WO2020233589A1 - 靶向cd19的全人源抗体及其应用 - Google Patents

靶向cd19的全人源抗体及其应用 Download PDF

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WO2020233589A1
WO2020233589A1 PCT/CN2020/091235 CN2020091235W WO2020233589A1 WO 2020233589 A1 WO2020233589 A1 WO 2020233589A1 CN 2020091235 W CN2020091235 W CN 2020091235W WO 2020233589 A1 WO2020233589 A1 WO 2020233589A1
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amino acid
acid sequence
antibody
cells
car
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WO2020233589A9 (zh
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谭涛超
戴振宇
贾向印
魏巧娥
杨永坤
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南京驯鹿医疗技术有限公司
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Priority to CA3141498A priority Critical patent/CA3141498A1/en
Priority to JP2022516254A priority patent/JP2022534135A/ja
Priority to CN202310120560.XA priority patent/CN116589579A/zh
Priority to EP20809306.2A priority patent/EP3974452A4/en
Priority to CN202080037449.9A priority patent/CN113840841B/zh
Priority to AU2020277525A priority patent/AU2020277525A1/en
Priority to US17/613,367 priority patent/US20220220200A1/en
Publication of WO2020233589A1 publication Critical patent/WO2020233589A1/zh
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Definitions

  • the present invention relates to a fully human antibody targeting CD19, and also relates to a chimeric antigen receptor (CAR) comprising a single chain antibody (scFv) of the fully human antibody.
  • CAR chimeric antigen receptor
  • the fully human antibody and its scFv and CAR can be used for the construction of CAR-T cells targeting CD19.
  • Kymriah is the world's first approved CAR-T treatment product for the treatment of acute lymphoblastic leukemia patients aged 3 to 25 and adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL).
  • Yescarta is the second CAR-T product approved for marketing in the world, used to treat adult relapsed or refractory B-cell lymphoma and non-Hodgkin's lymphoma.
  • Figure 1 shows the schematic molecular structure of commonly used CARs.
  • CAR-T technology has completely different treatment principles from traditional treatment methods such as surgery, chemotherapy, and radiotherapy, and has a revolutionary therapeutic effect on refractory and relapsed hematological tumor diseases, it has opened up a new era of tumor treatment.
  • CAR-T clinical trials have been carried out all over the world, and China and the United States are the countries with the most relevant clinical trials.
  • CD19 is indicated for B lymphocyte tumors. Because of its definite curative effect and controllable side effects, it is the most among the products currently on the market and under research. With the deepening of clinical research, more and more evidence shows that the short-term effect of CD19 CAR-T treatment is very good, but as time goes by, about 50% of patients will relapse. There are many reasons for recurrence, mainly divided into CD19 antigen-negative recurrence and positive recurrence. In the CD19 antigen-positive recurrence, the short duration of CAR-T cells in the patient's body is the main reason.
  • ADA anti-antibody
  • CTL killer T lymphocytes
  • a fully human antibody or single chain antibody or fragment thereof targeting CD19 wherein the light chain variable region of the fully human antibody includes LCDR1, LCDR2, and LCDR3, and the heavy chain variable region includes HCDR1 , HCDR2 and HCDR3, wherein said LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are selected from one of the following combinations:
  • LCDR1 amino acid sequence of LCDR1 is SSNIGAGYD;
  • the amino acid sequence of LCDR2 is ENT
  • the amino acid sequence of LCDR3 is QSYDSSLSGWRV;
  • HCDR1 The amino acid sequence of HCDR1 is GYSFTNSW;
  • HCDR2 The amino acid sequence of HCDR2 is IYPDDSDT;
  • amino acid sequence of HCDR3 is ARQSTYIYGGYYDT
  • the amino acid sequence of LCDR2 is YDD
  • the amino acid sequence of LCDR3 is AAWDDSLNGWV;
  • HCDR1 The amino acid sequence of HCDR1 is GYSFTSYW;
  • amino acid sequence of HCDR2 is IYPGDSDT
  • the amino acid sequence of HCDR3 is ARLSYSWSSWYWDF.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 8 and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 9; or the light chain may The variable region includes the amino acid sequence shown in SEQ ID NO: 11, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 12.
  • the single-chain antibody includes the amino acid sequence shown in SEQ ID NO: 7 or 10.
  • a chimeric antigen receptor targeting CD19 which includes a CD19-targeting single-chain antibody whose light chain variable region includes LCDR1, LCDR2, and LCDR3, and a heavy chain variable region Including HCDR1, HCDR2 and HCDR3, wherein said LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are selected from one of the following combinations:
  • LCDR1 amino acid sequence of LCDR1 is SSNIGAGYD;
  • the amino acid sequence of LCDR2 is ENT
  • the amino acid sequence of LCDR3 is QSYDSSLSGWRV;
  • HCDR1 The amino acid sequence of HCDR1 is GYSFTNSW;
  • HCDR2 The amino acid sequence of HCDR2 is IYPDDSDT;
  • amino acid sequence of HCDR3 is ARQSTYIYGGYYDT
  • the amino acid sequence of LCDR2 is YDD
  • the amino acid sequence of LCDR3 is AAWDDSLNGWV;
  • HCDR1 The amino acid sequence of HCDR1 is GYSFTSYW;
  • amino acid sequence of HCDR2 is IYPGDSDT
  • the amino acid sequence of HCDR3 is ARLSYSWSSWYWDF.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 8, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 9; or the light chain may The variable region includes the amino acid sequence shown in SEQ ID NO: 11, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 12. .
  • the single-chain antibody includes the amino acid sequence shown in SEQ ID NO: 7 or 10.
  • a modified T cell that expresses the aforementioned chimeric antigen receptor.
  • this document provides drugs for treating tumors expressing CD19 on the cell surface, which include the above-mentioned T cells.
  • nucleic acid molecule that encodes the aforementioned fully human antibody or single chain antibody or fragment thereof.
  • the sequence encoding the light chain variable region of the fully human antibody includes the nucleotide sequence shown in SEQ ID NO: 2, and the sequence encoding the heavy chain variable region includes the sequence shown in SEQ ID NO: 3.
  • the sequence encoding the light chain variable region of the human antibody includes the nucleotide sequence shown in SEQ ID NO: 5, and the sequence encoding the heavy chain variable region includes SEQ ID NO: 6 The nucleotide sequence shown.
  • sequence encoding the single-chain antibody includes the nucleotide sequence shown in SEQ ID NO: 1 or 4.
  • an expression vector including the aforementioned nucleic acid molecule.
  • the expression vector further includes the coding sequence of epidermal growth factor receptor (EGFR) or truncated form of EGFR (tEGFR).
  • EGFR epidermal growth factor receptor
  • tEGFR truncated form of EGFR
  • the fully human antibodies provided herein have lower immunogenicity than murine antibodies or humanized murine antibodies, and have better potential for antibody drugs or CAR-T applications. Compared with CAR-T cells using murine antibodies, CAR-T cells constructed using the fully human antibodies provided herein have better compatibility with the human body, and are beneficial to their long-term proliferation and survival in the body.
  • FIG. 1 is a schematic diagram of the structure of a chimeric antigen receptor (CAR) expressed on the cell surface.
  • CAR includes an extracellular binding region for binding to a specific target antigen (such as CD19), which is usually in the form of a single chain antibody (scFv); a hinge region between the cell membrane and the extracellular binding region; a cytoplasmic domain for transfection Direct the binding signal of the extracellular binding zone and activate the cell.
  • a specific target antigen such as CD19
  • scFv single chain antibody
  • a cytoplasmic domain for transfection Direct the binding signal of the extracellular binding zone and activate the cell.
  • Figure 2 shows the general process of screening specific antibodies targeting CD19 from the phage antibody library of the present invention.
  • Figure 3 shows the flow cytometric analysis results of cell clones obtained by knocking out Raji cells using different sgRNAs.
  • Figure 4 shows the results of enzyme-linked immunosorbent assay (ELISA) of the selected phage monoclonal and target antigen and control antigen.
  • Control 1 is FMC-63 (humanized mouse anti-human CD19 phage antibody clone);
  • Control 2 is a non-CD19 binding scFv phage antibody clone.
  • Figure 5 shows the results of flow cytometry analysis of partial phage monoclonal binding to Raji and Raji-CD19ko cells.
  • Figure 6 shows the results of flow cytometry analysis (MFI value) of the binding of the selected phage monoclonal to a variety of different CD19 positive and negative cell lines.
  • Control 1 is FMC-63 (humanized mouse anti-human CD19 phage antibody clone);
  • Control 2 is a negative control phage antibody clone.
  • Figure 7 shows the results of competitive binding experiments between FMC-63 antibody and #62, #78, FMC-63 phage.
  • Figure 8 shows a schematic diagram of the experimental principle of the reporter gene method for screening CAR molecules.
  • Figure 9 shows the activation of CAR-T cells constructed by the reporter gene method by various target cells, and the results are expressed in terms of the chemiluminescence intensity produced by luciferase.
  • Figure 10 shows the expression of CD19 on the surface of five target cells used in the reporter gene method experiment.
  • Figure 11 shows the results of CD107a degranulation of CAR-T cells by different target cells.
  • Figure 12 shows the killing results of CAR-T cells on multiple target cells (Nalm-6, Reh, Jvm-2, Jeko-1, Bv173, K562-CD19, K562, Thp-1 and Skm-1).
  • Figure 13 shows the killing of Nalm-6, Reh, Skm-1, and Thp-1 cells by CAR-T cells after stimulating CAR-T twice with Raji cells treated with mitomycin.
  • Figure 14 is a schematic diagram of the process of determining the affinity of FMC-63, #62, and #78scFv to the CD19 antigen.
  • Figure 15 shows the dynamic binding curves and KD, kon, and kdis parameters of FMC-63, #62, #78scFv and CD19 antigen affinity determination.
  • FIG 16 shows the experimental results of the membrane proteome array (MPA) of antibody #78.
  • Antibodies refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is correspondingly called an antigen.
  • the basic structure of an antibody molecule is a 4-mer composed of two identical heavy chains and two identical light chains. According to the conservative difference in amino acid sequence, the heavy chain and light chain are divided into the variable region (V) at the amino terminal and the constant region (C) at the carboxy terminal. The variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv).
  • variable region the composition and sequence of amino acid residues in certain regions are more changeable than other regions (framework regions, FR) in the variable region, called hypervariable regions (HVR), which are actually antibodies The key part that binds to the antigen. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDR). Both heavy chain and light chain have three complementary determining regions, which are called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively.
  • CDR complementarity-determining regions
  • Single chain antibody single chain fragment variable, scFv
  • scFv single chain fragment variable
  • Murine antibodies are antibodies produced by mice against specific antigens, and usually refer to antibodies produced by mouse B lymphocytes. In most cases, the murine antibody is a monoclonal antibody produced by hybridoma cells.
  • the fully human antibody of the present invention is obtained by screening from a human phage antibody library, which reduces immunogenicity compared with murine antibodies, and is more conducive to human therapeutic use.
  • Chimeric antigen receptor also known as chimeric T cell receptor or chimeric immune receptor
  • the chimeric antigen receptor usually consists of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signal domain.
  • the antigen-binding domain is a scFv sequence, which is responsible for recognizing and binding a specific antigen.
  • the intracellular signal domain usually includes an immunoreceptor tyrosine activation motif (ITAM), such as a signal transduction domain derived from a CD3 ⁇ molecule, which is responsible for activating immune effector cells and producing a killing effect.
  • ITAM immunoreceptor tyrosine activation motif
  • the chimeric antigen receptor can also include a signal peptide at the amino terminus which is responsible for the intracellular localization of the nascent protein, and a hinge region between the antigen binding domain and the transmembrane domain.
  • the intracellular signal domain may also include, for example, a costimulatory domain derived from a 4-1BB molecule.
  • CAR-T cells refer to T cells that express CAR, and are usually obtained by transducing T cells with an expression vector encoding CAR.
  • Commonly used expression vectors are viral vectors, such as lentiviral expression vectors.
  • Chimeric antigen receptor-modified T cells are not restricted by major histocompatibility complexes, and have specific targeted killing activity and the ability to continuously expand.
  • CD19 is a marker molecule on the surface of B lymphocytes and plays a role in regulating B cell activation and development. CD19 is not only expressed on normal B cells, but also on many malignant B cell tumors. This forms the basis for CAR-T targeting CD19 in the clinical treatment of B cell related tumors.
  • Bound monoclonal antibody were further identified with a variety of CD19 positive (Raji, JVM-2, K562-CD19) and negative cell lines (Raji-CD19ko, Jurkat, K562) by flow cytometric analysis (FACS), among which 2 clones ( #62 and #78) showed good specificity on multiple cell lines. These two clones (#62 and #78) were constructed into CAR-T format and subjected to in vitro functional experiments.
  • the phage antibody library can be contacted with cells expressing and not expressing the target antigen, respectively, and panning by binding conditions Select or identify specific antibodies.
  • a better way is to construct cells that knock out the gene encoding the target antigen. In this way, the difference between a pair of cells is mainly in this knock-out gene. This is an important application for cells in monoclonal antibody binding specificity identification and affinity panning.
  • CRISPR/Cas9 technology to knock out the CD19 gene on Raji cells with high expression of CD19, and obtained CD19-negative monoclonal Raji-CD19ko cells through monoclonal screening.
  • sgRNA1 to sgRNA6 design a variety of sgRNA sequences (sgRNA1 to sgRNA6), and conduct primer synthesis;
  • FACS detects the gene knockout efficiency of different sgRNAs, selects cell pools with higher knockout efficiency, and separates single clones by limiting dilution;
  • the CD19 knockout efficiency of cells corresponding to sgRNA5 is the highest, with 75.4% of CD19 knocked out. Therefore, we choose sgRNA5 electroporated cell pool for monoclonal screening.
  • the limiting dilution method was used to isolate single clones from the cell pool, and after the single clones were amplified, the CD19 expression of these clones was detected by FACS.
  • the results are shown in Table 2.
  • the CD19 expression of Raji-CD19ko-1, 11, and 14 clones is basically undetectable, and it can be considered as a single clone with successful knockout. These 3 clones were cultured and frozen. We used clone 1 in the subsequent study.
  • CD19 is an antigen normally expressed by B lymphocytes in the human body. For such antigens, the body will inactivate those B cells that can express CD19 antibodies through the mechanism of cloning and screening, resulting in a lack of such antigens in normal humans. Of high-affinity antibodies. Clone screening is the body's normal self-recognition and self-protection mechanism. However, the most commonly used form of phage antibody library is the natural library, which is constructed by directly cloning antibody genes in healthy human lymphocytes.
  • the semi-synthetic antibody library is composed of light chain and heavy chain FR1-FR3 from natural antibody sequence and artificially designed heavy chain CDR3, which can greatly increase the diversity of antibodies and improve the screening of high-affinity antibodies against normal antigens in the body (such as CD19) Opportunity.
  • BCMA-Fc was used as the negative panning protein
  • CD19-Fc was used as the positive panning protein to perform multiple rounds of panning to obtain a phage pool enriched with the target antibody clone.
  • the experimental steps are briefly described as follows:
  • the enriched phage pool can be used for subsequent monoclonal selection and ELISA/FACS identification.
  • the Raji-CD19ko cells prepared in Example 1 were used as negative panning cells, and Raji cells were used as positive panning cells to perform multiple rounds of panning to obtain a phage pool enriched with target antibody clones.
  • steps 1) to 5 usually 3 to 4 rounds of panning are required until the recovery rate of phage (the number of eluted phage/the number of input phage) has increased significantly.
  • the enriched phage pool can be used for the next step of monoclonal selection and ELISA/FACS identification.
  • Fully human phage antibody library including natural library and semi-synthetic library
  • Example 3 Using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) to screen from enriched phage pools Select specific clones
  • the phage pool enriched by the affinity panning step contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones.
  • specific clones we need to isolate a single clone from it, package it into a monoclonal phage, and conduct a preliminary screening of a large number of clones by enzyme-linked immunoassay (ELISA), and select the clones that specifically bind to the CD19 protein.
  • ELISA-specific clones flow cytometry is used for further screening to determine whether they can bind to the natural CD19 molecules on the cell surface.
  • the specific single-chain antibody sequence can be determined by DNA sequencing.
  • step 7 Add 100 ⁇ L of the cultured phage supernatant of step 1) to the wells coated with target antigen and control antigen, and bind for 2 hours at room temperature;
  • Monoclonal cells were randomly selected from the enriched phage antibody pool and packaged into phage.
  • the binding of the monoclonal phage to CD19-hFc-Bio protein and BCMA-hFc-Bio protein was detected by phage ELISA to find CD19-specific phage antibody clones.
  • the ELISA results of some clones are shown in Figure 4.
  • Control1 is FMC-63 (humanized mouse anti-human CD19 phage antibody clone);
  • Control2 is a non-CD19 binding scFv phage antibody clone.
  • clones #1, 4, 62, and 78 bind well to the target antigen CD19 (CD19-hFc-Bio), and do not bind to the control antigen BCMA (BCMA-hFc-Bio) and Streptavidin , Good specificity.
  • the clones #58 and #59 do not bind to the target antigen CD19 (CD19-hFc-Bio), irrelevant antigen BCMA (BCMA-hFc-Bio) and Streptavidin, which are negative clones.
  • the clones #79 and #81 can bind to the target antigen CD19 (CD19-hFc-Bio), but can also bind to the control antigen BCMA (BCMA-hFc-Bio) and are non-specific clones.
  • the FACS preliminary screening results of some clones are shown in Figure 5.
  • the #62 clone indicated by the arrow does not bind Raji-CD19ko, but binds to Raji cells, which is a specific clone; the other clones are non-specific (both two types of cells bind) or negative clones (no two types of cells bind).
  • ELISA and FACS preliminary screening we obtained a total of 13 specific clones.
  • Example 4 Using multiple cell lines to identify monoclonal specificity by FACS
  • the antibody used for treatment must have very good target specificity, only bind to the target antigen, and not any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will be different (Isomers or mutants) or binding ligands are not the same, we also need to investigate whether our antibodies can bind to various target protein-positive cells. In order to further analyze the specificity and universality of these monoclonals and find the best candidate clones, we further evaluated the specificity of the primary clones by flow cytometry.
  • the rest of the reagents are the same as the FACS preliminary screening.
  • Antibodies used for treatment must have very good target specificity.
  • flow cytometry to identify the multiple clones obtained in Example 3 on more cell lines. The results are shown in Table 5 and Figure 6.
  • Control 1 is FMC-63 (humanized mouse anti-human CD19 phage antibody clone);
  • Control 2 is a negative control phage antibody clone.
  • Clones #62 and #78 bind to all three CD19-positive cell lines, have high median fluorescence intensity (MFI), and do not bind to CD19-negative cell lines, have low MFI and good specificity.
  • MFI median fluorescence intensity
  • FMC63 is the most widely used anti-CD19 murine clone.
  • CAR-T therapy using this clone has obtained impressive clinical results, which may be related to the characteristics of the clone and the CD19 antigen-binding epitope.
  • the phage of the clone to be tested and the FMC63 antibody of different concentration gradients were mixed in advance and then combined with the positive target cell NALM6, by incubating the primary antibody mouse anti M13 antibody and the secondary antibody FITC horse anti mouse-IgG (H+L) After the antibody was tested, the FITC fluorescence intensity was determined to determine whether the FMC63 antibody had an effect on the binding of #62phage and #78 phage to the positive target cell NALM6, so as to determine whether the two clones have similar binding properties to the scFv derived from mouse FMC63.
  • Antibody gradient dilution According to the original concentration of FMC63 Ab and CD22 (clone: M971) antibody, dilute it to 400 ⁇ g/mL, and then dilute to 40 ⁇ g/mL, 4 ⁇ g/mL, 0.4 ⁇ g/mL, 0.04 ⁇ g/mL, 0.004 ⁇ g/mL, add 50 ⁇ L of antibody to each well.
  • Bacteriophage packaging and dilution as described above, KO7 infection and packaging of #62 phage, #78 phage and FMC63 phage, according to the original titer, were diluted to two concentrations of 4x10 10 pfu/mL and 2x10 10 pfu/mL, Add 50 ⁇ L of bacteriophage to each well and mix well with the antibody in advance.
  • FMC63Ab and CD22 (clone: M971) antibodies, independently expressed by IASO BIO
  • Example 4 In order to confirm whether these specific clones obtained in Example 4 can specifically recognize target cells and activate CAR-T cells after being constructed into CAR-T, we have developed an efficient CAR-T screening method, namely the reporter gene method.
  • the principle is as follows Shown in Figure 8.
  • the activation of CAR-T cells is achieved by CD3 ⁇ and costimulatory factors in the intracellular region of CAR molecules.
  • CD3 ⁇ can activate the NFAT signaling pathway in cells, which is a necessary condition for CAR-T cell activation. Therefore, the NFAT reporter gene method can be used to screen out CAR molecules that can activate the NFAT signaling pathway.
  • NFAT-ffLuc NFAT-ffLuc (ffLuc, firefly luciferase) reporter gene were used as reporter cells.
  • the CAR-encoding nucleic acid molecules are transiently expressed on the surface of reporter cells by plasmid electrotransformation.
  • the reporter cell expressing the CAR molecule is incubated with the target cell, the surface antigen of the target cell can specifically activate the CAR molecule, thereby activating the expression of the reporter gene.
  • the ability of CAR molecules to activate the NFAT signaling pathway can be evaluated.
  • the plasmid also includes a sequence encoding truncated EGFR (tEGFR), which can be used to mark cells that successfully express CAR when tEGFR is expressed on the cell surface.
  • tEGFR truncated EGFR
  • CMV-hRLuc internal reference plasmid blended with CAR-encoding nucleic acid molecules to calibrate the electroporation efficiency.
  • this reporter gene method Compared with the traditional method of packaging lentivirus and reinfecting T cells to prepare CAR-T to detect the function, this reporter gene method has simple steps and can quickly and efficiently evaluate the ability and specificity of candidate CAR molecules to recognize tumor cells.
  • Electrotransfer kit Celetrix, Cat. No. 1207;
  • Raji, JVM2 and REH are positive target cells expressing CD19 antigen, and they have different CD19 antigen expression density (as shown in Figure 9).
  • Raji-CD19ko and K562 serve as negative target cells and do not express CD19 antigen on their surface.
  • PXL092 is a positive reference CAR molecule encoding FMC63-bbz.
  • 57-1, 62-1, 69-1, 70-1 are the CAR molecules to be tested, and 62-1 is constructed using the phage 62 described above.
  • the background signal may come from the influence of the upstream promoter of the reporter gene, or it may be caused by the spontaneous activation of CAR molecules. Therefore, the higher the background signal, the greater the possibility of spontaneous activation of CAR molecules.
  • the reporter cell and the negative target cell are incubated together, if the CAR molecule does not bind non-specifically, the signal obtained should be the same as the Buffer group.
  • the CAR molecule will be specifically activated by the surface antigen of the positive target cell to generate a signal, and the signal intensity is related to the antigen density.
  • the figure shows that CAR molecule No.
  • CAR 62-1 has the ability to activate the NFAT signaling pathway similar to that of the control PXL092, and there is no obvious non-specific activation problem.
  • CAR molecules 57-1 and 69-1 can also activate the NFAT signaling pathway, but they are worse than PXL092.
  • CAR molecule No. 70-1 has a strong background activation signal, and its ability to be specific to positive target cells is weak.
  • CAR molecules with activating function on CAR-T cells obtained by preliminary screening by the reporter gene method described above needs further confirmation.
  • CD107a is a marker of intracellular microvesicles. When the granzyme-loaded microvesicles are fused with the cell membrane, CD107a on the cell membrane will increase. When monesin (purchased from BioLegend) is used to block its recovery , Can quantitatively reflect the strength of microvesicle release. When CAR-T is stimulated by the target antigen on the target cells, it will cause the release of granzyme, and the activation of T cells can be judged by the increase of CD107a by flow cytometry.
  • Alexa Fluor 488 anti-human EGFR BioLegend, Cat. No. 352908;
  • FITC-CD19 protein Acro Biosystems, Cat. No. CD9-HF251.
  • CAR-T cells were obtained by lentiviral transduction, and the CAR-T cells were cultured in vitro for 9-12 days and then subjected to CD107a degranulation experiment.
  • the CAR-T cells to be tested and target cells, monensin and CD107a antibody were incubated together for 3h, and the cell density of CAR-T cells and target cells were both 5 ⁇ 10 5 cells/mL. Then, after labeling the sample with CD8 antibody, EGFR antibody (or CD19-FITC protein), flow cytometry is performed.
  • luciferase When target cells are killed by CAR-T cells, luciferase will be released and quickly inactivated (the half-life of firefly luciferase is about 0.5h). If target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as target cells expand and luciferase continues to express. Therefore, the killing of target cells by CAR-T can be detected by the activity of luciferase.
  • RLU luciferase activity reading
  • the three sets of CAR-T cell samples (#62, #78 clones and CAR-T cells prepared by the control FMC63) can effectively kill positive target cells when the effective target ratio is 4:1. There was no obvious killing when T cells and positive target cells were incubated together. All CAR-T and T cell samples were not significantly killed when incubated with negative target cells. Therefore, the three sets of CAR-T samples can specifically kill CD19-positive target cells, and there is no non-specific killing of CD19-negative target cells.
  • the target cells (Raji) treated with mitomycin (Mitomycin) are mixed with CD19 CAR-T cells of different groups for 3 times of stimulation, then CAR-T cells and target cells are incubated and cultured to determine different scFv CAR-T Whether the killing ability of target cells changes after being stimulated repeatedly.
  • the three groups of CAR-T cell samples (#62, #78 clones and control FMC63) can effectively kill the positive target cells in a dose-dependent manner.
  • All CAR-T and T cell samples and negative target cells were not significantly killed when they were incubated together. Therefore, the three groups of CAR-T samples can specifically kill CD19-positive target cells after repeated stimulation of positive target cells, and there is no non-specific killing of CD19-negative target cells.
  • the affinity between CD19scFvs and antigen may have an important impact on the killing effect and survival time of CAR-T in patients.
  • ForteBio's Octet molecular interaction technology was used to determine it.
  • the biomembrane interference technology used in the Octet system is a label-free technology that provides high-throughput biomolecular interaction information in real time.
  • the instrument emits white light to the sensor surface and collects the reflected light.
  • the reflection spectrum of different frequencies is affected by the thickness of the optical film of the biosensor.
  • the reflected light of some frequencies forms constructive interference (blue), while others are destructive. Interference (red).
  • interferences are detected by the spectrometer and form an interference spectrum, which is displayed as the phase shift intensity (nm) of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the thickness of the biofilm on the sensor surface, from which the interaction of the biomolecules can be obtained. High-quality data to determine the kinetic parameters of the interaction between biomolecules (Kon, Kdis and KD), providing important information for the development process.
  • Affinity refers to the strength of the binding of a single molecule to its ligand. It is usually measured and reported by the equilibrium dissociation constant (KD).
  • KD equilibrium dissociation constant
  • the equilibrium dissociation constant can be used to evaluate and rank the strength of the interaction between two molecules.
  • the binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactant.
  • the smaller the KD value the greater the affinity of the antibody to its target.
  • FMC63, #62, and #78 can all bind to the CD19 antigen, and the affinity order is: FMC63>#78>#62.
  • Membrane Proteome Array is a cell-based high-throughput platform that can be used to identify targets for isolated antibodies and other ligands that bind to membrane proteins.
  • Membrane proteins account for about a quarter of all proteins encoded by the human genome, and are usually folded into a complex conformation structure that is difficult to retain outside the cell.
  • the key feature of MPA is that the membrane protein can be directly expressed and tested in the cell in its natural state, thereby maintaining its structural integrity and natural post-translational modification.
  • MPA utilizes the largest membrane protein library assembled to date, representing more than 5000 unique membrane proteins. Through the MPA platform, we conducted a #78 specific test to verify whether it will non-specifically bind to antigens other than CD19 and assess the risk of off-target effects.
  • MPA contains about 5000 different membrane protein clones, which account for more than 90% of the human membrane protein group. Each clone is overexpressed in HEK-293T cells containing a cDNA-containing plasmid, which is stored in a 384-well cell culture plate. Independent transfection in separate wells, 36h incubation time to ensure the expression of membrane protein.
  • each array plate includes positive and negative controls.
  • #78 antibody has no non-specific binding to most of the more than 5000 membrane proteins, but it can be observed that it has a high expression of SDC1, Frizzled 4, HTR5A HEK-293T The phenomenon of cell binding.
  • SDC1 Frizzled 4
  • HTR5A HTR5A HEK-293T
  • the phenomenon of cell binding In order to verify whether the binding is reproducible, we carried out a concentration gradient dilution of #78 antibody and repeated the experiment. The experimental results showed that #78 antibody can bind to HEK-293T cells with high expression of CD19 and positive control (ProteinA). And the average fluorescence intensity of its binding is dose-dependent.
  • #78 antibody does not bind to HEK-293T cells that highly express SDC1, Frizzled 4, TR5A at any concentration, which proves that #78 antibody has a better effect. Specificity, less risk of off-target in patients, and good safety.
  • the present invention uses fully human phage for antibody screening and directly obtains fully human monoclonal antibodies. Compared with traditional hybridoma technology, it saves the difficult steps of mouse antibody humanization, and fully human antibody has lower immunogenicity than humanized mouse antibody, which can be used in antibody drugs or CAR-T applications Have better potential.
  • phage antibodies that can bind to recombinantly expressed CD19 protein and Raji cells at the same time are enriched, and monoclonal antibodies that can specifically bind to the CD19 antigen on the cell membrane surface are selected.

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Abstract

提供了靶向CD19的全人源抗体或其单链抗体或片段。还提供了采用来自该全人源抗体的单链抗体(scFv)构建的嵌合抗原受体(CAR)。该全人源抗体和CAR可用于靶向CD19的CAR-T细胞的构建。与采用鼠源抗体的CAR-T细胞相比，所述的CAR-T细胞与人体具有更好的相容性，有利于其在体内的长期增殖和存活。

Description

靶向CD19的全人源抗体及其应用 技术领域
本发明涉及靶向CD19的全人源抗体，还涉及包括该全人源抗体的单链抗体(scFv)的嵌合抗原受体(CAR)。该全人源抗体及其scFv和CAR可用于靶向CD19的CAR-T细胞的构建。
背景技术
近年来，细胞免疫治疗技术、尤其是嵌合抗原受体T细胞(CAR-T)技术取得了突破性进展。2017年，诺华的Kymriah(Tisagenlecleucel)和Kite Pharma的Yescarta先后经美国FDA批准上市。Kymriah是全球首个获批的CAR-T治疗产品，用于治疗3至25岁的急性淋巴细胞白血病患者和治疗复发或难治性弥漫性大B细胞淋巴瘤(DLBCL)的成人患者。Yescarta是全球第二款获批上市的CAR-T产品，用于治疗成人复发或难治性B细胞淋巴瘤和非霍奇金淋巴瘤。图1显示了常用CAR的示意性分子结构。
由于CAR-T技术与手术、化疗、放疗等传统治疗方法完全不同的治疗原理以及对难治复发的血液肿瘤疾病有革命性的治疗效果，其开辟了肿瘤治疗的新时代。目前全世界已开展了大量CAR-T临床试验，其中中国和美国是相关临床试验最多的国家。
CD19作为CAR-T治疗的热门靶点之一，适应症是B淋巴细胞肿瘤。由于其疗效确定，副作用可控，在目前已上市和在研中的产品中是最多的。随着临床研究的深入，越来越多的证据表明，CD19 CAR-T治疗的短期效果很好，但随着时间的延长，大约50％的病人会复发。复发的原因很多，主要分为CD19抗原阴性的复发和阳性的复发。在CD19抗原阳性的复发中，CAR-T细胞在病人体内的存续时间短是主要的原因。根据对现有的CAR-T输注病人的研究，人体产生针对CAR-T使用的异源抗体的抗抗体(ADA)或杀伤性T淋巴细胞(CTL)，可能是导致部分病人体内CAR-T细胞迅速被清除的主要原因。
目前已经上市的2款产品及大多数临床实验中的产品，都是采用异源的抗体来识别CD19抗原。例如，Kymriah和Yescarta都采用了鼠源抗体，南京传奇的LCAR-B38M采用了羊驼来源的抗体。全人源抗体由于具有比异源抗体更低的免疫原性，在抗体药物开发领域已经成为一个主流的方向。同样道理，全人源的抗体应用于CAR-T产品，也能降低CAR-T细胞的免疫原性，延长CAR-T细胞在人体内的存活时间，增强CAR-T产品的长期疗效。因此，开发全人源的CD19抗体，对于研发下一代体内存续时间更长、长期疗效更好的CAR-T产品，有非常重要的意义。
发明内容
在一方面，本文提供了靶向CD19的全人源抗体或其单链抗体或片段，其中所述全人源抗体的轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3，其中所述LCDR1、LCDR2、LCDR3、HCDR1、HCDR2、和HCDR3选自如下组合之一：
(1)LCDR1的氨基酸序列为SSNIGAGYD；
LCDR2的氨基酸序列为ENT；
LCDR3的氨基酸序列为QSYDSSLSGWRV；
HCDR1的氨基酸序列为GYSFTNSW；
HCDR2的氨基酸序列为IYPDDSDT；
HCDR3的氨基酸序列为ARQSTYIYGGYYDT；
(2)LCDR1的氨基酸序列为SSNIGNNA；
LCDR2的氨基酸序列为YDD；
LCDR3的氨基酸序列为AAWDDSLNGWV；
HCDR1的氨基酸序列为GYSFTSYW；
HCDR2的氨基酸序列为IYPGDSDT；
HCDR3的氨基酸序列为ARLSYSWSSWYWDF。
在一些实施方案中，所述轻链可变区包括SEQ ID NO：8所示的氨基酸序列，所述重链可变区包括SEQ ID NO：9所示的氨基酸序列；或者所述轻链可变区包括SEQ ID NO：11所示的氨基酸序列，所述重链可变区包括SEQ ID NO：12所示的氨基酸序列。
在一些实施方案中，所述单链抗体包括SEQ ID NO：7或10所示的氨基酸序列。
另一方面，本文提供了靶向CD19的嵌合抗原受体，其包括靶向CD19的单链抗体，所述单链抗体的轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3，其中所述LCDR1、LCDR2、LCDR3、HCDR1、HCDR2、和HCDR3选自如下组合之一：
(1)LCDR1的氨基酸序列为SSNIGAGYD；
LCDR2的氨基酸序列为ENT；
LCDR3的氨基酸序列为QSYDSSLSGWRV；
HCDR1的氨基酸序列为GYSFTNSW；
HCDR2的氨基酸序列为IYPDDSDT；
HCDR3的氨基酸序列为ARQSTYIYGGYYDT；
(2)LCDR1的氨基酸序列为SSNIGNNA；
LCDR2的氨基酸序列为YDD；
LCDR3的氨基酸序列为AAWDDSLNGWV；
HCDR1的氨基酸序列为GYSFTSYW；
HCDR2的氨基酸序列为IYPGDSDT；
HCDR3的氨基酸序列为ARLSYSWSSWYWDF。
在一些实施方案中，所述轻链可变区包括SEQ ID NO：8所示的氨基酸序列，所述重链可变区包括SEQ ID NO：9所示的氨基酸序列；或者所述轻链可变区包括SEQ ID NO：11所示的氨基酸序列，所述重链可变区包括SEQ ID NO：12所示的氨基酸序列。。
在一些实施方案中，所述单链抗体包括SEQ ID NO：7或10所示的氨基酸序列。
另一方面，本文提供了经修饰的T细胞，其表达前述嵌合抗原受体。
另一方面，本文提供了治疗细胞表面表达CD19的肿瘤的药物，其包括上述T细胞。
另一方面，本文提供了分离的核酸分子，其编码前述全人源抗体或其单链抗体或片段。
在一些实施方案中，编码所述全人源抗体的轻链可变区的序列包括SEQ ID NO：2所示的核苷酸序列，编码重链可变区的序列包括SEQ ID NO：3所示的核苷酸序列；或者编码所述人源抗体的轻链可变区的序列包括SEQ ID NO：5所示的核苷酸序列，编码重链可变区的序列包括SEQ ID NO：6所示的核苷酸序列。
在一些实施方案中，编码所述单链抗体的序列包括SEQ ID NO：1或4所示的核苷酸序列。
另一方面，本文提供了包括前述核酸分子的表达载体。
在一些实施方案中，所述表达载体还包括表皮生长因子受体(EGFR)或截短形式的EGFR(tEGFR)的编码序列。
本文提供的全人源抗体比鼠源抗体或经人源化的鼠源抗体具有更低的免疫原性，在抗体药物或CAR-T应用上有更好的潜力。与采用鼠源抗体的CAR-T细胞相比，利用本文提供的全人源抗体构建的CAR-T细胞与人体具有更好的相容性，有利于其在体内的长期增殖和存活。
附图说明
图1为表达在细胞表面的嵌合抗原受体(CAR)的结构示意图。CAR包括细胞外结合区，用于与特定靶抗原(如CD19)结合，其通常为单链抗体(scFv)形式；细胞膜和细胞外结合区之间的铰链区；胞质结构域，用于转导细胞外结合区的结合信号并激活细胞。
图2显示了本发明从噬菌体抗体库筛选靶向CD19的特异抗体的大体流程。
图3显示利用不同sgRNA敲除Raji细胞所获得的细胞克隆的流式细胞分析结果。
图4显示了所淘选的噬菌体单克隆与靶抗原和对照抗原的酶联免疫吸附测定(ELISA)结果。Control 1为FMC-63(人源化小鼠抗人CD19噬菌体抗体克隆)；Control 2为非CD19结合scFv噬菌体抗体克隆。
图5显示了部分噬菌体单克隆与Raji和Raji-CD19ko细胞结合的流式细胞分析结果。
图6显示了所筛选的噬菌体单克隆与多种不同的CD19阳性和阴性细胞系的结合的流式细胞分析结果(MFI值)。Control 1为FMC-63(人源化小鼠抗人CD19噬菌体抗体克隆)；Control 2为阴性对照噬菌体抗体克隆。
图7显示了FMC-63抗体与#62、#78、FMC-63噬菌体的竞争结合实验结果。
图8显示了报告基因法筛选CAR分子的实验原理示意图。
图9显示了各种靶细胞对报告基因法构建的CAR-T细胞的激活情况，结果以荧光素酶产生的化学发光强度表示。
图10显示了报告基因法实验中所用的5种靶细胞的表面CD19表达情况。
图11显示了不同靶细胞对CAR-T细胞的CD107a脱粒作用结果。
图12显示了CAR-T细胞对多种靶细胞(Nalm-6、Reh、Jvm-2、Jeko-1、Bv173、K562-CD19、K562、Thp-1和Skm-1)的杀伤结果。
图13为用丝裂霉素处理后的Raji细胞刺激CAR-T 2次后，CAR-T细胞对Nalm-6、Reh、Skm-1、和Thp-1细胞的杀伤情况。
图14为FMC-63、#62、和#78scFv与CD19抗原的亲和力测定过程示意图。
图15显示了FMC-63、#62、#78scFv与CD19抗原的亲和力测定的动态结合曲线及KD、kon、kdis参数。
图16显示了#78抗体的细胞膜蛋白质组阵列(MPA)实验结果。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
抗体指由浆细胞(效应B细胞)分泌、被机体免疫系统用来中和外来物质(多肽、病毒、细菌等)的免疫球蛋白。该外来物质相应地称作抗原。抗体分子的基本结构是由2个相同重链和2个相同轻链组成的4聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。在可变区中，某些区域氨基酸残基的组成和排列次序比可变区内的其它区域(骨架区，FR)更易变化，称为高变区(HVR)，高变区实际 上是抗体与抗原结合的关键部位。由于这些高变区序列与抗原决定簇互补，故又称为互补决定区(complementarity-determining region,CDR)。重链和轻链均具有三个互补决定区，分别称为HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3。
单链抗体(single chain fragment variable,scFv)，是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠，来自重链和轻链的可变区通过非共价键相互作用形成Fv段，因而scFv能较好地保留其对抗原的亲和活性。
鼠源抗体是由鼠类针对特异抗原产生的抗体，通常指小鼠B淋巴细胞产生的抗体。在大多情况下，该鼠源抗体为杂交瘤细胞产生的单克隆抗体。本发明的全人源抗体是从人源噬菌体抗体库筛选获得，其相对于鼠源抗体降低了免疫原性，更利于人体的治疗用途。
嵌合抗原受体(CAR)，也称为嵌合T细胞受体、嵌合免疫受体，为一种工程化的蛋白受体分子，其可将期望的特异性赋予免疫效应细胞，例如与特定肿瘤抗原结合的能力。嵌合抗原受体通常由胞外抗原结合结构域、跨膜结构域和胞内信号结构域构成。多数情况下，抗原结合结构域为一段scFv序列，负责识别和结合特定的抗原。胞内信号结构域通常包括免疫受体酪氨酸活化基序(ITAM)，例如来源于CD3ζ分子的信号传导结构域，负责激活免疫效应细胞，产生杀伤作用。另外，嵌合抗原受体还可在氨基端包括负责新生蛋白在细胞内定位的信号肽，以及在抗原结合结构域和跨膜结构域之间包括铰链区。除了信号传导结构域，胞内信号结构域还可包括例如来源于4-1BB分子的共刺激结构域。
CAR-T细胞指表达CAR的T细胞，通常采用编码CAR的表达载体转导T细胞获得。常用的表达载体为病毒载体，例如慢病毒表达载体。经嵌合抗原受体修饰的T细胞(CAR-T)不受主要组织相容性复合体的限制，具有特异性靶向杀伤活性和持久扩增的能力。
CD19为B淋巴细胞表面标志分子，在调节B细胞活化和发育中起作用。CD19不仅在正常B细胞上表达，还在很多恶性B细胞肿瘤上表达，这构成了靶向CD19的CAR-T在临床上治疗B细胞相关肿瘤的基础。
研究概述：
我们应用大容量噬菌体抗体库筛选全人源的CD19特异抗体，并通过功能实验来评估这些抗体在CAR-T水平的肿瘤细胞杀伤有效性和安全性。最终，我们获得了若干特异性和有效性良好的全人源抗体克隆。这些全人源抗体会继续通过实验进一步评估，选出最优的候选克隆，应用于CD19 CAR-T产品开发。
我们使用不同的抗体库，经过重组CD19蛋白淘选和蛋白/细胞交替淘选，总共挑选了894个单克隆进行酶联免疫吸附测定(ELISA)初筛，其中176个克隆特异结合 CD19-hFc-Bio蛋白而不结合对照蛋白BCMA-hFc-Bio(蛋白淘选/ELISA初筛)。测序后得到了79种不同的单克隆序列。随后，我们通过流式细胞术(FACS)测定了这些单克隆与CD19阳性细胞系Raji和CD19敲除Raji细胞系(Raji-CD19ko)的结合情况，从中筛选到了13种能与细胞表面CD19抗原特异结合的单克隆抗体。将这13种抗体进一步与多种CD19阳性(Raji、JVM-2、K562-CD19)和阴性细胞系(Raji-CD19ko、Jurkat、K562)进行流式细胞分析(FACS)鉴定，其中2个克隆(#62和#78)在多个细胞系上都表现出良好的特异性。将这2个克隆(#62和#78)构建成CAR-T形式，进行体外功能实验。结果表明，这些克隆在CAR-T水平能被CD19阳性的细胞系激活NFAT信号通路，并表达CD107a蛋白(CAR-T细胞启动杀伤功能的标志物)，且能特异杀伤CD19阳性细胞系而不杀伤CD19阴性细胞系，具有与对照鼠源单抗FMC63CAR-T相似的活性与特异性。这些克隆的获得和初步功能验证为后续开发全人源的CD19 CAR-T产品奠定了基础。总体项目流程如图2所示。
上述2个克隆的测序结果如下：
SEQ ID NO：1#62 scFv DNA序列：765bp
Figure PCTCN2020091235-appb-000001
SEQ ID NO：2#62 VL DNA序列：339bp
Figure PCTCN2020091235-appb-000002
Figure PCTCN2020091235-appb-000003
SEQ ID NO：3#62 VH DNA序列：369bp
Figure PCTCN2020091235-appb-000004
SEQ ID NO：4#78 scFv DNA序列：753bp
Figure PCTCN2020091235-appb-000005
SEQ ID NO：5#78 VL DNA序列：333bp
Figure PCTCN2020091235-appb-000006
Figure PCTCN2020091235-appb-000007
SEQ ID NO：6#78 VH DNA序列：363bp
Figure PCTCN2020091235-appb-000008
SEQ ID NO：7#62 scFv氨基酸序列：255aa
Figure PCTCN2020091235-appb-000009
SEQ ID NO：8#62 VL氨基酸序列：113aa
Figure PCTCN2020091235-appb-000010
SEQ ID NO：9#62 VH氨基酸序列：123aa
Figure PCTCN2020091235-appb-000011
SEQ ID NO：10#78 scFv氨基酸序列:251aa
Figure PCTCN2020091235-appb-000012
Figure PCTCN2020091235-appb-000013
SEQ ID NO：11#78 VL氨基酸序列：111aa
Figure PCTCN2020091235-appb-000014
SEQ ID NO：12#78 VH氨基酸序列：121aa
Figure PCTCN2020091235-appb-000015
对应的抗原决定簇的氨基酸序列如下表所示：
表1抗原决定簇氨基酸序列
Figure PCTCN2020091235-appb-000016
以下结合具体实施例详细说明本发明。
实施例1.CD19敲除的细胞系Raji-CD19ko的构建
为了从噬菌体抗体库(全人源抗体库，包括天然库和半合成库)淘选特异性单克隆抗体，可以让该噬菌体抗体库分别与表达和不表达靶抗原的细胞接触，通过结合情况淘选或鉴定特异性抗体。较好的方式是构建敲除了靶抗原的编码基因的细胞。这样，一对细胞的差别主要在于这一个被敲除的基因。这对细胞将在单克隆抗体结合特异性鉴定以及亲和淘选方面都有很重要的应用。为此，我们用CRISPR/Cas9技术，将高表达CD19的Raji细胞上CD19基因敲除，并通过单克隆筛选，获得了CD19阴性的单克隆化的Raji-CD19ko细胞。
简要实验步骤如下：
1)设计多种sgRNA序列(sgRNA1至sgRNA6)，并进行引物合成；
2)PCR制备sgRNA转录模板；
3)sgRNA反转录及过柱纯化；
4)sgRNA/Cas9 RNP电转Raji细胞；
5)FACS检测不同sgRNA的基因敲除效率，挑选敲除效率较高的细胞池，用有限稀释法分离单克隆；
6)单克隆的FACS鉴定及敲除效率的分子生物学鉴定；
7)Raji-CD19ko细胞库构建及保存。
主要材料和试剂：
制备sgRNA所需oligoDNA，上海生工生物工程有限公司
TranscriptAid T7 High Yield Transcription Kit,Thermo,K0441
TrueCut TMCas9 Protein v2,thermo,A36498
PE anti-human CD19 Antibody,Biolegend,302254
实验结果：
如图3所示，sgRNA5对应的细胞CD19敲除效率最高，有75.4％的CD19被敲除。因此我们选择sgRNA5电转的细胞池进行单克隆的筛选。采用有限稀释法从该细胞池分离单克隆，等单克隆扩增起来后，通过FACS检测这些单克隆的CD19表达。结果如表2所示，其中Raji-CD19ko-1、11、14号克隆的CD19表达基本检测不到，可以认为是敲除成功的单克隆。将这3个克隆进行培养冻存。我们在随后的研究中使用克隆1。
表2经CD19敲除的Raji细胞的CD19表达情况
编号 活细胞下CD19+比例
Raji-CD19ko-1 0.055
Raji-CD19ko-7 37.5
Raji-CD19ko-8 42.8
Raji-CD19ko-11 0.074
Raji-CD19ko-14 0.28
Raji 98.2
实施例2.通过亲和淘选从噬菌体抗体库富集靶向CD19蛋白的特异抗体克隆
采用合适的负淘选和正淘选策略从噬菌体抗体库中富集我们所需的特异性抗体克隆。
噬菌体抗体库的构建
我们构建了半合成噬菌体抗体库，与天然库一起使用，解决天然库可能缺乏CD19高亲和力抗体克隆的问题。CD19是人体内B淋巴细胞正常表达的抗原，对于这类抗原，机体会通过克隆筛选的机制，使那些能表达CD19抗体的B细胞在发育过程中失活，导致正常人体内会缺乏这类抗原的高亲和力抗体。克隆筛选是机体正常的自我识别和自我保护机制。但是，最常用的噬菌体抗体库形式是天然库，它是通过直接克隆健康人淋巴细胞中抗体基因的方法构建的，其中很可能缺乏针对CD19这样人体内正常存 在抗原的抗体克隆。出于这方面的考虑，我们在构建抗体库的时候不仅构建了天然库，同时还构建了半合成抗体库。半合成抗体库由来自天然抗体序列的轻链以及重链FR1-FR3和人工设计的重链CDR3组成，可以大大增加抗体多样性，提高筛选到针对体内正常存在抗原(如CD19)的高亲和力抗体的机会。
CD19蛋白淘选
以BCMA-Fc作为负淘选蛋白，以CD19-Fc作为正淘选蛋白进行多轮淘选，获得富集目的抗体克隆的噬菌体池(pool)。实验步骤简述如下：
1)将带Fc标签的靶抗原(CD19-Fc)或者对照抗原(BCMA-Fc)包被高结合96孔ELISA板，并用封闭液封闭ELISA板；
2)加入噬菌体文库(含1x10 12个噬菌体颗粒)和对照抗原一起孵育，以便扣除非特异结合Fc标签或者封闭液成分的噬菌体抗体克隆；
3)孵育后将上清转移到包被好靶抗原的板中，继续孵育，使噬菌体和靶抗原结合；
4)用洗涤液洗涤固体载体的表面，将未结合的噬菌体洗去；
5)用洗脱液将阳性噬菌体从靶抗原上洗脱下来；
6)以洗脱后的噬菌体重新感染宿主菌XL1-blue，扩增回收的噬菌体。留少量样品梯度稀释，感染宿主菌，涂Amp抗性平板，计算回收噬菌体数量；
7)重复步骤1)至6)，通常需要进行3轮至4轮淘选，直到观察到噬菌体的回收率(洗脱噬菌体数/投入噬菌体数)有明显上升。
富集好的噬菌体池可用于进行随后的单克隆挑选以及ELISA/FACS鉴定。
Raji/Raji-CD19ko细胞淘选
以实施例1制备的Raji-CD19ko细胞作为负淘选细胞，以Raji细胞作为正淘选细胞进行多轮淘选，以获得富集了目的抗体克隆的噬菌体池。
简要实验步骤如下：
1)将经蛋白淘选后富集了特异性克隆的噬菌体池(含1x10 12个噬菌体颗粒)与1x10 7个负淘选细胞Raji-CD19ko混匀，在旋转混合仪上，室温孵育2小时。让与负淘细胞系结合的抗体克隆与这些细胞充分结合；
2)以1500rpm离心5分钟，沉淀细胞，将上清转移到新管中，与5x10 6个Raji细胞(CD19阳性细胞)混匀，在旋转混合仪上，室温结合2小时；
3)用PBS洗涤细胞6次，每次吸弃上清，重悬后1500rpm离心5分钟，以去除未结合的噬菌体；
4)用洗脱液将阳性噬菌体从靶抗原上洗脱下来；
5)用洗脱后的噬菌体重新感染宿主菌，扩增回收的噬菌体。留少量样品梯度稀释，感染宿主菌，涂Amp抗性平板，计算回收噬菌体数量；
6)重复步骤1)至5)，通常需要进行3至4轮淘选，直到观察到噬菌体的回收率(洗脱噬菌体数/投入噬菌体数)有明显上升。
富集好的噬菌体池可进行下一步的单克隆挑选及ELISA/FACS鉴定。
主要材料和试剂：
全人源噬菌体抗体库，包含天然库与半合成库；
辅助噬菌体KO7，Thermo/Invitrogen,18311019；
Human CD19(20-291)Protein,Fc Tag,ACRObiosystem,CD9-H5251；
Human BCMA/TNFRSF17 Protein,Fc Tag,ACRObiosystem,BC7-H5254；
High binding ELSIA plate,Costar，#3590
封闭液：PBS+3％BSA
漂洗液：PBS+0.1％Tween20
洗脱液：1mg/mL Trypsin in PBS
实验结果：
使用不同的抗体库，通过3轮蛋白淘选，每个淘选都观察到了回收率的显著上升(表3)，证明抗体克隆得到了有效富集。
表3蛋白淘选实验结果
Figure PCTCN2020091235-appb-000017
可以看到经过3轮淘选，不同的抗体库都得到了富集(第3轮回收率比上一轮显著提高)。但在后续的FACS实验中，我们从这些噬菌体池挑选的克隆都不能与高表达CD19抗原的Raji细胞系结合，即它们不能识别细胞表面天然状态的CD19抗原。因此，我们在随后的实验中采用了蛋白和细胞交替淘选的方法从另外的噬菌体抗体库分离特 异性抗体克隆。表4显示了利用重组CD19蛋白和Raji/Raji-CD19ko细胞系联合淘选的结果。从回收率来看，所有5个淘选都得到了富集，可以用于下一步挑选单克隆。
表4蛋白/细胞淘选实验结果
Figure PCTCN2020091235-appb-000018
实施例3.采用酶联免疫吸附测定(ELISA)和流式细胞术(FACS)从经富集的噬菌体池筛 选特异性克隆
目的和原理：通过亲和淘选步骤富集的噬菌体池中包含各种性质的噬菌体抗体：特异克隆、非特异克隆、以及阴性克隆。为了获得特异克隆，我们需要从中分离单克隆，包装成单克隆的噬菌体，并通过酶联免疫检测(ELISA)对大量单克隆进行初筛，从 中挑选到特异结合CD19蛋白的单克隆。对于这些ELISA特异的克隆，再利用流式细胞术进行进一步筛选，确定其能否结合细胞表面天然状态的CD19分子。特异的单克隆可以通过DNA测序确定其中包含的单链抗体序列。
在ELISA初筛中，通过链霉亲和素Streptavidin与生物素Biotin的结合，使得生物素化的靶蛋白(CD19-hFc-Bio)和对照蛋白(BCMA-hFc-Bio)在反应液中更接近于天然状态的抗原构象。只结合CD19-hFc-Bio而不结合BCMA-hFc-Bio和链霉亲和素Streptavidin的被认定为特异克隆。FACS初筛使用CD19高表达的细胞系Raji和敲除了CD19分子的Raji-CD19ko来进行，只结合Raji细胞且不结合Raji-CD19ko细胞的被认定为特异克隆。通过ELISA和FACS两种初筛，我们可以获得既能结合重组表达的CD19蛋白，又能识别细胞表面天然状态CD19分子的候选抗体，供随后进一步筛选。
ELISA实验简要步骤：
1)用深孔96孔板培养和包装单克隆噬菌体；
2)将Strepavidin用PBS稀释到2μg/mL，以100μL/孔加入到高结合酶标板中，室温结合2h；
3)弃掉包被液，每孔加入250μL封闭液，4℃封闭过夜；
4)250μL漂洗液洗板2次；
5)将带生物素标签的靶蛋白和对照蛋白用PBS稀释至2μg/mL，以100μg/孔加入预包被Strepavidin的酶标板中，室温结合1h；
6)250μL漂洗液洗板2次；
7)加入100μL步骤1)培养好的噬菌体上清到包被好靶抗原和对照抗原的孔，室温结合2h；
8)250μL漂洗液洗板4次；
9)加入1:2000稀释的mouse anti M13一抗，100μL/孔，室温孵育45min；
10)250μL漂洗液洗板4次；
11)加入1:1000稀释的HRP Donkey anti-Human IgG，100μL/孔，室温孵育45min；
12)250μL漂洗液洗板6次；
13)加入100μLTMB显色底物，显色5至10min；
14)加入100μL 2M H 2SO 4终止反应，在酶标仪上读取结果。
FACS初筛实验简要步骤：
1)用深孔96孔板培养和包装单克隆噬菌体；
2)Raji和Raji-CD19ko细胞用PBS洗2次，用PBS重悬成1x10 7/mL浓度，以50μL分装到96孔深孔板中；
3)每孔加入50μL包装好的单克隆噬菌体(1E11pfu/mL)，混匀后，4℃结合2h；
4)200μL PBS洗涤2次；
5)加入1:2000稀释的mouse anti M13一抗，100μL/孔，吹打混匀后，室温孵育45min；
6)200μL PBS洗涤2次；
7)加入1:300稀释的FITC horse anti mouse-IgG(H+L)，100μL/孔，吹打混匀后，室温孵育45min；
8)200μL PBS洗涤2次；最后用200μL PBS重悬细胞；
9)在流式细胞仪上检测样品FITC通道的荧光强度，分析结果。
主要材料和试剂：
辅助噬菌体KO7，Thermo/Invitrogen,18311019
Streptavidin,Pierce,21125
Biotinylated Human CD19,Fc Tag,ultra sensitivity(primary amine labeling),ACRObiosystem,CD9-H8259；
Biotinylated Human BCMA/TNFRSF17Protein,Fc Tag,Avi Tag(Avitag TM),ACRObiosystem,BC7-H82F0；
High binding ELSIA plate,Costar,#3590
Corning 96Well Clear Round Bottom TC-Treated Microplate,Costar,#3799
封闭液：PBS+3％BSA
漂洗液：PBS+0.1％Tween20
可溶型单组分TMB底物溶液,Tiangen,PA-107-02
Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225
HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306
FITC horse anti mouse-IgG(H+L),Vector,FI2000
实验结果：
从富集后的噬菌体抗体池随机挑选单克隆，包装成噬菌体后，通过噬菌体ELISA检测单克隆噬菌体与CD19-hFc-Bio蛋白、BCMA-hFc-Bio蛋白的结合，找到CD19特异的噬菌体抗体克隆。部分克隆的ELISA结果如图4所示。Control1为FMC-63(人源化小鼠抗人CD19噬菌体抗体克隆)；Control2为非CD19结合scFv噬菌体抗体克隆。从图中可知，#1、4、62、78号克隆与靶抗原CD19(CD19-hFc-Bio)结合良好，且不与对照抗原BCMA(BCMA-hFc-Bio)及链霉亲和素Streptavidin结合，特异性良好。#58和#59号克隆与靶抗原CD19(CD19-hFc-Bio)、不相关抗原BCMA(BCMA-hFc-Bio)及链霉亲和素Streptavidin都不结合，为阴性克隆。#79和#81号克隆能与靶抗原CD19(CD19-hFc-Bio)结合，但与对照抗原BCMA(BCMA-hFc-Bio)也可结合，为非特异性克隆。
部分克隆的FACS初筛结果如图5所示。其中箭头所指的#62号克隆不结合Raji-CD19ko，结合Raji细胞，是特异克隆；其它克隆是非特异(2种细胞都结合)或阴性克 隆(2种细胞都不结合)。通过ELISA检测和FACS初筛，我们总共获得13个特异性克隆。
实施例4.采用多个细胞系通过FACS鉴定单克隆特异性
实验目的和原理：用于治疗的抗体必须具有非常好的靶点特异性，只结合靶抗原，而不结合任何无关的抗原；另一方面，不同的细胞系上同一抗原的氨基酸序列会有差异(异构体或突变体)或结合的配体不一样，也需要考察我们的抗体能否与各种靶蛋白阳性的细胞都结合。为了进一步分析这些单克隆的特异性和普适性，寻找最佳的候选克隆，我们通过流式细胞术进一步评估初筛克隆的特异性。在这个实验中，我们采用多种CD19阳性的细胞系和多种CD19阴性的细胞系与这些单克隆噬菌体抗体进行反应，分析这些克隆是否可以结合不同的细胞系上的CD19抗原，以及是否与其它不表达CD19的细胞系有任何非特异的结合。通过这个实验，我们获得了若干具有优良特异性的克隆。这些克隆将用于构建成CAR-T的形式，通过CAR-T功能实验进一步进行筛选。
实验方法：与FACS初筛相同；
主要样品和试剂：
Raji细胞系，CD19阳性细胞系；
Raji-CD19ko细胞系，CD19阴性细胞系；
JVM2,CD19阳性细胞系；
Jurkat,CD19阴性细胞系；
K562-CD19,CD19阳性细胞系；
K562,CD19阴性细胞系；
其余试剂与FACS初筛相同。
实验结果：
用于治疗的抗体必须具有非常好的靶点特异性。为了进一步分析这些单克隆抗体的特异性，我们将实施例3获得的多个克隆在更多的细胞系上应用流式细胞术进行了鉴定。结果显示在表5和图6中。Control 1为FMC-63(人源化小鼠抗人CD19噬菌体抗体克隆)；Control2为阴性对照噬菌体抗体克隆。#62和#78号克隆与3种CD19阳性细胞系都结合，中位荧光强度(MFI)高，与CD19阴性细胞系都不结合，MFI低，特异性良好。而#50号及#52号克隆虽然与CD19阳性细胞系Raji结合，且不与CD19敲除Raji细胞(Raji-CD19ko)、CD19阴性细胞系Jurkat及CD19阴性细胞系K562结合，但它们不结合CD19阳性细胞系JVM-2及CD19阳性细胞系K562-CD19，说明其结合可能为非特异性结合，不符合实验需要。
表5采用多细胞系的FACS鉴定结果(MFI)
Figure PCTCN2020091235-appb-000019
实施例5.流式竞争法判断特异克隆的结合表位
实验目的和原理：
FMC63是应用最广的抗CD19鼠源克隆，利用该克隆进行的CAR-T疗法已经获得了令人印象深刻的临床结果，这可能与该克隆和CD19抗原结合表位的特性有关。为了确定我们所获得的特异克隆是否靶向与FMC63识别重叠的CD19表位，我们进行了该流式竞争实验。本实验将待测克隆的phage与不同浓度梯度的FMC63抗体预先混匀后再与阳性靶细胞NALM6进行结合，通过孵育一抗mouse anti M13抗体及二抗FITC horse anti mouse-IgG(H+L)抗体后检测FITC荧光强度确定FMC63抗体是否对#62phage及#78 phage与阳性靶细胞NALM6的结合有影响从而判断这两个克隆是否具有与鼠源FMC63衍生的scFv相似的结合特性。
流式竞争实验基本步骤：
1)抗体梯度稀释：根据FMC63 Ab及CD22(克隆：M971)抗体原始浓度将其稀释成400μg/mL，再依次稀释成40μg/mL、4μg/mL、0.4μg/mL、0.04μg/mL、0.004μg/mL，每孔各加入50μL抗体。
2)噬菌体的包装及稀释：如前述将#62噬菌体，#78噬菌体及FMC63噬菌体进行KO7感染和包装，根据原始滴度将其稀释成4x10 10pfu/mL及2x10 10pfu/mL两种浓度，每孔各加入50μL噬菌体，与抗体预先混匀。
3)将NALM6细胞用PBS洗一遍后用PBS重悬成6x10 6/mL，每孔加入50μL细胞及100μL预混好的抗体噬菌体混合液，混匀，4℃孵育1小时。
4)PBS洗板2次，加入PBS 1000x稀释的mouse anti M13抗体100μL/孔，4℃孵育0.5小时。
5)PBS洗板2次，加入PBS 100x稀释的FITC horse anti mouse-IgG(H+L)抗体100μL/孔，4℃孵育0.5小时。
6)PBS洗板2次，流式上机。
7)用FLOWJO软件分析，计算MFI并用graphpad prism软件绘制曲线。
主要样品和试剂：
靶细胞NALM6
FMC63Ab及CD22(克隆：M971)抗体，IASO BIO自主表达
辅助噬菌体KO7，Thermo/Invitrogen,18311019
Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225
FITC horse anti mouse-IgG(H+L),Vector,FI2000
实验结果：
结果显示在图7中。根据MFI可绘制出的曲线可知，随着FMC63抗体浓度的增加，#62、#78、FMC63噬菌体MFI均下降，出现竞争抑制且具有剂量依赖性，而CD22(克隆：M971)抗体对FMC63噬菌体MFI无明显影响，说明#62、#78及FMC63抗体与CD19抗原作用时结合表位有交叉，#62、#78及FMC63可能具有与CD19抗原相似的结合特性。
实施例6.报告基因法筛选CAR分子
实验目的和原理：
为了确认实施例4获得的这些特异克隆构建成CAR-T后，能不能特异识别靶细胞和激活CAR-T细胞，我们开发了一种高效CAR-T筛选方法，即报告基因法，其原理如图8所示。CAR-T细胞的激活是通过CAR分子胞内区中CD3ζ和共刺激因子来实现的，其中CD3ζ可以激活细胞中NFAT信号通路，这是CAR-T细胞激活的一个必要条件。因此，可以通过NFAT报告基因方法，筛选出具有激活NFAT信号通路功能的CAR分子。本实验中采用整合了NFAT-ffLuc(ffLuc，萤火虫荧光素酶)报告基因的Jurkat细胞作为报告细胞。将CAR编码核酸分子通过质粒电转的方式瞬时表达在报告细胞的表面。当表达CAR分子的报告细胞和靶细胞共孵育后，靶细胞表面抗原可以特异性激活CAR分子，进而激活报告基因的表达。然后，通过检测荧光素酶的活性，可以评价CAR分子激活NFAT信号通路的能力。该质粒还包括编码截短的EGFR(tEGFR)的序列，在tEGFR表达于细胞表面时，可用于标记成功表达了CAR的细胞。此外，由于不同CAR分子在电转时的效率不同，我们用与CAR编码核酸分子共混的内参质粒(CMV-hRLuc，编码海肾荧光素酶)来标定电转效率。通过相比包装慢病毒，再感染T细胞制备CAR-T来检测功能的传统方法，本报告基因法步骤简单，可以快速高效地初步评估候选CAR分子识别肿瘤细胞能力以及特异性。
报告基因法简要实验步骤：
1)将编码待测CAR分子的质粒和内参质粒按照固定比例混合后，用电转方法转染报告细胞；
2)转染后48h，取部分转染后的报告细胞，用PE-anti human EGFR antibody染色，通过流式方法检测CAR分子的表达情况；
3)转染后72h，将报告细胞和靶细胞按照1:1的比例混合后，分别铺入U底96孔板中孵育24h；其中每孔中加入3x10 4个报告细胞，每种靶细胞设置3个复孔；
4)孵育完成后，在4℃下以1000g离心5min，移除培养上清后，每孔用100μL裂解液裂解细胞，从中取20μL细胞裂解液用于荧光素酶活性检测。
主要样品和试剂：
靶细胞Raji、JVM2、REH、K562、Raji-CD19ko；
内参质粒pGL4.75，淼灵生物，P0211；
电转试剂盒,Celetrix,Cat.No.1207；
PE-anti human EGFR,BioLegend,Cat.No.352904；
双荧光素酶检测试剂盒，翊圣，11402ES60；
Corning 96 Well Clear Round Bottom TC-Treated Microplate,Costar,#3799。
实验结果：
通过报告基因方法，得到如图8所示结果。其中，Raji、JVM2和REH为表达CD19抗原的阳性靶细胞，它们具有不同的CD19抗原表达密度(如图9所示)。Raji-CD19ko和K562作为阴性靶细胞，表面不表达CD19抗原。PXL092为编码FMC63-bbz的阳性参照CAR分子。57-1、62-1、69-1、70-1为待测的CAR分子，其中62-1采用上文描述的62号噬菌体克隆构建。表面表达CAR分子的报告细胞在单独孵育时(Buffer组)，会产生较弱的背景信号。背景信号可能来源于报告基因上游启动子的影响，也可能是CAR分子自发激活产生的。因此背景信号越高，CAR分子产生自发激活的可能性越大。当报告细胞和阴性靶细胞共孵育时，如果CAR分子没有非特异性的结合，所得到的信号应该和Buffer组相同。当报告细胞和阳性靶细胞共孵育时，CAR分子会受阳性靶细胞表面抗原特异性的激活产生信号，并且信号强度和抗原密度相关。图中显示，62-1号CAR分子具有和对照PXL092相近的激活NFAT信号通路的能力，并且没有明显的非特异性激活问题。57-1和69-1号CAR分子也可以激活NFAT信号通路，但是相较PXL092要差一些。70-1号CAR分子有较强的背景激活信号，并且受阳性靶细胞特异性的能力很弱。
实施例7.CAR-T细胞的体外功能验证
实验目的和原理：
经上文描述的报告基因法初步筛选获得具有激活功能的CAR分子在CAR-T细胞上的功能需要进一步确认。为此，我们制备了这些克隆的慢病毒载体，并转导T细胞制备成CAR-T细胞。然后，通过CD107a脱粒实验(CD107a degranulation assay)和体外细胞杀伤实验(in vitro cytotoxicity assay)进行CAR-T细胞的体外生物学效力评估。通过 这些CAR-T水平的功能验证，我们最终选择到了有效性和安全性都理想的候选单链抗体克隆，进行下游的CAR-T产品开发。
CD107a脱粒实验
CD107a是细胞内微囊泡的标志物，当负载有颗粒酶的微囊泡与细胞膜融合后，细胞膜上的CD107a会增加，当用莫能酶素(monesin，购自BioLegend)阻断其回收时，可以定量反映微囊泡释放的强度。当CAR-T受靶细胞上靶抗原刺激后，会造成颗粒酶释放，并可通过流式检测CD107a的增加来判断T细胞的激活情况。
CD107a脱粒简要实验步骤：
1)将待测的CAR-T细胞和靶细胞分别在室温下以300g离心5min，弃上清后，用T细胞培养基重悬为1x10 6个细胞/mL；
2)在24孔板中，分别加入500μL待测的CAR-T细胞和500μL靶细胞，并混匀；
3)在每孔细胞中加入5μL PE/Cy7 mouse anti-human CD107a抗体和1μLmonensin，然后放入细胞培养箱中(37℃,5％CO 2)孵育3h；
4)孵育完成后，从24孔板中取出500μL细胞悬液，在4℃下300g离心5min后弃上清，用1mL PBS+1％HSA洗细胞2次；
5)用100μLPBS+1％HSA重悬细胞，并分别加入5μLAPC mouse anti-human CD8和5μLAlexa Fluor 488 anti-human EGFR抗体(或者FITC-CD19蛋白)，混匀后在冰上避光孵育20min；
6)孵育完成后，用1mL PBS+1％HSA洗细胞3次；用400μLPBS+1％HSA重悬后用流式细胞仪检测。
主要样品和试剂：
靶细胞Raji,REH,NALM6,K562-CD19,K562,Raji-CD19ko,；
Monensin,BioLegend,Cat.No.420701；
PE/Cy7 mouse anti-human CD107a,BD,Cat.No.561348；
APC mouse anti-human CD8,BD,Cat.No.555369；
Alexa Fluor 488 anti-human EGFR,BioLegend,Cat.No.352908；
FITC-CD19蛋白，Acro Biosystems,Cat.No.CD9-HF251。
实验结果：
通过慢病毒转导的方式获得CAR-T细胞，将该CAR-T细胞在体外培养9-12天后进行CD107a脱粒实验。待检测的CAR-T细胞和靶细胞、莫能酶素和CD107a抗体共同孵育3h，CAR-T细胞与靶细胞的细胞密度均为5×10 5个细胞/mL。然后用CD8抗体、EGFR抗体(或者CD19-FITC蛋白)标记样品后，进行流式检测。在FSC:SSC散点图中选取活细胞门(P1)，去除细胞碎片；在P1门中的细胞，经过FSC-H:SSC-A分析选取单分散细胞门(P2)；然后，在P2门中进一步选取CD8阳性的细胞(P3)；最后，在P3门 中，分析EGFR抗体或者CD19-FITC染色呈阳性的细胞(即CAR阳性细胞)中CD107a阳性的比例。分析结果如图11(三次独立实验的结果)所示，提示#62和#78号克隆的CAR-T细胞具有和对照CAR-T细胞(FMC63)相近的CD107a脱粒功能。
体外细胞杀伤实验
实验目的和原理：体外细胞杀伤实验采用Nalm-6、Reh、Jvm-2、Jeko-1、Bv173和K562-CD19作为CD19阳性靶细胞，K562、Thp-1和Skm-1细胞作为CD19阴性靶细胞，进行CD19 CAR-T细胞的抗原特异性杀伤能力评价。其中，以上细胞分别通过慢病毒转导方式，获得稳定表达萤火虫荧光素酶的靶细胞，因此样品中荧光素酶的活性可以反映靶细胞的数量。将CAR-T细胞和靶细胞共孵育培养。当靶细胞被CAR-T细胞杀伤时，荧光素酶会被释放并且很快失活(萤火虫荧光素酶半衰期约0.5h)。如果靶细胞没有被CAR-T细胞杀伤或者抑制，随着靶细胞的扩增和荧光素酶的持续表达，将会产生更多的荧光素酶。因此，可以通过荧光素酶的活性来检测CAR-T对靶细胞的杀伤情况。
体外细胞杀伤简要实验步骤：
1)将上述细胞分别在室温下以300g离心5min，弃上清后，用T细胞培养基重悬为2x10 5个细胞/mL；在96孔板的每孔中分别加入100μL靶细胞；
2)根据待测CAR-T样品的CAR阳性率和效靶比，分别在96孔板的每孔中加入100μLCAR-T细胞，并和靶细胞混匀；然后放入二氧化碳培养箱中孵育培养24h；
3)使用荧光素酶检测试剂盒分别检测每孔样品中的荧光素酶活性。
主要样品和试剂：
靶细胞Nalm-6、Reh、Jvm-2、Jeko-1、Bv173、K562-CD19、K562、Thp-1和Skm-1；
Steady-Glo Luciferase Assay System,Promega,Cat.No.E2520。
实验结果：
将CAR-T细胞样品和固定数量的靶细胞(1x10 4个)按照效靶比(E:T)为4:1的比例混合后，共同孵育24h，然后检测样品中的荧光素酶活性(RLU)。其中，control为只含有靶细胞的对照样品。由于荧光素酶活性可以反映靶细胞在样品中的数量，通过样品中荧光素酶活性的变化，可以得到CAR-T细胞对靶细胞的杀伤/抑制能力。荧光素酶活性读数(RLU)越低，靶细胞被杀伤的越多。
如图12所示，三组CAR-T细胞样品(#62、#78克隆以及对照FMC63制备的CAR-T细胞)在效靶比为4:1时，都可以有效的杀伤阳性靶细胞。当T细胞和阳性靶细胞共孵育时无明显杀伤。所有CAR-T和T细胞样品与阴性靶细胞共同孵育时都无明显杀伤。 因此，三组CAR-T样品都可以特异性的杀伤CD19阳性靶细胞，并且对CD19阴性靶细胞没有非特异性杀伤。
反复刺激后杀伤实验
实验目的和原理：
采用丝裂霉素(Mitomycin)处理过的靶细胞(Raji)与不同组别CD19 CAR-T细胞混合进行3次刺激后将CAR-T细胞和靶细胞共孵育培养，从而确定不同scFv CAR-T在被靶细胞持续多次刺激后的杀伤能力是否出现改变。
反复刺激后杀伤实验简要实验步骤：
1)取Raji细胞4x10 6细胞，300g，室温，离心5min；
2)完全培养基调整密度至0.2x10 6细胞/mL，加入4μL Mitomycin母液(5μg/μL)混匀后培养24h后待用。
3)取处理24h后的Raji-Mitomycin细胞，300g，离心换液，用PBS洗涤6次，CTS培养基重悬Raji-Mitomycin细胞，并计数且调整密度至2x10 6细胞/mL，待用。
4)依据CAR-TCAR阳性率，分别取1x10 5CAR+细胞，转入24孔板中；每孔CAR-T加入Raji-Mitomycin细胞50μL，调整效靶比(效应细胞以CAR+计)E:T＝1：1。用CTS完全培养基补液至培养终体积至500μL，混匀，37℃，5％CO 2培养72h并计数，再次用Mitomycin处理靶细胞(Raji)及检测CAR-TCAR阳性率，重复以上步骤3次后检测CAR-TCAR阳性率，与阳性靶细胞Nalm-6、Reh及阴性靶细胞Thp-1、Skm-1共孵育检测杀伤活性，步骤同体外细胞杀伤。
实验结果：
如图13所示，三组CAR-T细胞样品(#62、#78克隆以及对照FMC63)都可以有效的杀伤阳性靶细胞，且呈剂量依赖性，反复刺激后杀伤强度#78克隆>对照FMC63>#62克隆。当T细胞和阳性靶细胞共孵育时无明显杀伤。所有CAR-T和T细胞样品和阴性靶细胞共同孵育时都无明显杀伤。因此，三组CAR-T样品在阳性靶细胞反复刺激后都可以特异性的杀伤CD19阳性靶细胞，并且对CD19阴性靶细胞没有非特异性杀伤。
实施例8.抗CD19  scFvs亲和力的测定
实验目的和原理：
CD19scFvs与抗原间的亲和力大小可能对CAR-T在患者体内发挥杀伤作用及存续时间有着重要影响，为了确定这一重要性质，我们采用了ForteBio公司的Octet分子相互作用技术对其进行了测定。Octet系统所运用的生物膜干涉技术，是一种免标记技术，实时提供高通量的生物分子相互作用信息。该仪器发射白光到传感器表面并收集反射光，不同频率的反射光谱受到生物传感器的光膜层厚度的影响，一些频率的反射光形成了相长干涉(蓝色)，而另一些受到了相消干涉(红色)。这些干涉被光谱仪所检测到， 并形成一幅干涉光谱，并以干涉光谱的相位位移强度(nm)显示。因此，结合到传感器表面的分子一旦有数量上的增减，光谱仪便会实时地检测到干涉光谱的位移，而这种位移直接反应出传感器表面生物膜的厚度，从中可以获取生物分子相互作用的高质量的数据，从而进行生物分子间相互作用动力学参数测定(Kon,Kdis和KD)，为研发过程提供重要的信息。
简要实验步骤如图14所示：
1)用上样缓冲液(1×PBS，pH 7.4，0.01％BSA和0.02％Tween 20)将抗CD19 scFv-rFcs稀释至20μg/mL，并在生物传感器上上样，约0.8nM。
2)在60s平衡阶段后，在多种抗原浓度(400至12.5nM)下监测CD19抗原的结合动力学。在每个浓度下分别进行至160s结合和300s解离。
3)用10mMGlycine-HCl,pH1.5洗涤3次使芯片再生。
4)通过使用1：1结合位点模型(Biacore X-100评估软件)分析结合常数。
实验结果：
亲和力系指单个分子与其配体结合的强度，通常通过平衡解离常数(KD)进行测定和报告，平衡解离常数可用于评估两分子间相互作用的强度并对此进行排序。抗体与其抗原的结合是一个可逆的过程，结合反应的速率与反应物的浓度成正比。KD值越小，抗体对其靶标的亲和力越大。如图15示，FMC63、#62、#78均可与CD19抗原结合，且亲和力大小排序：FMC63>#78>#62。
实例9.细胞膜蛋白质组阵列(Membrane Proteome Array)
实验目的和原理：
膜蛋白质组阵列(MPA)是一个基于细胞的高通量平台，可用于鉴定与膜蛋白结合的孤立抗体和其他配体的靶标。膜蛋白约占人类基因组编码的所有蛋白的四分之一，通常折叠成构象复杂的结构，难以保留在细胞外。MPA的关键特征是是使膜蛋白可以直接在细胞内以天然状态单独表达和测试，从而保持其结构完整性和天然的翻译后修饰。MPA利用迄今已组装的最大的膜蛋白文库，代表了5000多种独特的膜蛋白。通过MPA平台，我们进行了#78的特异性检测，以验证其是否会与CD19之外的抗原发生非特异性的结合并评估其产生脱靶效应的风险。
简要实验步骤如下：
1)MPA包含约5000种不同的膜蛋白克隆，占人类膜蛋白组的90％以上，每个克隆都在含cDNA的质粒的HEK-293T细胞中过表达，该质粒在384孔细胞培养板的分开的孔中独立转染，36h的孵育时间以保证膜蛋白的表达。
2)在对MPA进行特异性测试之前，在表达阳性(ProteinA)和阴性(空载体转染)对照的HEK-293T和QT6细胞上确定用于筛选的#78抗体的测试浓度，使用AlexaFluor 647标记的二抗，通过流式细胞仪检测。
3)将Clone 78抗体稀释20μg/mL，并使用上述二抗在IntellicytiQue上测试整个蛋白质文库的结合活性。为了确保板对板的兼容性和可重复性，每个阵列板均包括阳性和阴性对照。
4)在连续稀释#78抗体后，第二次流式细胞术实验中再次确认了通过MPA筛选确定的每个靶标是否可重复的结合及是否具有剂量依赖性，并通过测序再次确认了靶标的同一性。
主要样品和试剂：
细胞系：HEK-293T,Q6T
Alexa Fluor靶标的同一性AffiniPure F(ab') 2Fragment Goat Anti-Rabbit IgG,Fc fragment specific,Jackson ImmunoResearch，111-606-046
Goat Serum,Sigma,G6767
实验结果：
如图16所示，在测试初筛阶段，#78抗体与5000多种膜蛋白中绝大多数蛋白都无非特异性的结合，但可观察到与高表达SDC1,Frizzled 4，HTR5A的HEK-293T细胞的结合现象。为了验证验证这一结合是否可重复，我们将#78抗体进行了浓度梯度稀释，并重复了该实验，实验结果显示#78抗体与高表达CD19及阳性对照(ProteinA)的HEK-293T细胞可以结合且其结合的平均荧光强度存在剂量依赖性，在验证实验中，#78抗体在任何浓度下均不与高表达SDC1、Frizzled 4、TR5A的HEK-293T细胞结合，证明#78抗体有较好的特异性，在患者体内脱靶风险较小，安全性好。
本发明使用全人源噬菌体进行抗体筛选，直接获得全人源的单克隆抗体。与传统杂交瘤技术相比，省却了困难的鼠源抗体人源化步骤，而且全人源抗体比人源化的鼠源抗体具有更低的免疫原性，在抗体药物或CAR-T应用上有更好的潜力。
在抗体筛选的过程中，我们发现直接使用重组表达的CD19蛋白筛选到的抗体克隆，都不能与高表达CD19的细胞系Raji结合。这可能是重组表达的CD19蛋白抗原与细胞膜表面的天然状态的CD19由于构象和可及抗原表位有很大的差别所致。为了克服这个问题，我们用CRISPR技术制备了CD19基因敲除的Raji-CD19ko细胞系。通过使用蛋白/细胞系交替淘选的方法，富集了能同时结合重组表达CD19蛋白和Raji细胞的噬菌体抗体，并从中筛选到了能特异结合细胞膜表面CD19抗原的单克隆抗体。
在开发流程上，通过噬菌体水平的抗体筛选/特异性鉴定，快速高效筛选到特异抗体克隆，后面直接衔接CAR-T功能测试，优选出最佳的候选抗体。这个流程跳过了费 时费力的抗体蛋白表达和功能鉴定实验，优化了以CAR-T开发为目的的抗体筛选流程，在保证研究质量的同时，提高了研发效率。
参考文献：
1.Albert T.Gacerez1,Benjamine Arellano1,and Charles L.Sentman,How chimeric antigen receptor design affects adoptive T cell therapy,Cell Physiol.2016 December；231(12):2590–2598.doi:10.1002/jcp.25419
2.Carolina Berger,Mary E.Flowers,Edus H.Warren,and Stanley R.Riddell,Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK–modified donor T cells after allogeneic hematopoietic cell transplanttation,Blood.2006Mar 15；107(6):2294-302.Epub 2005 Nov 10.
3.Daniel Sommermeyer1,Stanley R.Riddell et.al,Fully human CD19-specific chimeric antigen receptors for T-cell therapy,Leukemia.2017 October；31(10):2191–2199.doi:10.1038/leu.2017.57.
4.Gacerez AT,Hua CK,Ackerman ME,Sentman CL,Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6expression,Cancer Immunol Immunother.2018 May；67(5):749-759.doi:10.1007/s00262-018-2124-1
5.Jiasheng Wang,Yongxian Hu,and He Huang,Acute lymphoblastic leukemia relapse after CD19-targeted chimeric antigen receptor T cell therapy,Volume 102,PP1-10,December 2017 Journal of Leukocyte Biology
6.JieXua,Sai-Juan Chen et.al,Exploratory trial of a biepitopic CAR T-targeting B cell maturation antigen in relapsed/refractory multiple myeloma,Proc Natl Acad Sci U S A.2019 Apr 15.pii:201819745.doi:10.1073/pnas.1819745116
7.Phage display-methods and protocols，ISSN 1064-3745ISSN 1940-6029(electronic) Methods in Molecular Biology ISBN 978-1-4939-7446-7 ISBN 978-1-4939-7447-4(eBook),DOI 10.1007/978-1-4939-7447-4
8.Lamers CH,Gratama JW.et.al,Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells.Blood.2011 Jan 6；117(1):72-82.doi:10.1182/blood-2010-07-294520.
9.Marcela V.Maus1 and Carl H.June,Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy,Clin Cancer Res.2016 April 15；22(8):1875–1884.doi:10.1158/1078-0432.CCR-15-1433.
10.Sauna ZE,Rosenberg AS et.al,Evaluating and Mitigating the Immunogenicity of Therapeutic Proteins.Trends Biotechnol.2018 Oct；36(10):1068-1084
11.Rydzek J,Hudecek M et.al,tigen Receptor Library Screening Using a Novel NF- κB/NFAT Reporter Cell Platform，Mol Ther.2019 Feb 6；27(2):287-29.

Claims (13)

  1. 靶向CD19的全人源抗体或其单链抗体或片段，其中所述全人源抗体的轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3，所述LCDR1、LCDR2、LCDR3、HCDR1、HCDR2、和HCDR3选自如下组合之一：
    (1)LCDR1的氨基酸序列为SSNIGAGYD；
    LCDR2的氨基酸序列为ENT；
    LCDR3的氨基酸序列为QSYDSSLSGWRV；
    HCDR1的氨基酸序列为GYSFTNSW；
    HCDR2的氨基酸序列为IYPDDSDT；
    HCDR3的氨基酸序列为ARQSTYIYGGYYDT；
    (2)LCDR1的氨基酸序列为SSNIGNNA；
    LCDR2的氨基酸序列为YDD；
    LCDR3的氨基酸序列为AAWDDSLNGWV；
    HCDR1的氨基酸序列为GYSFTSYW；
    HCDR2的氨基酸序列为IYPGDSDT；
    HCDR3的氨基酸序列为ARLSYSWSSWYWDF。
  2. 如权利要求1所述的全人源抗体或其单链抗体或片段，其中所述轻链可变区包括SEQ ID NO：8所示的氨基酸序列，所述重链可变区包括SEQ ID NO：9所示的氨基酸序列；或者所述轻链可变区包括SEQ ID NO：11所示的氨基酸序列，所述重链可变区包括SEQ ID NO：12所示的氨基酸序列。
  3. 如权利要求1或2所述的全人源抗体或其单链抗体或片段，其中所述单链抗体包括SEQ ID NO：7或10所示的氨基酸序列。
  4. 靶向CD19的嵌合抗原受体，其包括靶向CD19的单链抗体，所述单链抗体的轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3，其中所述LCDR1、LCDR2、LCDR3、HCDR1、HCDR2、和HCDR3选自如下组合之一：
    (1)LCDR1的氨基酸序列为SSNIGAGYD；
    LCDR2的氨基酸序列为ENT；
    LCDR3的氨基酸序列为QSYDSSLSGWRV；
    HCDR1的氨基酸序列为GYSFTNSW；
    HCDR2的氨基酸序列为IYPDDSDT；
    HCDR3的氨基酸序列为ARQSTYIYGGYYDT；
    (2)LCDR1的氨基酸序列为SSNIGNNA；
    LCDR2的氨基酸序列为YDD；
    LCDR3的氨基酸序列为AAWDDSLNGWV；
    HCDR1的氨基酸序列为GYSFTSYW；
    HCDR2的氨基酸序列为IYPGDSDT；
    HCDR3的氨基酸序列为ARLSYSWSSWYWDF。
  5. 如权利要求4所述的嵌合抗原受体，其中所述轻链可变区包括SEQ ID NO：8所示的氨基酸序列，所述重链可变区包括SEQ ID NO：9所示的氨基酸序列；或者所述轻链可变区包括SEQ ID NO：11所示的氨基酸序列，所述重链可变区包括SEQ ID NO：12所示的氨基酸序列。
  6. 如权利要求4或5所述的嵌合抗原受体，其中所述单链抗体包括SEQ ID NO：7或10所示的氨基酸序列。
  7. 经修饰的T细胞，其表达权利要求4至6任一项的嵌合抗原受体。
  8. 治疗细胞表面表达CD19的肿瘤的药物，其包括权利要求7的T细胞。
  9. 分离的核酸分子，其编码权利要求1至3任一项的全人源抗体或其单链抗体或片段。
  10. 如权利要求9所述的核酸分子，其中编码所述全人源抗体的轻链可变区的序列包括SEQ ID NO：2所示的核苷酸序列，编码重链可变区的序列包括SEQ ID NO：3所示的核苷酸序列；或者编码所述全人源抗体的轻链可变区的序列包括SEQ ID NO：5所示的核苷酸序列，编码重链可变区的序列包括SEQ ID NO：6所示的核苷酸序列。
  11. 如权利要求9或10所述的核酸分子，其中编码所述单链抗体的序列包括SEQ ID NO：1或4所示的核苷酸序列。
  12. 包括权利要求10至11任一项的核酸分子的表达载体。
  13. 如权利要求12所述的表达载体，其还包括EGFR或截短形式的EGFR的编码序列。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022257984A1 (zh) * 2021-06-08 2022-12-15 南京驯鹿医疗技术有限公司 一种增强型嵌合抗原受体(car)细胞的制备及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891108B (zh) * 2022-05-30 2022-11-15 上海驯鹿生物技术有限公司 一种靶向bcma的全人源抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101233156A (zh) * 2005-06-20 2008-07-30 米德列斯公司 Cd19抗体及其用途
CN101636502A (zh) * 2006-12-13 2010-01-27 梅达雷克斯公司 结合cd19的人类抗体及其用途
CN104558181A (zh) * 2014-12-31 2015-04-29 四川大学 靶向b淋巴瘤细胞的人源单链抗体
CN106554414A (zh) * 2015-09-18 2017-04-05 科济生物医药（上海）有限公司 抗cd19全人抗体以及靶向cd19的免疫效应细胞
CN107531793A (zh) * 2015-10-13 2018-01-02 优瑞科生物技术公司 对人类cd19具有专一性的抗体药剂和其用途
CN107880128A (zh) * 2017-12-21 2018-04-06 常州费洛斯药业科技有限公司 一种抗cd19的全人源抗体或抗体片段及其方法和应用
CN109293774A (zh) * 2018-10-16 2019-02-01 南京医科大学 特异性结合cd19的全人源化抗体及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007002223A2 (en) * 2005-06-20 2007-01-04 Medarex, Inc. Cd19 antibodies and their uses
CL2007003622A1 (es) * 2006-12-13 2009-08-07 Medarex Inc Anticuerpo monoclonal humano anti-cd19; composicion que lo comprende; y metodo de inhibicion del crecimiento de celulas tumorales.

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101233156A (zh) * 2005-06-20 2008-07-30 米德列斯公司 Cd19抗体及其用途
CN101636502A (zh) * 2006-12-13 2010-01-27 梅达雷克斯公司 结合cd19的人类抗体及其用途
CN104558181A (zh) * 2014-12-31 2015-04-29 四川大学 靶向b淋巴瘤细胞的人源单链抗体
CN106554414A (zh) * 2015-09-18 2017-04-05 科济生物医药（上海）有限公司 抗cd19全人抗体以及靶向cd19的免疫效应细胞
CN107531793A (zh) * 2015-10-13 2018-01-02 优瑞科生物技术公司 对人类cd19具有专一性的抗体药剂和其用途
CN107880128A (zh) * 2017-12-21 2018-04-06 常州费洛斯药业科技有限公司 一种抗cd19的全人源抗体或抗体片段及其方法和应用
CN109293774A (zh) * 2018-10-16 2019-02-01 南京医科大学 特异性结合cd19的全人源化抗体及应用

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Phage display-methods and protocols", METHODS IN MOLECULAR BIOLOGY, ISBN: 978-1-4939-7446-7
ALBERT T. GACEREZLBENJAMINE ARELLANO!CHARLES L. SENTMAN: "How chimeric antigen receptor design affects adoptive T cell therapy", CELL PHYSIOL, vol. 231, no. 12, December 2016 (2016-12-01), pages 2590 - 2598, XP055753274, DOI: 10.1002/jcp.25419
CAROLINA BERGERMARY E. FLOWERSEDUS H. WARRENSTANLEY R. RIDDELL: "Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK-modified donor T cells after allogeneic hematopoietic cell transplantation", BLOOD, vol. 107, no. 6, 10 November 2005 (2005-11-10), pages 2294 - 302
CASTELLA, M. ET AL.: "Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions.", MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT., vol. 12, 31 March 2019 (2019-03-31), XP055716791, DOI: 20200729182944A *
DANIEL SOMMERMEYERSTANLEY R. RIDDELL: "Fully human CD19-specific chimeric antigen receptors for T-cell therapy", LEUKEMIA, vol. 31, no. 10, October 2017 (2017-10-01), pages 2191 - 2199, XP055482021, DOI: 10.1038/leu.2017.57
GACEREZ ATHUA CKACKERMAN MESENTMAN CL: "Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression", CANCER IMMUNOL IMMUNOTHER, vol. 67, no. 5, May 2018 (2018-05-01), pages 749 - 759, XP036491811, DOI: 10.1007/s00262-018-2124-1
JIASHENG WANGYONGXIAN HUHE HUANG: "Acute lymphoblastic leukemia relapse after CD 19-targeted chimeric antigen receptor T cell therapy", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 102, December 2017 (2017-12-01), pages 1 - 10
JIEXUASAI-JIIAN CHEN: "Exploratory trial of a biepitopic CAR T-targeting B cell maturation antigen in relapsed/refractory multiple myeloma", PROC NATL ACAD SCI U S A., 15 April 2019 (2019-04-15)
LAMERS CHGRATAMA JW: "Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells", BLOOD, vol. 117, no. 1, 6 January 2011 (2011-01-06), pages 72 - 82, XP055234814, DOI: 10.1182/blood-2010-07-294520
MARCELA VMAUS ICARL H: "June, Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy", CLIN CANCER RES., vol. 22, no. 8, 15 April 2016 (2016-04-15), pages 1875 - 1884
RYDZEK JHUDECEK M: "tigen Receptor Library Screening Using a Novel NF- B/NFAT Reporter", CELL PLATFORM, MOL THER., vol. 27, no. 2, 6 February 2019 (2019-02-06), pages 287 - 29
SAUNA ZEROSENBERG AS: "Evaluating and Mitigating the Immunogenicity of Therapeutic Proteins", TRENDS BIOTECHNOL, vol. 36, no. 10, October 2018 (2018-10-01), pages 1068 - 1084, XP085480958, DOI: 10.1016/j.tibtech.2018.05.008
See also references of EP3974452A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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