Armed anti-B7H3 CAR-T cells and their use in cancer therapy

Abstract

A chimeric antigen receptor (CAR) (anti-B7H3 CAR) that binds to B7 homolog 3 protein (B7H3), comprising (a) an extracellular antigen-binding moiety specific to human B7H3, (b) a costimulatory signaling domain, and (c) a cytoplasmic signaling domain. Also provided herein are immune cells expressing the anti-B7H3 CAR, optionally combined with armed polypeptides, and their use in cancer therapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits as of the filing date of U.S. Provisional Application No. 63 / 497,863, filed on 24 April 2023, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Chimeric antigen receptor (CAR-T) T cells are genetically engineered T cells that express artificial T cell receptors for use in immunotherapy. These artificial T cell receptors (known as chimeric antigen receptors) can specifically bind to disease cell antigens, such as cancer antigens. Upon binding to disease cells, CAR-T cells are activated and eliminate the disease cells.

[0003] B7 homolog 3 (B7H3), also known as differentiation cluster 276 (CD276), plays a crucial role in adaptive immunity, for example, by suppressing T cell activation and proliferation. In cancer, B7H3 functions as an immune checkpoint molecule that inhibits the anti-tumor immune response. The B7H3 protein has been found to be expressed in various tumor tissues, and its expression is strongly associated with undesirable therapeutic outcomes due to its role as an immune checkpoint molecule. Therefore, B7H3 may be a good target in cancer therapy. [Overview of the Initiative]

[0004] This disclosure is at least partially based on the development of potent anti-B7H3 chimeric antigen receptors, and CAR-T cells expressing such receptors, either alone or in combination with armed polypeptides (e.g., engineered armed polypeptides), which have shown high cytotoxicity and specificity against target cells (e.g., cancer cells) expressing B7H3 (either full-length or splicing variants). Exemplary anti-B7H3 CAR-T cells (e.g., armed) have also shown high antitumor activity in animal models.

[0005] Thus, in one aspect, the present disclosure features a chimeric antigen receptor (CAR) that binds to B7 homolog 3 protein (B7H3) (anti-B7H3 CAR), wherein the CAR comprises: (a) an extracellular antigen-binding portion specific for human B7H3; (b) a costimulatory signaling domain; (c) a cytoplasmic signaling domain; and optionally, (d) a hinge domain and / or transmembrane domain.

[0006] The extracellular antigen-binding portion specific for human B7H3 (a) may comprise a human heavy-chain antibody that contains the same heavy-chain complementarity-determining regions (CDRs) as the reference heavy-chain antibodies of BH-01, BH-02, or BH-03, all of whose sequence information is provided in Table 1 herein. In some examples, the human heavy-chain antibody comprises the amino acid sequence of SEQ ID NO: 4. In other examples, the human heavy-chain antibody comprises the amino acid sequence of SEQ ID NO: 8 or 9. In yet another example, the human heavy-chain antibody comprises the amino acid sequence of SEQ ID NO: 13.

[0007] Alternatively, the extracellular antigen-binding portion specific for human B7H3 of (a) may comprise a single-chain variable fragment (scFv) that contains a heavy-chain variable region (V H ) and a light-chain variable region (V L ). V H and V L each contain the same heavy-chain CDRs and light-chain CDRs as the reference antibodies of BH-04, BH-05, BH-06, BH-07, BH-08, BH-09, or BH-10, all of whose sequence information is provided in Table 1 herein. The V H and V L of the scFv fragment may be connected via a peptide linker (e.g., those disclosed herein).

[0008] In some examples, V H comprises the amino acid sequence of SEQ ID NO: 17 and V L comprises the amino acid sequence of SEQ ID NO: 21. Such an scFv containing V H / V L may comprise the amino acid sequence of SEQ ID NO: 112.

[0009] In some examples, V H This contains the amino acid sequence of SEQ ID NO: 25, V L This contains the amino acid sequence of SEQ ID NO: 29. Such V H / V L scFv containing this may contain the amino acid sequence of SEQ ID NO: 113.

[0010] In some examples, V H It contains the amino acid sequence of SEQ ID NO: 33, V L This contains the amino acid sequence of SEQ ID NO: 37. Such V H / V L scFv containing this may contain the amino acid sequence of SEQ ID NO: 114.

[0011] In some examples, V H It contains the amino acid sequence of SEQ ID NO: 41, V L This contains the amino acid sequence of SEQ ID NO: 45. Such V H / V L scFv containing this may contain the amino acid sequence of SEQ ID NO: 115.

[0012] In some examples, V H It contains the amino acid sequence of SEQ ID NO: 49, V L This contains the amino acid sequence of SEQ ID NO: 53. Such V H / V L scFv containing this may contain the amino acid sequence of SEQ ID NO: 116.

[0013] In some examples, V H It contains the amino acid sequence of SEQ ID NO: 57, V L This contains the amino acid sequence of SEQ ID NO: 61. Such V H / V L scFv containing this may contain the amino acid sequence of SEQ ID NO: 117.

[0014] In some examples, V H It contains the amino acid sequence of SEQ ID NO: 65, V L This contains the amino acid sequence of SEQ ID NO: 69. Such V H / VL scFv containing this may contain the amino acid sequence of SEQ ID NO: 118.

[0015] In some cases, the anti-B7H3 CARs disclosed herein comprise one of the human heavy chain antibody fragments disclosed herein (e.g., BH-02 or its derivatives). In some cases, the anti-B7H3 CAR comprises one of the scFv fragments disclosed herein (e.g., BH-07 or its derivatives). In some cases, the B7H3-specific extracellular antigen-binding moiety in the anti-B7H3 CARs disclosed herein may comprise one human heavy chain antibody fragment disclosed herein (e.g., derived from BH-02, which may comprise SEQ ID NOs. 8 or 9) and one scFv fragment disclosed herein (e.g., derived from BH-04, BH-05, BH-06, or BH-07, which may comprise the amino acid sequences of SEQ ID NOs. 112, 113, 114, or 115, respectively). The heavy chain antibody fragment and the scFv fragment may be linked via a peptide linker (e.g., one disclosed herein).

[0016] Any of the anti-B7H3 CAR constructs disclosed herein may include a co-stimulatory domain (b), which may be derived from a preferred co-stimulatory molecule, e.g., CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. For example, the co-stimulatory domain is the 4-1BB co-stimulatory domain (e.g., including the amino acid sequence of SEQ ID NO: 79). Alternatively, or in addition, the cytoplasmic signaling domain (c) of the anti-B7H3 CAR may be derived from CD3ζ (e.g., including the amino acid sequence of SEQ ID NO: 80).

[0017] The anti-B7H3 CARs disclosed herein may further include a hinge domain, a transmembrane domain, or a combination thereof. In some cases, the hinge and transmembrane domains may be located between the extracellular antigen-binding portion (a) and the costimulatory domain (b). For example, the hinge domain may include the amino acid sequence of SEQ ID NO: 75. Alternatively, or in addition, the transmembrane domain may include the amino acid sequence of SEQ ID NO: 78. In some cases, the anti-B7H3 CARs disclosed herein may further include a spacer located between the hinge domain and the transmembrane domain. Examples include SEQ ID NOs. 76 and 77 provided herein. In other cases, the anti-B7H3 CARs disclosed herein do not include a spacer fragment.

[0018] Exemplary anti-B7H3 CAR constructs containing human heavy chain antibodies disclosed herein may include the amino acid sequence of SEQ ID NO: 83, 85, or 87. Exemplary anti-B7H3 CAR constructs containing scFv fragments disclosed herein may include the amino acid sequence of SEQ ID NO: 89, 91, 93, 95, 97, 99, or 101. In some specific examples, the anti-B7H3 CAR may include the amino acid sequence of SEQ ID NO: 89 or 93. Exemplary anti-B7H3 CARs (biparatropic anti-B7H3 CARs) containing both heavy chain antibody fragments and scFv fragments disclosed herein may include the amino acid sequence of SEQ ID NO: 103, 105, 107, or 120.

[0019] Any of the anti-B7H3 CAR constructs disclosed herein may further comprise an N-terminal signal peptide. In some examples, the signal peptide may comprise the amino acid sequence of SEQ ID NO: 70. In other examples, the signal peptide may comprise the amino acid sequence of SEQ ID NO: 71.

[0020] A population of genetically engineered immune cells is also provided herein, which express one of the anti-B7H3 CAR constructs disclosed herein. In some embodiments, the genetically engineered immune cells may further express truncated EGFR and / or armed polypeptides (enhancing T cell functionality). In some cases, the truncated EGFR fragment or armed polypeptide comprises a signal peptide located at its N-terminus, which may enable secretion of the truncated EGFR or armed polypeptide by a host cell exhibiting such expression.

[0021] In some embodiments, genetically engineered immune cells co-express either the anti-B7H3 CAR and the truncated EGFR fragment disclosed herein. In some cases, the truncated EGFR fragment comprises the amino acid sequence of SEQ ID NO: 111.

[0022] In some embodiments, genetically engineered immune cells co-express one of the anti-B7H3 CARs and armed polypeptides disclosed herein. Examples of armed polypeptides include, but are not limited to, IL-2, IL-5, IL-15, costimulatory ligands, and anti-PDL1 antibodies. In some examples, the armed polypeptide may be an anti-PDL1 antibody, which may be an scFv fragment (e.g., containing the amino acid sequence of SEQ ID NO: 108). In some examples, the armed polypeptide may be an engineered IL-2 polypeptide, for example, containing the amino acid sequence of SEQ ID NO: 109.

[0023] In some cases, the armed polypeptide can be a fusion polypeptide comprising an anti-PDL1 antibody and an IL-2 polypeptide (e.g., those disclosed herein, e.g., an engineered IL-2 polypeptide such as SEQ ID NO: 109). In some examples, the fusion polypeptide comprises a single-chain variable fragment (scFv) that binds to PDL1 (e.g., comprising the amino acid sequence of SEQ ID NO: 108) and an engineered IL-2 polypeptide having reduced IL2Ra binding activity (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 109). In one specific example, the armed polypeptide comprises the amino acid sequence of SEQ ID NO: 110.

[0024] In some cases, the engineered immune cells disclosed herein co-express an anti-B7H3 CAR containing an extracellular antigen-binding moiety derived from BH-07 (e.g., containing the same heavy and light chain CDRs as BH-07), as well as an armed polypeptide containing an anti-PDL1 antibody and an IL-2 polypeptide. In one specific example, the engineered immune cells disclosed herein co-express an anti-B7H3 CAR containing the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 93, and an armed polypeptide containing the amino acid sequence of SEQ ID NO: 110. In some cases, the genetically engineered immune cells secrete the armed polypeptide.

[0025] In some cases, the manipulated immune cells include T cells, NK cells, macrophages, or a combination thereof. In some cases, the immune cells are substantially T cells (for example, at least 70% of the cells in the population are T cells).

[0026] In some cases, the manipulated immune cells may contain an anti-B7H3 CAR, as well as nucleic acids or sets of nucleic acids encoding armed polypeptides and / or truncated EGFR fragments expressed by the manipulated immune cells.

[0027] Furthermore, this disclosure provides a method for eliminating B7H3-expressing disease cells in a subject, comprising administering an effective amount of a population of genetically engineered immune cells disclosed herein to the subject in need. In some embodiments, the disease cells are cancer cells (e.g., expressing full-length B7H3, splicing variant B7H3, or both). In some embodiments, the subject of treatment is a human patient with B7H3-expressing cancer. Exemplary target cancers include, but are not limited to, lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, glioblastoma, HCC, RCC, gastric cancer, esophageal cancer, prostate cancer, bladder cancer, head and neck cancer, squamous cell carcinoma, and leukemia.

[0028] Furthermore, this disclosure provides any of the populations of anti-B7H3 CAR-T cells disclosed herein for use in the treatment of the targeted cancers disclosed herein, or for manufacturing pharmaceuticals for use in the treatment of the targeted cancers disclosed herein.

[0029] In another aspect, the present disclosure features a nucleic acid comprising a first nucleotide sequence encoding one of the anti-B7H3 CAR constructs disclosed herein, and optionally a second nucleotide sequence encoding an armed polypeptide (enhancing T cell functionality) or a truncated EGFR fragment. In some cases, the nucleic acid may further comprise a third nucleotide, located between the first and second nucleotide sequences, encoding a self-cleaving peptide (e.g., T2A). In some examples, the second nucleotide sequence encoding the truncated EGFR fragment or armed polypeptide may further encode a signal peptide located at the N-terminus of the armed polypeptide or truncated EGFR, thereby enabling the armed polypeptide or truncated EGFR to be secreted by a host cell exhibiting such expression.

[0030] In some embodiments, the second nucleotide sequence encodes an armed polypeptide. Examples include IL-2, IL-5, IL-15, a costimulatory ligand, and an anti-PDL1 antibody. In some cases, the armed polypeptide can be a fusion polypeptide comprising an anti-PDL1 antibody and an IL-2 polypeptide (e.g., an engineered IL-2 polypeptide, such as those disclosed herein). In some examples, the fusion polypeptide comprises a single-chain variable fragment (scFv) that binds to PDL1 (e.g., comprising the amino acid sequence of SEQ ID NO: 108) and an engineered IL-2 polypeptide having reduced IL2Ra binding activity (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 109). In one specific example, the armed polypeptide comprises the amino acid sequence of SEQ ID NO: 110. In other embodiments, the second nucleotide sequence encodes a truncated EGFR. In one example, the truncated EGFR is shown as SEQ ID NO: 111.

[0031] Furthermore, a series of nucleic acids (two distinct nucleic acid molecules) are also within the scope of this disclosure, the first nucleic acid encoding the anti-B7H3 CAR disclosed herein, and the second nucleic acid encoding an armed polypeptide and / or a truncated EGFR fragment.

[0032] Any nucleic acid or set of nucleic acids disclosed herein may be a set of expression vectors or expression vectors, such as viral vectors (e.g., lentiviral vectors), or a set of viral vectors.

[0033] In addition, any of the anti-B7H3 antibodies, anti-B7H3 CAR constructs, coding nucleic acids thereof, and their uses (e.g., therapeutic applications such as cancer treatment) disclosed herein are also within the scope of this disclosure.

[0034] Details of one or more embodiments of the present invention are shown in the following description. Other features or advantages of the present invention will be apparent from the following drawings and detailed descriptions of some embodiments, as well as from the appended claims.

[0035] The following drawings form part of this specification and are included to further demonstrate certain aspects of the disclosure, which can be better understood by referring to the drawings in conjunction with the detailed description of the specific embodiments shown herein. [Brief explanation of the drawing]

[0036] [Figure 1A] This graph shows the binding activity of exemplary anti-B7H3 scFv clones to full-length and splice variants of the B7H3 receptor on the cell surface, as determined by FACS analysis. The graph specifically shows the binding activity to full-length B7H3. [Figure 1B] This graph shows the binding activity of exemplary anti-B7H3 scFv clones to the full-length and splice variants of the B7H3 receptor on the cell surface, as determined by FACS analysis. The graph also shows the binding activity to the splice variant B7H3. [Figure 2A] This graph shows the binding activity of anti-B7H3 scFv against endogenous cancer cells expressing endogenous B7H3, as determined by FACS. It represents the binding activity of scFv against cancer lines with high B7H3 expression. [Figure 2B] This graph shows the binding activity of anti-B7H3 scFv against endogenous cancer cells expressing endogenous B7H3, as determined by FACS. It represents the binding activity of scFv against cancer lines with moderate B7H3 expression. [Figure 2C] This graph shows the binding activity of anti-B7H3 scFv against endogenous cancer cells expressing endogenous B7H3, as determined by FACS. It also shows the binding activity of scFv against cancer lines with low B7H3 expression. [Figure 2D] This graph shows the binding activity of anti-B7H3 scFv against endogenous cancer cells expressing endogenous B7H3, as determined by FACS. It compares BH-07 scFv binding activity against high, medium, and low B7H3-expressing cancer cell lines. [Figure 2E]This graph shows the binding activity of anti-B7H3 scFv against endogenous cancer cells expressing endogenous B7H3, as determined by FACS. It represents the dose-dependent binding activity of BH-07 scFv against cancer cell lines. [Figure 3A] This graph shows the epitope binning of anti-B7H3 VH antibodies against enobrituzumab by FACS. It shows the binding of anti-B7H3 VH antibodies BH-01 (top left), BH-02 (top right), and BH-03 (bottom) to CHOK1 cells expressing full-length B7H3, in the absence or presence of enobrituzumab. [Figure 3B] This graph shows the epitope binning of anti-B7H3 VH antibodies against enobrituzumab by FACS. It shows the binding of anti-B7H3VH antibodies BH-01 (top left), BH-02 (top right), and BH-03 (bottom) to CHOK1 cells expressing the splice variant B7H3, in the absence or presence of enobrituzumab. [Figure 3C] This graph shows the epitope binning of an anti-B7H3 VH antibody against enobrituzumab by FACS. It represents the binding of enobrituzumab to CHOK1 cells expressing full-length B7H3 or a splice variant of B7H3. [Figure 4A] This graph shows, using an SPR assay, that the exemplary anti-B7H3 scFv and VH antibodies shown bind to various epitopes of B7H3, specifically BH-02 against BH-04 or BH-05. [Figure 4B] This graph shows, using an SPR assay, that the exemplary anti-B7H3 scFv and VH antibodies shown bind to various epitopes of B7H3, specifically BH-01 against BH-04, BH-05, or BH-02. [Figure 5] This is a schematic diagram showing exemplary anti-B7H3 chimeric antigen receptor constructs and their co-expression with either EGFRt or armed polypeptides. [Figure 6A] This graph shows the cytotoxicity of anti-B7H3 VH CAR-T cells. CAR-T cell toxicity is indicated by the normalized total area of ​​GFP. [Figure 6B] This graph shows the cytotoxicity of anti-B7H3 VH CAR-T cells. The percentage represents cell death. [Figure 7] This graph shows the IFNγ secretion of anti-B7H3 VH CAR T cells when incubated with multiple B7H3 expression targets and cancer cell lines. [Figure 8] This graph shows the antigen-specific CTL activity of CAR-T cells expressing anti-B7H3 VH CAR EVLP25. [Figure 9A] This graph shows the bioactivity of anti-B7H3 scFv CAR T cells when engaged with various target cells. It also shows the percentage of CTL activity of CAR-T cell-expressed anti-B7H3 scFv cells against B7H3-negative, target-specific, and cancer cell strains. [Figure 9B] This graph shows the bioactivity of anti-B7H3 scFv CAR T cells during engagement with various target cells, specifically IFNγ secretion. [Figure 10A] This graph shows the proliferation of anti-B7H3 scFv CAR-T cells upon engagement with target cells. These are CAR-T cells expressing EPLV195. [Figure 10B] This graph shows the proliferation of anti-B7H3 scFv CAR-T cells upon engagement with target cells. These are CAR-T cells expressing EPLV196. [Figure 11A] This graph shows the bioactivity of CAR-T cells expressing biparatropic anti-B7H3 CAR. The percentage represents cell lysis. [Figure 11B] This graph shows the bioactivity of CAR-T cells expressing biparatropic anti-B7H3 CAR, specifically IFNγ secretion. [Figure 12A] This graph shows the bioactivity of CAR-T cells expressing exemplary anti-B7H3 CAR EPLV195. The percentage represents cell lysis. [Figure 12B] This graph shows the bioactivity of CAR-T cells expressing exemplary anti-B7H3 CAR EPLV195, specifically IFNγ secretion. [Figure 13A] This graph shows the cytotoxicity of anti-B7H3 CARs with different spacer fragments, as determined in in vitro CTL and rechallenge assays. The E:T ratio is 2.5:1. [Figure 13B] This graph shows the cytotoxicity of anti-B7H3 CARs with different spacer fragments, as determined in in vitro CTL and rechallenge assays. The E:T ratio is 1:1. [Figure 14A] This graph shows the cytotoxicity of CAR-T cells expressing anti-B7H3 CAR (EPLV326) or co-expressing anti-B7H3 CAR and armed polypeptide (armed CAR, EPLV330). The E:T ratio is 2.5:1. [Figure 14B] This graph shows the cytotoxicity of CAR-T cells expressing anti-B7H3 CAR (EPLV326) or co-expressing anti-B7H3 CAR and armed polypeptide (armed CAR, EPLV330). The E:T ratio is 1:1. [Figure 15] This figure shows the binding activity of the supernatants derived from armed and demilitarized EPC-002 CAR-T cells to PDL1 target cell lines. The supernatant of armed CAR-T cells (secreted armed, anti-PDL1 scFv-IL2 fusion) bound to the PDL1 / K562 cell line. The supernatant of demilitarized CAR-T cells did not show binding activity. [Figure 16A] This graph shows the antitumor activity and safety of CAR-T cells and armed CAR-T cells in A375 (melanoma cell) xenograft mice in primary tumors and tumor rechallenge trials. Tumor volume is shown. [Figure 16B] This graph shows the antitumor activity and safety of CAR-T cells and armed CAR-T cells in A375 (melanoma cell) xenograft mice in primary tumors and tumor rechallenge trials. Body weight is shown throughout the treatment. [Figure 17A] This graph shows the immunophenotyping of CAR-T cells and armed CAR-T cells produced for animal studies. The percentages represent T cell subtypes. [Figure 17B]This graph shows the immunophenotyping of CAR-T cells and armed CAR-T cells produced for animal testing. It represents PBMC immunophenotyping. [Figure 18A] This graph shows the immunophenotyping of PBMCs (Primary Body Cells) of CAR-T cells and armed CAR-T cells 20 days after treatment. The cells represented are human total CD3, CD8, and CAR+CD8+ and CAR+CD4+ T cells in the treatment group. [Figure 18B] This graph shows the immunophenotyping of PBMCs (Patient-Based Cells) of CAR-T cells and armed CAR-T cells 20 days after treatment. It also shows the immunophenotyping of human CD8+ T cells treated with 10E6 armed CAR-T cells. [Figure 19A] This graph shows the immunophenotyping of armed CAR-T cells in PBMCs and spleens 64 days after treatment with a 2E6 dose. The graph shows human whole CD4+ and CD8+ cells in PBMCs (upper left and upper right, respectively) and spleens (lower left and lower right, respectively) treated with EPC-002 armed CAR. [Figure 19B] This graph shows the immunophenotyping of armed CAR-T cells in PBMCs and spleen 64 days after treatment with a 2E6 dose. Immunophenotyping of human CAR+CD4+ and CAR+CD8+ T cells was performed in PBMCs and spleen treated with armed CAR-T cells. [Figure 20A] This graph shows the antitumor activity and safety of CAR-T armed CAR-T cells in low-dose A375 (melanoma cell) xenograft mice. Tumor volume is shown. [Figure 20B] This graph shows the antitumor activity and safety of CAR-T armed CAR-T cells in mice with low doses of A375 (melanoma cells) xenografts. Body weight is shown throughout the treatment. [Figure 21A] This graph shows the antitumor activity of EPC-002-armed CAR-T cells in an animal model with tumor cell rechallenge. The tumor volume is shown. [Figure 21B] This graph shows the antitumor activity of EPC-002-armed CAR-T cells in an animal model with tumor cell rechallenge. Body weight is shown throughout the treatment. [Figure 22A] This graph shows the pharmacokinetics and pharmacodynamics of EPC-002 CAR-T cells in an animal model. The tumor volume is also shown. [Figure 22B] This graph shows the pharmacokinetics and pharmacodynamics of EPC-002 CAR-T cells in an animal model. It also shows immunophenotypic analysis of the spleen of EPC-002-treated mice 7 days after treatment. [Figure 23A] This graph shows the antitumor activity of EPC-002-armed CAR-T cells in the H1975 NSCLC tumor mouse model. The tumor volume is shown. [Figure 23B] This graph shows the antitumor activity of EPC-002-armed CAR-T cells in the H1975 NSCLC tumor mouse model. Body weight is shown throughout the treatment. [Modes for carrying out the invention]

[0037] B7 homolog 3 (B7H3), also known as CD276, is a member of the B7 family that plays an immunomodulatory role in T cell responses. It is a type I transmembrane glycoprotein with two isoforms: B7H3 VC and B7H3 VCVC, depending on the structure of its extracellular domain. B7H3 VC contains a single extracellular V-like and C-like Ig domain pair, a transmembrane domain, and a cytoplasmic tail. B7H3 VCVC contains two identical pairs of V-like and C-like Ig domains in the extracellular domain (full-length B7H3), and this is the major isoform found in humans. The B7H3 VC isoform is also found in human cells due to alternative splicing (splicing variant). B7H3 has been reported to play an inhibitory role in T cell activation and proliferation, lead to tumor immune evasion, and influence both immune responses and tumor behavior through different signaling pathways. B7H3 expression has been found to be abnormally upregulated in many different cancer types and associated with poor prognosis. Zhao et al., J. Hematology & Oncology, 15(153):1-31(2022). Therefore, targeting B7H3 (full-length and / or splicing variants) is expected to be an effective approach in cancer treatment.

[0038] This disclosure reports the development of anti-B7H3 antibodies (in heavy chain antibody (e.g., VHH) form or single-chain variable fragment (scFv) form) that exhibit high specific binding activity to B7H3 (both full-length B7H3 and splicing variant B7H3), and anti-B7H3 CAR constructs containing such anti-B7H3 antibodies. CAR-T cells expressing anti-B7H3 CARs showed high cytotoxicity against B7H3+ target cells in vitro and exhibited antitumor activity in vivo. Furthermore, CAR-T cells administered to mice showed desired immunotyping over time, demonstrating the in vivo cellular persistence of such CAR-T cells. Therefore, the anti-B7H3 CAR-T cells disclosed herein are expected to have excellent therapeutic effects in cancer treatment.

[0039] Accordingly, anti-B7H3 antibodies, anti-B7H3 chimeric antigen receptors (CARs) constructed therefrom, engineered immune cells (e.g., T cells) expressing anti-B7H3 CARs, and optionally armed polypeptides (armed CARs) or truncated EGFRs, as well as their uses (e.g., therapeutic applications), are provided herein.

[0040] I. B7H3-specific chimeric antigen receptor (anti-B7H3 CAR) As used herein, the terms “chimeric antigen receptor” or “CAR” refer to an artificial immune cell receptor capable of binding to an antigen expressed by a target cell, e.g., human B7H3 (full-length and / or splicing variant). Generally, a CAR may comprise a fusion polypeptide comprising an extracellular antigen-binding domain (e.g., a single-chain variable fragment or scFv derived from an antibody specific to the target antigen), a costimulatory domain, and an intracellular signaling domain. In some cases, the fusion polypeptide may further comprise a hinge and a transmembrane domain located at the C-terminus of the extracellular antigen-binding domain. In some examples, the fusion polypeptide may further comprise a spacer between the hinge domain and the transmembrane domain. Alternatively, the fusion polypeptide may not comprise a spacer.

[0041] Typical antibody molecules usually have a heavy chain variable region (V) involved in antigen binding. H ) and light chain variable region (V L ) includes. V H Region and V L The domain can be further subdivided into hypervariable domains, also known as "complementarity-determining domains" ("CDRs"), which are dotted with more conserved domains known as "framework domains" ("FRs"). H and V LTypically, it consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The framework region and the scope of the CDRs can be precisely identified using methodologies known in the art, such as the Kabat definition, Chothia definition, AbM definition, and / or contact definition, all of which are well known in the art. For example, see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al. (1989) Nature 342:877, Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948, and Almagro, J. Mol. Recognit. 17:132-143 (2004). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0042] In some embodiments, the antibody portion disclosed herein has the same heavy chain and / or light chain complementary determination region (CDR) or the same V as the reference antibody. H and / or V L They can share a chain. The same V H and / or V L Two antibodies having a CDR mean that their CDRs are identical when determined by the same method (e.g., the Kabat method, Chothia method, AbM method, Contact method, or IMGT method known in the art; see, for example, bioinf.org.uk / abs / ). Such anti-B7H3 antibodies have the same V as the exemplary (reference) antibody described herein. H , same V LThey may have, or both.

[0043] In some embodiments, the antibody moieties disclosed herein may share a certain level of sequence identity with respect to a reference sequence. The "identity percentage" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc.Natl.Acad.Sci.USA 87:2264-68, 1990, as modified by Karlin and Altschul Proc.Natl.Acad.Sci.USA 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J.Mol.Biol.215:403-10, 1990. A BLAST protein search can be performed using the XBLAST program, with a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the target protein molecule. If a gap exists between two sequences, Gapped BLAST can be used, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the initial settings parameters for each program (e.g., XBLAST and NBLAST) can be used.

[0044] In some embodiments, the antibody moieties disclosed herein may have one or more amino acid variations relative to a reference antibody. The amino acid residue variations disclosed herein (e.g., in the framework region and / or CDR) may be conserved amino acid substitutions. As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size properties of the protein being substituted. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art. For example, they can be prepared by methods for modifying polypeptide sequences known to those skilled in the art, as found in references such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0045] Each anti-B7H3 CAR disclosed herein comprises an anti-B7H3 binding moiety (e.g., a heavy chain antibody fragment or an scFv fragment) or two anti-B7H3 binding moieties (e.g., one heavy chain antibody fragment and one scFv fragment) in its extracellular antigen-binding domain.

[0046] (A) Parent anti-B7H3 antibody In some embodiments, the anti-B7H3 antibody (parental anti-B7H3 antibody) for use in the construction of the anti-B7H3 CAR disclosed herein may be a human heavy chain antibody that may contain only the heavy chain variable region. Examples of human heavy chain antibodies include BH-01, BH-02, and BH-03 disclosed herein. For example, the human heavy chain antibody for use in the construction of the anti-B7H3 CAR may be BH-02 or a derivative thereof (see above disclosure). The amino acid sequences of these example human heavy chain antibodies, as well as their heavy chain complementarity determining regions (CDRs) determined according to the Kabat scheme, are provided in Table 1 below.

[0047] The anti-B7H3 binding moiety in any of the anti-B7H3 CARs disclosed herein is connected to the heavy chain variable domain (V) of an anti-B7H3 antibody via a peptide linker. H ) and light chain variable domain (V L It may be in scFv form, which is a fusion polypeptide containing ). In the scFv fragment, V H and V L The fragment can have any orientation. In some cases, scFv is from the N-terminus to the C-terminus, V L Fragments, peptide linkers, and V H It may include fragments. Alternatively, scFv is V from the N-terminus to the C-terminus. H Fragments, peptide linkers, and V L The fragment may include, and. In some examples, the scFv may further include an N-terminal signal peptide for directing the CAR containing the scFv to the cell surface. An exemplary anti-B7H3 scFv antibody fragment is BH-04-BH-10 disclosed herein. In one example, the scFv antibody fragment for use in constructing an anti-B7H3 CAR may be BH-07 or a derivative thereof (see disclosure above). The amino acid sequences of these exemplary scFv antibody fragments, as determined according to the Kabat scheme, as well as their heavy and light chain CDRs, are provided in Table 1 below. All of the enumerated anti-B7H3 antibodies disclosed herein, and their derivatives, are within the scope of this disclosure. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0048] The anti-B7H3 binding moiety derived from the reference antibody (e.g., heavy chain antibody fragment or scFv fragment) (listed in Table 1) refers to a binding moiety that has substantially the same structural and functional characteristics as the reference antibody. Structurally, the binding moiety has the same heavy chain and / or light chain complementary determinant region as the reference antibody, or the same V H Chain and / or V L The chain may be present. Alternatively, the binding region may have only a limited number of amino acid variations in one or more framework regions and / or one or more CDRs without significantly affecting binding affinity and binding specificity to the reference antibody. See the explanation below.

[0049] In some cases, the anti-B7H3 binding moiety may contain the same heavy chain CDR as one of the reference antibodies provided in Table 1 above (e.g., BH-02 or BH-07). Where applicable (e.g., in the context of scFv antibody fragments), the anti-B7H3 binding moiety may have the same light chain CDR as one of the reference antibodies provided in Table 1 above (e.g., BH-07). Such an anti-B7H3 binding moiety may have the same V as the reference antibody. H Chain and / or V LThe chain may be included. Alternatively, the anti-B7H3 binding moiety may contain amino acid variations in one or more framework regions relative to the corresponding framework region in the reference antibody. For example, the anti-B7H3 binding moiety may collectively contain up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions relative to the corresponding framework region in the reference antibody.

[0050] In some embodiments, the anti-B7H3 moiety may include a certain level of variation in one or more CDRs with respect to any of the reference antibodies (e.g., BH-02 or BH-07) provided in Table 1 above. For example, the anti-B7H3 moiety may include a certain level of variation in the reference antibody. H Compared to CDRs, it may contain heavy chain CDRs that have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively. Alternatively, or in addition, an anti-B7H3 antibody may be used as a reference antibody. L Compared to the CDR, the light chain CDR may have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively. As used herein, “individually” means that one CDR of an antibody shares the sequence identity shown to the corresponding CDR of a reference antibody (e.g., one of those in Table 1, such as BH-02 or BH-07). “Collectively” means that the three Vs of the combined antibody share at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. H or V L CDR corresponds to the three V values ​​of the combined reference antibody. H or V L This means that they share the same sequence identity as shown in the CDR.

[0051] In some cases, the anti-B7H3 moiety may collectively contain up to 10 amino acid variations (e.g., up to 9, 8, 7.6, 5, 4, 3, 2, or 1 amino acid variation) in one or more of the heavy chain CDRs and light chain CDRs, relative to the CDR of the reference antibody (e.g., BH-02 or BH-07) provided in Table 1. In some cases, the anti-B7H3 moiety may contain the same heavy chain CDR3 as the reference antibody and may contain one or more amino acid variations in one or more of the other heavy chain CDRs and light chain CDRs.

[0052] In some cases, the anti-B7H3 moieties disclosed herein may be any of the anti-B7H3 heavy chain antibodies provided in Table 1 above. For example, the anti-B7H3 binding moiety may contain the amino acid sequence of SEQ ID NO: 4, 8, 9, or 13. In a specific example, the anti-B7H3 binding moiety may contain the amino acid sequence of SEQ ID NO: 8 or 9 (e.g., SEQ ID NO: 8). Alternatively, the anti-B7H3 binding moiety may contain an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to the heavy chain antibody fragments provided in Table 1, e.g., SEQ ID NO: 4, 8, 9, or 13. Such anti-B7H3 binding moieties may contain variations only in the framework region compared to their parent counterpart.

[0053] In some cases, the anti-B7H3 binding moieties disclosed herein may be any of the scFv fragments provided in Table 1 above. For example, the anti-B7H3 scFv fragment may contain the amino acid sequence of SEQ ID NOs. 112, 113, 114, 115, 116, 117, or 118, or the same V H and V LIt may contain fragments. In a specific example, the anti-B7H3 scFv may contain the amino acid sequence of SEQ ID NO: 115. Alternatively, the anti-B7H3 moiety may contain an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to any of the scFv fragments provided in Table 1, e.g., SEQ ID NOs: 112, 113, 114, 115, 116, 117, or 118 (e.g., SEQ ID NO: 115). Such an anti-B7H3 binding moiety may contain variations only in the framework region compared to its parent counterpart. In other examples, the anti-B7H3 moiety disclosed herein is the same as those listed in Table 1, e.g., SEQ ID NOs: 112, 113, 114, 115, 116, 117, or 118. H and V L It may contain arrays, V H and V L It has reverse orientation of the fragments.

[0054] Any of the anti-B7H3 binding moieties disclosed herein (e.g., heavy chain antibody fragments provided in Table 1 such as SEQ ID NO: 8 or 9, scFv fragments provided in Table 1 such as SEQ ID NO: 115, or the reverse V) H and V L Their oriented counterparts (or derivatives thereof as disclosed herein) may be used to construct anti-B7H3 CAR constructs (including monospecific CARs and biparatopic CARs) as disclosed herein.

[0055] Any of the parental anti-B7H3 antibodies provided herein are also within the scope of this disclosure. In this context, the term “antibody” (used interchangeably in the plural) refers to an immunoglobulin molecule that can specifically bind to a target (here, human B7H3) via at least one antigen recognition site located within the variable region of the immunoglobulin molecule. The anti-B7H3 antibodies provided herein include not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also any other modified configurations of immunoglobulin molecules containing an antigen recognition site of the desired specificity, including their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins containing an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., VH-only antibodies), multispecific antibodies (e.g., bispecific antibodies), and glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies (e.g., antibody-drug conjugates or ADCs). Antibodies include any class of antibody, such as IgD, IgE, IgG, IgA, or IgM (or its subclasses), and an antibody does not have to be of any particular class. Depending on the amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional structures of the various classes of immunoglobulins are well known.

[0056] In some embodiments, anti-B7H3 antibodies are used for therapeutic purposes, for example, B7H3 +It can be formulated into pharmaceutical compositions for treating diseased cells or diseases involving the B7H3-mediated signaling pathway (e.g., cancer). To carry out such a therapeutic method, an effective amount of anti-B7H3 antibody or a pharmaceutical composition containing such antibody can be administered to a subject in need of treatment (e.g., a human patient) via a preferred route, such as intravenous infusion or subcutaneous injection.

[0057] Alternatively, such anti-B7H3 antibodies may be used to detect and optionally quantify B7H3 levels or B7H3+ cell levels in a biological sample using conventional methods, e.g., any immunohistochemical method known to those skilled in the art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985), Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting B7H3 expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Preferred assays are described in more detail elsewhere in this specification. The term “biological sample” means any biological sample obtained from an individual, cell line, tissue culture, or other cell source that potentially expresses B7H3. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0058] In some cases, the anti-B7H3 antibodies described herein can be conjugated to a detectable label, which may be any agent capable of directly or indirectly releasing a detectable signal. The presence or intensity of such a detectable signal indicates the presence or amount of the target antigen in the sample. Alternatively, a secondary antibody that is specific to the anti-B7H3 antibody or specific to the target antigen may be used in the methods disclosed herein.

[0059] (B) Other components of the chimeric antigen receptor construct In addition to the extracellular antigen-binding domains disclosed herein, any anti-B7H3 CAR may further comprise one or more intracellular signaling domains (e.g., co-stimulatory and cytoplasmic signaling domains), and optionally, a hinge domain, a spacer, a transmembrane domain, an N-terminal signal peptide, or a combination thereof. In some cases, the CAR can be co-expressed with armed polypeptides in host immune cells, thereby enhancing the physical and / or biological characteristics of the host immune cells. See, for example, the disclosures herein and related disclosures in WO2021 / 030633, WO2022 / 159771, U.S. Provisional Applications No. 63 / 340,294, and U.S. Provisional Applications No. 63 / 391,243, where each of these related disclosures is incorporated by reference for the subject matter and purposes referenced herein. In some cases, the anti-B7H3 CAR can be co-expressed with truncated EGFR, for example, as disclosed in Table 4 below. For example, a CAR coding sequence and a suicide gene may be configured in a two-cistronic expression cassette, and a CAR coding sequence and an arming gene may be linked via a self-cleaving peptide (e.g., P2A or T2A) coding sequence. Examples of CAR components are provided in Table 2 below. [Table 2]

[0060] (i) Signal transduction domain Any of the anti-B7H3 CAR constructs disclosed herein may comprise one or more intracellular signaling domains, typically containing a co-stimulatory domain and a cytoplasmic signaling domain. “Co-stimulatory signaling domain” refers to at least a fragment of a co-stimulatory signaling protein that mediates intracellular signaling to induce an immune response, such as effector function (secondary signaling). The cytoplasmic signaling domain may be any signaling domain involved in inducing cellular signaling (primary signaling) that results in the proliferation and / or activation of immune cells. The cytoplasmic signaling domains described herein are not known in the art of relaying co-stimulatory or secondary signals for the complete activation of immune cells.

[0061] In some embodiments, the co-stimulatory signaling domain and the cytoplasmic signaling domain are intended for use in the CAR constructs disclosed herein, which are introduced into T cells. In some cases, the co-stimulatory signaling domain may be derived from a co-stimulatory protein involved in the T cell response, e.g., a member of the B7 / CD28 family, a member of the TNF superfamily, a member of the SLAM family, or any other co-stimulatory molecule. Examples include, but are not limited to, 4-1BB, CD28, OX40, ICOS, CD40, CD40L, CD27, GITR, HVEM, TIM1, LFA1 (CD11a), or CD2. In a specific example, the co-stimulatory signaling domain is the 4-1BB signaling domain (e.g., Sequence ID No. 79 in Table 2 above).

[0062] The cytoplasmic signaling domain may contain an immune receptor tyrosine-based activation motif (ITAM) domain, or it may be ITAM-free. As used herein, "ITAM" refers to a conserved protein motif commonly found in the tails of signaling molecules expressed in many immune cells. An example of a cytoplasmic signaling domain is the signaling domain of CD3ζ, e.g., SEQ ID NO: 80.

[0063] (ii) Hinge and transmembrane domain In some cases, the anti-B7H3 CAR constructs disclosed herein may contain a transmembrane domain, which may be a hydrophobic alpha-helix spanning the membrane. The “transmembrane domain” may be a peptide fragment that is thermodynamically stable in the cell membrane, preferably the eukaryotic cell membrane. The transmembrane domain can provide stability to the CAR containing it. Exemplary transmembrane domains may be CD8 transmembrane domains or CD28 transmembrane domains. In one example, the transmembrane domain may include SEQ ID NO: 78 shown in Table 2 above.

[0064] Alternatively, or in addition, the CAR constructs disclosed herein may also include a hinge domain that may be located between the extracellular antigen-binding domain and the transmembrane domain, or between the transmembrane domain and the intracellular signaling domain. The hinge domain may function to provide flexibility to the CAR or its domain, or to prevent steric hindrance to the CAR or its domain. The hinge domain may contain 5 to 20 amino acid residues. In some embodiments, the hinge domain may be a CD8 hinge domain or an IgG hinge. Other hinge domains may be used. For example, the hinge domain may include Sequence ID No. 75 shown in Table 2 above.

[0065] In some examples, anti-B7H3 CARs may further include a spacer between the hinge domain and the transmembrane domain. Examples of such spacers are provided in Table 2 above (e.g., SEQ ID NO: 76 or SEQ ID NO: 77). Alternatively, anti-B7H3 CARs may lack such a spacer fragment between the hinge domain and the transmembrane domain.

[0066] (C) Anti-B7H3 chimeric antigen receptor In some embodiments, anti-B7H3 CAR constructs comprising an anti-B7H3 binding moiety and additional components disclosed herein, nucleic acids encoding such components, and host cells expressing such components are provided herein. An anti-B7H3 CAR may comprise (a) an extracellular binding domain which may be an anti-B7H3 scFv derived from any of the anti-B7H3 binding moieties, e.g., an anti-B7H3 human heavy chain antibody fragment or a reference antibody provided in Table 1 (e.g., BH-02 or BH-07), or a combination thereof; (b) a co-stimulatory signaling domain, such as those disclosed herein; and (c) a cytoplasmic signaling domain, such as those disclosed herein. An anti-B7H3 CAR may further comprise a hinge domain and a transmembrane domain located at the C-terminus of the extracellular antigen-binding domain. Optionally, an anti-B7H3 CAR may further comprise a spacer between the hinge domain and the transmembrane domain. In some cases, anti-B7H3 CARs may contain the amino acid sequences of SEQ ID NOs. 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 120. In one example, anti-B7H3 CARs may contain the amino acid sequence of SEQ ID NOs. 89 or 93 (e.g., SEQ ID NO. 93).

[0067] In some cases, the anti-B7H3 CAR constructs disclosed herein may further comprise an N-terminal signal peptide, e.g., SEQ ID NO: 70 or 71. Exemplary full-length anti-B7H3 CAR constructs (containing the signal peptide) may comprise the amino acid sequence of SEQ ID NO: 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, or 119 (e.g., SEQ ID NO: 88 or 92).

[0068] Exemplary anti-B7H3 CAR antibodies are provided in Table 3 below, all of which are within the scope of this disclosure. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0069] II. Engineered immune cells expressing anti-B7H3 CAR In some embodiments, genetically engineered immune cells, such as T cells, NK cells, or macrophages, having surface expression of any of the anti-B7H3 CAR constructs disclosed herein are provided herein.

[0070] (a) Armed CAR-T cells Any of the CAR-expressing immune cells disclosed herein may be engineered to include additional mechanisms for reprogramming the CAR-expressing cells to enhance their bioactivity and / or persistence, thereby enhancing their overall therapeutic effect. For example, CAR-expressing immune cells may be further engineered to express armed polypeptides to enhance the physical and / or biological characteristics of CAR-T cells. Such CAR-T cells are known as armed CAR-T cells that co-express one or more CAR constructs, as well as armed polypeptides that can enhance the characteristics of CAR-T cells, such as improving growth and / or persistence, enhancing efficacy, reducing toxicity, or a combination thereof.

[0071] Examples of armed polypeptides include, but are not limited to, preferred cytokines such as IL-2, IL-5, and / or IL-15, costimulatory ligands (e.g., CD80 or CD86), checkpoint inhibitors (e.g., anti-PD1 or anti-PDL1 antibody fragments), soluble receptors such as soluble PD1, TGFR2 traps, or VEGFR2 traps, and / or immune cell activating ligands (e.g., 4-1BBL). In some embodiments, the armed polypeptide may be a fusion polypeptide comprising, for example, a cytokine or a fragment thereof (e.g., IL2 or IL15 or a fragment thereof) and a checkpoint inhibitor (e.g., an anti-PDL1 fragment). Specific examples of armed polypeptides are provided in Table 4 below. [Table 4]

[0072] In some cases, the coding sequences for the CAR construct and the armed polypeptide may be located within the same expression cassette. The two coding sequences may be separated by a ribosome entry site (IRES) or a coding sequence for a self-cleaving peptide (e.g., P2A or T2A) to produce two distinct polypeptides (CAR and armed polypeptide). In other cases, two distinct expression cassettes may be used to express the CAR construct and armed polypeptide in armed CAR-T cells.

[0073] In some cases, armed polypeptides contain an N-terminal signal peptide, allowing them to be secreted from CAR-T cells. Alternatively, armed polypeptides can be expressed as intracellular or membrane-bound proteins.

[0074] In a specific example, the armed CAR-T cells disclosed herein are EPC-002 armed CAR-T cells that co-express an armed polypeptide comprising the amino acid sequence of EPLV326 anti-B7H3 CAR (SEQ ID NO: 93 or SEQ ID NO: 92) and SEQ ID NO: 110.

[0075] (b) Preparation of CAR-expressing immune cells Genetically engineered immune cells disclosed herein may be prepared by introducing one or more expression cassettes encoding any of the anti-B7H3 CAR constructs disclosed herein, optionally one or more armed polypeptides and / or truncated EGFRs, such as those disclosed herein, into suitable immune cells, and collecting the resulting engineered immune cells that express the CAR on the cell surface.

[0076] A population of immune cells as the starting parent cells can be obtained from any source, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, or tissues such as the spleen, lymph nodes, thymus, stem cells, or tumor tissue. Suitable sources for obtaining the desired type of host cells will be apparent to those skilled in the art. In some embodiments, the population of immune cells is derived from PBMCs. The desired type of host cell (e.g., T cells, NK cells, macrophages, or a combination thereof) can be proliferated within a population of cells obtained by co-incubating the cells with stimulating molecules. As a non-limiting example, anti-CD3 and anti-CD28 antibodies can be used for T cell proliferation. In some embodiments, a specific type of cell (e.g., T cells, NK cells, or macrophages) can be enriched from the immune cell population. Such enriched cell subpopulations can be proliferated and / or activated in vitro before being genetically engineered for the introduction of a CAR-coding expression cassette and / or an armed polypeptide-coding expression cassette (which may be the same expression cassette).

[0077] To construct immune cells expressing any of the anti-B7H3 CAR polypeptides described herein (e.g., those provided in Table 3), or optionally one or more armed polypeptides, such as those disclosed herein (e.g., those provided in Table 4), expression vectors for the stable or transient expression of the CAR polypeptides and optionally the armed polypeptides can be prepared by conventional methods and introduced into immune host cells. For example, nucleic acids encoding the CAR polypeptides and optionally the armed polypeptides can be cloned into one or more suitable expression vectors, such as viral vectors operably linked to a suitable promoter. Non-limiting examples of useful vectors of this disclosure include, for example, retroviral vectors, including gamma retroviral vectors, adeno-associated virus vectors (AAV vectors), and viral vectors, such as lentiviral vectors. Nucleic acids and vectors can be contacted with restriction enzymes under suitable conditions to create complementary ends on each molecule that can pair with each other and bind to ligases. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the nucleic acids encoding the CAR polypeptides and optionally the armed polypeptides. Synthetic linkers may contain nucleic acid sequences corresponding to specific restriction sites within the vector. The selection of expression vectors / plasmids / viral vectors depends on the type of host cell for the expression of CAR polypeptides, and optionally, armed polypeptides, but must be suitable for integration and replication in eukaryotic cells. Any such nucleic acids encoding CARs, and optionally, armed polypeptides, and expression vectors containing them are also within the scope of this disclosure.

[0078] Various promoters can be used for the expression of the CAR polypeptides described herein, and optionally for the expression of the armed polypeptides, including but not limited to the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian virus 40 (SV40) early promoter, or herpes simplex tk virus promoter. Additional promoters for the expression of the CAR polypeptides, and optionally for the armed polypeptides, include any constitutively active promoter in immune cells. Alternatively, any regulatory promoter may be used so that its expression can be regulated within the immune cell. In some embodiments, the promoter may be the pEF1α promoter.

[0079] In addition, the vector may contain, for example, some or all of the following: selectable marker genes such as neomycin or kanamycin genes for selecting stable or transient transfectants in host cells; enhancer / promoter sequences derived from the earliest genes of human CMV for high levels of transcription; transcription termination and RNA processing signals derived from SV40 for mRNA stability; ColE1 for the SV40 poliomavirus replication origin and proper episomal replication; internal ribosome binding sites (IRES), multiple versatile cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" (e.g., an inducible caspase such as HSV thymidine kinase or iCasp9) in case it induces a cause of death in the vector-carrying cells, as well as a reporter gene for evaluating CAR polypeptide expression.

[0080] In one particular embodiment, such a vector may also include a suicide gene. As used herein, the term “suicide gene” refers to a gene that causes the death of cells that express a suicide gene. A suicide gene may be a gene that confers sensitivity to a drug (e.g., a drug) to cells on which it expresses, causing the cells to die when they come into contact with or are exposed to the drug. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8.

[0081] The nucleic acids disclosed herein may comprise two coding sequences, one for one of the anti-B7H3 CAR constructs disclosed herein (e.g., those provided in Table 3), and the other for an armed polypeptide or truncated EGFR (e.g., those provided in Table 4). The two coding sequences may be configured such that the polypeptide encoded by the two coding sequences can be expressed as independent (and physically distinct) polypeptides. To achieve this objective, the nucleic acids described herein may comprise a third nucleotide sequence located between the first and second coding sequences. This third nucleotide sequence may, for example, encode a ribosome skipping site, a sequence that disrupts normal peptide bond formation. This mechanism results in the translation of an additional open reading frame from one messenger RNA. This third nucleotide sequence may, for example, encode a self-cleaving peptide such as a P2A, T2A, or F2A peptide (e.g., Kim et al., PLoS). See One.2011;6(4):e18556. See also Figure 5.

[0082] Any of the anti-B7H3 CAR polypeptides described herein, as well as any vectors comprising, optionally, an armed polypeptide and / or a nucleic acid sequence encoding a truncated EGFR, are also within the scope of this disclosure.

[0083] Such vectors, or CAR polypeptides contained therein, and optionally sequences encoding armed polypeptides or truncated EGFRs, can be delivered into host cells, such as host immune cells (e.g., T cells, NK cells, or macrophages), by any preferred method. Methods for delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection using reagents (e.g., liposomes), or viral transduction (e.g., retroviral transduction such as lentiviral transduction).

[0084] After introducing a vector encoding either the anti-B7H3 CAR polypeptide provided herein (e.g., provided in Table 3), or optionally the armed polypeptide and / or truncated EGFR also provided herein (e.g., provided in Table 4), into host cells, the cells may be cultured under conditions that allow for the expression of the CAR polypeptide and, optionally, the armed polypeptide and / or truncated EGFR. If the expression of the CAR polypeptide and / or armed polypeptide / truncategor EGFR is regulated by a regulating promoter, the host cells may be cultured under conditions in which the regulating promoter is activated. In some embodiments, the promoter is an inducible promoter, and the immune cells are cultured in the presence of the inducing molecule or under conditions in which the inducing molecule is produced. Determining whether the CAR polypeptide and / or armed polypeptide are expressed is obvious to those skilled in the art and can be assessed by any known method, e.g., detection of mRNA encoding the CAR polypeptide and / or armed polypeptide by quantitative reverse transcriptase PCR (qRT-PCR), or detection of the CAR or armed polypeptide protein by methods including Western blotting, fluorescence microscopy, and flow cytometry. Alternatively, the expression of functional CARs can be determined by their binding activity to target antigens, e.g., full-length B7H3 and / or splicing variant B7H3, and / or CTL activity.

[0085] Methods for preparing host cells expressing any of the CAR polypeptides and, optionally, armed polypeptides / truncate EGFR as described herein may also include ex vivo activation of the host cells. Activating host cells means stimulating the host cells to an activated state in which they can perform effector functions. The method of activating host cells will depend on the type of host cell used for expressing the CAR polypeptides and, optionally, the armed polypeptide and / or truncated EGFR. For example, T cells may be activated ex vivo in the presence of one or more molecules, including but not limited to anti-CD3 antibodies, anti-CD28 antibodies, IL-2, and / or phytohemoaglutinin. In other examples, NK cells may be activated ex vivo in the presence of one or more molecules, such as 4-1BB ligand, anti-4-1BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21, and / or K562 cells. In some embodiments, host cells expressing either the CAR polypeptides and, optionally, armed polypeptides (armed CAR cells) described herein (CAR-expressing cells) are activated ex vivo before administration to a subject. Determining whether the host cells are activated will be apparent to those skilled in the art and may involve evaluating cell activation, cytokine expression or secretion, and the expression of one or more cell surface markers related to cell morphology.

[0086] The methods described herein for preparing host cells expressing any of the CAR polypeptides and optionally armed polypeptides may include growing the host cells ex vivo. Growing the host cells may include any method that results in an increase in the number of cells expressing the CAR polypeptides and optionally armed polypeptides, for example, by enabling or stimulating the host cells to proliferate. The method for stimulating the host cell proliferation will depend on the type of host cell used for the expression of the CAR polypeptides and optionally armed polypeptides and will be apparent to those skilled in the art. In some embodiments, the host cells expressing any of the CAR polypeptides and optionally armed polypeptides described herein are grown ex vivo before administration to a subject.

[0087] In some embodiments, host cells expressing CAR polypeptides, and optionally armed polypeptides, are proliferated and activated ex vivo before administration of the cells to the target. Host cell activation and proliferation may be used to enable integration of the viral vector into the genome and expression of genes encoding the CAR polypeptides, and optionally armed polypeptides, as described herein. While mRNA electroporation may be more effective when performed on activated cells, activation and / or amplification may not be required.

[0088] In some cases, CAR polypeptides and / or armed polypeptides are transiently expressed in suitable host cells (e.g., for 3–5 days). Transient expression can be advantageous in the presence of potential toxicity and should be useful in the early stages of clinical trials for potential side effects.

[0089] (c) Pharmaceutical composition A pharmaceutical composition may be formed by mixing either the anti-B7H3 CAR disclosed herein, and optionally genetically engineered immune cells expressing armed polypeptides and / or truncated EGFRs, with a pharmaceutically acceptable carrier, which is also within the scope of this disclosure.

[0090] When used in connection with the compositions of this disclosure, the term “pharmaceutically acceptable” refers to a molecular entity and other components of such a composition that are physiologically acceptable and typically do not produce an adverse reaction when administered to a mammal (e.g., human). Preferably, as used herein, “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority or listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in animals, more specifically in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., nucleic acid, vector, cell, or therapeutic antibody) and that the composition does not adversely affect the subject to which it is administered. Any of the pharmaceutical compositions used in this method may contain a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution.

[0091] Pharmaceutically acceptable carriers containing buffers are well known in the art and may include phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or nonionic surfactants. For example, Remington: The Science and Practice of Pharmacy 20 th See Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0092] For additional examples of useful medications, also see Physician's Desk Reference, 59th sup.th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th sup.th edition, (2001), McGraw Hill, NY; and Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0093] III. Therapeutic applications Any of the genetically engineered immune cells (e.g., T cells, NK cells, and / or macrophages) expressing the anti-B7H3 CAR disclosed herein (e.g., those provided in Table 3), and optionally, armed polypeptides and / or truncated EGFR (e.g., those provided in Table 4), also disclosed herein, may be used for therapeutic purposes, for example, to eliminate undesirable cells expressing B7H3 (full-length and / or splicing variants). In some examples, the genetically engineered immune cells are armed CAR-T cells expressing any of the anti-B7H3 CAR constructs, such as those provided in Table 3 above, together with armed polypeptides, such as those provided in Table 4 above.

[0094] To carry out the methods described herein, an effective amount of any of the anti-B7H3 CARs described herein (e.g., those provided in Table 3 above), and optionally, immune cells (e.g., T lymphocytes, NK cells, or macrophages) expressing armed polypeptides and / or truncated EGFR (e.g., those provided in Table 4 above), or the pharmaceutical composition thereof, may be administered to a subject in need of treatment via a preferred route, such as intravenous administration. As used herein, an effective amount refers to the amount of each agent (e.g., CAR-T cells expressing anti-B7H3 CARs and optionally, armed polypeptides) that confers a therapeutic effect to the subject at the time of administration. Determining whether the amount of cells or compositions described herein has achieved a therapeutic effect will be obvious to those skilled in the art. The effective amount will vary, as recognized by those skilled in the art, depending on the specific condition being treated, the severity of the condition, the parameters of the individual patient (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of any concurrent therapy, the specific route of administration, and similar factors within the knowledge and expertise of the healthcare professional. In some embodiments, the effective dose reduces, alleviates, improves, enhances, reduces, or slows the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject requiring treatment is a human cancer patient.

[0095] As used herein, the term “therapeutically effective” as applied to dose or volume refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would have been effective if administered individually. In the context of this disclosure, “therapeutically effective” refers to an amount of a compound or pharmaceutical composition sufficient to delay, halt the progression of, alleviate, or reduce at least one of the symptoms of a disorder treated by the methods of this disclosure.

[0096] In some embodiments, the methods of this disclosure may be used to eliminate or inhibit disease cells expressing B7H3. Therefore, any of the immune cells disclosed herein may express B7H3 + B7H3 in cancer cells, etc. + It may be used to treat diseases associated with diseased cells. The method disclosed herein is B7H3 + It can be used to treat cancer cells, such as lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, glioblastoma, HCC, RCC, stomach cancer, esophageal cancer, prostate cancer, bladder cancer, head and neck cancer, squamous cell carcinoma, and cancers associated with leukemia. In some cases, the cancer is leukemia such as AML.

[0097] In some embodiments, any of the effective amounts of genetically engineered immune cells express the anti-B7H3 CAR disclosed herein (e.g., those provided in Table 3, such as EPLV195 or EPLV326), and optionally, armed polypeptides (e.g., those provided in Table 4, such as the fusion polypeptide of SEQ ID NO: 110) can be administered to a subject in need of treatment via a preferred route, e.g., intravenous infusion. + B7H3 in cancer cells, etc. + It may be a human patient with a disease associated with disease cells. In some cases, the human patient is B7H3 + The patient has cancer, including cancer cells. In some cases, a human patient may have lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, glioblastoma, HCC, RCC, stomach cancer, esophageal cancer, prostate cancer, bladder cancer, head and neck cancer, squamous cell carcinoma, or leukemia. In some cases, a human patient may have leukemia, such as acute myeloid leukemia (AML).

[0098] In some embodiments, the immune cells (e.g., NK and / or T cells) for use in the therapies disclosed herein may be autologous to the subject, i.e., the immune cells may be obtained from a subject in need of treatment, genetically engineered for the expression of CAR polypeptides, and then administered to the same subject. In one particular embodiment, the autologous immune cells (e.g., T lymphocytes, NK cells, or macrophages) are activated and / or proliferated ex vivo before reintroduction into the subject. Administration of autologous cells to a subject may result in a reduction of host cell rejection compared to administration of non-autologous cells.

[0099] Alternatively, genetically engineered immune cells (e.g., T cells, NK cells, or macrophages) can be allogeneic cells, i.e., cells obtained from a first subject, genetically engineered for the expression of anti-B7H3 CAR polypeptide, and administered to a second subject of the same species but different from the first subject. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient different from the donor. In certain embodiments, T lymphocytes are allogeneic T lymphocytes in which the expression of endogenous T cell receptors is inhibited or eliminated. In one particular embodiment, allogeneic T lymphocytes are activated and / or proliferated ex vivo before being introduced into a subject. T lymphocytes can be activated by any method known in the art, for example, in the presence of anti-CD3 / CD28, IL-2, and / or phytohemoaglutinin.

[0100] NK cells can be activated by any method known in the art in the presence of one or more agents selected from the group consisting of, for example, CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein, and K562 cell line. For example, see U.S. Patents 7,435,596 and 8,026,097 for a description of useful methods for growing NK cells. For example, NK cells used in the methods of this disclosure may be preferentially grown by exposure to cells that lack or have insufficient expression of major histocompatibility complex I and / or II molecules and have been genetically modified to express membrane-bound IL-15 and 4-1BB ligand (CDI37L). Examples of such cell lines include K562 [ATCC, CCL 243, Lozzio et al., Blood 45(3):321-334 (1975), Klein et al., Int. J. Cancer 18:421-431 (1976)], and Wilms tumor cell line HFWT [Fehniger et al., Int Rev Immunol 20(3-4):503-534 (2001), Harada H, et al., Exp Hematol Examples include, but are not limited to, the cell lines used (32(7):614-621(2004)), endometrial tumor cell line HHUA, melanoma cell line HMV-II, hepatoblastoma cell line HuH-6, lung small cell carcinoma cell lines Lu-130 and Lu-134-A, neuroblastoma cell lines NB19 and N1369, embryonal cancer cell lines from testicular NEC14, cervical cancer cell line TCO-2, and bone marrow metastatic neuroblastoma cell line TNB1 [Harada, et al., Jpn.J. Cancer Res 93:313-319(2002)]. Preferably, the cell lines used lack or express little to both MHCI and II molecules, such as K562 and HFWT cell lines. A solid support may be used instead of a cell line.Such a support preferably has at least one molecule bound to its surface that can bind to NK cells and induce a primary activation event and / or a proliferation response, or to a molecule having such an effect, thereby functioning as a scaffold. The support may have a CD137 ligand protein, a CD137 antibody, an IL-15 protein, or an IL-15 receptor antibody bound to its surface. Preferably, the support would have an IL-15 receptor antibody and a CD137 antibody bound to its surface.

[0101] According to this disclosure, the patient was approximately 10 5 ~10 9 Treatment can be achieved by infusing the patient with immune cells such as T lymphocytes and / or NK cells (e.g., EPLV195 or EPLV326) expressing the anti-B7H3 CAR polypeptide listed in Table 4 above, in therapeutically effective doses ranging from a range of CAR+ cells, as well as armed polypeptides (e.g., fusion polypeptides including SEQ ID NO: 110), and optionally, armed polypeptides listed in Table 4. Infusions can be repeated as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient but can be determined by the physician treating the particular patient. In some examples, approximately 10 6 Inject the initial dose of cells / kg, 10 8 The above cell / kg can be gradually increased.

[0102] The specific dosage regimens used in the methods described herein, i.e., dosage, timing, and repetitions, will depend on the specific subject and the subject's medical history. The appropriate dosage of CAR-expressing immune cells (e.g., armed CAR-T cells) used will depend on the type of cancer being treated, the severity and course of the disease, previous therapies, the patient's clinical history and response to immunotherapy, as well as the discretion of the attending physician.

[0103] In some embodiments, genetically engineered immune cells expressing any of the anti-B7H3 CAR constructs disclosed herein (e.g., armed CAR-T cells) may be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiotherapy, and gene therapy. Such therapies may be administered concurrently with or sequentially (in any order) with the immunotherapy described herein. When administered in combination with additional therapeutic agents, additive or synergistic effects may reduce the effective therapeutic dose of each agent.

[0104] IV. Kits for therapeutic use This disclosure also provides genetically engineered immune cells (e.g., T lymphocytes, NK cells, or macrophages) expressing the anti-B7H3 CAR disclosed herein (see Table 3), and optionally, kits for the use of the armed polypeptides and / or truncated EGFRs described herein. See, for example, Table 4. Such a kit may comprise one or more containers containing genetically engineered immune cells that can be formulated into a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0105] In some embodiments, the kits described herein include genetically engineered immune cells that can be grown in vitro. The immune cells may express any of the CARs disclosed herein, for example, any of the anti-B7H3 CARs, such as those provided in Table 3 above. The immune cells may be armed CAR-T cells that further express armed polypeptides (for example, those provided in Table 4 above).

[0106] In some embodiments, the kit may further include instructions for use in any of the methods described herein. The included instructions may include instructions for administering genetically engineered immune cells disclosed herein to a subject to achieve the intended activity, for example, eliminating target disease cells such as cancer cells expressing B7H3 (full-length and / or splicing variants). The kit may further include instructions for selecting a subject suitable for treatment based on whether the subject is in need of treatment.

[0107] Instructions for use of genetically engineered immune cells described herein generally include information regarding the dosage, dosage schedule, and route of administration for the intended treatment. Containers may be unit dosage forms, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions for use provided with the kits of this disclosure are typically found on the label or package insert. The label or package insert indicates that the genetically engineered immune cells are used to treat, delay the onset of, and / or alleviate, a disease or disorder associated with B7H3-positive disease cells in a subject.

[0108] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging. Packaging for use in combination with specific devices such as inhalers, nasal administration devices, or infusion devices is also intended. The kits may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). The container may also have a sterile access port.

[0109] The kit may optionally provide additional components such as buffers and interpretation information. Typically, the kit includes a container and a label or package insert(s) on or accompanying the container. In some embodiments, this disclosure provides a product containing the contents of the kit described above.

[0110] General technology Unless otherwise specified, the implementation of this invention will utilize conventional techniques within the scope of the art, including molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press; Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds.,1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985), Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985.

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[0111] Without further detail, it is expected that those skilled in the art can make the most of the present invention based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and in no way limit the remainder of this disclosure. All publications referenced herein are incorporated by reference for the purposes or subjects referenced herein.

[0112] Example 1: Discovery of human anti-B7H3 antibody This example demonstrates the development of exemplary human anti-B7H3 antibodies, including heavy chain antibodies (VH antibodies) and single-chain variable fragment (scFv) antibodies.

[0113] A. Development of exemplary human anti-B7H3 antibodies (i) Selection of human anti-B7H3 VH and scFv by mRNA display mRNA display technology was used to identify B7H3 VH and scFv conjugates from natural human VH and scFv libraries. In short, 10 8 VH and 1- 12~13The scFv DNA library was first transcribed into an mRNA library, and then translated into mRNA-VH or mRNA-scFv fusion libraries by covalent linking via a puromycin linker. This is similar to the procedure reported in U.S. Patent No. 6,258,558B1, the relevant disclosure of which is incorporated by reference for the subject and purposes referenced herein. The fusion libraries were first selected multiple times with human IgG (negative protein) to remove nonspecific binders, followed by selection for recombinant B7H3-Fc fusion protein and capture on Protein G magnetic beads. Full-length B7H3 (four Ig domains) and isoform (two Ig domains) were selected as controls in alternative rounds. The binders were eluted and then enriched by PCR amplification using library-specific oligonucleotides. In rounds 3–5, the VH and scFv libraries were selected on recombinant B7H3 / CHOK1 full-length and isoform cell lines. A total of four rounds of VH selection and six rounds of scFv selection were performed to generate a highly enriched pool of B7H3 VH and scFv binding for screening.

[0114] (ii) Identification and characterization of anti-B7H3 VH and scFv antibodies After 4–6 rounds of selection, VH and scFv libraries enriched with B7H3 were cloned into the bacterial periplasm expression vector pET22b and transformed into the top 10 competent cells. Each of the VH and scFv molecules was manipulated to have a C-terminal flag and a 6xHis tag for purification and assay detection. Clones from the top 10 cells were pooled, miniprep DNA was prepared, and then transformed into bacterial Rosetta II strain for expression. Single clones were harvested, grown, and induced in 0.1–0.5 mM IPTG in 96-well plates for expression. The supernatant was collected after 16–24 hours of induction at 30°C for assays to identify anti-B7H3 antibodies.

[0115] A B7H3 binding screening ELISA was developed for the identification of individual anti-B7H3 antibodies. Briefly, a 384-well plate was immobilized with human Fc and human B7H3-Fc (full length) at a final concentration of 2 ug / mL in 1×PBS in a total volume of 25 μL per well. The plate was incubated overnight at 4°C, followed by blocking with 80 μL of superblock per well for 1 hour. 25 μL of supernatant was added to the Fc and human B7H3 immobilized wells and incubated with shaking for 1 hour. B7H3 binding was detected by adding 25 μL of anti-frag HRP diluted 1:5000 in 1×PBST. Between each step, the plate was washed three times with 1×PBST in a plate washer. The plate was then developed with 20 μL of TMB substrate for 5 minutes and stopped by adding 20 μL of 2N sulfuric acid. The plates were read using an OD450nm Biotek plate reader, and binding and selectivity were analyzed using Excel bar graphs. Clones with B7H3 target binding over human Fc > 2x were subjected to DNA sequencing. Clones with unique sequences were produced and purified for further characterization.

[0116] (iii) Production of anti-B7H3 VH and scFv antibodies in E. coli Selective anti-B7H3 VH and scFv clones were selected from glycerol stock plates and grown overnight in 5 mL of culture in a permeable membrane Thomson 24-well plate. This culture, and all subsequent cultures described below, were grown at 37°C and 225 RPM with shaking in Terrific Broth Complete supplemented with 100 μg / mL carbenicillin and 34 μg / mL chloramphenicol, with the addition of 1:5,000 dilution of antifoaming agent-204 unless otherwise specified. Then, using this overnight starter culture, larger cultures, or starter cultures diluted 1:100, were inoculated into the specified production cultures and grown until the OD600 reached 0.5–0.8. At this point, the cultures were induced with a final concentration of 0.1 mM IPTG and incubated overnight at 30°C. The following day, the culture was rotated at 5,000 × g for 30 minutes to pelletize the cells, and then the supernatant was filtered and sterilized through a 0.2 μm sterile PES membrane.

[0117] For purification, 3 μL of GE Ni Sepharose Excel resin was used per 1 mL of filtered supernatant. Disposable 10 mL or 20 mL BioRad Econo-Pac columns were used. The resin was equilibrated with at least 20 column volumes (CV) of Buffer A (containing additional NaCl added to 1 × PBS, pH 7.4, 500 mM). The filtered supernatant was purified by gravity flow, either by controlling the flow rate to 1 mL / min or by pouring it twice onto the same packed resin bed. The column was then washed with the following buffers: 10 CV Buffer A, 20 CV Buffer B (containing additional NaCl at 1 × PBS, pH 7.4, 500 mM, and 30 mM imidazole). Endotoxin removal was performed as an optional step if necessary, using two Detox buffers. For the purification of 250 mL of expression culture, the antibody-conjugated column was sequentially washed with 20CV buffer C (1×PBS pH 7.4, additional 500 mM NaCl, 1% Tx114), 20CV buffer D (1×PBS pH 7.4, additional 500 mM NaCl, 1% Tx100 + 0.2% TNBP), and 40CV buffer E (1×PBS pH 7.4, additional 500 mM NaCl). The protein was eluted in a total of six fractions (0.5 CV pre-elution, 5×1 CV elution) with elution buffer F (1×PBS pH 7.4, additional 500 mM NaCl, and 500 mM imidazole). The fractions were subjected to the Bradford assay (100 μl diluted Bradford solution + 10 μl sample). The bright blue fractions were pooled. Protein concentration was measured by the A280 elongation coefficient. The purity of the purified antibody was analyzed using an SDS-PAGE gel.

[0118] B. Characterization of exemplary human anti-B7H3 antibodies (i) Determination of anti-B7H3 VH and scFv antibody binding affinity by ELISA To determine the EC50 of the anti-B7H3 antibody, an ELISA assay was developed. Briefly, a 384-well plate was immobilized with the anti-human B7H3-Fc recombinant protein at a final concentration of 2 μg / mL in 1×PBS in a total volume of 25 μL per well. The plate was incubated overnight at 4°C, followed by blocking with 80 μL of superblock per well for 1 hour. Purified anti-B7H3 VH or scFv was titrated 2x sequentially from 200 nM. 25 μL of diluted VH or scFv was added to the human B7H3 immobilized wells and incubated for 1 hour with shaking. B7H3 binding was detected by adding 25 μL of anti-frag HRP diluted 1:5000 in 1×PBST. Between each step, the plate was washed three times with 1×PBST in a plate washer. Next, the plates were developed with 20 μL of TMB substrate for 5 minutes and stopped by adding 20 μL of 2N sulfuric acid. The plates were read with an OD450 nm Biotek plate reader and then plotted using Prism 8.1 software. EC50 was calculated and is shown in Table 5 below. The VH and scFv clones, with the exception of the BH-03 clone, showed moderate to high affinity binding to the B7H3 recombinant protein. [Table 5]

[0119] (ii) Binding kinetics of anti-B7H3 VH and ScFv antibodies to B7H3 by SPR The dynamic analysis of anti-B7H3 VH and scFv was evaluated using SPR technique with Biacore T200. The assay was performed using Biacore T200 control software version 2.0. Fc fusion proteins were captured in the assay using a Protein A sensor chip. For each cycle, 1 ug / mL of human B7H3-Fc protein was captured at a flow rate of 10 μL / min for 60 seconds on flow cell 2 in 1×HBSP buffer on the Protein A sensor chip. Purified anti-B7H3 VH or scFv with HIS tags, serially diluted 2-fold, was injected at a flow rate of 30 μL / min for 150 seconds into both reference flow cell 1 and B7H3-Fc capture flow cell 2, followed by washing for 300 seconds. The flow cells were then regenerated with glycine pH2 buffer (GE) at a flow rate of 30 μL / min for 60 seconds. Eight concentration points ranging from 300 to 0 nM per anti-B7H3 VH or scFv were assayed in a 96-well plate.

[0120] The kinetics of VH and scFv binding to the B7H3 protein were analyzed using Biacore T200 evaluation software version 3.0. Specific binding response units originated from the subtraction of binding from B7H3 capture flow cell 2 to reference flow cell 1. on , K off The KD values ​​were calculated for the selected ScFv antibodies and are shown in Table 6. Antibodies were generated by live cell-based selection in full-length and splice forms of the B7H3 recombinant cell line, and lower KD values ​​were observed in this assay format due to the different epitope exposures on cells and as soluble proteins. [Table 6]

[0121] (iii) Conjugation of VH and ScFv antibodies to B7H3-positive cell lines by FACS analysis CHOK1 cells were transfected with constructs encoding full-length and splice variant isoforms of the human B7H3 sequence with a C-terminal flag and Myc tag in a pCMV6-Entry vector. A polyclonal, drug-resistant pool of B7H3 target-expressing cells was obtained using the G418 drug selection process. B7H3 target-expressing cells were sorted by FACS to obtain a B7H3 target-expressing polyclonal pool. High-expression B7H3 / CHOK1 and splice variant B7H3 / CHOK1 cell lines were then used in selection and screening assays. B7H3 expression levels in a panel of B7H3 / CHOK1 and B7H3-expressing cancer cell lines were quantified by FACS using MESF microsphere beads (Bangs Laboratories, Inc., 647) for standard calibration according to the manufacturer's protocol. B7H3 receptor counts are summarized in Table 7. [Table 7]

[0122] To determine the binding selectivity and affinity of anti-B7H3 VH and scFv antibodies to cells expressing B7H3, 200 nM purified VH and scFv antibodies were diluted in complete medium and incubated on ice with recombinant B7H3 / CHOK1 cell lines (full-length, splicing variant) and CHOK1 cells in 96-well plates for 1 hour. The cells were spun down at 1200 rpm for 6 minutes at 4°C to remove the primary antibody. The cells were then washed once with 200 μL of complete medium per well. The samples were detected with pre-mixed anti-His biotin streptavidin Alexa fluor647 by adding 100 μL of diluted secondary antibody and incubated in the dark for 30 minutes at 4°C. The samples were spun down at 1200 rpm for 5 minutes at 4°C and washed twice with 200 μL of 1×PBS per well. The samples were reconstituted in 200 μL of 1×PBS and read using an Attune NxT flow cytometer. Analysis was performed using Attune NxT software, plotting overlaid histograms of anti-B7H3 VH and scFv binding to both negative and target cell lines. Anti-B7H3 VH and scFv showed selective binding to B7H3 / CHOK1 cells but not to CHOK1 parental cells (not shown). Full-length and isoform B7H3 cell binding affinities to VH and ScFv antibodies were also generated using the serially diluted VH and scFv described above. All anti-B7H3 VH and scFv showed binding to both isoforms of B7H3 cell lines with similar affinities, as seen in Figures 1A–1B. 50 The values ​​were calculated and are shown in Table 8. [Table 8]

[0123] The results demonstrate that exemplary anti-B7H3 antibodies (either in heavy chain antibody form or scFv form) can bind to both isoforms of the B7H3 receptor.

[0124] (iv) Binding of anti-B7H3 VH and scFv to cancer cells expressing endogenous B7H3 Cell binding activity with cancer cell lines expressing B7H3 was also assayed by FACS according to the method described above. 200 nM anti-B7H3 VH or scFv was tested for binding ability to cell lines expressing recombinant B7H3 (full-length or splice variant) or cancer cell lines expressing endogenous B7H3. As shown in Figures 2A-2C, the tested scFv antibodies showed binding activity that canceled out cells expressing high, medium, and low levels of endogenous B7H3. BH-07 scFv showed strong binding to most endogenous B7H3 cell lines, exhibiting moderate to high affinity (Figures 2D-2E and Table 9). [Table 9]

[0125] (v) Epitope binding of anti-B7H3 VH and scFv antibodies to enobrituzumab by FACS Purified anti-B7H3 VH antibody was titrated from 100–1000 nM in 2-fold serial dilutions. Each diluted sample was mixed with 20 nM enobrituzumab IgG antibody and then incubated with either full-length or isoform B7H3 / CHOK1 cells at 4°C for 1 hour, while the diluted VH was assayed in parallel. The cells were spun down at 1200 rpm for 5 minutes at 4°C. The samples were detected with pre-mixed anti-His biotin streptavidin Alexa fluor647 by adding 100 μL of diluted secondary antibody and incubated in the dark at 4°C for 30 minutes. The samples were spun down at 1200 rpm for 5 minutes at 4°C and washed twice with 200 μL of 1×PBS per well. The resulting samples were reconstituted in 200 μL of 1×PBS and read using an Attune NxT cytometer. The analysis was performed using Attune NxT software, and then plotted using Prism 8.1 software.

[0126] As shown in Figures 3A–3C, enobrituzumab does not compete with the anti-B7H3 VH antibody for binding to both isoforms of B7H3 expressed on CHOK1 cells, indicating that exemplary anti-B7H3 VH antibodies bind to different epitopes for enobrituzumab.

[0127] (vi) Epitope binning of anti-B7H3 VH and scFv antibodies by surface plasmon resonance (SPR) To develop the biparatopic B7H3 CAR, the binding epitopes of anti-B7H3 VH and scFv were evaluated. An epitope binning assay was developed using Biacore T200. Briefly, human B7H3-Fc tagged protein was immobilized on the protein A sensor chip FC2 at a flow rate of 10 μL / min for 60 seconds at 1 μg / mL. 300 nM VH1 or scFv1 was injected into FC1 and FC2 at a flow rate of 30 μL / min for 90 seconds to reach binding saturation, followed by the injection of 300 nM VH2 or scFv2 at the same flow rate and time. Data were analyzed using Biacore T200 evaluation software version 3.0. Dual baselines were established for analysis. Binding response units were calculated by subtracting FC1 from FC2. Figures 4A–4B show competitive binding across multiple VH and scFv antibodies. The epitope binning data is summarized in Table 10 below. [Table 10]

[0128] (vii) Anti-B7H3 VH antibody thermal stability test The scFv melting temperature was measured using Protein Thermal Shift® Dye (Thermo Fisher, 4461146). Test VH sample reactions were prepared according to the manufacturer's instructions. The plates or strips were then placed in a Quant Studio3 instrument and the method described above was performed. Step 1: 100% ramp plate to 25.0°C in 2 minutes, and finally Step 2: 1% ramp plate to 99.0°C in 2 minutes. The samples and subsequent Tm were then analyzed (and Tm calculated) using QuantStudio Design and Analysis Software and Protein Thermal Shift Software 1.3. The Tm values ​​for exemplary anti-B7H3 antibodies are summarized in Table 11. [Table 11]

[0129] Example 2: Construction of an anti-B7H3 chimeric antigen receptor This example demonstrates the construction of an anti-B7H3 chimeric antigen receptor (CAR) using exemplary anti-B7H3 antibodies disclosed herein, including VH and scFv forms.

[0130] A.CAR Structure Three anti-B7H3 VH antibodies (BH-01, BH-02, and BH-03) and four anti-B7H3 scFv antibodies (BH-04 to BH-08) were selected to construct anti-B7H3 monospecific CARs. From N-terminus to C-terminus, the CAR constructs provided herein include an anti-B7H3 VH or scFv antigen fragment, a flag tag, an IgG4 hinge, a spacer (optional), a CD28 transmembrane domain, a costimulatory domain from 4-1BB, and an intracellular signaling domain from CD3ζ. The spacer fragment has a variable length as shown for candidate screening and optimization. Some CAR constructs include only the hinge domain without the spacer fragment. The CAR constructs also include an N-terminal signal peptide to facilitate their positioning on the cell surface. Biparatopic anti-B7H3 CAR constructs containing two anti-B7H3 antibody fragments disclosed herein were prepared according to the guidance provided above.

[0131] In some cases, the coding sequence of the CAR construct is linked to the coding sequence of membrane-bound EGFRt via the coding sequence of the T2A cleavage linker and secretory signaling. In other cases, the coding sequence of the CAR construct is linked to the coding sequence of an armed polypeptide (e.g., a fusion polypeptide containing anti-PDL1 scFv and engineered human IL-2) via the coding sequence of the T2A cleavage linker. Expression cassettes of exemplary CAR constructs, either alone or in combination with FGFRt or armed polypeptides, are shown in Figure 5. Sequences of exemplary anti-B7H3 CAR constructs and their functional fragments are provided in Tables 1-4.

[0132] Nucleic acids encoding CAR were cloned into lentiviral vectors, either alone or in combination with EGFRt or armed polypeptides, according to standard molecular biological methods. The resulting lentiviral vectors were co-transfected with Expi HEK293 cells using polyethyleneimine (PEI) transfection reagent and LV-MAX packaging mix, according to the manufacturer's protocol. Transfected cells were grown at 37°C for 72 hours with shaking at 8% CO2 level. The supernatant was centrifuged at 3200 rpm at room temperature for 10 minutes and collected by vacuum filtration using a 0.45 μm PES membrane. The viruses were concentrated by ultracentrifugation (Beckman Coulter) at 12,000 rpm for 1 hour at 4°C. The pellet was then resuspended in lentiviral stabilizer, immediately divided into equal portions, and stored at -80°C.

[0133] B.B7H3 monospecific CAR-T cell transduction and expansion PBMCs were isolated from fresh healthy donor LRS chambers using density gradient centrifugation with lymphhoprep and the SepMate50 kit from Stemcell Technology. CD3+ Pan T cells were then isolated from the PBMCs using the EasySep human T cell isolation kit, according to the Stemcell Technology protocol. Pan T cells were activated with human T activator CD3 / CD28 DynaBeads in a 1:1 bead-to-cell ratio for 24 hours, and then transduced with lentivirus in the presence of DynaBeads and 1 mg / mL protamine sulfate. Spinoculation was performed at 300 g for 2 hours at 25°C. Cells and viruses were incubated at 37°C for 24 hours. The following day, cells were removed from the beads and viruses. Cells were grown for 4–9 days in 5% human serum containing recombinant human IL15 and IL7 (Peprotech) in X-vivo15 (Lonza) medium. The culture medium was changed every 2-3 days, and fresh cytokines were added.

[0134] C. Characterization of anti-B7H3 CAR-T cells (i) Surface manifestation CAR surface expression was evaluated by surface staining with an anti-flag-tagged antibody directly conjugated with Mix-n-stain AF647. Briefly, 100,000 lentiviral transducer T cells were incubated with 0.1 μL of anti-flag-AF647 in the dark for 1 hour with shaking at 4°C. The cells were spun down at 1,300 rpm for 5 minutes, the supernatant was removed, and the cells were washed with 200 μL of 1×PBS. The resulting samples were reconstituted in 200 μL of 1×PBS. Surface expression percentages were quantified by reading the fluorescently stained cells using an Attune NxT flow cytometer. CAR expression for different CAR constructs ranged from 50–85%.

[0135] (ii) Anti-B7H3 VH CAR activity Isolation, viral transduction, and T cell proliferation of human PBMCs and Pan T cells were described above. VH CARs with different spacers were constructed. See Table 3 for sequence information. To screen for different CAR activities, a real-time image-based CTL activity assay was performed using GFP-engineered glioblastoma cell line U87 MG. Briefly, CAR-transduced T cells and non-transduced T cells from the same donor were incubated with 10,000 U87MG-GFP in RPMI1640 medium containing 10% FBS, in a 10:1 effector (CAR-T) to target cells (cancer cell line) ratio. No cytokines were added. The assay was run for 42 hours, and GFP of target cells was imaged and quantified using a Cytation 5 scanner. Data were further analyzed using Prism 8 software.

[0136] Figure 6A shows the CTL activity of VH CAR-T cells using different sequences and spacers. The percentage of target cell killing endpoints by different CAR-T cells were calculated as shown in Figure 6B. Three CAR constructs, EPLV23 (BH-03 medium spacer), EPLV25 (BH-02 medium spacer), and EPLV47 (BH-02 long spacer), showed potent CTL activity. EPLV24 (BH-1 moderate spacer) and EPLV30 (BH-1, no spacer) showed moderate CTL activity. The binding activity of the VH CAR constructs was also confirmed by evaluating cytokine release by CAR-T cells expressing B7H3 when incubated with multiple B7H3-expressing cancer cell lines, including U87MG (glioma cell line), A172 (glioblastoma), and Raji (human B lymphoblastoma cell line). Non-transduced donor T cells (UTDs) were used as a control. IFNγ was detected using the Human IFNγ Duoset ELISA Kit (R&D System). Recombinant IFNγ was serially diluted and measured in the assay to construct a standard curve. Supernatant IFNγ and recombinant IFNγ were assayed according to the manufacturer's protocol. Data were analyzed using Prism 8.0 software. High levels of IFNγ secretion by VH CAR-T cells upon target cell engagement were observed, which is consistent with the CTL activity described above. Figure 7.

[0137] EPLV25 was further evaluated by CTL assays for selectivity against parental CHOK1-GFP, B7H3 / CHOK-1 GFP, U87 MG-CHOK1, and UTD controls at an effector-to-target ratio of 10:1. EPLV25 showed specific and potent B7H3 target cell-killing activity (Figure 8). CAR-T cells produced from multiple donors have been shown to exhibit dose-dependent activity of EPLV25.

[0138] (iii) Anti-B7H3 scFv CAR activity Five B7H3 scFv CAR-T cells were produced in accordance with the disclosures provided herein. Their CTL activity and cytokine secretion capacity were analyzed as described herein. Surface expression of anti-B7H3 scFv CAR constructs was detected on CAR-T cells containing anti-flag-tagged antibodies, as shown in Table 12 below. All CAR constructs showed expression levels greater than 80%. [Table 12]

[0139] CAR-T cells were co-incubated for 64 hours at an effector-to-target cell ratio of 5:1 with engineered CHOK1 cells expressing GFP, full-length or splice variant B7H3, HEK293 cells, or U87MG cells (expressing B7H3 receptors at different levels). Untransduced T cells (UTDs) from the same donor were used as controls. Potent and specific CAR-T mediated CTL activity was observed in co-culture with T cells expressing anti-B7H3 CARs EPLV195, EPLV196, or EPLV199. Activity correlated with B7H3 receptor expression levels in target cells. Nonspecific cytotoxic activity was observed in CAR-T cells expressing EPLV197 or EPLV198, which may be due to tonic signaling, Figure 9A. CAR-T cells also produced high levels of IFNγ upon target cell engagement, results consistent with CTL activity and specificity, Figure 9B.

[0140] (iv) scFv CAR-T cell proliferation during target cell engagement To further investigate CAR-T cell expansion upon target cell engagement, a 3-day proliferation assay was performed using EPLV195 and EPLV196 as examples. Transduced T cells were labeled with Cell Trace Far Red at a final concentration of 1 μM. 20,000 labeled T cells were co-cultured with 20,000 CHOK1, B7H3 / CHOK1 (full-length and splice variant), and U87-MG target cells, respectively, in an E:T ratio of 1:1. The assay was set up in RPMI medium with 10% FBS, and fresh medium was added to the cells every two days. No cytokines were added to the medium during the assay. CAR-T proliferation was analyzed using an Attune NxT flow cytometer. CAR-T cells demonstrated target cell-specific expansion upon engagement over 3 days, correlating with target expression levels on the cells. CAR-T cells expressing EPLV195 showed higher cell dilation capacity than CAR-T cells expressing EPLV196, Figures 10A-10B.

[0141] (v) Characterization of anti-B7H3 biparatopic CAR activity The anti-B7H3 VH and scFv antibodies disclosed herein demonstrate broad binding epitopes to B7H3. From initial VH CAR and scFv CAR screening (see disclosure above), potent and specific VH and scFv CAR constructs were identified. Biparatopic CAR constructs containing tandem VH-scFv binding moieties, and CAR-T cells expressing such constructs, were constructed to search for CAR constructs with enhanced activity.

[0142] Four anti-B7H3 biparatopic CAR constructs were constructed, including: EPLV235 (BH-02 and BH-04), EPLV236 (BH-02 and BH-05), EPLV237 (BH-02 and BH-06), and EPLV238 (BH-02 and BH-07). Structural information for these biparatopic CAR constructs is provided in Table 3 above. CAR-T cells expressing such anti-B7H3 CARs were produced. Their CTL activity and cytokine secretion capacity were evaluated as described above. Expression levels of the anti-B7H3 biparatopic CAR constructs on primary T cells obtained from different donors ranged from 30 to 85%. Exemplary results from CAR-T cells derived from one donor are provided in Table 13 below. [Table 13]

[0143] Anti-B7H3 CAR-T cells were co-incubated with GFP / CHOK1, B7H3 / CHOK1, HEK293, or U87 MG target cells. CTL activity and IFNγ secretion were evaluated as described herein. As an example, EPLV236 showed potent and specific target cell-killing activity and high levels of IFNγ release, as shown in Figures 11A-11B.

[0144] Example 3: Characterization of anti-B7H3 CAR candidates This embodiment illustrates the characterization of an exemplary anti-B7H3 CAR construct.

[0145] Characterization of A.EPLV195 scFv CAR For further characterization, EPLV195 expression, CTL activity, and cytokine release of CAR-T cells derived from multiple donors were tested in the presence of multiple target and control cell lines with different effector-to-target cell ratios, as described. EPLV195 was confirmed to have robust surface expression in T cells (e.g., approximately 96% in CAR-T cells derived from a single donor), and CAR-T cells expressing this anti-B7H3 CAR exhibited potent and specific target cell-killing activity and cytokine release. Figures 12A–12B show the results obtained from EPLV195-expressing CAR-T cells prepared from a single donor.

[0146] B.EPLV195 scFv CAR Spacer Optimization and In Vitro Sustainability Evaluation The space between the scFv-binding domain and the transmembrane domain in a CAR construct is known to be important for providing an optimal immune synapse between CAR-expressing immune cells and target tumor cells, and for mediating the efficacy of antitumor activity. To optimize anti-B7H3 CAR constructs, candidate anti-B7H3 CAR constructs without spacers, culture medium spacers, or longer spacers between the hinge and the transmembrane domain in the CAR construct were constructed and evaluated according to the disclosures provided herein (see Figure 5). The sequences of these CAR constructs are provided in Table 3 as SEQ ID NOs: 88, 90, and 92 (including the signal peptide).

[0147] CAR-T cells expressing the anti-B7H3 CAR construct described above were incubated with the A375-GFP cell line for 72 hours at an effector-to-target cell ratio of 2.5:1 or 1:1 without additional cytokines. The CAR-T cells were rechallenged twice with A375 cancer cells, and each rechallenge was incubated for an additional 72 hours without the addition of CAR-T cells or cytokines. Similar cytotoxic activity was observed in the CAR-T cells during the first 72 hours of the assay. In the first rechallenge assay, CAR-T cells expressing the anti-B7H3 CAR without a spacer (SEQ ID NO: 92) showed better CTL activity than CAR-T cells expressing the CAR construct with a long spacer (SEQ ID NO: 88). CAR-T cells expressing the CAR without a spacer (SEQ ID NO: 92) or the culture medium spacer (SEQ ID NO: 90) showed similarly potent and sustained CTL activity. Figures 13A-B.

[0148] C. CAR-T cells co-expressing anti-B7H3 scFv CAR candidate and armed polypeptide A multi-mechanism armed polypeptide containing an anti-PDL1 scFv fused with an engineered IL2 was engineered to co-express with an exemplary anti-B7H3 scFv CAR (EPLV326) via a T2A-cleavable linker. The armed polypeptide contains a secretory signal at its N-terminus. The sequences of the armed polypeptide and the anti-B7H3 CAR are provided in Tables 3 and 4. Further details of the armed polypeptide are also referred to in U.S. Provisional Applications 63 / 340,294 and 63 / 391,243, the relevant disclosures of which are incorporated by reference to the subject and purposes referenced herein. The anti-PDL1 scFv fragment in the armed polypeptide blocks the PDL1 immune checkpoint and localizes the armed polypeptide to tumor cells. The engineered IL-2 fragment therein significantly reduces IL2Rα binding activity and suppresses Treg cell activation. In addition, IL2 binding to IL2Rβ / γ was also fine-tuned to selectively activate central memory T cells at a reduced activity and slower rate. During transduction and CAR-T activation, the armament is secreted outside the CAR-T cells, activating CAR-T cells and other bystander immune cells, thereby improving efficacy and persistence by encountering multiple mechanisms of the suppressive tumor microenvironment.

[0149] The in vitro activity of candidate CARs and armed CARs (co-expression of CAR and armed polypeptide) was compared using the CTL assay and A375 cancer cell rechallenge assay described above. No significant differences were observed in the 2.5:1 or 1:1 effector-to-target cell ratios in the 5-round killing and rechallenge assay. Figures 14A and 14B.

[0150] The supernatants from co-cultures of CAR-T cells expressing anti-B7H3 CAR (EPLV326) and CAR-T cells expressing both CAR and armed polypeptide (EPLV330) were also tested for binding activity to PDL1 / K562 cells. The results are shown in Figure 15.

[0151] Example 4: In Vivo Efficacy of Anti-B7H3 CAR-T Cells in a Xenograft Mouse Model To evaluate the antitumor activity of EPC-002 armed CAR-T cells (co-expressing the EPLV326 anti-B7H3 CAR and the arming polypeptide disclosed herein), 6- to 8-week-old female NCG mice (Charles River Laboratories, Wilmington, MA) were subcutaneously inoculated in the right hind flank with 5 × 10 6 A375 human melanoma tumor cells suspended in a 1:1 mixture of serum-free medium and Matrigel (Corning, Glendale, Arizona). When the tumors reached an average volume of approximately 120 mm 3 The mice were randomized into five treatment groups. The treatment groups (n = 5 mice / group) were as follows: (1) vehicle, (2) EPC-002 unarmed CAR-T at a 2E6 CAR-T cell dose, (3) EPC-002 unarmed CAR-T at a 10E6 CAR-T cell dose, (4) EPC-002 armed CAR-T cells at a 2E6 CAR-T cell dose, and (5) EPC-002 armed CAR-T cells at a 10E6 CAR-T cell dose.

[0152] Treatments were administered by intravenous injection on the day of randomization and again 9 days later. Tumor volume and mouse body weight were recorded twice weekly and plotted against time for each group. All animals treated with EPC-002 armed CAR-T cells demonstrated complete tumor regression 23-27 days after the first treatment with CAR-T.

[0153] No weight loss was observed, and no adverse clinical signs were noted. Subsequently, these animals were subcutaneously inoculated in the left hind flank with 5 × 10 6The A375 tumor cells were re-challenged. Five naive, age-matched female NCG mice were simultaneously inoculated with A375 tumor cells to serve as a control cohort. Once tumors became palpable, tumor volume and body weight were recorded twice weekly. While the control-treated mice showed tumor growth, EPC-002-armed CAR-T demonstrated sustained antitumor activity. Figures 16A and 16B. All mice from both the 2E6 and 10E6 dose levels in the EPC-002-armed CAR-T treatment group showed complete tumor regression.

[0154] CAR-T cell phenotype is associated with the persistence of antitumor activity. Blood was collected from mice 20 days after CAR-T treatment. T cell and CAR-T cell phenotypes were analyzed using a FACS assay with a panel of antibodies that detect T cell differentiation markers. Briefly, transduced T cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, and anti-CD62L, as described above. Analysis was performed using Attune NxT software. Human CD3, CD4, and CD8-positive CAR-T cells and Tn, Tscm, Tcm, and Tem cells were gated.

[0155] Figures 17A-17B characterize the subtypes of transduced CAR-T cells before treatment. On day 20, dose-dependent proliferation of total T cells, CD8 T cells, and EPC-002 armed CAR-T cells was observed in the armed CAR-T treatment group. Over 70% of CD8+ T cells exhibited naive and central memory T cell phenotypes, Figures 18A-18B. In addition, both CD4+, CD8+ T cells, and EPC-002 armed CAR-T cells were found to be homing to the spleen on day 64. Over 90% of CD8+ armed CAR-T cells were central memory T cells in PBMCs and the spleen, indicators of persistence, Figures 19A-19B.

[0156] Example 5: Dose-response of antitumor activity of EPC-002 armed CAR-T cells To evaluate the minimum effective dose of EPC-002 armed CAR-T cells, 6-8 week old female NCG mice (Charles River Laboratories, Wilmington, MA) were given 5 × 10⁶ doses of EPC-002 armed CAR-T cells suspended in 1:1 serum-free medium and Matrigel (Corning, Glendale, Arizona). 6 Individual A375 human melanoma tumor cells were subcutaneously inoculated. The tumor was approximately 110 mm. 3 When the mean tumor volume was reached, the mice were randomized into four treatment groups. The treatment groups (n=5 mice / group) were as follows: (1) vehicle, (2) EPC-002-armed CAR-T with a dose of 0.3E6 CAR+T cells, (3) EPC-002-armed CAR-T with a dose of 1E6 CAR+T cells, and (4) EPC-002-armed CAR-T with a dose of 3E6 CAR-T cells. Treatment was administered by intravenous injection on the day of randomization and again 7 days later. Tumor volume and mouse body weight were recorded twice weekly and plotted against time for each group. All animals in the EPC-002 treatment groups showed complete tumor regression 25-28 days after treatment with low-dose CAR-T cells, Figure 20A. The mean body weight of each treatment group increased progressively over time, Figure 20B.

[0157] Example 6: Antitumor activity of EPC-002-armed CAR-T cells with tumor rechallenge To evaluate the minimum effective dose of EPC-002 armed CAR-T cells, 6-8 week old female NCG mice (Charles River Laboratories, Wilmington, MA) were given 5 × 10⁶ doses of EPC-002 armed CAR-T cells suspended in 1:1 serum-free medium and Matrigel (Corning, Glendale, Arizona). 6 Individual A375 human melanoma tumor cells were subcutaneously inoculated. The tumor was approximately 110 mm. 3When the average volume was reached, the mice were randomized into four treatment groups. The treatment groups (n = 5 mice / group) were as follows: (1) vehicle (PBS), (2) EPC-002 (0.3E6 CAR+T cell dose), (3) EPC-002 (1E6 CAR+T cell dose), and (4) EPC-002 (3E6 CAR-T cell dose). Treatments were administered by intravenous injection on the day of randomization and again 7 days later. Tumor volume and mouse body weight were recorded twice a week and plotted against time for each group. All animals administered 1E6 or 3E6 CAR+T cells showed complete tumor regression 25 - 28 days after the first treatment with CAR-T. Four out of five animals administered 0.3E6 CAR+T cells showed complete tumor regression. Forty days after the first CAR-T cell treatment, mice previously given a dose of 0.3E6 CAR+T cells were rechallenged with Capan-2 pancreatic tumor cells (1 × 10 6 tumor cells and Matrigel) in the left flank. As a control for tumor growth, five age-matched naive NCG mice were also inoculated with Capan-2 cells in the left flank. Tumor volume and mouse body weight were recorded once a week and plotted against time for each group. Capan-2 tumor growth was prevented in mice previously treated with CAR T cells (Figure 21A). No significant weight loss was observed over time in any of the treatment groups (Figure 21B).

[0158] Example 7: Pharmacokinetics and Pharmacodynamics of EPC-002 CAR-T Cells To evaluate tumor infiltration and in vivo expansion of EPC-002 CAR-T cells, 6 - 8-week-old female NCG mice (Charles River Laboratories, Wilmington, MA) were subcutaneously inoculated in the right hind flank with 5 × 10 6 A375 human melanoma tumor cells suspended in a 1:1 mixture of serum-free medium and Matrigel (Corning, Glendale, Arizona). When the tumors were approximately 350 mm 3When the mean volume was reached, mice were randomized into two treatment groups (n=9 mice / group): (1) vehicle (PBS) and (2) EPC-002 (3E6 CAR+ cells). Tumors and spleens were collected from three animals in each group on days 3, 7, and 10 after the start of treatment for immunophenotyping or immunohistochemical analysis. Tumor volume was also captured twice weekly from all mice in the study. Tumor volume in mice treated with EPC-002 CAR-T cells showed a significant reduction over time compared to mice treated with vehicle controls. Figure 22A. Immunohistochemical staining of CAR constructs demonstrated EPC-002 infiltration into A375 tumors on day 10 post-treatment, and CD4 and CD8 T cell markers demonstrated that unmanipulated immune cells infiltrated and expanded within the tumor microenvironment. Immunophenotypic analysis detected the presence of primarily naive human CD4 and CD8 cells in the spleens of EPC-002-treated mice on day 7 post-treatment. Figure 22B.

[0159] Example 8: Antitumor activity of EPC-002-armed CAR-T cells in the H1975 NSCLC tumor model To evaluate the antitumor effects of EPC-002-armed CAR-T cells against NSCLC, female NCG mice aged 6-8 weeks (Charles River Laboratories, Wilmington, MA) were subjected to 1.3 × 10⁶ doses suspended in 1:1 serum-free medium and Matrigel (Corning, Glendale, Arizona). 6 Individual H1975 human NSCLC tumor cells were subcutaneously inoculated. The tumor was approximately 95 mm in size. 3 When the mean tumor volume was reached, the mice were randomized into three treatment groups, each containing 5 mice. The treatment groups were: (1) vehicle (PBS), (2) EPC-002 (2E6 CAR+T cell dose), and (3) EPC-002 (10E6 CAR+T cell dose). Treatment was administered by intravenous injection on the day of randomization. Tumor volume and mouse body weight were recorded twice a week and plotted against time for each group. All animals treated with EPC-002 showed tumor regression (Figure 23A) and maintained body weight throughout the study period (Figure 23B).

[0160] Other Embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly stated, each feature disclosed is merely an example of a general set of equivalent or similar features.

[0161] From the above description, those skilled in the art will readily recognize the essential features of this disclosure and can make various changes and modifications to the invention to suit various uses and conditions without departing from its spirit and scope. Accordingly, other embodiments are also within the scope of the claims.

[0162] Equal portions While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for carrying out the function and / or obtaining one or more of the results and / or advantages described herein, and each of such variations and / or modifications will be considered to fall within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on the particular application(s) to which the teachings of the present invention are used. Those skilled in the art will be able to recognize or confirm many equivalents to the particular embodiments of the present invention described herein by means of routine experimentation alone. Therefore, it should be understood that the embodiments described herein are presented merely as examples, and embodiments of the present invention may be carried out in ways other than those specifically described or claimed, within the scope of the appended claims and their equivalents. Embodiments of the present invention in this disclosure cover each of the individual features, systems, articles, materials, kits and / or methods described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the inventions of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not inconsistent with each other.

[0163] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated herein by reference, and / or the ordinary meanings of the terms defined.

[0164] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they refer to, and in some cases the entire document may be included.

[0165] In this specification and in the claims, the indefinite articles "a" or "an" should be understood to mean "at least one" unless explicitly stated otherwise.

[0166] As used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, that is, elements that may coexist in some cases and, in other cases, elements that exist exclusively. Similarly, any multiple elements listed in “and / or” should be interpreted as “one or more” of the elements thus combined. Other elements other than those specifically identified by the “and / or” clause may exist at their discretion, whether related to the specifically identified elements or not. Thus, as a non-restrictive example, a reference to “A and / or B” when used in combination with unrestrictive language such as “including” may, in one embodiment, refer to A only (optionally including elements other than B), in another embodiment, refer to B only (optionally including elements other than A), in yet another embodiment, refer to both A and B (optionally including other elements), and so on.

[0167] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, meaning that it includes not only at least one element from a number of elements or a list of elements, but also two or more elements and, optionally, additional items not listed. Only terms that clearly indicate the opposite meaning, such as “one of” or “exactly one of” or, when used in the claims, “consisting of,” would refer to including exactly one element from a number of elements or a list. In general, where used herein, the term “or” should be interpreted only as indicating an exclusive choice (i.e., “one or the other, but not both”) when preceded by an exclusive term such as “either,” “one of,” “one of,” or “exactly one of.” Where used in the claims, “essentially consisting of” should have the usual meaning as used in the field of patent law.

[0168] As used herein in this specification and in the claims, the phrase “at least one” should be understood to mean at least one element selected from any one or more elements in a list of elements, but not necessarily including at least one of all elements specifically enumerated in that list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the existence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether or not they are related to the specifically identified elements, at the discretion of the system. Therefore, as a non-restrictive example, “at least one of A and B” (or equivalently, “at least one of A or B,” or equivalently, “at least one of A and / or B”) could mean, in one embodiment, at least one A (and optionally including elements other than B) in which B is absent and which is of any choice one or more; in another embodiment, at least one B (and optionally including elements other than A) in which A is absent and which is of any choice one or more; and in yet another embodiment, at least one A and at least one B (and optionally including other elements) in which B is of any choice one or more.

[0169] Furthermore, if any method claimed in this specification includes two or more steps or operations, it should be understood that, unless explicitly stated otherwise, the order of the steps or operations of the method is not necessarily limited to the order in which the steps or operations of the method are listed.

Claims

1. A chimeric antigen receptor (CAR) (anti-B7H3 CAR) that binds to B7 homolog 3 protein (B7H3), (a) An extracellular antigen binding site specific to human B7H3, (b) Co-stimulatory signaling domain and (c) comprising a cytoplasmic signaling domain, The extracellular antigen-binding portion of (a) (i) A human heavy chain antibody containing the same heavy chain complementation determining region (CDR) as the reference antibody of BH-01, BH-02, or BH-03, and / or (ii) Heavy chain variable region (V Ｈ ) and light chain variable region (V Ｌ A single-chain variable fragment (scFv) containing the V Ｈ and the V Ｌ However, a single-chain variable fragment (scFv) containing the same heavy and light chain CDRs as the reference antibody of BH-04, BH-05, BH-06, BH-07, BH-08, BH-09, or BH-10, Contains anti-B7H3 CAR.

2. The anti-B7H3 CAR according to claim 1, wherein the extracellular antigen-binding portion of (a) in the CAR comprises the human heavy chain antibody comprising the amino acid sequence of SEQ ID NO: 4, 8, 9, or 13.

3. The extracellular antigen-binding portion of (a) in the CAR includes the scFv, (i) The above V Ｈ This includes sequence number 17, and the V Ｌ Does it include sequence number 21? (ii) The above V Ｈ This includes sequence number 25, and the V Ｌ Does it include sequence number 29? (iii) the V Ｈ includes SEQ ID NO: 33, and the V Ｌ includes SEQ ID NO: 37 or (iv) The VH includes sequence number 41 and the VL includes sequence number 45, (v) The above V Ｈ This includes sequence number 49, and the V Ｌ Does it include sequence number 53? (vi) The above V Ｈ This includes sequence number 57, and the V Ｌ This includes sequence number 61, or (vii) The above V Ｈ This includes sequence number 65, and the V Ｌ This includes sequence number 69, The anti-B7H3 CAR according to claim 1.

4. The anti-B7H3 CAR according to any one of claims 1 to 3, wherein the extracellular antigen-binding portion of (a) in the CAR comprises both the human heavy chain antibody and the scFv.

5. The anti-B7H3 CAR according to claim 4, wherein the human heavy chain antibody is derived from BH-02, which optionally contains the amino acid sequence of SEQ ID NO: 8 or 9, and the scFv is derived from BH-04, BH-05, BH-06, or BH-07, which optionally contains the amino acid sequence of SEQ ID NO: 112, 113, 114, or 115.

6. The anti-B7H3 CAR according to any one of claims 1 to 5, wherein the co-stimulatory domain of (b) is from a co-stimulatory molecule selected from the group consisting of CD28, 4-1BB, OX40, ICOS, CD27, CD40, and CD40L.

7. The anti-B7H3 CAR according to any one of claims 1 to 6, wherein the cytoplasmic signaling domain of (c) is derived from CD3ζ.

8. An anti-B7H3 CAR according to any one of claims 1 to 7, further comprising a hinge domain, a transmembrane domain, or a combination thereof, wherein the hinge domain and / or the transmembrane domain is optionally located between (a) the extracellular antigen-binding portion and (b) the costimulatory domain.

9. The anti-B7H3 CAR according to claim 8, wherein the hinge domain comprises the amino acid sequence of SEQ ID NO: 75 and / or the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

78.

10. The anti-B7H3 CAR according to claim 8 or 9, further comprising a spacer located between the hinge domain and the transmembrane domain, wherein the spacer optionally comprises the amino acid sequence of SEQ ID NO: 76 or 77.

11. The anti-B7H3 CAR according to claim 1, comprising any one amino acid sequence from sequence numbers 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, and 120, and optionally comprising the amino acid sequence of sequence number 89 or 93.

12. The anti-B7H3 CAR according to any one of claims 1 to 11, further comprising an N-terminal signal peptide.

13. The anti-B7H3 CAR according to claim 12, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 70 or 71.

14. A nucleic acid comprising a first nucleotide sequence encoding the anti-B7H3 CAR according to any one of claims 1 to 13.

15. The nucleic acid according to claim 14, further comprising a second nucleotide sequence encoding an armed polypeptide or a truncated EGFR fragment that enhances T cell functionality, and optionally a third nucleotide encoding a self-cleaving peptide located between the first nucleotide sequence and the second nucleotide sequence.

16. The nucleic acid according to claim 15, wherein the second nucleotide sequence further encodes a signal peptide located at the N-terminus of the armed polypeptide or the truncated EGFR.

17. The nucleic acid according to claim 15 or 16, wherein the second nucleotide sequence encodes the armed polypeptide selected from the group consisting of IL-2, IL-5, IL-15, a costimulatory ligand, an anti-PDL1 antibody, and a fusion polypeptide comprising the anti-PDL1 antibody, and optionally the anti-PDL1 antibody is a single-chain variable fragment (scFv).

18. The nucleic acid according to claim 17, wherein the second nucleotide sequence encodes the fusion polypeptide comprising the anti-PDL1 antibody and the IL-2 polypeptide, and the anti-PDL1 antibody is an scFv fragment optionally comprising the amino acid sequence of SEQ ID NO:

108.

19. The nucleic acid according to claim 18, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

110.

20. The nucleic acid according to claim 15 or 16, wherein the second nucleotide sequence optionally encodes the abbreviated EGFR, comprising the amino acid sequence of sequence number 111.

21. The nucleic acid according to any one of claims 14 to 20, wherein the nucleic acid is an expression vector, or optionally a viral vector.

22. A population of genetically modified immune cells, wherein the genetically modified immune cells express the anti-B7H3 CAR described in any one of claims 1 to 13.

23. The population of genetically modified immune cells according to claim 22, wherein the immune cells include T cells, NK cells, macrophages, or a combination thereof, and optionally, the immune cells are T cells.

24. A population of genetically modified immune cells according to claim 22 or 23, further expressing the armed polypeptide and / or truncated EGFR according to any one of claims 15 to 20.

25. The population of genetically modified immune cells according to any one of claims 22 to 24, wherein the genetically modified immune cells comprise (a) the anti-B7H3 CAR, and optionally (b) a nucleic acid or set of nucleic acids encoding the armed polypeptide and / or the truncated EGFR.

26. The population of genetically modified immune cells according to claim 25, wherein the genetically modified immune cells include the nucleic acid described in any one of claims 14 to 21.

27. A method for eliminating B7H3-expressing disease cells in a subject, comprising administering an effective amount of a population of genetically modified immune cells described in any one of claims 22 to 26 to the subject in need thereof.

28. The method according to claim 27, wherein the diseased cells are cancer cells.

29. The method according to claim 27 or 28, wherein the subject is a human patient having B7H3-expressing cancer.

30. The method according to claim 29, wherein the cancer is a B7H3-positive cancer, and optionally, the cancer is a leukemia that is lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, glioblastoma, HCC, RCC, gastric cancer, esophageal cancer, prostate cancer, bladder cancer, head and neck cancer, squamous cell carcinoma, and optionally, acute myeloid leukemia (AML).

31. An antibody that binds to human B7 homolog 3 protein (B7H3) (anti-B7H3 antibody), (a) A human heavy chain antibody containing the same heavy chain complementation determining region (CDR) as the reference antibody of BH-01, BH-02, or BH-03, or (b) Heavy chain variable region (V Ｈ ) and light chain variable region (V Ｌ ) and the above V Ｈ and the V Ｌ However, the heavy chain variable region (V) contains the same heavy chain and light chain CDR as the reference antibody of BH-04, BH-05, BH-06, BH-07, BH-08, BH-09, or BH-10. Ｈ ) and light chain variable region (V Ｌ Anti-B7H3 antibodies, including ).

32. The anti-B7H3 antibody according to claim 31, comprising the human heavy chain antibody of (a), wherein the antibody optionally includes the amino acid sequence of SEQ ID NO: 4, 8, 9, or 13.

33. The antibody is (b) the V Ｈ and the V Ｌ Includes, (i) The above V Ｈ This includes sequence number 17, and the V Ｌ Does it include sequence number 21? (ii) The above V Ｈ This includes sequence number 25, and the V Ｌ Does it include sequence number 29? (iii) The above V Ｈ This includes sequence number 33, and the V Ｌ Does it contain sequence number 37? (iv) The VH includes sequence number 41 and the VL includes sequence number 45, (v) The above V Ｈ This includes sequence number 49, and the V Ｌ Does it include sequence number 53? (vi) The above V Ｈ This includes sequence number 57, and the V Ｌ This includes sequence number 61, or (vii) The above V Ｈ This includes sequence number 65, and the V Ｌ This includes sequence number 69, The anti-B7H3 antibody according to claim 31.

34. A nucleic acid or a series of nucleic acids that collectively encode the anti-B7H3 antibody according to any one of claims 31 to 33.

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