Scfv targeting b7-h3 and uses thereof

Abstract

This invention relates to the field of biomedicine, specifically to CAR-T therapy targeting solid tumors. It primarily involves antibody panning to identify ScFv cells specifically targeting B7-H3, then designing different CAR-T structures by combining them with chimeric antigen receptors. The indications for solid tumors (including but not limited to lung cancer and ovarian cancer) are determined through in vitro functional validation and in vivo pharmacodynamic evaluation. Simultaneously, its activity and applications in different detection methods (including but not limited to pathological immunohistochemistry and flow cytometry) are verified.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to scFv targeting B7-H3 and its applications. Background Technology

[0002] B7-H3 (also known as CD276) is a co-activating or co-inhibiting immunoregulatory protein of the B7 family of immune checkpoints, playing a crucial role in tumor invasion and metastasis. Multiple studies have shown that B7-H3 is abnormally highly expressed in various solid tumors, including lung cancer, ovarian tumors, and prostate cancer, and patients with negative B7-H3 protein expression have significantly higher survival rates than those with positive expression. While B7-H3 RNA expression levels are generally high in normal, healthy tissues, its protein expression is limited, primarily distributed in the intestine, liver, kidneys, and lymph nodes. Furthermore, B7-H3 is closely related to tumor development and metastasis, being expressed in tumor-associated vessels (TAVs) and the stroma. Therefore, B7-H3 can not only avoid off-target toxicity during immunotherapy, preventing damage to normal tissues and organs and ensuring clinical safety, but also holds promise for disrupting the tumor microenvironment and inhibiting angiogenesis, making it an ideal target for immunotherapy.

[0003] Single-chain antibodies (scFvs) are antibodies composed of variable regions of the heavy and light chains linked by a short peptide (linker) of 15–20 amino acids. scFvs can better retain their affinity activity for antigens and have the characteristics of small molecular weight, strong penetrability, and weak antigenicity, playing an important role and having broad application prospects in the clinical diagnosis, treatment, and prevention of diseases.

[0004] Therefore, providing scFvs that target B7-H3 with high specificity and high affinity is of great practical significance. Summary of the Invention

[0005] In view of this, the present invention provides scFvs targeting B7-H3 and their applications. The present invention mainly utilizes phage display technology to screen scFvs with high specificity and high affinity targeting B7-H3, then designs different CAR-T structures by combining them with chimeric antigen receptors, and verifies their killing ability against lung cancer and ovarian cancer through in vitro functional validation and in vivo pharmacodynamic evaluation. The activity and application of the scFvs screened by the present invention in different detection fields have also been verified.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides an scFv for identifying B7H3, comprising any one of the following:

[0008] (I) The CDR1, CDR2, and CDR3 of the heavy chain variable region of the scFv have the amino acid sequences shown in SEQ ID No. 6, 7, and 8, respectively; and

[0009] The CDR1, CDR2, and CDR3 of the light chain variable region of the scFv have, in sequence, the amino acid sequence shown in SEQ ID No. 4, the AAS sequence, and the amino acid sequence shown in SEQ ID No. 5; or

[0010] (II) The CDR1, CDR2, and CDR3 of the heavy chain variable region of the scFv have amino acid sequences as shown in SEQ ID No. 13, 14, and 15, respectively; and

[0011] The CDR1, CDR2, and CDR3 of the light chain variable region of the scFv have, in sequence, the amino acid sequence shown in SEQ ID No. 11, GAS, and the amino acid sequence shown in SEQ ID No. 12; or

[0012] (III) The CDR1, CDR2, and CDR3 of the heavy chain variable region of the scFv have amino acid sequences as shown in SEQ ID No. 6, 7, and 19, respectively; and

[0013] The CDR1, CDR2, and CDR3 of the light chain variable region of the scFv have, in sequence, the amino acid sequence shown in SEQ ID No. 18, the AAS sequence, and the amino acid sequence shown in SEQ ID No. 5; or

[0014] (IV) A sequence based on any of the amino acid sequences shown in (I) to (III) that has undergone substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0015] (V) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (I) to (IV).

[0016] In some specific embodiments of the present invention, the scFv includes any of the following:

[0017] (i) The heavy chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 2; and

[0018] The light chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 1; or

[0019] (ii) The heavy chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 10; and

[0020] The light chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 9; or

[0021] (iii) The heavy chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 17; and

[0022] The light chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 16; or

[0023] (iv) A sequence based on any of the amino acid sequences shown in (i) to (iii) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0024] (v) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (i) to (iv).

[0025] In some specific embodiments of the present invention, the light chain variable region and the heavy chain variable region are connected by a linker peptide.

[0026] The linker peptide has the following characteristics:

[0027] (vi) An amino acid sequence as shown in SEQ ID No. 20; or

[0028] (vii) A sequence based on the amino acid sequence shown in (vi) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0029] (viii) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in (vi).

[0030] In some specific embodiments of the present invention, the scFv includes any of the following:

[0031] (1) The scFv has an amino acid sequence as shown in SEQ ID No. 3; or

[0032] (2) The scFv has an amino acid sequence as shown in SEQ ID No. 41; or

[0033] (3) The scFv has an amino acid sequence as shown in SEQ ID No. 42; or

[0034] (4) A sequence based on any of the amino acid sequences shown in (1) to (3) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0035] (5) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (1) to (4).

[0036] Secondly, the present invention also provides a nucleic acid molecule encoding the scFv.

[0037] In some specific embodiments of the present invention, the nucleic acid molecule is chemically modified. For example, in some embodiments, one or more thymidines of the nucleic acid molecule are replaced with uridines. In some embodiments, one or more uridines of the nucleic acid molecule are replaced with thymidines. In some embodiments, one or more guanosines of the nucleic acid molecule are replaced with creatinine. In some embodiments, the chemical modification is to replace one or more nucleotides of the nucleic acid molecule with their corresponding nucleotide derivatives. For example, in some embodiments, one or more uridines of the nucleic acid molecule are respectively replaced by one or more of the following: 5-(carboxyhydroxymethyl)uridine (chm5u), 5-carboxymethylaminomethyluridine (cmnm5u), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2u), dihydrouridine (dhu), 2'-O-methylpseudouridine (fm), 1-methylpseudouridine (m1f), 3-(3-amino-3-carboxy-propyl)uridine ((acp3)u), uridine-5-oxyacetic acid (o5u), uridine-5-oxyacetic acid methyl ester (mv), 5-methoxycarbonylmethyl ester. 5-Methoxycarbonylmethyl-2-thiouracil (mcm5s2u), 5-Methoxyuridine (mo5u), 5-Methyl-2-thiouridine (s2t), 2-Thiouridine (s2u), 4-Thiouridine (s4u), 5-Methyluridine (m5u), 2'-O-methyl-5-methyluridine (tm), 2'-O-methyluridine (um), 5-Methylaminomethyluridine (mam5u), 5-Methylaminomethyl-2-thiouridine (mam5s2u), pseudouridine (p), and 5-Methoxycarbonylmethyl-2-thioguanosine (mcm5s2u). In some embodiments, one or more guanosines of the nucleic acid molecule are replaced by one or more of the following: wybutoxosine (osyw), wybutoxosine (yw), 1-methylinosine (m1i), 2'-O-methylguanosine (gm), 1-methylguanosine (m1g), 2,2-dimethylguanosine (m22g), 2-methylguanosine (m2g), 7-methylguanosine (m7g), and β,D-galactosoQ nucleoside (galq), Q nucleoside (q), and β,D-mannose Q nucleoside (manq).In some embodiments, one or more adenosines of the nucleic acid molecule are replaced by one or more of the following: N6-isopentenyl adenosine (i6a), 1-methyl adenosine (m1a), 2-methyl adenosine (m2a), N6-methyl adenosine (m6a), 2-methylthio-N6-isopentenyl adenosine (ms2i6a), N-((9-β-D-furanoribosyl-2-thiomethylpurine-6-yl)carbamoyl)threonine (ms2t6a), N-((9-β-D-furanoribosyl-6-yl)N-methylcarbamoyl)threonine (mt6a), N-((9-β-D-furanoribosyl-6-yl)carbamoyl)threonine (t6a), and β,Q nucleoside (q) and D-mannoseQ nucleoside (man q). In some embodiments, one or more cytidines of the nucleic acid molecule are replaced by one or more of the following: 4-acetylcytidine (ac4c), 2'-O-methylcytidine (cm), 3-methylcytosine (m3c), N4-methylcytidine (m4c), 5-methylcytidine (m5c), β, and 2-thiocytidine (s2c). In some embodiments, the chemical modification includes 2'-O-methylation on the ribose of the nucleotide and / or 3'-thiophosphate bond modification between nucleotides. In some embodiments, the chemical modification includes one or more of the following: 2'-O-methylation on the ribose of the first three nucleotides at the 5' end, 2'-O-methylation on the ribose of the last three nucleotides at the 3' end, 3'-thiophosphate bond modification between the first three nucleotides at the 5' end, and / or 3'-thiophosphate bond modification between the last three nucleotides at the 3' end.

[0038] Thirdly, the present invention also provides a protein assembly including the scFv, hinge region, transmembrane region, and intracellular signaling domain.

[0039] In some specific embodiments of the present invention, the scFv is connected to the transmembrane region via the hinge region;

[0040] The hinge region is derived from IgG, and the hinge region includes, but is not limited to, one or a combination of two or more of the hinge regions of IgG1, IgG4, IgD, CD4, CD7, CD8α or CD28 molecules.

[0041] The transmembrane region includes, but is not limited to, one or a combination of two or more of the transmembrane regions of ICOS, TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, PD-1, or CD226 molecules.

[0042] The intracellular signaling functional domain includes a co-stimulatory domain and a primary signal transduction domain.

[0043] The co-stimulatory domains include, but are not limited to, CD27, CD28, 4-1BB, OX40, 2B4, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 1a, LFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-l, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or a combination of one or more of the co-stimulatory domains of molecules that specifically bind to CD83;

[0044] The primary signal transduction domains include, but are not limited to, one or a combination of two or more of the primary signal transduction domains of the CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 or DAP12 molecules.

[0045] In some specific embodiments of the present invention, the protein combination includes:

[0046] (A) The transmembrane region has an amino acid sequence as shown in SEQ ID No. 22, 29; or

[0047] (B) The hinge region has an amino acid sequence as shown in SEQ ID No. 21, 36, 39, 40; or

[0048] (C) The co-stimulatory domain has the amino acid sequences shown in SEQ ID No. 23, 30, and 35;

[0049] (D) The primary signal transduction domain has an amino acid sequence as shown in SEQ ID No. 24;

[0050] or

[0051] (E) A sequence based on any of the amino acid sequences shown in (A) to (D) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0052] (F) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with any of the amino acid sequences shown in (A) to (D).

[0053] In some specific embodiments of the present invention, the protein combination includes CAR-1, CAR-2, CAR-3, CAR-4, CAR-5, CAR-6, CAR-7, CAR-8, CAR-9, CAR-10, CAR-11 and / or CAR-12;

[0054] (A) The CAR-1, CAR-2, CAR-3, CAR-4, CAR-5, CAR-6, CAR-7, CAR-8, CAR-9, CAR-10, CAR-11, and CAR-12 respectively have the amino acid sequences shown in SEQ ID No. 27, 31, 43, 44, 45, 46, 25, 34, 38, 37, 32, and 33; or

[0055] (B) A sequence based on the amino acid sequence shown in (A) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0056] (C) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in (A) or (B).

[0057] Fourthly, the present invention also provides gene elements, including the nucleic acid molecule.

[0058] Fifthly, the present invention also provides an artificial vector comprising the genetic element and an acceptable auxiliary agent or vector.

[0059] In a sixth aspect, the present invention also provides viral particles, including the artificial carrier.

[0060] In a seventh aspect, the present invention also provides engineered cells, including any of the following:

[0061] (a) the aforementioned scFv; and / or

[0062] (b) the aforementioned nucleic acid molecules; and / or

[0063] (c) the protein combination described above; and / or

[0064] (d) the aforementioned gene element; and / or

[0065] (e) the aforementioned artificial carrier; and / or

[0066] (f) Transducing the viral particles.

[0067] In some specific embodiments of the present invention, the engineered cells include, but are not limited to, NK cells, T cells, macrophages, DC cells, and related precursor cells.

[0068] Eighthly, the present invention also provides the use of any of the following in detecting B7H3 expression and / or preparing products for detecting B7H3 expression:

[0069] I) the aforementioned scFv; and / or

[0070] II) The aforementioned nucleic acid molecules; and / or

[0071] III) the protein combination described above; and / or

[0072] IV) the aforementioned gene elements; and / or

[0073] V) The artificial carrier mentioned above.

[0074] In some specific embodiments of the present invention, the product includes a kit, a detection antibody, and / or a B7H3 molecular tracer reagent.

[0075] Ninthly, the present invention also provides a product, characterized in that it comprises:

[0076] I) the aforementioned scFv; and / or

[0077] II) The aforementioned nucleic acid molecules; and / or

[0078] III) the protein combination described above; and / or

[0079] IV) the aforementioned gene elements; and / or

[0080] V) The artificial carrier; and / or

[0081] VI), the aforementioned virus particles; and / or

[0082] VII), the engineered cells described above.

[0083] In a tenth aspect, the present invention also provides the use of any of the following in the preparation of medicaments for the prevention and / or treatment of diseases:

[0084] 1) the aforementioned scFv; and / or

[0085] 2) The aforementioned nucleic acid molecules; and / or

[0086] 3) The protein combination mentioned above; and / or

[0087] 4) the aforementioned gene element; and / or

[0088] 5) the aforementioned artificial carrier; and / or

[0089] 6) the aforementioned virus particles; and / or

[0090] 7) The engineered cells mentioned above.

[0091] In some specific embodiments of the present invention, the disease includes diseases with positive expression of B7H3.

[0092] In some specific embodiments of the present invention, the disease includes a tumor; the tumor includes one or more of lung cancer, ovarian cancer, colorectal cancer, prostate cancer, or pancreatic cancer.

[0093] In an eleventh aspect, the present invention also provides a medicament or combination of medicaments, comprising any of the following and pharmaceutically acceptable excipients or adjuvants:

[0094] 1) the aforementioned scFv; and / or

[0095] 2) The aforementioned nucleic acid molecules; and / or

[0096] 3) The protein combination mentioned above; and / or

[0097] 4) the aforementioned gene element; and / or

[0098] 5) the aforementioned artificial carrier; and / or

[0099] 6) the aforementioned virus particles; and / or

[0100] 7) The engineered cells mentioned above.

[0101] In a twelfth aspect, the present invention also provides a method for preventing and / or treating tumors, comprising applying any of the following:

[0102] i) the engineered cells described above; and / or

[0103] ii) the drug or combination of drugs mentioned above.

[0104] In some specific embodiments of the present invention, the tumor includes a B7H3-positive tumor; preferably, the tumor includes one or more of lung cancer, ovarian cancer, colorectal cancer, prostate cancer, or pancreatic cancer.

[0105] This invention primarily utilizes phage display technology to screen scFvs with high specificity and high affinity targeting B7-H3. These scFvs can be used for B7H3 recognition, in detection kits, and for detecting B7H3 expression using immunohistochemistry, flow cytometry, and other techniques. The scFvs can also serve as drugs or drug combinations for treating B7H3. Furthermore, Fc structures or bispecific antibodies can be designed based on these scFvs for the treatment of B7H3-positive tumors. Additionally, the scFvs can be used as an extracellular recognition domain, combined with extracellular segments, intracellular T cell co-stimulatory signals, etc., to form chimeric antigen receptors (CARs) or as extracellular recognition portions of similar fusion protein structures. Different designed CAR structures can be expressed on T cells, including αβT cells or γδT cells, to form CAR-T cells; they can also be expressed on genetically modified T cells, such as universal T cells, to form universal or allogeneic CAR-T cells (UCAR-T); they can be expressed on NK cells to form CAR-NK cells; or they can be expressed on macrophages or other immune cells such as Tregs. Attached Figure Description

[0106] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0107] Figure 1A This demonstrates the ability of different ScFv samples to recognize B7H3 using chemiluminescence detection. Figure 1B This demonstrates the recognition capabilities of different ScFv methods for B7H3. Figure 1C This demonstrates the recognition capabilities of different ScFv methods for B7H3.

[0108] Figure 2 SDS-PAGE purification and identification are shown;

[0109] Figures 3A-3DThe expression of B7H3 in DLD-1, LoVo, PANC-1, and LNCap cells was detected.

[0110] Figure 4 The flow cytometry staining results of PK-01-C09, PK-02-A08, and PK-02-A09, which were finally selected with good atopy, are shown.

[0111] Figure 5 The ligand-analyte interaction kinetics fitting curve;

[0112] Figure 6 This demonstrates the specificity detection of the ScFv membrane protein array during screening.

[0113] Figure 7A This study demonstrates the detection of PK-01-C09 in blood samples containing B7H3-positive tumor cells. Specifically, blood-NCG-secondary antibody is used to label NCG mouse blood samples with a secondary antibody, and blood-NCG-PK-01-C09 is used to label NCG mouse blood samples with PK-01-C09. The commercial antibody, secondary antibody, and PK-01-C09 are all used to label B7-H3+ SKOV3 breast cancer cells. Figure 7B The results of detecting B7H3 expression in B7H3-positive lung cancer cells A549(B7H3+) using commercial antibodies, secondary antibodies, and PK-01-C09 are shown.

[0114] Figure 8A This demonstrates the testing of a fixed SK-OV-3 (B7-H3+) smear using PK-01-C09. Figure 8B For the detection of PK-01-C09 on smears of fixed B7-H3 negative cells;

[0115] Figure 9 Flow cytometry was used to detect the positive expression of CAR-T cells.

[0116] Figure 10A The results of CAR-T cells with different CAR structures constructed from PK-01-C09, PK-02-A08, and PK-02-A09 killing lung cancer cells A549(B7H3+) are shown. Figure 10B The results of CAR-T cells with different CAR structures constructed using PK-01-C09 killing lung cancer cells A549(B7H3+) were shown. Figure 10C The study showed the killing effect of CAR-T cells with different CAR structures constructed from PK-01-C09 on human ovarian cancer cells SK-OV-3(B7H3+).

[0117] Figure 11The efficacy of CAR-T cells expressing different CAR structures constructed with PK-01-C09 against lung cancer cells was demonstrated. Compared with the CT group, ****P<0.0001;

[0118] Figure 12 The efficacy of CAR-T cells expressing different CAR structures constructed with PK-01-C09 against ovarian cancer cells was demonstrated. Compared with the CT group, **P=0.0039, P<0.01;

[0119] Figure 13A This demonstrates its in vivo efficacy against lung cancer cells (A549(B7-H3+-Luc-GFP)). Figure 13B The curve showing the tumor volume change of lung cancer cells (A549(B7-H3+-Luc-GFP)) is shown.

[0120] Figure 14 The curve shows the tumor volume change of ovarian cancer cells (SK-OV-3(B7-H3+-Luc-GFP)). Detailed Implementation

[0121] This invention discloses a scFv targeting B7-H3 and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0122] Terminology

[0123] CAR-T: Defined as chimeric antigen receptor T cells, these are genetically engineered cells that can endow T lymphocytes with specificity for a specific antigenic epitope, thereby enhancing the T lymphocyte's ability to recognize antigen signals and activate.

[0124] Phage display technology is defined as a biotechnology that inserts the DNA sequence of a foreign protein or polypeptide into an appropriate position in the structural gene of the phage coat protein, so that the foreign gene is expressed along with the expression of the coat protein, and the foreign protein is displayed on the surface of the phage as the phage is reassembled.

[0125] ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a highly sensitive assay based on immunological reactions, combining the specific reaction of antigens and antibodies with the highly efficient catalytic action of enzymes on substrates. Because the antigen-antibody reaction takes place in the wells of a solid-phase carrier—a polystyrene microtiter plate—excess free reactants can be removed by washing after each reagent is added and incubated, thus ensuring the specificity and stability of the test results. In practical applications, various methods and procedures can be implemented through different designs, including indirect methods for antibody detection, double-antibody sandwich methods for antigen detection, and antigen competition methods for detecting small molecule antigens or haptens, etc. The double-antibody sandwich method and the indirect method are the most commonly used.

[0126] Flow cytometry: This fluorescence-based detection technique allows for the simultaneous determination of multiple properties, such as cell population count and protein abundance, from single cells suspended in solution. It is a powerful tool for the rapid, quantitative, and accurate determination of cell characteristics and provides excellent interpretation of cell population heterogeneity. In cell biology, a common application of flow cytometry is the detection and quantification of cell types present in a sample. Cell detection is possible because different cell types express different proteins on their surfaces. Antibodies specifically target these proteins, effectively adding a colorimetric marker to distinguish one cell type from another.

[0127] Membrane protein array technology: This technology utilizes principles of cell biology, molecular biology, and biophysics to study the structure, function, and interactions of proteins on the cell membrane. Its principle involves using protein chip technology to express membrane proteins directly within the cell in their natural state, maintaining their structural integrity and natural post-translational modifications. This results in a high-throughput cell array used to identify the target specificity of antibodies, drugs, or other ligands that bind to membrane proteins. This provides a comprehensive cross-reactivity assessment for new regulatory requirements in IND applications for CAR-T and antibody drugs. Furthermore, this technology can be extended to multiple fields such as new target discovery, viral mechanism analysis, and receptor de-arresting.

[0128] Immunohistochemical staining involves binding fluorescent or chromogenic chemicals to antibodies. Utilizing the specific binding between antigens and antibodies in immunology, it detects the presence of target antigens in cells or tissues. This method can be used not only to determine the degree of antigen expression but also to observe the specific location of antigen expression. Immunohistochemical staining is a crucial method for basic research, prevention, and diagnosis. Due to its specificity, sensitivity, simplicity, and speed, it is widely used in hospital pathology departments, typically for screening cancers using specific tumor markers.

[0129] Chimeric antigen receptors (CARs): A CAR is a group of engineered peptides or proteins that, when present in immune effector cells, bind to specific antigens on target cells and generate intracellular signals upon recognition of the antigen, activating downstream pathways in the cell containing the receptor to initiate the killing action of the immune effector cells on the target cells. CARs typically include at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain specifically recognizes antigens. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments or natural ligands that bind to their homologous receptors, artificially designed target-specific recognition domains such as fibronectin type III (FN3) domain combinations, and designed ankyrin repeat proteins (DARPins) that recognize specific targets. The "signal transduction domain" typically contains an immune-receptor tyrosine-based activation motif (ITAM), whose basic composition is YXXL / V. Here, Y represents tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to its corresponding ligand, the tyrosine residue in the ITMA linked to it can be phosphorylated by a protein tyrosine kinase (PTK) connected to the cell membrane, thereby recruiting other free intracellular protein kinases or adaptor proteins to transduce activation signals into the cell. In some embodiments, the "signal transduction domain" is selected as the intracellular signal transduction domain of TCRζ (CD3ζ) or FcεRIγ. As used herein, the "co-stimulatory domain," also known as the "co-stimulatory signaling domain," is primarily used to provide co-stimulatory signals to enhance the capabilities of immune cells, including, for example, enhancing the proliferation, survival, and / or development of memory cells. In some embodiments, the "co-stimulatory domain" is selected from intracellular domains of CD28, 4-1BB (CD137), OX40 (CD134), etc. As used herein, the "transmembrane domain," also known as a "transmembrane region," refers to a thermodynamically stable protein structural region anchored within the cell membrane. Transmembrane domains can be obtained from natural proteins, such as transmembrane domains derived from the T cell receptor (TCR). In some embodiments, the transmembrane domain is selected from transmembrane domains of CD4, CD8α, CD28, and CD3ζ.

[0130] "Chimeric antigen receptors" or "CARs" can have various structures, such as those containing secretible or membrane-expressed cytokines or antibody gene sequences; and those containing structures that can be regulated for activation or inactivation, including: suicide switches such as inducible caspase-9 (iCasp9), thymidine kinase (HSV-TK) and suicide epitopes in herpes simplex virus, truncated EGFR (EGFRt), and Fas-FasL apoptosis structures; and induced CAR structures such as: peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv linked to antibody, streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structures, and biotin-biding immune receptors. The receptor (BBIR) system; the "logic gate" regulatory system that binds to the SynNotch receptor, etc.

[0131] This invention primarily utilizes phage display technology to screen for scFvs with high specificity and high affinity targeting B7-H3. These scFvs can be used for B7H3 recognition, in detection kits, and for detecting B7H3 expression using immunohistochemistry, flow cytometry, and other techniques. The scFvs can also serve as drugs or drug combinations for treating B7H3. Furthermore, Fc structures or bispecific antibody designs can be constructed based on these scFvs for the treatment of B7H3-positive tumors. The content and construction methods of 202110206096.7 are entirely incorporated herein by reference. The scFvs described in this application can replace the CD96ScFv or PD1ScFv in the CD96ScFv holes-PD1ScFv knobes and CD96ScFv-(G4S)4-PD1ScFv-Fc structures disclosed in 202110206096.7, or simply use the structural framework to form a combination of B7H3 scFvs with other scFvs or ligands. ScFv can also be used as an extracellular recognition domain to bind extracellular segments, intracellular T cell co-stimulatory signals, etc., to form chimeric antigen receptors (CARs) or to serve as the extracellular recognition portion of similar fusion protein structures. CARs with different designed structures can be expressed on T cells, including αβT cells or γδT cells, to form CAR-T cells. They can also be expressed on genetically modified T cells, such as universal T cells, to form universal or allogeneic CAR-T cells (UCAR-T). The structural design of UCARs is described in patents 201710983276.X, 202111652734.4, 202111652757.5, 202111657691.9, and 202111657717.X regarding the design of universal or allogeneic CAR structures. They can also be expressed on NK cells to form CAR-NK cells, or on macrophages or other immune cells such as Treg cells.

[0132] The three scFvs (PK-01-C09, PK-02-A08, and PK-02-A09) selected by this invention and their corresponding CAR structure sequences are shown in the table below:

[0133] Table 1

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] The ScFv screened in this invention can effectively kill lung and ovarian cancer when used in the construction of CARs. In addition to lung and ovarian cancer, it is also effective against B7H3-positive tumors such as colorectal cancer, prostate cancer, and pancreatic cancer. The ScFv protected by this invention can also be used for the treatment or detection of these indications.

[0145] The raw materials and reagents used in the B7-H3-targeting scFv and its application provided by this invention are all commercially available.

[0146] The present invention will be further illustrated below with reference to the embodiments:

[0147] Example 1: ScFv selection

[0148] 1) Construction of a fully human single-chain antibody library

[0149] PBMCs were separated using Ficoll separation solution. The Ficoll solution was slowly added to normal human blood to maintain a clear separation interface between the Ficoll solution and the blood. A 50 mL centrifuge tube containing the blood and separation solution was centrifuged at approximately 15°C for 20 minutes. After centrifugation, the liquid surface separated into four layers: an upper layer of plasma mixture, a lower layer of erythrocytes and granulocytes, and a middle layer of Ficoll solution. At the boundary between the upper and middle layers, there was a narrow band of white, cloudy layer dominated by PBMCs, which was the PBMC cell layer. The upper plasma mixture was carefully aspirated using a sterile Pasteur pipette, and then the PBMCs were aspirated using a new sterile Pasteur pipette to obtain the separated PBMCs.

[0150] Total RNA was extracted using conventional methods and reverse transcribed into cDNA. Based on the similarity of the heavy and light chain germline gene sequences, degenerate primers were designed at both ends of the variable region, with VL preceding VH and the middle linked by a flexible linker. PCR was used to obtain the heavy chain and light chain variable region gene fragments of the antibody. The scFv nucleic acid fragment was amplified using conventional overlap PCR (PCR method referenced from *Molecular Cloning: A Laboratory Manual* (3rd edition), Joe Sambrook and David Russell, Science Press, USA). The scFv nucleic acid fragment was ligated into the phage vector pComb3xssXSS, and the product was transformed into TGI strains using an electroporator to obtain a fully human single-chain antibody library.

[0151] 2) Preparation of a phage-displayed fully human single-chain antibody library

[0152] Add the bacterial culture to fresh SOC liquid medium with ampicillin at a concentration of 100 μg / mL, and resuscitate at 37°C and 250 rpm. Incubate until OD600 ≈ 0.5, then add VSCM13 helper phage at a VCSM13:bacteria ratio of 50:1, mix thoroughly, incubate for 30 min, and then continue incubation on a shaker. Centrifuge the culture and discard the supernatant. Resuspend the precipitate in SOB medium containing 50 μg / mL ampicillin and 50 μg / mL kanamycin, and incubate overnight at 30°C and 250 rpm. Centrifuge the bacterial culture at 4°C and 8000 rpm, collect the supernatant, add 1 / 5 volume of 20% PEG 8000 2.5 mmol / L NaCl solution, incubate on ice, then centrifuge at 4°C and 12000 rpm. Resuspend the precipitated phage in PBS, centrifuge at 4°C and 15000 rpm, and collect the supernatant.

[0153] 3) Antigen panning

[0154] B7-H3 protein (Human B7-H3(4Ig) / B7-H3b Protein, Fc Tag, ACRO) was co-incubated with proteinG magnetic beads to prepare B7-H3-proteinG-coupled magnetic beads. These beads were then extracted into the prepared phage display library. After three rounds of co-incubation, washing, and elution, specific monoclonal antibodies against the B7-H3 antigen were enriched.

[0155] 4) Antigen detection

[0156] After the initial screening, the final batch of single-clone plaques were selected for chemiluminescence immunoassay. Specifically, DLD-1 and DLD-1-B7H3 cells were screened at a concentration of 5 × 10⁻⁶ cells / mL. 5After seeding cells / well in a 96-well plate, add 100 μL of phage culture to each well and incubate at 4°C for 30 min. Wash twice with PBS, centrifuge and discard the supernatant. Add Anti-M13-HRP secondary antibody (HRP / Anti-M13 Monoclonal Conjugate, Beijing Baokewei Food Safety Biotechnology Co., Ltd.) to each well and incubate at 4°C for 30 min. Wash with PBS, then add 25 μL of ECL reagent kit (Smart-ECL Super, Changzhou Tiandi Renhe) Solution I to each well to resuspend the cells. Transfer to the detection plate and add 25 μL of Solution I to each well. II. Immediately place the sample in a microplate reader to detect fluorescence. The results showed that the phage clones with higher detection values ​​were PK-01-C09, PK-02-A04, PK-02-A05, PK-02-A08, PK-02-A09, PK-02-A10, PK-02-B01, PK-02-B02, PK-02-B05, PK-02-C02, PK-02-C03, PK-02-C06, PK-02-C12, PK-02-D02, and PK-02-D04. Figure 1A , Figure 1B , Figure 1C As shown.

[0157] 5) Expression and purification of ScFv

[0158] The plasmid of the positive clone was mixed with competent bacteria and incubated on ice for 5 min, then heat-shocked at 42℃ for 90 s. After another ice incubation, it was spread onto LB agar plates containing ampicillin and incubated overnight at 37℃. Single colony plaques were picked and incubated in LB medium at 37℃. When the OD of the bacterial culture reached 0.5-1.0, an inducer was added to induce expression. The bacterial cells were collected by centrifugation and the precipitate was resuspended in PBS. After sonication and lysis buffer treatment, the precipitate was discarded and the supernatant was collected. After filtration through 0.22 μm, ScFv was purified and collected. SDS-PAGE was used to identify the purification effect of ScFv. The results showed that the purity was good before concentration (1) and after concentration (2). Figure 2 As shown.

[0159] 6) Flow cytometry staining specificity detection

[0160] The expression of B7H3 in human colorectal cancer epithelial cells (DLD-1), human colorectal cancer cells (LoVo), human pancreatic cancer cells (PANC-1), and human prostate cancer cells (LNCap) was detected by flow cytometry using commercially available antibodies. Specifically, two tubes of each cell type (DLD-1, LoVo, PANC-1, and LNCap) were placed in EP tubes, with 1.0 x 10⁻⁶ cells per tube. 6Cells were resuspended in 200 μL PBS, and then 3 μL of commercial B7H3 antibody (Biolegend, 351006) and its isotype control antibody (Biolegend, 400122) were added for labeling at 4℃ for 30 min. After centrifugation at 350g for 5 min, the cells were washed and resuspended in PBS and then analyzed by flow cytometry. The results are as follows: Figure 3A , 3B As shown in Figures 3C and 3D. The results indicate that human colorectal cancer cell lines DLD-1, LoVo, LNCap, and PANC-1 all express B7H3. This suggests that the B7H3-recognizing ScFv of this application can be applied to human colorectal cancer, human prostate cancer, and human pancreatic cancer, or to prepare detection reagents, kits, or molecular tracers for detecting B7H3 in the aforementioned tumors or tumor cells.

[0161] Then, B7H3-positive DLD-1 cells were used for specific detection. The experiment was divided into two groups: a blank control group (Blank-DLD-1), a secondary antibody-labeled group (PBS-DLD-1), and the ScFv groups obtained by screening (i.e., the above-mentioned PK-01-C09, PK-02-A04, PK-02-A05, PK-02-A08, PK-02-A09, PK-02-A10, PK-02-B01, PK-02-B02, PK-02-B01, PK-02-B05, PK-02-C02, PK-02-C03, PK-02-C06, PK-02-C12, PK-02-D02, PK-02-D04). Each group was aliquoted into 1.5mL Eppendorf tubes, 5×10⁻⁶ per tube. 5 Cells were analyzed. DLD-1 cells were labeled with 30 μg / mL of each ScFv group selected by the applicant, and labeled as PK-01-C09-DLD-1, PK-02-A08-DLD-1, and PK-02-A09-DLD-1, respectively. After incubation at 4℃ for 1 h, the cells were washed and resuspended, then 30 μL of Anti-His-647 fluorescent secondary antibody (GenScript, A01802-100 μg) was added, and the cells were incubated at 4℃ in the dark for 30 min. The cells were then washed twice with PBS, centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL of PBS and transferred to detection tubes for analysis. The positive rate and mean fluorescence intensity (MFI) of flow cytometry were measured. Simultaneously, the clones were sequenced to exclude duplicates, and three ScFv lines with good specificity—PK-01-C09, PK-02-A08, and PK-02-A09—were finally selected.

[0162] Figures 3A-3D To identify B7H3 positive expression in DLD-1, LoVo, PANC-1, and LNCap cells using commercial antibodies.Figure 4 The flow cytometry staining results for the finally selected PK-01-C09, PK-02-A08, and PK-02-A09 with good specificity.

[0163] The obtained ScFv sequences are shown in Table 1.

[0164] Those skilled in the art should know that there are multiple ways to define a Complementary Determination Region (CDR). The LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 described in this application are defined by the IMGT system. LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are collectively referred to as Complementary Determination Regions (CDRs). Besides the IMGT system definition, different CDR definition methods can also be combined (CDR1 and CDR2 are defined by one method, and CDR3 by another). Specific different CDR definition methods are as follows:

[0165] Table 2

[0166]

[0167] in:

[0168] The amino acid numbering in the Kabat system definition follows the Kabat numbering system (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0169] The amino acid numbering in the Chothia system definition follows the Chothia numbering system (see, for example, Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0170] The amino acid numbering in the MacCallum system definition follows the MacCallum numbering system (see MacCallum et al., J.Mol.Biol.262:732-745(1996); Abhinandan and Martin, Mol.Immunol.,45:3832-3839(2008)).

[0171] The amino acid numbering in the IMGT system definition follows the IMGT numbering system (see, for example, Lefranc MP. (2013) IMGT Unique Numbering. In: Dubitzky W., Wolkenhauer O., Cho KH., Yokota H. (eds) Encyclopedia of Systems Biology. Springer, New York, NY; https: / / doi.org / 10.1007 / 978-1-4419-9863-7_127).

[0172] 5 The amino acid numbering in the AHo system definition (see, for example, Honegger and Plückthun, J.Mol.Biol., 309:657-670 (2001)) is based on the AHo numbering system.

[0173] Because CDRs have different definitions, those skilled in the art can define CDRs according to different methods used in LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of this application, thereby obtaining CDRs that are sequence-differentiated from the same combination of heavy chain variable regions and light chain variable regions as in this application. Those skilled in the art should understand that even if the sequences of CDR combinations obtained from the antibodies of this application through different definitions are different from those of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of this application, antibodies containing such CDR combinations are still undoubtedly covered within the scope of this application.

[0174] Example 2: Affinity of ScFv targeting B7H3

[0175] use The streptavidin (SA) biosensor was cured according to the instruction manual using PK-01-C09-biotin (biotin labeling using EZ-Link). TMThe Sulfo-NHS-Biotin kit (proceded according to the instructions) was used, and affinity was detected using a FortebioOctet K2 molecular interaction analyzer. The concentration gradients of analyte B7-H3 were 500, 250, 125, and 62.5 nm. The ligand (PK-01-C09-biotin) bound to the sensor for 120 s, the ligand bound to the analyte for 120 s, and dissociated for 180 s. Data processing involved leveling other analyte concentration gradients using an analyte-free reference, removing oversaturated, low, or abnormal data, and retaining at least four valid gradients. Experimental results showed that the maximum response value of PK-01-C09-biotin to B7-H3 during the binding phase was >= 0.05 nm, and the response value during the dissociation phase decreased with time, indicating significant specific binding. The equilibrium dissociation constant (KD) was 4.8 nM. The ligand-analyte interaction kinetic parameters were calculated based on the fitted model. Figure 5 As shown:

[0176] Example 3: Specific recognition and detection of B7H3 by PK-01-C09

[0177] Membrane protein arrays were used to detect the specific recognition of B7H3 by PK-01-C09. Membrane protein array technology uses HEK-293T cells as the tool cells to perform high-throughput transient transfection of 5900 plasmids, and then uses PK-01-C09 as a label to screen for membrane proteins recognized by PK-01-C09.

[0178] HEK-293T cells were used as the cell line for high-throughput transient transfection of 5900 plasmids. Thirty-two negative control wells were included, with hB7H3 as a positive control. Typical proteins from different human plasma membrane protein types (such as type I transmembrane protein CD7, type II transmembrane protein CD70, type III transmembrane protein BCMA, and multi-transmembrane protein GPRC5D) were selected as quality control controls. After transient transfection for more than 40 hours, the ZsGreen or mCherry fluorescence values ​​of all samples were detected using a microplate reader to confirm plasmid transfection and expression. The cells were then incubated with PK-01-C09 at a working concentration of 0.5 μg / ml. Chemiluminescence values ​​were detected using a Smart-ECL Super assay kit and a microplate reader to preliminarily assess the risk and screen for human plasma membrane proteins that pose a risk of binding to the test substances. Simultaneously, flow cytometry was used to transiently transfect and validate human plasma membrane proteins suspected of risk in the second screening phase and negative controls. The results showed that, under the experimental conditions, the test substance exhibited a targeted binding relationship with the B7-H3 protein, but no clear targeted binding relationship with any of the 5899 human plasma membrane proteins. (Results are as follows...) Figure 6 As shown.

[0179] Example 4: ScFv targeting B7H3 for B7H3 detection

[0180] 1) Used for B7H3 expression detection

[0181] A549 (B7-H3+) cells, SK-OV-3 (B7-H3+) cells, and NCG mouse blood samples (blood-NCG) were each aliquoted into 1.5 mL Eppendorf tubes, 1 × 10⁻⁶ per tube. 6 Cells were centrifuged at 350g for 5 min, and the supernatant was discarded. Cells were resuspended in 100 μL of PBS, and 1 μL of commercially available B7-H3 antibody (Biolegend, 351006), a secondary antibody detection antibody (Anti-His-647, GenScript, A01802-100 μg) as a negative control, and the antibody PK-01-C09 selected in this application (60 μg / ml) were added. Cells were incubated at 4°C in the dark for 30 min. After centrifugation at 350g for 5 min, the supernatant was discarded, and cells were resuspended in 100 μL of PBS. Secondary antibody (Anti-His-647, GenScript, A01802-100 μg) was added, and cells were incubated at 4°C in the dark for 30 min. After washing cells with PBS, the supernatant was discarded, and cells were resuspended again in 200 μL of PBS. The positive rate was then detected by flow cytometry.

[0182] The results are as follows Figure 7A , Figure 7B As shown, Figure 7A The blood-NCG-secondary antibody is used to label and detect NCG mouse blood samples, and the blood-NCG-PK-01-C09 is used to label NCG mouse blood samples with PK-01-C09. The commercial antibody, secondary antibody and PK-01-C09 are all used to label B7-H3+ ovarian cancer cells SKOV3 cells. Figure 7B This report presents the results of detecting B7H3 expression in B7H3-positive lung cancer cells A549 (B7H3+) using commercial antibodies, secondary antibodies, and PK-01-C09. The results show that the PK-01-C09 protected in this application can recognize B7H3 expression with good specificity, recognizing only human B7H3 protein and not mouse blood samples. These results indicate that the B7H3-targeting ScFv protected in this application can be used for detecting B7H3 expression in ovarian cancer, ovarian cancer cells, lung cancer, and lung cancer cells, or for preparing detection reagents, kits, or molecular tracer reagents for detecting B7H3 in ovarian cancer, ovarian cancer cells, lung cancer, and lung cancer cells.

[0183] 2) Used for pathological sample testing

[0184] SK-OV-3 (B7-H3+) cells and B7-H3 negative cells (mouse colorectal cancer cells CT26.CL25, Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains) were fixed in 4% paraformaldehyde solution. The cells were then subjected to a process including drying, antigen retrieval, removal of catalase activity, antigen blocking, and administration of primary antibody (PK-01-C09) and secondary antibody (Anti-6X His). The tissue was incubated with Abcam (ab213204) and subjected to DAB staining. The staining was then observed under a microscope, followed by hematoxylin staining to examine the tissue staining. Finally, the tissue was dehydrated, cleared, and mounted with resin. Results are as follows: Figure 8A and 8B As shown, Figure 8A For the testing of fixed SK-OV-3 (B7-H3+) smears using PK-01-C09, Figure 8B The results of the assay using PK-01-C09 on smears of fixed B7-H3 negative cells showed that PK-01-C09 can detect B7H3 positive samples. These results indicate that the B7H3-targeting ScFv protected in this application can be used for the detection of B7H3 expression in human ovarian cancer and ovarian cancer cells, or for the preparation of diagnostic reagents, kits, or molecular tracers for detecting B7H3 in human ovarian cancer and ovarian cancer cells.

[0185] Example 5: Application of screened ScFv in CAR-T

[0186] The selected ScFv PK-01-C09, PK-02-A08, and PK-02-A09 were introduced into a viral expression system containing a CD8 hinge transmembrane domain, a 4-1BBZ co-activation domain, and a CD3ζ signaling domain for CAR structure design. Furthermore, considering the specificity of the tumor microenvironment, SIRP and Tight fusion proteins were introduced to construct plasmids, completing the CAR structure design, as shown in Table 3.

[0187] Table 3 CAR Structural Design 1

[0188]

[0189]

[0190] Among them, 8h is derived from the human CD8α hinge region sequence, 8TM is derived from the human CD8α transmembrane region sequence, BB is derived from the intracellular region sequence of 4-1BB, also known as CD137, CD3ζ (sometimes written as CD3Z) is derived from the intracellular region sequence of human CD3ζ, BBZ is an abbreviation for the combination of BB and CD3ζ, 7h is derived from the human CD7 hinge region sequence, G4h is derived from the human IgG4 hinge region sequence, G4H12 is derived from the optimized hinge region of human IgG4, 28TM is derived from the human CD28 transmembrane region sequence, 28 is derived from the human CD28 intracellular region sequence, 28Z is an abbreviation for the combination of the intracellular region sequence from human CD28 and the intracellular region sequence from human CD3ζ, 2B4Z is an abbreviation for the combination of the intracellular region sequence from human 2B4 and the intracellular region sequence from human CD3ζ; P2A is a self-cleaving peptide, specifically 2A peptide. A type of peptide that connects two or more gene reading frames, which can be two or more genes expressed independently at the same time.

[0191] 1) The corresponding structure was constructed using a lentiviral expression vector to prepare lentivirus. PBMCs (in some embodiments, these can be apheresis blood, cryopreserved and resuscitated PBMCs, or freshly isolated PBMCs) and CD3-positive T lymphocytes obtained from PBMCs (in some embodiments, these CD3-positive T lymphocytes can be αβT cells, γδT cells, or a mixed cell population) were conjugated with CD3 / CD28 antibodies using Dynabeads. TM CD3 / CD28, 40203D) activated T cells, and after activation, B7H3 CAR-T cells were prepared by transducing a lentiviral vector targeting B7H3. The cells were cultured for 7–21 days and then harvested.

[0192] CAR-T cell expression was detected by flow cytometry. Results showed that CAR structural expression was good in all groups, such as... Figure 9 As shown.

[0193] 2) Using A549(B7H3+) and SK-OV-3(B7H3+) as target cells, CAR-T cells with different CAR structures constructed from different ScFv were selected as effector cells. These cells were plated at a 1:1 ratio with target cells and co-incubated for 24 hours. In vitro killing assays were then performed, and the co-culture supernatant was used to detect cytokine secretion. The results are as follows: Figure 10A , Figure 10B , Figure 10C As shown. Among them. Figure 10AThe killing effect of CAR-T cells with different CAR structures constructed from PK-01-C09, PK-02-A08, and PK-02-A09 on lung cancer cells A549(B7H3+) was investigated. The results showed that CAR-T cells with different CAR structures constructed from PK-01-C09, PK-02-A08, and PK-02-A09 had good in vitro function. Figure 10B The killing effect of CAR-T cells with different CAR structures constructed for PK-01-C09 on lung cancer cells A549(B7H3+) showed that the PK-01-C09 protected in this application can be used to construct chimeric antigen receptors and can be used as an antigen recognition region in any chimeric antigen receptor or fusion protein structure. Figure 10C The results of CAR-T cells with different CAR structures constructed using PK-01-C09 killing human ovarian cancer cells SK-OV-3(B7H3+) showed that chimeric antigen receptor T cells expressing PK-01-C09, which is protected in this application, can be used to treat lung cancer and ovarian cancer, and can be used to prepare one or two drugs or drug combinations for the treatment of lung cancer and ovarian cancer.

[0194] The above results indicate that the PK-01-C09, PK-02-A08, and PK-02-A09 protected in this application can be used to construct the extracellular recognition region of a chimeric antigen receptor, and can be used to prepare one or more drugs or drug combinations for treating lung cancer, ovarian cancer, colorectal cancer, prostate cancer, or pancreatic cancer. Cells expressing the PK-01-C09, PK-02-A08, and PK-02-A09 protected in this application can be used to prepare one or more drugs or drug combinations for treating lung cancer, ovarian cancer, colorectal cancer, prostate cancer, or pancreatic cancer.

[0195] Using A549 (B7H3+) and SK-OV-3 (B7H3+) as target cells, CAR-T cells expressing different structures constructed from PK-01-C09 were validated in multiple batches. The results are as follows: Figure 11 and 12 As shown. Figure 11 The killing efficacy of CAR-T cells expressing different CAR structures constructed for PK-01-C09 against lung cancer cells was evaluated, p<0.0001; Figure 12 The killing efficacy of CAR-T cells expressing different CAR structures constructed for PK-01-C09 against ovarian cancer cells was investigated. Specific data are shown in Table 4, which presents the cell killing data.

[0196] Table 4 Cell Killing Data

[0197]

[0198]

[0199] The results show that the PK-01-C09 protected in this application can be used to construct the extracellular recognition region of a chimeric antigen receptor, and can be used to prepare one or two drugs or drug combinations for treating lung cancer and ovarian cancer. Cells expressing the PK-01-C09 protected in this application can be used to prepare one or two drugs or drug combinations for treating lung cancer and ovarian cancer.

[0200] 3) In vivo pharmacodynamic evaluation

[0201] Immunodeficient mice (NCG) aged 6-8 weeks were selected and subcutaneously or subcutaneously + intraperitoneally inoculated with A549 (B7-H3+-Luc-GFP) and SK-OV-3 (B7-H3+-Luc-GFP) cells (the tumor cells were derived from ATCC) at a rate of 1.0E+06 cells / mouse. After randomization using SPSS software, B7-H3 CAR-T cell preparations were injected intraperitoneally. The CT group received non-CAR-expressing T cells infused with the same dose and culture protocol as the CAR-T cells. CAR1, CAR2, CAR5, and CAR6 were CAR-T cells prepared using the construction and culture protocol described in Example 5. In vivo pharmacodynamics was evaluated by periodically monitoring changes in mouse fluorescence values ​​and tumor volume growth using small animal in vivo imaging and tumor measurement techniques.

[0202] The results are shown in Figures 13 and 14. Figure 13A To assess the in vivo efficacy against lung cancer cells (A549(B7-H3+-Luc-GFP)), Figure 13B The curve shows the tumor volume change of lung cancer cells (A549(B7-H3+-Luc-GFP)). Figure 14 The image shows the tumor volume change curve for ovarian cancer cells (SK-OV-3(B7-H3+-Luc-GFP)). The results indicate that the PK-01-C09 and PK-02-A09 cells we screened can be used for CAR construction, and cells expressing PK-01-C09 and PK-02-A09 can effectively kill lung and ovarian cancers.

[0203] In summary: The PK-01-C09, PK-02-A08, and PK-02-A09 cells we screened can be used to construct CARs that effectively kill tumors in lung and ovarian cancer, and can be applied to treat various tumors expressing B7H3. They can also be used to prepare drugs or drug combinations for treating tumors expressing B7H3. Cells expressing our screened PK-01-C09, PK-02-A08, and PK-02-A09 cells can be used to prepare drugs or drug combinations for tumor cells expressing B7H3. The tumor cells are lung and ovarian cancer, but can also be B7H3-positive tumors such as colorectal cancer, prostate cancer, and pancreatic cancer. The ScFv protected by this application can also be used for the treatment or detection of these indications.

[0204] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The scFv for identifying B7H3, characterized in that, Including any of the following: (I) The CDR1, CDR2, and CDR3 of the heavy chain variable region of the scFv have the amino acid sequences shown in SEQ ID No. 6, 7, and 8, respectively; and The CDR1, CDR2, and CDR3 of the light chain variable region of the scFv have, in sequence, the amino acid sequence shown in SEQ ID No. 4, the AAS sequence, and the amino acid sequence shown in SEQ ID No. 5; or (II) The CDR1, CDR2, and CDR3 of the heavy chain variable region of the scFv have amino acid sequences as shown in SEQ ID No. 13, 14, and 15, respectively; and The CDR1, CDR2, and CDR3 of the light chain variable region of the scFv have, in sequence, the amino acid sequence shown in SEQ ID No. 11, GAS, and the amino acid sequence shown in SEQ ID No. 12; or (III) The CDR1, CDR2, and CDR3 of the heavy chain variable region of the scFv have amino acid sequences as shown in SEQ ID No. 6, 7, and 19, respectively; and The CDR1, CDR2, and CDR3 of the light chain variable region of the scFv have, in sequence, the amino acid sequence shown in SEQ ID No. 18, the AAS sequence, and the amino acid sequence shown in SEQ ID No. 5; or (IV) A sequence based on any of the amino acid sequences shown in (I) to (III) that has undergone substitution, deletion, addition, and / or replacement of one or more amino acids; or (V) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (I) to (IV).

2. The scFv as described in claim 1, characterized in that, Including any of the following: (i) The heavy chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 2; and The light chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 1; or (ii) The heavy chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 10; and The light chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 9; or (iii) The heavy chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 17; and The light chain variable region of the scFv has an amino acid sequence as shown in SEQ ID No. 16; or (iv) A sequence based on any of the amino acid sequences shown in (i) to (iii) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (v) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (i) to (iv).

3. The scFv as described in claim 1 or 2, characterized in that, The light chain variable region and the heavy chain variable region are connected by a linker peptide. The linker peptide has the following characteristics: (vi) The amino acid sequence as shown in SEQ ID No. 20; or (vii) A sequence based on the amino acid sequence shown in (vi) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (viii) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in (vi).

4. The scFv as described in any one of claims 1 to 3, characterized in that, Including any of the following: (1) The scFv has an amino acid sequence as shown in SEQ ID No. 3; or (2) The scFv has an amino acid sequence as shown in SEQ ID No. 41; or (3) The scFv has an amino acid sequence as shown in SEQ ID No. 42; or (4) A sequence based on any of the amino acid sequences shown in (1) to (3) by substitution, deletion, addition and / or replacement of one or more amino acids; or (5) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (1) to (4).

5. A nucleic acid molecule encoding the scFv as described in any one of claims 1 to 4.

6. A protein assembly, characterized in that, Includes the scFv, hinge region, transmembrane region, and intracellular signaling domain as described in any one of claims 1 to 4.

7. The protein combination as described in claim 6, characterized in that, The scFv is connected to the transmembrane region via the hinge region; The hinge region is derived from IgG, and the hinge region includes, but is not limited to, one or a combination of two or more of the hinge regions of IgG1, IgG4, IgD, CD4, CD7, CD8α or CD28 molecules. The transmembrane region includes, but is not limited to, one or a combination of two or more of the transmembrane regions of ICOS, TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, PD-1, or CD226 molecules. The intracellular signaling functional domain includes a co-stimulatory domain and a primary signal transduction domain; The co-stimulatory domains include, but are not limited to, CD27, CD28, 4-1BB, OX40, 2B4, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 1a, LFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-l, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or a combination of one or more of the co-stimulatory domains of molecules that specifically bind to CD83; The primary signal transduction domains include, but are not limited to, one or a combination of two or more of the primary signal transduction domains of the CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 or DAP12 molecules.

8. The protein combination as described in claim 6 or 7, characterized in that, (A) The transmembrane region has an amino acid sequence as shown in SEQ ID No. 22, 29; or (B) The hinge region has an amino acid sequence as shown in SEQ ID No. 21, 36, 39, 40; or (C) The co-stimulatory domain has the amino acid sequences shown in SEQ ID No. 23, 30, and 35; (D) The primary signal transduction domain has an amino acid sequence as shown in SEQ ID No. 24; or (E) A sequence based on any of the amino acid sequences shown in (A) to (D) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (F) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with any of the amino acid sequences shown in (A) to (D).

9. The protein combination according to any one of claims 6 to 8, characterized in that, The protein combination includes CAR-1, CAR-2, CAR-3, CAR-4, CAR-5, CAR-6, CAR-7, CAR-8, CAR-9, CAR-10, CAR-11 and / or CAR-12; (A) The CAR-1, CAR-2, CAR-3, CAR-4, CAR-5, CAR-6, CAR-7, CAR-8, CAR-9, CAR-10, CAR-11, and CAR-12 respectively have the amino acid sequences shown in SEQ ID No. 27, 31, 43, 44, 45, 46, 25, 34, 38, 37, 32, and 33; or (B) A sequence based on the amino acid sequence shown in (A) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (C) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in (A) or (B).

10. A gene element, characterized in that, Includes the nucleic acid molecules as described in claim 5.

11. An artificial carrier, characterized in that, Includes the gene element as described in claim 10, and acceptable adjuvants or vectors.

12. Viral particles, characterized in that, Including the artificial carrier as described in claim 11.

13. Engineered cells, characterized in that, Includes any of the following: (a) the scFv as described in any one of claims 1 to 4; and / or (b) the nucleic acid molecule as described in claim 5; and / or (c) the protein combination as described in any one of claims 6 to 9; and / or (d) the gene element as described in claim 10; and / or (e) the artificial carrier as described in claim 11; and / or (f) Transduction of viral particles as described in claim 12.

14. The engineered cell as described in claim 13, characterized in that, The engineered cells include, but are not limited to, NK cells, T cells, macrophages, DC cells, and related precursor cells.

15. The following are applications in detecting B7H3 expression and / or preparing products for detecting B7H3 expression: I) scFv as described in any one of claims 1 to 4; and / or II) The nucleic acid molecule as described in claim 5; and / or III) The protein combination as described in any one of claims 6 to 9; and / or IV) The gene element as described in claim 10; and / or V) The artificial carrier as described in claim 11.

16. The application as described in claim 15, characterized in that, The products include kits, detection antibodies, and / or B7H3 molecular tracer reagents.

17. The product, characterized in that, include: I) The scFv as described in any one of claims 1 to 4; and / or II) The nucleic acid molecule as described in claim 5; and / or III) The protein combination as described in any one of claims 6 to 9; and / or IV) The gene element as described in claim 10; and / or V) The artificial carrier as described in claim 11; and / or VI) Virus particles as described in claim 12; and / or VII) The engineered cells as described in claim 13 or 14.

18. The following are applications in the preparation of medicaments for the prevention and / or treatment of diseases: 1) The scFv as described in any one of claims 1 to 4; and / or 2) The nucleic acid molecule as described in claim 5; and / or 3) The protein combination as described in any one of claims 6 to 9; and / or 4) The gene element as described in claim 10; and / or 5) The artificial carrier as described in claim 11; and / or 6) Virus particles as described in claim 12; and / or 7) The engineered cells as described in claim 13 or 14.

19. The application as described in claim 18, characterized in that, The diseases mentioned include those with positive B7H3 expression.

20. The application as described in claim 19, characterized in that, The disease includes tumors; the tumors include one or more of lung cancer, ovarian cancer, colorectal cancer, prostate cancer, or pancreatic cancer.

21. A drug or combination of drugs, characterized in that, Includes any of the following and pharmaceutically acceptable excipients or adjuvants: 1) The scFv as described in any one of claims 1 to 4; and / or 2) The nucleic acid molecule as described in claim 5; and / or 3) The protein combination as described in any one of claims 6 to 9; and / or 4) The gene element as described in claim 10; and / or 5) The artificial carrier as described in claim 11; and / or 6) Virus particles as described in claim 12; and / or 7) The engineered cells as described in claim 13 or 14.

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