Trop2 antibody or antigen binding fragment thereof and application thereof

Abstract

The invention relates to a Trop2 antibody or an antigen binding fragment thereof and application thereof, and belongs to the technical field of biological immunotherapy, the antibody has good safety and a Trop2-targeting treatment effect, can be used for preparing a Trop2-targeting immune effector cell, and provides a treatment or improvement approach for diseases related to Trop2 expression.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a Trop2 antibody or antigen binding fragment thereof and application thereof. BACKGROUND

[0002] For tumors, traditional surgical resection, chemotherapy, and radiotherapy can damage normal tissues and have limitations and limited effects. In recent years, targeted therapy has emerged, which, at the cellular and molecular levels, designs corresponding therapeutic drugs for specific oncogenic sites. The drugs enter the body and specifically bind to the oncogenic sites to cause effects, resulting in specific death of tumor cells without damaging normal tissue cells around the tumor.

[0003] Trop2 is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, and is also known as human trophoblast cell surface antigen 2. Trop2 is composed of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail, is a single-pass transmembrane glycoprotein, and has a size of 35.7KD. Trop2 is a type of calcium ion channel signal transducer. The N-terminal of Trop2 protein is an extracellular domain (Trop2 EC), which is connected with an intracellular short tail (Trop2 IC) through a one-way transmembrane helix (TM), and is thus fixed to the cell membrane. The cytoplasmic tail has a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence, indicating that PIP2 plays an important role in the signal transduction of Trop2. In addition to the PIP2 binding motif, it also contains conserved tyrosine and serine phosphorylation sites. Trop2 is not expressed or lowly expressed in normal tissues, but is overexpressed in many malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer. Trop2 can promote the occurrence, invasion, metastasis, and diffusion of tumors, and plays a key role in the growth of tumors. Therefore, Trop2 is considered to be a target for tumor immunization.

[0004] Chimeric antigen receptor (Chimeric Antigen Receptor-T cell, CAR-T) T cells are T cells that are genetically modified to recognize specific target antigens in an MHC-unrestricted manner and are continuously activated and expanded. The 2012 International Cell Therapy Association Annual Meeting pointed out that biological immune cell therapy has become the fourth means of treating tumors in addition to surgery, radiotherapy, and chemotherapy, and is a new type of immunotherapy method for specific antigens on the surface of tumor cells. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune responses, and significantly improve the survival status of patients.

[0005] Chimeric antigen receptors (CARs) are the core components of CAR-T, which endow T cells with the ability to recognize tumor antigens in a HLA-independent manner, which makes CAR-modified T cells able to recognize a wider range of targets compared to natural T cell surface receptors TCR. The basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv fragment of the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signaling domain. The selection of target antigens is a critical determinant for the specificity, effectiveness and safety of genetically modified T cells.

[0006] As an important component of CAR, single-chain antibodies are traditionally used in mouse-derived antibodies, but the heterogeneity of mouse antibodies can cause human anti-mouse antibody reaction (HAMA), which leads to the rapid clearance of CAR-T in the circulatory system and loss of efficacy.

[0007] There is a need to develop more Trop2 binding molecules with good therapeutic effect and safety to treat Trop2-mediated diseases more safely and efficiently. SUMMARY

[0008] The present application provides a Trop2 binding molecule comprising an anti-Trop2 antibody or an antigen binding fragment thereof, the Trop2 binding molecule comprising a heavy chain variable region comprising a heavy chain complementarity determining region HCDR as shown in any one of SEQ ID NO: 1-3, 7-9, 13-15, 19-21, and a light chain variable region comprising a light chain complementarity determining region LCDR as shown in any one of SEQ ID NO: 4-6, 10-12, 16-18, 22-24, In one or more embodiments, the CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region are selected from any one of the following: (1) HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 3, and LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; (2) HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12; (3) HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, and LCDR3 of SEQ ID NO: 18; (4) HCDR1 of SEQ ID NO: 19, HCDR2 of SEQ ID NO: 20, HCDR3 of SEQ ID NO: 21, LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 23, and LCDR3 of SEQ ID NO: 24.

[0009] In one or more embodiments, the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID NOs: 25, 27, 29, and 31; the amino acid sequence of the light chain variable region is set forth in any one of SEQ ID NOs: 26, 28, 30, and 32; In one or more embodiments, the sequences of the heavy chain variable region and the light chain variable region are selected from any one of the following: (1) when the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 25, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 26; (2) when the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 27, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 28; (3) when the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 29, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 30; (4) when the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 31, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 32.

[0010] In one or more embodiments, the antibody is at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody; the antigen binding fragment is at least one of a Fab, a F(ab'), a F(ab')2, a single chain antibody scFv, or a disulfide-linked Fv (sdFv).

[0011] In another aspect, the present application provides a chimeric antigen receptor comprising an optional signal peptide sequence, the Trop2 binding molecule described in any one of the embodiments herein, a hinge region, a transmembrane region, and an intracellular region.

[0012] In one or more embodiments, the intracellular region comprises an intracellular costimulatory domain and / or an intracellular signaling domain.

[0013] In one or more embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a signal peptide, the Trop2 binding molecule described in any one of the embodiments herein, a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain.

[0014] The present application also provides a nucleic acid molecule having a sequence selected from any one of: (1) a coding sequence of the Trop2 binding molecule or the chimeric antigen receptor described in any one of the embodiments herein; (2) a complement of (1).

[0015] In one or more embodiments, the fragment is a primer.

[0016] The present application also provides a nucleic acid construct comprising the nucleic acid molecule described herein.

[0017] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

[0018] The present application also provides a host cell selected from: (1) expressing and / or secreting the Trop2 binding molecule or the chimeric antigen receptor described in any one of the embodiments herein; (2) comprising the nucleic acid molecule described herein; and / or (3) comprising the nucleic acid construct described herein.

[0019] In one or more embodiments, the host cell is an immune effector cell, preferably a T cell.

[0020] The present application also provides a method of producing a Trop2 binding molecule of any of the embodiments herein, comprising: culturing a host cell described herein under conditions suitable for production of a Trop2 binding molecule (e.g., an anti-Trop2 antibody or antigen binding fragment thereof, a monovalent or multivalent anti-Trop2 antibody, or a multi-specific anti-Trop2 antibody), and optionally purifying the Trop2 binding molecule from the culture.

[0021] The present application also provides a pharmaceutical composition comprising a Trop2 binding molecule, nucleic acid molecule, nucleic acid construct, or host cell of any of the embodiments herein, and a pharmaceutically acceptable excipient.

[0022] In one or more embodiments, the pharmaceutical composition is used to treat a Trop2 expression-related disease or condition.

[0023] The present application also provides use of a Trop2 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct, or host cell of any of the embodiments herein in the manufacture of an activated immune cell (e.g., a T cell).

[0024] The present application also provides use of a Trop2 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct, or host cell of any of the embodiments herein in the manufacture of a medicament for preventing or treating a Trop2 expression-related disease or condition.

[0025] In one or more embodiments, the disease or condition is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, and ovarian cancer.

[0026] The present application also provides a method of treating or preventing a Trop2 expression-related disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of a Trop2 binding molecule or host cell of any of the embodiments herein, or a pharmaceutical composition of any of the embodiments herein.

[0027] The present application also provides a kit for detecting Trop2, for example, to assess the efficacy of a drug treatment or to diagnose cancer, the kit comprising a Trop2 binding molecule, nucleic acid molecule, nucleic acid construct, or host cell of any of the embodiments herein.

[0028] In one or more embodiments, the kit further comprises a reagent for detecting binding of Trop2 to the Trop2 binding molecule. For example, a reagent for detecting the binding by enzyme-linked immunoassay.

[0029] In one or more embodiments, the detection agent is a detectable label, such as biotin, which can be attached to the Trop2 binding molecule. The detectable label can be attached to the Trop2 binding molecule or present separately in a kit.

[0030] The present application also provides a non-diagnostic method for detecting the presence of Trop2 in a sample, comprising incubating a sample with a Trop2 binding molecule as described in any of the embodiments herein, and detecting the binding of Trop2 to the Trop2 binding molecule, thereby determining the presence of Trop2 in the sample. The detection is by an enzyme-linked immunoassay.

[0031] The present application also provides the use of a Trop2 binding molecule as described in any of the embodiments herein in the manufacture of a kit for detecting Trop2 in a sample, assessing the effectiveness of a drug treatment, or diagnosing cancer.

[0032] The present application has the following advantages: The present application provides an antibody specifically recognizing Trop2 and a CAR modified cell containing the antibody, which has good therapeutic effect and safety in targeting Trop2, and the Trop2 CAR-T cell constructed has outstanding killing effect on breast cancer cells, especially breast cancer cells with high expression of Trop2, which is significantly better than other Trop2 antibodies screened at the same period and CAR-T cells constructed from known other Trop2 antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. Other related drawings can also be obtained by those skilled in the art without creative labor.

[0034] Figure 1 SDS electrophoresis map of recombinant human Trop2-avi-his antigen protein.

[0035] Figure 2 SDS-PAGE map of antibody protein of different clones.

[0036] Figure 3 ELISA map of antibody binding with Trop2 antigen of different clones.

[0037] Figure 4 Structural schematic diagram of CAR of different clones.

[0038] Figure 5Flow cytometry detection chart of tonic signal for different clones.

[0039] Figure 6 CAR positive rate expression of Trop2 CAR-T cells for different clones.

[0040] Figure 7 Expression of target cell surface Trop2.

[0041] Figure 8 Killing experiment results of Trop2 CAR-T cells for different clones on positive target cells MDA-MB-231 and control target cells MDA-MB-231-Trop2KO.

[0042] Figure 9 Killing experiment results of Trop2 CAR-T cells for different clones on positive target cells MDA-MB-468 and control target cells MDA-MB-468-Trop2KO.

[0043] Figure 10 Killing experiment results of Trop2 CAR-T cells for different clones on target cells HCC1395 overexpressing Trop2 and control target cells HCC1395.

[0044] Figure 11 Killing experiment results of L3F4 Trop2 CAR-T cells and control T cells on target cells HCC1395 overexpressing Trop2. DETAILED DESCRIPTION

[0045] The present inventors have made extensive and in-depth research, and through a large number of screening, found a class of anti-Trop2 antibodies and antigen binding fragments thereof, which can specifically recognize Trop2, bind to Trop2 with high affinity, and have good functional activity.

[0046] Specifically, the present application first constructs a human Trop2 protein expression vector, expresses and purifies human Trop2 protein in eukaryotic cells, then constructs a human natural antibody phage display library, and screens to obtain anti-Trop2 antibodies with good targeting and safety, which can specifically bind to the extracellular domain of human Trop2.

[0047] The present application also provides a chimeric antigen receptor (CAR) containing the anti-Trop2 antibody. The vector containing the coding sequence of the CAR is used to infect immune cells, and immune effector cells with significant killing ability to tumor cells overexpressing Trop2 can be obtained, which can be applied to treat or improve diseases related to Trop2 expression, especially the killing effect on HCC1395 overexpressing Trop2 is very significant.

[0048] Antibody Herein, a "Trop2 binding molecule" is a protein that specifically binds to Trop2, including but not limited to, an antibody, a heavy chain antibody, a nanobody, or an antigen binding fragment thereof.

[0049] Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions with 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" and "immunoglobulin" are used interchangeably.

[0050] A conventional "antibody" contains a basic 4-chain antibody unit, which is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at its N-terminus a variable domain (VH) followed by three constant domains (CH1, CH2, and CH3) and a hinge region between the CH1 and CH2 domains. Each light chain has at its N-terminus a variable domain (VL) followed by a constant domain (CL) at its other end. The variable domains of the heavy and light chains are held together in pairs to form an antigen binding site. See, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, eds., Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6, for a more detailed description of the structure and properties of different classes of antibodies. The light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the CH sequence and function, the gamma and alpha classes can be further divided into subclasses, e.g., in humans the following subclasses are expressed: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0051] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding sites.

[0052] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, and are used to confer antigen binding specificity and particular antigenic specificity of a particular antibody. However, the variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 in the heavy chain variable region and LCDR1, LCDR2, and LCDR3 in the light chain variable region. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of the naturally occurring heavy and light chains each comprise four FR regions, FR1, FR2, FR3, and FR4, mostly from beta-sheet conformation, connected by three HVRs. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. Generally, the structure of a light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of a heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not participate directly in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity. There are various numbering schemes for the variable regions of antibodies, including: Chothia, Kabat, IMGT, and Contact. The IMGT numbering scheme is used illustratively herein.

[0053] The "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors (FcR) located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of each of the two heavy chains; the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. While the boundaries of the Fc region of immunoglobulin heavy chains might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino-terminus at heavy chain position C226 or P230 to the carboxy-terminus of the

[0054] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and / or variable region of the intact antibody. Antibody fragments preferably are antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, and Fv fragments, disulfide-linked Fvs; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments capable of increasing the half-life of the antibody by chemical modification or by incorporation into a liposome. Antigen binding fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, and expression in a host cell of a nucleic acid encoding the fragment.

[0055] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute most of the antigen- binding specificity and diversity of an 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, although at a lower affinity than the entire binding site. "Single-chain Fv" also is abbreviated "sFv" or "scFv" and is an antibody fragment that comprises the VHand VLdomains of antibody, linked by a synthetic linker into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VHand VLdomains, which enables the sFv to form the desired structure for antigen binding.

[0056] The term "monoclonal antibody" as used herein 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 can 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, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, which does not

[0057] Monoclonal antibodies also include "chimeric" antibodies 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(s) 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.

[0058] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain sequence derived from non-human immunoglobulin. Thus, "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions have been exchanged for sequences found in human antibodies. Typically in a humanized antibody, 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. The 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 whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with a genetically engineered immune system. In the present application, antibodies, single chain antibodies, and the like include humanized variants of each of the recited antibodies.

[0059] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques disclosed herein for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries.

[0060] In some embodiments, the present application also provides antibodies or antigen binding fragments thereof that bind to the same epitope on human Trop2 as the antigen binding region of any of the anti-Trop2 antibodies of the present application, i.e., antibodies or antigen binding fragments thereof that are capable of cross- competing with the antigen binding region of any of the antibodies of the present application for binding to Trop2.

[0061] In the present application, the Trop2 binding molecule comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDRs as set forth in any one of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, and a light chain variable region comprising light chain complementarity determining regions LCDRs as set forth in any one of SEQ ID NOs: 4-6, 10-12, 16-18, 22-24. Preferably, the CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region of the Trop2 binding molecule of the present application are selected from any one of the following: (a) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3, and LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, LCDR3 as set forth in SEQ ID NO: 6; (b) HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 8, HCDR3 as set forth in SEQ ID NO: 9, and LCDR1 as set forth in SEQ ID NO: 10, LCDR2 as set forth in SEQ ID NO: 11, LCDR3 as set forth in SEQ ID NO: 12; (c) HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 14, HCDR3 as set forth in SEQ ID NO: 15, and LCDR1 as set forth in SEQ ID NO: 16, LCDR2 as set forth in SEQ ID NO: 17, LCDR3 as set forth in SEQ ID NO: 18; (d) HCDR1 as set forth in SEQ ID NO: 19, HCDR2 as set forth in SEQ ID NO: 20, HCDR3 as set forth in SEQ ID NO: 21, and LCDR1 as set forth in SEQ ID NO: 22, LCDR2 as set forth in SEQ ID NO: 23, LCDR3 as set forth in SEQ ID NO: 24.

[0062] Preferably, the amino acid sequence of the heavy chain variable region of the Trop2 binding molecule of the present application is as set forth in any one of SEQ ID NOs: 25, 27, 29, and 31; and the amino acid sequence of the light chain variable region is as set forth in any one of SEQ ID NOs: 26, 28, 30, and 32.

[0063] The Trop2 binding molecules described herein can be monovalent or multivalent antibodies, or multispecific antibodies, comprising one, two, or more of the anti-Trop2 antibodies or antigen binding fragments described herein. The multispecificity can be to Trop2 and another antigen, or to two different epitopes of Trop2.

[0064] The present application also includes derivatives and analogs of the antibodies. By "derivatives" and "analogs" is meant polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. Derivatives or analogs of the present application can be (i) polypeptides having substitution groups at one or more amino acid residues, or (ii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, e.g., a polyethylene glycol, or (iii) polypeptides formed by fusion of additional amino acid sequences to either the N- or C-terminus of the polypeptide (such as a leader or secretion sequence or a sequence for purification of the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). These derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0065] One of skill in the art can make alterations to the antibody sequences of the present application of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids without substantially affecting the activity of the antibody, to obtain variants of the sequences of the antibody or functional fragments thereof. These variants include, but are not limited to, deletions from, insertions into, and / or substitutions of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, as well as the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids to the C-terminus and / or the N-terminus of the antibody. In the art, conservative substitutions of similar or analogous properties are commonly made to proteins without changing the function of the protein. For example, substitutions of amino acids of similar properties in the FR and / or Fc regions. Amino acid residues which can be conservatively substituted are well known in the art. Such substituted amino acid residues can or can not be encoded by the genetic code. Also, for example, the addition of one or several amino acids to the C-terminus and / or the N-terminus of the protein usually does not change the function of the protein. These are all considered to be within the scope of the present application.

[0066] Variants of the antibodies described herein include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibodies of the application under conditions of high or low stringency, and polypeptides or proteins obtained using antisera against the antibodies of the application. In some embodiments, the sequences of the variants of the application can have at least 95%, 96%, 97%, 98%, or 99% identity to the sequences from which they are derived. The sequence identity of the sequences described in the application can be measured using sequence analysis software, for example, the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. The application also includes molecules having the variable region of the heavy chain or the variable region of the light chain of an antibody with CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0067] The antibodies of the application can be prepared using methods conventional in the art, such as the hybridoma technique. The single chain antibodies of the application can be prepared using methods conventional in the art, such as the phage display technique well known in the art. Alternatively, the antibodies or single chain antibodies of the application can be expressed in other cell lines. A suitable mammalian host cell can be transformed with a sequence encoding an antibody of the application, and the host cell is then cultured and the antibody is purified. Transformation can be performed using 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, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei, among others. Mammalian cell lines useful as hosts for expression are well known in the art, including, but 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., Hep G2), and the like.

[0068] CAR The application 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-Trop2 binding molecule described herein, a hinge region, a transmembrane region, and an intracellular region. The intracellular region comprises one or more intracellular costimulatory domains and / or one or more intracellular signaling domains. The "hinge region," "transmembrane region," and "intracellular region" herein can be selected from the sequences of the hinge region, transmembrane region, and intracellular region in known CAR-T technology.

[0069] The optional signal peptide on the CAR can be selected as desired. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired location 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 can 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 sequences of which are within the knowledge of one of skill in the art. A CD8 signal peptide suitable for use in the application can be any of the various human CD8 signal peptide sequences commonly used in CARs in the art. In certain embodiments, the CD8 signal peptide has an amino acid sequence comprising the sequence set forth in SEQ ID NO: 33 and a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 38.

[0070] The hinge region of the chimeric antigen receptor is located between the extracellular antigen binding region and the transmembrane region, and is a segment of amino acids that is often found between two domains of a protein and can allow flexibility of the protein and movement of the two domains relative to each other. The hinge region can 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, can also be used in the chimeric antigen receptors described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptors described herein. Exemplarily, the hinge region of the CAR is selected from a CD8a hinge region, an IgD hinge region, an IgGl 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. A CD8a hinge region suitable for use in the application can be any of the various human CD8a hinge region sequences commonly used in CARs in the art. In certain embodiments, the human CD8a hinge region comprises the sequence set forth in SEQ ID NO: 34 and a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 39.

[0071] The transmembrane region of the chimeric antigen receptor can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell domain. The transmembrane region can be of natural or synthetic origin. The transmembrane region can be selected from the transmembrane region of the following proteins: CD3 epsilon, CD4, CD5, CD8a, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, the alpha, beta, or zeta chain of the T cell receptor. A human CD8a transmembrane region suitable for use in the application can be any of the various human CD8a transmembrane region sequences commonly used in CARs in the art. In certain embodiments, the human CD8a transmembrane region has an amino acid sequence comprising the sequence set forth in SEQ ID NO: 35 and a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 40.

[0072] The intracellular signaling region (or intracellular signaling domain) is responsible for the activation of at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. While it is generally possible to utilize the entire intracellular signaling domain, in many cases it is unnecessary to use the entire chain. In terms of using a truncated portion of the intracellular signaling domain, such truncated portion can be used in place of the complete chain so long as it transduces an effector function signal. Thus, the intracellular signaling domain includes any truncated form of the intracellular signaling domain sufficient to transduce an effector function signal. The intracellular signaling domain of the CAR can be selected as desired, including but not limited to an intracellular signaling domain derived from at least one of CD3 zeta, FcR gamma (FCER1G), FcR beta (FcsRib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling region is derived from a human CD3 zeta intracellular signaling region. Further, the human CD3 zeta intracellular signaling region has the amino acid sequence set forth in SEQ ID NO: 37, and the nucleotide sequence comprises the sequence set forth in SEQ ID NO: 42.

[0073] In addition to stimulation by an antigen-specific signal, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, as well as activation of effector functions of the cell. The "co-stimulatory domain" can be the cytoplasmic portion of a 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 with a co-stimulatory ligand to mediate a co-stimulatory response by the immune cell, such as but not limited to proliferation and survival. Suitable intracellular co-stimulatory domains can be selected as desired, including having an intracellular domain of a co-stimulatory signaling molecule, for example at least one derived from an intracellular domain of 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, FcsRIy, MyD88, and 41BBL. In certain embodiments, the 4-1BB co-stimulatory domain has the amino acid sequence set forth in SEQ ID NO: 36, and the nucleotide sequence comprises the sequence set forth in SEQ ID NO: 41.

[0074] The above-mentioned parts of the chimeric antigen receptor of the present application, such as CD8 signal peptide, anti-Trop2 single-chain antibody, CD8a hinge region, CD8a transmembrane region, CD3 zeta intracellular signaling domain, 4-1BB costimulatory domain, etc., can be directly connected to each other, or can be connected by linker sequences. The linker sequence can be a linker sequence known in the art suitable for antibodies, such as a linker sequence containing G and S. Generally, the linker contains one or more motifs repeated before and after. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence, without inserting amino acid residues between the repeats. The linker sequence can comprise 1, 2, 3, 4 or 5 repeat motifs. The length of the linker can be 3-25 amino acid residues, such as 3-15, 5-15, 10-20 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 generally 2-20, such as 2-15, 2-10, 2-8. In addition to glycine and serine, the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. In certain embodiments, the linker sequence is (GGGGS)n, where n is an integer from 1 to 5.

[0075] In an exemplary embodiment, the CAR contains, in order from N-terminus to C-terminus, a CD8 signal peptide, an anti-Trop2 antibody or antigen-binding fragment thereof described herein, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0076] It should be understood that in the process of gene cloning, it is often necessary to design suitable restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, which does not affect the activity of the sequence of interest. In order to construct a fusion protein, facilitate the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable region within the protein, such as, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino terminus or carboxyl terminus of the CAR of the present application can also contain one or more polypeptide fragments as a protein tag. Any suitable tag can be used herein. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-Tag II, AU1, EE, T7, 4A6, epsilon, B, gE and Ty1. These tags can be used for protein purification.

[0077] The antigen recognition region in the CAR of the present application can be a variant of the aforementioned anti-Trop2 antibody or functional fragment sequence thereof. Furthermore, other parts of the CAR can also be subject to sequence changes, resulting in a mutant having 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 retaining the biological activity of the CAR (e.g. activation of T cells). The sequence identity between two aligned sequences can be calculated using, for example, BLASTp of NCBI.

[0078] Mutants also include amino acid sequences having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence of the CAR described in any of the embodiments, while still retaining the biological activity of the CAR. The several mutations typically refer to 1-10, for example 1-8, 1-5 or 1-3. The substitutions are preferably conservative substitutions. For example, in the art, a conservative substitution is typically made when a similar or similar property of an amino acid is used, without changing the function of the protein or polypeptide. "Similar or similar properties of amino acid residues" include, for example, families of amino acid residues with similar side chains, which include amino acids with basic side chains (e.g. lysine, arginine, histidine), amino acids with acidic side chains (e.g. aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g. alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with β-branching side chains (e.g. threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more positions in the polypeptides of the present application with another amino acid residue from the same side chain class will not substantially affect its activity.

[0079] Nucleic acid The present application also provides polynucleotides encoding the above-mentioned antibodies or CARs. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The present application also includes degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e. nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0080] Accordingly, the present application also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. In particular, the present application relates to polynucleotides which hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing at low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS at 60°C; or (2) hybridization in the presence of a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll at 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological function and activity as the mature polypeptides.

[0081] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain or the light chain can be fused with an expression tag (such as 6His) to form a fusion protein. The sequence of the CAR can also be obtained as above. Alternatively, the sequence of each part of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region or intracellular region) can be obtained as above and then ligated to obtain the full-length CAR.

[0082] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually by cloning it into a vector, then transforming into cells, and then separating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules in isolated form. At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be completely obtained by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis. The parts of the CAR can be cloned into vectors sequentially or can be cloned after being integrated into the full-length CAR.

[0083] The present application also relates to nucleic acid constructs comprising the polynucleotide sequences described herein, and one or more control sequences operably linked to the sequences. The polynucleotide sequences described herein can be manipulated in a variety of ways to produce the antibody or CAR described herein. Manipulation of the nucleic acid constructs prior to their insertion into a vector can be used to direct the expression of the antibody or CAR. Techniques for modifying polynucleotide sequences using recombinant DNA methods are known in the art.

[0084] The regulatory sequences can be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutated, truncated, and hybrid promoters, and can be derived from genes either homologous or heterologous to the host cell. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence which is capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous Sarcoma Virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the use of inducible promoters can also be contemplated. The use of inducible promoters provides a molecular switch which can turn on the expression of a polynucleotide sequence operably linked to the inducible promoter at times when expression is desired, and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0085] The regulatory sequences can also be a suitable transcription terminator sequence, a sequence recognized by a host cell for termination of 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 can be used in the present application. The regulatory sequences can also be a suitable leader sequence, an untranslated region of an mRNA 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 leader sequence which is functional in the host cell of choice can be used in the present application.

[0086] In certain embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integrating vector. Expression of the polynucleotide sequences of the application is typically achieved by operably linking the polynucleotide sequences of the application to an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of expression of the desired nucleic acid sequences. Integrating vectors contain components that allow integration of the target sequences into the genome of the cell. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences that are useful for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. The sequences, which in certain embodiments are collectively referred to as "flanking sequences," generally 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 insertion of nucleic acids encoding antibodies to be expressed, and optional marker elements.

[0087] Furthermore, the type of vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus, and cosmid, and can be changed depending on the host cell to be introduced. Viral vector technology is well known in the art and described in, for example, 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.

[0088] To assess expression of the CAR polypeptide or portion thereof, the vector introduced into the cell can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cells from the population of cells sought to be transfected or infected by the viral vector.

[0089] 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, particularly immune cells, preferably immune effector cells. Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 or Sf9; animal cells, such as CHO, COS7, 293 cells, and the like.

[0090] An "immune effector cell" is an immune cell that can perform an immune effector function. In some embodiments, an immune effector cell expresses at least FcyRIII and performs an 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 cell is selected from the group consisting of at least one of an immune cell differentiated from a pluripotent stem cell or an embryonic stem cell, a T lymphocyte, a NK cell, a peripheral blood mononuclear cell (PBMC), and a hematopoietic stem cell. More preferably, the immune effector cell is a T lymphocyte (also referred to as a T cell). In some embodiments, the T cell can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cell produces IL-2, IFN, and / or TNF when expressing a chimeric antigen receptor and binds to a target cell. In some embodiments, the CD8+ T cell lyses an antigen-specific target cell when expressing a chimeric antigen receptor and binds to a target cell.

[0091] T cells suitable for use in the present application can be of various types from various sources. For example, the T cells can be derived from PBMCs of a patient with a malignant solid tumor (e.g., pancreatic cancer). In certain embodiments, after the T cells are obtained, they can be stimulated for activation with an appropriate amount (e.g., 30-80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in a medium containing an appropriate amount (e.g., 30-80 IU / ml, such as 50 IU / ml) of IL2 for use.

[0092] Methods of introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be introduced into the cells by physical, chemical, or biological means. When the host is a prokaryote, such as E. coli, cells that have been made competent by the CaCI2method can be harvested after exponential growth and treated with CaCI2, using procedures well known in the art. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. can be used. In some embodiments, the transduced or transfected immune effector cells are propagated ex vivo after introduction of the nucleic acid or vector.

[0093] The transformants obtained can be cultured in conventional methods to express the antibodies or CARs encoded by the genes of the present application. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional media. The culture is performed under conditions suitable for the growth of the host cell. When the host cell has grown to an appropriate cell density, the selected promoter is induced using a suitable method (e.g., temperature shift or chemical induction), and the cells are cultured for an additional period of time.

[0094] The polypeptides in the above methods can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0095] Uses and methods By constructing a single-chain antibody library, the inventors screened single-chain antibodies that can bind to Trop2. Using these single-chain antibodies, the inventors constructed CARs and CAR-T cells, which were verified by cell level experiments to have strong immune function, better CD107a expression, IFN-γ and IL-2 secretion, and specific killing function on target cells, and the in vivo efficacy was significantly improved.

[0096] All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare a medicament for preventing or treating various conditions and diseases described herein, which are diseases or conditions associated with Trop2 expression, diseases or conditions directly or indirectly caused by abnormal expression of Trop2, and generally diseases caused by overexpression of Trop2, such as cancer, including but not limited to: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, and ovarian cancer.

[0097] The present application also includes a class of cell therapy, including expressing the CAR described herein in immune cells (such as T cells), and administering a therapeutically effective amount of the cells to a recipient in need thereof, the cells being capable of killing tumor cells of the recipient. Compared with antibody therapy, CAR-T cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control. The anti-tumor immune response caused by CAR-T cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells induce an immune response specific to the antigen-binding portion in the CAR.

[0098] The antibodies, nucleic acids, or CAR-modified cells of the present application can be administered alone or in combination with diluents and / or with other components such as relevant cytokines or cell populations as a pharmaceutical composition. In this regard, the pharmaceutical compositions can be prepared by mixing the active agent having the desired degree of purity with optional pharmaceutically acceptable carriers, in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and can comprise 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 surface active or lubricating agents. In addition, to be useful for in vivo administration, the pharmaceutical compositions must be sterile. The pharmaceutical compositions can be rendered sterile by filtration through a sterile filtration membrane.

[0099] In some embodiments, the pharmaceutical compositions can contain at least one additive of a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, a growth inhibitory agent, and an active agent required for the particular indication to be treated. The specific amount of additive added can be adjusted as necessary. The pharmaceutical compositions of the present application can be administered in an "immunologically effective amount," an "anti-tumor effective amount," a "tumor-inhibiting effective amount," or a "therapeutic amount." "Treatment" refers to the adoption of a therapeutic regimen by a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of infiltration of cancer cells into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth). When referring to an "immunologically effective amount," an "anti-tumor effective amount," a "tumor-inhibiting effective amount," or a "therapeutic amount," the precise amount of the composition of the present application to be administered can be determined by a physician with consideration for a patient's (subject's) age, body weight, tumor size, extent of infection or metastasis, and individuality of disease. Generally, the pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, preferably 10 5 to 10 6 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in the art of immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by monitoring the patient's disease signs and adjusting the treatment accordingly by a person skilled in the medical arts.

[0100] Administration of the compositions can be carried out in any convenient manner, including by spray, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the present application are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present application are preferably administered by intravenous injection. The compositions of T cells can be injected directly into a tumor, lymph node or site of infection.

[0101] In some embodiments of the present application, the CAR-T cells of the present application 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 can be combined with radiation or chemotherapy agents known in the art to treat mesothelin-mediated diseases.

[0102] As used herein, "anti-tumor effect" refers to a biological effect that can be indicated by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0103] "Patient," "subject," "individual," and the like are used interchangeably herein to 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.

[0104] Diagnosis, Detection and Kits The binding molecules of the present application, due to their high affinity for Trop2, can be used in assays, such as binding assays, to detect and / or quantify Trop2 expressed in tissues or cells. The binding molecules, such as single chain antibodies, can be used in research to further study the role of Trop2 in disease. Methods of detecting Trop2 are generally as follows: obtaining a sample of cells and / or tissue; detecting the level of Trop2 in the sample.

[0105] The Trop2 binding molecules of the present application can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or conditions associated with Trop2. The present application provides for detecting the presence of Trop2 in a sample using classical immunohistological methods known to those of skill 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.

[0106] For diagnostic applications, the binding molecules, e.g., single chain antibodies, are typically labeled with a detectable label group. Suitable label groups include, but are not limited to, the following: a radioisotope or radionuclide (e.g.,3H,14C,15N,35S,90Y,99Tc,111In,125I,131I), a fluorescent group (e.g., FITC, rhodamine, lanthanide phosphors), an enzymatic group (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotinyl group, or a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag), an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent. Various methods for labeling proteins are known in the art and can be used to practice the present application.

[0107] Another aspect of the present application provides a method of detecting the presence of a test molecule that competes with the antibodies of the present application for binding to Trop2. An 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 the test molecule. An increase in the amount of free antibody (i.e., antibody that is not bound to Trop2) 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 label group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0108] The present application also provides a test kit for detecting the level of Trop2, which comprises an antibody recognizing the Trop2 protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The test kit can be an in vitro diagnostic device.

[0109] The present application will be illustrated hereinafter in the manner of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present application. The methods and materials used in the examples are conventional in the art unless otherwise specified. Examples

[0110] Example 1: Construction of recombinant human Trop2 protein expression vector and eukaryotic expression

[0111] 1. Synthesis of gene sequence and construction of protein expression vector The human Trop2 protein sequence was downloaded from Uniport (https: / / www.uniprot.org / uniprot), optimized using an online codon optimization tool (http: / / www.jcat.de / #opennewwindow), and then sent to Sangon Biotech to synthesize the Trop2 extracellular gene sequence. Simultaneously, avi-tag and 6×his-tag nucleic acid sequences were added to the 3' end of this gene sequence, and the fusion gene sequence encodes the amino acid sequence shown in SEQ ID NO:51. The sequence of the fusion gene is shown in SEQ ID NO:52. Using molecular cloning, the splicing product was cloned into pTT5 using the TaKaRa seamless cloning kit to obtain the expression vector.

[0112] 2. Expression and purification of recombinant human Trop2 protein Five days after transfecting 293T cells (ATCC) with the obtained expression vector, the culture supernatant was collected, and recombinant human Trop2 protein was purified using AKTA Explorer100 (GE). The Trop2 protein was stained with Coomassie Brilliant Blue after SDS-PAGE electrophoresis, revealing a size of approximately 36 kDa. The results are as follows: Figure 1 As shown.

[0113] Example 2: Preparation of anti-human Trop2 antibody

[0114] 1. Construction of scFv phage display library A two-step method was used to construct a human natural antibody phage display library, which involved ligating the light chain variable region gene and the heavy chain variable region gene of the human natural antibody into the phage display vector in two steps.

[0115] 1) Gene amplification of variable regions in the κ and λ chains. Using human PBMC cDNA as a template, the human immunoglobulin κ chain variable region gene was amplified by PCR using forward primers (3z3-1-F) and reverse primers (Huvksc-R) for the κ chain variable region, and the human immunoglobulin λ chain variable region gene was amplified by PCR using forward primers (3z3-1-F) and reverse primers (HuvLR-1) for the λ chain variable region. The PCR reaction conditions were as follows: 98℃ pre-denaturation for 45 seconds followed by temperature cycling: 98℃ denaturation for 15 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 23 seconds, for 30 cycles, with a final extension at 72℃ for 5 minutes. After PCR products were subjected to 1.5% agarose gel electrophoresis, the approximately 330 bp κ chain variable region gene fragment and λ chain variable region gene fragment were recovered using a gel extraction kit (Promega).

[0116] 2) Construction of κ-chain and λ-chain libraries The κ chain gene, λ chain gene and phagemid pcomb3sc were double-enzyme cut by NheI-HF and SalI-HF DNA endonuclease (NEB), and the κ chain gene and λ chain gene were recovered directly by a gel recovery kit after enzyme cutting. The pcomb3sc vector after enzyme cutting was subjected to 1% agarose gel electrophoresis, and a 4000bp vector fragment was recovered by a gel recovery kit (Promega). The κ chain gene and λ chain gene were connected to the pcomb3sc vector by a T4 DNA ligase kit (Invitrogen), 1000ng of pcomb3sc, 200ng of κ and λ, respectively, multiple reaction tubes were prepared, 50ul / tube, 16℃ connection overnight, and a small amount of ligation product was subjected to agarose gel electrophoresis to detect the ligation efficiency. The ligation product was desalted by a MECK MILLIPOREF microporous filter membrane. The desalted ligation product was electroporated into self-made TG1 electrotransformation competent cells to obtain a κ chain library and a λ chain library. The κ chain library and the λ chain library after electroporation were amplified overnight, and the κ chain library plasmid and the λ chain library plasmid were extracted by a plasmid large extraction kit (NucleoBond Xtra Maxi EF) the next day.

[0117] 3) Heavy chain variable region VH gene amplification The human immunoglobulin heavy chain variable region forward primer (VH-MIX 1 / 7) and the reverse primer (VH-MIX R), the human immunoglobulin heavy chain variable region forward primer (VH-MIX3) and the reverse primer (VH-MIX R), and the human immunoglobulin heavy chain variable region forward primer (VH-MIX4) and the reverse primer (VH-MIX R) were used to amplify the human immunoglobulin VH variable region gene with human PBMC cDNA as a template. The PCR reaction conditions were as follows: 98℃ pre-denaturation for 45 seconds, followed by temperature cycling, 98℃ denaturation for 15 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 23 seconds, 30 cycles, and 72℃ final extension for 5 minutes. The PCR product was subjected to 1% agarose gel electrophoresis, and the 330bp VH gene fragment was recovered by a gel recovery kit (Promega).

[0118] 4) Construction of scFv library The recovered VH fragments were mixed in equal proportions, and both the kappa chain library plasmid and the lambda chain library plasmid were subjected to single enzyme digestion with Sfil DNA endonuclease, 50°C for 16h, and the digested VH genes were directly recovered by column using a gel recovery kit, while the digested kappa chain library and lambda chain library were subjected to 1% agarose gel electrophoresis, and the vector fragments of about 4000bp were recovered using a gel recovery kit (Promega). The VH genes were ligated into the kappa chain and lambda chain vector libraries using a T4 DNA ligase kit (Invitrogen), i.e. pcomb3sc-lambda / kappa (Sfil) 1000ng, VH amplify mix (sfil) 200ng, multiple reaction tubes were prepared, 50ul / tube, 16°C overnight, and a small amount of the ligation product was subjected to agarose gel electrophoresis to detect the ligation efficiency. The ligation product was desalted using a MECK MILLIPOREF microporous filter membrane.

[0119] The ligation product was added to the self-made TG1 electrotransformation competent cells, and then an electrotransformation instrument was used for electroporation. 50ul of the bacterial solution was taken and gradient diluted with PBS for 10 2 -10 5 times. 10ul of each gradient dilution was streaked on Amp / 2YT plates, incubated at 37°C overnight, and the phage antibody library size was counted and calculated. The remaining electrotransformation bacteria were supplemented with 2YT to 500ml, 100mg / ml ampicillin stock solution was added at 1:1000, and the culture was incubated at 30°C, 220rpm overnight. Finally, more than 3E10 scFv immune libraries were obtained. The antibody library after electroporation was amplified overnight, the library bacteria were collected by centrifugation, and the final concentration was 20% glycerol-80°C storage.

[0120] 5) Phage display library The frozen part of the human natural antibody scFv library strain was inoculated into 2YT medium, the inoculation density was 0.1OD, and the bacterial solution was cultured at 37°C, 220rpm. After about 1.5 hours, the bacterial density reached 0.6OD, at which time 20 times the number of M13KO7 phage was added and allowed to stand for 30 minutes for infection, and then incubated at 30°C, 220rpm overnight. The next day, the bacterial solution was centrifuged at 10000g, and the culture supernatant was collected. 1 / 4 volume of 5x PEG / NaCl solution (20% PEG8000, 2.5M NaCl) was added to the culture supernatant, mixed well, and then ice bathed for 1 hour. After ice bathing, the precipitate was collected by centrifugation at 8000g for 10 minutes, and the precipitate was dissolved in 10% glycerol / PBST to obtain the human natural antibody phage display library. The OD268 was measured, and it was aliquoted into 1.5ml centrifuge tubes, 6OD / tube, and stored at-80°C.

[0121] 2. Panning Trop2 scFv antibodies 1) Recombinant human Trop2 protein conjugated to streptavidin magnetic beads The avi-tag of recombinant human Trop2 protein was biotinylated using a biotinylation kit (Genecopoeia) according to the kit instructions. 10 μg of the biotinylated recombinant protein was added to 100 μL of streptavidin magnetic beads (DynaBeads 280) that had been washed three times with PBS and incubated for 1 hour at room temperature on a rotating shaker at 20 rpm. The beads were then washed three times with PBS.

[0122] 2) Blocking of phage library and negative magnetic beads One vial each of the Kappa-scFv library and Lambda-scFv library was thawed at room temperature and each was added to 200 μL of 5% BSA / PBST and incubated for 1 hour at room temperature on a rotating shaker at 20 rpm. These phage were designated Input 1. At the same time, 100 μL of DynaBeads 280 that had not been conjugated to protein were washed three times with PBS and then added to 1 mL of 1% BSA / PBS and incubated for 1 hour at room temperature on a rotating shaker at 20 rpm.

[0123] 3) Blocking of positive magnetic beads The Trop2-conjugated magnetic beads were added to 1 mL of 1% BSA / PBS and incubated for 1 hour at room temperature on a rotating shaker at 20 rpm.

[0124] 4) Negative panning To remove antibodies that interact with the magnetic beads, negative panning was performed. The BSA-blocked phage library (Input 1) and magnetic beads that had not been conjugated to antigen were mixed and incubated for 1 hour at room temperature on a rotating shaker at 20 rpm. After the incubation, the phage and magnetic bead mixture was placed on a magnetic stand until the beads were all adhered to the wall. The supernatant was then transferred to a new EP tube.

[0125] 5) Positive panning The above-mentioned closed coupling of Trop2 protein magnetic beads were added to the phage supernatant after negative selection for positive selection, 20 rpm, room temperature rotation closed for 1 hour. After incubation, the magnetic beads were washed with 1 mL PBST (0.1% Tween-20 in PBS) and the washing was repeated 10 times. After washing, 1 mL of 100 mM glycine (pH 2.0) was added and placed on a rotary shaker at a speed of 20 rpm for 10 minutes. After elution, the EP tube was placed on a magnetic stand, and after the magnetic beads were all attached to the wall, the eluate was transferred to a new EP tube. 0.2 mL of 1M Tris-HCl solution (pH 8.0) was added to the eluate for neutralization. The neutralized eluate was added to 30 mL of TG1 bacterial solution with OD600 of about 0.6 and incubated for 30 minutes, then 20 times the number of bacteria were added M13KO7 phage, incubated for 30 minutes, and finally 100 mL of 2YT medium and 100 μg / mL of ampicillin and kanamycin were added. The culture was incubated at 30°C, 220 rpm overnight. The next day, the phage was harvested according to the above method for harvesting the phage library, and the phage obtained at this time was Input2.

[0126] 6) Repeat positive selection According to the above selection method, 2 times of repetition were carried out, that is, Input2 was subjected to the next round of negative selection and positive selection to obtain Input3. The difference is that after the eluate obtained by the selection of Input3 infects TG1, no M13KO7 is added, but 10 μl of bacterial solution is gradient diluted with PBS, and 10 μl of each of the three dilution gradients is plated on 2YT / Amp plates, and cultured at 30°C overnight. The remaining bacterial solution was cultured at 30°C, 220 rpm overnight. 3 、10 4 、10 5 Three dilution gradients of 100 μl of bacterial solution were plated on 2YT / Amp plates and cultured at 30°C overnight. The remaining bacterial solution was cultured at 30°C, 220 rpm overnight.

[0127] 7) ELISA screening of positive antibodies The TG1 monoclonal in the above flat plate was randomly picked with a toothpick into an 800 μL deep well plate containing 10x autoinduction 2YT / Amp, and the deep well plate was covered with a gas permeable membrane. After incubation at 37°C, 220 rpm for 3 hours, and then at 30°C, 220 rpm overnight, the recombinant human Trop2 protein was coated in the ELISA plate at 100 ng per well. The next day, 50 μl of bacteria was taken from the deep well plate, and the remaining 4000 rpm centrifuged for 10 minutes to remove the culture medium in the well and retain the bacterial pellet. 100 μl of TES solution (20% sucrose, 0.1 mM EDTA, 50 mM Tris-HCl, pH 8.0) was added to each well, and the bacterial pellet was resuspended by shaking. After 30 minutes of ice bath, 200 μL of ultrapure water was added and mixed for 30 minutes. After shaking, the 4000 rpm centrifuged for 10 minutes, the supernatant solution in the deep well plate was the periplasmic cavity extract containing the antibody. The ELISA plate was washed three times with a plate washer, then 200 μL of 1% BSA / PBS was added and incubated at 37°C for 1 hour. The blocking solution in the ELISA plate was removed, 100 μL of the above periplasmic cavity extract was added, and incubated at 37°C for 1 hour (a control of trastuzumab was also prepared at the same time). The plate was washed three times with a plate washer, 1% BSA / PBS was added, and incubated at 37°C for 1 hour. The plate was washed three times with a plate washer, 100 μL of TMB color developing solution was added, and incubated at 37°C for 10 minutes. 100 μL of stop solution was added to terminate the reaction. The OD450 value was read using an enzyme-labeled instrument, and the clones with a reading value higher than 3 times the background value were subjected to Sanger sequencing to obtain the antibody gene sequence.

[0128] 8) Verification of positive clones According to the sequencing results, the clones with large differences in CDR3 amino acid sequences were selected for re-inoculation and overnight induction. The selected clones were re-verified for their ability to bind Trop2 according to the above ELISA method. Finally, four better antibody sequences, L2A8, L3G1, K2G2, and L3F4, were obtained.

[0129] The amino acid sequence of the heavy chain variable region of L2A8 is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region sequence is shown in SEQ ID NO: 26. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are GYSFTNYD (SEQ ID NO: 1), INPNSGNT (SEQ ID NO: 2), VRGRSYYYDKRGHYLNWYFDT (SEQ ID NO: 3), SSDVGGYNY (SEQ ID NO: 4), DV (SEQ ID NO: 5), and GSYTSNNIPWV (SEQ ID NO: 6), respectively.

[0130] The amino acid sequence of the heavy chain variable region of L3G1 is shown as SEQ ID NO: 27, and the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: 28. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 sequences are GYSFTSYW (SEQ ID NO: 7), IYPGDSDT (SEQ ID NO: 8), ARLGGSYYYYGMDV (SEQ ID NO: 9), SSDFFNYHF (SEQ ID NO: 10), DV (SEQ ID NO: 11), SSYTNSGTWV (SEQ ID NO: 12), respectively.

[0131] The amino acid sequence of the heavy chain variable region of K2G2 is shown as SEQ ID NO: 29, and the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: 30. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 sequences are GYTFTDYY (SEQ ID NO: 13), INPDSGGT (SEQ ID NO: 14), ARGGLLPSSDWSSYYNGMNV (SEQ ID NO: 15), QRVSSF (SEQ ID NO: 16), AA (SEQ ID NO: 17), QQGYSTPPT (SEQ ID NO: 18), respectively.

[0132] The amino acid sequence of the heavy chain variable region of L3F4 is shown as SEQ ID NO: 31, and the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: 32. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 sequences are GFTFDNYA (SEQ ID NO: 19), ISWNSGTI (SEQ ID NO: 20), AKVRLRYFDWGSFDY (SEQ ID NO: 21), RSNVLFNT (SEQ ID NO: 22), GT (SEQ ID NO: 23), SAWDDSLNGYV (SEQ ID NO: 24), respectively.

[0133] Example 3: ELISA verification of Trop2 protein level

[0134] 1. Construction of PTT5-Trop2 plasmid and protein expression The Trop2 antibody screened in Example 2 was constructed into pTT5 expression vector by homologous recombination, and the correct clone was prepared for plasmid transfection of 293F cells to express protein. The Trop2 antibody protein is about 110 kilodaltons, and after SDS-PAGE electrophoresis, it shows a clear main band by Coomassie blue staining. Due to the influence of glycosylation, it is larger than the actual size. The results are shown in Figure 2 .

[0135] 2. ELISA detection of Trop2 antibody protein Commercial (ACRO) and self-produced Trop2 antigen was coated in a 96-well plate, 100 ng / 100 μl / well. The coated plate was placed at four degrees overnight. Control wells without antigen and control wells without primary antibody were set up. CD70 antigen was set up as a negative control. The next day, the plate was washed three times with 1x PBST (0.05% tween 20), dried, and blocked with 1% BSA in PBS, 200 μl / well, at 37°C for 1 h. After blocking, the plate was washed three times with a plate washer, then 100 ng / 100 μl / well of our own antibody was added, incubated at 37°C for 1 h. Then HRP-FC (1:500) was diluted in blocking solution, 100 μL / well, incubated at 37°C for 1 h, and the plate was washed three times with a plate washer. After washing, 100 μl / well of TMB color developing solution was added, incubated at 37°C for 15 min, and finally 100 μl / well of Stop Solution was added. The value was read on an OD450 enzyme marker. It was found that all Trop2 antibodies had good binding to commercial and self-expressed antigens and did not bind to the control CD70 antigen. The results are shown in Figure 3 .

[0136] Example 4: Preparation of retrovirus stock containing anti-human Trop2 chimeric antigen receptor elements

[0137] 1. Preparation of chimeric antigen receptor targeting human Trop2 antigen The chimeric antigen receptor sequence containing a signal peptide, a single-chain antibody scFv against human Trop2 antigen, a hinge region, a transmembrane region, and an intracellular signal segment was synthesized or cloned, and its structure is as follows Figure 4The amino acid and nucleotide sequences of the signal peptide are shown in SEQ ID NO: 33 and 38, respectively; the amino acid and nucleotide sequences of the CD8 hinge region are shown in SEQ ID NO: 34 and 39, respectively; the amino acid and nucleotide sequences of the CD8 transmembrane region are shown in SEQ ID NO: 35 and 40, respectively; the amino acid and nucleotide sequences of the 41BB intracellular co-stimulatory domain are shown in SEQ ID NO: 36 and 41, respectively; the amino acid and nucleotide sequences of the intracellular CD3z signaling domain are shown in SEQ ID NO: 37 and 42, respectively; the amino acid sequences of the scFv of L2A8, L3G1, K2G2 and L3F4 are shown in SEQ ID NO: 43-46, respectively, and the nucleotide sequences are shown in SEQ ID NO: 47-50, respectively.

[0138] The retroviral plasmids expressing the chimeric antigen receptors of the L2A8, L3G1, K2G2 and L3F4 clones were constructed using the retroviral vector MSGV as the backbone vector. The correct clones were selected, inoculated into 200 ml 2YT medium, shaken overnight, and the plasmid was completed according to the NucleoBond Xtra Maxi EF kit instructions.

[0139] 2. Retrovirus packaging 1) The retrovirus was packaged with the cationic polymer PEI, and the process is as follows: 18 μl of PEI and 3 μg of the retrovirus packaging master plasmid were diluted with 300 μl of serum-free Opti-MEM (Gibco), respectively; then the PEI / Opti-MEM™ was added to the plasmid / Opti-MEM™ mixture, vortexed to mix, and allowed to stand at room temperature for 10 minutes; the plasmid-PEI complex was added to the pre-plated 293V cells (4E6). The medium was changed 16 h after transfection, and the first viral supernatant was collected 48 h later and stored at 4°C. The second viral supernatant was collected 72 h later, combined with the first collected, filtered with a 0.45 μm filter, aliquoted into 1.5 ml centrifuge tubes, 1 ml / tube, and stored at -80°C for standby.

[0140] 2) Virus titer detection 293T cells were digested and treated, 1E5 cells per well were plated in TC-treated 24-well plates, and the above packaged virus liquid was added to the plated cells at 5 μl, 15 μl, 45 μl, and 135 μl per well, and polybrene was added at 1:2000, and the volume of each well was supplemented with the corresponding medium to 1 ml. Seal with sealing film, centrifuge at 32°C, 2500 rpm for 90 min. Flow cytometry was used to detect the CAR positive rate 72h after infection, and the virus titer was calculated. The results are shown in Table 1.

[0141] Table 1 Virus name Positive rate (%) Virus volume (μl) Cell plating number (number) Titer (TU / mL) L2A8 20.4 5 1.00E+05 4.08E+06 L3G1 14.8 5 1.00E+05 2.96E+06 K2G2 12.8 45 1.00E+05 2.84E+05 L3F4 16.8 5 1.00E+05 3.36E+06

[0142] Example 5: Trop2 Tonic signal detection Jurkat-Nur77-GFP reporter cell lines carrying the Trop2 CAR gene were infected with the virus, and the infected cells were transferred to a 37°C, 5% CO2 cell incubator for 2-3 days. The infected Jurkat-Nur77-GFP reporter cells, negative control cells 293T, and target cells MDA-MB-231 were counted, and the corresponding volume was taken according to the required number of cells in the experimental design, centrifuged at 500g for 5 min. The density of all cells was adjusted to 8x10 6 cells / ml, and 4x10 5 cells / 50 μl was added, and the experimental groups were as follows: A. Background group: 50 μL Trop2 CAR-Jurkat-Nur77-GFP cell suspension, add 50 μl R10 complete medium to 100 μl; B. Negative control group: 50 μl Trop2 CAR-Jurkat-Nur77-GFP cell suspension, add 50 μl 293T negative control (NC) cells; C. Positive control group: 50 μl Trop2 CAR-Jurkat-Nur77-GFP cells, add 25 μl 4 μM PMA and 25 μl 4 μM Ionomycin; D. Sample group: 50 μl Trop2 CAR-Jurkat-Nur77-GFP cells, add 50 μl MDA-MB-231 positive target cells.

[0143] Incubate the above groups of samples with good sample at 37°C in a 5% CO2 incubator for 4 hours. After incubation, transfer the 96-well plate cells to a 1.5 ml EP tube, add 1 ml PBS to wash the cells, centrifuge at 500g for 5 minutes, and discard the supernatant. Stain with primary antibody 0.5 μg bio-Trop2 / test (self-made) for 30 minutes at 4°C in the dark; wash the cells after staining; prepare the secondary antibody BV421 Streptavidin (1: 500, Biolegend) and Zombie Red™ Fixable Viability (1: 500, Biolegend) in PBS, 100 μl per sample, and stain for 30 minutes at 4°C in the dark.

[0144] After staining, wash the cells, resuspend the cells in 100 μl PBS, and detect on the machine. The results are shown in Figure 5 The positive control group has a certain proportion of GFP + expressing cells, indicating that the experimental system can successfully detect Tonic signals; in addition to K2G2-CAR Jurkat, there are also GFP + cells in the background control group and the negative control group of other CAR Jurkats, indicating that the background Tonic signal exists in other CAR structures; and in other CAR Jurkat sample groups, the proportion of GFP + cells increased to a certain extent, indicating that other CARs can be activated specifically by Trop2 + target cells. However, there are no GFP + positive cell populations in the background control group, negative control group, and sample group of K2G2-CAR Jurkat, indicating that although K2G2-CAR has no background Tonic signal, it cannot be activated by Trop2 + target cells, and it is speculated that the tumor killing function of this structure is poor (CAR Jurkat full name: CAR Jurkat-Nur77-GFP).

[0145] Example 6: Preparation of Trop2 CAR-T cells and determination of CAR positive rate

[0146] 1. PBMC resuscitation relief, sorting activation Take one PBMC, check the patient's individual identification code, and then resuscitate. Adjust the cell density to 1~5×10 6cells / mL. After 2 hours of relief, the PBMCs were gently blown, filtered with a 70-μm cell screen, transferred to a 50-ml centrifuge tube, centrifuged at room temperature at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with an appropriate amount of CAR-T medium, 200 μl was taken for counting with NC200, and the viability, CD3 + cell count was calculated. According to the counting results, an appropriate amount of CD3 + cell suspension was taken to a 50-ml centrifuge tube, centrifuged at 20°C, 470 g for 5 min. After centrifugation, the supernatant was discarded, and the cells were resuspended with 3.6 ml of DPBS, and the density was adjusted to 1×10 7 live cells, and then transferred to a 5-ml sterile flow tube. According to the ratio of CD3 / CD28 magnetic beads to CD3+ cells 1:1, the amount of magnetic beads was calculated, and the magnetic bead amount =

CD3 + cell count / 4×10 5

[0147] 2. Virus infection and CAR-T cell preparation The virus stock solution with MOI = 1 was added to the 24-well plate coated with Retronectin in advance, and centrifuged at 32°C, 2000 g for 2 h. After centrifugation, the supernatant was discarded, and 1 ml of activated T cells (5×10 5 / ml, IL-2 300 IU / ml) was added to each well, and centrifuged at 32°C, 1000 g for 10 min. After centrifugation, the 24-well plate was transferred to a carbon dioxide incubator for culture at 37°C, 5% CO2. The CAR-T cells cultured for 24 h were removed from the incubator, the magnetic beads were removed by the magnetic stand, and the cell density was adjusted to 3×10 5 / ml for expansion culture.

[0148] 3. CAR positive rate detection The CAR positive rate was detected after seven days of CAR-T cell preparation. 1×106 Cells were centrifuged to remove the medium, washed once with 500 μl PBS, and resuspended with 100 μl PBS. Biotin-labeled Trop2 antigen (0.3 μg / test) was added and incubated at 4°C for 30 min. After incubation, the cells were washed once with 1x PBS and then added with secondary antibody BV421-SA (1:500, BioLegend), APC-cy7-CD3 (1:100, BioLegend), Zombie Red™ Fixable Viability (1:500, Biolegend), and incubated at 4°C for 30 min in the dark. After incubation, the cells were washed with 500 μl PBS, resuspended with 100 μl PBS, and detected by flow cytometry. The results of CAR-T positive rate flow cytometry analysis are shown in Figure 6 Figure 6, and the expression of each CAR protein in T cells was observed, except that the K2G2 positive rate was slightly lower, and the positive rates of other CARs were about 60%.

[0149] Example 7: In vitro killing experiment

[0150] For positive target cells MDA-MB-231, MDA-MB-468, HCC1395 overexpressing Trop2 and negative target cells MDA-MB-231-Trop2KO, MDA-MB-468-Trop2KO and HCC1395 (the expression of Trop2 in each target cell is shown in Figure 7 ), CAR-T cells were co-cultured with Trop2 positive target cells MDA-MB-231-Luc-GFP, MDA-MB-468-Luc-GFP, HCC1395-Trop2-Luc-GFP (target cells 1x10 4 / 100 μl / well) and Trop2 negative control target cells MDA-MB-231-Trop2 KO-Luc-GFP, MDA-MB-468-Trop2 KO-Luc-GFP, HCC1395-Luc-GFP (target cells 1x10 4 / 100 μl / well) at an effector target ratio of 3:1 in a 96-well white plate (Jiamian Biotech), and negative controls co-incubated with NT cells and groups without effector cells were set up. After co-incubation at 37°C for 19 h, the co-incubated cells were centrifuged at 500g for 5 min, 90 μl of supernatant was removed, and then luciferase Bright-Glo TM luciferaseAssay stystem (Promega) reagent 50 μl / well was added. After co-incubation for 10 min, the killing efficiency was detected: killing efficiency = (1-experimental well fluorescence value / control well fluorescence value) x 100%. The results are shown inFigure 8 , Figure 9 , Figure 10 and Figure 11 The expression of Trop2 on each target cell was combined to obtain the short-term killing results: K2G2 had little killing on high expression MDA-MB-468 and low expression MDA-MB-231, which may be related to the low expression and inactivation of CAR, and the structure of CAR needs to be optimized according to the characteristics of K2G2 antibody; the killing function of other clones on Trop2 knock-out negative target cells was inhibited to a certain extent; L3F4 was better than other clones in killing Trop2 overexpressed cells HCC1395.

[0151] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A Trop2 binding molecule, characterized in that, comprises an anti-Trop2 antibody or antigen binding fragment thereof, the Trop2 binding molecule comprising a heavy chain variable region comprising heavy chain complementarity determining regions HCDRs set forth in any one of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, and a light chain variable region comprising light chain complementarity determining regions LCDRs set forth in any one of SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, Preferably, the CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region are selected from any one of the following: (1) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, LCDR3 of SEQ ID NO: 6; (2) HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, and LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, LCDR3 of SEQ ID NO: 12; (3) HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, and LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, LCDR3 of SEQ ID NO: 18; (4) HCDR1 of SEQ ID NO: 19, HCDR2 of SEQ ID NO: 20, HCDR3 of SEQ ID NO: 21, and LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 23, LCDR3 of SEQ ID NO: 24, Preferably, the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID NOs: 25, 27, 29, and 31, and the amino acid sequence of the light chain variable region is set forth in any one of SEQ ID NOs: 26, 28, 30, and 32, Preferably, the sequences of the heavy chain variable region and the light chain variable region are selected from any one of the following: (1) when the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 25, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 26; (2) when the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 27, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 28; (3) when the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 29, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 30; (4) when the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 31, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 32, Preferably, the antibody is at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, and the antigen-binding fragment is at least one of a Fab, a F(ab'), a F(ab')2, a single-chain antibody scFv, or a disulfide-linked Fv (sdFv).

2. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an optional signal peptide, the Trop2 binding molecule of claim 1, a hinge region, a transmembrane region, and an intracellular region, Preferably, the intracellular region comprises a signaling region and / or a costimulatory signaling region, Preferably, from N-terminal to C-terminal, the chimeric antigen receptor comprises a signal peptide, the Trop2 binding molecule of claim 1, a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain.

3. A nucleic acid molecule, characterized in that, which has a sequence selected from any one of the following: (1) a coding sequence of the Trop2 binding molecule of claim 1 or the chimeric antigen receptor of claim 2; (2) a complement of (1).

4. A nucleic acid construct, characterized in that, which comprises the nucleic acid molecule of claim 3, Preferably, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

5. A host cell, characterized in that, selected from: (1) expressing and / or secreting the Trop2 binding molecule of claim 1 or the chimeric antigen receptor of claim 2; (2) comprising the nucleic acid molecule of claim 3; and / or (3) comprising the nucleic acid construct of claim 4, Preferably, the host cell is an immune effector cell.

6. The host cell of claim 5, wherein, The immune effector cell is a T cell.

7. A method of producing the Trop2 binding molecule of claim 1 or the chimeric antigen receptor of claim 2, comprising: culturing the host cell of claim 5 under conditions suitable for producing the Trop2 binding molecule, and optionally purifying the Trop2 binding molecule or chimeric antigen receptor from the culture.

8. A pharmaceutical composition comprising the Trop2 binding molecule of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4, or the host cell of claim 5, and a pharmaceutically acceptable excipient.

9. Use of the Trop2 binding molecule of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4, or the host cell of claim 5 in the manufacture of an activated T cell, or in the manufacture of a medicament for preventing or treating a disease or condition associated with Trop2 expression, Preferably, the disease or condition is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, and ovarian cancer.

10. A kit for detecting Trop2, said kit comprising a Trop2 binding molecule of claim 1, a nucleic acid molecule of claim 3, a nucleic acid construct of claim 4, or a host cell of claim 5, Preferably, the kit further comprises a reagent for detecting the binding of Trop2 to the Trop2 binding molecule; more preferably, the reagent for detecting binding is a detectable label that is capable of being linked to the Trop2 binding molecule.

Images