CN114805582B

CN114805582B - anti-Trop 2 nano antibody and application thereof

Info

Publication number: CN114805582B
Application number: CN202210744603.7A
Authority: CN
Inventors: 王海鹰; 熊青卉; 王彦; 胡红明
Original assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-10-04
Anticipated expiration: 2042-06-29
Also published as: WO2024002252A1; CN114805582A

Abstract

The invention relates to an anti-Trop 2 nano antibody and application thereof. The invention provides a Trop2 binding molecule, which comprises an anti-Trop 2 nano antibody or an antigen binding fragment thereof, wherein a CDR (complementary determining region) of the anti-Trop 2 nano antibody comprises a CDR1, a CDR2 and a CDR3. The invention also provides a chimeric antigen receptor containing the Trop2 binding molecule and an expression cell thereof. The antibodies and cells described herein have good therapeutic effects targeting Trop2.

Description

anti-Trop 2 nano antibody and application thereof

Technical Field

The invention relates to the technical field of biological immunotherapy, in particular to an anti-Trop 2 nano antibody and application thereof.

Background

Trop2 is a Cell Surface glycoprotein expressed by the tactd 2 gene, and is collectively called human Trophoblast Cell Surface glycoprotein antigen 2 (tropiblast Cell Surface Antigens 2). Trop2 consists of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail, is single transmembrane glycoprotein, has the size of 35.7KD, and is a calcium ion channel signal converter. The Trop2 protein is N-terminal to an extracellular domain (Trop 2 EC) that is immobilized to the cell membrane by a unidirectional transmembrane helix (TM) linked to an intracellular short tail (Trop 2 IC). Its cytoplasmic tail has a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP 2) binding sequence, suggesting that PIP2 plays an important role in signal transduction by Trop2. In addition to the PIP2 binding motif, it contains conserved tyrosine and serine phosphorylation sites.

The Trop2 in a normal tissue is not expressed or is under-expressed, is over-expressed in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer and the like, can promote the occurrence, invasion, metastasis, diffusion and the like of tumors, and plays a key role in the growth process of the tumors, so the Trop2 is considered as a target point of a tumor immunotherapy candidate.

Chimeric Antigen Receptor T cells (CAR-T) refer to T cells that are genetically modified to recognize a specific Antigen of interest in an MHC-unrestricted manner and to continuously activate expanded T cells. The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors except for surgery, radiotherapy and chemotherapy, and is a novel immunotherapy method for specific antigens on the surface of tumor cells. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients.

Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. CAR consists of three major domains: an extracellular antigen binding domain, a transmembrane domain, and an intracellular signal domain. The antibody is an important component of CAR, and has a decisive influence on CAR-T to exert specific efficient killing effect and reduce toxicity. Antibodies that are capable of binding to the antigen of interest with high specificity, while having low immunogenicity, are therefore the focus of the development of CAR-T products.

In the peripheral blood of alpaca, there is a naturally light chain-deficient antibody that contains only one heavy chain Variable region (VHH) and two conventional CH2 and CH3 regions. The cloned and expressed VHH structure has structural stability equivalent to that of the original heavy chain antibody and binding activity to an antigen, and is the smallest unit which is known to bind to a target antigen. The VHH crystal is 2.5nm, 4nm long and has a molecular weight of only 15KDa, so the VHH crystal is also called a Nanobody (Nb). VHH has extremely high solubility, easy manufacture and modification, short circulation half-life period, high tissue penetrability, difficult aggregation, high temperature resistance, strong acid, strong alkali and other denaturation conditions, is suitable for prokaryotic expression and various eukaryotic expression systems, and is widely used in the fields of development of therapeutic antibody medicaments, diagnostic reagents, affinity purification matrixes, scientific research and the like. The nanobody has unique properties such as small molecular weight, good water solubility, strong stability, strong antigen recognition capability, easy production and the like, is more and more popular as a diagnostic tool in recent years, and is also considered to be an important basis for CAR-T and targeted drug delivery.

There are no reports of anti-Trop 2 nanobodies and CAR-T cells containing the same, as described in the present application.

Disclosure of Invention

The invention provides a Trop2 binding molecule, which comprises an anti-Trop 2 nano antibody or an antigen binding fragment thereof, wherein a Complementarity Determining Region (CDR) of the anti-Trop 2 nano antibody comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises a sequence shown in SEQ ID NO. 1, the CDR2 comprises a sequence shown in SEQ ID NO. 2, and the CDR3 comprises a sequence shown in SEQ ID NO. 3.

In one or more embodiments, the heavy chain variable region sequence of the anti-Trop 2 nanobody is as shown in any one of SEQ ID NOs 4-5.

In one or more embodiments, FR1 of the anti-Trop 2 nanobody may be selected from FR1 of a VHH set forth in any one of SEQ ID NOs 4-5, FR2 may be selected from FR2 of a VHH set forth in any one of SEQ ID NOs 4-5, FR3 may be selected from FR3 of a VHH set forth in any one of SEQ ID NOs 4-5, and FR4 may be selected from FR4 of a VHH set forth in any one of SEQ ID NOs 4-5.

In one or more embodiments, the Trop2 binding molecule is a monovalent or multivalent nanobody or single domain antibody, or a multispecific nanobody or single domain antibody comprising one, two, or more anti-Trop 2 nanobodies or antigen binding fragments thereof.

In one or more embodiments, the multivalent binding molecule or multispecific binding molecule is linked to a plurality of anti-Trop 2 nanobodies or antigen-binding fragments thereof through a linker. The linker consists of 1-15 amino acids selected from G and S.

In one or more embodiments, the nanobody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.

In one or more embodiments, the nanobody further comprises a heavy chain constant region.

In one or more embodiments, the heavy chain constant region is a constant region of a camelid heavy chain antibody, comprising CH2 and CH3.

In one or more embodiments, the CH2 and CH3 are CH2 and CH3 of a human IgG Fc, e.g., CH2 and CH3 of IgG 1.

In one or more embodiments, the heavy chain constant region is a constant region of a chondrorelbine heavy chain antibody, comprising CH1, CH2, CH3, CH4, and CH5.

In one or more embodiments, the Trop2 binding molecule of any of the embodiments of the invention is a chimeric antibody or a fully human antibody; preferably fully human antibodies.

In another aspect, the invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a Trop2 binding molecule according to any of the embodiments herein, a hinge region, a transmembrane region, and an intracellular region.

In one or more embodiments, the intracellular region comprises an intracellular co-stimulatory domain and/or an intracellular signaling domain.

In one or more embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a signal peptide, a Trop2 binding molecule of any of the embodiments described herein, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

The invention also provides a nucleic acid molecule having a sequence selected from any one of:

(1) A coding sequence for a Trop2 binding molecule or chimeric antigen receptor of any one of the embodiments herein;

(2) The complement of (1);

(3) A fragment of 5 to 50bp of any one of (1) or (2).

In one or more embodiments, the fragment is a primer.

The invention also provides a nucleic acid construct comprising a nucleic acid molecule as described herein.

In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

The invention also provides a host cell selected from the group consisting of:

(1) Expressing and/or secreting a Trop2 binding molecule or chimeric antigen receptor according to any of the embodiments herein;

(2) Comprising a nucleic acid molecule as described herein; and/or

(3) Comprising a nucleic acid construct as described herein.

In one or more embodiments, the host cell is an immune effector cell, preferably a T cell.

The invention also provides a method of producing a Trop2 binding molecule of any of the embodiments herein, comprising: culturing a host cell as described herein under conditions suitable for the production of a Trop2 binding molecule (e.g., a nanobody or antigen-binding fragment thereof, a monovalent or multivalent nanobody or single domain antibody, or a multispecific nanobody or single domain antibody), and optionally purifying the Trop2 binding molecule from the culture.

The invention also provides a pharmaceutical composition comprising a Trop2 binding molecule, nucleic acid construct or host cell of any of the embodiments described herein, and a pharmaceutically acceptable excipient.

In one or more embodiments, the pharmaceutical composition is for treating a disease or condition associated with Trop2 expression.

The invention also provides the use of a Trop2 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell as described in any embodiment herein in the preparation of an activated immune cell (e.g., a T cell).

The invention also provides the use of a Trop2 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell according to any one of the embodiments herein in the preparation of a medicament for the prevention or treatment of a disease or condition associated with Trop2 expression.

In one or more embodiments, the disease or condition is selected from one or more of the following: breast, stomach, colorectal, pancreatic, prostate, cervical, head and neck, lung, esophageal, kidney, bladder, uterus and ovary cancer.

The invention also provides a method of treating or preventing a disease or condition associated with Trop2 expression, comprising administering to a patient in need thereof a therapeutically effective amount of a Trop2 binding molecule or host cell according to any of the embodiments of the invention, or a pharmaceutical composition according to any of the embodiments of the invention.

The invention also provides a kit for detecting Trop2, for example for assessing the efficacy of a drug treatment or diagnosing cancer, comprising a Trop2 binding molecule, nucleic acid construct or host cell according to any one of the embodiments described herein.

In one or more embodiments, the kit further comprises reagents for detecting the binding of Trop2 to the Trop2 binding molecule. The bound reagent is detected, for example, by enzyme-linked immunosorbent assay.

In one or more embodiments, the agent that detects binding is a detectable label, such as biotin, that can be attached to a Trop2 binding molecule. The detectable label is attached to the Trop2 binding molecule or is present separately in a kit.

The invention also provides a non-diagnostic method for detecting the presence of Trop2 in a sample, which comprises: incubating a Trop2 binding molecule as described in any of the embodiments herein with a sample, and detecting binding of Trop2 to the Trop2 binding molecule, thereby determining the presence of Trop2 in the sample. The detection is enzyme-linked immunosorbent assay detection.

The invention also provides the use of a Trop2 binding molecule as described in any embodiment herein in the preparation of a kit for detecting Trop2 in a sample, assessing the efficacy of a drug treatment or diagnosing cancer.

The invention has the following beneficial effects:

the invention provides a novel nano antibody for specifically recognizing Trop2 and a CAR modified cell containing the same, wherein the antibody and the cell have good treatment effect of targeting Trop2, and a novel treatment or improvement way is provided for diseases related to Trop2 expression.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the SDS electrophoresis of recombinant human Trop2-avi-his antigen protein.

Fig. 2 is a schematic of different clones, trop2 CAR.

FIG. 3 shows the positive rates of CAR expression for different cloned Trop2CAR-T cells.

FIG. 4 shows CD107a expression of different cloned Trop2CAR-T cells.

Fig. 5 is the INF γ secretion from different cloned Trop2CAR-T cells at a potency target ratio of 10.

FIG. 6 is INF γ secretion from different cloned Trop2CAR-T cells at a potency target ratio of 2.

FIG. 7 shows IL-2 secretion from different cloned Trop2CAR-T cells at a target-to-effect ratio of 10.

FIG. 8 shows IL-2 secretion from different cloned Trop2CAR-T cells at a potency target ratio of 2.

FIG. 9 shows the results of experiments on killing target cells BxPC3-LUC-GFP by different cloned Trop2CAR-T cells.

FIG. 10 shows the results of killing experiments on target cells U251-LUC-GFP by different cloned Trop2CAR-T cells.

Detailed Description

The inventor finds a class of anti-Trop 2 nano antibodies and antigen binding fragments thereof through extensive and intensive research, can specifically recognize Trop2, can be combined with the Trop2 with high affinity, and has good functional activity.

Specifically, the invention utilizes Trop2 protein to immunize alpaca to obtain a high-quality single-domain antibody gene library. Then screening an antibody gene library by using a phage display technology, thereby obtaining the Trop2 specific single domain antibody gene. Then, the gene is transferred into mammalian cells, and an antibody strain which can be expressed in mammalian cells with high efficiency and high specificity is obtained. The antibody or the antigen binding fragment thereof has good safety and targeting property, and can specifically bind to the extracellular domain of human Trop2.

The invention also provides Chimeric Antigen Receptors (CARs) containing the nanobodies. The carrier containing the CAR coding sequence is used for infecting immune cells, so that immune effector cells with remarkable killing capacity on tumor cells over expressing Trop2 can be obtained, and the immune effector cells can be applied to treating or improving diseases related to Trop2 expression, thereby laying a foundation for treating Trop2 positive tumors.

Antibodies

Herein, a "Trop2 binding molecule" is a protein that specifically binds to Trop2, including, but not limited to, antibodies, heavy chain antibodies, nanobodies, or antigen-binding fragments thereof.

Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single chain molecules, as well as antibody fragments, particularly antigen binding fragments, e.g., fab, F (ab') 2, fd, and Fv. Herein, "antibody" is used interchangeably with "immunoglobulin".

A traditional "antibody" contains a basic 4-chain antibody unit, a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH 1, CH2 and CH3 for each of the alpha and gamma chains) and four (CH 1, CH2, CH3 and CH4 for the mu and epsilon isotypes) constant domains (CH) and a Hinge region (Hinge) located between the CH1 and CH2 domains. Each light chain has a variable domain at the N-terminus (VL) followed by a constant domain at its other end (CL). The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies see e.g. Basic and Clinical Immunology, eighth edition, daniel p. Sties, abba i. Terr and Tristram g. Parsolw editions, appleton & Lange, norwalk, CT,1994, page 71 and chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types called kappa and lambda, depending on their constant domain amino acid sequences. The γ and α classes can be further divided into subclasses based on the relatively small differences in CH sequence and function, for example humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA1, and IgA2.

As used herein, a "heavy chain antibody" is an antibody derived from a camelid or chondrocypristine organism. In contrast to the 4-chain antibody described above, the heavy chain antibody lacks the light and heavy chain constant region 1 (CH 1), and comprises only 2 heavy chains consisting of a variable region (VHH) linked to the constant region by a hinge-like structure and other constant regions. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a chondrosarco heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). Antigen-binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. The heavy chain antibody may have CH2 and CH3 of human IgG Fc by fusion with the constant region of human IgG Fc.

As used herein, the terms "single domain antibody", "anti-Trop 2 single domain antibody", "heavy chain variable region domain of a heavy chain antibody", "VHH", are used interchangeably and all refer to single domain antibodies that specifically recognize and bind to Trop2. Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs. Preferably, the single domain antibody of the present invention has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3. Single domain antibodies are the smallest functional antigen-binding fragment. Typically, a single domain antibody consisting of only one heavy chain variable region is constructed by obtaining an antibody naturally lacking the light and heavy chain constant region 1 (CH 1) and then cloning the variable region of the heavy chain of the antibody.

Herein, "nanobody" refers to an antibody that contains a VHH described herein. It may be a heavy chain antibody as described above, a multivalent or multispecific antibody comprising a plurality of VHHs, or a recombinant antibody obtained by recombining a VHH and an antibody Fc (e.g., CH2 and CH3 or CH2, CH3 and CH 4). The binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more different specific single domain antibodies is a multispecific single domain antibody. A multivalent single domain antibody or multispecific single domain antibody is linked to multiple single domain antibodies by a linker. The linker typically consists of 1-15 amino acids selected from G and S.

Herein, heavy chain antibodies and antibodies (classical four chain antibodies) are intended to distinguish different combinations of antibodies. Due to the structural similarity of the two, the following structural descriptions for antibodies apply to heavy chain antibodies as well as to light chains.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain an antigen binding site.

The term "variable" refers to the situation where certain segments in the variable domains differ widely in antibody sequence. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domains. Instead, it is concentrated in three regions called hypervariable regions (HVRs), both in the light and heavy chain variable domains, namely HCDR1, HCDR2, HCDR3 (which may be abbreviated as CDR1, CDR2, CDR3 in heavy chain antibodies) of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region, respectively. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions (FR 1, FR2, FR3, and FR 4), which largely adopt a β -sheet conformation, connected by three HVRs that form loops connecting, and in some cases forming part of, the β -sheet structure. The HVRs in each chain are held together in close proximity by the FR region and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. Typically, the light chain variable region has the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the heavy chain variable region has the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity. There are a number of labeling schemes for the variable regions of antibodies, including: chothia, kabat, IMGT, and Contact. The IMGT labeling scheme is used herein exemplarily.

"Fc region" (crystallizable fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or to the first component of the classical complement system (C1 q). In IgG, igA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of an antibody; the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as the stretch of sequence from the amino acid residue at heavy chain position C226 or P230 to the carboxy-terminus, where the numbering is according to the EU index, as in Kabat. As used herein, an Fc region may be a native sequence Fc or a variant Fc.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or variable regions of an intact antibody. The antibody fragment is preferably an antigen-binding fragment of an antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2, fd, and Fv fragments, disulfide-linked Fv; a diabody; a linear antibody; a single chain antibody molecule; a scFv-Fc fragment; multispecific antibodies formed from antibody fragments; and any fragment that should be able to increase half-life by chemical modification or by incorporation into liposomes. Antigen-binding fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by expression from host cells containing the antigen-binding fragment.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) are highlighted from the folding of these two domains, contributing the amino acid residues for antigen binding and conferring antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with less avidity than the entire binding site. "Single-chain Fv", which may also be abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL domains of an antibody, joined as a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv forms the desired antigen binding structure. For heavy chain antibodies or nanobodies, scFv is VHH.

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having part or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence, single cell sequencing methods.

Monoclonal antibodies also include "chimeric" antibodies herein in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged with sequences found in a human antibody. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). CDRs, some or all of which are encoded by nucleic acids derived from a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to produce an antibody, the specificity of which is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, e.g., using mice with genetically engineered immune systems. In the present invention, antibodies, single domain antibodies, heavy chain antibodies, and the like, include humanized variants of each of the antibodies.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

In some embodiments, the invention also provides nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof (e.g., single domain antibodies VHH) that bind to the same epitope on human Trop2 as the antigen-binding region of any anti-Trop 2 nanobody of the invention, i.e., nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that are capable of cross-competing with the antigen-binding region of any nanobody of the invention for binding to Trop2.

In the invention, the anti-Trop 2 single-domain antibody has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3.

FR1, FR2, FR3 and FR4 of the anti-Trop 2 single domain antibody described herein can be independently selected from FR1, FR2, FR3 and FR4 of the single domain antibody shown in any one of SEQ ID NOS 4-5, respectively. Preferably, the amino acid sequence of the anti-Trop 2 single domain antibody is as shown in any one of SEQ ID NO's 4-5.

When the single domain antibody is linked to a heavy chain constant region, the nanobody is a heavy chain antibody comprising a single domain antibody as described herein. The heavy chain constant region may be a constant region of a camelid heavy chain antibody, comprising CH2 and CH3. Preferably, the antibody constant region is derived from: a constant region derived from any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD, more preferably a constant region derived from any one of IgG1, igG2, igG3, and IgG 4. In one or more embodiments, the heavy chain constant region is CH2 and CH3 of a human IgG Fc, e.g., CH2 and CH3 of IgG 1.

The Trop2 binding molecule described herein may be a monovalent or multivalent nanobody or single domain antibody, or a multispecific nanobody or single domain antibody comprising one, two, or more anti-Trop 2 nanobodies or single domain antibodies described herein. The multispecific may be for Trop2 and another antigen, or for two different epitopes of Trop2.

The invention also includes derivatives and analogs of the antibodies. "derivatives" and "analogs" refer to polypeptides that retain substantially the same biological function or activity as the antibodies of the invention. The derivative or analogue of the invention may be (i) a polypeptide having a substituent group in one or more amino acid residues, or (ii) a polypeptide formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, for example polyethylene glycol, or (iii) a polypeptide formed by fusing an additional amino acid sequence to the sequence of the polypeptide (such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a6 His-tag). Such derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

One skilled in the art can alter one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more) amino acids of the antibody sequences of the invention to obtain variants of the antibody or functional fragment thereof sequences without substantially affecting the activity of the antibody. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. Conservative substitutions with amino acids of similar or similar properties are not known in the art to alter the function of the protein. Such as substituting amino acids with similar properties in the FR and/or Fc regions. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. All of which are considered to be included within the scope of the present invention.

Variants of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing to DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera raised against the antibody of the present invention. In some embodiments, the sequence of a variant of the invention may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it was derived. The sequence identity described in the present invention can be measured using sequence analysis software. For example the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. The invention also includes those molecules having the heavy chain variable region of an antibody with CDRs which are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

Antibodies of the invention can be prepared using methods conventional in the art, such as hybridoma technology. Nanobodies of the present invention may be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or nanobodies of the present invention may be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention, followed by culture of the host cells and purification of the antibodies. Transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus, among others. Mammalian cell lines useful as hosts for expression are well known in the art and include, but are not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hepG 2), and the like.

CAR

The invention also provides a Chimeric Antigen Receptor (CAR) targeting Trop2. The CAR contains an optional signal peptide sequence, an antigen recognition region, i.e., an anti-Trop 2 binding molecule described herein, a hinge region, a transmembrane region, and an intracellular region. Wherein the intracellular region comprises one or more intracellular co-stimulatory domains and/or one or more intracellular signal domains. The "hinge region", "transmembrane region" and "intracellular region" herein may each be selected from the sequences of hinge, transmembrane and intracellular regions known in the CAR-T art.

The optional signal peptide on the CAR can be selected as desired. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. The signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to integrate and anchor to the lipid bilayer; the signal peptide may also be a membrane localization signal peptide. Exemplary signal peptides are, for example, a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, or a light chain signal peptide, the sequence of which is within the knowledge of one skilled in the art. CD8 signal peptides suitable for use in the present invention can be various human CD8 signal peptide sequences commonly used in the art for CARs. In certain embodiments, the amino acid sequence of the CD8 signal peptide comprises the sequence set forth in SEQ ID NO 6.

The hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region, is a segment of amino acids that typically exists between two domains of a protein, and can allow for the flexibility of the protein and the movement of the two domains relative to each other. The hinge region may be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as an IgG, igA, igM, igE, or IgD antibody) may also be used for the chimeric antigen receptors described herein. Non-naturally occurring peptides may also be used as the hinge region of the chimeric antigen receptor described herein. Illustratively, the hinge region of the CAR is selected from a CD8 a hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, or an IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of one of skill in the art. Suitable CD8 a hinge regions for use in the present invention can be various human CD8 a hinge region sequences commonly used in the art for CARs. In certain embodiments, the human CD8 a hinge region comprises the sequence set forth in SEQ ID NO 7.

The transmembrane region of the chimeric antigen receptor may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the domain of a cellular phospholipid bilayer. The transmembrane region may be of natural or synthetic origin. The transmembrane region may be selected from the transmembrane regions of the following proteins: CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, alpha, beta or zeta chain of T cell receptor. The human CD8 a transmembrane region suitable for use in the invention can be a variety of human CD8 a transmembrane region sequences commonly used in CARs in the art. In certain embodiments, the amino acid sequence of the human CD8 a transmembrane region comprises the sequence set forth in SEQ ID NO 8.

The intracellular signaling region (or intracellular signaling region) is responsible for the activation of at least one normal effector function of an immune effector cell expressing the chimeric antigen receptor. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. While the entire intracellular signaling region can generally be utilized, in many cases, the use of the entire strand is not necessary. For use of a truncated portion of an intracellular signaling region, such a truncated portion may be used in place of the entire strand, so long as it transduces effector function signals. Thus, an intracellular signaling region includes any truncated form of an intracellular signaling region sufficient to transduce an effector function signal. The intracellular signaling domain of the CAR can be selected as desired, including but not limited to, intracellular signaling domains derived from at least one of CD3 ζ, fcR γ (FCER 1G), fcR β (fcepsilon Rib), CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66 d. Preferably, the intracellular signaling region is derived from a human CD3 ζ intracellular signaling region. Further, the human CD3 zeta intracellular signal region has the amino acid sequence shown in SEQ ID NO 10.

In addition to stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cells. The "co-stimulatory domain" may be the cytoplasmic portion of the co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Suitable intracellular co-stimulatory domains, including those with co-stimulatory signaling molecules, can be selected as desired, such as at least one of the intracellular domains derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, fc ε RI γ, myD88, and 41 BBL. In certain embodiments, the amino acid sequence of the 4-1BB co-stimulatory domain comprises the sequence set forth in SEQ ID NO 9.

The aforementioned portions forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, the anti-Trop 2 nanobody, the CD8 α hinge region, the CD8 α transmembrane region, the CD3 ζ intracellular signal region, the 4-1BB co-stimulatory region, and the like, may be directly linked to each other, or may be linked via a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, for example 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (a), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and the like. In certain embodiments, the linker sequence is a (GGGGS) n linkage, where n is an integer from 1 to 5.

In an exemplary embodiment, the CAR comprises, in order from N-terminus to C-terminus, a CD8 signal peptide, an anti-Trop 2 nanobody or antigen-binding fragment thereof described herein, a CD8 a hinge region, a CD8 a transmembrane region, a 4-1BB co-stimulatory domain, a CD3 ζ intracellular signal domain. In specific embodiments, an exemplary CAR having the structure described above is set forth in any one of SEQ ID NOS 11-12.

It will be appreciated that in gene cloning procedures it is often necessary to design appropriate cleavage sites which will introduce one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-terminus or the carboxy-terminus of a CAR of the invention may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag can be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

The antigen recognition region in the CAR of the invention may be a variant of the aforementioned anti-Trop 2 nanobody, or a functional fragment thereof. In addition, other portions of the CAR may also be subject to sequence changes, resulting in mutants that have at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retain the biological activity of the CAR (e.g., activated T cells). Sequence identity between two aligned sequences can be calculated using, for example, BLASTp by NCBI.

Mutants also include: an amino acid sequence having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence of the CAR according to any of the embodiments, while still retaining the biological activity of the CAR. The number of mutations usually means within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids that are similar or analogous in performance are not generally known in the art to alter the function of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in the polypeptide of the invention will not substantially affect its activity.

Nucleic acids

The invention also provides polynucleotides encoding the above antibodies or CARs. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences which encode the same amino acid sequence but differ in nucleotide sequence.

Thus, the present invention also relates to polynucleotides which hybridize to the above-described polynucleotide sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding a denaturing agent upon hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1%; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possible method is to synthesize the sequence of interest by artificial synthesis, especially if the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6 His) can be fused together to form a fusion protein. The sequence of the CAR can also be obtained as above. Alternatively, the full length of the CAR can be obtained by obtaining the sequences of the various portions of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) as described above and then ligating them together.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis. The parts of the CAR can be cloned sequentially into the vector or can be integrated into a full-length CAR and then cloned.

The invention also relates to nucleic acid constructs comprising a polynucleotide sequence as described herein, and one or more control sequences operably linked to the sequence. The polynucleotide sequences of the invention can be manipulated in a variety of ways to ensure expression of the antibody or CAR. The nucleic acid construct may be manipulated prior to insertion into the vector depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV 40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the EB virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, inducible promoters are also contemplated. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter during a period of expression and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of a polynucleotide sequence of the invention is typically achieved by operably linking the polynucleotide sequence of the invention to an expression vector. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence. The integration vector contains the components for integrating the target sequence into the genome of the cell. These vectors may be used to transform an appropriate host cell so that it can express the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting nucleic acid encoding an antibody to be expressed, and optional marker elements.

Furthermore, the type of vector is not limited, and for example, plasmids, phagemids, phage derivatives, animal viruses and cosmids may be changed depending on the host cell to be introduced. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, new York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

To assess the expression of the CAR polypeptide or portion thereof, the vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector.

Cells

Host cells suitable for introduction of the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, in particular immune cells, preferably immune effector cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cell animal cells, etc.

An "immune effector cell" is an immune cell that can perform an immune effector function. In some embodiments, the immune effector cells express at least Fc γ RIII and perform ADCC effector function. Examples of immune effector cells that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), natural Killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, the immune effector cells are selected from: culturing at least one of differentiated immune cells, T lymphocytes, NK cells, peripheral Blood Mononuclear Cells (PBMCs) and hematopoietic stem cells from pluripotent stem cells or embryonic stem cells. More preferably, the immune effector cell is a T lymphocyte (homo T cell). In some embodiments, the T cells can be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or a combination thereof. In some embodiments, the T cells produce IL-2, IFN, and/or TNF when expressing the chimeric antigen receptor and binding to the target cell. In some embodiments, the CD8+ T cells lyse antigen-specific target cells when expressing the chimeric antigen receptor and binding to the target cells.

T cells suitable for use in the present invention may be of various types from various sources. For example, T cells may be derived from PBMCs of patients with malignant solid tumors (e.g., pancreatic cancer). In certain embodiments, after T cells are obtained, activation may be stimulated with an appropriate amount (e.g., 30 to 80ng/ml, such as 50 ng/ml) of CD3 antibody prior to culturing in a medium containing an appropriate amount (e.g., 30 to 80IU/ml, such as 50 IU/ml) of IL2 for use.

Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into the cells by physical, chemical, or biological methods. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like. In some embodiments, the transduced or transfected immune effector cells are propagated ex vivo following introduction of the nucleic acid or vector.

The obtained transformant can be cultured by a conventional method to express the antibody or CAR encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Use and method

By constructing a nanobody library, the inventors screened nanobodies that can bind to Trop2. By utilizing the nano-antibodies, the inventor constructs CAR and CAR-T cells, and the CAR-T cells have strong immune function, better CD107a expression, IFN-gamma and IL-2 secretion and specific killing function on target cells through cell level experiments, and have obvious in-vivo drug effect.

All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein are useful in the preparation of medicaments for the prevention or treatment of various conditions and diseases described herein, which are diseases or conditions associated with Trop2 expression, referring to diseases caused directly or indirectly by aberrant Trop2 expression, typically diseases caused by Trop2 overexpression, such as cancer, including but not limited to: breast, gastric, colorectal, pancreatic, prostate, cervical, head and neck, lung and ovarian cancers.

The invention also includes a class of cell therapies comprising expressing a CAR described herein in immune cells (e.g., T cells), and administering to a recipient in need thereof a therapeutically effective amount of cells that are capable of killing tumor cells in the recipient. CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control compared to antibody therapy. The anti-tumor immune response elicited by the CAR-T cells can be an active or passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells induce an immune response specific for the antigen-binding portion in the CAR.

The antibodies, nucleic acids, or CAR-modified cells of the invention can be administered alone or as a pharmaceutical composition in conjunction with diluents and/or with other components such as relevant cytokines or cell populations. In this regard, the pharmaceutical compositions can be prepared in lyophilized formulations or aqueous solutions by mixing the active agent having the desired purity with an optional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and may include at least one of buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and surfactants. Furthermore, in order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. The pharmaceutical composition may be sterilized by filtration through a sterile filtration membrane.

In some embodiments, the pharmaceutical composition may contain: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, a growth inhibitory agent, and at least one additive of an active agent required for the particular indication to be treated. The specific addition amount of the additive can be adjusted according to actual needs. The pharmaceutical compositions of the present invention may be administered in an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount" amount. "treatment" refers to the subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis and individual variability of the condition of the patient (subject). In general, a pharmaceutical composition comprising a T cell described herein can be in the range of 10 ⁴ To 10 ⁹ Dosage of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Dosage of individual cells/kg body weight. T cell compositions may also be administered multiple times at these doses. The cells can be treated by using a known injection in immunotherapyAdministration is by the art (see, e.g., rosenberg et al, new Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, treatment may be in combination with radiation or chemotherapeutic agents known in the art for the treatment of mesothelin-mediated diseases.

Herein, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

"patient," "subject," "individual," and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

Diagnostics, assays and kits

The binding molecules of the invention are useful in assays, e.g., binding assays, for detecting and/or quantifying Trop2 expressed in tissues or cells due to its high affinity for Trop2. Binding molecules such as single domain antibodies may be used in studies to further investigate the role of Trop2 in disease. The method of detecting Trop2 is roughly as follows: obtaining a cell and/or tissue sample; the level of Trop2 in the sample is detected.

The Trop2 binding molecules of the invention are useful for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with Trop2. The present invention provides for the detection of the presence of Trop2 in a sample using classical immunohistological methods known to those skilled in the art. Detection of Trop2 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of Trop2 include ELISA, FACS, RIA, and the like.

For diagnostic applications, the binding molecules, such as single domain antibodies, are typically labeled with a detectable labeling group. Suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin groups, or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging), or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used to carry out the present invention.

Another aspect of the invention provides a method of detecting the presence of a test molecule that competes with an antibody of the invention for binding to Trop2. One example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of Trop2 in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody that does not bind to Trop 2) would indicate that the test molecule is able to compete with the antibody for binding to Trop2. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.

The invention also provides a detection kit for detecting the Trop2 level, which comprises an antibody for identifying the Trop2 protein, a lysis medium for dissolving a sample, and general reagents and buffers required by detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.

The invention will be elucidated hereinafter by means of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise indicated, all those materials and methods conventional in the art.

Examples

Example 1: construction and eukaryotic expression of recombinant human Trop2 protein expression vector

1. Synthesis of Gene sequence and construction of expression vector for protein

The human Trop2 protein sequence (https:// www.uniprot.org/uniprot) is downloaded from Uniport, and is optimized by a codon online optimization tool (http:// www.jcat.de/# opennewwindow), and then is handed in to the Trop2 extracellular segment gene sequence synthesized by the genetic engineering. At the same time, the 3' end of the gene sequence is added with the nucleic acid sequences of avi-tag and 6 × his-tag, the fusion gene sequence encodes the amino acid sequence shown in SEQ ID NO. 15, and the nucleotide sequence of the fusion gene is shown in SEQ ID NO. 16. The expression vector is obtained by cloning the splicing product into pTT5 by using a TaKaRa seamless cloning kit through molecular cloning.

2. Expression and purification of recombinant human Trop2 protein

After 293T cells (ATCC) were transfected with the obtained expression vector for 5 days, culture supernatants were collected and the recombinant human Trop2 protein was purified using AKTA Explorer 100 (GE). The Trop2 protein was electrophoresed by SDS-PAGE and stained with Coomassie Brilliant blue to show a size of about 36 kilodaltons, and the results are shown in FIG. 1.

Example 2: preparation of anti-Trop 2VHH antibodies

VHH phage display library construction

A nano antibody phage display library is constructed by adopting a one-step method, namely the alpaca nano antibody VHH gene is connected into a phage display carrier.

1) Alpaca immunization

And (3) alpaca selection: the alpaca with good health, mental state and body form is selected, and the selected alpaca has bright wool color and no injury and discomfort symptoms. Selecting animals, and pre-culturing for about 1 week to eliminate some unqualified animals, so that later experiments can be carried out smoothly.

The immunization scheme comprises the following steps: the alpaca was selected and the animals were assured to be fit and the immunization experiment was started after the ear number was recorded. A total of 4 immunizations were performed. The immunization protocol was as follows: d0, taking 10mL of blood before immunization as negative serum control, mixing 0.5mg of antigen and 1mL of CFA uniformly, and injecting subcutaneously; d21, mixing 0.25mg of antigen with 1ml of CFA uniformly and then injecting subcutaneously; d28, taking 10ml of blood; d42, mixing 0.25mg of antigen with 1ml of CFA uniformly and injecting subcutaneously; d49, collecting 50mL of peripheral blood to separate lymphocytes; d63, mixing 0.25mg of antigen with 1ml of CFA uniformly and then injecting subcutaneously; d70, collecting 50mL of peripheral blood to separate lymphocytes.

And (3) serum detection:

A. the antigen was diluted to 2. Mu.g/mL with 0.05M carbonate buffer (pH 9.6) and coated overnight at 4 ℃ in 100. Mu.L/well;

B. discarding the coating solution, washing for 3 times by PBST, adding 5% skimmed milk of 300 mu L into each hole, and sealing for 1 h at 37 ℃;

pbst was washed 3 times, 100 μ L/well serum dilutions (two-fold dilution from 1;

PBST washing 5 times, respectively adding horseradish peroxidase labeled goat anti-Alpaca secondary antibody (diluted by PBS according to 1W), 100 μ L/hole, 37 ℃ incubation for 45 min;

pbst wash plates 5 times. Adding TMB color developing solution for color development, 100 μ L/well, 37 deg.C, 5min, adding stop solution to stop reaction, 50 μ L/well, and measuring optical density at 450 nm.

2) cDNA Synthesis

Extracting total RNA of PBMC: dissolving peripheral blood lymphocytes preserved by Trizol on ice, transferring to a 1.5mL centrifuge tube, adding 1/5 volume of chloroform, shaking and mixing uniformly, standing at room temperature for 5 minutes, and centrifuging at 12000g for 15 minutes; transferring the centrifuged supernatant to a new centrifuge tube, adding isopropanol with the same volume into the new centrifuge tube, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 4 ℃ at 12000g, washing the precipitate with 75% ethanol, centrifuging for 5 minutes at 4 ℃ at 7500g, removing the supernatant, drying the precipitate at room temperature, and dissolving the precipitate in a proper amount of RNase-free water. RNA extraction purity was analyzed from A260/280 to prepare RNA transcripts.

cDNA Synthesis: using the SuperScript IV First-Strand Synthesis System kit, cDNA was obtained by reverse transcription and stored at-80 ℃.

3) VHH gene amplification

The VHH-CH2 gene was PCR amplified using the PBMC cDNA as template using the forward primer (VHH-F) and the reverse primer (CH 2-R) under the following PCR conditions: after pre-denaturation at 98 ℃ for 45 seconds, temperature cycling is carried out, denaturation at 98 ℃ for 15 seconds, annealing at 58 ℃ for 20 seconds, extension at 72 ℃ for 45 seconds, cycling for 30 times, and final extension at 72 ℃ for 7 minutes. After the PCR product was electrophoresed through 1.5% agarose gel, a 750bp band of interest was recovered using a gel recovery kit (Promega). The VHH gene was PCR amplified using forward primers (VHH-CH 2-F) and reverse primers (VHJ-R) with VHH-CH2 as template, the PCR reaction conditions were as follows: pre-denaturation at 98 ℃ for 45 seconds followed by temperature cycling, denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 20 seconds, extension at 72 ℃ for 45 seconds, cycling for 30 times, and final extension at 72 ℃ for 7 minutes. After the PCR product was electrophoresed through 1.5% agarose gel, a 400bp band of interest was recovered using a gel recovery kit (Promega).

4) VHH library construction

Phagemid vector pcomb3xTT and VHH gene are subjected to single enzyme digestion by SfiI DNA endonuclease, enzyme digestion is carried out for 16h at 50 ℃, after enzyme digestion, pcomb3xTT vector is subjected to 1% agarose gel electrophoresis, and 4000bp vector fragment is recovered by using a gel recovery kit (Promega). The enzyme-cut VHH gene is directly recovered by passing through a column (400 bp) by using a gel recovery kit. The VHH genes were ligated into phagemid vectors using the T4 DNA ligase kit (Invitrogen), ligated overnight at 16 ℃ and ligation efficiencies were checked by agarose gel electrophoresis using a small amount of ligation product. The ligation product was desalted using MECK MILLIPOREF Millipore filter.

The ligation product was added to a home-made TG1 electroporation competent cell, followed by electroporation using an electrotransformation apparatus. 50 μ L of the suspension was taken out and diluted with PBS in a gradient 10 ² -10 ⁵ And (4) multiplying. 10 u L each gradient dilution in Amp/2YT plate flow line, 37 degrees C were incubated overnight, counting and statistics of phage antibody library size. The rest of the electrically transformed bacteria are supplemented with 2YT to 500ml, added with 100 muAmpicillin (g/mL) was incubated overnight at 220rpm at 30 ℃. Finally, a VHH immune library exceeding 9E9 is obtained. And (4) performing overnight amplification on the antibody library after electric shock transformation, centrifuging to collect library thalli, and adding glycerol with the final concentration of 20% to 80 ℃ for storage.

Inoculating frozen partial natural antibody phage display library strains into a 2YT culture set, wherein the inoculation density is 0.1OD, culturing the bacterial liquid at 37 ℃ and 220rpm for about 1.5 hours until the bacterial liquid density reaches 0.6OD, adding M13KO7 phage with the number 20 times that of the bacterial cells, standing for 30 minutes for infection, and then culturing at 30 ℃ and 220rpm overnight. The next day, 10000g of the culture broth were centrifuged, the culture supernatant was collected, and 1/4 volume of PEG/NaCl solution (20% PEG8000,2.5M NaCl) was added to the culture supernatant, followed by mixing and ice-cooling for 1 hour. And after the ice bath is finished, centrifuging at 8000 g for 10 minutes, collecting the precipitate, and dissolving the precipitate by 10% of Glycerol/PBST to obtain the VHH phage display library. OD268 was measured and dispensed into 1.5mL centrifuge tubes, 6 OD/tube and stored at-80 ℃.

2. Panning anti-Trop 2VHH antibodies

1) Biotinylation of recombinant human Trop2 protein

And (3) performing biotin modification on the avi-tag of the recombinant human Trop2 protein by using a biotinylation kit (Yijinhua) according to the kit specification to obtain the biotinylated Trop2 protein. Mu.g of the biotin-modified recombinant protein was added to 100. Mu.L of streptavidin magnetic beads (DynaBeads 280) washed 3 times with PBS, and the mixture was coupled on a rotary shaker at 20rpm for 1 hour at room temperature and washed 3 times with PBS.

2) Closed phage library and negative magnetic beads

Taking one VHH lib, melting at room temperature, adding 200 mu L of 5% BSA/PBST, placing on a rotary table at the speed of 20rpm, rotating and sealing at room temperature for 1 hour, and taking the phage as Input1. After washing the same 100. Mu.L of unconjugated protein DynaBeads 280 with PBS 3 times, 1mL of 1% BSA/PBS was added and incubated for 1 hour with rotation under the above conditions.

3) Closed positive magnetic bead

To the above Trop 2-coupled beads, 1ml of 1% BSA/PBS was added at 20rpm, and spin-blocked for 1 hour at room temperature.

4) Negative panning

To remove the antibodies that interacted with the magnetic beads, a negative panning was necessary. The BSA blocked phage library was mixed with magnetic beads not coupled with antigen, and incubated for 1 hour with rotation under the above conditions. After incubation, the phage-magnetic bead mixture was placed on a magnetic frame, and after the beads were attached to the wall, the supernatant was transferred to a new EP tube.

5) Positive panning

And adding the closed magnetic beads coupled with the Trop2 protein into the phage supernatant subjected to negative panning for positive panning, rotating at the room temperature for 1 hour at 20 revolutions per minute. After incubation, the beads were washed with 1mL PBST (0.1% Tween-20 in PBS) and the washing was repeated 10 times. After the end of the washing, 1mL of 100mM glycine (pH 2.0) was added, and the mixture was placed on a rotary shaker at a speed of 20rpm and subjected to rotary elution for 10 minutes. After the elution is finished, the EP tube is placed on a magnetic frame, and the eluent is transferred to a new EP tube after the magnetic beads are attached to the wall. To the eluate was added 0.2mL of 1M Tris-HCl solution (pH 8.0) for neutralization. The neutralized eluate was added to 30mL of TG1 bacterial solution with OD600 of about 0.6 and allowed to stand for 30 minutes, then M13KO7 phage was added in an amount of 20 times the number of cells, allowed to stand for 30 minutes, finally 100mL of 2YT medium and ampicillin and kanamycin at a final concentration of 100. Mu.g/mL were added, and cultured overnight at 30 ℃ and 220 rpm. The next day, phages were harvested as described above for the phage library, at which time the resulting phages were Input2.

6) Repeat positive panning

The panning method was repeated 2 times, i.e. Input2 was subjected to the next round of negative panning and positive panning to obtain Input3. Except that after TG1 was infected with the eluate obtained by panning with Input3, 10. Mu.L of the resulting suspension was diluted with PBS in a gradient manner without adding M13KO7, and 10 of the resulting suspension was collected ³ 、10 ⁴ 、10 ⁵ Three dilution gradient each 100 u L bacterial liquid coating 2YT/Amp plate, 30 degrees C were cultured overnight, the remaining bacterial liquid 30 degrees C, 220rpm culture overnight.

7) ELISA screening of Positive antibodies

Randomly picking TG1 single clone in the above plate with toothpick into 800. Mu.L deep well plate containing 10 × self-induced 2YT/Amp, coating the deep well plate with air permeable membrane, culturing at 37 deg.C and 220rpm for 3 hr, and culturing at 30 deg.C and 220rpm overnight. The ELISA plate was coated with 100ng of recombinant human Trop2 protein per well. The next day, taking 50 μ L of the deep-hole plate for conserving bacteria, centrifuging the rest at 4000rpm for 10 minutes, removing the culture medium in the hole, keeping the bacterial precipitation, adding 100 μ L of TES solution (20% sucrose, 0.1mM EDTA, 50mM Tris-HCl, pH 8.0) into each hole, oscillating to resuspend the bacterial, carrying out ice bath for 30 minutes, adding 200 μ L of ultrapure water, oscillating and mixing uniformly for 30 minutes, centrifuging at 4000rpm for 10 minutes after oscillation is finished, and obtaining the supernatant solution in the deep-hole plate, namely the periplasmic cavity extract containing the antibody. The ELISA plates were washed three times with a plate washer, then 200. Mu.L of 1% BSA/PBS was added and blocked at 37 ℃ for 1 hour. The blocking solution in the ELISA plate was removed, 100. Mu.L of the above periplasmic cavity extract was added, incubation was performed at 37 ℃ for 1 hour, washing was performed 3 times using a plate washing machine, 100. Mu.L of Chicken anti-HA HRP (1% BSA/PBS) was added, incubation was performed at 37 ℃ for 1 hour, washing was performed 3 times using a plate washing machine, 100. Mu.L of TMB developing solution was added, development was performed at 37 ℃ for 10 minutes, and 100. Mu.L of Stop solution was added. And (3) reading the OD450 value by using a microplate reader, and carrying out Sanger sequencing on the clone with the reading value being 3 times higher than the background value to obtain the gene sequence of the antibody.

8) Verification of Positive clones

And selecting clones with larger difference of the CDR3 amino acid sequences of the antibodies according to the sequencing result, re-inoculating and inducing overnight, and verifying whether the selected clones can be combined with the Trop2 again according to the ELISA method. Finally, antibody sequences such as 1A4 and 1F2 are obtained. The amino acid sequence of the heavy chain variable region of 1A4 is shown as SEQ ID NO. 4, and the amino acid sequence of the heavy chain variable region of 1F2 is shown as SEQ ID NO. 5.

Example 3: preparation of retrovirus stock solution containing anti-human Trop2 chimeric antigen receptor element

1. Preparation of CAR targeting human Trop2 antigen

A chimeric antigen receptor sequence containing a single domain antibody VHH of an anti-human Trop2 antigen, a hinge region, a transmembrane region and an intracellular signal segment is synthesized or cloned by genes, and the structure of the chimeric antigen receptor sequence is shown in figure 2. According to the difference of loading VHH, the chimeric antigen receptors are named as 1A4-BBz and 1F2-BBz respectively, the amino acid sequences are shown as SEQ ID NO. 11 and SEQ ID NO. 12 respectively, and the nucleotide sequences are shown as SEQ ID NO. 13 and SEQ ID NO. 14 respectively.

The retroviral vector MSGV is used as a framework vector to construct a retroviral plasmid for expressing the chimeric antigen receptor cloned by 1A4 and 1F 2. Selecting clones with correct sequencing, inoculating a bacterial solution into a 200ml 2YT culture medium, shaking the bacteria overnight, and completing large plasmid extraction according to the instruction of a NucleoBond Xtra Maxi EF kit.

2. Retroviral packaging

Packaging of retrovirus with cationic polymer PEI the procedure is as follows: mu.l PEI and retroviral packaging plasmid (6. Mu.g virus master plasmid, gag-pol 3.8. Mu.g, vsvg 1.5. Mu.g) were diluted with 600. Mu.l DMEM without serum, respectively; adding PEI/DMEM into the plasmid/DMEM mixture, vortex, shaking and mixing uniformly, and standing for 15 minutes at room temperature; plasmid-PEI complexes were added to pre-plated 293T cells. The solution was changed 16h after transfection, the first virus supernatant was collected after 48h, the second virus supernatant was collected after 72h and filtered with 0.45 μm filter and dispensed into 1.5mL centrifuge tubes, 1 mL/tube and stored at-80 ℃ for future use.

Example 4: production of Trop2CAR-T cells and determination of CAR positivity

PBMC isolation and activation

And taking a PBMC, checking that the individual identification code of the patient is correct, and then resuscitating. Cell density was adjusted to 1X 10 with X-VIVO complete Medium ⁶ and/mL. The recovered overnight PBMC after recovery was gently blown up, filtered by a 70 μm cell screen, transferred into a 50ml centrifuge tube, centrifuged at room temperature and 1500rpm for 5min, and the supernatant was discarded. Resuspending the cells with an appropriate amount of DPBS, mixing 20 μ l with trypan blue 1, counting the viability and CD3+ cells, then taking the desired volume of cells, centrifuging at 1500rpm at room temperature for 5min, and discarding the supernatant for sorting. The amount of magnetic beads was calculated according to the ratio of CD3/CD28 magnetic beads to CD3+ cells 1, the amount of magnetic beads = [ CD3+ cell number/4 × 10 = ⁵ μ l. And (3) cleaning magnetic beads: taking one sterile flow tube, adding 2ml of DPBS, adding magnetic beads, standing on a magnetic frame for 1min, and removing the supernatant. Removing the magnetic frame from the flow tube, taking DPBS or X-VIVO15 with the same volume as the cell suspension, re-suspending, adding the cell suspension, and centrifuging the magnetic beads and the cells in 15mlAfter mixing the tubes, incubate on a rotary mixer. Incubate at room temperature for 30min. After incubation was complete, the cells were gently transferred to a sterile flow tube, and the 15ml centrifuge tube was rinsed with 1ml DPBS and the rinse was incorporated into the same flow tube. And (4) moving the sterile flow tube to a magnetic frame, standing for 1min, and then absorbing and discarding unadsorbed liquid. The sterile flow tubes were removed from the magnetic frame, the cells were resuspended in 1ml CAR-T medium, and the tube walls were rinsed 2 times with CAR-T medium, all collected and transferred to the same centrifuge tube. Cell density was adjusted to 1X 10 with CAR-T medium ⁶ ml, IL-2 is added to the final concentration of 200IU/ml, and the mixture is placed on a table for 37 ℃ and 5% CO ₂ The incubator was used for two days.

2. Infection and culture of virus stock solution

The activated T cells were adjusted to 5X 10 ⁵ PermL, 1mL of T cells and 1mL of virus stock were added to a 24-well plate, 2. Mu.l of polybrene was added to each well, and centrifuged at 32 ℃ and 2500rpm for 1.5h. The supernatant was discarded and 1ml of T cell medium (containing IL-2 300IU/ml) was added to each well. The plates were incubated at 37 ℃ in 5% CO ₂ Culturing in an incubator. Transferring to 6-well plate 24h after infection, observing cell density every day, and timely supplementing T cell culture solution containing IL-2 300IU/ml to maintain T cell density at 1 × 10 ⁶ Cells were expanded at around/ml.

CAR Positive Rate detection

Retroviral-infected T lymphocytes tested CAR positivity 72h after viral infection. 1X 10 samples of the chimeric antigen receptor group containing 1A4 and 1F2 clones and the negative uninfected control group NT ⁶ The cells were centrifuged to remove the medium, washed once with 500. Mu.l PBS and resuspended in 100. Mu.l in a flow-through loading tube (BD). Biotin-labeled Trop2 antigen (1. After one washing of the cells with PBS, secondary antibody PE-SA streptavidin (BioLegend) was added as 1. After washing the cells with 500. Mu.l PBS, the cells were resuspended in 200. Mu.l PBS and tested on the machine, and the results of the CAR-T positive rate flow analysis are shown in FIG. 3.

Example 5: functional analysis based on anti-human Trop2CAR-T cells

1. Anti-human Trop2CAR-T cell CD107a expression assay

CAR-T cells containing different antibody clones were compared to target cells (Trop 2-positively expressed human pancreatic cancer cell line BxPC 3), control target cells (human glioma cells U251) at a potency-to-target ratio of 1 (3 × 10 for both effector and target cells) ⁵ One) mixed at 37 ℃ with 5% CO ₂ After the incubation in the incubator for 4 hours, the ratio of the number of CD107 a-expressing cells to the number of CD3+ cells in each group of samples was measured by flow. The degranulation response of CAR-T cells after stimulation by target cells was evaluated. The results of flow analysis of CD107a expression are shown in figure 4.

2. Detection of anti-human Trop2CAR-T cell cytokine secretion capacity

CAR-T cells containing different antibody clones were compared to target cells (Trop 2-positively expressed cell line BxPC 3) and control target cells (Trop 2-negatively expressed cell line U251) at an effective target ratio of 10 ⁴ And) after 24 hours of co-incubation, the supernatant was collected and assayed for secretion of IFN-. Gamma.and IL-2 by ELISA (enzyme-linked immunosorbent assay). IFN-gamma and IL-2 detection is detected by using an Aibixin Kit (Human IFN-gamma ELISA Kit and Human IL-2 ELISA Kit), and the experimental steps are carried out according to the product instruction. The results of IFN- γ secretion are shown in FIGS. 5 and 6. The results of IL-2 secretion are shown in FIGS. 7 and 8.

3. Anti-human Trop2CAR-T cytotoxicity assay

CAR-T killing toxicity experiments CAR-T cell function in vitro was assessed by testing the killing effect of CAR-T cells on target cells in vitro. At different effective target ratios (at 3X 10) ⁴ Taking each target cell as a benchmark, the effective target ratio is respectively 10 and 2). After overnight incubation at 37 ℃,100 μ l of luciferase reaction substrate was added to the culture system, the fluorescence value was measured, and the killing efficiency was calculated by the following equation: killing efficiency = (fluorescence value of positive control well-fluorescence value of experimental well)/fluorescence value of positive control well) × 100%. The experimental grouping and analysis results are shown in fig. 9 and 10.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shanghai Hengrunheng Dasheng Biotech Co., ltd

<120> anti-Trop 2 nano-antibody and application thereof

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gctccaacta tcgcaagtca gcccctgtca ctgcgccctg aagcctgtcg ccctgctgcc 540

gggggagctg tgcatactcg gggactggac tttgcctgtg atatctacat ctgggcgccc 600

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaggttc 660

agtgtcgtga agagaggccg gaagaagctg ctgtacatct tcaagcagcc tttcatgagg 720

cccgtgcaga ctacccagga ggaagatgga tgcagctgta gattccctga agaggaggaa 780

ggaggctgtg agctgagagt gaagttctcc cgaagcgcag atgccccagc ctatcagcag 840

ggacagaatc agctgtacaa cgagctgaac ctgggaagac gggaggaata cgatgtgctg 900

gacaaaaggc ggggcagaga tcctgagatg ggcggcaaac caagacggaa gaacccccag 960

gaaggtctgt ataatgagct gcagaaagac aagatggctg aggcctactc agaaatcggg 1020

atgaagggcg aaagaaggag aggaaaaggc cacgacggac tgtaccaggg gctgagtaca 1080

gcaacaaaag acacctatga cgctctgcac atgcaggctc tgccaccaag a 1131

<210> 15

<211> 271

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

His Thr Ala Ala Gln Asp Asn Cys Thr Cys Pro Thr Asn Lys Met Thr

1 5 10 15

Val Cys Ser Pro Asp Gly Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu

20 25 30

Gly Ser Gly Met Ala Val Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu

35 40 45

Leu Leu Lys Ala Arg Met Ser Ala Pro Lys Asn Ala Arg Thr Leu Val

50 55 60

Arg Pro Ser Glu His Ala Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro

65 70 75 80

Asp Cys Asp Pro Glu Gly Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr

85 90 95

Ser Val Cys Trp Cys Val Asn Ser Val Gly Val Arg Arg Thr Asp Lys

100 105 110

Gly Asp Leu Ser Leu Arg Cys Asp Glu Leu Val Arg Thr His His Ile

115 120 125

Leu Ile Asp Leu Arg His Arg Pro Thr Ala Gly Ala Phe Asn His Ser

130 135 140

Asp Leu Asp Ala Glu Leu Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu

145 150 155 160

His Pro Lys Phe Val Ala Ala Val His Tyr Glu Gln Pro Thr Ile Gln

165 170 175

Ile Glu Leu Arg Gln Asn Thr Ser Gln Lys Ala Ala Gly Asp Val Asp

180 185 190

Ile Gly Asp Ala Ala Tyr Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser

195 200 205

Leu Phe Gln Gly Arg Gly Gly Leu Asp Leu Arg Val Arg Gly Glu Pro

210 215 220

Leu Gln Val Glu Arg Thr Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro

225 230 235 240

Lys Phe Ser Met Lys Arg Leu Thr Ala Ser Gly Leu Asn Asp Ile Phe

245 250 255

Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His His

260 265 270

<210> 16

<211> 813

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

cacaccgccg cccaggacaa ctgcacctgc cccaccaaca agatgaccgt gtgcagcccc 60

gacggccccg gcggccgctg ccagtgccgc gccctgggca gcggcatggc cgtggactgc 120

agcaccctga ccagcaagtg cctgctgctg aaggcccgca tgagcgcccc caagaacgcc 180

cgcaccctgg tgcgccccag cgagcacgcc ctggtggaca acgacggcct gtacgacccc 240

gactgcgacc ccgagggccg cttcaaggcc cgccagtgca accagaccag cgtgtgctgg 300

tgcgtgaaca gcgtgggcgt gcgccgcacc gacaagggcg acctgagcct gcgctgcgac 360

gagctggtgc gcacccacca catcctgatc gacctgcgcc accgccccac cgccggcgcc 420

ttcaaccaca gcgacctgga cgccgagctg cgccgcctgt tccgcgagcg ctaccgcctg 480

caccccaagt tcgtggccgc cgtgcactac gagcagccca ccatccagat cgagctgcgc 540

cagaacacca gccagaaggc cgccggcgac gtggacatcg gcgacgccgc ctactacttc 600

gagcgcgaca tcaagggcga gagcctgttc cagggccgcg gcggcctgga cctgcgcgtg 660

cgcggcgagc ccctgcaggt ggagcgcacc ctgatctact acctggacga gatccccccc 720

aagttcagca tgaagcgcct gaccgctagc ggtctgaacg acatcttcga ggctcagaaa 780

atcgaatggc acgaacatca tcaccatcac cat 813

Claims

1. A Trop2 binding molecule comprises an anti-Trop 2 nanobody or an antigen binding fragment thereof, wherein a CDR of a Complementarity Determining Region (CDR) of the anti-Trop 2 nanobody comprises a CDR1, a CDR2 and a CDR3, wherein the CDR1 is a sequence shown in SEQ ID NO. 1, the CDR2 is a sequence shown in SEQ ID NO. 2, and the CDR3 is a sequence shown in SEQ ID NO. 3.

2. The Trop2 binding molecule according to claim 1, wherein the Trop2 binding molecule further has characteristics selected from one or more of:

the heavy chain variable region sequence of the anti-Trop 2 nano antibody is shown in any one of SEQ ID NO 4-5,

the Trop2 binding molecule is a monovalent or multivalent nanobody or single domain antibody comprising one, two or more anti-Trop 2 nanobodies or antigen-binding fragments thereof,

the nano antibody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody,

the nanobody further comprises a heavy chain constant region,

the Trop2 binding molecule is a chimeric antibody or a fully human antibody.

3. A chimeric antigen receptor comprising an optional signal peptide sequence, a Trop2 binding molecule of any one of claims 1 to 2, a hinge region, a transmembrane region, and an intracellular region.

4. The chimeric antigen receptor according to claim 3, wherein the intracellular domain comprises an intracellular costimulatory domain and/or an intracellular signaling domain.

5. The chimeric antigen receptor according to claim 3, which comprises, in order from N-terminus to C-terminus, a signal peptide, the Trop2 binding molecule of any one of claims 1 to 2, a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain.

6. A nucleic acid molecule having the characteristics of any one or more of:

(1) Encoding the Trop2 binding molecule of any one of claims 1 to 2 or the chimeric antigen receptor of any one of claims 3 to 5;

(2) (ii) a sequence encoding a Trop2 binding molecule of any one of claims 1 to 2 or a chimeric antigen receptor of any one of claims 3 to 5;

(3) The sequence is the complement of (2).

7. A nucleic acid construct comprising the nucleic acid molecule of claim 6.

8. The nucleic acid construct of claim 7, wherein the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

9. A host cell selected from the group consisting of:

(1) Expressing and/or secreting a Trop2 binding molecule of any one of claims 1 to 2 or a chimeric antigen receptor of any one of claims 3 to 5;

(2) Comprising the nucleic acid molecule of claim 6; and/or

(3) Comprising the nucleic acid construct of any one of claims 7-8.

10. The host cell of claim 9, wherein the host cell is an immune effector cell.

11. The host cell of claim 10, wherein the host cell is a T cell.

12. A method of producing a Trop2 binding molecule of any one of claims 1 to 2 or a chimeric antigen receptor of any one of claims 3 to 5, comprising: culturing the host cell of any one of claims 9-11 under conditions suitable for the production of a Trop2 binding molecule, and optionally purifying the Trop2 binding molecule or chimeric antigen receptor from the culture.

13. A pharmaceutical composition comprising a Trop2 binding molecule of any one of claims 1 to 2, a chimeric antigen receptor of any one of claims 3 to 5, a nucleic acid molecule of claim 6, a nucleic acid construct of any one of claims 7 to 8 or a host cell of any one of claims 9 to 11 and a pharmaceutically acceptable excipient.

14. Use of a Trop2 binding molecule of any one of claims 1 to 2, a chimeric antigen receptor of any one of claims 3 to 5, a nucleic acid molecule of claim 6, a nucleic acid construct of any one of claims 7 to 8 or a host cell of any one of claims 9 to 11in the preparation of an activated immune cell, or in the preparation of a medicament for the prevention or treatment of a disease or condition associated with Trop2 expression, selected from one or more of: breast, stomach, colorectal, pancreatic, prostate, cervical, head and neck, lung, esophageal, kidney, bladder, uterus and ovary cancer.

15. A kit for detecting Trop2, said kit comprising a Trop2 binding molecule of any one of claims 1 to 2, a nucleic acid molecule of claim 6, a nucleic acid construct of any one of claims 7 to 8, or a host cell of any one of claims 9 to 11.

16. The kit of claim 15, further comprising reagents for detecting the binding of Trop2 to the Trop2 binding molecule.

17. The kit of claim 16, wherein the reagent that detects Trop2 binding to the Trop2 binding molecule is a detectable label that can be linked to a Trop2 binding molecule.

18. Use of a Trop2 binding molecule according to any one of claims 1 to 2 in the preparation of a kit for detecting Trop2 in a sample, assessing the efficacy of a drug therapy or diagnosing a cancer associated with Trop2 expression, said cancer being selected from one or more of the following: breast, stomach, colorectal, pancreatic, prostate, cervical, head and neck, lung, esophageal, kidney, bladder, uterus and ovary cancer.