Patent Application
20220242974
The present invention relates to an anti-PD-L1 nanobody. The present invention also relates to an anti-PD-L1 Nanobody Fc fusion protein. The present invention also relates to the application of the anti-PD-L1 Nanobody. The present invention also relates to the application of the anti-PD-L1 Nanobody Fc fusion protein. The present invention belongs to the field of biomedicine.
In classical immune surveillance theory, the immune system can recognize tumor antigens and eliminate them. If the immune system can completely eliminate tumor cells, then immune clearance can be carried out stably. If tumor cells evade the clearance of the immune system through mutation, the immune system will rebalance. During this process, the immunogenicity of tumor cells gradually decreased. The ability of tumor cells to proliferate is weakened under the pressure of immune system, which makes it more difficult to detect tumor cells.
Activation of oncogene causes tumor cells to alter themselves and tumor microenvironment, which breaks the balance between immune system and tumor cells. When the immune system and tumor cells enter the escape stage, the malignant degree of tumor cells will increase, and the loss of MEW molecules in tumor cells will prevent them from being recognized and eliminated by immune cells. Tumor microenvironment can also inhibit the immune system by releasing immunosuppressive factors, such as IL-10 and TGF-β. Immunosuppressive proteins (such as programmed death ligand-1, PD-L1) are also highly expressed on the surface of tumor cells. When effector T cells combine with tumor cells, PD-L1 interacts with PD-1 and induces apoptosis of T cells, which is one of the main reasons for tumor tolerance to immune system. Then tumors grow rapidly and metastasize. If the host immune system is artificially activated and redirected to tumor cells, the tumor tissue can be cleared theoretically, and the theory of immunotherapy has been widely proved in clinical treatment.
Immunotherapy can be divided into two categories: specific therapy and non-specific therapy. Specific therapies in the category include the following treatment strategies: Tumor vaccines activate antigens against patients by injecting tumor-activated immune cells. Tumor vaccines include inactivated tumor cell vaccine, tumor antigen vaccine, tumor DNA vaccine, dendritic cell (DC) vaccine and bacterial vaccine. Specific ACT immunotherapy mainly includes three treatment methods:
Tumor infiltrating lymphocytes (TIL): Lymphocytes isolated from tumor tissues and cultured in vitro. TIL can secrete IL-2 with specific anti-tumor ability.
T cell receptor (TCR) therapy: T cells recognize tumor antigen through its single chain antibody fragment (scFv), and the single chain antibody fragment TCR is cloned into normal T cells through viral vector. Therefore, normal T cells become specific tumor killing T cells.
CAR-T: T cells are genetically modified to obtain T cells with tumor-specific receptors. Different from conventional T cell recognition mechanism, CAR-T cell recognition of tumor antigen is not limited by MHC molecule. Therefore, CAR-T cells can overcome the immune escape mechanism of tumor by increasing costimulatory signal molecules, and enhance the killing ability of T cells to tumor cells.
In nonspecific ACT immunotherapy, there are two main therapeutic methods: lymphokine activated killer (LAK) cell therapy and cytokine induced killer (CIK) cell therapy. LAK cell therapy: On the one hand, LAK cells stimulate immune cells in peripheral blood lymphocytes by IL-2, including NK cells and T cells. On the other hand, LAK cells enhance the recognition ability of target cells by overexpressing FAS ligands, and kill tumor cells by releasing perforin and granzyme.
CIK cell therapy: CIK cells are derived from peripheral blood lymphocytes (PBL) of patients or healthy people, and expanded in ex vivo by the stimulation of anti-CD3 antibody, IFN-γ and IL-2. CIK cells play an anti-tumor role mainly through FasL and perforin.
Immune checkpoint is a protective molecule in human immune system, which prevents inflammatory injury caused by excessive activation of T cells in normal body. Tumor cells can use this characteristic to over-express immune checkpoint molecules, inhibit the immune response of the body, escape the monitoring and killing of the human immune system, and thus promote the growth of tumor cells. Immune checkpoint inhibitor therapy can inhibit the activity of immune checkpoint in tumor microenvironment, reactivate the immune response of T cells to tumor, and achieve anti-tumor effect. The complete activation of a T cell is regulated by a “double signal” system: the first signal comes from the specific binding of its own TCR (T cell receptor) to MHC of antigen, that is, the T cell recognizes antigen; The second signal comes from a co-stimulatory molecule, which is involved in the interaction between the co-stimulatory molecule expressed by antigen presenting cells (APC) and the corresponding receptor or ligand (e.g. CD28) on the surface of the T cell. For example, CD28-B7 is a positive costimulatory signal, while CTLA4-B7 pathway and PD-1/PD-L1 pathway are the main negative costimulatory molecules. After tumor cells invade, this inhibition pathway is favored by tumor cells to inhibit T cell activation, thereby evading the clearance of immune system.
PD-1 (CD279) was first reported in 1992. Human PD-1 encoding gene PDCD1 was located at 2q37.3, with a total length of 2097bp, which was composed of 6 exons. The translation product was PD-1 precursor protein composed of 288 amino acids, and the mature protein was obtained by cutting the signal peptide composed of the first 20 amino acids. PD-1 comprises an extracellular immunoglobulin variable region IgV domain, a hydrophobic transmembrane domain and an intracellular domain. The N-terminal ITIM motif of the intracellular tail domain contains two phosphorylation sites, while the C-terminal is an ITSM motif. PD-1 is a membrane protein belonging to CD28 immunoglobulin superfamily, which is mainly expressed on the surface of activated T cells, and has low abundance expression in CD4-CD8-T cells of thymus, activated NK cells and monocytes. PD-1 has two ligands, PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) of B7 protein family. The amino acid sequences of PD-L1 and PD-L2 are 40% identical. The main difference between them lies in the different expression patterns. PD-L1 is constitutively low expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells and islet cells) and immunological privileged sites (such as placenta, testis and eyes). Inflammatory cytokines such as type I and type II interferon, TNF-α and VEGF can all induce PD-L1 expression. PD-L2 is expressed only in activated macrophages and dendritic cells. After PD-1 binds to PD-L1 on activated T cells, the ITSM motif of PD-1 undergoes tyrosine phosphorylation, which leads to dephosphorylation of downstream protein kinases Syk and PI3K, and inhibits the activation of downstream AKT and ERK pathways, and finally inhibits the transcription and translation of genes and cytokines needed for T cell activation, thus playing a negative role in regulating T cell activity.
In tumor cells, tumor cells and tumor microenvironment negatively regulate T cell activity and inhibit immune response by up-regulating PD-L1 expression and binding with PD-1 on the surface of tumor-specific CD8+ T cells. Tumor cells can up-regulate PD-L1 expression through the following four ways: 1. Amplification of PD-L1 encoding gene (9p24.1); 2. The activation of EGFR, MAPK, PI3K-Akt signaling pathway and HIF-1 transcription factors can up-regulate the expression of PD-L1 at transcription level; 3. Induction of EBV (EBV positive gastric cancer and nasopharyngeal carcinoma show high expression of PD-L1); 4. Epigenetic regulation. In tumor microenvironment, the stimulation of inflammatory factors such as interferon-y can also induce the expression of PD-L1 and PD-L2. Inflammatory factors can induce other cells in tumor microenvironment, including macrophages, dendritic cells and stromal cells to express PD-L1 and PD-L2, while tumor infiltrating T cells that can recognize tumor antigens can secrete interferon-y, and then induce up-regulation of PD-L1 expression. This process is called “adaptive immune resistance”, and tumor cells can protect themselves through this mechanism. There are more and more evidences show that tumors use PD-1-dependent immunosuppression to escape from immunity. High expression of PD-L1 and PD-L2 has been found in various solid tumors and hematological malignancies. In addition, the expression of PD-Ls has a strong correlation with the poor prognosis of tumor cells, which proves that it includes esophageal cancer, gastric cancer, renal cancer, ovarian cancer, bladder cancer, pancreatic cancer and melanoma.
At present, the therapeutic monoclonal antibodies of PD-1 approved by FDA are Nivolumab (Opdivo, September 2014), Pembrolizumab (Keytruda, December 2014) and Cemiplimab (Libtayo, September 2018), and the therapeutic monoclonal antibodies of PD-L1 are Atezolizumab (Tecentriq, September 2014), avelumab (Bavencio, May 2016) and Duravulumab (Imfinzi, May 2017). The approved indications are shown in the following table.
In addition, PD-1 monoclonal antibodies such as Pidilizumab, AMP-224, AMP-514 and PDR001, and PD-L1 monoclonal antibodies such as BMS-936559 and CK-301 are under development and clinical trials.
However, the affinity of existing monoclonal antibodies has not reached the ideal state, and because of their large size, they have strong immunogenicity.
The purpose of the present invention is to provide an anti-PD-L1 nanobody and its Fc fusion protein and application to solve the above-mentioned problems.
The present invention provides an anti-PD-L1 nanobody, comprising at least one VHH fragment, which comprises three amino acid fragments of CDR1, CDR2 and CDR3, and CDR1, CDR2 and CDR3 are respectively selected from the following sequences:
Further, the anti-PD-L1 nanobody of the present invention is characterized in that its sequence is as shown in any of SEQ ID No:1 to SEQ ID No:41.
The present invention also provides an Fc fusion protein of the anti-PD-L1 nanobody, comprising the anti-PD-L1 nanobody of claim 1 or 2 and an Fc segment, and the sequence of the Fc segment is as shown in SEQ ID No: 42.
Furthermore, the anti-PD-L1 nanobody of the present invention is characterized in that in its sequence, except for CDR1, CDR2 and CDR3, 80% of the amino acid sequence is identical to the sequence shown in any of SEQ ID No: 1 to SEQ ID No: 41.
The present invention also provides an application of the anti-PD-L1 nanobody in preparing a reagent for blocking the binding of PD-L1 and PD-1.
The invention also provides a use of the Fc fusion protein of the anti-PD-L1 nanobody in preparing a reagent for blocking the binding of PD-L1 and PD-1.
Further, the anti-PD-L1 nanobody of the invention is characterized in that the dosage is 20 ug/ml to 0.000128 ug/ml.
Further, the anti-PD-L1 nanobody of the invention is humanized and has a sequence as shown in any of SEQ ID No: 100 to SEQ ID No:105.
The invention also provides an Fc fusion protein of humanized anti-PD-L1 nanobody, comprising the anti-PD-L1 nanobody and an Fc segment of claim 8, and the sequence of the Fc segment is as shown in SEQ ID No:42.
Use of any one of the above-mentioned nanobody or its Fc fusion protein in the preparation of a medicine for treatment of cancer, infection or immunomodulatory diseases.
Use of any one of the above-mentioned nanobodies in the preparation of drugs for inhibiting tumor growth.
Use of any one of the above-mentioned nanobodies or its Fc fusion protein, wherein cancer or tumor is selected from the following tissues or sites: colorectal, breast, ovary, pancreas, stomach, esophagus, prostate, kidney, cervix, bone marrow cancer, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberant, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.
The nanobody and the Fc fusion protein of the present invention have strong specificity, high affinity, low immunogenicity to human and remarkable anti-tumor effects.
The technical scheme of the present invention is described in further detail below.
In order to facilitate screening, 41 clones were transformed into PD-L1l-FC fusion protein with human IgG1 Fc at C terminal. The reconstructed plasmid was expressed in HEK293 cells and purified by protein A affinity chromatography. Besides QP1121-FC, a total of 40 candidate PD-L1-FC fusion proteins were obtained, and the sequences consisted of the C terminal of the nanobody shown in SEQ ID NO:1-41 connected with the Fc segment of SEQ ID NO:42. The number of Fc fusion protein of the corresponding nanobody is formed by adding Fc suffix after the corresponding nanobody.
The results are shown in
Table 5 shows EC50 values for each antibody corresponding to the experimental results in
Table 6 shows the EC50 values of each antibody corresponding to the experimental results in Fig.
Table 7 shows the EC50 values of each antibody corresponding to the experimental results in
Table 8 shows the EC50 values of each antibody corresponding to the experimental results in Fig. 2D.
The results are shown in
Table 9 shows the IC50 values of each antibody corresponding to the experimental results in
Table 10 shows the IC50 values of each antibody corresponding to the experimental results in
Table 11 shows the IC50 values of each antibody corresponding to the experimental results in
5. The curve of PD-L1 nanobody human Fc fusion protein blocking PD-1/PD-L1 interaction, detected by competitive ELISA and biotin
The results are shown in
Table 12 shows the IC50 values of each antibody corresponding to the experimental results in
Table 13 shows the IC50 values of each antibody corresponding to the experimental results in
Table 14 shows the IC50 values of each antibody corresponding to the experimental results in
Table 15 shows the IC50 values of each antibody corresponding to the experimental results in
Table 16 shows the IC50 values of each antibody corresponding to the experimental results in
Table 17 shows the IC50 values of each antibody corresponding to the experimental results in
Table 18 shows the IC50 values of each antibody corresponding to the experimental results in
The results are shown in
Table 19 shows the EC50 values of each antibody corresponding to the experimental results in
Table 20 shows the EC50 values of each antibody corresponding to the experimental results in
Table 20 shows the EC50 values of each antibody corresponding to the experimental results in
By comparing IMGT human antibody heavy light chain variable region germline gene database and MOE software, the heavy and light chain variable region germline genes with high homology with QP1162 (SEQ ID No:35) and QP1166 (SEQ ID No:39) were selected as templates, and the CDRs of camel antibody were transplanted into the corresponding human templates to form the variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then some important amino acid residues were selected for reversion mutation combination. The amino acid residues were identified and annotated by Kabat numbering system.
Primers were designed for PCR to construct the VH gene fragment of each humanized antibody, and then homologously recombined with the expression vector pQD with signal peptide and constant region gene (FC) fragment to construct the full-length expression vector VH-FC-pQD.
Using online software DNAWorks (v3.2. 2) (http://helixweb.nih.gov/dnaworks/), multiple primers were designed to synthesize VH/VK gene fragments: 5′-30 bp signal peptide+VH+30 bp FC-3′. According to the instructions of TaKaRa Primer STAR GXL DNA polymerase, the VH/VK gene fragments were amplified by two-step PCR with the primers designed above. Construction and digestion of expression vector pQD with signal peptide and constant region gene (FC) fragments. The expression vector pQD with signal peptide and constant region gene (FC) fragments was designed and constructed by using restriction enzymes such as BsmBI, which have different recognition sequences and digestion sites. Vector was digested by BsmBI, and the gel was cut and recycled for later use. The expression vector VH-FC-pQD was constructed by recombination. VH containing the recombinant gene fragment and BsmBI digestion recovery expression vector pQD (with signal peptide and constant region gene (FC) fragment) were added into DHSH competent cells at a ratio of 3:1, respectively, ice bath at 0° C. for 30 min, heat shock at 42° C. for 90 seconds, adding 5 times volume of LB medium, incubating at 37° C. for 45 min, coating LB-Amp plate, culturing overnight at 37° C. Monoclones were picked out and sent for sequencing to obtain each target clone.
The sequences and protein expression numbers of variable regions of light and heavy chains of each clone humanized design are as follows. In this table, the antibody fused the constant region of human IgG1-FC at its C terminal:
At the same time, the humanized pre-chimeric antibody and control antibody were designed and cloned as shown in the following table:
The cell density of 293E was maintained at 0.2-3×106/ml, the culture medium (GIBCO Freestyle 293 expression medium) was used in the maintenance stage, and the cells to be transfected were centrifuged and changed medium one day before transfection, and the cell density was adjusted to 0.5-0.8×106/ml. On the day of transfection, the density of 293E cells was 1-1.5×106/ml. The plasmid and transfection reagent PEI were prepared. The amount of plasmid needed to be transfected was 100 ug/100 ml cells, and the mass ratio of PEI to plasmid was 2:1. The plasmid and PEI were mixed evenly and left standing for 15 min, but not more than 20 min. The mixture of plasmid and PEI was slowly added into 293E cells, and cultured in a shaking table at 8% CO2, 120 rpm and 37° C. On the fifth day of transfection, the supernatant was collected by centrifuging at 4700 rpm for 20min in a horizontal centrifuge.
Protein A affinity chromatography purification
The equilibrium solution was used to pass through the column, at least 3 CV, with an actual volume of 20 ml, ensuring that the pH and conductivity of the solution flowing out of the final instrument were consistent with those of the equilibrium solution, and the flow rate was 1 ml/min. After centrifugation, the supernatant of culture solution was used to pass through the column, and 40 ml of sample was loaded at a flow rate of 0.33 ml/min. The equilibrium solution was used to pass through the column, at least 3 CV, with an actual volume of 20 ml, ensuring that the pH and conductivity of the solution flowing out of the final instrument were consistent with those of the equilibrium solution, and the flow rate was 0.33 ml/min. The elution was used to pass through the column, and the elution peak (PAC-EP) was collected when UV280 was up to 15 mAU and stopped when UV280 was down to 15 mAU at a flow rate of 1 ml/min. After sample collection, PAC-EP was adjusted to neutral with pH adjusting solution.
Coated with antibody QP1162/QP320/QP321/QP322/QP1166/QP323/QP324/QP325 at 0.75 ug/ml, QP11801181 at 1.5 ug/ml, 50 ul/well, overnight at 4° C. Washed with PBS for 3 times. Blocking: 3% BSA 250 ul/well, RT 1 h. After incubation, 2 ug/ml biotin-PDL1-FC was diluted to different concentrations at 1:4, and incubated at room temperature for 1 h. Washed with PBST for 3 times and with PBS for 3 times. Incubation of secondary antibody: HRP-strepavidin (1:5000) 50 ul/well, washed with PBST for 6 times, and with PBS for 3 times. Development: TMB 100 ul/well, developed for 10 min. Terminated with 2M H2SO4 50 ul/well. The results are shown in
Coated with protein QP1138 (PD1-FC) at 2 ug/ml 50 ul/well, overnight at 4° C. Washed with PBS for 3 times. Blocking: 3% BSA 250 ul/well, incubated at room temperature for 1 h. 2 ug/ml of biotin-PDL1-FC and different concentrations of QP1120 15 ug/ml were prepared respectively, QP11801181 30 ug/ml was diluted at 1:3, mixed in equal volume, and incubated at room temperature for 1 h. Washed with PBST for 3 times and with PBS for 3 times. Incubation of secondary antibody: HRP-strepavidin (1:5000) 50 ul/well, washed with PBST for 6 times, and with PBS for 3 times. Development: TMB 100 ul/well, developed for 10 min. Terminated with 2M H2SO4 50 ul/well. The results are shown in
Affinity detected by surface plasmon resonance (SPR)
Biacore T200 (GE) was used to determine the affinity of the molecules with human PD-L1 and cynoPD-L1 proteins
The antigen information is as follows:
The anti-CLDN18.2/anti-PD-L1 bispecific antibody molecule QP3711461 was constructed and designed as shown in
1. Identification of PD-L1 Function in vitro (Mixed Lymphocyte Reaction (MLR))
Preparation of DC (donor1) cells: PBMCs were resuscitated, and monocytes were isolated with EasySep™ Human Monocyte Isolation Kit (Stemcell 19359), rhGM-CSF (1000 U/ml) and rhIL4 (500 U/ml) were added, and the cells were cultured at 37° C. for 6 days to induce to iDC. The half medium was changed every 2-3 days, and rhGM-CSF (1000 U/ml) and rhIL4 (500 U/ml) were supplemented at the same time. Cells were collected and centrifuged at 300×g for 5 min, then re-suspended in the medium with rhGM-CSF (1000U/ml) and rhIL4 (500 U/ml), and LPS (1 μg/ml) was added. Cells were cultured at 37° C. for 1 day to induce to mature DC. The cells were collected and counted for later use.
Preparation of T (donor2) cells: PBMCs were resuscitated and CD4+ T cells were isolated with EasySep™ Human CD4+ T Cell Isolation Kit (Stemcell 17952).
Preparation of antibody: 6 concentrations of antibodies (initial concentration of 10 ug/ml) were diluted by 1:5 gradient in medium. DC cells and T cells were mixed at a ratio of 1:10, and different concentrations of antibodies were added, then mixed cultivation. The expression of IL-2 in the culture supernatant was detected on the second day, and the expression of IFNg in the culture supernatant was detected on the fifth day.
In mixed lymphocyte reaction test, QP3711461 showed obvious antibody concentration dependence on the concentration of IFN γ and IL-2 produced by activated T cells. The biological function of PD-L1 antibody in QP3711461 was proved. As shown in
2. Identification of PD-L1 Function in vivo
Mouse colon cancer cell line MC38-hPDL1 was used in subcutaneous model of transgenic mouse C57BL/6-hPDL1, to evaluate the efficacy of anti-PD-L1 nanobody in vivo.
Experiment Methods: MC38-hPDL1 cells (which were knocked out mouse PDL1 and expressed human PDL1) of mouse colon cancer cell in logarithmic growth phase were inoculated subcutaneously in the right flank of C57BL/6-hPDL1 mice after the culture medium was removed and washed twice with PBS, inoculation volume: 5×10 5/100 μL/mouse. After inoculation, the mice were observed and the growth of tumor was monitored. On the 8th day after inoculation, when the average tumor volume reached 92.9 mm3, they were randomly divided into 4 groups with 9 mice in each group according to the tumor volume. The day of grouping was defined as D0, and administration began on D0.
Experimental results are shown in
The anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461 was administered at 4 mpk, 10 mpk, 25 mpk, BIW×3, i.p. On the 27th day after administration, the average tumor volume of PBS group (negative control group) reached 1445.20 mm3, the average tumor volume of QP3711461 (4mpk) group was 477.00 mm3, TGI=72.00%, the average tumor volume of QP3711461 (10 mpk) group was 279.97 mm3, TGI=86.14%, and the average tumor volume of QP3711461 (25 mpk) group was 293.96 mm3, TGI=85.14%. There was significant difference in tumor volume between each administrating group and PBS group (t test, P<0.01).
The experiment showed that the anti-PD-L1 molecule in the anti-CLDN18.2/anti-PD-L1 bispecific antibody of the invention had superior anti-tumor ability in the MC38-hPDL1 model of immune targets humanized transgenic mice.
The anti-PD-L1 nanobody numbers and corresponding sequences provided by the invention are as follows.
TDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYWGQ
DGVTYYADSVKGRFTISQDDAKNTLYLQMDSLKPEDTAMYYCASNGMCGQYWALEDEY
KYWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
KYADSVKGRFTISQDNAKNTLNLQMDSLKPEDTAMYFCAGRSYTNCRDGPPSASHYSHWG
TYANSVKGRFTISKDKAEDTVYLQMNNLKPEDTAMYVCKTFSCRNRGGAYLADAWGQGT
NYASSVKGRFTISKDNAKDTVYLQMNNLKPEDTAMYVCKTFSCRNRGGSYLPDTWGQGT
NTYYADSVKGRFTISQDNAKNTVYLRMNSLKPEDTAMYYCAADTRAALWYIGPLNSDQY
NTWGQGTQVTVSS
TYVDSVKGRFTISRDNTNKNSYTLTLTMNNLNPEDTAMYYCAATSQLGFWAQKLWEAIRD
GTWSPSTTDFGFWGRGTQVTVSS
TKYADSVKGRFTISRDDAKNTLYLQMNSLKPEDTAVYYCAASDWSRLYKIYWLDDNYYV
RWGQGTQVTVSS
TYADSVKDRFTISRDNAKNTVYLQMNSLKPEDTAMYYCAAGQALLWASLRQTSYQFWGQ
PIAYADSVKGRFTISRDNAKNTLYLQMTSLKSEDTALYYCARGWYFSGDYVPMTQGTQVT
NTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
TYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQYNI
DTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQY
NIWGQGTQVTVSS
NTNYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQY
NIWGQGTQVTVSS
NTSYADSVKGRFTISQDNAKNTVYLQTNSLKPEDTAMYYCAADTRAAFWYIGPLNSHQYN
IWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNLVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQY
NIWGQGTQVTVSS
NTAYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKDTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTHYADSVKGRFAISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
TYYADSVKGRFTISQDNAKNTVYLQMHSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYN
LWGQGTQVTVSS
NTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWSIGPLNSDQYN
IWGQGTQVTVSS
NTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQY
NIWGQGTQVTVSS
NTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDSAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYN
SWGQGTQVTVSS
NTYYADPVKGRFTISQDNAKNLVYLQMNSLKPEDTAMYYCAADTRAAFWNIGPLNSDQY
NIWGQGTQVTVSS
NTYYADSVKGRFTISQDNAKNTVYLRMNSLKPEDTAMYYCAADTRAALWYIGPLNSDQY
NTWGQGTQVTVSS
NTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQYN
IWGQGTQVTVSS
TYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADARAAFWYIGPLNSDQYNI
NTDYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNSDQY
NIWGQGTQVTVSS
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYW
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHAGFSRYW
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYW
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYW
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYW
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAARDFGYCTASWVHEGFSRYW
SYTDPVKGRFTISQDNAKNTLYLQMNSLKPGDTAMYYCAARDFGYCTASWVHEGFSRYW
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910567277.5 | Jun 2019 | CN | national |
| 202010574992.4 | Jun 2020 | CN | national |
This application contains a sequence listing submitted in Computer Readable Form (CRF). The CFR file containing the sequence listing entitled “PBA4085215 ST25.txt”, which was created on Dec. 27, 2021, and is 91,722 bytes in size. The information in the sequence listing is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/098051 | 6/24/2020 | WO | 00 |
