Screening and application of targeted CD5 fully humanized single-chain antibody

Abstract

The invention belongs to the technical field of cellular immune engineering, and particularly relates to a full-human-derived polypeptide for recognizing CD5 and application of the full-human-derived polypeptide. The polypeptide specifically recognizes CD5 and is good in affinity performance, and the polypeptide specifically targeting CD5 can mark CD5 positive cells, can be combined with CD5 antigens and the CD5 positive cells and can serve as a detection reagent of CD5 molecules and an in-vivo tracing marking reagent. The polypeptide specifically targeting CD5 provided by the invention can also be used in an extracellular antigen recognition region of a chimeric antigen receptor, is used in an immune cell treatment drug targeting CD5, and is used for killing malignant tumors expressing CD5. The polypeptide capable of specifically recognizing CD5 is a natural full-humanized antibody source, compared with a mouse antibody, a chimeric antibody and a humanized antibody, the immunogenicity of the polypeptide is greatly reduced, and the safety can be guaranteed to the maximum extent in clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a fully human ScFv targeting CD5 and an application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) is an artificial receptor that simulates TCR function. CAR molecules generally contain an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain. The intracellular signaling domain is usually the CD3ζ chain or FcRγ, or is connected to one or more co-stimulatory molecules, such as 4-1BB, CD28, and ICOS (CD278).

[0003] CD5 is a T-cell surface glycoprotein, also known as lymphocyte antigen T1 / LEU1 and LEU1. It is phosphorylated on tyrosine residues by LYN, so that CD5 can create binding sites for PTPN6 / SHP-1. It is frequently expressed in about 85% of T-cell malignancies as well as some B-cell malignancies. It acts as an inhibitory receptor for both the T-cell receptor (TCR) and the B-cell receptor (BCR).

[0004] The extracellular antigen recognition domain can also be called the extracellular antigen binding domain, which can specifically recognize tumor surface antigens. The CAR molecule uses the characteristics of specific recognition of tumor antigens by the antigen binding domain, and transmits immune cell activation signals through the hinge region and transmembrane region. The most common extracellular antigen recognition domain is the ScFv (single-chain antibody) that recognizes tumor antigens. The selection of ScFv is crucial for the function of CAR. Therefore, for immunotherapy targeting CD5, the screening of anti-CD5 antibodies or anti-CD5 ScFv is very necessary. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a fully humanized single-chain antibody targeting CD5 and its use in detection reagents and anti-tumor drugs. The ScFv provided by the present invention can specifically recognize CD5, recognize and detect CD5 expressed by cells and its ratio, and can be used as a detection reagent for CD5 or an in vivo tracer marker molecule; the ScFv provided by the present invention can be used as the antigen recognition domain of the chimeric antigen receptor to recognize CD5 on the surface of the tumor, construct a chimeric antigen receptor immune cell, and can effectively inhibit the growth of tumors in the body, and the anti-tumor effect of expressing CD5 targets is significant. In addition, it can be used as an effective ingredient of anti-tumor drugs for malignant tumors with high CD5 expression, and effectively control the proliferation of malignant tumors with high CD5 expression.

[0006] The antigen-binding polypeptide targeting CD5 comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3; specifically, any combination of the following is shown:

[0007] (1) the amino acid sequence of L-CDR1 is QRVASSY, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYNTYPFT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLSNYYYGYGT; or

[0008] (2) the amino acid sequence of L-CDR1 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYGSSPLT, the amino acid sequence of H-CDR1 is GFTFSSYA, the amino acid sequence of H-CDR2 is ISSDGRNK, and the amino acid sequence of H-CDR3 is ARGLLYDYGLDV;

[0009] (3) the amino acid sequence of L-CDR1 is QSISSW, the amino acid sequence of L-CDR2 is KAS, the amino acid sequence of L-CDR3 is QQLNSYPYT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLDYGDYGYYYGMDV;

[0010] (4) The amino acid sequence of L-CDR1 is QGISSA, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQFNSYPLT, the amino acid sequence of H-CDR1 is GFTFSSYE, the amino acid sequence of H-CDR2 is ISSSGSTI, and the amino acid sequence of H-CDR3 is ARGYDYGFAFDI.

[0011] The antigen-binding polypeptide targeting CD5 and the amino acids in the light chain variable region of the single-chain antibody are shown in SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29 or SEQ ID NO.31.

[0012] The antigen-binding polypeptide targeting CD5 and the amino acids in the heavy chain variable region of the single-chain antibody are shown in SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30 or SEQ ID NO.32.

[0013] The single-chain antibody that recognizes CD5 comprises a heavy chain variable region and a light chain variable region, and comprises a sequence shown in any of the following:

[0014] (1) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.26; or

[0015] (2) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.28; or

[0016] (3) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.29, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.30; or

[0017] (4) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.31, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.32; or

[0018] (5) In some embodiments, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.35 or SEQ ID NO.36 or SEQ ID NO.37 or SEQ ID NO.38.

[0019] Furthermore, the antigen-binding polypeptide targeting CD5 also comprises a Linker sequence. In some embodiments, the Linker amino acid sequence is GSTSGSGKPGSGEGSTKG or (GGGGS)n, n=1-5, and the amino acid sequence is shown in SEQ ID NO.33 or SEQ ID NO.34.

[0020] Furthermore, the antigen-binding polypeptide targeting CD5 further comprises a Linker sequence, preferably the sequence shown in SEQ ID NO:33.

[0021] An antibody that recognizes CD5, said antibody comprising the CDRs of SEQ ID NOs: 1-24.

[0022] An antibody that recognizes CD5 may also contain a light chain sequence of SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, or SEQ ID NO.31.

[0023] An antibody that recognizes CD5 may also contain a heavy chain sequence of SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, or SEQ ID NO.32.

[0024] Another object of the present invention is to provide a chimeric antigen receptor targeting CD5.

[0025] The chimeric antigen receptor comprises an extracellular antigen recognition domain that can recognize CD5 antigen, and the extracellular antigen recognition domain that recognizes CD5 antigen comprises CDRs of SEQ ID NO: 1-24; specifically, any combination of the following:

[0026] (1) the amino acid sequence of L-CDR1 is QRVASSY, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYNTYPFT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLSNYYYGYGT; or

[0027] (2) the amino acid sequence of L-CDR1 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYGSSPLT, the amino acid sequence of H-CDR1 is GFTFSSYA, the amino acid sequence of H-CDR2 is ISSDGRNK, and the amino acid sequence of H-CDR3 is ARGLLYDYGLDV;

[0028] (3) the amino acid sequence of L-CDR1 is QSISSW, the amino acid sequence of L-CDR2 is KAS, the amino acid sequence of L-CDR3 is QQLNSYPYT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLDYGDYGYYYGMDV;

[0029] (4) The amino acid sequence of L-CDR1 is QGISSA, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQFNSYPLT, the amino acid sequence of H-CDR1 is GFTFSSYE, the amino acid sequence of H-CDR2 is ISSSGSTI, and the amino acid sequence of H-CDR3 is ARGYDYGFAFDI.

[0030] In some embodiments, the extracellular antigen recognition domain that recognizes the CD5 antigen may also contain a light chain sequence of SEQ ID NO.25 or SEQ ID NO.27 or SEQ ID NO.29 or SEQ ID NO.31, and a heavy chain amino acid sequence of SEQ ID NO.26 or SEQ ID NO.28 or SEQ ID NO.30 or SEQ ID NO.32.

[0031] In some embodiments, the extracellular antigen recognition domain that recognizes the CD5 antigen comprises an amino acid sequence as shown in SEQ ID NO.35 or SEQ ID NO.36 or SEQ ID NO.37 or SEQ ID NO.38.

[0032] In some embodiments, the extracellular antigen recognition domain that recognizes the CD5 antigen comprises a linker sequence of SEQ ID NO.33 or SEQ ID NO.34.

[0033] The chimeric antigen receptor further comprises a hinge region, a transmembrane region and an intracellular recognition domain, and the hinge can be derived from: human IgG4, human CD8, human CD4, human CD28, human CD7, etc.

[0034] Furthermore, the hinge region preferably comprises an amino acid sequence as shown in SEQ ID NO. 40 or a functional variant thereof.

[0035] Furthermore, the transmembrane region may be derived from: α or β chain of T cell receptor, CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 and CD86, etc.

[0036] The transmembrane region preferably comprises an amino acid sequence as shown in SEQ ID NO. 39 or a functional variant thereof.

[0037] The chimeric antigen receptor also comprises an intracellular signaling domain, which comprises a signaling region and a co-stimulatory domain; the intracellular signaling domain comprises a signaling region, which is derived from a primary cytoplasmic signaling sequence containing ITAM, including but not limited to protein molecules such as CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278), FcεRI, CD66d, DAP10 and DAP12.

[0038] The signal transduction region is derived from CD3ζ, and its amino acid sequence is shown in SEQ ID NO.42.

[0039] The chimeric antigen receptor further comprises an intracellular signaling domain, which comprises a signal transduction region and a co-stimulatory domain; the co-stimulatory domain may be derived from: CD28, 41BB, OX40, CD27, DAP10, 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc.

[0040] Furthermore, the co-stimulatory domain comprises an amino acid sequence as shown in SEQ ID NO.41 or a functional variant thereof.

[0041] The chimeric antigen receptor described above further comprises a leader peptide, which comprises an amino acid sequence as shown in SEQ ID NO. 43 or a functional variant thereof. The CD5-targeted antibody or chimeric antigen receptor is positioned on the cell membrane under the guidance of the leader peptide. As known to those skilled in the art, all other signal peptides that can be used in the field of CAR or antibodies can be used in the present invention, such as the extracellular segment generally comprising a CD8α or GM-CSFRα leader peptide.

[0042] The present invention also aims to provide a nucleic acid sequence encoding the aforementioned chimeric antigen receptor.

[0043] In some embodiments, the nucleic acid sequence is combined with a vector as a target gene and transfected into immune cells to obtain engineered immune cells.

[0044] The nucleic acid sequence of the chimeric antigen receptor comprises the nucleic acid sequence of the antigen-binding polypeptide targeting CD5 as shown in SEQ ID NO.52 or SEQ ID NO.53 or SEQ ID NO.54 or SEQ ID NO.55.

[0045] Furthermore, the nucleic acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.48 or SEQ ID NO.49 or SEQ ID NO.50 or SEQ ID NO.51.

[0046] The present invention also aims to provide a multi-nucleic acid vector.

[0047] Furthermore, the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector, and a plasmid.

[0048] In certain embodiments, the lentiviral vector is selected from the group consisting essentially of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0049] In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR encompassed by the present invention, and a right (3') retroviral LTR.

[0050] In certain embodiments, the chimeric antigen receptor targeting CD5 or the multi-target CAR described above comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

[0051] In certain embodiments, the nucleic acid sequence encoding the aforementioned chimeric antigen receptor comprises an optimized Kozak sequence.

[0052] In certain embodiments, the gene expression vector may comprise a secretory anti-PD-1 ScFv; in certain embodiments, the gene expression vector comprises a PD-1 conjugated transduction peptide (such as a PD-1-CD28-CD137-CD3 signal structure); in certain embodiments, the gene expression vector may comprise a plurality of CAR combinations, such as a combination of two CARs targeting different antigens or different recognition sites of the same antigen.

[0053] The present invention also aims to provide an engineered cell, wherein the cell expresses any of the following sequence combinations:

[0054] (1) the amino acid sequence of L-CDR1 is QRVASSY, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYNTYPFT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLSNYYYGYGT; or

[0055] (2) the amino acid sequence of L-CDR1 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYGSSPLT, the amino acid sequence of H-CDR1 is GFTFSSYA, the amino acid sequence of H-CDR2 is ISSDGRNK, and the amino acid sequence of H-CDR3 is ARGLLYDYGLDV;

[0056] (3) the amino acid sequence of L-CDR1 is QSISSW, the amino acid sequence of L-CDR2 is KAS, the amino acid sequence of L-CDR3 is QQLNSYPYT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLDYGDYGYYYGMDV;

[0057] (4) The amino acid sequence of L-CDR1 is QGISSA, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQFNSYPLT, the amino acid sequence of H-CDR1 is GFTFSSYE, the amino acid sequence of H-CDR2 is ISSSGSTI, and the amino acid sequence of H-CDR3 is ARGYDYGFAFDI.

[0058] In some embodiments, the engineered cells express a polypeptide comprising a sequence that recognizes the CD5 antigen, such as SEQ ID NO.25 or SEQ ID NO.27 or SEQ ID NO.29 or SEQ ID NO.31, and a sequence such as SEQ ID NO.26 or SEQ ID NO.28 or SEQ ID NO.30 or SEQ ID NO.32; in some embodiments, the cells express a chimeric antigen receptor, and the chimeric antigen receptor comprises an extracellular antigen recognition domain that can recognize the CD5 antigen, and the extracellular antigen recognition domain that recognizes the CD5 antigen comprises the CDR of the aforementioned sequence (1)-(4); in some embodiments, the extracellular antigen recognition domain that recognizes the CD5 antigen comprises a sequence that can also comprise a light chain sequence of SEQ ID NO.25 or SEQ ID NO.27 or SEQ ID NO.29 or SEQ ID NO.31, and a sequence such as SEQ ID NO.26 or SEQ ID NO.28 or SEQ ID NO.30 or SEQ ID NO.32; in some embodiments, the cell further comprises a nucleic acid sequence for expressing a chimeric antigen receptor, in some embodiments, the nucleic acid sequence of the chimeric antigen receptor comprises a nucleic acid sequence of an antigen-binding polypeptide targeting CD5 as shown in SEQ ID NO.52 or SEQ ID NO.53 or SEQ ID NO.54 or SEQ ID NO.55; in some embodiments, the nucleic acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO.48 or SEQ ID NO.49 or SEQ ID NO.50 or SEQ ID NO.51.

[0059] The engineered cells are immune cells such as T cells, T cell precursors or NK cells, DC cells, macrophages, etc.

[0060] Preferably, the immune cells are T lymphocytes.

[0061] The present invention further aims to provide a cell product, which comprises any of the engineered cells described above.

[0062] Furthermore, the cell product also includes other active agents that can enhance the expression activity of CAR.

[0063] In certain embodiments, the active agent is an immunosuppressant such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation.

[0064] In certain specific embodiments, the activating agent for enhancing the activity of CAR-expressing cells can be an activating agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to initiate immune effector responses in some embodiments. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as PD1 CAR.

[0065] Further, a variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab, pembrolizumab, pidilizumab, and atezolizumab.

[0066] In particular, additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, and trastuzumab. neimatinib, cetuximab, panitumumab, catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitini b, pazopanib, sunitinib b), sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, pona tini b, rad otini b, bosutinibb), lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, and denileukin.

[0067] Furthermore, the cell product can also be used in combination with some therapeutic means, and the therapeutic means can be surgery, chemotherapy, and radiation.

[0068] The present invention further aims to provide a pharmaceutical composition, which comprises any of the above-mentioned polypeptides or single-chain antibodies, the nucleic acid sequence of a chimeric antigen receptor or any of the above-mentioned expression vectors or any of the above-mentioned engineered cells.

[0069] The present invention also aims to provide a use of any of the above-mentioned polypeptides or single-chain antibodies, chimeric antigen receptors or nucleic acid sequences of chimeric antigen receptors or expression vectors or engineered cells or cell products or pharmaceutical compositions in drugs for tumors expressing CD5 targets.

[0070] Specifically, the anti-tumor drugs include but are not limited to anti-renal cancer drugs, anti-prostate cancer drugs, anti-thymic tumor drugs, anti-ovarian cancer drugs, anti-glioblastoma drugs, anti-pancreatic cancer drugs, anti-bladder cancer drugs, anti-nasopharyngeal cancer drugs, anti-gastric cancer drugs, anti-colorectal cancer drugs and other malignant solid tumor drugs.

[0071] In the present invention, the term "functional variant" generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the chimeric antigen receptor) and having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity therewith. In certain embodiments, the variant of the amino acid sequence has substantially the same function therewith. The beneficial effects of the present invention are

[0072] The ScFv provided by the present invention can specifically recognize CD5, recognize and detect CD5 expressed by cells and its ratio, and can be used as a detection reagent for CD5 or a tracer marker molecule in vivo.

[0073] The ScFv provided by the present invention can be used as the antigen recognition domain of the chimeric antigen receptor to recognize CD5 on the surface of the tumor, construct a chimeric antigen receptor immune cell, effectively inhibit the growth of tumors in vivo, and has a significant effect on tumors expressing CD5 targets. In addition, it can be used as an effective ingredient of anti-tumor drugs for malignant tumors with high CD5 expression, effectively controlling the proliferation of malignant tumors with high CD5 expression.

[0074] The ScFv in the CD5 chimeric antigen receptor provided by the present invention is a natural fully human antibody, and its sequence is completely derived from the human antibody gene library. Compared with mouse antibodies, chimeric antibodies and humanized antibodies, its immunogenicity is greatly reduced and its toxic side effects are relatively small. Its later application in clinical efficacy will be more stable and have higher affinity, and its safety and therapeutic effect can be guaranteed to the greatest extent.

[0075] The CD5 chimeric antigen receptor provided by the present invention can specifically recognize human CD5 protein, and is of great significance for the treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : Four clones, JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03, were detected to have specific binding with human CD5 antigen.

[0077] Figure 2: Detection capability verification of 4 ScFv proteins JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 against target cells expressing CD5; Figure 2A Flow cytometry staining of Jurkat cells for JK-01-A11 protein; Figure 2B Flow cytometry staining of Jurkat cells for JK-01-B04 protein; Figure 2C Flow cytometry staining of Jurkat cells for JK-01-B06 protein; Figure 2D Flow cytometry staining of Jurkat cells with JK-02-H03 protein.

[0078] Figure 3 : In vitro killing detection of CAR2 and CAR4.

[0079] Figure 4 : In vitro killing detection of CAR2 and CAR3.

[0080] Figure 5 :Verification of the in vivo effectiveness of CAR2.

[0081] Figure 6 : Verification of CAR2 persistence in vivo, where the higher the copy number, the better the persistence. DETAILED DESCRIPTION

[0082] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0083] Example 1 Screening of ScFv sequences that specifically recognize CD5

[0084] Construction of a fully human phage antibody library

[0085] Ficoll separation solution is used to separate PBMC, and Ficoll separation solution is slowly added to normal human blood so that Ficoll separation solution and normal human blood maintain a clear separation interface. The centrifuge tube containing the blood and separation solution is centrifuged. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, wherein there is a narrow band of white cloud layer mainly composed of PBMC at the junction of the upper and middle layers, i.e., the PBMC cell layer. Carefully suck off the plasma mixture on the upper layer with a sterile Pasteur pipette, and then suck PBMC with a new sterile Pasteur pipette to obtain separated PBMC.

[0086] Total RNA was extracted by conventional methods and reverse transcribed into cDNA, and primers were designed according to the method in Sblattero, D., Bradbury, A., (1998) A definitive set of oligonucleotide primers for amplifying human Vregions. Immunotechnology 3, 271-278, wherein VL was in front or in the back, VH was in the back or in the front, and they were connected in the middle by a flexible linker. PCR was used to obtain the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody, and the ScFv nucleic acid fragment was amplified by conventional overlapping PCR (the PCR method refers to "Molecular Cloning: A Laboratory Manual" (3rd edition), Joe Sambrook, David Russell, Science Press, USA), the ScFv nucleic acid fragment was connected to the phagemid vector pComb3xss, and the product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

[0087] The library bacterial solution was added to fresh LB liquid medium for recovery, cultured to OD600≈0.5, and then VSCM13 helper phage was added at a multiplicity of infection of VCSM13: bacteria = 50:1, mixed thoroughly, and continued to be cultured in a shaker after standing. The culture was centrifuged and the supernatant was discarded, and the precipitate was resuspended in ampicillin and kanamycin double resistance SOB medium and cultured overnight. The bacterial solution was centrifuged for 20 minutes, the supernatant was collected and PEG 80002.5mol / L NaCl solution was added, incubated on ice for 1 hour, and then centrifuged for 30 minutes. The precipitated phage was resuspended in PBS and filtered with a 0.22μm filter membrane to obtain the phage library.

[0088] Antigen panning

[0089] Patent 202211370708.7 introduces the steps of antigen panning into this application, based on the antibodies of the antigens described in this application:

[0090] The CD5 protein with Fc tag is co-incubated with proteinG magnetic beads to prepare CD5-proteinG coupled magnetic beads, which are then put into the prepared phage panning of the fully human single-chain antibody library. After 3-4 rounds of co-incubation, washing and elution, specific monoclonal antibodies against the antigen can be enriched.

[0091] The above-described fully human antibody preparation method was used to screen and obtain the ScFv sequences shown in Table 1 below:

[0092] Table 1 Single chain antibody targeting CD5:

[0093] The bold and underlined sequences in the above table are the CDR sequences of the screened ScFv.

[0094] After the panning, single clone screening was performed. The results were as follows Figure 1 As shown, the binding force between CD5 ScFv and antigen is detected. The horizontal axis is different clone numbers, and the vertical axis is the color value after the screened ScFv binds to the antigen, indicating the ability to bind to the antigen. The higher the color value, the stronger the binding strength. The results show that the four clones numbered JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 were finally screened out and specifically bind to the human CD5 antigen.

[0095] The above four candidate ScFv phage sequences were purified and expressed to obtain ScFv polypeptide proteins. After being identified as normal by SDS-PAGE electrophoresis, 5% trehalose and mannitol were added respectively and the concentrations were tested, and the proteins were frozen at -80°C.

[0096] The above ScFv was obtained using prokaryotic expression and purification methods:

[0097] The pComb3xss plasmids constructed with the above ScFv gene sequences were transformed into Rosetta gami B competent cells, spread on LB plates containing ampicillin resistance, and cultured at 37°C overnight; the next day, single clones were picked and placed in 4ml LB medium containing ampicillin resistance for 8h, and then the bacterial solution was transferred to 200mL LB medium for further culture. When the bacterial solution OD reached 0.5-1.0, IPTG with a final concentration of 1mM was added, and expression was induced at 37°C for 12h. The bacteria were collected by centrifugation and PBS was added to resuspend the precipitate, ultrasonically broken, centrifuged for 10min, the precipitate was discarded, and the supernatant was collected for protein purification, which was purified using a GE Ni Sepharose excel purification column.

[0098] Example 2 Recognition of CD5 antigen by ScFv protein

[0099] Jurkat cells endogenously expressing CD5 antigen were divided into 1.5mL Ep tubes, 5e5-1e6 cells / tube, centrifuged at 4℃300g for 5min, supernatant discarded, 50μL 30ug / ml positive clone ScFv solution was added to resuspend, incubated at 4℃ for 1h, then 1ml PBS was added to resuspend the cells, centrifuged at 4℃300g for 5min, supernatant discarded, 30μl Anti-His-647 fluorescent secondary antibody was added and resuspended, incubated at 4℃ in the dark for 30min, finally resuspended with 1ml PBS and washed twice at 4℃300g for 5min, supernatant discarded and resuspended with 100μL PBS. All cell tubes were tested for flow staining positive rate and mean fluorescence intensity (MFI), and the results are shown in Figure 2. Figure 2A The flow cytometry staining of Jurkat cells by JK-01-A11 protein. Figure 2B Flow cytometry staining of Jurkat cells with JK-01-B04 protein. Figure 2C The flow cytometry staining of Jurkat cells by JK-01-B06 protein. Figure 2DThe JK-02-H03 protein was used for flow cytometry staining of Jurkat cells; the results showed that JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 could all recognize CD5 expressed on the cell surface. Combined with Example 1, JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 could be used as CD5 antigen recognition reagents for CD5 antigen detection reagents or detection kits or in vivo tracing or protein expression detection combination products.

[0100] Example 3 Targeting CD5 ScFv Function and Application Verification in CAR Structure

[0101] In order to verify the function of the screened fully human antibodies, we constructed CAR vectors to prepare CAR-NK cells to verify the ability of the screened fully human antibodies to recognize CD5 expressed on cells and kill tumor cells expressing CD5.

[0102] 1) Screening for ScFv with excellent killing ability against tumor cells expressing CD5

[0103] Construction of CD5-targeting CAR plasmid

[0104] The ScFv sequence was obtained by PCR amplification, and then connected to the CAR vector structure by restriction endonuclease digestion, including 8h (CD8 hinge region), 8TM (CD8 transmembrane region), BB (intracellular signaling domain of CD137), and Z (CD3ζ chain) co-stimulatory domain. The constructed lentiviral vector was successfully constructed after verification by sequencing comparison.

[0105] CAR structure: ScFv(CD5)-8h-8TM-BBZ

[0106] The sequences of the present invention are shown in Table 2 below:

[0107] The present embodiment packages the lentivirus using the calcium phosphate method to package the lentivirus, referring to the Molecular Cloning Experiment Guide (third edition, J. Sambrook et al.), specifically: using 293T cells cultured to the optimal state, the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5 centrifuge tube in a certain ratio, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture medium, and the liquid is changed to 10mL DMEM medium containing 10% FBS again after 3-5h, and the cell supernatant is collected after 48h or 72h, and the virus is purified. The purified virus is packaged and stored at -80°C for standby use. The virus infects CHO cells, and Protein-L is used to detect total CAR expression 48h after infection, and the positive rate is calculated. The positive rate calculation formula is: titer (Tu / ml) = 1e5×positive rate×dilution factor×1000÷virus volume (uL), the virus titer is calculated according to the standard curve, and the result is expressed in TU / mL. The virus titer results of JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 prepared by using the CAR structure constructed by the antigen recognition region recognizing CD5 are shown in Table 3 below:

[0108] Table 3: Titers of viruses containing CD5-CAR gene FWf CAR Number Amino acid sequence Virus titer (Tu / ml) JK-01-A11 CAR1 SEQ ID NO.44 6.71E+08 JK-01-B04 CAR2 SEQ ID NO.45 5.86E+08 JK-01-B06 CAR3 SEQ ID NO.46 6.04E+08 JK-02-H03 CAR4 SEQ ID NO.47 6.62E+08

[0109] From Table 3 above, it can be found that JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 can be applied to CAR structure design and CAR construction.

[0110] Lymphocytes were separated by gradient centrifugation; after centrifugation, the second layer of white lymphocytes was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After obtaining PBMC, CD56 antibody-labeled Microbeads were used for magnetic sorting to obtain CD56-positive NK cells. NK cells can be transduced with lentivirus 24 hours after activation. Three days after transduction, the positive rate of CAR-NK needs to be detected using CD5-6×His (ACRO-Biosystems) (primary antibody) and anti-his-AF647 (GenScript) (secondary antibody). The results of the positive rate detection of CAR-NK labeled with CD3-PE / Cy7 and CD56-BV510 (BioLegend) for NK cells are shown in Table 4.

[0111] Table 4: CD5-CAR-T positive rate test results Plasmid number CAR positive rate (%) CAR-1 11.64 CAR-2 58.82 CAR-3 69.75 CAR-4 20.89

[0112] It can be seen that JK-01-A11, JK-01-B04, JK-01-B06, and JK-02-H03 can all be applied to CAR construction, and the constructed CAR structures can be normally expressed in NK cells; the preferred CAR structures constructed by JK-01-B04 and JK-01-B06 have better expression positivity rates.

[0113] CD5-NALM6-Luc-GFP cells were used as positive target cells, and NALM6-Luc-GFP negative target cells were used. CAR-NK cells and target cells were plated on target cells at a ratio of 1:1 or 1:4, and killing was detected by luciferase after 24 hours. Luciferase principle: During the detection, after the target cells are lysed with lysis buffer, the luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using fluorescence value is: CAR-NK cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group). The results are as follows Figure 3 and Figure 4 As shown, Figure 3 These are the killing results of CAR2 and CAR4 against CD5-positive cells: CD5-NALM6-Luc-GFP and CD5-negative cells: NALM6-Luc-GFP, where the horizontal axis represents different CAR structures and the vertical axis represents the killing percentage. Figure 4 The killing of CAR2 and CAR3 against CD5-positive cells: CD5-NALM6-Luc-GFP, CD5-negative cells: NALM6-Luc-GFP, and the specific killing rates are shown in Table 5 below. The results show that CAR2, CAR3, and CAR4 can kill CD5-positive target cells, but not CD5-negative target cells, indicating that JK-01-B04, JK-01-B06, and JK-02-H03 can be applied to the antigen recognition region of the CAR structure targeting CD5, and the constructed CAR structure can function on NK cells.

[0114] Table 5: In vitro killing data of CAR-2, CAR-3, and CAR-4

[0115] The cell supernatant was collected 24 hours after killing to detect the IFN-γ secretion ability of CAR-NK cells after being stimulated by target cells. The collected supernatant was tested for IFN-γ secretion using ELISA (enzyme-linked immunosorbent assay). The results are shown in Table 6 below:

[0116] Table 6: Cellular IFN-γ secretion data of CAR-2, CAR-3, and CAR-4 (unit: pg / ml) The results showed that compared with CNK, CAR-2, CAR-3, and CAR-4 modified CAR-NK cells could all detect significant IFN-γ secretion after target cell stimulation. This showed that JK-01-B04, JK-01-B06, and JK-02-H03 could be applied to the antigen recognition region of the CAR structure targeting CD5, and the constructed CAR structure could function on NK cells.

[0117] Example 4 In vivo efficacy evaluation of NK cells modified with CAR-2, CAR-3, and CAR-4

[0118] CD5+ cells CD5-NALM6-Luc-GFP were used as positive target cells and injected into NCG mice through the tail vein at a dose of 1e6 / mouse for pre-tumor formation. The pre-tumor formation time was 1 day. CAR-2 modified NK was cultured in vitro for 13 days and then frozen; after the pre-tumor formation was completed, the CAR-NK was resuscitated and injected into the pre-tumored NCG mice through the tail vein at a dose of 4e6 / mouse. NK cells that were not modified by CAR were injected with the same dose and method as the control group. Thereafter, the fluorescent substrate of luciferase was injected intraperitoneally once every 7 days, and the tumor load was detected by in vivo imaging using a small animal in vivo imaging device. The results are shown as follows: Figure 5 The results showed that CAR-2 modified NK cells have superior in vivo efficacy compared with unmodified NK cells.

[0119] In addition, submandibular blood was collected every 7 days after CAR-NK transfusion, and the copy number of BBz gene in the blood was detected by qPCR. Figure 6 As shown, the copy number of BBz gene in mouse blood, the results showed that CAR-2 modified NK cells have better in vivo persistence than unmodified NK cells.

[0120] It should be noted that although the present invention takes NK cells and CAR-NK cells as examples, based on the content disclosed in the present invention, technicians in the field can obviously infer that other immune cells such as T cells, macrophages, DC cells and related prerequisite cells can also express the engineered immune cells described herein containing CAR for CD5, and specifically recognize CD5, and play a killing role against malignant tumors expressing CD5. The chimeric antigen receptor constructed by the polypeptide or ScFv that recognizes CD5 as an extracellular antigen recognition domain or one of the extracellular antigen recognition domains described in this application can be expressed in T cells, NK cells, macrophages, DC cells and related prerequisite cells; it can be co-expressed with fusion proteins of any structure in T cells, NK cells, macrophages, DC cells and related precursor cells, and used for drugs or drug combinations for malignant tumors expressing CD5 for tumor treatment.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An antigen-binding polypeptide targeting CD5, characterized in that The antigen-binding polypeptide comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3; specifically, any combination of the following is shown: (1) the amino acid sequence of L-CDR1 is QRVASSY, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYNTYPFT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, and the amino acid sequence of H-CDR3 is ARLSNYYYGYGT; or (2) the amino acid sequence of L-CDR1 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQYGSSPLT, the amino acid sequence of H-CDR1 is GFTFSSYA, the amino acid sequence of H-CDR2 is ISSDGRNK, The amino acid sequence of H-CDR3 is ARGLLYDYGLDV; (3) the amino acid sequence of L-CDR1 is QSISSW, the amino acid sequence of L-CDR2 is KAS, the amino acid sequence of L-CDR3 is QQLNSYPYT, the amino acid sequence of H-CDR1 is GYSFTSYW, the amino acid sequence of H-CDR2 is IYPGDSDT, The amino acid sequence of H-CDR3 is ARLDYGDYGYYYGMDV; (4) the amino acid sequence of L-CDR1 is QGISSA, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQFNSYPLT, the amino acid sequence of H-CDR1 is GFTFSSYE, the amino acid sequence of H-CDR2 is ISSSGSTI, The amino acid sequence of H-CDR3 is ARGYDYGFAFDI.

2. The polypeptide of claim 1, comprising a light chain variable region represented by amino acids as shown in SEQ ID NO. 25 or SEQ ID NO. 27 or SEQ ID NO. 29 or SEQ ID NO.

31.

3. The polypeptide of claim 1, comprising a heavy chain variable region having amino acids as shown in SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, or SEQ ID NO.

32.

4. The polypeptide according to claim 1 is a single-chain antibody that recognizes CD5, characterized in that: Contains any of the following sequences: (1) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.26; or (2) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.28; or (3) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.29, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.30; or (4) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.31, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.32; or (3) The single-chain antibody, the amino acid sequence of which is shown in SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37 or SEQ ID NO.

38.

5. The single-chain antibody recognizing CD5 according to claim 4, further comprising a linker sequence, the amino acid sequence of which is shown as SEQ ID NO.33 or SEQ ID NO.

34.

6. A kit for detecting CD5 expression, characterized in that: Comprising the antigen-binding polypeptide of claims 1-5.

7. A chimeric antigen receptor, characterized in that It comprises an extracellular antigen recognition domain that can recognize CD5 antigen, and the extracellular antigen recognition domain comprises the antigen-binding polypeptide targeting CD5 as described in claims 1-5.

8. The chimeric antigen receptor according to claim 7, characterized in that It also contains a hinge region, a transmembrane region and an intracellular signaling domain.

9. The chimeric antigen receptor according to claim 8, characterized in that The hinge region amino acid sequence is shown in SEQ ID NO.

40.

10. The chimeric antigen receptor according to claim 9, characterized in that It also comprises a transmembrane region, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO.

39.

11. The chimeric antigen receptor of claim 10, wherein the intracellular signaling domain comprises an amino acid sequence derived from CD3ζ such as the sequence shown in SEQ ID NO.

42.

12. The chimeric antigen receptor of claim 11, wherein the intracellular signaling domain further comprises a costimulatory signaling domain having an amino acid sequence such as that shown in SEQ ID NO.

41.

13. A nucleic acid sequence, characterized in that Encoding the chimeric antigen receptor according to claim 7.

14. The nucleic acid sequence encoding the chimeric antigen receptor according to claim 13, characterized in that: The antigen recognition nucleic acid sequence is shown as SEQ ID NO.52 or SEQ ID NO.53 or SEQ ID NO.54 or SEQ ID NO.

55.

15. The nucleic acid sequence encoding the chimeric antigen receptor according to claim 14, characterized in that: The nucleic acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.48 or SEQ ID NO.49 or SEQ ID NO.50 or SEQ ID NO.

51.

16. An expression vector comprising the nucleic acid sequence of any one of claims 13-15.

17. An engineered cell, characterized in that The cell expresses the polypeptide or single-chain antibody according to any one of claims 1 to 5, or the chimeric antigen receptor according to any one of claims 7 to 12, or the cell comprises the nucleic acid or vector according to claims 13 to 16.

18. A cell product, characterized in that The cell product comprises the engineered cells of claim 17.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide or single-chain antibody according to any one of claims 1-5 or the nucleic acid or vector according to claims 13-16, the engineered cell according to any one of claim 17 or the cell product according to claim 18.

20. Use of the polypeptide or single-chain antibody according to any one of claims 1-5, the nucleic acid or vector according to claims 13-16, the engineered cell according to any one of claim 17, the cell product according to claim 18, the pharmaceutical composition according to claim 19, or the chimeric antigen receptor according to claims 7-12 in the preparation of a drug for tumors expressing CD5 targets.

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