CD7-targeted single-domain antibody and application thereof

Abstract

The invention discloses a CD7-targeted single-domain antibody and an application thereof. The single-domain antibody comprises an amino acid sequence which has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with any one amino acid sequence as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The fully human single-domain antibody provided by the invention can specifically recognize and bind human CD7 protein and CD7 + cells, and has very strong affinity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a single-domain antibody targeting CD7 and application thereof. BACKGROUND

[0002] CD7, also known as GP40, is a 90kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily, which is mainly highly expressed on T cells and natural killer cells (NK cells) and involved in T cell activation, proliferation and intercellular signal transduction. CD7 can also be continuously expressed on some malignant tumor cells such as acute leukemia and T cell lymphoma, and thus is considered as a potential tumor treatment target.

[0003] Clinical observation found that the high expression of CD7 in some patients with acute T lymphoblastic leukemia (T-ALL) and peripheral T cell lymphoma has a certain specificity and is related to disease progression. In addition, CD7 is only expressed in limited normal hematopoietic stem cells and bone marrow progenitor cells, so targeting CD7 is expected to selectively eliminate tumor cells while preserving normal immune function. Studies have attempted to construct CD7-specific antibodies or antibody derivatives for in vitro cytotoxicity experiments and animal model verification, indicating that this target has high therapeutic potential.

[0004] However, most of the existing CD7 antibodies are traditional full-length antibodies or chimeric antibodies based on murine sources, which have large molecular weight, strong immunogenicity and limited tissue penetration ability, limiting their application in solid tumor treatment. In order to overcome the above problems, the development of small molecule, low immunogenicity and high specificity and affinity CD7 single-domain antibodies has become a research focus. SUMMARY

[0005] The purpose of the present application is to provide a single-domain antibody targeting CD7 and application thereof to overcome the deficiencies in the prior art.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is:

[0007] The first aspect is to provide a single-domain antibody targeting CD7, which comprises CDR1, CDR2 and CDR3.

[0008] CDR1 is an amino acid sequence as shown in SEQ ID NO: 1, CDR2 is an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 3; or

[0009] wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 4, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 5, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 6; or

[0010] wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 7, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 8, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 9; or

[0011] wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 10, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 11, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 12; or

[0012] wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 13, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 14, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 15; or

[0013] wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 16, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 17, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 18.

[0014] In some embodiments, the single-domain antibody targeting CD7 provided herein comprises CDR1, CDR2, and CDR3; wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 1, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 2, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 3.

[0015] In some embodiments, the single-domain antibody targeting CD7 provided herein comprises CDR1, CDR2, and CDR3; wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 4, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 5, and CDR6 is an amino acid sequence as set forth in SEQ ID NO: 3.

[0016] In some embodiments, the single-domain antibody targeting CD7 provided herein comprises CDR1, CDR2, and CDR3; wherein CDR1 is an amino acid sequence as set forth in SEQ ID NO: 7, CDR2 is an amino acid sequence as set forth in SEQ ID NO: 8, and CDR3 is an amino acid sequence as set forth in SEQ ID NO: 9.

[0017] In some embodiments, the single-domain antibody targeting CD7 provided by the present application comprises CDR1, CDR2 and CDR3; wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 10, CDR2 is an amino acid sequence as shown in SEQ ID NO: 11, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 12.

[0018] In some embodiments, the single-domain antibody targeting CD7 provided by the present application comprises CDR1, CDR2 and CDR3; wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 13, CDR2 is an amino acid sequence as shown in SEQ ID NO: 14, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 15.

[0019] In some embodiments, the single-domain antibody targeting CD7 provided by the present application comprises CDR1, CDR2 and CDR3; wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 16, CDR2 is an amino acid sequence as shown in SEQ ID NO: 17, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 18.

[0020] In the technical solutions disclosed by the present application, the specific amino acid sequence information of SEQ ID NO: 1 to SEQ ID NO: 18 is shown in Table 1.

[0021] Table 1

[0022] SEQ ID NO: amino acid sequence 1 DYYMN 2 GIGSGGGNTYYADSVKS 3 APGGWLSRYFDL 4 DYAMN 5 WINPGNGSPSYAKKFQG 6 SGGAFDY 7 GYYMH 8 WMNPNSGNTGYAQEFQG 9 DLGPMAFDI 10 GYYMH 11 IINPSGGSTSYAQKFQG 12 ALGWYGSGSNDY 13 NAWMR 14 IINPSGGSTSYAQKFQG 15 HDWFEP 16 NAWMR 17 IINPSGDSTSYAQKFQG 18 HDWFDP

[0023] The single-domain antibody or CDR region thereof provided by the present application encompasses the CDR defined by the above numbering scheme or other known numbering schemes. For example, those skilled in the art can understand that the CDR region determined by any numbering scheme, as long as it is the same as, or contains the CDR region of the present application, falls within the protection scope of the present application.

[0024] The single-domain antibody targeting CD7 provided by the present application comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences shown in Table 2 or comprising the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0025] In some embodiments, the CD7 binding single domain antibodies provided herein comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the CD7 binding single domain antibodies provided herein comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the CD7 binding single domain antibodies provided herein comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the CD7 binding single domain antibodies provided herein comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the CD7 binding single domain antibodies provided herein comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the CD7 binding single domain antibodies provided herein comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24.

[0026] In some embodiments, the CD7 binding single domain antibodies provided herein comprise any one of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0027] In some embodiments, the single-domain antibody targeting CD7 provided by the present application is an amino acid sequence as set forth in SEQ ID NO: 19. In some embodiments, the single-domain antibody targeting CD7 provided by the present application is an amino acid sequence as set forth in SEQ ID NO: 20. In some embodiments, the single-domain antibody targeting CD7 provided by the present application is an amino acid sequence as set forth in SEQ ID NO: 21. In some embodiments, the single-domain antibody targeting CD7 provided by the present application is an amino acid sequence as set forth in SEQ ID NO: 22. In some embodiments, the single-domain antibody targeting CD7 provided by the present application is an amino acid sequence as set forth in SEQ ID NO: 23. In some embodiments, the single-domain antibody targeting CD7 provided by the present application is an amino acid sequence as set forth in SEQ ID NO: 24.

[0028] Table 2

[0029]

[0030] The second aspect is to provide a nucleic acid molecule encoding the single-domain antibody of the first aspect of the present application.

[0031] In some embodiments, the nucleic acid provided by the present application is in the form of DNA or RNA. In some embodiments, the DNA provided by the present application includes cDNA, genomic DNA, or artificially synthesized DNA. In some embodiments, the DNA provided by the present application is single-stranded or double-stranded DNA. In some embodiments, the DNA provided by the present application is coding strand or non-coding strand DNA.

[0032] The third aspect is to provide a vector comprising the nucleic acid molecule of the second aspect of the present application.

[0033] In some embodiments, the vector is a plasmid, a lentivirus vector, an adenovirus vector, an AAV, a retrovirus vector, a transposon, or a combination thereof.

[0034] The fourth aspect is to provide a construct comprising the single-domain antibody targeting CD7 of the first aspect of the present application, further comprising one or more other residues, groups, moieties, or binding units, and connected by one or more linkers.

[0035] The fifth aspect is to provide a pharmaceutical composition comprising the single-domain antibody targeting CD7 of the first aspect of the present application, or the nucleic acid molecule of the second aspect, the vector of the third aspect, or the construct of the fourth aspect, and one or more pharmaceutically acceptable excipients and / or carriers.

[0036] The sixth aspect is to provide the use of the above-mentioned pharmaceutical composition in the treatment of tumors or cancers.

[0037] In some embodiments, the cancer or tumor refers to a cancer or tumor associated with CD7 expression.

[0038] A seventh aspect provides a diagnostic reagent comprising the single-domain antibody targeting CD7 according to the first aspect, or the nucleic acid molecule according to the second aspect, the vector according to the third aspect, or the construct according to the fourth aspect.

[0039] Compared with the prior art, the present application has the following technical effects:

[0040] The fully human single-domain antibody provided by the present application can specifically recognize and bind to human CD7 protein and CD7 + cells, and has extremely strong affinity. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The figure shows the phage enrichment of the single-domain antibody phage display library screening targeting CD7.

[0042] Figure 2 The figure shows the results of detecting the binding of the single-domain antibody to CD7 protein and PD1 protein by ELISA.

[0043] Figure 3 The figure shows the competition of n701, n702, n703, n704, n706 and n705.

[0044] Figure 4 The figure shows the binding results of 40nM single-domain antibody to the CD7 highly-expressed cell line Jurkat.

[0045] Figure 5 The figure shows the binding results of 20nM single-domain antibody to the CD7 highly-expressed cell line Jurkat. DETAILED DESCRIPTION

[0046] In order to enable a more complete understanding of the application, some definitions are listed below. The above definitions are intended to include grammatical equivalents.

[0047] An "antibody molecule" in the present invention means a protein consisting of one or more domains encoded by all or a portion of a recognized immunoglobulin gene. The recognized immunoglobulin genes, e.g., in humans, include the kappa (K), lambda (λ), and heavy chain loci, which include numerous variable region genes and several constant region genes, mu (μ), delta (δ), gamma (γ), epsilon (ε), alpha (α), respectively, that encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. An antibody in the present invention is meant to include whole antibodies, individual chains thereof, and all portions, domains or fragments thereof, as well as natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic or other purposes as further defined below. The term "antibody" includes antibody fragments, which are well known in the art, such as Fab, Fab', F(ab')2, Fv, scFv, or antigen binding domains of antibodies (e.g., VHH domains or VH / VL domains), or those produced by modification of whole antibodies or resynthesized using recombinant DNA techniques. The term "antibody" includes monoclonal as well as polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing antibodies, or inhibitory antibodies, or stimulatory antibodies. Antibodies of the present invention can be non-human antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

[0048] Specifically included within the definition of "antibody" are aglycosylated antibodies. Preferably, the "aglycosylated antibodies" used in the present invention mean antibodies that lack the attachment of a sugar at position 297 in the Fc region, where numbering is according to the EU system of Kabat. The aglycosylated antibodies can be deglycosylated antibodies, which are antibodies from which the Fc carbohydrate has been removed, e.g., by chemical or enzymatic means. Alternatively, the aglycosylated antibodies can be aglycosylated or unglycosylated antibodies, which are antibodies that do not express an Fc carbohydrate, e.g., by mutating one or more residues that encode the glycosylation pattern or by expression in an organism that does not attach sugars to proteins, such as bacteria.

[0049] "IgG" in the present invention means a polypeptide belonging to the class of antibodies that is essentially encoded by the recognized immunoglobulin gamma gene. In humans, this class includes IgGl, IgG2, IgG3, and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b, IgG3. "Immunoglobulin (Ig)" in the present invention means a protein consisting of one or more polypeptides essentially encoded by an immunoglobulin gene. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have many structural forms, including, but not limited to, full-length antibodies, antibody fragments, and individual immunoglobulin domains. The Ig domains known in the IgG class of antibodies are VH, Cγi, Cγ2, Cγ3, VL, and CL.

[0050] A single domain antibody in the present invention refers to a fragment of antibody molecule consisting of a single immunoglobulin variable region, which is capable of recognizing and binding to an epitope of an antigen independently without pairing with a complete heavy chain and light chain. The variable region can be derived from naturally occurring heavy chain antibodies (such as VHH of camelid) or light chain antibodies (such as VNAR of shark IgNAR), or obtained by artificial modification or engineering from the heavy chain or light chain variable region of conventional immunoglobulin. A single domain antibody usually has a smaller molecular weight (about 12-15 kDa), high stability, good water solubility and antigen binding specificity, and can be used independently for diagnosis, treatment or as a molecular module to further construct multispecific antibodies, fusion proteins or other immunomolecules.

[0051] An "antigen" used in the present invention means a compound, composition or substance that can stimulate antibody production or T cell response in an animal body, including compositions injected or absorbed into the animal body, which can be a protein, a sugar, a lipid or other pathogen.

[0052] An "antigen epitope" used in the present invention refers to a structure or region on an antigen molecule that can be specifically bound by an immune recognition molecule. The immune recognition molecule includes but is not limited to an antibody, a single domain antibody, an antibody fragment, a T cell receptor or a functional derivative thereof. The antigen epitope can be a naturally occurring region on a protein, a polypeptide, a polysaccharide, a lipid, a nucleic acid or a derivative thereof, or a functional region obtained by chemical modification, artificial synthesis or molecular engineering. The epitope can be a linear epitope (composed of consecutive amino acids or monomer sequences), or a conformational epitope (composed of monomer residues that are spatially adjacent after three-dimensional folding of the antigen molecule, but not consecutive in the primary sequence). The antigen epitope is not limited to a natural structure, and also includes: an epitope variant mutated, modified or optimized; an amino acid sequence with partial homology or conservative substitution; a functional binding region formed by computational design or artificial construction.

[0053] An "immunoglobulin single variable domain" used in the present invention means an immunoglobulin variable domain capable of specifically binding to an antigen epitope without pairing with other immunoglobulin variable domains. An example of the immunoglobulin single variable domain in the meaning of the present invention is a "domain antibody", such as a VH domain antibody in the present invention.

[0054] A "heavy chain variable region (VH)" used in the present invention means a region of an immunoglobulin heavy chain near the N-terminal amino acid sequence that varies greatly, which is the heavy chain variable region domain in a conventional 4-chain antibody. It is particularly used to distinguish VHH antibodies of camelids.

[0055] "Identity" as used herein means the similarity between nucleotide or amino acid sequences, also referred to as sequence identity. Sequence identity is typically measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences. A homologue or variant will have a relatively high degree of sequence identity when aligned using standard methods. Sequence alignment methods for comparison are well known in the art. The sequence identity between a sequence described herein and a sequence having identity thereto can be at least 75%, 80%, 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Various programs and alignment algorithms are described in: Smith and Waterman, Adv Appl. Math., 2:482, 1981; Needlema and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73: 237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 16: 10881-10890, 1988; and Altschul et al., Nature Genet., 1994, 6: 119-129.

[0056] NCBI Basic Local Alignment Search Tool (BLAST™) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet at the address www.ncbi.nlm.nih.gov. BLAST programs for sequence analysis programs blastp, blastn, blastx, tblastn and tblastx are available on the internet.

[0057] The term "fully human antibody" as used herein refers to an antibody molecule whose variable and constant regions are derived from human immunoglobulin gene repertoires, and does not contain any amino acid sequences from non-human species. The fully human antibody includes, but is not limited to, intact immunoglobulin molecules, single chain antibodies, single domain antibodies, antibody fragments (such as Fab, F(ab')2, scFv, VH, VL, etc.), and antibody derivatives fused to other molecules or protein domains. The fully human antibody can be obtained in a variety of ways, including but not limited to, from animals or cells carrying or expressing human immunoglobulin genes; from human antibody libraries by phage display, yeast display, mammalian cell display or other in vitro display systems; directly isolated, expanded or cloned from human B cells, plasma cells or immune cells; generated by artificial synthesis, genetic engineering or computer-aided design, and the sequences correspond to human immunoglobulin gene repertoires.

[0058] The term "amino acid" as used herein means one of the 20 naturally occurring amino acids or any unnatural analog thereof, which can be at a specifically designated position. The term "protein" as used herein means at least two covalently linked amino acids, and includes proteins, polypeptides, oligopeptides, and peptides. The protein can be composed of naturally occurring and peptide bonds, or of synthetic peptide mimetic structures, i.e., "analog." Thus, the term "amino acid" or "peptide residue" as used herein means both naturally occurring and synthetic amino acids. For example, for purposes of the present application, homophenylalanine, citrulline and norleucine are considered to be amino acids for purposes of the present application. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain can be in the (R) or (S) configuration. In preferred embodiments, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions can be made, for example, to prevent or delay in vivo degradation.

[0059] The term "nucleic acid" as used herein means a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds. Thus, the term includes nucleotide polymers in which the nucleotides and linkages between them include non-naturally occurring synthetic analogs, such as, but not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. For example, these polynucleotides can be synthesized using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, typically no more than about 50 nucleotides. It will be appreciated that, when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" is substituted for "T."

[0060] The present application uses conventional symbols to describe nucleotide sequences: the left-hand end of single-stranded nucleotide sequences is the 5' end; the left-hand direction in double-stranded nucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the coding strand.

[0061] "Plasmid" as used herein in the present application means a plasmid artificially constructed on the basis of a natural plasmid for adaptation to laboratory manipulation. The nucleic acid molecule can be introduced into a host cell, thereby producing a transformed host cell. The vector can include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication, and can also include one or more selectable marker genes and other genetic elements known in the art.

[0062] "Host cell" as used herein in the present application means a host cell that receives an exogenous gene in transformation and transduction (infection).

[0063] "Pharmaceutically acceptable carrier" as used herein in the present application means a conventional pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, E W Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more antibodies, and additional agents.

[0064] Construct: As used herein, "construct" refers to an antibody or antigen-binding fragment thereof linked to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, and the like. The linkage can be a covalent bond or a non-covalent interaction, such as by way of electrostatic forces. A variety of linkers known in the art can be employed to form the construct. Additionally, the construct can be provided as a fusion protein, which can be expressed from a polynucleotide encoding the construct. As used herein, "fusion protein" refers to a protein produced by joining two or more genes or gene fragments that originally coded for separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

[0065] “Encoding” as used in the application means the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in processes having determinate sequences of nucleotides, or to yield products having determinate sequences of amino acids and that are biologically and otherwise functionally characterized. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene results in a protein, whether or not the mRNA has ever been actually synthesized. The coding strand of a known sequence (which has the same nucleotide sequence as the mRNA and is usually provided in sequence listings) and the non-coding strand (which has the sequence of the mRNA and is complementary to the coding strand) can be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise indicated, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. Nucleotide sequences which encode proteins and RNA can include introns.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term “comprising” means “including.” All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including the

[0067] Standard recombinant DNA and molecular cloning techniques used in the examples are well known in the art (Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience) and are applied to the materials and methods for microbial growth well known in the art. The main chemicals, biological reagents are purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc.

[0068] The application will be further described below in connection with the drawings and specific embodiments, but not as a limitation of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0069] Screening of CD7 specific single domain antibody (VH) of Example 1

[0070] Taking human VH 3-23 subfamily germ line antibody as a template, the heavy chain CDR regions (CDR1, CDR2, CDR3) from all antibody subfamilies of healthy adults, newborns, cancer patients and AIDS patients are introduced by design and antibody engineering technology to construct a super large full human single domain antibody library with a practical measuring library capacity of 1.0x10 12 The phage display single domain antibody library is used to screen antibodies against biotin-labeled CD7 protein. The biotin-labeled CD7 protein is fixed on a streptavidin-coated magnetic column, and 10 12 phage-displayed antibodies are incubated with 5, 4, 2 and 1 micrograms of antigen for two hours at room temperature in 1, 2, 3 and 4 rounds respectively, and 10 12 phages are used in each round of screening. The enrichment of antibodies is detected by polyclonal phage ELISA. The phages of the first, second, third and fourth rounds are incubated with the coated protein, and the binding of the phages and the protein is detected by anti-phage HRP-coupled antibody. According to the polyclonal phage ELISA results (as shown in Figure 1 , very significant enrichment is obtained after the third and fourth rounds of screening. The phages obtained in the two rounds of screening are used to infect TG1 cells, and single clones are randomly selected for monoclonal phage ELISA, and the enriched single domain antibodies are further sequenced and identified.

[0071] The specific steps of monoclonal ELISA are as follows: the single clones obtained in the third and fourth rounds of screening are selected into 96-well shaking plates, which are shaken at 37 degrees Celsius and 220 rpm for 2-3 hours, and then 1:1000 IPTG is added for overnight induction at 30 degrees Celsius. CD7 protein is coated in a 96-well half-hole plate (Corning) at a concentration of 100 micrograms per well, and incubated at 4 degrees Celsius overnight. After washing three times with 0.05% PBST, 3% milk is added for blocking for 1 hour, and then washed three times with 0.05% PBST. Then, the bacterial solution shaken in the previous step is added as the primary antibody, and incubated at 37 degrees Celsius for 1.5 hours. Then, it is washed three times with 0.05% PBST, and then 1:5000 diluted anti-Flag-HRP (Sigma-Aldrich) secondary antibody is added, and incubated at 37 degrees Celsius for 45 minutes. It is washed five times with 0.05% PBST, and then ABTS (Invitrogen) is added for color development. The OD405 value is read on an enzyme-labeled instrument, and the OD value of the negative control is more than 5 times as a positive single clone. The bacterial solution of the positive single clone is sent to a sequencing company for sequencing.

[0072] Detection of the binding ability of single domain antibodies of Example 2 to CD7 protein

[0073] Positive single domain antibody expression and purification: According to the sequencing results, the sequence-enriched monoclonal antibodies were selected, and the preparation of their soluble expression products was basically carried out according to the literature (Cell Host Microbe. 2017. 22(4): 471-483.e5.). Specifically: the plasmid was transformed into HB2151 competent cells, a single colony was picked from an overnight grown ampicillin plate, inoculated into SB bacterial culture solution, and expressed for 12-14 hours under the condition of 30 degrees IPTG induction. After the bacteria were harvested and broken, the single domain antibodies were purified from them using Ni-TNA (Yeasen Biotech).

[0074] Activity detection: 100 ng of CD7 protein was coated in a 96-well half-hole plate (Corning) at 4°C overnight, washed three times with 0.05% PBST, blocked with 3% milk for 1 hour, then washed three times with 0.05% PBST, added to the single domain antibody diluted in a 3-fold concentration gradient starting from 2 micromoles, incubated for 1.5 hours, washed three times with 0.05% PBST, incubated with anti-FLAG-HRP antibody (Sigma-Aldrich) for 45 minutes, then washed five times with 0.05% PBST, and added ABTS (Invitrogen) for color development. At the same time, 100 ng of PD-1 protein was coated in a 96-well half-hole plate (Corning) to detect the binding activity with the single domain antibody in the same way.

[0075] The results show that single domain antibodies n701, n702, n703, n704, n705, and n706 have strong binding ability to CD7 protein and do not have non-specific binding to PD-1 protein Figure 2 ).

[0076] Example 3 BLI-based tandem antibody epitope competition experiment

[0077] In this example, to evaluate whether the single domain antibodies bind to the same or different epitopes of CD7, a tandem epitope competition experiment based on the biolayer interference technology (BLI) was used for analysis. In the experiment, AR2G type sensors were used as the fixed platform, and the CD7 protein was first coupled to the sensor surface through the EDC / NHS chemical activation method. The buffer used in the experiment was phosphate buffered saline (PBST) containing 0.02% Tween.

[0078] In the epitope competition experiment, first inject the first single domain antibody, bind with the fixed antigen on the sensor surface to form an antigen-antibody complex, and after the binding signal reaches a stable platform, inject the first antibody and the second antibody mixed solution in equal amounts to detect whether it can further bind to the complex. Among them, the first antibody is n701, n702, n703, n704, n706, and the second antibody is n705.

[0079] The results show that n704 and n706 have a strong competitive relationship with n705, and n701, n702, n703 have a weak competitive relationship with n705. Figure 3

[0080] Example 4: Detection of binding activity of single domain antibody to CD7 high expression cell line

[0081] Flow cytometry was used to detect the binding of single domain antibody to Jurkat cell line with high expression of CD7. The specific implementation steps are as follows: 40 nanomolar or 20 nanomolar single domain antibody and anti-FLAG-PE antibody (BioLegend) were incubated at 4 degrees Celsius in the dark for 30 minutes, and then transferred to 10 6 Jurkat cells and incubated at 4 degrees Celsius in the dark for 30 minutes. Multicolor flow cytometry (BD LSR Fortessa) was used to detect the binding of single domain antibody to cells. The results show that single domain antibodies n701, n702, n703, n704, n705, and n706 have strong binding ability to Jurkat cells. Figures 4-5

[0082] The above is only the preferred embodiment of the present application, and does not limit the implementation and protection scope of the present application. For those skilled in the art, it should be realized that any equivalent replacement and obvious changes made according to the content of the present application and the drawings should be included in the protection scope of the present application.​​

Claims

1. A single-domain antibody targeting CD7, characterized in that, The single-domain antibody comprises CDR1, CDR2 and CDR3; wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 1, CDR2 is an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 3; or wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 4, CDR2 is an amino acid sequence as shown in SEQ ID NO: 5, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 6; or wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 7, CDR2 is an amino acid sequence as shown in SEQ ID NO: 8, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 9; or wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 10, CDR2 is an amino acid sequence as shown in SEQ ID NO: 11, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 12; or wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 13, CDR2 is an amino acid sequence as shown in SEQ ID NO: 14, and CDR3 is an amino acid sequence as shown in SEQ ID NO: 15; or wherein CDR1 is an amino acid sequence as shown in SEQ ID NO: 16, CDR2 is an amino acid sequence as shown in SEQ ID NO: 17, and CDR3 is an amino acid sequence as shown in SEQ ID NO:

18.

2. The single-domain antibody of claim 1, wherein, The single-domain antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO:

24.

3. The single-domain antibody of claim 1, wherein, The single-domain antibody comprises any one of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO:

24.

4. A nucleic acid molecule, characterized in that, A nucleic acid sequence encoding the single-domain antibody as claimed in any one of claims 1-3.

5. A vector comprising the nucleic acid molecule as claimed in claim 4.

6. The carrier of claim 5, wherein, The vector is one of a plasmid, a lentivirus vector, an adenovirus vector, an AAV, a retrovirus vector, a transposon.

7. A construct, characterized in that, The single-domain antibody targeting CD7 as claimed in any one of claims 1-3, further comprises one or more other residues, groups, moieties or binding units, and is connected by one or more linkers. The single-domain antibody targeting CD7 as claimed in any one of claims 1-3, further comprises one or more other residues, groups, moieties or binding units, and is connected by one or more linkers.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the single-domain antibody targeting CD7 according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the vector according to any one of claims 5-6, or the construct according to claim 7, and one or more pharmaceutically acceptable excipients and / or carriers.

9. Use of the single-domain antibody targeting CD7 according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the vector according to any one of claims 5-6, or the construct according to claim 7, in the manufacture of a medicament for treating a tumor or cancer.

10. A diagnostic reagent, characterized by, The pharmaceutical composition comprises the single-domain antibody targeting CD7 according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the vector according to any one of claims 5-6, or the construct according to claim 7, and one or more pharmaceutically acceptable excipients and / or carriers.

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