Humanized antibodies targeting CD7 and uses thereof

Abstract

The present invention provides humanized antibodies targeting CD7, as well as multispecific antibodies, chimeric antigen receptors, antibody conjugates, pharmaceutical compositions and kits comprising the same, and their use in the diagnosis / treatment / prevention of diseases associated with CD7 expression.

Description

Humanized antibodies targeting CD7 and their applications Technical Field

[0001] This invention belongs to the field of immunotherapy. More specifically, this invention relates to humanized antibodies targeting CD7, and their use in the prevention and / or treatment and / or diagnosis of diseases. Background Technology

[0002] CD7 is a cell surface glycoprotein with a molecular weight of approximately 40 kDa, belonging to the immunoglobulin superfamily. CD7 is expressed in most T cells, NK cells, myeloid cells, T-cell acute lymphoblastic leukemia / lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. It has been reported that CD7 acts as a co-stimulatory signal during T cell activation by binding to its ligand K12 / SECTM1. Furthermore, it has been reported that disrupting CD7 in mouse T progenitor cells still results in normal T cell development and homeostasis, suggesting that CD7 does not appear to have a critical impact on T cell development and function, making it a very suitable therapeutic target for T-cell acute lymphoblastic leukemia (T-ALL). In fact, CD7 has been extensively studied as a target for cytotoxic molecules in the treatment of leukemia and lymphoma.

[0003] The present invention aims to provide a humanized antibody targeting CD7, and its use in disease prevention and / or treatment and / or diagnosis. Summary of the Invention

[0004] In a first aspect, the present invention provides a humanized CD7 antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6, and the light chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 8, 11, 14, and 17, and the heavy chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 9, 12, 15, and 18.

[0005] In one embodiment, the CD7 humanized antibody comprises a light chain variable region selected from SEQ ID NO: 8, 11, 14 and 17 and a heavy chain variable region selected from SEQ ID NO: 9, 12, 15 and 18.

[0006] In one embodiment, the amino acid sequence of the CD7 humanized antibody is selected from SEQ ID NO: 10, 13, 16, and 19. The present invention also provides a nucleic acid molecule encoding the above-described CD7 humanized antibody. Therefore, in one embodiment, the nucleic acid molecule encoding the CD7 humanized antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the nucleotide sequences selected from SEQ ID NO: 20-23, and the CD7 humanized antibody encoded therefrom is capable of specifically binding to the CD7 antigen. Preferably, the nucleic acid molecule encoding the CD7 humanized antibody is selected from SEQ ID NO: 20-23.

[0007] In another aspect, the present invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising the CD7 humanized antibody as described above and one or more second antibodies or their antigen-binding portions that specifically bind to other antigens.

[0008] In one embodiment, the second antibody or its antigen-binding portion may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0009] The present invention also provides a vector comprising a nucleic acid molecule encoding the above-mentioned humanized CD7 antibody or multispecific antibody, and a host cell expressing the humanized CD7 antibody or multispecific antibody.

[0010] In another aspect, the present invention also provides a chimeric antigen receptor comprising the CD7 humanized antibody described herein, a transmembrane domain, and an intracellular signal transduction domain. Preferably, the chimeric antigen receptor further comprises one or more co-stimulatory domains. More preferably, the chimeric antigen receptor comprises the CD7 humanized antibody as provided herein or a multispecific antibody containing the CD7 humanized antibody, a CD8α transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain.

[0011] The present invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting CD7 as defined above, and a vector comprising said nucleic acid molecule.

[0012] The present invention also provides cells comprising a chimeric antigen receptor targeting CD7 as defined above, preferably immune cells, such as T cells, NK cells, NKT cells, macrophages, and dendritic cells.

[0013] In another aspect, the present invention also provides an antibody conjugate comprising a CD7 humanized antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

[0014] In one embodiment, the structural portion for extending the half-life is selected from: albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind to human serum albumin. In one embodiment, the detectable marker is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents. In one embodiment, the drug is selected from cytotoxins and immunomodulators.

[0015] In another aspect, the present invention also provides a detection kit comprising the humanized antibody, multispecific antibody, antibody-drug conjugate, or chimeric antigen receptor described in the present invention.

[0016] In another aspect, the present invention also provides a pharmaceutical composition comprising the humanized antibody, chimeric antigen receptor, multispecific antibody or antibody-drug conjugate described herein, and one or more pharmaceutically acceptable excipients.

[0017] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with CD7 expression, comprising administering to a subject a humanized antibody, chimeric antigen receptor, multispecific antibody, antibody-drug conjugate, or pharmaceutical composition as described above.

[0018] Invention Details Unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] As used herein, the term "antibody" has the broadest meaning as understood by those skilled in the art and includes monoclonal antibodies (comprising complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting desired biological activity. The antibodies described in this invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).

[0020] Typically, a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds, with each light chain connected to its respective heavy chain via disulfide bonds, forming a "Y"-shaped structure. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain variable region contains three complementation-determining regions (CDRs): CDR-H1, CDR-H2, and CDR-H3, and the heavy chain constant region contains three constant structural domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain variable region contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, and the light chain constant region contains one constant structural domain, CL. Within the heavy / light chain variable regions, the CDRs are separated by more conserved frame regions (FRs). The variable regions of the heavy / light chains are responsible for the recognition and binding of antigens, while the constant regions mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

[0021] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using many numbering schemes well-known in the art, including: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (“Kabat” numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp. Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0022] The boundaries of a given CDR or FR can vary depending on the protocol used for identification. For example, the Kabat protocol is based on structure alignment, while the Chothia protocol is based on structural information. Both the Kabat and Chothia protocols number antibodies based on the length of the most common antibody region sequences, where insertions are indicated by insert letters (e.g., "30a") and deletions occur in some antibodies. These two protocols place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact protocol is based on the analysis of complex crystal structures and is similar to the Chothia numbering protocol in many ways. The AbM protocol is a compromise between the Kabat and Chothia definitions, based on the protocol used by the Oxford Molecular AbM antibody modeling software.

[0023] Therefore, unless otherwise specified, it should be understood that the “CDR” of a given antibody or its region (such as its variable region) encompasses the CDRs defined by any of the above-described protocols or other known protocols. For example, in specifying that a particular CDR (e.g., CDR3) contains a given amino acid sequence, it should be understood that such a CDR may also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above-described protocols or other known protocols. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) encompasses the FRs defined by any of the above-described protocols or other known protocols.

[0024] As used herein, a "humanized" antibody refers to an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR and all or substantially all of the FR amino acid residues are derived from a human FR. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization to generally reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody derived from CDR residues) for example, to restore or improve antibody specificity or affinity.

[0025] Humanized antibodies and their preparation methods are well known to those skilled in the art, see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008). Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best fit" method; frame regions of common sequences of human antibodies derived from specific subgroups of light or heavy chain variable regions; human mature (somatic mutation) frame regions or human germline frame regions; and frame regions obtained by screening FR libraries.

[0026] As used herein, the term "antibody fragment" or "antigen-binding portion" refers only to a portion of a complete antibody, generally containing the antigen-binding site of the complete antibody and thus retaining the ability to bind antigens. Examples of antibody fragments in this invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragments, Fd', Fv fragments, scFv, disulfide-linked Fv (sdFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, linear antibodies, "dimers" having two antigen-binding sites, single-domain antibodies, nanobodies, natural ligands of said antigens, or functional fragments thereof. Therefore, the term "antibody" in this invention encompasses antibody fragments as defined above.

[0027] In one embodiment, the humanized CD7 antibody of the present invention is an scFv. "Single-chain antibody" and "scFv" are used interchangeably herein and refer to an antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker. An optimal linker length and / or amino acid composition can be selected. The linker length significantly affects the folding and interaction of the variable regions of the scFv. In fact, using a shorter linker (e.g., between 5 and 10 amino acids) can prevent intra-chain folding. Regarding the selection of connector size and composition, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. U.S. A. 90:6444-6448; U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794; and PCT Publications WO2006 / 020258 and WO2007 / 024715, the entire contents of which are incorporated herein by reference. The scFv may contain VH and VL connected in any order, such as VH-connector-VL or VL-connector-VH.

[0028] In one embodiment, the present invention provides a humanized CD7 antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequences selected from SEQ ID NO: 8, 11, 14, and 17, and the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 9, 12, 15, and 18.

[0029] In one embodiment, the CD7 humanized antibody comprises a light chain variable region selected from SEQ ID NO: 8, 11, 14 and 17 and a heavy chain variable region selected from SEQ ID NO: 9, 12, 15 and 18.

[0030] In one embodiment, the amino acid sequence of the CD7 humanized antibody is shown in SEQ ID NO: 10, 13, 16, and 19. As used herein, the term “sequence identity” refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having identical sequences have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0031] In one aspect, the present invention also provides a multispecific antibody (preferably a bispecific or trispecific antibody) comprising the humanized CD7 antibody as described above, which further comprises one or more second antibodies that specifically bind to other antigens.

[0032] As used herein, the term "multispecific" refers to an antigen-binding protein having multiple epitope specificities (i.e., the ability to specifically bind to two, three, or more different epitopes on a single biomolecule or the ability to specifically bind to epitopes on two, three, or more different biomolecules). As used herein, the term "bispecific" indicates that an antigen-binding protein has two different antigen-binding specificities.

[0033] In one implementation, the second antibody may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0034] Therefore, in one embodiment, the second antibody targets antigens selected from the following: BCMA, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24. HLA-DR, tendinin, angiogenic factors, VEGF, PIGF, ED-B fibronectin, oncogenes, oncogene products, CD66a-d, necrosis antigen, Ii, IL-2, T101, TAC, IL-6, ROR1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, ErbB dimer, EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor C family 5D (GPRC5D), HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, melanoma preferential expression antigen (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, CD171, G250 / CAIX, HLA-A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-α, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, dual antigens, antigens associated with universal tags, cancer-testis antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, VEGF-R2, cancer Embryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, liver glycoside B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclins, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, cytokinin, AFP, p53, cyclin (D1), CS-1, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule, MAGE-A1, MAGEA3, cyclins (such as cyclin A1 (CCNA1)) and / or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV and / or other pathogens; and / or neoeptopes or neoantigens.

[0035] In another aspect, this invention relates to nucleic acid molecules encoding the humanized CD7 antibody or multispecific antibody of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

[0036] In one embodiment, the nucleic acid molecule encoding the CD7 humanized antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequences selected from SEQ ID NO: 20-23, and the CD7 humanized antibody encoded therein is capable of specifically binding to the CD7 antigen. Preferably, the nucleic acid molecule encoding the CD7 humanized antibody is as shown in SEQ ID NO: 20-23.

[0037] The nucleic acids of the present invention may also be in the form of a vector, which may be present in and / or part of a vector, such as a plasmid, a sticky-terminal plasmid, or a YAC. The vector may be, in particular, an expression vector, providing a means for expressing CD7 humanized antibodies in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). This expression vector typically contains at least one nucleic acid molecule of the present invention operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of said regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the CD7 humanized antibodies of the present invention include, but are not limited to, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0038] In another aspect, the present invention also provides host cells expressing the humanized CD7 antibody, multispecific antibody, and / or containing the nucleic acid or vector of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0039] Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0040] Suitable fungal cells include cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0041] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0042] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0043] Chimeric antigen receptor In another aspect, the present invention also provides a recombinant receptor comprising the humanized CD7 antibody as described above, such as a recombinant TCR receptor or a chimeric antigen receptor. Preferably, the present invention also provides a chimeric antigen receptor comprising the humanized CD7 antibody as described above.

[0044] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that typically includes a ligand-binding domain (e.g., the antigen-binding portion of an antibody), a transmembrane domain, an optional co-stimulatory domain, and an intracellular signal transduction domain, all connected by a linker. CARs can utilize the antigen-binding properties of antibodies to specifically and reactively redirect T cells and other immune cells to a selected target in a non-MHC-restricted manner.

[0045] In one embodiment, the present invention provides a chimeric antigen receptor comprising a humanized CD7 antibody as described above or a multispecific antibody containing said humanized CD7 antibody, a transmembrane domain, and an intracellular signal transduction domain.

[0046] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric antigen receptor on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells) and guides the cellular response of immune cells against target cells. Transmembrane domains can be natural or synthetic and can be derived from any membrane-binding or transmembrane protein. When a chimeric antigen receptor binds to a target antigen, the transmembrane domain enables signal transduction. Transmembrane domains particularly suitable for use in this invention can be derived from, for example, the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, transmembrane domains can be synthetic and may primarily contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8α chain or CD28, and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:24 or 26, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:25 or 27.

[0047] As used herein, the term "intracellular signaling domain" refers to a protein portion that transduces effector functional signals and directs the cell to perform a specified function. In one embodiment, the intracellular signaling domain of the chimeric antigen receptor of the present invention may be an intracellular region sequence of a T-cell receptor and a co-receptor that, upon antigen receptor binding, act together to initiate signal transduction, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signaling domain may contain a number of immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting embodiments of the intracellular signaling domain of the present invention include, but are not limited to, intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signal transduction domain of the CAR of the present invention may include an intracellular CD3ζ region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:32 or 34, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:33 or 35.

[0048] In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antibody. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be wholly or partially derived from natural molecules, such as the extracellular regions of CD8, CD4, or CD28, or wholly or partially derived from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1, more preferably a CD8α, CD28, or IgG4 hinge, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:40, 42, or 44, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:41, 43, or 45.

[0049] In one embodiment, the chimeric antigen receptor may further include one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain derived from a co-stimulatory molecule, comprising the entire intracellular portion of the co-stimulatory molecule or a functional fragment thereof. A "co-stimulatory molecule" refers to a homologous binding partner that specifically binds to a co-stimulatory ligand on a T cell, thereby mediating a co-stimulatory response (e.g., proliferation) of the T cell. Co-stimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting embodiments of the co-stimulatory domains of the present invention include, but are not limited to, co-stimulatory signal transduction domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the co-stimulatory domain of the CAR of the present invention is derived from 4-1BB, CD28, or 4-1BB+CD28. In one embodiment, the 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:30, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:31. In one embodiment, the CD28 co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:28, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:29.

[0050] In one embodiment, the CAR of the present invention may further comprise a signal peptide such that, when expressed in cells such as T cells, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long, hydrophobic amino acid segment with a tendency to form a single α-helix. At the terminal end of the signal peptide, there is typically an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave the peptide during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, etc. In one embodiment, the signal peptide used in this invention is derived from B2M or CD8α, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:36 or 38, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:37 or 39.

[0051] In one embodiment, the CAR contains a humanized CD7 antibody as provided herein or a multispecific antibody containing said humanized CD7 antibody, a CD8α transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain. In this embodiment, the CAR may further contain a signal peptide from B2M, CD8α, IgG1, or GM-CSFRα.

[0052] The present invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting CD7 as defined above, and a vector comprising said nucleic acid molecule.

[0053] As used herein, the term "vector" is a medium nucleic acid molecule used to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule may, for example, be replicated and / or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids into the cell interior by, for example, homologous recombination or using a hybrid recombinase with a specific target site sequence. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences (e.g., those encoding the chimeric antigen receptor polypeptide of the present invention) in a suitable host cell and for the translation of their mRNA. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia virus, Raul's sarcoma virus (RSV, polyomavirus, and adeno-associated virus (AAV) etc.), bacteriophages, phage particles, granules, and artificial chromosomes (including BAC and YAC). The vector itself is typically a nucleic acid molecule, usually consisting of a DNA sequence containing an insert (transgenic) and a larger sequence serving as the vector's "backbone." Engineered vectors typically also include an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is required), a selection marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally include elements such as a promoter, polyA tail, 3' UTR, enhancer, terminator, insulator, operon, selection marker, reporter gene, target sequence, and / or protein purification tag. In one specific embodiment, the vector is an in vitro transcription vector.

[0054] Engineered immune cells In one aspect, the present invention also provides engineered immune cells expressing the CAR described herein.

[0055] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and / or NKT cells. In one embodiment, the immune cell is derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Preferably, the immune cell is a T cell. The T cell can be any T cell, such as cultured T cells, such as primary T cells, or T cells derived from cultured T cell lines such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be at any developmental stage, including but not limited to CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. Various techniques known to those skilled in the art, such as Ficoll isolation, can be used to obtain T cells from the subject's blood.

[0056] Nucleic acid sequences encoding chimeric antigen receptors can be introduced into immune cells using conventional methods known in the art, such as transduction, transfection, and transformation. "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of virus-mediated nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow material uptake. Transfection can be performed using calcium phosphate, via electroporation, via cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their carriers inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and into non-animal eukaryotic cells (including plant cells). Therefore, transformation is a genetic alteration in bacteria or non-animal eukaryotic cells, resulting from direct uptake from their surroundings via the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be achieved artificially. For transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques may include heat shock-mediated uptake, fusion with bacterial protoplasts of intact cells, microinjection, and electroporation. After introducing nucleic acids or vectors into immune cells, those skilled in the art can amplify and activate the resulting immune cells using conventional techniques.

[0057] In one embodiment, to reduce the risk of graft-versus-host disease, the engineered immune cells further comprise at least one gene selected from the following whose expression is suppressed or silenced: CD52, GR, dCK, TCR / CD3 genes (e.g., TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA), and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTP. N22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD 3. SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. Preferably, the engineered immune cells further comprise at least one gene selected from the following whose expression is suppressed or silenced: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.

[0058] Methods for suppressing gene expression or silencing genes are well known to those skilled in the art. For example, gene expression can be suppressed using antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA / miRNA, trans dominant-negative proteins (TNPs), chimeric / antibody conjugates, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Alternatively, gene silencing can also be achieved by mediating DNA breaks through, for example, broad-spectrum nucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.

[0059] In one embodiment, a plurality of immune cells are provided, each of which is engineered to express one or more chimeric antigen receptors. For example, in some embodiments, one immune cell is engineered to express a chimeric antigen receptor that binds to and / or targets CD7 (e.g., a CAR comprising the humanized CD7 antibody described in this invention), and another cell is engineered to express a chimeric antigen receptor that binds to and / or targets other antigens. In one embodiment, the immune cells may also express a multispecific chimeric antigen receptor that targets one or more antigens, including CD7. For example, such a multispecific chimeric antigen receptor may comprise a multispecific antibody targeting CD7, or simultaneously comprise the humanized CD7 antibody described in this invention and an antibody targeting other antigens. In such embodiments, the plurality of engineered immune cells may be administered together or separately. In one embodiment, the plurality of immune cells may be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.

[0060] Antibody conjugates In one aspect, the present invention provides an antibody conjugate comprising a humanized CD7 antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, a radioisotope, a structural portion for extended half-life, a detectable marker, and a drug.

[0061] In one embodiment, the present invention provides an antibody conjugate comprising a humanized CD7 antibody and an Fc as defined herein. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, including native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, generated by digesting an intact antibody. The immunoglobulin source that generates the native Fc is preferably derived from humans. The native Fc fragment consists of monomeric polypeptides that can be linked covalently (e.g., by disulfide bonds) or non-covalently into dimer or multimer forms. Depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), the native Fc molecule has 1-4 intermolecular disulfide bonds between its monomeric subunits. An example of a natural Fc is a disulfide-linked dimer produced by digesting IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). As used herein, the term “natural Fc” generally refers to monomeric, dimeric, and polymeric forms. A “variant Fc” is an amino acid sequence that differs from the amino acid sequence of a “natural” or “wild-type” Fc due to at least one “amino acid modification” as defined herein, also referred to as an “Fc variant.” Therefore, “Fc” also includes single-chain Fc (scFc), i.e., a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, capable of naturally folding into a functional dimer Fc region. In one embodiment, the Fc is preferably a human immunoglobulin Fc, more preferably a human IgG1 Fc.

[0062] In one embodiment, the present invention provides an antibody conjugate comprising a humanized CD7 antibody as defined herein and a radioactive isotope. Examples of radioactive isotopes that can be used in the present invention include, but are not limited to, At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 , 99m Tc, 123 I, 18 F and 68 Ga.

[0063] In one embodiment, the present invention provides an antibody conjugate comprising a CD7 humanized antibody as defined in the present invention and a structural portion for extending half-life, wherein the structural portion for extending half-life is selected from albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind to human serum albumin.

[0064] In one embodiment, the present invention provides an antibody conjugate comprising a humanized CD7 antibody as defined herein and a detectable marker. The term "detectable marker" herein refers to a compound that generates a detectable signal. For example, a detectable marker may be an MRI contrast agent, a scintillation scanning contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, or an optical imaging contrast agent. Examples of detectable markers include fluorophores (such as fluorescein, Alexa, or anthocyanins), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as horseradish peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotic resistance genes (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.), and contrast agents (such as nanoparticles or gadolinium). Those skilled in the art can select appropriate detectable markers based on the detection system used.

[0065] In one embodiment, the present invention provides an antibody-drug conjugate comprising a humanized CD7 antibody as defined herein and a drug, such as a cytotoxin or immunomodulator, conjugated to said humanized CD7 antibody (i.e., an antibody-drug conjugate). Typically, the drug is covalently linked to the antibody and is usually dependent on a linker. In one embodiment, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulator. Examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin, cis-dichlorodiamineplatin(II) (DDP), cisplatin, carboplatin, zolrubicin, doxorubicin, detoxin, carminoxetine, idarubicin, epirubicin, mitoxantrone, and actinomycin. Bleomycin D, bleomycin, salinomycin, sclerosomycin, atrazocin (AMC), vincristine, vinblastine, paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthradinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferon, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vorciclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogues, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., G-CSF and (GM-CSF), interferons (e.g., interferon-α, interferon-β, and interferon-γ), stem cell growth factors named "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0066] reagent kits and pharmaceutical compositions In another aspect, the present invention also provides a detection kit comprising the humanized antibody, multispecific antibody, antibody-drug conjugate, or chimeric antigen receptor described in the present invention.

[0067] In another aspect, the present invention also provides a pharmaceutical composition comprising the humanized antibody, chimeric antigen receptor, multispecific antibody or antibody-drug conjugate described herein, and one or more pharmaceutically acceptable excipients.

[0068] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adhesion agents, flow aids, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension modifiers. Those skilled in the art know how to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients used in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. Typically, the selection of a suitable excipient depends in particular on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0069] The pharmaceutical compositions according to the invention are suitable for administration via a variety of routes. Typically, administration is performed via parenteral delivery. Parenteral delivery methods include local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0070] The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly as ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for the desired method of administration. Processes known in this invention for manufacturing pharmaceuticals may include, for example, conventional mixing, dissolving, granulation, coating, grinding, emulsification, encapsulation, embedding, or lyophilization processes. Pharmaceutical compositions containing, for example, immune cells as described herein, are generally provided in solution form and preferably contain pharmaceutically acceptable buffers.

[0071] The pharmaceutical compositions according to the invention can also be administered in combination with one or more other pharmaceutical agents suitable for treating and / or preventing the disease to be treated. Preferred examples of pharmaceutical agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, and orlistatine E. E) vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases and RNases; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. Furthermore, the pharmaceutical compositions of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0072] Therapeutic / Preventive / Diagnostic Uses In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with CD7 expression, comprising administering to a subject a humanized antibody, chimeric antigen receptor, multispecific antibody, antibody-drug conjugate, or pharmaceutical composition as described above.

[0073] In one implementation, diseases associated with CD7 expression include non-solid tumors (such as hematologic malignancies, e.g., leukemia and lymphoma) and solid tumors. Hematologic malignancies are cancers of the blood or bone marrow, including but not limited to acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Solid tumors include: lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, T-lymphoblastic leukemia / lymphoma (T-ALL / LBL), early pro-T lymphoblastic leukemia (ETP-ALL), extranodal NK / T-cell lymphoma, small lymphoblastic lymphoma (SLL), and spinal dysplasia. Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas; they can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, pancreatic cancer, ovarian cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, melanoma, renal cancer, laryngeal cancer, soft tissue cancer, gastric cancer, testicular cancer, colon cancer, esophageal cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, thyroid cancer, and ureteral cancer. In one embodiment, diseases associated with CD7 expression are preferably selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), T-lymphoblastic lymphoma (T-LBL), early pro-T lymphoblastic leukemia (ETP-ALL), and extranodal NK / T-cell lymphoma.

[0074] The present invention will now be described in detail with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are merely illustrative and do not constitute any limitation on the present invention. Where there is no contradiction, the embodiments and features described in this application can be combined with each other. Attached Figure Description

[0075] Figure 1: Shows the scFv expression level in CAR-T cells containing CD7 humanized antibodies.

[0076] Figure 2: This shows the killing effect of CAR-T cells containing humanized CD7 antibodies on target cells.

[0077] Figure 3 shows the cytokine release levels after CAR-T cells containing humanized CD7 antibodies were co-cultured with target cells. Detailed Implementation

[0078] Example 1. Preparation of CD7 Humanized Antibody Humanized antibody was prepared based on mouse CD7 scFv (clone m189). Clone m189 contains CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6, and its amino acid sequence is shown in SEQ ID NO: 7. The specific method for preparing the humanized antibody is as follows: First, sequence similarity searches were performed using Ig BLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ) and IMGT (Immune Gene Database IMGT: http: / / www.imgt.org), and antibodies with high sequence similarity to both the light and heavy chains were selected as templates for the humanized antibody from the search results. Then, the heavy chain CDR and light chain CDR from the mouse CD7 scFv were transplanted into the framework of the humanized antibody template with high sequence similarity, followed by reverse mutation to ensure the affinity and specificity of the humanized antibody. The final amino acid sequence of the CD7 humanized single-chain antibody (scFv) is shown in Table 1 below.

[0079] Table 1. Sequence of humanized CD7 scFv

[0080] Example 2. CAR-T cells containing a humanized CD7 antibody were prepared to synthesize sequences encoding the following proteins and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), catalog number: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 38), CD7 humanized scFv (selected from SEQ ID NO: 10, 13, 16, 19), CD8α hinge region (SEQ ID NO: 40), CD8α transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 30), and CD3ζ intracellular signal transduction domain (SEQ ID NO: 32). The correct insertion of the target sequences was confirmed by sequencing. The amino acid sequence of anti-CD7 scFv contained in hCAR7-1 CAR is shown in SEQ ID NO: 10; the amino acid sequence of anti-CD7 scFv contained in hCAR7-2 CAR is shown in SEQ ID NO: 13; the amino acid sequence of anti-CD7 scFv contained in hCAR7-3 CAR is shown in SEQ ID NO: 16; and the amino acid sequence of anti-CD7 scFv contained in hCAR7-4 CAR is shown in SEQ ID NO: 19.

[0081] Add 3 ml of Opti-MEM (Gibco, catalog number 31985-070) to a sterile tube to dilute the above plasmid. Then, add the packaging vector psPAX2 (Addgene, catalog number 12260) and the envelope vector pMD2.G (Addgene, catalog number 12259) in a plasmid:viral packaging vector:viral envelope vector ratio of 4:2:1. Next, add 120 μL of X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001), mix immediately, and incubate at room temperature for 15 min. Then, add the plasmid / vector / transfection reagent mixture dropwise to a culture flask of 293T cells. Collect the virus at 24 and 48 hours, combine them, and then ultracentrifuge (25000g, 4℃, 2.5 h) to obtain concentrated lentivirus.

[0082] Since CD7 is also expressed on T cells, the inventors knocked out CD7 in T cells using the CRISP / Cas9 system to avoid mutual killing between CAR-T cells. After 1 day of culture, DynaBeads CD3 / CD28 CTS were used. TM(Gibco, catalog number 40203D) T cells were activated and cultured at 37°C and 5% CO2 for 1 day. Then, concentrated lentivirus was added, and after continuous culture for 3 days, CAR T cells expressing different humanized CD7 scFv were obtained. Unmodified wild-type T cells were used as negative controls (NT).

[0083] Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab')2Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (Jackson Immunoresearch, catalog number 115-065-072) was used as the primary antibody, and APC Streptavidin (BD Pharmingen, catalog number 554067) or PE Streptavidin (BD Pharmingen, catalog number 554061) was used as the secondary antibody. The expression levels of scFv on hCAR7-1 T cells, hCAR7-2 T cells, hCAR7-3 T cells, and hCAR7-4 were detected by flow cytometry. The results are shown in Figure 1.

[0084] It can be seen that the humanized CD7 scFv in the CAR T cells prepared by this invention can be effectively expressed.

[0085] Example 3: Killing effect of CAR T cells on target cells and cytokine release

[01] 3.1 Killing effect of CAR-T cells on target cells

[02] When T cells kill target cells, the number of target cells decreases. When T cells are co-cultured with target cells expressing luciferase, the number of target cells decreases, and the secretion of luciferase also decreases. Luciferase catalyzes the conversion of luciferin into oxidized luciferin, and during this oxidation process, bioluminescence is generated, the intensity of which depends on the level of luciferase expressed by the target cells. Therefore, the detected fluorescence intensity can reflect the killing ability of T cells against target cells.

[0086]

[03] To test the killing ability of CAR-T cells against target cells, firstly, 1x10 4 Jurkat target cells carrying the luciferin gene were seeded into 96-well plates. CAR T cells and NT cells were then seeded into 96-well plates at effector-to-target ratios of 1:1, 0.5:1, and 0.25:1 for co-culture. Fluorescence values ​​were measured using a microplate reader after 16-18 hours. The killing efficiency was calculated using the formula: (mean fluorescence value of target cells - mean fluorescence value of sample cells) / mean fluorescence value of target cells × 100%, and the results are shown in Figure 2.

[0087]

[04] It can be seen that, compared with NT, the CAR T cells of the present invention have specific killing effects on target cells.

[0088]

[05] 3.2 Cytokine Release from CAR-T Cells

[06] When T cells kill target cells, the number of target cells decreases while cytokines are released. Following the steps below, enzyme-linked immunosorbent assay (ELISA) was used to determine the release level of cytokine IFNγ when the CAR T cells of the present invention kill target cells.

[0089]

[07] (1) Collect the cell co-culture supernatant

[08] at 1x10 5 Target cells were seeded into 96-well plates, and then CAR T and NT cells (negative control) were co-cultured with target cells at a 1:1 ratio. The cell co-culture supernatant was collected after 18-24 hours.

[0090]

[09] (2) ELISA detection of IFNγ secretion in supernatant

[10] A 96-well plate was coated with a capture antibody, Purified anti-human IFN-γ Antibody (Biolegend, catalog number 506502), and incubated overnight at 4°C. Then the antibody solution was removed, and 250 μL of PBST (containing 0.1% Tween 1XPBS) solution containing 2% BSA (sigma, catalog number V900933-1kg) was added and incubated at 37°C for 2 hours. Then the plate was washed 3 times with 250 μL of PBST (containing 0.1% Tween 1XPBS). 50 μL of cell co-culture supernatant or standard was added to each well and incubated at 37°C for 1 hour. Then the plate was washed 3 times with 250 μL of PBST (containing 0.1% Tween 1XPBS). Then, 50 μL of the detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, catalog number ab25017) was added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with 250 μL of PBST (containing 0.1% Tween in 1X PBS). HRP Streptavidin (Biolegend, catalog number 405210) was then added, and the plate was incubated at 37°C for 30 minutes. The supernatant was discarded, and the plate was washed five times with 250 μL of PBST (containing 0.1% Tween in 1X PBS). 50 μL of TMB substrate solution was added to each well. The reaction was allowed to proceed in the dark at room temperature for 30 minutes, after which 50 μL of 1 mol / L H2SO4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, the absorbance at 450 nm was measured using a microplate reader, and the cytokine levels were calculated based on a standard curve (plotted according to the readings and concentrations of the standards). The results are shown in Figure 3.

[0091]

[11] It can be seen that the cytokine release water of the CAR T cells of the present invention is significantly higher than that of the control NT cells.

[0092] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A humanized antibody targeting CD7, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6, and the CD7 humanized antibody comprises a light chain variable region selected from SEQ ID NO: 8, 11, 14, and 17 and a heavy chain variable region selected from SEQ ID NO: 9, 12, 15, and 18.

2. The humanized antibody of claim 1, wherein the amino acid sequence of the CD7 humanized antibody is selected from SEQ ID NO: 10, 13, 16 and 19.

3. A nucleic acid molecule encoding the humanized antibody as described in claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein it has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequences selected from SEQ ID NO: 20-23, and wherein the CD7 humanized antibody encoded therefrom is capable of specifically binding to the CD7 antigen.

5. A multispecific antibody comprising the humanized antibody of claim 1 or 2 and one or more second antibodies or their antigen-binding portions that specifically bind to other antigens.

6. The multispecific antibody of claim 5, wherein the second antibody or its antigen-binding portion is selected from full-length antibodies, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibodies, biantibodies, or sdAb.

7. A vector comprising a nucleic acid molecule encoding a humanized antibody as described in claim 1 or 2 or a multispecific antibody as described in claim 5 or 6.

8. A host cell expressing the humanized antibody of claim 1 or 2 or the multispecific antibody of claim 5 or 6.

9. A chimeric antigen receptor comprising the humanized antibody of claim 1 or 2 or the multispecific antibody of claim 5 or 6, a transmembrane domain, and an intracellular signal transduction domain.

10. The chimeric antigen receptor of claim 9, further comprising a co-stimulatory domain selected from CD28 or 4-1BB.

11. The chimeric antigen receptor of claim 10, comprising the humanized antibody of any one of claims 1-2 or the multispecific antibody of claim 5 or 6, a CD8α transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain.

12. An engineered immune cell comprising the chimeric antigen receptor as described in any one of claims 9-11.

13. The engineered immune cells of claim 12, wherein the cells are selected from T cells, NK cells, NKT cells, macrophages, and dendritic cells.

14. An antibody-drug conjugate comprising the humanized antibody of claim 1 or 2 or the multispecific antibody of claim 5 or 6, and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

15. The antibody-drug conjugate of claim 14, wherein the structural portion for extending the half-life is selected from: albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides bound to human serum albumin; the detectable marker is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents; and the drug is selected from cytotoxins and immunomodulators.

16. A detection kit comprising the humanized antibody of claim 1 or 2, the multispecific antibody of claim 5 or 6, the chimeric antigen receptor of any one of claims 9-11, or the antibody conjugate of claim 14 or 15.

17. A pharmaceutical composition comprising the humanized antibody of claim 1 or 2, the multispecific antibody of claim 5 or 6, the chimeric antigen receptor of any one of claims 9-11, or the antibody-drug conjugate of claim 14 or 15, and one or more pharmaceutically acceptable excipients.

18. Use of the humanized antibody of claim 1 or 2, the multispecific antibody of claim 5 or 6, the chimeric antigen receptor of any one of claims 9-11, the antibody-drug conjugate of claim 14 or 15, or the pharmaceutical composition of claim 17 in the preparation of a medicament for treating and / or preventing and / or diagnosing diseases related to CD7 expression.

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