Patent Application
20240002505
The present application claims priority to the Chinese patent application No. 202011200628.8 filed on Nov. 1, 2020 in the CNIPA, which is incorporated herein by reference in its entirety.
The present application includes a sequence listing in .txt format submitted electronically. This .txt file contains a sequence listing named “CD5 CAR_ST25.txt” created on Nov. 1, 2020, and is approximately 126 kilobytes in size. The sequence listing contained in the .txt file is part of the specification and is incorporated herein by reference in its entirety.
The present application relates to the field of biomedicine, in particular to a fully human antibody capable of specifically binding to CD5 protein, a fully human chimeric antigen receptor and applications thereof.
In recent years, chimeric antigen receptor T-cell (CAR-T) technology has made breakthroughs, especially great success has been made in targets for B-cell malignancies. As represented by anti-CD19 CAR-T, so far, three products, Kymriah, Yescarta and Tecartus, have been approved by the FDA (US) for marketingE[1]. Clinical trials have confirmed that CAR-T cells specifically targeting CD19 molecules can effectively target and treat B-cell malignancies, including relapsed/refractory B-ALL, CLL, and B-cell lymphoma[2-9]. According to literature reports, the effective rate of CD19 CAR-T in treatment of relapsed/refractory ALL can be as high as 90%, and the effective rate for CLL and some B-cell lymphomas is greater than 50%. Although CAR-T therapy has achieved great success in the field of treatment of B-cell malignancies, research on CAR-T therapy and application thereof in T-cell malignancies are very limited.
T-cell malignancies include acute T-lymphocytic leukemia (T-ALL) and T-cell lymphoma (TCL). T-ALL is a blood disease caused by abnormal proliferation of T lymphocytes, which is aggressive and progresses rapidly. T-cell lymphoma is a malignancy of T-cells that can develop in lymphoid tissues (such as lymph nodes and spleen) or outside lymphoid tissues (such as gastrointestinal tract, liver, nasal cavity, and skin), it accounts for about 10-15% of non-Hodgkin's lymphoma, and this proportion is higher in China. CD5 is constitutively expressed on lymphocyte precursors, mature T-cells and a portion of mature B-cells (B1 cells)[10][10]. CD5 is highly expressed in about 85% of T-ALLs and about 75% of peripheral T-cell lymphomas. In addition, CD5 is also frequently expressed in mantle cell lymphoma, chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia cells (HCL). At present, T-cell malignancies have a high recurrence rate and poor prognosis after radiotherapy and chemotherapy. They are clinically intractable hematological malignancies, and there is an urgent need to develop cell therapy drugs for this disease. In normal cells, the expression of CD5 is limited to mature T-cells and some subtypes of B-cells, and the biological characteristics of CD5 antigen allow CD5 CAR T cells to produce effective anti-tumor activity against T-ALL and T lymphoma cells in vitro and in vivo[12]. Therefore, CD5 can serve as a safe and reliable target for T-cell tumors.
Researchers such as Mamonkin M developed a second-generation CD5 CAR using the scFv of murine monoclonal antibody H65. The results of clinical trials show that the CD5 CAR-T cells are safe in patients with r/r CD5+T-ALL and T-cell non-Hodgkin's lymphoma (T-NHL) who have been treated by multiple lines in the past, and can produce clinical efficacy without completely eliminating T-cells. More importantly, depletion of malignant T-cells by CD5 CAR-T cells may allow patients who were previously ineligible for transplantation to undergo HSCT (hematopoietic stem cell transplantation). However, according to the existing clinical follow-up data, the murine scFv CAR-T may suffer relapse of CD5+ malignant diseases after infusion, and the limited duration of CAR-T in patients may be related to the production of anti-murine antibodies, while fully human scFv may be able to solve the problem of short duration of CD5 CAR-T in patients. The development of a fully human CD5 antibody is of great significance for the development of the next generation of CAR-T products that last longer in vivo and have better long-term efficacy.
The present application provides a fully human antibody that can specifically bind to CD5, a fully human chimeric antigen receptor and applications thereof.
In one aspect, the present application provides a CD5-targeting fully human antibody or a single chain antibody or fragment thereof, wherein the fully human antibody comprises a heavy chain variable region (HCVR) and/or a light chain variable region (LCVR), and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3 and/or LCDR1, LCDR2 and LCDR3 are selected from one of the following combinations:
In some embodiments, the amino acid sequence of the heavy chain variable region and/or the light chain variable region is selected from any one of the following combinations:
In some embodiments, the amino acid sequence of the heavy chain variable region and/or the light chain variable region is selected from any one of the following combinations:
In certain embodiments, the fully human antibody comprises an amino acid sequence as set forth in SEQ ID NO: 28, 34, 40, 88, 91 or 94.
In another aspect, the present application provides a nucleic acid molecule encoding the above-mentioned fully human antibody or single-chain antibody or fragment thereof.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 25-27, 31-33, 37-39, and 79-87.
In another aspect, the present application provides an expression vector comprising the above-mentioned nucleic acid molecule.
In another aspect, the present application provides a host cell comprising the above-mentioned expression vector.
In another aspect, the present application provides a pharmaceutical composition comprising the above-mentioned fully human antibody or single chain antibody or fragment thereof, and a pharmaceutically acceptable carrier or diluent.
In another aspect, the present application provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of the above-mentioned fully human antibody or single-chain antibody or fragment thereof, host cell, or pharmaceutical composition to a patient in need thereof, to eliminate, inhibit or reduce CD5 activity, thereby preventing, alleviating, improving or inhibiting the disease or condition.
In some embodiments, the disease or condition is a cancer.
In some embodiments, the cancer is a T-cell malignancy.
In another aspect, the present application provides an antibody or fragment competing for the same epitope with the above-mentioned fully human antibody or single-chain antibody or fragment thereof.
In another aspect, the present application provides a kit for detecting CD5 protein in a sample, wherein the kit comprises the above-mentioned fully human antibody or single-chain antibody or fragment thereof.
In another aspect, the present application provides use of the above-mentioned fully human antibody or single chain antibody or fragment thereof or host cell in the preparation of a drug for eliminating, inhibiting or reducing CD5 activity, thereby preventing, alleviating, improving or inhibiting a disease or condition.
In some embodiments, the disease or condition is a cancer.
In some embodiments, the cancer is a T-cell malignancy or a B-cell malignancy.
In some embodiments, the T-cell malignancy is selected from T-cell acute lymphocytic leukemia (T-ALL) and T-cell lymphoma (TCL), and the B-cell malignancy is selected from chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (B-MCL).
In another aspect, the present application includes a chimeric antigen receptor (CAR), wherein the CAR comprises a CD5 binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from the following combinations: (1) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 43, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 44, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45; (2) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 49, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 50, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 51; (3) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 55, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 56, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 57; (4) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 61, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 62, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 63; (5) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 67, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 68, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 69; or (6) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 75.
In some embodiments, the antibody further comprises a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3), the amino acid sequences of the LCDR1, LCDR2, LCDR3 are selected from the following combinations: (1) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 46, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 47, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 48; (2) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 52, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 53, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 54; (3) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 58, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 59, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 60; (4) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 64, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 65, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 66; (5) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 70, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 71, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 72; or (6) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 76, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 77, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 78.
In some embodiments, the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 comprised in the antibody are selected from any one of the following combinations: (1) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 43, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 44, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 45, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 46, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 47, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 48; (2) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 49, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 50, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 51, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 52, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 53, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 54; (3) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 55, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 56, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 57, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 58, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 59, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 60; (4) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 61, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 62, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 63, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 64, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 65, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 66; (5) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 67, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 68, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 69, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 70, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 71, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 72; or (6) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 75, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 76, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 77, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 78.
In some embodiments, the antibody comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 30, 36, 42, 90, 93 or 96. In some embodiments, the antibody comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 29, 35, 41, 89, 92 or 95. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 28, 34, 40, 88, 91 or 94 or a functional variant thereof.
In some embodiments, the transmembrane domain of the CAR comprises a polypeptide selected from the following proteins: u, R or (chains of T-cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises an amino acid sequence as set forth in SEQ ID NO: 6 or a functional variant thereof.
In some embodiments, the co-stimulatory domain of the CAR comprises a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40, and ICOS. In some embodiments, the co-stimulatory domain comprises an amino acid sequence as set forth in SEQ ID NO: 8 or a functional variant thereof.
In some embodiments, the intracellular signaling domain of the CAR comprises a signaling domain from CD3ζ. In some embodiments, the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 10 or a functional variant thereof.
In some embodiments, the CAR further comprises a hinge region connecting the antibody and the transmembrane domain. In some embodiments, the hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 4 or a functional variant thereof.
In some embodiments, the CAR is also linked to a CD8α signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence as set forth in SEQ ID NO: 2 or a functional variant thereof.
In some embodiments, the CAR is also linked to a cleaving peptide. In some embodiments, the cleaving peptide comprises an amino acid sequence from a T2A peptide. In some embodiments, the cleaving peptide comprises an amino acid sequence as set forth in SEQ ID NO: 12 or a functional variant thereof. In some embodiments, the CAR is also linked to a truncated EGFR molecule (tEGFR) by linking to the cleaving peptide and a CSF2RA signal peptide. In some embodiments, the truncated EGFR molecular comprises an amino acid sequence as set forth in SEQ ID NO: 16 or a functional variant thereof. The CSF2RA signal peptide comprises an amino acid sequence as set forth in SEQ ID NO: 14 or a functional variant thereof.
In some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 22-24, 98, 100 or 102 or a functional variant thereof.
In another aspect, the present application further includes an isolated nucleic acid molecule encoding the CAR of the present application.
In another aspect, the present application further includes an isolated nucleic acid molecule encoding CAR, which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19-21, 25-27, 31-33, 37-39, 79-87, 97, 99 or 101 or a functional variant thereof.
In another aspect, the present application further includes a vector comprising the nucleic acid molecule of the present application. In some embodiments, the vector is selected from a plasmid, a retroviral vector and a lentiviral vector.
In another aspect, the present application further includes an immune effector cell comprising the CAR of the present application, the nucleic acid molecule of the present application, or the vector of the present application. In some embodiments, the immune effector cell is selected from a T lymphocyte and a natural killer (NK) cells In some embodiments, CD5 on the immune effector cell is not expressed.
In another aspect, the present application further includes a method for preparing an immune effector cell, which comprises knocking out the CD5 gene of the immune effector cell, and introducing the vector of the present application into the immune effector cells. In some embodiments, the knocking out is carried out using the sgRNA of any one of SEQ ID NOs: 103-106.
In another aspect, the present application further includes a pharmaceutical composition, which comprises the immune effector cells of the present application and a pharmaceutically acceptable adjuvant.
In another aspect, the present application further includes use of the CAR, the nucleic acid molecule, the vector, or the immune effector cell in the preparation of a drug for treating a disease or condition associated with the expression of CD5. In some embodiments, the disease or condition associated with the expression of CD5 is a cancer or malignancy.
In another aspect, the present application further relates to use of the CAR, the nucleic acid molecule, the vector, or the immune effector cell or the pharmaceutical composition in the treatment of a disease or condition associated with the expression of CD5. In some embodiments, the disease or condition associated with the expression of CD5 is a cancer or malignancy. In another aspect, the present application further includes a method of treating a disease or condition associated with the expression of CD5, which comprises administering a therapeutically effective amount of the immune effector cell of the present application, or the pharmaceutical composition of the present application to a patient in need thereof. In some embodiments, anti-EGFR antibodies such as cetuximab can be further administered to patients in need thereof to inhibit the effect of the immune effector cell or the pharmaceutical composition, wherein the patients in need thereof include those patients who have serious adverse reactions to the immune effector cell or pharmaceutical composition of the present application.
Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention in which the present application is involved. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.
The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in the specification. The CAR of the present application can specifically bind to CD5, the CAR-T cells prepared by using the CAR can stably express the CAR, and the CAR-T cells prepared by using the CAR have a high CAR-positive rate. In addition, the CAR can promote the release of cytokines and can be used for treating diseases or conditions associated with the expression of CD5.
The implementation of the present application will employ, unless expressly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, within the skill of the art. Descriptions of these methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed., 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated in July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I&II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames&S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y, 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and journal monographs such as Advances in Immunology.
Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present application, the following terms are defined below.
In the present application, the term “chimeric antigen receptor” (CAR) is a fusion protein of the variable region of a single-chain antibody and a T-cell signaling molecule. It allows T-cells to recognize specific antigens in a non-MHC-restricted manner and play a killing role. CAR is the core component of chimeric antigen receptor T-cells (CAR-Ts), and it can comprise a tumor-associated antigen (TAA) binding region, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, and can further comprise a hinge region between the tumor-associated antigen binding region and the transmembrane domain. In the present application, the CAR can be a genetically engineered chimeric protein capable of redirecting the cytotoxicity of immune effector cells to B-cells, which combines antibody-based antigen (such as CD5) specificity with the T-cell receptor activation intracellular domain. T-cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing the target antigen. For descriptions of CAR and CAR T-cells, see, for example, Sadelain M, Brentjens R, Rivi'ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4):388-398; Turtle C J, Hudecek M, Jensen M C, Riddell S R. Engineered T-cells for anti-cancer therapy. Curr Opin Immunol. 2012; 24(5):633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T-cells. Immunol Rev. 2014; 257(1):107-126; and WO2013154760, WO2016014789.
In the present application, the term “CD5” is a type I transmembrane glycosylated protein that plays an important role in the negative regulation of T-cell receptor signaling and promotes the survival of normal and malignant lymphocytes. CD5 is one of the characteristic surface markers of T-cell malignancies, and 80% of T-cell acute lymphocytic leukemia (T-ALL) and peripheral T-cell lymphomas express CD5. In the present application, the CD5 may be human CD5, whose GenBank accession number is NM_014207.4. The CD5 proteins may also include fragments of CD5, such as the extracellular domain and fragments thereof.
In the present application, the term “CD5 binding domain” generally refers to the extracellular domain of CD5 CAR, which can specifically bind to an antigen. For example, the CD5 extracellular binding domain may comprise a chimeric antigen receptor capable of specifically binding to a CD5 polypeptide expressed on a human cell, an anti-CD5 antibody or an antigen-binding fragment thereof. The terms “binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “extracellular antigen-specific binding domain” are used interchangeably in the present application, and a CAR with the ability to specifically bind to a target antigen of interest (e.g., CD5) is provided. The CD5 binding domain may be of natural, synthetic, semi-synthetic or recombinant origin.
In the present application, the term “antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv). In the present application, a “fragment” of an antibody may refer to an antigen-binding fragment of an antibody, for example, Fab, Fab′ and (Fab′)2 fragments, etc. The term “antibody” also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof.
Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain can be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors which include various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
In the present application, the term “single-chain antibody”, which may also be called scFv, refers to an antibody consisting of one chain composed of a heavy chain variable region and a light chain variable region connected by a linker peptide.
“Murine antibody” is an antibody produced by murine against a specific antigen, usually referring to an antibody 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 application is selected from a human phage antibody library, which reduces the immunogenicity compared with the murine antibody, and is more conducive to the therapeutic use in the human body.
The “fully human antibody or single-chain antibody or fragment thereof” of the present application generally refers to any form of antigen-binding molecule capable of binding to a target antigen. For example, the antigen-binding molecule can be a protein or a polypeptide, including, for example, an antibody and its antigen-binding fragments, single-chain scFv antibodies, single-domain antibodies, various fusions and conjugates constructed based on scFv, such as scFv-Fc antibodies, immunoconjugates, antibody drug conjugates (ADCs), multi/bispecific antibodies, chimeric antigen receptors (CARs).
“Epitope” refers to a portion of a molecule that is bound by an antigen-binding protein (e.g., an antibody). Epitopes may comprise non-contiguous portions of the molecule (e.g., in polypeptides, amino acid residues that are not contiguous in the primary sequence of the polypeptide, but are sufficiently close to each other in the trivalent and tetravalent structure of the polypeptide to be bound by the antigen binding protein).
In the present application, the term “transmembrane domain” generally refers to a domain in CAR that passes through the cell membrane, which is connected to the intracellular signaling domain and plays a role in transmitting signals. In the present invention, the transmembrane domain may be CD28 transmembrane domain.
In the present application, the term “co-stimulatory domain” generally refers to an intracellular domain that can provide immune co-stimulatory molecules which are cell surface molecules required for an effective response of lymphocytes to antigens. The co-stimulatory domain may include the co-stimulatory domain of CD28, and may also include the co-stimulatory domain of the TNF receptor family, such as the co-stimulatory domain of OX40 and 4-1BB.
In the present application, the term “hinge region” generally refers to the connecting region between the antigen-binding region and the binding region of an immune cell Fc receptor (FcR). In the present invention, the hinge region may be CD28 hinge region.
In the present application, the term “intracellular signaling domain” generally refers to the component of CAR for signal transduction in cells, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: it may be selected from CD3ζ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.
In the present application, the term “CD8α signal peptide” generally refers to a short (5-30 amino acids in length) peptide chain that guides the transfer of newly synthesized proteins to the secretory pathway.
In the present application, the term “cleaving peptide” refers to a self-cleaving 2A peptide, which can realize the function of cleaving proteins through ribosome skipping instead of proteolytic hydrolysis, and may include T2A, F2A and P2A, etc.
In the present application, the term “marker detection signal” generally refers to a gene, protein or other molecules with a known function or sequence that can function as a specific marker and emit a detectable signal. The marker detection signal can be a fluorescent protein, such as: GFP, RFP, and YFP. The marker detection signal may be EGFRt. The terms “EGFRt” and “tEGFR” are used interchangeably in the present invention and refer to a gene encoding a truncated human epidermal growth factor receptor polypeptide, which lacks the distal membrane EGF binding domain and cytoplasmic signaling tail, but retains extracellular epitopes recognized by anti-EGFR antibodies. EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker which has a function of genetically modifying cells. In the present application, it can be used as a marker molecule for CAR-T cells, which is used to clear the anti-EGFR antibody (for example, cetuximab) mediated ADCC pathway (cetuximab mediated ADCC pathway) of CAR-T cells in vivo if necessary (see U.S. Pat. No. 8,802,374B2), that is, it is used as a safety switch during clinical transformation.
In the present invention, the term “CSF2RA signal peptide”, that is, colony stimulating factor 2 receptor subunit alpha signal peptide, is a peptide chain that can guide the expression of newly synthesized proteins on the surface of CAR-T cells.
In the present application, “anti-EGFR antibody” refers to an antibody that has the ability to trigger antibody dependent cell-mediated cytotoxicity, allowing immune cells to attack CAR-T cells with truncated epidermal growth factor receptor (EGFRt), and assist in the removal of CAR-T cells. The anti-EGFR antibody can be used when patients have severe adverse reactions after infusion of CAR-T or when removal of CAR-T cells is required. It can assist in the removal of CAR-T cells and alleviate symptoms related to CAR-T treatment. The anti-EGFR antibodies include, but are not limited to, cetuximab, panitumumab, necitumumab and nimotuzumab.
In the present application, “sequence identity” generally refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-by-amino acid basis over a comparison window. “Percent sequence identity” can be calculated by comparing two optimally aligned sequences over a comparison window and determining the number of positions of identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) present in two sequences to obtain the number of matching positions, and dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity. Optimal alignment for purposes of determining percent sequence identity can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequences of interest.
In some embodiments, the fully human antibody provided by the present application further comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) with an sequence as set forth in any one of SEQ ID NO: 28, 34, 40, 88, 91 or 94.
Those skilled in the art can understand that, on the basis of the specific sequences provided herein, the corresponding variants of the antibodies targeting CD5 provided herein can be obtained by substituting, deleting, adding a few amino acids, and verifying or selecting the resultant product for its binding ability with the corresponding antigen CD5 or its biological activity, and these variants should also be included within the scope of the present invention. For example, the fully human antibody or single-chain antibody or fragment thereof of the present application may have at least one and no more than 10, or no more than 5, 4, 3, 2 or 1 amino acid changes in the full-length or CDR sequence.
Those skilled in the art can also understand that, on the basis of the specific heavy chain variable region sequences provided herein, an antibody light chain library (such as a human phage light chain library) can be selected by using CD5 as the antigen, so as to obtain light chain variable regions matched with the heavy chain variable regions while maintaining CD5 binding ability. Anti-CD5 antibody molecules obtainable in this way are also included in the scope of the present invention.
In some embodiments, the antigen binding molecules of the present application may further comprise post-translational modifications. Examples of post-translational protein modifications include: phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination, biotinylation, or addition of polypeptide side chains or hydrophobic groups. Thus, a modified soluble polypeptide may comprise non-amino acid components such as lipoids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is the modification by sialylation, the attachment of one or more sialic acid groups to a polypeptide. The sialic acid group improves the solubility and serum half-life of the protein, while reducing the possible immunoheritability of the protein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.
The term “functional variant” of a protein or polypeptide sequence in the present application refers to a sequence having amino acid changes compared to the parent sequence by 1 or more, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions. Functional variants substantially retain the biological properties of the protein or polypeptide sequence before the change. In one aspect, the present application encompasses variants of any protein or polypeptide sequence of the present application. In some embodiments, a functional variant of a protein or polypeptide sequence retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the parent sequence before the change. The functional variants described in the present invention may be functional variants of signal peptide, antibody, hinge region, transmembrane domain, co-stimulatory domain, intracellular signaling domain, cleaving peptide, CSF2RA signal peptide, and EGFRt. When referring to functional variants of antibodies, functional variants such as antibody variable regions (such as VH or VL), antibody constant regions (such as CH or CL), heavy chain CDR regions (HCDR1, HCDR2 or HCDR3), and light chain CDR regions (LCDR1, LCDR2 or LCDR3) are also included. Amino acid substitutions, deletions and/or insertions may occur in either the heavy chain CDR region or the light chain CDR region, or the heavy chain FR region or the light chain FR region, or the heavy chain constant region or the light chain constant region, wherein the variant substantially retains the biological properties of the previous antibody molecule before the change. For antibodies, biological activities include, for example, antigen binding ability. In some embodiments, functional variants of antibodies comprise amino acid changes that do not result in the antibody variant losing binding to the antigen, but optionally confer properties such as increased affinity for the antigen and different effector functions. It is understood that the antibody heavy chain variable region or light chain variable region, or the CDR regions can be altered individually or in combination. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes are present in one or more or all three heavy chain CDRs. In some embodiments, the amino acid changes may be amino acid substitutions, such as conservative substitutions. In some embodiments, the functional variant has at least 80%, 85%, 90% or 95% or 99% or more amino acid identity to the parent. Similarly, a “functional variant” of a nucleic acid molecule in the present application refers to a nucleic acid molecule that encodes the same amino acid sequence as the parent nucleic acid molecule.
In the present application, the term “isolated” generally means that an antibody is one that has been separated from components of its natural environment. In some embodiments, antibodies are purified to have a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). A review of methods for assessing antibody purity can be found in Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.
In the present application, the term “nucleic acid molecule” generally refers to isolated nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length isolated from their natural environment or artificially synthesized. The nucleic acid molecule of the present application can be isolated. For example, it may be produced or synthesized by (i) amplification in vitro, such as amplification by polymerase chain reaction (PCR), (ii) cloning and recombination, (iii) purification, for example by fractionation through enzyme digestion and gel electrophoresis, or (iv) synthesis, for example by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies or antigen-binding fragments thereof can be prepared by various methods known in the art, including, but not limited to, manipulations using restriction fragments or using overlap extension PCR of synthetic oligonucleotides. For specific operations, refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.
In the present application, the term “vector” generally refers to a nucleic acid molecule capable of self-replicating in a suitable host for transferring an inserted nucleic acid molecule into and/or between host cells. The vector may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA. The vector also includes a vector having various functions as described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector. In the present application, one or more of the nucleic acid molecules may be comprised in the vector. In addition, other genes may be comprised in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also comprise expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequences are regulatable elements. The specific structure of the expression control sequence may vary depending on the species or the function of the cell type, but generally includes 5′ non-transcribed sequences and 5′ and 3′ non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, capping sequence, and CAAT sequence. For example, the 5′ non-transcribed expression control sequence may comprise a promoter region which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid. The vectors of the present application may be selected from plasmids, retroviral vectors and lentiviral vectors. The plasmids, retroviral vectors and lentiviral vectors of the present application may comprise CAR.
In the present application, the term “plasmid” generally refers to DNA molecules other than chromosomes or nucleoids in organisms such as bacteria and yeast, which exist in the cytoplasm and have the ability to replicate autonomously, enabling them to maintain a constant number of copies in daughter cells and express the genetic information they carry. Plasmids are used as vectors of genes in genetic engineering research.
In the present application, the term “retroviral vector” generally refers to a type of RNA virus whose genetic information is stored on ribonucleic acid, and most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, a gene for the proteins that make up the center and structure of the virus; pol, a gene for the reverse transcriptase enzyme; and env, a gene for the proteins that make up the viral capsid. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes that it carries into the host cell genome, for example, integrate CAR molecules into the host cell.
In the present application, the term “lentiviral vector” generally refers to a type of diploid RNA viral vector belonging to retroviruses. The lentiviral vector is based on the genome of lentivirus. Multiple sequence structures related to viral activity in the genome of lentivirus are removed to make it biologically safe, and then a vector prepared from the sequence and expression structure of the target gene required for the experiment is introduced into the genome skeleton. Compared with other retroviruses, lentiviral vectors have a wider range of hosts, have the ability to infect both dividing and non-dividing cells, and for some cells that are difficult to transfect, such as primary cells, stem cells and undifferentiated cells, can greatly improve the transduction efficiency of the target gene (see Chen Chen and Wan Haisu, “Lentiviral vectors and their research progress, Chinese Journal of Lung Cancer 17.12 (2014): 870-876. PMC). Through lentiviral vector transfection, the lentiviral vector can randomly and stably integrate its own genome and the foreign genes that it carries into the host cell genome, for example, integrate CAR molecules into the host cell.
In the present application, the term “transposon” generally refers to a discrete segment of DNA containing a transposase gene, flanked by terminal inverted repeats (TIRs) containing transposase binding sites. Transposases can bind to TIRs and transfer the transposon to a new site. The transposon of the present application is a two-component system consisting of a plasmid carrying CAR (transposon) and another plasmid carrying a transposase. The transposon can be introduced into target cells by means of electrotransduction and the like. For example, first, the two components are electroporated into peripheral blood mononuclear cells (PBMCs), and the expressed transposase acts on the terminal inverted repeats (TIRs) on both sides of the CAR for cleavage of the CAR (transposon) and subsequent integration into the TA dinucleotide sequence in the genome of the target cell (e.g., T-cell). After transposition and stable genomic integration, the CAR protein can be expressed on the surface of the target cell (see Cheng Zhang, Jun Liu, Jiang F Zhong, et al. Engineering CAR-T cells. Biomarker Research. 2017, 5:22).
In the present application, the term “gene editing” generally refers to the technology of site-directed modification of the genome, which may include those based on zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (Cas9) (CRISPR/Cas9), etc. It enables highly efficient targeted modification of the genome by adding, removing or changing genetic materials at specific positions in the genome. The gene editing of the present application may include introducing CAR molecules into the genome of recipient cells through gene editing technologies (such as CRISPR-Cas9).
In the present application, the term “immune effector cell” generally refers to immune cells involved in clearing foreign antigens and performing effector functions in an immune response. For example, plasma cells, cytotoxic T-cells, NK cells, APSC pluripotent cells, mast cells, etc. are included.
In the present application, the term “pharmaceutically acceptable adjuvant” generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.
In the present application, the term “cancer” generally refers to or describes a physiological condition in mammals that is typically characterized by dysregulation of cell proliferation or survival. In the present application, hyperproliferative diseases referred to as cancers include, but are not limited to, solid tumors such as those occurring in the breast, respiratory tract, brain, reproductive organs, digestive tract, urethra, eye, liver, skin, head and neck, thyroid, and parathyroid glands, and their distant metastases. Such diseases also include lymphomas, sarcomas, and leukemias. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic keratinomas, cerebellar and cerebral astrocytomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors. Tumors of the male genitalia include, but are not limited to, prostate and testicular cancers. Tumors of the female genitalia include, but are not limited to, cancers of the endometrium, cervix, ovary, vagina, and vulva, and uterine tumors. Gastrointestinal tumors include, but are not limited to, cancers of the anus, colon, colorectum, esophagus, gallbladder, stomach, pancreas, rectum, small intestine, and salivary glands. Tumors of the urethra include, but are not limited to, cancers of the bladder, penis, kidney, renal pelvis, ureter, and urethra. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without the fibrolamellar variation), cholangiocarcinoma (intrahepatic biliary cancer), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, cancers of the larynx/hypopharynx/nasopharynx/oropharynx, and cancers of the lips and mouth. Lymphatic cancers include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcomas, and rhabdomyosarcomas. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.
When referring to pharmaceutical compositions, “pharmaceutically acceptable carrier” is used to refer to substances such as solid or liquid diluents, fillers, antioxidants, and stabilizers, which are safe for administration, and which are suitable for administration to humans and/or animals without undue adverse side effects, while being suitable for maintaining the viability of the drug or active agent therein.
A “therapeutically effective amount” refers to an amount of an active compound or cell sufficient to elicit the biological or medical response desired by a clinician in a subject. The “therapeutically effective amount” can be determined by those skilled in the art according to the administration route, the subject's body weight, age, condition and other factors. For example, a typical daily dose may range from 0.01 mg to 100 mg of active ingredient per kg of body weight for an antibody. The administration mode of the antibody of the present application includes but is not limited to injection, such as intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal injection and the like.
The term “and/or” should be understood as any one of the alternatives or both of the alternatives.
As used in the present application, the term “comprise” or “include” means including stated elements, integers or steps, but not excluding any other elements, integers or steps. In the present application, when the term “comprise” or “include” is used, unless otherwise specified, situations consisting of the mentioned elements, integers or steps are also covered. For example, when referring to an antibody variable region that “comprises” a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.
In the present application, the term “about” generally refers to a variation with the range of 0.5%-10% above or below a specified value, such as a variation within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
Anti-CD5 Antibody
The present invention uses fully human phages for antibody selection to directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, it dispenses with the difficult step of humanizing murine antibodies. Also, fully human antibodies have lower immunogenicity than humanized murine antibodies and have better potential in antibody drugs (including monoclonal antibodies, biclonal antibodies, ADC, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), detection reagents and other applications.
The present invention uses the method of antigen protein panning, which can efficiently enrich antibodies that simultaneously bind to recombinant CD5 and natural CD5 on the cell membrane, greatly reducing the difficulty of later antibody selection and improving efficiency.
We used a large-capacity phage antibody library to select fully human CD5-specific antibodies, and evaluated the specificity of these antibodies at the phage level by ELISA and FACS experiments. Finally, we obtained several fully human antibody clones with good specificity.
With different antibody libraries and through recombinant CD5 protein panning, we selected 184 monoclonal antibodies for preliminary selection by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), among which 93 clones specifically bound to CD5-Fc-Bio protein and CD5-expressing positive cell Jurkat, but not to control protein CD19-Fc-Bio and CD5-expressing negative cell Raji. After sequencing, 64 different monoclonal sequences were obtained. Subsequently, we identified these 64 antibodies through flow cytometry (FACS) with various CD5 positive (Jurkat, CCRF-CEM) and negative (Raji, NALM6) cell lines, and through enzyme-linked immunosorbent assay (ELISA) with CD5 proteins (Kactus-CD5-Fc-Bio, Acro-CD5-his, SB-CD5-his-Bio), non-related proteins (Kactus-BAFFR-his-Bio, Kactus-CD19-FC-Bio, SA) from different companies, of which 49 clones showed good binding and specificity on multiple cell lines and various protein antigens. The acquisition of these clones laid the foundation for the subsequent development of fully human CD5 CAR-T products or antibody drugs. The overall project flow is shown in
Chimeric Antigen Receptor
In the present application, the CAR may comprise an extracellular domain specifically binding to CD5, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain. In the present application, the extracellular domain of the CAR may comprise the single-chain scFv antibody of the present invention. For example, the single chain antibody may be linked to the transmembrane domain via a hinge region, such as CD8 hinge. In the present application, the CAR can be used to transduce immune effector cells (such as T-cells) and be expressed on the cell surface. Therefore, the present application can also provide T-cells expressing the chimeric antigen receptor, and the use of the T-cells and/or the CAR in the preparation of drugs for treating CD5-related diseases.
In the present application, the chimeric antigen receptor (CAR) may comprise a CD5-binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain. In the present application, the CD5 binding domain may comprise an antibody specifically binding to CD5 or a fragment thereof. The antibody may comprise a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3), and the amino acid sequences of HCDR1-3 are set forth in SEQ ID NOs: 43-45, 49-51, 55-57, 61-63, 67-69 or 73-75. The antibody may also comprise a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2) and a light chain complementarity determining region 3 (LCDR3), and the amino acid sequences of LCDR1-3 are set forth in SEQ ID NOs: 46-48, 52-54, 58-60, 64-66, 70-72 or 76-78. In the present application, the antibody may comprise a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 30, 36, 42, 90, 93 or 96. In the present application, the antibody may comprise a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 29, 35, 41, 89, 92 or 95.
In the present application, the antibody may be a single chain antibody. In some embodiments, the antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 28, 34, 40, 88, 91 or 94 or a functional variant thereof. For example, the single-chain antibody may include scFv10 having a sequence as set forth in SEQ ID NO: 28; the single-chain antibody may include scFv32 having a sequence as set forth in SEQ ID NO: 34; the single-chain antibody may include scFv35 having a sequence as set forth in SEQ ID NO: 40; the single-chain antibody may include scFv6 having a sequence as set forth in SEQ ID NO: 88; the single-chain antibody may include scFv7 having a sequence as set forth in SEQ ID NO: 91; and the single-chain antibody may include scFv9 having a sequence as set forth in SEQ ID NO: 94.
For example, the single-chain antibody of the present application may be scFv10 (originally numbered as clone 10), the sequence of which is set forth in SEQ ID NO: 28. The amino acid sequences of LCDR1-3 of the single-chain antibody scFv10 are set forth in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively; the amino acid sequence of VL is set forth in SEQ ID NO: 29; the amino acid sequences of HCDR1-3 are set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively; and the amino acid sequence of VH is set forth in SEQ ID NO: 30. The single-chain antibody of the present application may be scFv32 (originally numbered as clone 32), the sequence of which is set forth in SEQ ID NO: 34. The amino acid sequences of LCDR1-3 of the single-chain antibody scFv32 are set forth in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, respectively; the amino acid sequence of VL is set forth in SEQ ID NO: 35; the amino acid sequences of HCDR1-3 are set forth in SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, respectively; and the amino acid sequence of VH is set forth in SEQ ID NO: 36. The single-chain antibody of the present application may be scFv35 (originally numbered as clone 35), the sequence of which is set forth in SEQ ID NO: 40. The amino acid sequences of LCDR1-3 of the single-chain antibody scFv35 are set forth in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively; the amino acid sequence of VL is set forth in SEQ ID NO: 41; the amino acid sequences of HCDR1-3 are set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively; and the amino acid sequence of VH is set forth in SEQ ID NO: 42. The single-chain antibody of the present application may be scFv6 (originally numbered as clone 6), the sequence of which is set forth in SEQ ID NO: 88. The amino acid sequences of LCDR1-3 of the single-chain antibody scFv6 are set forth in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; the amino acid sequence of VL is set forth in SEQ ID NO: 89; the amino acid sequences of HCDR1-3 are set forth in SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, respectively; and the amino acid sequence of VH is set forth in SEQ ID NO: 90. The single-chain antibody of the present application may be scFv7 (originally numbered as clone 7), the sequence of which is set forth in SEQ ID NO: 91. The amino acid sequences of LCDR1-3 of the single-chain antibody scFv7 are set forth in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively; the amino acid sequence of VL is set forth in SEQ ID NO: 92; the amino acid sequences of HCDR1-3 are set forth in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively; and the amino acid sequence of VH is set forth in SEQ ID NO: 93. The single-chain antibody of the present application may be scFv9 (originally numbered as clone 9), the sequence of which is set forth in SEQ ID NO: 94. The amino acid sequences of LCDR1-3 of the single-chain antibody scFv9 are set forth in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, respectively; the amino acid sequence of VL is set forth in SEQ ID NO: 95; the amino acid sequences of HCDR1-3 are set forth in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; and the amino acid sequence of VH is set forth in SEQ ID NO: 96.
The CAR of the present application may include a transmembrane domain, and the transmembrane domain may comprises a polypeptide selected from the following proteins: u, R or (chains of T-cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain may comprise an amino acid sequence as set forth in SEQ ID NO: 6 or a functional variant thereof. For example, the transmembrane domain of the present application may include CD8a, the sequence of which is set forth in SEQ ID NO: 6.
In the present application, the co-stimulatory domain may comprise a polypeptide selected from the following proteins: CD28, 4-1BB, OX40 and ICOS. In the present application, the co-stimulatory domain may comprise an amino acid sequence as set forth in SEQ ID NO: 8 or a functional variant thereof.
The CAR described in the present application may include an intracellular signaling domain, which can comprise a signaling domain from CD3ζ. In the present application, the intracellular signaling domain may comprises an amino acid sequence as set forth in SEQ ID NO: 10 or a functional variant thereof.
The CAR of the present application may include a hinge region, and the hinge region may connect the antibody and the transmembrane domain. In the present application, the hinge region may comprise an amino acid sequence as set forth in SEQ ID NO: 4 or a functional variant thereof.
The CAR of the present application may include a signal peptide, and for example, the signal peptide may be located at the N-terminus of the extracellular domain that specifically binds to CD5. The signal peptide may comprise an amino acid sequence as set forth in SEQ ID NO: 2 or a functional variant thereof. For example, the signal peptide may be CD8a signal peptide, the sequence of which is set forth in SEQ ID NO: 2.
In the present application, the CAR can also be linked to a cleaving peptide. In the present application, the cleaving peptide may comprise an amino acid sequence derived from a T2A peptide. In the present application, the cleaving peptide may comprise an amino acid sequence as set forth in SEQ ID NO: 12 or a functional variant thereof. For example, the cleaving peptide can be T2A, the sequence of which is set forth in SEQ ID NO: 12.
In the present application, the CAR can also be connected to an EGFRt fragment, and the EGFRt fragment can be used for signal detection, or used as a molecular switch for CAR-T cells.
In the present application, the CAR may comprise an amino acid sequence as set forth in SEQ ID NO: 22-24, 98, 100 or 102 or a functional variant thereof. For example, the CAR can be selected from CAR001, the sequence of which is set forth in SEQ ID NO: 22. For another example, the CAR can be selected from CAR002 having a sequence as set forth in SEQ ID NO: 23; the CAR can be selected from CAR003 having a sequence as set forth in SEQ ID NO: 24; the CAR can be selected from CAR004 having a sequence as set forth in SEQ ID NO: 98; the CAR can be selected from CAR005 having a sequence as set forth in SEQ ID NO: 100; and the CAR can be selected from CAR006 having a sequence as set forth in SEQ ID No: 102.
In some embodiments, the CAR of the present application may sequentially include a CD5 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus. Among others, the CAR may include a CD5 binding domain, and the sequence of the CD5 binding domain is set forth in SEQ ID NO: 28. Among others, the CD5 binding domain may include HCDR1-3, whose sequences are set forth in SEQ ID NOs: 43-45, respectively; and, the CD5 binding domain may include LCDR1-3, whose sequences are set forth in SEQ ID NOs: 46-48, respectively. For example, the CAR may include CAR001 or the CAR of the present application having the same LCDR1-3 and HCDR1-3 as CAR001. The CD5 binding domain may include a heavy chain variable region having a sequence as set forth in SEQ ID NO: 30; and, the CD5 binding domain may also include a light chain variable region having a sequence as set forth in SEQ ID NO: 29. For example, the CAR may include CAR001 or the CAR of the present application having the same light chain variable region and heavy chain variable region as CAR001. A linker peptide may also be included between the light chain variable region and the heavy chain variable region, the sequence of which is set forth in SEQ ID NO: 18. For example, 25 the CAR may include CAR001 or the CAR of the present application having the same linker peptide as CAR001. The transmembrane domain may include a transmembrane domain from CD8a, the sequence of which may be as set forth in SEQ ID NO: 6. For example, the CAR may include CAR001 or the CAR of the present application having the same transmembrane domain as CAR001. The co-stimulatory domain may comprise a co-stimulatory structure from 4-1BB, the sequence of which may be as set forth in SEQ ID NO: 8. For example, the CAR may include CAR001 or the CAR of the present application having the same co-stimulatory domain as CAR001. The intracellular signaling domain may comprise a signaling domain from CD3ζ, the sequence of which is set forth in SEQ ID NO: 10. For example, the CAR may include CAR001 or the CAR of the present application having the same intracellular signaling domain as CAR001. The CAR can also include a hinge region, which may be located at the C-terminus of the CD5 binding domain and at the N-terminus of the transmembrane domain, and the sequence of the hinge region may be as set forth in SEQ ID NO: 4, for example. For example, the CAR can include CAR001 or the CAR of the present application having the same hinge region as CAR001. The CAR can also be linked to a signal peptide, which can be located at the N-terminus of the CAR, and the sequence of the signal peptide can be as set forth in SEQ ID NO: 2.
The CAR can also be linked to a cleaving peptide, such as T2A. The cleaving peptide can be located at the C-terminus of the intracellular signal transduction domain, the sequence of which can be as set forth in SEQ ID NO: 12. The CAR can also be linked to CSF2RA signal peptide, which can be located before EGFRt, and the sequence of the CSF2RA signal peptide can be as set forth in SEQ ID NO: 14, for example. The CAR can also be linked to a marker detection signal, which can be located at the C-terminus of the CAR (or, the cleaving peptide). The marker detection signal can be selected from the group consisting of GFP, RFP, YFP or EGFRt, and the sequence of EGFRt can be as set forth in SEQ ID NO: 16, for example.
For example, the CAR of the present application can be CAR001, the amino acid sequences of its LCDR1-3 are set forth in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively; the amino acid sequence of its VL is set forth in SEQ ID NO: 29; the amino acid sequences of its HCDR1-3 are set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively; the amino acid sequence of its VH is set forth in SEQ ID NO: 30; the sequence of the linker peptide between VH and VL is set forth in SEQ ID NO: 18; its hinge region is set forth in SEQ ID NO: 4; its transmembrane domain is set forth in SEQ ID NO: 6; its co-stimulatory domain is 4-1BB co-stimulatory domain, as set forth in SEQ ID NO: 8; and its CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 10. The CAR001 further comprises a cleaving peptide as set forth in SEQ ID NO: 12, a CSF2RA signal peptide as set forth in SEQ ID NO: 14, and EGFRt as set forth in SEQ ID NO: 16; and a CD8a signal peptide as set forth in SEQ ID NO: 2.
For example, the CAR of the present application can be CAR002, the amino acid sequences of its LCDR1-3 are set forth in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, respectively; the amino acid sequence of its VL is set forth in SEQ ID NO: 35; the amino acid sequences of its HCDR1-3 are set forth in SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, respectively; the amino acid sequence of its VH is set forth in SEQ ID NO: 36; the sequence of the linker peptide between VH and VL is set forth in SEQ ID NO: 18; its hinge region is set forth in SEQ ID NO: 4; its transmembrane domain is set forth in SEQ ID NO: 6; its co-stimulatory domain is 4-1BB co-stimulatory domain, as set forth in SEQ ID NO: 8; and its CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 10. The CAR002 further comprises a cleaving peptide as set forth in SEQ ID NO: 12, a CSF2RA signal peptide as set forth in SEQ ID NO: 14, and EGFRt as set forth in SEQ ID NO: 16; and a CD8a signal peptide as set forth in SEQ ID NO: 2.
For example, the CAR of the present application can be CAR003, the amino acid sequences of its LCDR1-3 are set forth in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively; the amino acid sequence of its VL is set forth in SEQ ID NO: 41; the amino acid sequences of its HCDR1-3 are set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively; the amino acid sequence of its VH is set forth in SEQ ID NO: 42; the sequence of the linker peptide between VH and VL is set forth in SEQ ID NO: 18; its hinge region is set forth in SEQ ID NO: 4; its transmembrane domain is set forth in SEQ ID NO: 6; its co-stimulatory domain is 4-1BB co-stimulatory domain, as set forth in SEQ ID NO: 8; and its CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 10. The CAR003 further comprises a cleaving peptide as set forth in SEQ ID NO: 12, a CSF2RA signal peptide as set forth in SEQ ID NO: 14, and EGFRt as set forth in SEQ ID NO: 16; and a CD8a signal peptide as set forth in SEQ ID NO: 2.
For example, the CAR of the present application can be CAR004, the amino acid sequences of its LCDR1-3 are set forth in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; the amino acid sequence of its VL is set forth in SEQ ID NO: 89; the amino acid sequences of its HCDR1-3 are set forth in SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, respectively; the amino acid sequence of its VH is set forth in SEQ ID NO: 90; the sequence of the linker peptide between VH and VL is set forth in SEQ ID NO: 18; its hinge region is set forth in SEQ ID NO: 4; its transmembrane domain is set forth in SEQ ID NO: 6; its co-stimulatory domain is 4-1BB co-stimulatory domain, as set forth in SEQ ID NO: 8; and its CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 10. The CAR004 further comprises a cleaving peptide as set forth in SEQ ID NO: 12, a CSF2RA signal peptide as set forth in SEQ ID NO: 14, and EGFRt as set forth in SEQ ID NO: 16; and a CD8a signal peptide as set forth in SEQ ID NO: 2.
For example, the CAR of the present application can be CAR005, the amino acid sequences of its LCDR1-3 are set forth in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively; the amino acid sequence of its VL is set forth in SEQ ID NO: 92; the amino acid sequences of its HCDR1-3 are set forth in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively; the amino acid sequence of its VH is set forth in SEQ ID NO: 93; the sequence of the linker peptide between VH and VL is set forth in SEQ ID NO: 18; its hinge region is set forth in SEQ ID NO: 4; its transmembrane domain is set forth in SEQ ID NO: 6; its co-stimulatory domain is 4-1BB co-stimulatory domain, as set forth in SEQ ID NO: 8; and its CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 10. The CAR005 further comprises a cleaving peptide as set forth in SEQ ID NO: 12, a CSF2RA signal peptide as set forth in SEQ ID NO: 14, and EGFRt as set forth in SEQ ID NO: 16; and a CD8a signal peptide as set forth in SEQ ID NO: 2.
For example, the CAR of the present application can be CAR006, the amino acid sequences of its LCDR1-3 are set forth in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, respectively; the amino acid sequence of its VL is set forth in SEQ ID NO: 95; the amino acid sequences of its HCDR1-3 are set forth in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; the amino acid sequence of its VH is set forth in SEQ ID NO: 96; the sequence of the linker peptide between VH and VL is set forth in SEQ ID NO: 18; its hinge region is set forth in SEQ ID NO: 4; its transmembrane domain is set forth in SEQ ID NO: 6; its co-stimulatory domain is 4-1BB co-stimulatory domain, as set forth in SEQ ID NO: 8; and its CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 10. The CAR006 further comprises a cleaving peptide as set forth in SEQ ID NO: 12, a CSF2RA signal peptide as set forth in SEQ ID NO: 14, and EGFRt as set forth in SEQ ID NO: 16; a CD8a signal peptide as set forth in SEQ ID NO: 2.
Nucleic Acid, Vector, Cell, Preparation Method and Composition
In another aspect, the present application provides an isolated nucleic acid molecule encoding the CAR of the present application. The isolated nucleic acid molecule encoding CAR of the present application can comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19-21, 25-27, 31-33, 37-39, 79-87, 97, 99 or 101 or a functional variant thereof. The nucleic acid molecule of the present application can be isolated. For example, it may be produced or synthesized by (i) amplification in vitro, such as amplification by polymerase chain reaction (PCR), (ii) cloning and recombination, (iii) purification, for example by fractionation through enzyme digestion and gel electrophoresis, or (iv) synthesis, for example by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
In another aspect, the present application provides a vector comprising the nucleic acid molecule. In the present application, the vector can be selected from one or more of plasmids, retroviral vectors and lentiviral vectors. The lentiviral vector of the present application may comprise CAR. For example, lentiviral vector of the present application can comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19-21, 25-27, 31-33, 37-39, 79-87, 97, 99 or 101 or a functional variant thereof. In addition, other genes may be comprised in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also comprise expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequences are regulatable elements. The specific structure of the expression control sequence may vary depending on the species or the function of the cell type, but generally includes 5′ non-transcribed sequences and 5′ and 3′ non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, capping sequence, and CAAT sequence. For example, the 5′ non-transcribed expression control sequence may comprise a promoter region which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid. One or more nucleic acid molecules of the present application can be operably linked to the expression control element. Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector, including vector scFv plasmid and/or CAR plasmid.
In another aspect, the present application provides an immune effector cell comprising the CAR, the nucleic acid molecule, or the vector of the present application. In the present application, the immune effector cells may be mammalian cells. In the present application, the immune effector cells may be selected from T lymphocytes and natural killer (NK) cells.
In another aspect, the present application provides a method for preparing immune effector cells, which comprises knocking out the CD5 gene of the immune effector cells, and introducing the vector of the present application into the immune effector cells. For example, the vectors of the present application can be introduced into the immune effector cells, such as T lymphocytes or natural killer (NK) cells. In some embodiments, each type of cells or each cell may comprise one or more of the vectors of the present application. In some embodiments, each type of cells or each cell may comprise multiple (e.g., 2 or more) or multiple types of (e.g., 2 or more types of) vectors of the present application. In the present application, for introducing the vector into immune effector cells, the vector of the present application can be introduced into the cells by methods known in the art. For example, immune effector cells can be transfected with retroviral vectors to integrate the viral genome with CAR molecules into the host genome to ensure long-term and stable expression of the target gene. For another example, transposons can be used and introduced into target cells through CAR (transposon)-carrying plasmids and transposase-carrying plasmids. As another example, CAR molecules can be added to the genome through gene editing (such as CRISPR/Cas9). In the present application, the vector carrying the CAR molecule of the present application can be introduced into the cells by methods known in the art, such as electroporation, and liposome transfection (lipofectamine 2000, Invitrogen).
In another aspect, the present application provides a pharmaceutical composition, which comprises the immune effector cells and a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionic surfactants, etc. In the present application, the pharmaceutical composition can be formulated for administration orally, intravenously (e.g., intravenous injection, I.V.), intramuscularly (e.g., intramuscular injection, I.M.), in situ at the site of the tumor, by inhalation, rectally, vaginally, transdermally or via a subcutaneous depot.
Pharmaceutical Use
In another aspect, the present application provides use of the antibody, the CAR, the nucleic acid molecule, the vector, or the immune effector cell in the preparation of a drug for treating a disease or condition associated with the expression of CD5. In the present application, the disease or condition associated with the expression of CD5 is a cancer or malignancy. In some embodiments, the cancer or malignancy may be selected from a T-cell malignancy or a B-cell malignancy. Among them, the T-cell malignancy can be selected from T-cell acute lymphocytic leukemia (T-ALL), T-cell lymphoma (TCL) (such as peripheral T-cell lymphoma, cutaneous T-cell lymphoma (CTCL), T-cell non-Hodgkin's Lymphoma (T-NHL)); the malignant B-cell neoplasm may be selected from chronic lymphocytic leukemia (B-CLL) (e.g. hairy cell leukemia cell (HCL)), mantle cell lymphoma (B-MCL), diffuse large B-cell lymphoma (DLBCL).
In another aspect, the present application provides use of the antibody, the CAR, the nucleic acid molecule, the vector, or the immune effector cell in the treatment of a disease or condition associated with the expression of CD5.
In another aspect, the present application provides a method for treating a disease or condition associated with the expression of CD5, which comprises administering the antibody, the CAR, the nucleic acid molecule, the vector, or the immune effector cell to a patient.
Without intending to be bound by any theory, the following examples are only to illustrate the working mode of the chimeric antigen receptor, vector, cell, and composition of the present application, and are not intended to limit the scope of the invention of the present application.
Research Overview
The researchers use fully human phages for antibody selection to directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, it dispenses with the difficult step of humanizing murine antibodies, and fully human antibodies have lower immunogenicity than humanized murine antibodies, providing better potential in the development of CAR-T. After obtaining antibody clones that specifically bind to the cell surface CD5 antigen and CD5 recombinant protein, the applicant conducted in-depth research, constructed these clones and the control clone H65 onto the second-generation CAR structure, then packaged them with lentivirus and transfected them into T-cells, and at the level of CAR-T cells, selected fully human CD5 antibody clones and candidate CAR-T molecules with stronger functions than the control clone H65 in terms of target cell activation and killing, and target cell stimulation and proliferation.
In addition, according to the literature reports, in most constructed tumor animal models, re-emerging tumor cells retain CD5 expression, which suggests that tumor recurrence is not due to the loss of antigen, and that failure to eradicate all xenografts is due to the poor persistence of CD5 CAR-T cells in mice[13]. The present application uses CRISPR technology to knock out the CD5 gene in the jurkat cell line, and successfully selects sgRNAs with high knockout efficiency. The experimental results show that CD5-knockout CAR-T cells can expand normally, and maintain the function of killing CD5-positive target cells, while minimizing the self-activation and suicide of CD5 CAR-T, ensuring its persistence and effectiveness in clinical verification.
Appropriate negative panning and positive panning strategies were used to enrich the specific antibody clones we need from the phage antibody library.
Construction of Phage Antibody Library
The phage antibody libraries we constructed include natural libraries, semi-synthetic libraries and single-domain libraries. The semi-synthetic phage antibody library, used together with the natural library, solves the problem that the natural library may lack CD5 high-affinity antibody clones. The single-domain phage antibody library is an antibody library composed only of the variable region amino acids of heavy chain antibodies, which have similar or higher specificity and affinity than traditional antibodies although their molecular weight is only 12-15 kDa. In addition, single-domain antibodies have attracted much attention because of their stable physical and chemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.
CD5 Protein Panning
Using CD5-Fc-Bio as the positive panning protein and CD19-Fc-Bio as the negative panning protein, multiple rounds of panning were performed to obtain a phage pool enriched with the target antibody clones. The experimental steps are briefly described as follows:
The enriched phage pool can be used for subsequent monoclonal selection and ELISA/FACS selection.
Main Materials and Reagents:
Experimental Results:
Using different antibody libraries, through 3 rounds of protein panning, a significant increase in the recovery rate was observed in each round of panning (Table 1), proving that antibody clones were effectively enriched.
It can be seen that after three rounds of panning, different antibody libraries were enriched (the recovery rate of the third round was significantly higher than that of the previous round).
Purpose and Principle:
The phage pool enriched by the affinity panning step contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. In order to obtain specific clones, we need to isolate monoclones, package the monoclones into monoclonal phages, and conduct preliminary selection on a large number of monoclones by enzyme-linked immunoassay (ELISA) and flow cytometry (FACS) to select monoclones specifically binding to both CD5 protein and CD5 positive cell line Jurkat. The specific monoclones are further determined by DNA sequencing to determine unique antibody sequences contained therein.
In the primary selection by ELISA, through the binding of streptavidin and biotin, the biotinylated target protein (CD5-Fc-Bio) is closer to the natural antigen conformation in the reaction solution. Those that only bind to CD5-Fc-Bio but not to the control antigen CD19-Fc-Bio are identified as specific clones. FACS primary selection is carried out using the positive cell line Jurkat with high expression of CD5 and the cell line Raji negative for CD5, and those that only bind to Jurkat cells but not to Raji cells are identified as specific clones. Through ELISA and FACS, we can obtain candidate antibodies that can bind to recombinantly expressed CD5 proteins and recognize the natural CD5 molecules on the cell surface for subsequent further selection.
Brief Steps of ELISA Primary Screening Experiment:
Brief Steps of FACS Primary Screening Experiment:
Main Materials and Reagents:
Experimental Results:
Monoclones were randomly selected from the enriched phage antibody pool, packaged into phages, and the binding of the monoclonal phages to CD5-Fc-Bio protein and control protein CD19-Fc-Bio was detected by phage ELISA to find CD5-specific phage antibody clones. The ELISA results of some clones are shown in
The results of FACS primary selection of antibody clones corresponding to ELISA are shown in
Through ELISA detection and FACS primary selection, we obtained a total of 93 ELISA and FACS double-positive clones with good specificity, and then we sequenced the 93 double-positive clones with good specificity, and obtained 64 different monoclone sequences after sequencing. Then, these 64 monoclones with different sequences were further tested for the binding specificity of the candidate clones through FACS identification with multiple cell lines and ELISA identification with various antigens.
Experimental Purpose and Principle:
Antibodies used for therapy must have very good target specificity, binding only to the target antigen and not to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will vary (isomers or mutants) or the bound ligands will be different, and it is also necessary to investigate whether our antibodies can bind to various cells positive for the target protein. In order to further analyze the specificity and universality of these monoclones and find the best candidate clones, we further evaluated the specificity of the primarily selected clones by flow cytometry. In this experiment, we used a variety of CD5-positive cell lines and a variety of CD5-negative cell lines to react with these monoclonal phage antibodies, and analyzed whether these clones could bind to the CD5 antigen on different cell lines and whether they had any non-specific binding to other cell lines that do not express CD5. Through this experiment, we obtained several clones with excellent specificity.
Main Samples and Reagents:
The rest of the reagents were the same as the FACS primary selection.
Experimental Results:
Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified the unique specific clones obtained in Example 2 on more antigens and cell lines by using ELISA and flow cytometry. The results are shown in
Experimental Purpose and Principle:
Antibodies used for therapy must have very good target specificity, binding only to the target antigen and not to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen produced by different companies will vary (isomers or mutants), and it is also necessary to investigate whether our antibodies can bind to various target proteins. In order to further analyze the specificity and universality of these monoclones and find the best candidate clones, we further evaluated the specificity of the primarily selected clones by enzyme linked immunosorbent assay (ELISA). In this experiment, we used CD5 antigens purchased from different companies and various CD5-unrelated antigens to react with these monoclonal phage antibodies, and analyzed whether these clones could bind to different CD5 antigens and whether they had any non-specific binding to other CD5-unrelated antigens. Through this experiment, we obtained several clones with excellent specificity.
Main Samples and Reagents:
The rest of the reagents were the same as in the ELISA primary screening.
Experimental Results:
Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified multiple clones obtained in Example 2 on various antigens using enzyme-linked immunosorbent assay (ELISA). As shown in
The following table lists the amino acid and nucleic acid sequence numbers corresponding to the heavy chain variable region (HCVR), light chain variable region (LCVR), ScFv and CDR sequences of some selected anti-CD5 antibodies:
(1) Experiment Purpose and Principle:
All mature T-cells express the CD5 antigen on the surface, while the CD5 CAR-T without CD5 knockout developed by Mamonkin M and other researchers has been reported to have a certain degree of suicide1, and survive in the patient for a limited period of time, which greatly limits the disease remission time of the patient and the application of the CAR-T product. In order to solve this problem, the present application uses the CRISPR/Cas9 technology to knock out the CD5 antigen on the surface of T-cells, which minimizes the self-activation and suicide of CD5 CAR-T, and ensure its persistence and effectiveness.
In order to select sgRNAs with high knockout efficiency, we firstly use different sgRNAs to knock out the CD5 gene in jurkat cells that naturally highly express CD5, so as to determine the efficient sgRNAs for knocking out the CD5 antigen of T-cells and obtain jurkat-CD5ko pool with high knockout efficiency, and then obtained CD5-negative monoclonal jurkat-CD5ko cells through monoclonal selection. In this way, the difference between jurkat and jurkat-CD5ko cells lies in this knocked-out gene. This pair of cells will have important applications in the identification of monoclonal antibody binding specificity and CAR-T functional verification.
(2) The Brief Experimental Steps are as Follows:
As shown in
The present application uses lentiviral transfection to make the knockout T-cells express CAR. For the preparation process of CAR-T, please refer to the patent (Zhou J, Liu J, Hu G, et al. Chimeric antigen receptor (car) binding to bcma, and uses thereof: U.S. patent application Ser. No. 16/650,580[P]. 2020-8-6.). The lentiviral vector is based on the genome of lentivirus. Multiple sequence structures related to viral activity in the genome of lentivirus are removed to make it biologically safe, and then a vector prepared from the sequence and expression structure of the target gene required for the experiment is introduced into the genome skeleton. Compared with other retroviruses, lentiviral vectors have a wider range of hosts, have the ability to infect both dividing and non-dividing cells, and for some cells that are difficult to transfect, such as primary cells, stem cells and undifferentiated cells, can greatly improve the transduction efficiency of the target gene (see Chen Chen and Wan Haisu, “Lentiviral vectors and their research progress, Chinese Journal of Lung Cancer 17.12 (2014): 870-876. PMC). Through lentiviral vector transfection, CAR molecules can be integrated into host cells.
The T-cells were knocked out of CD5 and transfected with the CAR lentivirus with the structure shown in
The brief experimental steps for CD5 antigen expression detection and EGFRt expression detection of CAR-T/T cells are as follows:
Main Materials and Reagents:
Experimental Results:
As shown in
Experimental Purpose and Principle:
In the present application, a variety of specific antibody clones targeting CD5 protein that are enriched, selected and identified from the phage antibody library by affinity panning are constructed on the second-generation CAR, and considering the fact that the scFv that binds to the CD5 antigen may not have good activation function, its function on CAR-T cells needs to be further confirmed to select the most active CAR molecules. To this end, we prepare lentiviral vectors of these CAR molecular clones, and transduce T-cells to prepare CAR-T cells. Then, the in vitro biological efficacy of CAR-T cells is evaluated by CD107a degranulation assay and in vitro cytotoxicity assay. Through these functional verifications at CAR-T level, CAR molecules with ideal efficacy and safety are finally selected for downstream CAR-T product development.
CD107a Degranulation Assay
CD107a is a marker for intracellular microvesicles, and CD107a on the cell membrane increases after granzyme-loaded microvesicles fuse with the cell membrane, and when its recovery is blocked by monesin (purchased from BioLegend), the strength of microvesicle release can be quantitatively reflected. When CAR-T is stimulated by the target antigen on the target cell, it will cause the release of granzyme, and the activation of T-cells can be judged by the increase of CD107a by flow cytometry.
Brief Experimental Steps of CD107a Degranulation:
Main Samples and Reagents:
Experimental Results:
The CAR-T cells were obtained by lentiviral transduction, and CD107a degranulation assay was performed after the CAR-T cells were cultured in vitro for 9-12 days. The CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibody for 4 h, and the cell density of both CAR-T cells and target cells was 5×105 cells/mL. Then, after the samples were labeled with anti-CD8 antibody and anti-EGFR antibody, flow cytometry was performed. Analysis was conducted with Flowjo software. In the scatter plot, living cell gate (P1) was selected and cell debris was removed; for the cells in the P1 gate, a single scattered cell gate (P2) was selected after analysis; then, CD8 positive cells were further selected in the P2 gate (P3); finally, in the P3 gate, the proportion of CD107a-positive cells in positive cells (i.e., CAR-positive cells) found through anti-EGFR antibody staining was analyzed. The analysis results are shown in
In Vitro Cytotoxicity Assay
The purpose and principle of the assay: in the in vitro cytotoxicity assay, CCRF-CEM and MOLT4 were used as CD5-positive target cells, and K562 and RAJI cells are used as CD5-negative target cells to evaluate the antigen-specific killing ability of CD5 CAR-T cells. Among others, the above cells are respectively transduced by lentivirus to obtain target cells stably expressing firefly luciferase, so the activity of luciferase in the sample can reflect the number of target cells. The CAR-T cells and target cells are co-incubated and cultured. When target cells are killed by CAR-T cells, luciferase is released and quickly inactivated (firefly luciferase has a half-life of about 0.5 h). If the target cells are not killed or inhibited by CAR-T cells, more luciferases will be produced as the target cells proliferate and continue to express luciferase. Therefore, the killing of target cells by CAR-T can be detected by the activity of luciferase.
Brief Steps of In Vitro Cytotoxicity Assay
Main Samples and Reagents:
Experimental Results:
The CAR-T cell sample and a fixed number of target cells (1×104) were mixed according to different effector-target ratios (E:T), and incubated together for 24 h, and then the luciferase activity (RLU) in the sample was detected. Among them, CD5 ko T-cells were the control sample comprising only the target cells. Since the luciferase activity can reflect the number of target cells in the sample, the killing/inhibiting ability of CAR-T cells on target cells can be obtained through the change of luciferase activity in the sample. The lower the luciferase activity readout (RLU), the more target cells are killed.
As shown in
Repeated Stimulation Proliferation Assay
Experimental Purpose and Principle:
Mitomycin-treated target cells (CCRF-CEM) were mixed with different groups of CD5 CAR-T cells for 4 stimulations, and then the CAR-T cells and target cells were co-incubated to determine the proliferation ability of different scFv CAR-T cells after being repeatedly stimulated by the target cells.
Brief Steps of Cytotoxicity Assay after Repeated Stimulation:
Experimental Results:
As shown in
Experimental Purpose and Principle:
The affinity between CD5 scFvs and the antigen may also have an important impact on the killing effect and duration of CAR-T in patients. In order to determine this important property, the present application uses the Octet molecular interaction technology of ForteBio to determine it. The biofilm interferometry 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 surface of the sensor and collects the reflected light. The reflected light spectrum of different frequencies is affected by the thickness of the optical film layer of the biosensor. Some frequencies of reflected light form constructive interference (blue), while others form destructive interference (red). These interferences are detected by the spectrometer and form an interference spectrum, which is demonstrated by 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 shift of the interference spectrum in real time, and this shift directly reflects the thickness of the biofilm on the sensor surface, from which high-quality data of biomolecular interaction can be obtained, so as to determine the kinetic parameters (Kon, Kdis and KD) of biomolecular interactions, providing important information for the research and development process.
Brief Experimental Steps:
Experimental Results:
Affinity refers to the strength of the binding of a single molecule to its ligand and is usually measured and reported by the equilibrium dissociation constant (KD), which is used to assess 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 reactants. The smaller the KD value, the greater the affinity of the antibody for its target. As shown in Table 3 and
The foregoing detailed description has been offered by way of explanation and exemplification, and it not intended to limit the scope of the appended claims. Variations of the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.
The specific sequences in the Examples of the present application are as follows:
CDRs of Scfv10 (Clone 10) Antibody in CAR001:
CDRs of Scfv32 (Clone 32) Antibody in CAR002:
CDRs of Scfv35 (Clone 35) Antibody in CAR003:
CDRs of Scfv6 (Clone 6) Antibody in CAR004:
CDRs of Scfv7 (Clone 7) Antibody in CAR005:
CDRs of Scfv9 (Clone 9) Antibody in CAR006:
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011200628.8 | Nov 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/127995 | 11/1/2021 | WO |
