions on the basis of the domain 2 of the LAG-3 protein: 188, 192, 196, 197, 172, 175, 177, 178, 183, 185, 186, 187, 189, 190, 195, 199, 203, 208, 210, 211, 212, 214, 216, 218, 198, 201, 207 and 209, preferably, mutations at one or more of the following positions on the basis of the domain 2 of the LAG-3 protein: 177, 183, 185, 186, 187, 190, 195, 197, 198, 199, 201, 207, 212, 214 and 218, or preferably, mutations at one or more of the following positions on the basis of the domain 2 of the LAG-3 protein: 183, 185, 186, 187, 190, 195, 197, 199, 201, 207 and 212, wherein the numbering of the amino acid positions corresponds to the numbering of the sequence set forth in SEQ ID NO: 63, and preferably, the sequence of the domain 2 of the LAG-3 protein is set forth in SEQ ID NO: 11; preferably, the LAG-3 protein comprises the domain 1 and the domain 2, and optionally the domain 3 and/or the domain 4;
2. The LAG-3 protein mutant according to item 1, comprising one or more of the following mutations on the basis of the domain 2 of the LAG-3 protein: R188A, R192A, H196A, H197A, P172A, P175A, S177A, V178A, N183A, G185A, Q186A, G187A, V189A, P190A, P195A, L199A, F203A, Q208A, S210A, P211A, M212A, S214A, P216A, G218A, H198G, H198L, H198M, H198W, H198Y, H198V, E201R, E201N, E201D, E201Q, E201H, E201G, E201F, E201S, P207R, P207D, P207E, P207I, P207M, P207S, P207T, P207Y and V209T, preferably, P207E, P207I, P207R, P207D, M212A, P207M, S177A, P207T, Q186A, G187A, E201D, E201G, H197A, H198Y, G185A, L199A, N183A, P190A, P195A, S214A, P207Y, G218A, H198W and H198V, or preferably, N183A, G185A, Q186A, G187A, P190A, P195A, H197A, L199A, E201D, E201G, P207E, P207I, P207R, P207D, M212A and P207M.
3. The LAG-3 protein mutant according to item 1 or 2, wherein the mutation present in the domain 2 of the LAG-3 protein is selected from the group consisting of: R188A; R192A; H196A; H197A; P172A; P175A; S177A; V178A; N183A; G185A; Q186A; G187A; V189A; P190A; P195A; L199A; F203A; Q208A; S210A; P211A; M212A; S214A; P216A; G218A; H198G; H198L; H198M; H198W; H198Y; H198V; E201R; E201N; E201D; E201Q; E201H; E201G; E201F; E201S; P207R; P207D; P207E; P207I; P207M; P207S; P207T; P207Y; V209T; P207E and M212A; P207E and E201D; P207I and E201G; E201D and Q186A; H197A and E201G; P207I, E201D and Q186A; E201D, Q186A and P195A; P207E, Q186A and E201G; P207E, E201D, P195A and H197A; P207I, M212A, E201D and N183A; and P207E, M212A, E201G and N183A, N183A, G185A, Q186A, G187A, P190A, P195A, L199A and E201D; or preferably, the mutation is selected from the group consisting of: N183A; G185A; Q186A; G187A; P190A; P195A; L199A; E201D; E201G; P207E and M212A; P207E and E201D; P207I and E201G; E201D and Q186A; H197A and E201G; and P207I, E201D and Q186A;
4. An LAG-3 fusion protein, comprising a structure as follows: a structural unit 1—a structural unit 2, wherein the structural unit 1 is selected from LAG-3 D1-D2, LAG-3 D1-D2-D3, or LAG-3 D1-D2-D3-D4,
5. An LAG-3 fusion protein dimer or multimer, comprising the LAG-3 fusion protein according to item 4, wherein the structural unit 1 in the LAG-3 fusion protein dimer or multimer is identical or different.
6. The LAG-3 fusion protein dimer or multimer according to item 5, wherein the LAG-3 fusion protein dimer or multimer is an LAG-3 fusion protein dimer, and the structural unit 1 is selected from LAG-3 D1-D2, LAG-3 D1-D2-D3, or LAG-3 D1-D2-D3-D4,
7. The LAG-3 fusion protein according to item 4, wherein the LAG-3 D1, D2, D3, D4, and the structural unit 2 are connected with or without a linker, and preferably the linker is selected from a sequence set forth in any one of SEQ ID NOs: 6-9.
In some embodiments, the linker is flexible. In some other embodiments, the linker is rigid. In some embodiments, the linker may be a linker derived from a native multidomain protein or a linker peptide conventionally used in the art. In some embodiments, the linker may be designed using a linker design database and a computer program.
8. A conjugate, comprising the LAG-3 protein mutant according to any one of items 1-3 and a conjugated moiety, or comprising the LAG-3 fusion protein according to item 4 and a conjugated moiety, or comprising the LAG-3 fusion protein dimer or multimer according to item 5 or 6 and a conjugated moiety, wherein the conjugated moiety is a purification tag (e.g., His-tag, Fc-tag), a detectable label, a drug, a prodrug, a toxin, a cytokine, a protein (e.g., an enzyme), a virus, a lipid, a biological response modulator (e.g., an immunomodulator), PEG, a hormone, a polypeptide, an oligonucleotide, a diagnostic agent, a cytotoxic agent, or a combination thereof, preferably, the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.
9. A pharmaceutical composition, comprising the LAG-3 protein mutant according to any one of items 1-3, or the fusion protein according to item 4, or the LAG-3 fusion protein dimer or multimer according to item 5 or 6, or the conjugate according to item 8, wherein preferably, the pharmaceutical composition further comprises at least one drug for treating a cancer or an infectious disease; preferably the drug is selected from a chemotherapeutic drug, an immunotherapeutic drug, or a combination thereof; preferably, the drug is selected from a radiotherapeutic agent, a chemotherapeutic agent (e.g., paclitaxels, anthracyclines, gemcitabine), a therapeutic antibody (e.g., rituximab, cetuximab, edrecolomab, trastuzumab, an anti-PD-1 antibody, an anti-PD-L1 antibody), a cytokine, a polypeptide, an antimetabolite, or a combination thereof;
10. Use of the LAG-3 protein mutant according to any one of items 1-3, or the fusion protein according to item 4, or the LAG-3 fusion protein dimer or multimer according to item 5 or 6, or the conjugate according to item 8 in modulating an immune response or immunostimulation or treating or diagnosing a cancer or Parkinson's disease, or in preparing a medicament, an immunostimulant or an adjuvant for modulating an immune response or treating or diagnosing a cancer or Parkinson's disease.
In some embodiments, the pharmaceutical composition is administered in combination, preferably simultaneously or sequentially, with additional therapeutic or prophylactic regimens, e.g., radiation therapy, chemotherapy, or immunotherapy.
In some embodiments, preferably, the chemotherapeutic agent is an alkylating agent, an antimetabolite, an antibiotic, a botanical agent, and/or a hormonal agent, preferably cyclophosphamide, pemetrexed, a platinum-based drug such as cisplatin, carboplatin or oxaliplatin, doxorubicin, paclitaxel, a vinca alkaloid, an anthracycline, gemcitabine, tamoxifen, megestrol, goserelin, asparaginase, and/or a fluorouracil antineoplastic agent.
11. A nucleic acid molecule, comprising a nucleic acid sequence encoding the LAG-3 protein mutant according to any one of items 1-3, or the fusion protein according to item 4, or the LAG-3 fusion protein dimer or multimer according to item 5 or 6, or the conjugate according to item 8, or a complement thereof.
12. A vector, comprising the nucleic acid molecule according to item 11.
13. A host cell, comprising the nucleic acid molecule according to item 11 or the vector according to item 12.
14. A method of treating a disease, comprising administering to a subject in need a therapeutically effective amount of the LAG-3 protein mutant according to any one of items 1-3, or the fusion protein according to item 4, or the LAG-3 fusion protein dimer or multimer according to item 5 or 6, or the conjugate according to item 8, or the pharmaceutical composition according to item 9.
15. A kit, comprising the LAG-3 protein mutant according to any one of items 1-3, or the fusion protein according to item 4, or the LAG-3 fusion protein dimer or multimer according to item 5 or 6, or the conjugate according to item 8, or the pharmaceutical composition according to item 9, wherein preferably, the kit further comprises an antibody that specifically recognizes the LAG-3 protein; optionally, the antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.
It will be appreciated that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described hereinafter (as in the examples) may be combined with each other to constitute a new or preferred embodiment. Due to the limited space, such embodiments are not described herein. The terms referred to in the present invention have the conventional meanings understood by those skilled in the art. Where a term has two or more definitions as used and/or acceptable in the art, the definitions of the terms used herein are intended to include all meanings.
The LAG-3 molecule consists of 3 moieties: an extracellular region, a transmembrane region, and an intracellular region. The extracellular region consists of 4 immunoglobulin domains: D1 (the domain 1 of the LAG-3 protein), D2 (the domain 2 of the LAG-3 protein), D3 (the domain 3 of the LAG-3 protein), and D4 (the domain 4 of the LAG-3 protein). The D1 region is a member of the V immunoglobulin superfamily (IgSF), and the D2, D3 and D4 regions are members of the C2 IgSF. The D1 domain comprises an extra loop consisting of 30 amino acids rich in proline, which is reported to be involved in the interaction between LAG-3 and major histocompatibility complex class II (MHCII). In 2019, Chen Lieping et al. found that FGL1 is a ligand of LAG-3 for the T cell inhibitory function, and through deletion of specific domains, it was demonstrated that the D1 and D2 of the LAG-3 are the major domains interacting with FGL1 (Wang et al., Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3, Cell (2019)). The LAG-3 binds to MHCII and FGL1 dependent on the D1 and D2. It will be appreciated by those of ordinary skills in the art that a partial or intact LAG-3 protein comprising the D1 and D2 domains of the LAG-3 may achieve the interaction with FGL1 or MHCII, e.g., a partial LAG-3 protein comprising the D1, D2 and D3 domains, or an intact LAG-3 protein comprising the D1, D2, D3 and D4 domains.
In one embodiment of the present invention, the D1 has a sequence set forth in SEQ ID NO: 10; the D2 has a sequence set forth in SEQ ID NO: 11 or set forth in the domain 2 mutant of the present invention, the domain 2 mutant being defined as the domain 2 of the LAG-3 protein mutant according to any one of items 1-3; the D3 has a sequence set forth in SEQ ID NO: 12; and the D4 has a sequence set forth in SEQ ID NO: 13.
In one embodiment of the present invention, the D1-D4 domains of the LAG-3 are defined according to the Uniprot database, the sequences of the D1, D2, D3 and D4 of the LAG-3 differ from the D1, D2, D3 and D4 sequences of the present invention (SEQ ID NOs: 10, 11, 12 and 13) in the N and C termini, as seen in Uniprot P18627 (SEQ ID NO: 64), wherein the sequence definitions of D1-D4 are as follows: D1, amino acids 37-167; D2, amino acids 168-252; D3, amino acids 265-343; D4, amino acids 348-419. It will be appreciated by those of ordinary skills in the art that the D1-D4 domains of the LAG-3 protein defined according to the Uniprot database, exert the same function as the corresponding D1-D4 (SEQ ID NOs: 10-13) of the present invention.
LAG-3 (CD223) is known to induce the maturation of monocyte-derived dendritic cells in vitro and to induce CD41 helper T cell responses and CD8 T cell responses in vivo as an immunotherapeutic adjuvant. Further information on LAG-3 and use thereof as an immunostimulant can be found in the works of TRIEBELE et al., TRIEBEL et al., and HUARD et al. Some soluble forms of LAG-3 are capable of binding to MHCII molecules and inducing dendritic cell maturation and migration to secondary lymphoid organs where they are capable of initiating naive CD4− helper cells and CD8 cytotoxic T cells leading to tumor rejection. Recently, a recombinant soluble human LAG-3-Ig fusion protein has been demonstrated to activate a wide range of effector cells, e.g., inducing monocyte-macrophages to secrete cytokines/chemokines, in both innate and acquired immune responses.
In one preferred embodiment of the present invention, the LAG-3 protein fragment is selected from any one of the following:
In the present invention, the structural units for forming the dimer may be selected from, for example, an Fc fragment, c-JUN, c-FOS, VL-CL, and VH-CH1, wherein the VL-CL and VH-CH1 are paired to form an Fab fragment specific for an antigen, and the c-JUN and c-FOS are paired to form a leucine zipper.
In a specific embodiment, the Fc fragment has a sequence set forth in SEQ ID NO: 1; when the VL-CL has a sequence set forth in SEQ ID NO: 4, the VH-CH1 has a sequence set forth in SEQ ID NO: 5, or when the VL-CL has a sequence set forth in SEQ ID NO: 61, the VH-CH1 has a sequence set forth in SEQ ID NO: 62; the c-JUN-His has a sequence set forth in SEQ ID NO: 2, and the c-FOS-His has a sequence set forth in SEQ ID NO: 3.
In the present invention, the structural unit for forming a trimer is the T4 fibritin foldon domain.
It will be appreciated by those of ordinary skills in the art that structural units capable of being used to form a dimer or multimer known in the art can all be used to form the dimer or multimer of the present invention.
In some embodiments, the LAG-3 protein mutant, LAG-3 fusion protein, or LAG-3 fusion protein dimer or multimer of the present invention may be conjugated with a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent or PEG. The LAG-3 protein mutant, LAG-3 fusion protein, or LAG-3 fusion protein dimer or multimer of the present invention can be linked or fused to a therapeutic agent, and the therapeutic agent may comprise a detectable label such as a radioactive label, an immunomodulator, a hormone, an enzyme, a polypeptide, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent that may be a drug or a toxin, an ultrasound enhancing agent, a nonradioactive label, a combination thereof and other such ingredients known in the art.
In the present invention, linker denotes linker, linker1 denotes linker1, linker2 denotes linker2, and linker3 denotes linker 3.
Compared with the prior art, the present invention has one or more of the following beneficial effects:
The LAG-3 protein mutant features a high expression level, improved purity, excellent biological activity and specificity, significant in-vitro and in-vivo anti-tumor bioactivity, and good stability. The dimer with the LAG-3-Fab structure (such as LAG3 D1-D2-D3-D4-Fab and LAG3 D1-D2-Fab) of the present invention retains the activity of the LAG-3 moiety and the Fab moiety, and has good stability.
The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are only for illustrating the present invention, and should not be construed as limitations to the scope of the present invention. This present invention may be implemented in many other ways than those described herein, and it will be apparent to those skilled in the art that similar modifications can be made without departing from the spirit of the present invention. Therefore, the protection scope of the present invention is defined by the claims, rather than limited by the specific examples disclosed below.
Gene fragments of the D1 and D2 domains of the LAG-3 were acquired by genetic synthesis. The Fc gene was added to the 3′ moiety of the D2 domain by a linker and the gene of LAG-3 D1-D2-Fc was inserted between polyclonal enzyme cleavage sites of eukaryotic expression vector pCDNA3.1 to give the corresponding eukaryotic expression vector (as shown in
Plasmids were extracted according to a conventional plasmid extraction method and used for chemical transfection of CHO-S cells (gibco). The transfected cells were cultured on a shaker at 5% s CO2/37° C. for 7-10 days. The supernatant was harvested by 3000×g centrifugation and filtered through a 0.22 m filter. The LAG-3 D1-D2-Fc wild type fusion protein and mutants thereof were obtained by purification through protein A affinity chromatography. The concentration of the purified protein was determined by UV absorbance at 280 nm and the corresponding extinction coefficient, and the expression level of the proteins was calculated. The expression level of some mutants was significantly higher than that of the wild type, as shown in Table 2.
The multimer content of the LAG-3 D1-D2-Fc wild type and the mutants thereof was determined by high performance size exclusion chromatography (HPLC-SEC). Compared with those of the wild type, some mutants demonstrated significantly reduced multimer content and improved purity, as shown in Table 3. SDS-PAGE showed that the molecular weight was consistent with the theoretical value 110 WD.
1. Binding capacity of LAG-3 D1-D2-Fc wild type and mutant thereof to human FGL1 (hFGL1)
The binding capacity of the LAG-3 D1-D2-Fc wild type and the mutants thereof to human FGL1 (hFGL1) was determined by ELISA. The samples were prepared into 10 μg/mL coating solutions by using PBS buffer, added to a plate (100 μg/well), and incubated overnight at 4° C. The next day, the remaining coating solution was discarded. PBST (PBS containing 0.1% Tween 20) was added at 300 μL/well for washing once, and 3% BSA was added at 300 μL/well for blocking the wells at 37° C. for 1 h. After 1 wash with 300 μL/well of PBST, serially diluted human FGL1-His antigen (ACRO, FG1-H52Hy) solutions were added at 100 μL/well and the plate was incubated at 37° C. for 1 h. After 3 washes with 300 L/well of PBST, diluted 6×His tag antibody [GT359] (HRP) (GeneTex, GTX628914-01) was added at 100 μL/well and the plate was incubated at 37° C. for 1 h. After 5 washes with 300 μL/well of PBST, a TMB chromogenic solution was added (100 μL/well). Finally, the reaction was stopped by adding 2 M HCl and the samples were detected on a microplate reader (Molecular Devices, SPECTRA Max plus 384) for OD450.
The results are shown in
2. Binding capacity of LAG-3 D1-D2-Fc wild type and mutant thereof to human MHCII The direct and competitive binding capacities of LAG-3 D1-D2-Fc wild type and mutants thereof to human MHCII were determined by FACS using Daudi cells (from CCTCC-GDC097) as positive cells of human MHCII.
Direct binding: Daudi cells were collected by centrifugation, resuspended in a buffer (PBS+1% FBS), and added at 1×105 cells/100 μL/well in a 96-well plate. Then the mixture was centrifuged at 350×g for 5 min, and the supernatant was discarded. The samples were diluted to 2000 nM with a buffer and serially 3- or 4-fold diluted to the 11th concentration. The samples were added to a 96-well plate at 100 μL/well, resuspended, incubated at 4° C. in the dark for 1 h, and centrifuged. The supernatant was discarded. The samples were washed twice with the buffer, resuspended in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304), incubated at 4° C. in the dark for 30 min, then washed twice with the buffer, resuspended in 100 μL of buffer, and loaded on a flow cytometer (BD Accuri™ C6) for detection.
The results are shown in
Competitive binding: Daudi cells were collected by centrifugation, resuspended in a buffer (PBS+1% FBS), and added at 1×105 cells/100 μL/well in a 96-well plate. Then the mixture was centrifuged at 350×g for 5 min, and the supernatant was discarded. The samples were diluted to 4000 nM with a buffer and serially 3- or 4-fold diluted to the 11th concentration. 30 μL of serially diluted sample was taken and mixed with an equal volume of 4000 nM IP321-PE (the IMP321 sequence is derived from SEQ ID NO: 17 in Patent No. US20110008331A1). The mixture was then added into a 96-well plate at 50 μL/well, resuspended, incubated in the dark for 30 min at 4° C., washed twice with a buffer, resuspended in 50 μL of buffer, and loaded on a flow cytometer (BD Accuri™ C6) for detection.
The results are shown in
Through the binding capacity to MHCII, FGL1 and the like, the mutants demonstrated significant in vitro and in vivo anti-tumor bioactivity.
The stability of the samples was evaluated using differential scanning calorimetry (DSC). Compared with the wild type W1161-WT, the mutants WS447 and WS451 demonstrated improved Tm1 values, as shown in Table 4.
The samples were diluted to 0.5 mg/mL, added into 1.5-mL EP tubes at 100 μL/tube, and incubated in a water bath at 40° C. for thermal acceleration for 14 days. The start of the test was designated as D0, the 7th day was designated as D7, and the 14th day was designated as D14. After two weeks, the D0, D7, and D14 samples were subjected to SDS-PAGE.
As shown in
The specific structure of LAG-3 D1-D2-D3-D4-Fc (
The results are shown in Table 5-2. The mutants A7817, A7820, A7836, and A7842 demonstrated improved expression and purity as compared with the wild type. The mutations E201D, E201G, P207I, and M212A produced similar effect on the domain D2 in the LAG-3 D1-D2-D3-D4-Fc fusion protein (A7817, A7820, A7836, and A7842) as the effect in the LAG-3 D1-D2-Fc fusion protein (WS447, WS451, WS488, and WS343). Compared with the wild type, the expression and purity were significantly improved. Similarly, for the LAG-3 D1-D2-D3-D4-Fc fusion protein, other mutations in the D2 domain (such as N183A, G185A, Q186A, G187A, H197A, H198Y, L199A, P207R, P207T, M212A, P211A, etc.) also increased the expression or purity compared with the wild type, and the effect (improvement in expression or purity) was similar to that produced in the LAG-3 D1-D2-Fc fusion protein.
The binding capacity of the LAG-3 D1-D2-D3-D4-Fc wild type and the mutants thereof to human FGL1 was determined by ELISA using the same method as in Example 3. As shown in
The direct binding capacity of LAG-3 D1-D2-D3-D4-Fc wild type and mutants thereof to human MHCII was determined by FACS using Daudi cells as positive cells of human MHCII using the same method as in Example 3. As shown in
Through the binding capacity to MHCII, FGL1 and the like, the mutants demonstrated significant in vitro and in vivo anti-tumor bioactivity.
The stability of the samples was evaluated using differential scanning calorimetry (DSC). Compared with the wild type IMP321, the mutants A7817 and A7820 demonstrated improved Tm1 values, as shown in Table 6.
The samples were diluted to 0.5 mg/mL, added into 1.5-mL EP tubes at 100 μL/tube, and incubated in a water bath at 40° C. for thermal acceleration for 14 days. The start of the test was designated as D0, and the 14th day was designated as D14. After two weeks, the D0 and D14 samples were subjected to SDS-PAGE. As shown in
A schematic of the LAG-3 D1-D2-LZ is shown in
The results are shown in Table 7-2. The expression and purity of the mutants WS447-LZ, WS451-LZ, WS488-LZ, and WS343-LZ were improved as compared with the wild type. Furthermore, the mutations E201D, E201G, P207I, and M212A produced similar effect on the domain D2 in the LAG-3 D1-D2-LZ fusion protein (WS447-LZ, WS451-LZ, WS488-LZ, and WS343-LZ) as the effect in the LAG-3 D1-D2-Fc fusion protein (WS447, WS451, WS488, and WS343). Compared with the wild type, the expression and purity were significantly improved.
Similarly, for the LAG-3 D1-D2-LZ fusion protein, other mutations in the D2 domain (such as N183A, G185A, Q186A, G187A, H197A, H198Y, L199A, P207R, P207T, M212A, P211A, etc.) also increased the expression or purity compared with the wild type, and the effect (improvement in expression or purity) was similar to that produced in the LAG-3 D1-D2-Fc fusion protein.
The binding capacity of the LAG-3 D1-D2-LZ wild type and the mutants thereof to human FGL1 was determined by ELISA using the same method as in Example 3. As shown in
The direct binding capacity of LAG-3 D1-D2-D3-D4-Fc wild type and mutants thereof to human MHCII was determined by FACS using Daudi cells as positive cells of human MHCII using the same method as in Example 3. As shown in
The stability of the samples was evaluated using differential scanning calorimetry (DSC). Compared with the wild type W1161-LZ, the mutants WS447-LZ and WS451-LZ demonstrated improved Tm1 values, as shown in Table 8.
An illustrative schematic of the LAG-3-Fab is shown in
The activity of the LAG-3 moiety in the LAG-3-Fab wild type and the mutants thereof, including the binding capacities to human FGL1 and human MHCII, were determined by ELISA and FACS using the method as in Example 3. The change trend induced by the mutations in the LAG3-Fab was basically the same as that in the LAG-3 D1-D2-Fc.
The activity of two moieties of the LAG-3-Fab, including the LAG-3 moiety and the anti-PD-L1 or anti-PD-1 Fab moiety, was detected by Biacore. An antigen was fixed on a CM5 chip by amino coupling, wherein the antigen coupling amount was 800 RU. The sample was diluted to an initial concentration using 1×HBS-EP+buffer, and then serially 2-fold diluted to four different concentrations. The diluted samples were loaded on a detection system in ascending order of concentration for detection, wherein the binding flow rate was 30 μL/min, the binding time was 120 s, and the dissociation time was 300 s. The chip was regenerated with a pH1.5Glycine solution, wherein the regeneration flow rate was L/min and the regeneration time was 30 s. After the detection was completed, data fitting was conducted on the resultant pattern by using the Biacore T200 Evaluation Software in a 11 binding fitting mode to obtain an equilibrium dissociation equilibrium constant (KD).
The results are shown in Table 10. Y103-4A, Y103-7A, Y103-4B, and Y103-7B demonstrated strong binding capacity to hFGL1 antigen and PD-L1 (SB, Cat. No. 10084-H08H) or PD-1 (SB, Cat. No. HPLC-10377-H08H) antigen and higher affinity for hFGL1 as compared with IMP321. All of these exhibited significant in vitro and in vivo anti-tumor bioactivity.
The stability of the samples was evaluated using differential scanning calorimetry (DSC). Compared with IMP321, Y103-4A and Y103-4B demonstrated improved Tm1 values, and compared with W1161-WT, Y103-7A and Y103-7B demonstrated higher Tm1 values, as shown in Table 11.
The samples were diluted to 0.5 mg/mL, added into 1.5-mL EP tubes at 100 μL/tube, and incubated in a water bath at 40° C. for thermal acceleration for 14 days. The start of the test was designated as D0, and the 14th day was designated as D14. After two weeks, the D0 and D14 samples were subjected to SDS-PAGE. As shown in
LAG-3 D1-D2-D3-Fc and mutants thereof were constructed, and the specific structure was D1-D2-D3-Linker-Fc. The mutation sites of the mutants were E201D, E201G, P207I, and M212A, and the sequences of the linker and Fc are respectively set forth in SEQ ID NO: 6 and SEQ ID NO: 1. Similar effects were also achieved by repeating the experimental procedures of Examples 2-4 above, i.e., the expression and purity of the mutants were significantly improved as compared to the wild type (>50% for expression and >15% for purity), and the affinity and stability were also improved. Through the binding capacity to MHCII, FGL1 and the like, the mutants demonstrated significant in vitro and in vivo anti-tumor bioactivity.
The point mutations validated in the above examples were combined to give fusion protein constructs LAG-3 D1-D2-Fc, LAG-3 D1-D2-D3-D4-Fc, LAG-3 D1-D2-LZ, and LAG-3-Fab. The specific combinations of mutations are shown in Table 12 below. The specific sequences can be found in Examples 1-12 above, and the sequences of VL-CL and VH-CH1 in the LAG-3-Fab are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The structures of LAG-3 D1-D2-Fc and LAG-3 D1-D2-D3-D4-Fc are shown in
JAWSII cells in the logarithmic growth phase (from ATCC® CRL-11904™) were seeded on a 96-well plate at 5×104 cells/100 μL/well. The sample was diluted to 500 nM with a buffer and serially 5-fold diluted to nine different concentrations. The diluted samples were then added to a 96-well plate at 100 μL/well. The 96-well plate was incubated in an incubator at 5% CO2/37° C. for 48 h, and centrifuged at 300×g for 5 min. The cell supernatant was collected, and the expression of mTNF-α in the cell supernatant was detected with an mTNF-α ELISA kit (R&D, DY410-05).
The results are shown in
A
ESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMY
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110503020.0 | May 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/091464 | 5/7/2022 | WO |
