Anti-CD276 and anti-CD99 logic-gated chimeric antigen receptors for cancer treatment

Abstract

This disclosure provides compositions and methods for logic-gate CAR-T therapies for treating cancers such as DIPG, Ewing sarcoma, and AML, including logic pairs of CAR polypeptides comprising anti-CD276 CAR polypeptide and anti-CD99 CAR polypeptide.

Description

[Technical Field]

[0001] This disclosure provides compositions and methods for logic-gate CAR-T therapies for treating cancers such as DIPG, Ewing sarcoma, and AML, including logic pairs of CAR polypeptides comprising anti-CD276 CAR polypeptide and anti-CD99 CAR polypeptide.

[0002] Related applications This application claims priority to and benefit from U.S. Provisional Patent Application No. 63 / 489,724, filed March 10, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] government support This invention was made with government support under K12 CA086913-18, awarded by the National Cancer Institute. The government has certain rights in this invention.

[0004] Sequence List The sequence listing XML accompanying this application is provided electronically in XML file format and is incorporated herein by reference. The XML file containing the sequence listing XML is named "UNCO-064_001WO_SeqList". The XML file is 87,472 bytes in size, was created on March 10, 2024, and filed electronically through the USPTO Patent Center. [Background technology]

[0005] Diffuse pontine glioma (DIPG) is the most aggressive type of childhood brain tumor and the leading cause of brain tumor-related death in children. DIPG diffusely invades the pons, which makes the tumor surgically inoperable and results in a median survival time of 11 months for children with this tumor. Radiotherapy provides only temporary relief, and chemotherapy is ineffective. For the same reasons, the 5-year survival rate has remained constant at 0% since 1950. Recent studies have shown that somatic Lys27Met substitution at histone 3.3 (H3.3K27M mutation) occurs in over 85% of patients with DIPG and is associated with poor survival. While these studies provide crucial insights into driver mutations, they have yet to lead to novel therapeutic options. Therefore, there is a critical need to identify and validate more effective biology-based therapies to target DIPG, including DIPG specifically arising from the presence of the H3K27M mutation. [Overview of the project]

[0006] In recent years, chimeric antigen receptor (CAR) T cells targeting DIPG have shown promise in preclinical models and are in development for clinical trials. However, a major obstacle to the use of CAR T cell therapy for the treatment of solid tumors (e.g., DIPG) is the absence of truly "tumor-only" antigens. The vast majority of target antigens are also expressed by multiple normal cells, which creates a risk of targeting normal cells in addition to cancer cells. In the context of DIPG, single-antigen-targeted CAR-T cells have limited efficacy due to low antigen density on tumors, which can lead to off-target toxicity and tumor evasion. For example, preliminary data have shown that CD56 CAR-T cells effectively target DIPG cells but cause neurotoxicity in mice due to cross-reactivity with normal brain cells. Furthermore, CD99 CAR-T cells initially demonstrate complete clearance of tumor tissue volume in DIPG xenograft models, but tumor recurrence can occur. While we do not wish to be limited by theory, since they also express the target antigen CD99, relapse is likely to be due to a lack of CAR-T cells (i.e., cell fratricide). Therefore, there is a need in the art for improved CAR-T cell therapy for DIPG. This disclosure addresses this need by providing a "logical pair" of CAR polypeptides (as well as accompanying methods and compositions) for use in the treatment of various cancers, including DIPG.

[0007] In addition to DIPG, the logical pairs of CAR polypeptides of this disclosure, as well as their associated methods and compositions, can also be used in the treatment of other cancers such as acute myeloid leukemia (AML), Ewing's sarcoma, posterior fossa type A (PFA) ependymoma, and atypical teratomatous rhabdoid tumor (ATRT).

[0008] AML is a blood cancer in which the bone marrow of a subject produces abnormal myeloblasts, red blood cells, or platelets. AML is one of the most common forms of acute leukemia in adults. The increase in AML cells in the bone marrow and blood can rapidly lead to infection, anemia, excessive bleeding, and death. Venetoclax, a BCL-2 inhibitor, has emerged in recent years as a key component of therapy for acute myeloid leukemia (AML). The current FDA-approved standard of care for the majority of patients who are too old or unsuitable for aggressive chemotherapy is venetoclax in combination with azacitidine ("Ven / aza treatment") or a hypomethylating agent such as decitabine. It is estimated that about 70% of these patients achieve complete remission (CR) of their disease with Ven / aza treatment. However, it is estimated that about 30% of patients do not respond to treatment with ven / aza and are unable to achieve CR. Therefore, there is an unmet need in the art for improved compositions and methods for treating AML, including in subjects that would not respond to treatment with Ven / aza.

[0009] Ewing's sarcoma is a type of cancer that primarily affects bone or soft tissue. While it can occur in any bone, it is most commonly found in the hip bone, ribs, or long bones (e.g., femur, tibia, or humerus). The tumor may also affect the surrounding muscles and soft tissues. Ewing's sarcoma cells can also metastasize (spread) to other parts of the body, including the bone marrow, lungs, kidneys, heart, adrenal glands, and other soft tissues. As the second most common type of bone cancer affecting children and young adults, Ewing's sarcoma accounts for about 1 percent of childhood cancers. Approximately 225 children and adolescents are diagnosed with Ewing's sarcoma each year in the United States. While Ewing's sarcoma can occur at any point in childhood, it most commonly develops during adolescence when bones are growing rapidly. Ewing's sarcoma most frequently occurs in children between the ages of 10 and 20. Over the past 40 years, both local therapy and multi-agent adjuvant chemotherapy have achieved significant progress in the treatment of localized disease, improving the 5-year survival rate from less than 20% to over 70%, although the recurrence rate remains high. However, metastatic disease, which is mostly localized and asymptomatic, is present in almost all cases. Approximately 25% of patients with initially localized disease eventually relapse. There is no standard of care for relapsed, refractory Ewing sarcoma, and survival rates are less than 30% in patients with solitary lung metastases and less than 20% in patients with bone and bone marrow lesions. Therefore, there is an unmet need in the art for improved compositions and methods for treating Ewing sarcoma, including in subjects with relapsed, refractory disease.

[0010] [Overview of the prefecture] This disclosure provides a pair of chimeric antigen receptor (CAR) polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, wherein the anti-CD276 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD276 antigen-binding domain comprising an anti-CD276 scFv comprising the amino acid sequence of SEQ ID NO: 12; (iii) a hinge domain comprising a CD28 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 40; (iv) a transmembrane domain comprising a CD28 transmembrane polypeptide comprising the amino acid sequence of SEQ ID NO: 44; and the anti-CD99 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD99 antigen-binding domain comprising an anti-CD99 scFv comprising the amino acid sequence of SEQ ID NO: 38; (iii) a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (iv) (v) comprising a transmembrane domain containing a CD8 transmembrane polypeptide containing the amino acid sequence of SEQ ID NO: 46; and an activation domain containing a CD3ζ activating polypeptide containing the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 63, and the anti-CD99 CAR contains the amino acid sequence of SEQ ID NO: 65.

[0011] This disclosure provides a pair of CAR polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, wherein the anti-CD276 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD276 antigen-binding domain comprising an anti-CD276 scFv comprising the amino acid sequence of SEQ ID NO: 12; (iii) a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (iv) a transmembrane domain comprising a CD8 transmembrane polypeptide comprising the amino acid sequence of SEQ ID NO: 46; and a costimulatory domain comprising a 4-1BB costimulatory polypeptide comprising the amino acid sequence of SEQ ID NO: 50; and the anti-CD99 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD99 antigen-binding domain comprising an anti-CD99 scFv comprising the amino acid sequence of SEQ ID NO: 38; (iii) a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (iv) (v) comprising a transmembrane domain containing a CD8 transmembrane polypeptide containing the amino acid sequence of SEQ ID NO: 46; and an activation domain containing a CD3ζ activating polypeptide containing the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 64, and the anti-CD99 CAR contains the amino acid sequence of SEQ ID NO: 65.

[0012] This disclosure provides a pair of chimeric antigen receptor (CAR) polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, wherein the anti-CD99 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD99 antigen-binding domain comprising an anti-CD99 scFv comprising the amino acid sequence of SEQ ID NO: 38; (iii) a hinge domain comprising a CD28 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 40; (iv) a transmembrane domain comprising a CD28 transmembrane polypeptide comprising the amino acid sequence of SEQ ID NO: 44; and the anti-CD276 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD276 antigen-binding domain comprising an anti-CD276 scFv comprising the amino acid sequence of SEQ ID NO: 12; (iii) a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (iv) (v) comprising a transmembrane domain containing a CD8 transmembrane polypeptide containing the amino acid sequence of SEQ ID NO: 46; and an activation domain containing a CD3ζ activating polypeptide containing the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 61, and the anti-CD99 CAR contains the amino acid sequence of SEQ ID NO: 62.

[0013] This disclosure provides a pair of CAR polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, wherein the anti-CD99 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD99 antigen-binding domain comprising an anti-CD99 scFv comprising the amino acid sequence of SEQ ID NO: 38; (iii) a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (iv) a transmembrane domain comprising a CD8 transmembrane polypeptide comprising the amino acid sequence of SEQ ID NO: 46; and a costimulatory domain comprising a 4-1BB costimulatory polypeptide comprising the amino acid sequence of SEQ ID NO: 50; and the anti-CD276 CAR polypeptide comprises, from N-terminus to C-terminus, (i) a signal peptide; (ii) an anti-CD276 antigen-binding domain comprising an anti-CD276 scFv comprising the amino acid sequence of SEQ ID NO: 12; (iii) a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (iv) (v) comprising a transmembrane domain containing a CD8 transmembrane polypeptide containing the amino acid sequence of SEQ ID NO: 46; and an activation domain containing a CD3ζ activating polypeptide containing the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 60, and the anti-CD99 CAR contains the amino acid sequence of SEQ ID NO: 62.

[0014] This disclosure provides a nucleic acid molecule comprising at least one nucleic acid sequence encoding an anti-CD276 CAR polypeptide from a pair of CAR polypeptides of the Disclosure; and at least one nucleic acid sequence encoding an anti-CD99 CAR polypeptide from a pair of CAR polypeptides of the Disclosure. In some embodiments, the at least one nucleic acid sequence encoding the anti-CD276 CAR polypeptide and the at least one nucleic acid sequence encoding the anti-CD99 CAR polypeptide are separated by at least one nucleic acid sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a P2A self-cleaving peptide.

[0015] This disclosure provides vectors comprising nucleic acid molecules of this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an AAV vector or a lentiviral vector.

[0016] This disclosure provides cells expressing the CAR polypeptide pairs of this disclosure. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells, NK cells, NK-like cells, NKT cells, or cytokine-induced killer (CIK) cells. In some embodiments, the immune cells are T cells.

[0017] This disclosure provides a population of cells of the present disclosure.

[0018] This disclosure provides a method for treating cancer in a subject, comprising the step of administering one or more amounts of a population of cells of this disclosure to the subject.

[0019] This disclosure provides a method for preventing cancer metastasis in a subject, comprising the step of administering one or more amounts of a population of cells of this disclosure.

[0020] In some embodiments, the method further includes the step of administering at least one additional therapy to the subject. In some embodiments, the at least one additional therapy includes at least one of radiotherapy, chemotherapy, and surgery.

[0021] In some cases, the cancer is diffuse pontine glioma (DIPG), acute myeloid leukemia (AML), Ewing's sarcoma, posterior fossa type A (PFA) ependymoma, or atypical teratomatous rhabdoid tumor (ATRT).

[0022] Any of the embodiments described above, or any embodiment described herein, may be combined with any other embodiment.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. In this specification, the singular also includes the plural unless the context clearly indicates otherwise; for example, the terms “a,” “an,” and “the” are understood to be singular or plural, and the term “or” is understood to be inclusive. For example, “an element” means one or more elements. Throughout this specification, the word “contains,” or variations such as “contains” or “contains,” will be understood to imply the inclusion of a specified element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. "Approximately" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all figures provided herein are modified by the term "approximately."

[0024] Similar or equivalent methods and materials may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. References cited herein are not deemed to be prior art to the claimed invention. In case of any inconsistency, this specification, including definitions, shall prevail. In addition, materials, methods, and examples are illustrative and not intended to be limiting. Other features and advantages of this disclosure will be apparent from the detailed description and claims below.

[0025] The above and further features will be more clearly understood from the following detailed description when interpreted in conjunction with the attached drawings. [Brief explanation of the drawing]

[0026] [Figure 1A] This figure shows the genetic engineering of a dual-antigen CAR. Figure 1A shows a schematic diagram of a CAR designed so that only binding to both antigens on the same cell leads to the complete activation of the CAR. [Figure 1B] This figure shows the genetic manipulation of a dual antigen CAR. Figure 1B shows two types of logic gate type CAR constructs. [Figure 2] This figure shows that the CD99 / CD276 logic gate CAR construct protects single-antigen T cells and inhibits T cell sibling killing. Figure 2 shows ELISA measurements of cytokine production in CD99-only expressing T cells after treatment with either mock CAR-T cells, single-antigen CD99-BBz CAR-T cells, single-antigen CD276-BBz CAR-T cells, or CD99 / CD276 logic gate CAR-T cells. [Figure 3] This figure shows that the CD99 / CD276 logic gate type CAR construct protects single-antigen T cells and inhibits T cell sibling killing. Figure 3 shows the percentage survival rate of CD99-expressing CAR-T cells over time after transduction using each CAR construct. [Figure 4A] This figure shows the functionality of single-antigen CAR-T cell therapy for DIPG. Figure 4A shows XCELLigence real-time cell proliferation measurements of DIPG007 tumor cells treated with single-antigen CD99 CAR-T cells and single-antigen CD276 CAR-T cells compared with DIPG007 tumor cells treated with single-antigen CD19 CAR-T cells. [Figure 4B] This figure shows the functionality of single-antigen CAR-T cell therapy for DIPG. Figure 4B shows ELISA measurements of cytokine production in DIPG007 cells after treatment with single-antigen CD19 CAR-T cells, single-antigen CD99 CAR-T cells, or single-antigen CD276 CAR-T cells. [Figure 4C]This figure shows the functionality of single-antigen CAR-T cell therapy for DIPG. Figure 4C shows ELISA measurements of cytokine production in DIPG BT245 cells treated with single-antigen CD19 CAR-T cells or single-antigen CD99 CAR-T cells. [Figure 5A] This figure shows the functionality of CD99 / CD276 logic gate type CAR-T cells against DIPG in vitro. Figure 5A shows the XCELLigence real-time cell proliferation measurement of DIPG007 tumor cells treated with CD99 / CD276 logic gate type CAR-T cells compared with DIPG007 tumor cells treated with single antigen CD19 CAR-T cells. [Figure 5B] This figure shows the in vitro functionality of CD99 / CD276 logic gate CAR-T cells against DIPG. Figure 5B shows ELISA measurements of cytokine production in DIPG007 cells after treatment with single-antigen CD19 CAR-T cells or CD99 / CD276 logic gate CAR-T cells. [Figure 5C] This figure shows the functionality of CD99 / CD276 logic gate type CAR-T cells against DIPG in vitro. Figure 5C shows CAR-T cell death as measured by the increase in caspase 3 / 7 incucyte reagent when co-cultured with RAJI cells expressing only CD99. [Figure 6A] This figure shows the functionality of CD99 / CD276 logic gate CAR-T cells against DIPG in vivo. Figure 6A shows Kaplan-Meier survival analysis of BT245 pontine-containing DIPG tumor mouse models after single-dose systemic infusion of single-antigen CD19 CAR-T cells, single-antigen CD99 CAR-T cells, single-antigen CD276 CAR-T cells, or CD99 / CD276 logic gate CAR-T cells. [Figure 6B]This figure shows the functionality of CD99 / CD276 logic gate CAR-T cells against DIPG in vivo. Figure 6B shows Kaplan-Meier survival analysis of DIPG007 pontine-containing DIPG tumor mouse models after single-dose systemic infusion of single-antigen CD19 CAR-T cells, single-antigen CD99 CAR-T cells, single-antigen CD276 CAR-T cells, or CD99 / CD276 logic gate CAR-T cells. [Figure 7] This figure shows the functionality of CD99 / CD276 logic gate type CAR-T cells against PFA EPN in vitro. Figure 7 shows the XCELLigence real-time cell proliferation measurement of PFN EPN MAF928 cells treated with CD99 / CD276 logic gate type CAR-T cells compared with PFN EPN MAF928 tumor cells treated with single antigen CD19 CAR-T cells (mock). [Figure 8] This figure shows the in vitro functionality of CD99 / CD276 logic gate type CAR-T cells against PFA EPN. Figure 8 shows ELISA measurements of cytokine production in PFN EPN MAF928 cells after treatment with single antigen CD19 CAR-T cells or CD99 / CD276 logic gate type CAR-T cells. [Figure 9] This figure shows the Kaplan-Meier survival analysis of ATRT tumor mouse models after a single-dose systemic infusion of single-antigen CD19 CAR-T cells or CD99 / CD276 logic-gate type CAR-T cells. [Figure 10] This figure shows the functionality of CD99 / CD276 logic gated CAR-T cells against Ewing sarcoma in vivo. Figure 10 shows Kaplan-Meier survival analysis of Ewing sarcoma tumor mouse models after single-dose infusion of single-antigen CD19 CAR-T cells, single-antigen CD99 CAR-T cells, single-antigen CD276 CAR-T cells, or CD99 / CD276 logic gated CAR-T cells. [Figure 11]This figure shows the antitumor efficacy of CD99 / CD276 logic gate type CAR-T cells against CD276-low expressing K562 cells. Figure 11 shows tumor volume measured by caliper over time after CAR-T cell injection. [Modes for carrying out the invention]

[0027] Anti-CD276 CAR polypeptide This disclosure provides an anti-CD276 CAR polypeptide comprising, essentially comprising, or comprising, an anti-CD276 antigen-binding domain, a transmembrane domain, and at least one costimulatory domain, from the N-terminus to the C-terminus.

[0028] The disclosure also provides an anti-CD276 CAR polypeptide comprising, essentially consisting of, or consisting of, an anti-CD276 antigen-binding domain, a transmembrane domain, and an activating domain, from N-terminus to C-terminus.

[0029] In some embodiments, the anti-CD276 CAR polypeptides of the present disclosure may further include a hinge domain located between the anti-CD276 antigen-binding domain and the transmembrane domain. Thus, the present disclosure provides anti-CD276 CAR polypeptides comprising, essentially comprising, or comprising, an anti-CD276 antigen-binding domain, a hinge domain, a transmembrane domain, and at least one costimulatory domain from N-terminus to C-terminus. The present disclosure also provides anti-CD276 CAR polypeptides comprising, essentially comprising, or comprising, an anti-CD276 antigen-binding domain, a hinge domain, a transmembrane domain, and an activating domain from N-terminus to C-terminus.

[0030] In some embodiments, the anti-CD276 CAR polypeptides of the present disclosure may further include a signal peptide at the N-terminus of the anti-CD276 CAR polypeptide. Thus, the present disclosure provides anti-CD276 CAR polypeptides comprising, essentially comprising, or comprising, a signal peptide, an anti-CD276 antigen-binding domain, a transmembrane domain, and at least one costimulatory domain, from N-terminus to C-terminus. The present disclosure also provides anti-CD276 CAR polypeptides comprising, essentially comprising, or comprising, a signal peptide, an anti-CD276 antigen-binding domain, a transmembrane domain, and an activating domain, from N-terminus to C-terminus. As described above, the anti-CD276 CAR polypeptides of the present disclosure may also include a hinge domain. Thus, the present disclosure provides anti-CD276 CAR polypeptides comprising, essentially comprising, or comprising, a signal peptide, an anti-CD276 antigen-binding domain, a hinge domain, a transmembrane domain, and at least one costimulatory domain, from N-terminus to C-terminus. The disclosure also provides an anti-CD276 CAR polypeptide comprising, essentially consisting of, or consisting of, a signal peptide, an anti-CD276 antigen-binding domain, a hinge domain, a transmembrane domain, and an activating domain, from N-terminus to C-terminus.

[0031] The anti-CD276 antigen-binding domain, transmembrane domain, costimulatory domain, signal peptide, hinge domain, and activation domain of the anti-CD276 CAR polypeptides of this disclosure may be selected from any of the anti-CD276 antigen-binding domain, transmembrane domain, costimulatory domain, signal peptide, hinge domain, and activation domain of the anti-CD276 CAR polypeptides described in more detail herein.

[0032] The following are exemplary anti-CD276 CAR polypeptides of the present disclosure.

[0033] This disclosure provides an anti-CD276 CAR polypeptide comprising, essentially comprising, or comprising, a signal peptide, an anti-CD276 antigen-binding domain containing anti-CD276 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and an activation domain containing a CD3ζ activating polypeptide, from N-terminus to C-terminus. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide.

[0034] This disclosure provides an anti-CD276 CAR polypeptide comprising, essentially, or consisting thereof, an anti-CD276 antigen-binding domain comprising a signal peptide, an anti-CD276 scFv, wherein the anti-CD276 scFv comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 12, a hinge domain comprising a CD8 hinge polypeptide, wherein the CD8 hinge polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 42, a transmembrane domain comprising a CD8 transmembrane polypeptide, wherein the CD8 transmembrane polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 46, and an activation domain comprising a CD3ζ activating polypeptide, wherein the CD3ζ activating polypeptide comprises an activation domain comprising an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 52. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide, which contains an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 58.

[0035] This disclosure provides an anti-CD276 CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD276 antigen-binding domain containing anti-CD276 scFv, wherein the anti-CD276 scFv comprises an anti-CD276 antigen-binding domain containing the amino acid sequence of SEQ ID NO: 12, a hinge domain containing a CD8 hinge polypeptide, the CD8 hinge polypeptide comprising a hinge domain containing the amino acid sequence of SEQ ID NO: 42, a transmembrane domain containing a CD8 transmembrane polypeptide, the CD8 transmembrane polypeptide comprising a transmembrane domain containing the amino acid sequence of SEQ ID NO: 46, and an activation domain containing a CD3ζ activating polypeptide, wherein the CD3ζ activating polypeptide comprises, essentially consists of, or consists of an activation domain containing the amino acid sequence of SEQ ID NO: 52. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide, the GMCSF signal peptide comprising the amino acid sequence of SEQ ID NO: 58.

[0036] In some embodiments, the anti-CD276 CAR polypeptide comprises, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 62.

[0037] This disclosure provides an anti-CD276 CAR polypeptide comprising, essentially comprising, or comprising, a signal peptide, an anti-CD276 antigen-binding domain containing an anti-CD276 scFv, a hinge domain containing a CD28 hinge polypeptide, a transmembrane domain containing a CD28 transmembrane polypeptide, and at least one costimulatory domain containing at least one CD28 costimulatory polypeptide, from N-terminus to C-terminus. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide.

[0038] This disclosure relates to an anti-CD276 antigen-binding domain comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD276 scFv, wherein the anti-CD276 scFv comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 12, a hinge domain comprising a CD28 hinge polypeptide, wherein the CD28 hinge polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 40, a transmembrane domain comprising a CD28 transmembrane polypeptide, wherein the CD28 transmembrane polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 44, and at least one costimulatory domain comprising at least one CD28 costimulatory polypeptide, wherein the at least one CD28 costimulatory polypeptide comprises at least one costimulatory domain comprising an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 48, or is essentially composed of, or composed of, an anti-CD276 The present invention provides a CAR polypeptide. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide, the GMCSF signal peptide having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 58.

[0039] This disclosure provides an anti-CD276 CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD276 antigen-binding domain containing anti-CD276 scFv, wherein anti-CD276 scFv comprises an anti-CD276 antigen-binding domain containing the amino acid sequence of SEQ ID NO: 12, a hinge domain containing a CD28 hinge polypeptide, a transmembrane domain containing a CD28 transmembrane polypeptide, a transmembrane domain containing the amino acid sequence of SEQ ID NO: 40, and at least one costimulatory domain containing at least one CD28 costimulatory polypeptide, wherein the at least one CD28 costimulatory polypeptide comprises, essentially consists of, or consists of at least one costimulatory domain containing the amino acid sequence of SEQ ID NO: 48. In some embodiments of the anti-CD276 CAR polypeptide described above, the signal peptide may include a GMCSF signal peptide, the GMCSF signal peptide containing the amino acid sequence of SEQ ID NO: 58.

[0040] In some embodiments, the anti-CD276 CAR polypeptide comprises, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 63.

[0041] The disclosure also provides an anti-CD276 CAR polypeptide comprising, essentially consisting of, or consisting of, a signal peptide, an anti-CD276 antigen-binding domain containing an anti-CD276 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and at least one costimulatory domain containing at least one 4-1BB costimulatory polypeptide, from N-terminus to C-terminus. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide.

[0042] This disclosure also includes, essentially consists of, or consists of, an anti-CD276 antigen-binding domain comprising a signal peptide, an anti-CD276 scFv, wherein the anti-CD276 scFv comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 12, a hinge domain comprising a CD8 hinge polypeptide, wherein the CD8 hinge polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 42, a transmembrane domain comprising a CD8 transmembrane polypeptide, wherein the CD8 transmembrane polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 46, and at least one costimulatory domain comprising at least one 4-1BB costimulatory polypeptide, wherein the at least one 4-1BB costimulatory polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 50. CAR polypeptides are also provided. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may include a GMCSF signal peptide, the GMCSF signal peptide having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 58.

[0043] The disclosure also provides an anti-CD276 CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD276 antigen-binding domain comprising anti-CD276 scFv, the anti-CD276 scFv comprising the amino acid sequence of SEQ ID NO: 12, a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42, a transmembrane domain comprising a CD8 transmembrane polypeptide, the CD8 transmembrane polypeptide comprising the amino acid sequence of SEQ ID NO: 46, and at least one costimulatory domain comprising at least one 4-1BB costimulatory polypeptide, the at least one 4-1BB costimulatory polypeptide comprising, essentially comprising, or comprising at least one costimulatory domain comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments of the aforementioned anti-CD276 CAR polypeptide, the signal peptide may comprise a GMCSF signal peptide, the GMCSF signal peptide comprising the amino acid sequence of SEQ ID NO: 58.

[0044] In some embodiments, the anti-CD276 CAR polypeptide comprises, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 64.

[0045] Anti-CD99 CAR polypeptide The disclosure also provides anti-CD99 CAR polypeptides comprising, essentially consisting of, or consisting of, an anti-CD99 antigen-binding domain, a transmembrane domain, and an activating domain, from N-terminus to C-terminus.

[0046] The disclosure also provides an anti-CD99 CAR polypeptide comprising, essentially consisting of, or consisting of, an anti-CD99 antigen-binding domain, a transmembrane domain, and at least one costimulatory domain, from N-terminus to C-terminus.

[0047] In some embodiments, the anti-CD99 CAR polypeptides of the present disclosure may further include a hinge domain located between the anti-CD99 antigen-binding domain and the transmembrane domain. Thus, the present disclosure provides anti-CD99 CAR polypeptides comprising, essentially comprising, or comprising an anti-CD99 antigen-binding domain, a hinge domain, a transmembrane domain, and an activating domain from N-terminus to C-terminus. The present disclosure also provides anti-CD99 CAR polypeptides comprising, essentially comprising, or comprising an anti-CD99 antigen-binding domain, a hinge domain, a transmembrane domain, and at least one costimulatory domain from N-terminus to C-terminus.

[0048] In some embodiments, the anti-CD99 CAR polypeptides of the present disclosure may further include a signal peptide at the N-terminus of the anti-CD99 CAR polypeptide. Thus, the present disclosure provides anti-CD99 CAR polypeptides comprising, essentially comprising, or comprising, a signal peptide, an anti-CD99 antigen-binding domain, a transmembrane domain, and an activation domain from N-terminus to C-terminus. The present disclosure also provides anti-CD99 CAR polypeptides comprising, essentially comprising, or comprising, a signal peptide, an anti-CD99 antigen-binding domain, a transmembrane domain, and at least one costimulatory domain from N-terminus to C-terminus. As described above, the anti-CD99 CAR polypeptides of the present disclosure may also include a hinge domain. Thus, the present disclosure provides anti-CD99 CAR polypeptides comprising, essentially comprising, or comprising, a signal peptide, an anti-CD99 antigen-binding domain, a hinge domain, a transmembrane domain, and an activation domain from N-terminus to C-terminus. The disclosure also provides an anti-CD99 CAR polypeptide comprising, essentially consisting of, or consisting of, a signal peptide, an anti-CD99 antigen-binding domain, a hinge domain, a transmembrane domain, and at least one costimulatory domain, from N-terminus to C-terminus.

[0049] The anti-CD99 antigen-binding domain, transmembrane domain, costimulatory domain, signal peptide, hinge domain, and activation domain of the anti-CD99 CAR polypeptides of this disclosure may be selected from any of the anti-CD99 antigen-binding domain, transmembrane domain, costimulatory domain, signal peptide, hinge domain, and activation domain of the anti-CD99 CAR polypeptides described in more detail herein.

[0050] The following are exemplary anti-CD99 CAR polypeptides of this disclosure.

[0051] This disclosure provides an anti-CD99 CAR polypeptide comprising, essentially comprising, or comprising, a signal peptide, an anti-CD99 antigen-binding domain containing anti-CD99 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and an activation domain containing a CD3ζ activating polypeptide, from N-terminus to C-terminus. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide.

[0052] This disclosure provides an anti-CD99 CAR polypeptide comprising, essentially, or consisting thereof, an anti-CD99 antigen-binding domain comprising a signal peptide, an anti-CD99 scFv, wherein the anti-CD99 scFv comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 38, a hinge domain comprising a CD8 hinge polypeptide, wherein the CD8 hinge polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 42, a transmembrane domain comprising a CD8 transmembrane polypeptide, wherein the CD8 transmembrane polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 46, and an activation domain comprising a CD3ζ activating polypeptide, wherein the CD3ζ activating polypeptide comprises an activation domain comprising an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 52. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide, the CD8 signal peptide having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 56.

[0053] This disclosure provides an anti-CD99 CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD99 antigen-binding domain containing anti-CD99 scFv, wherein the anti-CD99 scFv comprises an anti-CD99 antigen-binding domain containing the amino acid sequence of SEQ ID NO: 38, a hinge domain containing a CD8 hinge polypeptide, a hinge domain containing the amino acid sequence of SEQ ID NO: 42, a transmembrane domain containing a CD8 transmembrane polypeptide, a transmembrane domain containing the amino acid sequence of SEQ ID NO: 46, and an activation domain containing a CD3ζ activating polypeptide, wherein the CD3ζ activating polypeptide comprises, essentially consists of, or consists of an activation domain containing the amino acid sequence of SEQ ID NO: 52. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide, and the CD8 signal peptide may include the amino acid sequence of SEQ ID NO: 56.

[0054] In some embodiments, the anti-CD99 CAR polypeptide comprises, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 65.

[0055] This disclosure provides an anti-CD99 CAR polypeptide comprising, essentially comprising, or comprising, a signal peptide, an anti-CD99 antigen-binding domain containing an anti-CD99 scFv, a hinge domain containing a CD28 hinge polypeptide, a transmembrane domain containing a CD28 transmembrane polypeptide, and at least one costimulatory domain containing at least one CD28 costimulatory polypeptide, from N-terminus to C-terminus. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide.

[0056] This disclosure provides an anti-CD99 CAR polypeptide comprising, essentially, or consisting thereof, an anti-CD99 antigen-binding domain comprising a signal peptide, an anti-CD99 scFv, wherein the anti-CD99 scFv comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 38, a hinge domain comprising a CD28 hinge polypeptide, wherein the CD28 hinge polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 40, a transmembrane domain comprising a CD28 transmembrane polypeptide, wherein the CD28 transmembrane polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 44, and at least one costimulatory domain comprising at least one CD28 costimulatory polypeptide, wherein the at least one CD28 costimulatory polypeptide comprises an amino acid sequence at least 97%, 98%, or 99% identical to SEQ ID NO: 48, or at least one costimulatory domain comprising at least one CD28 costimulatory polypeptide. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide, the CD8 signal peptide having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 56.

[0057] This disclosure provides an anti-CD99 CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD99 antigen-binding domain containing anti-CD99 scFv, wherein the anti-CD99 scFv comprises an anti-CD99 antigen-binding domain containing the amino acid sequence of SEQ ID NO: 38, a hinge domain containing a CD28 hinge polypeptide, a transmembrane domain containing a CD28 transmembrane polypeptide, a transmembrane domain containing the amino acid sequence of SEQ ID NO: 40, and at least one costimulatory domain containing at least one CD28 costimulatory polypeptide, wherein the at least one CD28 costimulatory polypeptide comprises, essentially consists of, or consists of at least one costimulatory domain containing the amino acid sequence of SEQ ID NO: 48. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide, the CD8 signal peptide containing the amino acid sequence of SEQ ID NO: 56.

[0058] In some embodiments, the anti-CD99 CAR polypeptide comprises, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 61.

[0059] The disclosure also provides anti-CD99 CAR polypeptides comprising, essentially consisting of, or consisting of, a signal peptide, an anti-CD99 antigen-binding domain containing an anti-CD99 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and at least one costimulatory domain containing at least one 4-1BB costimulatory polypeptide, from N-terminus to C-terminus. In some embodiments of the aforementioned anti-CD99 CAR polypeptides, the signal peptide may include a CD8 signal peptide.

[0060] The disclosure also provides an anti-CD99 CAR polypeptide comprising, essentially comprising, or comprising, at least one costimulatory domain comprising, at least one 4-1BB costimulatory polypeptide polypeptide comprising, at least one 4-1BB costimulatory polypeptide, at least one costimulatory polypeptide comprising, at least one 4-1BB costimulatory polypeptide, at least one costimulatory domain comprising, at least one 4-1BB costimulatory polypeptide, at least one 4-1BB costimulatory polypeptide, at least one 4-1BB costimulatory polypeptide, at least one 4-1BB costimulatory polypeptide, at least one 4-1BB costimulatory polypeptide, at least In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may include a CD8 signal peptide, the CD8 signal peptide having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 56.

[0061] The disclosure also provides an anti-CD99 CAR polypeptide comprising, from N-terminus to C-terminus, a signal peptide, an anti-CD99 antigen-binding domain comprising anti-CD99 scFv, the anti-CD99 scFv comprising the amino acid sequence of SEQ ID NO: 38, a hinge domain comprising a CD8 hinge polypeptide comprising the amino acid sequence of SEQ ID NO: 42, a transmembrane domain comprising a CD8 transmembrane polypeptide, the CD8 transmembrane polypeptide comprising a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 46, and at least one costimulatory domain comprising at least one 4-1BB costimulatory polypeptide, the at least one 4-1BB costimulatory polypeptide comprising at least one costimulatory domain comprising the amino acid sequence of SEQ ID NO: 50, or comprising essentially these, or comprising these. In some embodiments of the aforementioned anti-CD99 CAR polypeptide, the signal peptide may comprise a CD8 signal peptide, the CD8 signal peptide comprising the amino acid sequence of SEQ ID NO: 56.

[0062] In some embodiments, the anti-CD99 CAR polypeptide comprises, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 60.

[0063] antigen-binding domain In some embodiments, the antigen-binding domain of the CAR polypeptide of the present disclosure may include an antibody or antigen-binding fragment specific to a congener antigen of the CAR polypeptide (i.e., either CD99 or CD276).

[0064] Therefore, in some embodiments, the anti-CD276 antigen-binding domain may include, essentially consist of, or be composed of, an anti-CD276 antibody or its antigen-binding fragment. Any suitable anti-CD276 antibody known in the art, such as that described in U.S. Patent No. 1,0730,941, can be used.

[0065] In some embodiments, the anti-CD276 antigen-binding domain may contain, essentially consist of, or be composed of an anti-CD276 scFv. Any suitable anti-CD276 scFv known in the art, including those described in or derived from U.S. Patent No. 1,0562,952, may be used.

[0066] In some embodiments, anti-CD276 scFv may include CDHR1 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 1; CDRH2 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 2; CDRH3 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 3; CDRL1 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 4; CDRL2 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 5; and CDHL3 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 6.

[0067] In some embodiments, the anti-CD276 scFv may include CDHR1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, CDRH3 having the amino acid sequence of SEQ ID NO: 3, CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDHL3 having the amino acid sequence of SEQ ID NO: 6.

[0068] In some embodiments, anti-CD276 scFv may include a VH domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 7. Thus, the nucleic acid sequence encoding the VH domain may comprise, essentially consist of, or be comprised of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 8.

[0069] In some embodiments, anti-CD276 scFv may include a VL domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 9. Thus, the nucleic acid sequence encoding the VL domain may comprise, essentially consist of, or be comprised of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 10.

[0070] In some embodiments, anti-CD276 scFv may include a linker domain that connects the VL domain and the VH domain, the linker domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 11.

[0071] In some embodiments, the anti-CD276 scFv may include a VH domain containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-CD276 scFv may include a VL domain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD276 scFv may include a linker domain that connects the VL domain and the VH domain, the linker domain containing the amino acid sequence of SEQ ID NO: 11.

[0072] In some embodiments, anti-CD276 scFv contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 12. Thus, the nucleic acid sequence encoding anti-CD276 scFv contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 13.

[0073] In some embodiments, the anti-CD99 antigen-binding domain may include, essentially consist of, or consist of an anti-CD99 antibody or its antigen-binding fragment. Any suitable anti-CD99 antibody may be used, including those known in the art, such as those described in PCT international application PCT / US2023 / 067715 (published as international publication 2023 / 235767) and U.S. patent 8710194.

[0074] In some embodiments, the anti-CD99 antigen-binding domain may contain, essentially consist of, or be composed of an anti-CD99 scFv. Any suitable anti-CD99 scFv may be used, including those known in the art, such as those described in PCT international application PCT / US2023 / 067715 (published as international publication 2023 / 235767) and U.S. Patent No. 8710194 and those derived therefrom.

[0075] In some embodiments, anti-CD99 scFv may include CDHR1 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 14; CDRH2 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 15; CDRH3 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 16; CDRL1 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 17; CDRL2 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 18; and CDHL3 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 19.

[0076] In some embodiments, the anti-CD99 scFv may include CDHR1 having the amino acid sequence of SEQ ID NO: 14, CDRH2 having the amino acid sequence of SEQ ID NO: 15, CDRH3 having the amino acid sequence of SEQ ID NO: 16, CDRL1 having the amino acid sequence of SEQ ID NO: 17, CDRL2 having the amino acid sequence of SEQ ID NO: 18, and CDHL3 having the amino acid sequence of SEQ ID NO: 19.

[0077] In some embodiments, anti-CD99 scFv may include a VH domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 20. Thus, the nucleic acid sequence encoding the VH domain may comprise, essentially consist of, or be comprised of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 21.

[0078] In some embodiments, anti-CD99 scFv may include a VL domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 22. Thus, the nucleic acid sequence encoding the VL domain may comprise, essentially consist of, or be comprised of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 23.

[0079] In some embodiments, anti-CD99 scFv may include a linker domain that connects the VL domain and the VH domain, the linker domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 24.

[0080] In some embodiments, the anti-CD99 scFv may include a VH domain containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-CD99 scFv may include a VL domain containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD99 scFv may include a linker domain that connects the VL domain and the VH domain, the linker domain containing the amino acid sequence of SEQ ID NO: 24.

[0081] In some embodiments, anti-CD99 scFv contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 25. Thus, a nucleic acid sequence encoding anti-CD99 scFv contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 26.

[0082] In some embodiments, anti-CD99 scFv may include CDHR1 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 27; CDRH2 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 28; CDRH3 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 29; CDRL1 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 30; CDRL2 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 31; and CDHL3 having an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 32.

[0083] In some embodiments, the anti-CD99 scFv may include CDHR1 having the amino acid sequence of SEQ ID NO: 27, CDRH2 having the amino acid sequence of SEQ ID NO: 28, CDRH3 having the amino acid sequence of SEQ ID NO: 29, CDRL1 having the amino acid sequence of SEQ ID NO: 30, CDRL2 having the amino acid sequence of SEQ ID NO: 31, and CDHL3 having the amino acid sequence of SEQ ID NO: 32.

[0084] In some embodiments, anti-CD99 scFv may include a VH domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 33. Thus, the nucleic acid sequence encoding the VH domain may comprise, essentially consist of, or be comprised of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 34.

[0085] In some embodiments, anti-CD99 scFv may include a VL domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 35. Thus, the nucleic acid sequence encoding the VL domain may comprise, essentially consist of, or be comprised of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 36.

[0086] In some embodiments, anti-CD99 scFv may include a linker domain that connects the VL domain and the VH domain, the linker domain comprising an amino acid sequence that is at least 97%, 98%, or 99% identical to SEQ ID NO: 37.

[0087] In some embodiments, the anti-CD99 scFv may include a VH domain containing the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-CD99 scFv may include a VL domain containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD99 scFv may include a linker domain that connects the VL domain and the VH domain, the linker domain containing the amino acid sequence of SEQ ID NO: 37.

[0088] In some embodiments, anti-CD99 scFv contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 38. Thus, a nucleic acid sequence encoding anti-CD99 scFv contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 39.

[0089] As used herein, “antibody” means either a monoclonal or polyclonal antibody. The term “monoclonal antibody” as used herein means an antibody produced by a single clone of a B cell and bound to the same epitope. In contrast, “polyclonal antibody” means a group of antibodies produced by different B cells and bound to different epitopes of the same antigen. A complete antibody typically consists of four polypeptides: two identical copies of a heavy (H) polypeptide and two identical copies of a light (L) polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CHL CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have a similar overall structure, and each region contains four framework regions whose sequences are relatively conserved. The framework region is linked by three complementarity-determining regions (CDRs). These three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody responsible for antigen binding.

[0090] The terms “antibody fragment,” “antibody fragment,” “functional fragment of an antibody,” “antigen-binding portion,” and “antigen-binding fragment” are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see Holliger et al. (2005) Nat. Biotech. 23(9):1126-29 in general). An antibody fragment preferably comprises, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or a combination thereof.

[0091] Examples of antibody fragments, but not limited to, include: (i) Fab fragments, which are monovalent fragments containing VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the stalk region; (iii) Fv fragments, which contain the VL and VH domains of a single arm of the antibody; (iv) single-chain Fv(scFv), which are monovalent molecules containing two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al. (1988), Science 242: 423-6; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83; and Osbourn et al. (1998) Nat. Biotechnol. 16: 778-81); and (v) Diabodies are polypeptide chain dimers, each containing a VH linked to VL by a peptide linker that is too short to enable pairing between VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimeric molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application Publication No. 2009 / 0093024Al.

[0092] Accordingly, in some embodiments, the antigen-binding domain of the CAR polypeptide described herein comprises an scFv including a VH domain and a VL domain, wherein the VH comprises CDRH1, CDRH2, and CDRH3, each containing the amino acid sequences of CDRH1, CDRH2, and CDRH3 of anti-CD276 or anti-CD99 antibodies known in the art, and the VL comprises CDRL1, CDRL2, and CDRL3, each containing the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the same anti-CD276 or anti-CD99 antibodies known in the art. The determination of the CDR region is well within the scope of the skills of the art. In some embodiments, it is understood that the CDR may be a combination of Kabat and Chothia CDRs (also referred to as a “combined CR” or “extended CDR”).

[0093] In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In other embodiments, the CDR is an IMGT CDR. In other words, in embodiments containing two or more CDRs, the CDR may be a combination of Kabat, Chothia, IMGT, or any combination thereof.

[0094] transmembrane domain The anti-CD99 CAR polypeptide and the anti-CD276 CAR polypeptide of the present disclosure are intended to be expressed on the surface membrane of cells. Therefore, the anti-CD99 CAR polypeptide and the anti-CD276 CAR polypeptide of the present disclosure may contain transmembrane domains.

[0095] As will be understood by those skilled in the art, a transmembrane domain can be any protein structure that is thermodynamically stable within a membrane. Typically, this is an α-helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portions of the anti-CD99 CAR polypeptide and anti-CD276 CAR polypeptide of this disclosure. The presence and span of the protein's transmembrane domain can be determined by those skilled in the art using the DeepTMHMM algorithm (see Hallgren et al. bioRxiv (2022). DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. https: / / doi.org / 10.1101 / 2022.04.08.487609) or any equivalent algorithm known in the art.

[0096] Transmembrane domains can be derived from either natural or synthetic sources. Transmembrane domains can be derived from any membrane-bound or transmembrane protein. As a non-limiting example, transmembrane polypeptides may be partial sequences or subunits of T cell receptors such as α, β, γ, or δ; polypeptides constituting the CD3 complex; IL-2 receptor p55 (a chain), p75 (β chain), or γ chain; Fc receptors, particularly Fcγ receptor III or subunit chains of CD proteins. Alternatively, transmembrane domains may be synthetic and may contain, essentially consist of, or be composed of hydrophobic residues such as leucine and valine.

[0097] In some embodiments, the transmembrane domain includes, essentially consists of, or can consist of a CD8 transmembrane domain.

[0098] In some embodiments, the CD8 transmembrane domain contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 46. Thus, the nucleic acid sequence encoding the CD8 transmembrane domain contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 47.

[0099] In some embodiments, the transmembrane domain includes, essentially consists of, or may consist of the CD28 transmembrane domain.

[0100] In some embodiments, the CD28 transmembrane domain contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 44. Thus, the nucleic acid sequence encoding the CD28 transmembrane domain contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 45.

[0101] Co-stimulatory domain In some embodiments, the co-stimulatory domain of the CAR polypeptide of this disclosure may include an intracellular domain of a co-stimulatory molecule or a fragment thereof.

[0102] As used herein, "costimulatory molecule" refers to a congenital binding partner on an immune cell, such as a T cell, that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the cell, such as proliferation, but is not limited to this. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, and CD83.

[0103] A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a congeneral costimulatory signaling molecule on an immune cell, such as a T cell, and thereby provides signals that mediate T cell responses, including, but are not limited to, proliferation activation and differentiation, in addition to the primary signal provided by, for example, the binding affinity of the TCR / CD3 complex to MHC molecules carrying peptides. Examples of costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (igand) (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to CD276. Co-stimulatory ligands also include antibodies that specifically bind to co-stimulatory molecules presented on T cells, such as ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, CD276, and CD83, among others.

[0104] In some embodiments, the co-stimulatory domain may include, essentially consist of, or be composed of, an intracellular domain of 4-1BB(CD137) or a fragment thereof. Such a co-stimulatory domain is referred to herein as a 4-1BB co-stimulatory polypeptide. Accordingly, the co-stimulatory domain of the CAR polypeptide of this disclosure may include, essentially consist of, or be composed of at least one 4-1BB co-stimulatory polypeptide.

[0105] In some embodiments, the 4-1BB costimulatory polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 50. Thus, the nucleic acid sequence encoding the 4-1BB costimulatory polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 51.

[0106] In some embodiments, the co-stimulatory domain may include, essentially consist of, or be composed of, an intracellular domain of CD28 or a fragment thereof. Such a co-stimulatory domain is referred to herein as a CD28 co-stimulatory polypeptide. Accordingly, the co-stimulatory domain of the CAR polypeptide of this disclosure may include, essentially consist of, or be composed of at least one CD28 co-stimulatory polypeptide.

[0107] In some embodiments, the CD28 costimulatory polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 48. Thus, the nucleic acid sequence encoding the CD28 costimulatory polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 49.

[0108] Activation Domain In some embodiments, the activation domain of the CAR polypeptide of this disclosure includes, essentially consists of, or may consist of, a cytoplasmic signaling sequence that initiates antigen-dependent primary activation. As will be understood by those skilled in the art, the primary cytoplasmic signaling sequence includes, essentially consists of, or may consist of, a signaling motif known as an immunoreceptor tyrosine activation motif (ITAM). ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d.

[0109] In some embodiments, the activating domain may include, essentially consist of, or be composed of a CD3ζ activating polypeptide.

[0110] In some embodiments, the CD3ζ-activated polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 52. Thus, the nucleic acid sequence encoding the CD3ζ-activated polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 53.

[0111] Hinged Domain In some embodiments, the hinge domain of the CAR polypeptide in this disclosure refers to a polypeptide located between the antigen-binding domain and the transmembrane domain. While we do not wish to be bound by theory, the hinge domain may offer greater flexibility and access to the antigen-binding domain.

[0112] In some embodiments, the hinge domain may contain, essentially consist of, or be composed of up to 300 amino acids.

[0113] In some embodiments, the hinge domain may contain, essentially consist of, or be composed of 10 to 100 amino acids or 25 to 50 amino acids.

[0114] In some embodiments, the hinge domain can be derived from all or part of naturally occurring molecules, such as all or part of CD8, CD4, CD28, 4-1BB, or the extracellular region of IgG (particularly the hinge region of IgG), or all or part of the constant region of the antibody heavy chain.

[0115] In some embodiments, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or the entire sequence may be a synthetic hinge sequence. In some embodiments, the hinge domain may contain, essentially consist of, or be composed of a partial sequence of CD8α, IgG1, or FcγRIIIα.

[0116] In some embodiments, the hinge domain may contain, essentially consist of, or be composed of the CD28 hinge polypeptide.

[0117] In some embodiments, the CD28 hinge polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 40. Thus, the nucleic acid sequence encoding the CD28 hinge polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 41.

[0118] In some embodiments, the hinge domain may contain, essentially consist of, or be composed of the CD8 hinge polypeptide.

[0119] In some embodiments, the CD8 hinge polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 42. Thus, the nucleic acid sequence encoding the CD8 hinge polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 43.

[0120] Signal peptide As used herein, the term “signal peptide” means a peptide that functions to direct the polypeptide to which it is linked toward the secretory pathway. As will be understood by those skilled in the art, in the context in which the polypeptide of this disclosure includes both a signal peptide and a transmembrane domain, the signal peptide functions to direct the polypeptide toward the secretory pathway so that the polypeptide is embedded in a membrane, preferably a cell membrane. Signal peptides are well known in the art. Therefore, the signal peptide in the polypeptide of this disclosure may be any signal peptide known in the art that is sufficient to direct the polypeptide to which it is linked toward the secretory pathway.

[0121] In some embodiments, the signal peptide may include, essentially consist of, or be composed of a CD8 signal peptide.

[0122] In some embodiments, the CD8 signal peptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 56. Thus, the nucleic acid sequence encoding the CD8 signal peptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 57.

[0123] In some embodiments, the signal peptide may include, essentially consist of, or be composed of a GMCSF signal peptide.

[0124] In some embodiments, the GMCSF signal peptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 58. Thus, the nucleic acid sequence encoding the GMCSF signal peptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 59.

[0125] Logical pairs of CAR polypeptides This disclosure provides logical pairs of CAR polypeptides, which include, essentially consist of, or consist of the anti-CD276 CAR polypeptide and the anti-CD99 CAR polypeptide of this disclosure.

[0126] While we do not wish to dwell on theory, pairing anti-CD276 CAR polypeptides with anti-CD99 CAR polypeptides enables the production of "logic-gate" CAR T cells. These logic-gate CAR T cells express a logical pair of CAR polypeptides and achieve full activation only when they encounter cells expressing both CD99 and CD276. Such T cells are also referred to herein as CD99 / CD276 logic-gate CAR-T cells.

[0127] While I don't want to get bogged down in theory, since co-expression of CD99 and CD276 is a more specific marker for certain types of cancer cells (e.g., DIPG cells) compared to either CD99 or CD276 alone, such logic-gate CAR T cells exhibit relatively low extra-target toxicity. In other words, if logic-gate CAR T cells encounter non-cancerous cells expressing only CD99 and not CD276, or non-cancerous cells expressing only CD276 and not CD99, the logic-gate CAR T cells will not be activated by these non-cancerous cells, and thus will miss the non-cancerous cells.

[0128] In some embodiments of the logical pairs of CAR polypeptides, one of the paired CAR polypeptides has an intracellular domain containing one or more costimulatory domains and not containing an activating domain, while the other CAR polypeptide in the pair has an intracellular domain containing an intracellular domain and not containing any costimulatory domains. In other words, compared to the “conventional CAR form,” the logical pairs of CAR polypeptides of this disclosure separate the costimulatory and activating domains onto separate molecules. While we do not wish to dwell on theory, by separating the costimulatory and intracellular domains onto separate CAR molecules (e.g., CD99 and CD276) that are specific to different antigens, both CAR molecules must mate to both antigens on target cells in order to achieve complete activation of CAR T cells.

[0129] In some embodiments of the logical pair of CAR polypeptides, the logical anti-CD276 CAR polypeptide comprises one or more costimulatory domains, and the logical anti-CD99 CAR polypeptide comprises an activating domain. In some embodiments, the one or more costimulatory domains comprise a CD28 costimulatory polypeptide and / or a 4-1BB costimulatory polypeptide. In some embodiments, the activating domain comprises one or more CD3ζ activating polypeptides.

[0130] In some embodiments of the logical pair of CAR polypeptides, the logical anti-CD99 CAR polypeptide comprises one or more costimulatory domains, and the logical anti-CD276 CAR polypeptide comprises an activating domain. In some embodiments, the one or more costimulatory domains comprise a CD28 costimulatory polypeptide and / or a 4-1BB costimulatory polypeptide. In some embodiments, the activating domain comprises one or more CD3ζ activating polypeptides.

[0131] Accordingly, this disclosure provides compositions comprising logical pairs of CAR polypeptides of this disclosure. In some embodiments, the composition takes the form of cells or populations of cells expressing logical pairs of CAR polypeptides. In some embodiments, the composition takes the form of nucleic acid molecules (e.g., vectors) comprising sequences encoding logical pairs of CAR polypeptides. These compositions are described in further detail herein.

[0132] In some embodiments, a logical pair of CARs can be expressed from N-terminus to C-terminus as a dicistronic CAR polypeptide comprising, essentially comprising, or comprising a first CAR polypeptide, a self-cleaving peptide, and a second CAR polypeptide. Accordingly, the Disclosure provides a dicistronic CAR polypeptide comprising, essentially comprising, or comprising an anti-CD276 CAR polypeptide, a self-cleaving peptide, and an anti-CD99 CAR polypeptide from the Disclosure, from N-terminus to C-terminus. The Disclosure also provides a dicistronic CAR polypeptide comprising, essentially comprising, or comprising an anti-CD99 CAR polypeptide, a self-cleaving peptide, and an anti-CD276 CAR polypeptide from the Disclosure, from N-terminus to C-terminus.

[0133] In some embodiments, a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 66. Thus, a nucleic acid sequence encoding a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 67.

[0134] In some embodiments, a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 68. Thus, a nucleic acid sequence encoding a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 69.

[0135] In some embodiments, a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 70. Thus, a nucleic acid sequence encoding a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 71.

[0136] In some embodiments, a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 72. Thus, a nucleic acid sequence encoding a dicistronic CAR polypeptide contains, essentially consists of, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 73.

[0137] Functional variants of the polypeptides disclosed herein This disclosure also considers functional variants of CAR polypeptides disclosed and described herein. The term “functional variant” as used herein means a CAR polypeptide having substantial or significant sequence identity or similarity to the parent CAR polypeptide, such that the functional variant retains the biological activity of the parent CAR polypeptide from which it is a variant. Functional variants include, for example, variants of CAR polypeptides described herein (parent CAR polypeptides) that retain, to a similar, equal, or greater degree of ability to recognize target cells than the parent CAR polypeptide. With reference to the parent CAR, a functional variant may, for example, be at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identical in amino acid sequence to the parent CAR.

[0138] A functional mutant may, for example, include the amino acid sequence of a parent CAR having at least one conserved amino acid substitution. Alternatively, or additionally, a functional mutant may include the amino acid sequence of a parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional mutant. The non-conserved amino acid substitution can enhance the biological activity of the functional mutant so that its biological activity is increased compared to that of the parent CAR.

[0139] Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as resulting in only slight changes. These slight changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on an examination of its hydrophobicity and charge. Amino acids with similar hydropathic indices can be substituted, and the protein function may still be preserved. In some embodiments, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that will result in proteins that retain biological function. Examining the hydrophilicity of amino acids in the context of polypeptides allows for the calculation of the maximum local mean hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, fully incorporated herein by reference.

[0140] Substitutions of amino acids with similar hydrophilicity values ​​can produce polypeptides that retain biological activity, such as immunogenicity. Substitutions can be made using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity of an amino acid are influenced by its specific side chain. Consistent with these observations, it is understood that amino acid substitutions compatible with biological function depend on the relative similarity of amino acids, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties, and especially on the side chains of those amino acids.

[0141] As used herein, “conservative” amino acid substitutions may be defined as set forth in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions introduced by modifications of the polynucleotides encoding the polypeptides of this disclosure. Amino acids can be classified according to their physical properties and contributions to secondary and tertiary protein structures. A conservative substitution is the substitution of one amino acid for another amino acid having similar properties. Exemplary conservative substitutions are listed in Table A.

[0142] [Table 1]

[0143] Alternatively, the conserved amino acids can be grouped as shown in Table B, as described by Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0144] [Table 2]

[0145] Alternatively, exemplary conservative substitutions are listed in Table C.

[0146] [Table 3]

[0147] It should be understood that the polypeptides of this disclosure are intended to include polypeptides having one or more amino acid residue insertions, deletions, or substitutions, or any combination thereof, as well as modifications other than amino acid residue insertions, deletions, or substitutions. The polypeptides or nucleic acids of this disclosure may contain one or more conservative substitutions.

[0148] As used throughout this disclosure, the term "two or more" in the context of the aforementioned amino acid substitutions means two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteen, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twenty or more of the specified amino acid substitutions. The term "two or more" may mean two, three, four, or five of the specified amino acid substitutions.

[0149] Nucleic acid molecules of the present disclosure This disclosure provides nucleic acid molecules comprising, essentially consisting of, or comprising one or more nucleic acid sequences encoding the anti-CD99 CAR polypeptide of this disclosure.

[0150] This disclosure provides nucleic acid molecules comprising, essentially consisting of, or comprising one or more nucleic acid sequences encoding the anti-CD276 CAR polypeptide of this disclosure.

[0151] This disclosure provides nucleic acid molecules comprising, essentially comprising, or comprising one or more nucleic acid sequences encoding the anti-CD276 CAR polypeptide of this disclosure and one or more nucleic acid sequences encoding the anti-CD99 CAR polypeptide of this disclosure. In other words, this disclosure provides nucleic acid molecules comprising, essentially comprising, or comprising one or more nucleic acid sequences encoding logical pairs of CAR polypeptides. Accordingly, this disclosure provides nucleic acid molecules comprising, essentially comprising, or comprising one or more nucleic acid sequences encoding the dicistronic CAR polypeptide of this disclosure.

[0152] In embodiments of the present disclosure in which the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-CD276 CAR polypeptide and a nucleic acid sequence encoding an anti-CD99 CAR polypeptide, these nucleic acid sequences may be in either order, i.e., the nucleic acid molecule may contain, from 5' to 3', the nucleic acid sequence encoding the anti-CD276 CAR polypeptide and the nucleic acid sequence encoding the anti-CD99 CAR polypeptide of the present disclosure, or the nucleic acid molecule may contain, from 5' to 3', the nucleic acid sequence encoding the anti-CD99 CAR polypeptide and the nucleic acid sequence encoding the anti-CD276 CAR polypeptide of the present disclosure.

[0153] In embodiments of the present disclosure in which the nucleic acid molecule comprises two or more nucleic acid sequences encoding polypeptides (for example, a nucleic acid sequence encoding the anti-CD276 CAR polypeptide and a nucleic acid sequence encoding the anti-CD99 CAR polypeptide), the nucleic acid sequences encoding different polypeptides may be separated by one or more nucleic acid sequences encoding self-cleaving peptides.

[0154] As will be understood by those skilled in the art, the amino acid sequences of self-cleaving peptides and the nucleic acid sequences encoding said amino acid sequences are known in the art. Non-limiting examples of self-cleaving peptides include 2A self-cleaving peptide sequences such as T2A peptides, E2A peptides, F2A peptides, and P2A peptides.

[0155] In some embodiments of the nucleic acid molecules of this disclosure, the sequence encoding a self-cleaving peptide is a nucleic acid sequence encoding a P2A self-cleaving peptide. As will be understood by those skilled in the art, a P2A self-cleaving peptide may contain, essentially consist of, or have an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 54. Accordingly, a nucleic acid sequence encoding a P2A self-cleaving peptide may contain, essentially consist of, or have an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 55.

[0156] Accordingly, the Disclosure provides nucleic acids comprising, essentially consisting of, or comprising, a nucleic acid sequence encoding the anti-CD276 CAR polypeptide of the Disclosure, a nucleic acid sequence encoding the P2A self-cleaving peptide, and a nucleic acid sequence encoding the anti-CD99 CAR polypeptide, from 5' to 3'. The Disclosure also provides nucleic acids comprising, essentially consisting of, or comprising, a nucleic acid sequence encoding the anti-CD99 CAR polypeptide, a nucleic acid sequence encoding the P2A self-cleaving peptide, and a nucleic acid sequence encoding the anti-CD276 CAR polypeptide, from 5' to 3'.

[0157] This disclosure provides nucleic acid molecules that contain, essentially consist of, or consist of a nucleic acid sequence that is identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) a nucleic acid sequence identical to SEQ ID NO: 67.

[0158] This disclosure provides nucleic acid molecules that contain, essentially consist of, or consist of a nucleic acid sequence that is identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) a nucleic acid sequence to SEQ ID NO: 69.

[0159] This disclosure provides nucleic acid molecules that contain, essentially consist of, or consist of a nucleic acid sequence that is identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) a nucleic acid sequence identical to SEQ ID NO: 71.

[0160] This disclosure provides nucleic acid molecules that contain, essentially consist of, or consist of a nucleic acid sequence that is identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) a nucleic acid sequence identical to, SEQ ID NO: 73.

[0161] This disclosure also provides a vector comprising at least one nucleic acid molecule of this disclosure. In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be an AAV vector or a lentiviral vector.

[0162] This disclosure also encompasses all nucleic acid molecules that are complementary to the nucleic acid molecules described in detail herein.

[0163] cell This disclosure provides cells expressing at least one anti-CD276 CAR polypeptide and at least one anti-CD99 CAR polypeptide of this disclosure. In other words, this disclosure provides cells expressing logical pairs of the CAR polypeptides of this disclosure. Since they can be used in methods to treat cancer as described below, these cells expressing logical pairs of the CAR polypeptides of this disclosure are also referred to herein as “therapeutic cells.”

[0164] Accordingly, the present disclosure provides a population of cells (also referred to herein as a large number of cells) expressing at least one anti-CD276 CAR polypeptide and at least one anti-CD99 CAR polypeptide of the present disclosure. In other words, the present disclosure provides a population of cells expressing logical pairs of the CAR polypeptides of the present disclosure.

[0165] In some embodiments of the cells of this disclosure, the expression levels of anti-CD276 CAR polypeptide and anti-CD99 CAR polypeptide may be the same. In some embodiments of the cells of this disclosure, the expression levels of anti-CD276 CAR polypeptide and anti-CD99 CAR polypeptide may be different.

[0166] The Disclosure provides a population of cells in which at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% of the cells in the population express both the anti-CD276 CAR polypeptide of the Disclosure and the anti-CD99 CAR polypeptide of the Disclosure.

[0167] In some embodiments, the cells described above may be immune cells.

[0168] As used herein, “immune cells” means hematopoietic cells that are functionally involved in initiating and / or carrying out innate and / or adaptive immune responses.

[0169] Examples of immune cells include, but are not limited to, T cells (e.g., regulatory T cells, CAR T cells, CD8 + CAR T cells, CD4 + CAR T cells, CD4 + T cells, CD8 + T cells, peripheral blood (PB)-derived T cells, umbilical cord blood (UCB)-derived T cells, or γδ T cells), NK cells, NK-like cells, invariant NK cells, NKT cells, cytokine-induced killer (CIK) cells, stem cells (e.g., mesenchymal stem cells (MSC), hematopoietic stem cells, hematopoietic progenitor cells, or induced pluripotent stem (iPSC) cells). In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0170] In some aspects, the cells are T cells.

[0171] In some aspects, the cells are NK cells.

[0172] In some aspects, the above cells can be tumor infiltrating lymphocytes (“TIL”). As will be understood by those skilled in the art, TIL refers to a population of white blood cells that have left the bloodstream of a subject and migrated into the tumor. Populations of TIL include, but are not limited to, T helper 17 cells (Thl7, CD4 + IL17 + T cells), cytotoxic T cells (Tc17, CD8 + IL17 + T cells) and regulatory T cells (Treg, CD4 + CD25 + Foxp3 +Examples include T cells, natural killer (NK) cells, dendritic cells, and M1 macrophages. As will be understood by those skilled in the art, TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into the subject.

[0173] Immune cells can be enriched / purified from any tissue in which they exist, including, but not limited to, blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgery, and tissues obtained via biopsy procedures. The tissues / organs from which immune cells are enriched, isolated, and / or purified can be isolated from both living and non-living subjects, the non-living subjects being organ donors. Isolated immune cells can be used directly or stored for a period of time, such as by freezing. In the case of TILs, the TIL population can be enriched / purified from tumor samples obtained from the subject.

[0174] This disclosure also provides pharmaceutical compositions comprising therapeutic cells or a number of therapeutic cells as described herein. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical compositions of this disclosure may further comprise one or more further pharmaceutically active polypeptides and / or compounds. The pharmaceutical compositions of this disclosure may be formulated for a specific route of administration, e.g., intravenous or intrathecal infusion.

[0175] As used herein, “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” and “pharmaceutically acceptable diluent” refer to any substance that, when combined with the active ingredient, enables the ingredient to retain its biological activity and be non-reactive with the subject’s immune system. Examples, but not limited to, include standard pharmaceutical carriers such as phosphate-buffered saline, water, oil / water emulsions, and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or ordinary (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington’s Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0176] How to use This disclosure provides a method for treating cancer in a subject, comprising the step of administering one or more types of therapeutic cells of this disclosure to the subject.

[0177] This disclosure provides one or more types of numerous therapeutic cells for use in the treatment of cancer in subjects.

[0178] This disclosure provides the use of one or more types of multiple therapeutic cells for use in the manufacture of pharmaceuticals for the treatment of cancer.

[0179] This disclosure provides a method for preventing cancer metastasis in a subject, comprising the step of administering a large number of one or more types of therapeutic cells of this disclosure to the subject.

[0180] This disclosure provides one or more types of numerous therapeutic cells for use in the prevention of cancer metastasis in subjects.

[0181] This disclosure provides the use of one or more types of multiple therapeutic cells for use in the manufacture of pharmaceuticals for the prevention of cancer metastasis.

[0182] In some embodiments, multiple or numerous therapeutic cells are administered to the subject in a therapeutically effective dose.

[0183] In some embodiments, multiple or numerous therapeutic cells are administered intravenously. In some embodiments, multiple or numerous therapeutic cells are administered intrathecally. In some embodiments, multiple or numerous therapeutic cells are administered intratumorally. In some embodiments, multiple or numerous therapeutic cells are administered intrapleurally, intraperitoneally, or intrathoracically.

[0184] This disclosure provides a method for treating cancer in a subject, comprising the step of administering one or more of the therapeutic cells of this disclosure and at least one additional therapy to the subject. In some embodiments, the at least one additional therapy may be selected from immunotherapy, stem cell transplantation, anticancer therapy, chemotherapy, targeted therapy, radiotherapy, or any combination thereof. In some embodiments, the at least one additional therapy is radiotherapy. In some embodiments, the at least one additional therapy is chemotherapy. In some embodiments, the at least one additional therapy includes administration of a combination of venetoclax and azacitidine. In some embodiments, the subject may be pre-treated with radiotherapy prior to administration of the therapeutic cells of this disclosure.

[0185] In some embodiments, at least one additional therapy and one or more of the therapeutic cells of this disclosure may be administered in close proximity in time.

[0186] As used herein, the term “temporal proximity” means that the administration of one therapeutic agent (e.g., one or more therapeutic cells as disclosed herein) occurs within a period before or after the administration of another therapeutic agent (e.g., radiotherapy), such that the therapeutic effects of one therapeutic agent overlap with those of the other therapeutic agent. In some embodiments, the therapeutic effects of one therapeutic agent completely overlap with those of the other therapeutic agent. In some embodiments, “temporal proximity” means that the administration of one therapeutic agent occurs within a period before or after the administration of the other therapeutic agent, such that a synergistic effect is observed between the two therapeutic agents. “Temporal proximity” can vary depending on various factors, including, but are not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to whom the therapeutic agent is to be administered; the disease or condition to be treated or improved; the therapeutic outcome to be achieved; the dosage, frequency, and duration of administration of the therapeutic agent; the pharmacokinetics and pharmacodynamics of the therapeutic agent; and the route through which the therapeutic agent is administered. In some embodiments, “temporal proximity” means within 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, or 8 weeks. In some embodiments, multiple doses of one therapeutic agent may occur in temporal proximity to a single dose of another therapeutic agent. In some embodiments, temporal proximity may vary during a treatment cycle or within a drug regimen.

[0187] The terms "cancer" and "cancerous" mean or describe a physiological condition in mammals typically characterized by uncontrolled cell proliferation.

[0188] In some embodiments, cancer can be a solid tumor. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of the pancreas, colon, cecum, stomach, brain, head, neck, ovaries, kidneys, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.

[0189] In some aspects, cancer is a hematological cancer. Exemplary hematological tumors include, but are not limited to, tumors of the bone marrow, T or B cell malignancies, myeloid malignancies, leukemia, lymphoma, blastoma, and myeloma.

[0190] Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0191] Cancer can be, but is not limited to, the following histological types: neoplasms, malignant tumors; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; piloerector carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; cord-like adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial adenomatous polyposis adenocarcinoma; solid tumors; malignant carcinoid tumors; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; phosinophilic carcinoma; phosinophilic carcinoma; phosinophilic carcinoma Acidic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant male germ cell tumor; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; achromatic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanoma; nodular melanoma; malignant melanoma in a giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma Malignant mesothelioma; undifferentiated germ cell tumor; fetal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paraosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastoma; malignant ameloblastoma; ameloblastoma; malignant pineal glandoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma;Neuroblastoma; retinoblastoma; olfactory neuroma; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; T lymphoblastic leukemia; T lymphoblastic lymphoma; B cell leukemia; Hodgkin's disease; Hodgkin's lymphoma; lateral granuloma; small lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other identified Non-Hodgkin lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-incisional nuclear cell NHL; Bulky lesion NHL; Mantle cell lymphoma; AIDS-associated lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia, acute lymphoblastic leukemia (ALL); acute lymphoblastic leukemia; acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasm; chronic myeloblastic leukemia; diffuse large B-cell lymphoma (DLBCL); peripheral T-cell lymphoma (PTCL); or anaplastic large cell lymphoma (ALCL). In some embodiments, cancer includes liquid tumors. In some embodiments, the fluid tumor is leukemia or lymphoma. In some embodiments, the leukemia or lymphoma is B-cell leukemia or B-cell lymphoma.

[0192] In some embodiments, cancer is characterized by the expression of both CD276 and CD99 in cancerous cells.

[0193] In some cases, the cancer is diffuse pontine glioma (DIPG).

[0194] In some cases, the cancer is acute myeloid leukemia (AML).

[0195] In some cases, the cancer is Ewing's sarcoma.

[0196] In some manifestations, the cancer is an ependymoma. In some manifestations, the cancer is a posterior fossa type A (PFA) ependymoma.

[0197] In some cases, the cancer is an atypical teratomatoid rhabdoid tumor (ATRT).

[0198] In some cases, the cancer is NK / T-cell lymphoma.

[0199] In some manifestations, cancer is a large granular lymphocytic leukemia.

[0200] In some forms, cancer is chronic myeloid leukemia.

[0201] In some cases, the cancer is glioblastoma.

[0202] In some forms, cancer is a glioma.

[0203] In some forms, cancer is an ependymoma.

[0204] In some cases, cancer is neuroblastoma.

[0205] In some embodiments, the cancer is glioblastoma multiforme (GBM). Therefore, in some embodiments, the subject may be an adult with glioblastoma multiforme.

[0206] In some embodiments, the cancer is recurrent cancer. Therefore, the cancer can be selected from recurrent DIPG, recurrent AML, and recurrent Ewing's sarcoma.

[0207] In some embodiments, cancer is refractory to treatment using therapies other than the therapeutic cells of the Disclosure. Therefore, cancer can be selected from refractory DIPG, refractory AML, and refractory Ewing sarcoma.

[0208] In some manifestations, the cancer is metastatic. Therefore, the cancer can be selected from metastatic DIPG, metastatic AML, and metastatic Ewing sarcoma.

[0209] The terms “subject” and “patient” are used interchangeably herein. In some embodiments, the subject treated according to the methods described herein is a human patient. In some embodiments, the subject is male. In some embodiments, the subject is female.

[0210] In some embodiments of the methods of this disclosure, the subject may be at least about 5 years old, or at least about 10 years old, or at least about 15 years old, or at least about 18 years old, or at least about 20 years old, or at least about 25 years old, or at least about 30 years old, or at least about 35 years old, or at least about 40 years old, or at least about 45 years old, or at least about 50 years old, or at least about 55 years old, or at least about 60 years old, or at least about 65 years old, or at least about 70 years old, or at least about 75 years old, or at least about 80 years old, or at least about 85 years old, or at least about 90 years old, or at least about 95 years old, or at least about 100 years old.

[0211] In some aspects of the methods of this disclosure, the subject may be approximately 5 years old, or approximately 10 years old, or approximately 15 years old, or approximately 18 years old, or approximately 20 years old, or approximately 25 years old, or approximately 30 years old, or approximately 35 years old, or approximately 40 years old, or approximately 45 years old, or approximately 50 years old, or approximately 55 years old, or approximately 60 years old, or approximately 65 years old, or approximately 70 years old, or approximately 75 years old, or approximately 80 years old, or approximately 85 years old, or approximately 90 years old, or approximately 95 years old, or approximately 100 years old or younger.

[0212] In some aspects of the method of this disclosure, the subject may be approximately 1 to 20 years old, or approximately 2 to 10 years old, or approximately 5 to 9 years old, or approximately 10 to 15 years old.

[0213] In some embodiments, the subject may have received at least one prior therapy; that is, prior to the administration of the therapeutic cells of the Disclosure, the subject has received at least one prior therapy. In some embodiments, the subject may be unresponsive to at least one prior therapy. In some embodiments, the subject may have initially responded to at least one prior therapy but subsequently became unresponsive to at least one prior therapy over the course of treatment. In some embodiments, at least one prior therapy continues to be administered to the subject after the administration of the therapeutic cells of the Disclosure.

[0214] In some embodiments, at least one prior therapy may be selected from surgery, immunotherapy, stem cell transplantation, anticancer therapy, chemotherapy, targeted therapy, radiotherapy, or any combination thereof. In some embodiments, at least one prior therapy is radiotherapy. In some embodiments, at least one prior therapy is chemotherapy. In some embodiments, at least one prior therapy includes administration of a combination of venetoclax and azacitidine.

[0215] In some embodiments, at least one prior treatment is surgical. In some embodiments, the surgical procedure involves amputation of one or more body parts. Accordingly, the present disclosure provides a method for preventing cancer metastasis in a subject having Ewing's sarcoma, the subject having previously undergone amputation of one or more body parts to treat Ewing's sarcoma.

[0216] As used herein, the terms “to treat” or “to heal” describe the management and care of a patient for the purpose of combating a disease, condition, or disorder, and include the administration of the compounds of this disclosure, or their pharmaceutically acceptable salts, polymorphs, or solvates, for the purpose of reducing the symptoms or complications of the disease, condition, or disorder, or for eliminating the disease, condition, or disorder. The term “to treat” may also include the treatment of cell or animal models in vitro.

[0217] In this specification, "prevent," "prevent," etc., refer to stopping the onset of a disease, condition, or disorder, or one or more of its symptoms or complications.

[0218] The terms “effective dose” and “therapeutic dose” for cells, drugs, or compounds are used in their broadest sense to mean a quantity of cells, active drugs, or compounds that are non-toxic but sufficient to provide the desired effect or benefit.

[0219] In some embodiments, the therapeutic cells of the present disclosure are at least about 0.1 × 10⁶ 6 cells / kg, or at least about 0.25 × 10⁻⁶ 6 cells / kg, or at least about 0.5 × 10⁻⁶ 6 cells / kg, or at least about 0.75 × 10⁶ 6 cells / kg, or at least about 1 × 10⁶ 6 cells / kg, or at least about 1.25 × 10⁻⁶ 6 cells / kg, or at least about 1.5 × 10⁶ 6 cells / kg, or at least about 1.75 × 10⁻⁶ 6 cells / kg, or at least about 2 × 10⁶ 6 cells / kg, or at least about 2.25 × 10⁻⁶ 6 cells / kg, or at least about 2.5 × 10⁻⁶ 6 cells / kg, or at least about 2.75 × 10⁻⁶ 6 cells / kg, or at least about 3 × 10⁶ 6 It can be administered to the subject at a dose of cells / kg.

[0220] In some embodiments, the therapeutic cells of this disclosure are approximately 0.1 × 10⁻⁶ 6 Cells / kg, or approximately 0.25 × 10⁻⁶ 6 Cells / kg, or approximately 0.5 × 10⁻⁶ 6 Cells / kg, or approximately 0.75 × 10⁻⁶ 6 cells / kg, or approximately 1 × 10⁶ 6 Cells / kg, or approximately 1.25 × 10⁻⁶ 6 Cells / kg, or approximately 1.5 × 10⁻⁶ 6 Cells / kg, or approximately 1.75 × 10⁻⁶6 Cells / kg, or approximately 2 × 10⁻⁶ 6 Cells / kg, or approximately 2.25 × 10⁻⁶ 6 Cells / kg, or approximately 2.5 × 10⁻⁶ 6 Cells / kg, or approximately 2.75 × 10⁻⁶ 6 Cells / kg, or approximately 3 × 10⁻⁶ 6 It can be administered to the subject at a dose of cells / kg.

[0221] In some embodiments, the therapeutic cells of this disclosure are approximately 5 × 10 6 Cells, or approximately 10 x 10 6 Cells, or approximately 15 x 10 6 Cells, or approximately 20 x 10 6 Cells, or approximately 25 x 10 6 Cells, or approximately 30 x 10 6 Cells, or approximately 35 x 10 6 Cells, or approximately 40 x 10 6 Cells, or approximately 45 x 10 6 Cells, or approximately 50 x 10 6 Cells, or approximately 55 x 10 6 Cells, or approximately 60 x 10 6 Cells, or approximately 65 x 10 6 Cells, or approximately 70 x 10 6 Cells, or approximately 75 x 10 6 Cells, or approximately 80 x 10 6 Cells, or approximately 85 x 10 6 Cells, or approximately 90 x 10 6 Cells, or approximately 95 x 10 6 Cells, or approximately 100 x 10 6 Cells, or approximately 10⁵ × 10⁵ 6 Cells, or approximately 110 × 10 6 1 piece, or approximately 115 x 10 6 Cells, or approximately 120 x 10 6 Cells, or approximately 125 × 10 6 Cells, or approximately 130 x 10 6 Cells, or approximately 135 × 10 6 Cells, or approximately 140 x 10 6 Cells, or approximately 145 × 10 6 Cells, or approximately 150 x 10 6cells, or about 155×10 6 cells, or about 160×10 6 cells, or about 165×10 6 cells, or about 170×10 6 cells, or about 175×10 6 cells, or about 180×10 6 cells, or about 185×10 6 cells, or about 190×10 6 cells, or about 195×10 6 cells, or about 200×10 6 cells, or about 205×10 6 cells, or about 210×10 6 cells, or about 215×10 6 cells, or about 220×10[[ID=并27]] 6 cells, or about 225×10 6 cells, or about 230×10 6 cells, or about 235×10 6 cells, or about 240×10 6 cells, or about 245×10 6 cells, or about 250×10 6 cells, or about 300×10 6 cells, or about 350×10 6 cells, or about 400×10 6 cells, or about 450×10 6 cells, or about 500×10 6 cells, or about 600×10 6 cells, or about 700×10 6 can be administered in an amount not exceeding about cells, or about 155×10

[0222] Note: There seems to be a formatting issue in the original text around line 27 where "并27" is present. It's not clear what that should be. I've translated it as is, but it might need to be corrected in the original source for a more accurate translation.The term “benefit” is used in its broadest sense and refers to any desired effect, specifically including clinical benefits as defined herein. Clinical benefits can be measured by evaluating various endpoints, e.g., inhibition of disease progression to a certain extent, including slowing and complete cessation; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition of disease cell infiltration into adjacent peripheral organs and / or tissues (i.e., reduction, slowing, or complete cessation); inhibition of disease spread (i.e., reduction, slowing, or complete cessation); reduction of autoimmune responses that may or may not result in regression or ablation of disease lesions; reduction to a certain extent of one or more symptoms associated with the disorder; increased duration of asymptomatic period after treatment, e.g., progression-free survival; increased overall survival; higher response rates; and / or a reduction in mortality at a given time point after treatment.

[0223] Cell generation method disclosed herein This disclosure provides therapeutic cells and methods for generating a large number of therapeutic cells.

[0224] Accordingly, the present disclosure provides a method for generating a plurality of cells expressing a logical pair of the CAR polypeptide of the present disclosure, comprising the steps of (a) obtaining a plurality of cells from a subject; and (b) introducing one or more nucleic acid molecules of the present disclosure into the plurality of cells, wherein the one or more nucleic acid molecules comprise one or more nucleic acid sequences encoding a logical pair of the CAR polypeptide of the present disclosure.

[0225] In applications where cells are obtained from a subject and subsequently returned to the subject therapeutically through transplantation, these cells are called "autologous." In applications where cells are obtained from a first subject and subsequently transplanted into a different second subject, these cells are called "allogeneic."

[0226] The term "introduction" refers to the presentation of a nucleic acid molecule to a cell in a manner that allows the nucleic acid molecule to gain access to the interior of the host cell. The methods of this disclosure do not depend on any specific method for introducing a nucleic acid molecule into a host cell, but solely on the polynucleotide construct gaining access to the interior of a single host cell. Methods for introducing nucleic acid molecules (e.g., vectors) into bacteria, plants, fungi, and animals, including but not limited to stable transformation methods, transient transformation methods, and virus-mediated methods, are known in the art. For example, lentiviral and retroviral transduction methods can be used. As will be understood by those skilled in the art, these methods may include the use of retronectin to enhance the efficiency of transduction.

[0227] The cell generation method described above may further include a step of expanding and growing the cells before step (b), after step (b), or both before and after step (b). Cell expansion and growth can be carried out using any cell expansion and growth method known in the art.

[0228] The cell generation method described above may further include a step of culturing cells before step (b), after step (b), or both before and after step (b). The cell culture step can be carried out using any cell culture method known in the art.

[0229] The manufacturing method described above may further include, after step (b), a step of enriching cells expressing the logical pair of CAR polypeptides of the present disclosure. Enrichment can be carried out by contacting a plurality of cells with an affinity reagent that binds to one of the CAR polypeptides of the logical pair of CAR polypeptides. In some embodiments, the affinity reagent is an antibody that binds to one of the CAR polypeptides of the logical pair. In some embodiments, after enrichment, at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99%, or at least about 100% of the cells express a logical pair of the CAR polypeptides of the Disclosure.

[0230] kit This disclosure provides a kit comprising any of the compositions described herein, or any combination of the compositions described herein.

[0231] Accordingly, the Disclosure provides a kit comprising (i) the anti-CD99 CAR polypeptide of the Disclosure; (ii) the anti-CD276 CAR polypeptide of the Disclosure; (iii) logical pairs of CAR polypeptides of the Disclosure; (iv) cells of the Disclosure; (v) a population of cells of the Disclosure; (vi) nucleic acid molecules of the Disclosure; or any combination thereof.

[0232] General methods and definitions Methods for gene delivery and cell modification As used herein, “vector” means a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell, as defined in some embodiments. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0233] Those skilled in the art will be well capable of constructing vectors for the expression of the antigen receptors of this disclosure through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Vectors include, but are not limited to, retroviral vectors (e.g., derived from Moloney's mouse leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors including their replication-capable, replication-deficient, and gutless forms, adeno-associated virus (AAV) vectors, Simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, and Epsomian virus vectors. Examples include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), such as humper virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey mouse sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and group B adenovirus enadenotucirev vectors.

[0234] Viral vector Viral vectors encoding antigen receptors, cytokines, and / or functional effector elements can be provided in certain embodiments of the methods of this disclosure. In the production of recombinant viral vectors, non-essential genes are typically replaced by genes or coding sequences for heterologous (or non-innate) proteins. Viral vectors are a type of expression construct that utilizes viral sequences to introduce nucleic acids and, optionally, proteins, into cells. The ability of certain viruses to infect cells or enter them via receptor-mediated endocytosis, and their ability to integrate into the host cell genome to stably and efficiently express viral genes, has made them attractive candidates for the delivery of exogenous nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain embodiments of the present invention are described below.

[0235] In some embodiments of the methods of this disclosure, introducing nucleic acid sequences and / or genome-editing constructs into immune cells ex vivo, in vivo, in vitro, or in situ involves a viral vector. In some embodiments, the viral vector is a non-integrating non-chromosomal vector. Examples of non-integrating non-chromosomal vectors include, but are not limited to, adeno-associated viruses (AAVs), adenoviruses, and herpesviruses. In some embodiments, the viral vector is an integrating chromosomal vector. Examples of integrating chromosomal vectors include, but are not limited to, adeno-associated vectors (AAVs), lentiviruses, and gamma-retroviruses.

[0236] In some embodiments of the methods of the present disclosure, introducing a nucleic acid sequence and / or a genome editing construct into immune cells ex vivo, in vivo, in vitro or in situ involves a combination of vectors. Exemplary non-limiting combinations of vectors include viral and non-viral vectors, multiple non-viral vectors, or multiple viral vectors. Exemplary but non-limiting combinations of vectors include combinations of DNA-derived and RNA-derived vectors, combinations of RNA and reverse transcriptase, combinations of transposons and transposases, combinations of non-viral vectors and endonucleases, and combinations of viral vectors and endonucleases.

[0237] In some embodiments of the methods of the present disclosure, genome modification, which includes introducing a nucleic acid sequence and / or a genome editing construct into immune cells ex vivo, in vivo, in vitro or in situ, results in stable integration of the nucleic acid sequence, transient integration of the nucleic acid sequence, site-specific integration of the nucleic acid sequence, or biased integration of the nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0238] In some embodiments of the methods of the present disclosure, genome modification, which includes introducing nucleic acid sequences and / or genome editing constructs into immune cells ex vivo, in vivo, in vitro or in situ, results in stable integration of the nucleic acid sequences. In some embodiments, stable chromosomal integration can be random integration, site-specific integration, or biased integration. In some embodiments, site-specific integration can be non-assisted or assisted. In some embodiments, assisted site-specific integration is co-delivered with a site-specific nuclease. In some embodiments, the site-specific nuclease comprises a transgene having 5' and 3' nucleotide sequence extensions that include percent homology to regions upstream and downstream of the site of genome integration. In some embodiments, a transgene having homology nucleotide extensions enables genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. In some embodiments, site-specific integration occurs at a safe harbor site. A safe harbor site of the genome provides for integration of new genetic material in a manner that ensures that newly inserted gene elements function reliably (e.g., are expressed at therapeutically effective levels) and do not cause deleterious changes to the host genome that pose a risk to the host organism. Possible genomic safe harbors include, but are not limited to, the intron sequence of the human albumin gene, the adeno-associated virus site 1 (AAVS1), the naturally occurring integration site of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

[0239] In some embodiments, site-directed transgene integration occurs at a site that interferes with the expression of the target gene. In some embodiments, interference with target gene expression occurs through site-directed integration at introns, exons, promoters, gene elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-directed integration include, but are not limited to, any immunosuppressive genes and genes involved in allo-rejection.

[0240] In some embodiments, site-directed transgene integration occurs at sites that result in enhanced expression of the target gene. In some embodiments, enhanced target gene expression occurs through site-directed integration at introns, exons, promoters, gene elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0241] Adjustment element Expression cassettes included in useful vectors in this disclosure include, in particular, eukaryotic transcription promoters functionally linked to protein-coding sequences (in the 5'-3' direction), splicing signals including intervening sequences, and transcription termination / polyadenylation sequences. Promoters and enhancers that regulate the transcription of protein-coding genes in eukaryotic cells are composed of multiple gene elements. Cellular mechanisms can collect and integrate regulatory information transmitted by each element, thereby enabling various genes to develop unique, often complex, patterns of transcriptional regulation. Promoters used in the context of this disclosure include constitutive, inducible, and tissue-specific promoters.

[0242] origin of replication To propagate the vector in host cells, the vector may contain one or more origin sites (often referred to as "ori"), for example, nucleic acid sequences corresponding to the oriP of EBV as described above, which are specific nucleic acid sequences where replication begins, or genetically modified oriPs with similar or enhanced function during programming. Alternatively, origins of replication or autonomous replication sequences (ARS) of other extrachromosomal replicating viruses as described above can be used.

[0243] Other methods of nucleic acid delivery In addition to viral delivery of nucleic acids encoding antigen receptors, the following are additional methods for recombinant gene delivery to a given cell (e.g., NK cells), and are therefore discussed in this disclosure.

[0244] The introduction of nucleic acid molecules such as DNA or RNA into immune cells as described herein can be carried out using any preferred method for nucleic acid delivery relating to cell transformation, as described herein or as would be known to those skilled in the art. Such methods include, but are not limited to, ex vivo transfection, injection including microinjection; electroporation; calcium phosphate precipitation; using DEAE-dextran followed by polyethylene glycol; direct sonication; liposome-mediated transfection and receptor-mediated transfection; lipid nanoparticle transfection; microprojectile bombardment; stirring with silicon carbide fibers; Agrobacterium-mediated transformation; direct DNA delivery such as drying / inhibition-mediated DNA incorporation, and any combination of such methods. Through the application of these and other techniques, organelles, cells, tissues, or organisms can be transformed stably or transiently.

[0245] Generally, gene transfer systems can include transposon-based or virus-based integration systems.

[0246] In some embodiments, the gene transfer system includes a transposon system. DNA transposons can move via a non-replicating "cut and paste" mechanism. This mechanism requires recognition of two inverted terminal repeats (ITRs) by a catalytic enzyme, i.e., a transposase, which can cleave its target and consequently detach the DNA transposon from its donor template. Upon cleavage, the DNA transposon can subsequently integrate into acceptor DNA, which is cleaved by the same transposase. In some of their native configurations, DNA transposons may have two adjacent ITRs and may contain a gene encoding a transposase that catalyzes the transposition. As will be understood by those skilled in the art, transposon systems offer numerous advantages over nucleic acid integration, for example, compared to viral vectors. For example, transposons can carry relatively large cargo, which can be advantageous for delivering one or more of the CARs, functional effector elements, and / or cytokines disclosed herein to immune cells (e.g., NK cells). Furthermore, transposons can be used, for example, with CRISPR tools (e.g., along with cargo), thereby enabling multiple gene manipulation of cells.

[0247] Chimeric antigen receptor Chimeric antigen receptors are also known as artificial cell receptors, chimeric cell receptors, or chimeric immune receptors. As will be understood by those skilled in the art, chimeric antigen receptors (CARs) are genetically modified receptors that transplant selected specificities into immune effector cells. CARs typically have an extracellular domain (ectodomain), a transmembrane domain, and an intracellular (endodomain). In some embodiments, the ectodomain includes an antigen-binding domain and a hinge domain, the antigen-binding domain specifically binding to an antigen of particular interest in the treatment of a specific disease or disorder (e.g., an antigen localized on a specific cancer cell or an antigen localized on an infected cell). In some embodiments, the antigen is a protein expressed on the surface of a cell (e.g., on the surface of a cancer cell or an infected cell).

[0248] As will be understood by those skilled in the art, CARs are available in various different "forms," ​​sometimes referred to as different "generations" of CARs (see, for example, Hiltensperger M, Krackhardt AM. Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells. Front Immunol. 2023 Mar 6;14:1121030. doi: 10.3389 / fimmu.2023.1121030. PMID: 36949949; PMCID: PMC10025359). Therefore, as used herein, the term genetically engineered CAR encompasses any of the forms / generations known in the art.

[0249] In non-limiting examples, CARs may be in the form of universal CARs, such as those disclosed in PCT Publication No. 2012 / 082841 and U.S. Patents No. 9,233,125 and 10,973,893. As will be understood by those skilled in the art, in a universal CAR system, the extracellular domain of the CAR specifically binds to a common “tag” molecule. Such CARs may be referred to as “anti-tag CARs.” These tag molecules can then be fused to antigen-targeting molecules (e.g., antibodies), thereby allowing the anti-tag CAR to indirectly bind to target cells of interest via the tag fusion protein. In this way, a subject may be administered a single population of cells expressing anti-tag CARs and, subsequently, various different tag fusion proteins that can specifically direct the anti-tag CAR cells to various target cells.

[0250] Genetically modified T cell receptors As will be understood by those skilled in the art, T cell receptors (TCRs) are heterodimeric cell surface proteins of the immunoglobulin superfamily associated with the invariant CD3 complex, which is involved in mediating signal transduction. TCRs exist as αβ and γδ heterodimers, which are structurally similar but have very different anatomical localizations and possibly functions. The extracellular portion of the innate heterodimeric PTCR consists of two polypeptide chains, each having a membrane-proximal constant domain and a membrane-distal variable domain. Each of the constant and variable domains contains an intrachain disulfide bond. The variable domain contains a highly pleomorphic loop, similar to the complementarity-determining region (CDR) of an antibody. As used herein, the term “genetically engineered T cell receptor” means a TCR designed to specifically bind to an antigen of particular interest in the treatment of a particular disease or disorder (e.g., an antigen localized on a specific cancer cell or an antigen localized on an infected cell). In some embodiments, the antigen is a protein expressed on the surface of a cell (e.g., on the surface of a cancer cell or an infected cell). As will be understood by those skilled in the art, genetically engineered TCRs are available in a variety of different “forms” (see, for example, Hiltensperger M, Krackhardt AM. Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells. Front Immunol. 2023 Mar 6;14:1121030. doi: 10.3389 / fimmu.2023.1121030. PMID: 36949949; PMCID: PMC10025359). Thus, as used herein, the term genetically engineered TCR encompasses any of the forms known in the art.

[0251] definition As will be understood by those skilled in the art, "CD99" refers to the protein known in the art as differentiation cluster 99, also known as MIC2.

[0252] As will be understood by those skilled in the art, "CD276" refers to the protein known in the art as differentiation cluster 276, also known as B7 homolog 3 (B7-H3).

[0253] As used herein, the term “antigen” refers to a molecule that can be bound by antibodies, T cell receptors, chimeric antigen receptors, and genetically modified immune receptors. Antigens can be widely used to induce humoral immune responses and / or cellular immune responses that lead to the generation of B and / or T lymphocytes.

[0254] The terms “tumor-associated antigen,” “tumor antigen,” and “cancer cell antigen” are interchangeable herein. In each case, these terms refer to proteins, glycoproteins, or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0255] As used herein, the term “part” means, when used in reference to a polypeptide or peptide, a fragment of a polypeptide or peptide. In some embodiments, a “part” of a polypeptide or peptide retains at least one function and / or activity of the full-length polypeptide or peptide from which it is derived. For example, in some embodiments, if a full-length polypeptide binds to a given ligand, a part of the full-length polypeptide also binds to the same ligand.

[0256] The terms "protein" and "polypeptide" are used interchangeably within this specification.

[0257] As is well known in the art, when the terms "nucleic acid molecule", "polynucleotide", or "nucleic acid" are used interchangeably herein, they mean a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can include modified nucleotides such as methylated nucleotides and their analogs. When present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The nucleotide sequence can be interrupted by non-nucleotide components.

[0258] The term "exogenous", when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, means a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or, in relation to a cell, the term means a cell that has been isolated and then introduced into a cell population or organism by artificial or natural means. Exogenous nucleic acids can be derived from a different organism or cell, or can be one or more additional copies of a nucleic acid that naturally exists within the organism or cell. Exogenous cells can be derived from a different organism or from the same organism. By way of non-limiting example, an exogenous nucleic acid is one that is at a chromosomal location different from where it exists in a natural cell, or one that has a nucleic acid sequence adjacent to it that is different from that found naturally in any other manner. The term "exogenous" is used interchangeably with the term "heterologous".

[0259] The terms "expression construct" or "expression cassette" are used to refer to nucleic acid molecules that can be directed to transcription. An expression construct includes at least one transcriptional regulatory element (such as a promoter, enhancer, or a functionally equivalent structure) that directs gene expression in at least one desired cell type, tissue, or organ. Additional elements, such as transcription termination signals, may also be included.

[0260] A gene, polynucleotide, coding region, sequence, nucleic acid sequence, segment, fragment, or transgene that codes for a specific protein is a piece of nucleic acid molecule that, when controlled by appropriate regulatory sequences, is transcribed and optionally translated into a gene product, such as a polypeptide, in vitro or in vivo. The coding region can exist in cDNA, genomic DNA, or RNA form. If in DNA form, the nucleic acid molecule can be single-stranded (i.e., sense strand) or double-stranded. The coding region boundary is determined by a 5' (amino) start codon and a 3' (carboxy) translation termination codon. Genes include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is usually located 3' relative to the gene sequence.

[0261] The term "cell" is used herein in its broadest sense within the art, and refers to a structural unit of tissue in a multicellular organism, surrounded by a membrane structure that separates it from the outside, possessing the ability to self-replicate, and containing genetic information and the mechanisms for expressing it. Cells as used herein may be naturally occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0262] As used herein, the term "antibody" means monoclonal or polyclonal antibodies. The term "monoclonal antibody" as used herein means an antibody produced by a single clone of B cells and that binds to the same epitope. In contrast, a "polyclonal antibody" means a population of antibodies produced by different B cells and that binds to different epitopes of the same antigen. A complete antibody typically consists of four polypeptides: two identical copies of the heavy chain (H) polypeptide and two identical copies of the light chain (L) polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have a similar overall structure, and each region contains four framework regions whose sequences are relatively conserved. The framework regions are linked by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable regions" of the antibody responsible for antigen binding. [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​These are MHC class I-restricted T cells, however, some cytotoxic T cells are CD4 + In some embodiments, the T cells of this disclosure are CD4 + or CD8 + That is the case.

[0264] The activated state of a T cell is when the T cell is "resting" (i.e., in the G phase of the cell cycle). o This defines whether a T cell is in the remission phase or is "activated" to proliferate after appropriate stimulation, such as stimulation by OKT3 antibody, PHA, or PMA, or upon recognition of its specific antigen. The "phenotype" of a T cell (e.g., naive, central memory, effector memory, lysing effector, help effector (THI and TH2 cells), and regulatory effector) describes the function the cell exerts when activated. Healthy donors have T cells of each of these phenotypes, and they are primarily in a quiescent state. Naive T cells proliferate when activated and subsequently differentiate into memory T cells or effector T cells. They can then become quiescent again until the next activation to exert their novel function, and their phenotype can change again. Effector T cells will sequester upon activation and antigen-specific effector function.

[0265] Natural killer T cells (NKT cells) bridge the gap between the adaptive immune system and the innate immune system, so as not to be confused with the natural killer cells of the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (WIC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. When activated, these cells can exhibit functions belonging to both Th and Tc cells (i.e., cytokine production and release of cytolytic / cytotoxic molecules). They can also recognize and eliminate some tumor cells and herpesvirus-infected cells.

[0266] Natural killer cells ("NK cells") are a type of cytotoxic lymphocyte in the innate immune system. In some cases, NK cells provide the first line of defense against viral infection and / or tumorigenesis. NK cells can detect MHC presented on infected or cancerous cells, triggering cytokine release, which in turn induces lysis and apoptosis. NK cells can also detect stressed cells in the absence of antibodies and / or MHC, thereby enabling a rapid immune response.

[0267] The term "culturing" means the in vitro maintenance, differentiation, and / or proliferation of cells in a suitable culture medium. "Enriched" means a composition containing cells that constitute a higher percentage of the total cells than would be found in the tissues in which it exists in an organism.

[0268] When used throughout this disclosure, the identity between two sequences can be determined by using a standalone executable BLAST engine program (bl2seq) for BLAST searching of two sequences, which can be read from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (incorporated herein by reference in its entirety, Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250). The term “identity” or “identity,” when used in the context of two or more nucleic acid or polypeptide sequences, means the specified percentage of residues that are the same across each specified region of the sequences. In some embodiments, sequence identity is determined over the entire length of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing them across a specified region, determining the number of positions where identical residues exist in both sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more misaligned ends, and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0269] Exemplary Embodiments Embodiment 1. An anti-CD276 CAR polypeptide comprising an anti-CD276 antigen-binding domain, a transmembrane domain, and at least one costimulatory domain, from the N-terminus to the C-terminus.

[0270] Embodiment 2. An anti-CD276 CAR polypeptide comprising an anti-CD276 antigen-binding domain, a transmembrane domain, and at least one activating domain, from the N-terminus to the C-terminus.

[0271] Embodiment 3. An anti-CD276 CAR polypeptide from any of the prior embodiments, further comprising a hinge domain located between an anti-CD276 antigen-binding domain and a transmembrane domain.

[0272] Embodiment 4. An anti-CD276 CAR polypeptide of any one of the prior embodiments, further comprising a signal peptide at its N-terminus.

[0273] Embodiment 5. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the anti-CD276 antigen-binding domain comprises an anti-CD276 scFv.

[0274] Embodiment 6. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the anti-CD276 antigen-binding domain comprises CDHR1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, CDRH3 having the amino acid sequence of SEQ ID NO: 3, CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDHL3 having the amino acid sequence of SEQ ID NO: 6.

[0275] Embodiment 7. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the anti-CD276 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 12.

[0276] Embodiment 8. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the transmembrane domain includes a CD8 transmembrane domain.

[0277] Embodiment 9. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 46.

[0278] Embodiment 10. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the transmembrane domain includes a CD28 transmembrane domain.

[0279] Embodiment 11. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44.

[0280] Embodiment 12. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein at least one co-stimulatory domain comprises at least one 4-1BB co-stimulatory polypeptide.

[0281] Embodiment 13. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein at least one 4-1BB co-stimulatory polypeptide comprises the amino acid sequence of SEQ ID NO: 50.

[0282] Embodiment 14. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein at least one co-stimulatory domain comprises at least one CD28 co-stimulatory polypeptide.

[0283] Embodiment 15. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein at least one CD28 co-stimulatory polypeptide comprises the amino acid sequence of SEQ ID NO: 48.

[0284] Embodiment 16. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the signal peptide comprises a CD8 signal peptide.

[0285] Embodiment 17. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 56.

[0286] Embodiment 18. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the signal peptide comprises a GMCSF signal peptide.

[0287] Embodiment 19. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the GMCSF signal peptide comprises the amino acid sequence of SEQ ID NO: 58.

[0288] Embodiment 20. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the hinge domain comprises a CD8 hinge domain.

[0289] Embodiment 21. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 42.

[0290] Embodiment 22. An anti-CD276 CAR polypeptide from any one of the prior embodiments, wherein the hinge domain comprises a CD28 hinge domain.

[0291] Embodiment 23. An anti-CD276 CAR polypeptide of any one of the prior embodiments, wherein the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 40.

[0292] Embodiment 24. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the activating domain comprises a CD3ζ-activating polypeptide.

[0293] Embodiment 25. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD3ζ-activating polypeptide comprises the amino acid sequence of SEQ ID NO: 52.

[0294] Embodiment 26. An anti-CD276 CAR polypeptide from any one of the prior embodiments, comprising, from N-terminus to C-terminus, a signal peptide containing a GMSCF signal peptide, an anti-CD276 antigen-binding domain containing an anti-CD276 scFv, a hinge domain containing a CD28 hinge polypeptide, a transmembrane domain containing a CD28 transmembrane polypeptide, and at least one costimulatory domain containing at least one CD28 costimulatory polypeptide.

[0295] Embodiment 27. An anti-CD276 CAR polypeptide of any one of the prior embodiments, comprising the amino acid sequence of SEQ ID NO: 63.

[0296] Embodiment 28. An anti-CD276 CAR polypeptide from any one of the prior embodiments, comprising, from N-terminus to C-terminus, a signal peptide containing a GMCSF signal peptide, an anti-CD276 antigen-binding domain containing an anti-CD276 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and at least one costimulatory domain containing at least one 4-1BB costimulatory polypeptide.

[0297] Embodiment 29. An anti-CD276 CAR polypeptide of any one of the prior embodiments, comprising the amino acid sequence of SEQ ID NO: 64.

[0298] Embodiment 30. An anti-CD276 CAR polypeptide from any one of the prior embodiments, comprising, from N-terminus to C-terminus, a signal peptide containing a GMCSF signal peptide, an anti-CD276 antigen-binding domain containing an anti-CD276 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and at least one activation domain containing at least one CD3ζ activating polypeptide.

[0299] Embodiment 31. An anti-CD276 CAR polypeptide of any one of the prior embodiments, comprising the amino acid sequence of SEQ ID NO: 62.

[0300] Embodiment 32. An anti-CD99 CAR polypeptide comprising an anti-CD99 antigen-binding domain, a transmembrane domain, and an activating domain from the N-terminus to the C-terminus.

[0301] Embodiment 33. An anti-CD99 CAR polypeptide comprising an anti-CD99 antigen-binding domain, a transmembrane domain, and a costimulatory domain from the N-terminus to the C-terminus.

[0302] Embodiment 34. An anti-CD99 CAR polypeptide from any of the prior embodiments, further comprising a hinge domain located between an anti-CD99 antigen-binding domain and a transmembrane domain.

[0303] Embodiment 35. An anti-CD99 CAR polypeptide of any one of the prior embodiments, further comprising a signal peptide at its N-terminus.

[0304] Embodiment 36. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the anti-CD99 antigen-binding domain comprises an anti-CD99 scFv.

[0305] Embodiment 37. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the anti-CD99 antigen-binding domain comprises CDHR1 having the amino acid sequence of SEQ ID NO: 27, CDRH2 having the amino acid sequence of SEQ ID NO: 28, CDRH3 having the amino acid sequence of SEQ ID NO: 29, CDRL1 having the amino acid sequence of SEQ ID NO: 30, CDRL2 having the amino acid sequence of SEQ ID NO: 31, and CDHL3 having the amino acid sequence of SEQ ID NO: 32.

[0306] Embodiment 38. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the anti-CD99 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 38.

[0307] Embodiment 39. An anti-CD99 CAR polypeptide of any one of the prior embodiments, wherein the transmembrane domain includes a CD8 transmembrane domain.

[0308] Embodiment 40. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 46.

[0309] Embodiment 41. An anti-CD99 CAR polypeptide of any one of the prior embodiments, wherein the transmembrane domain includes a CD28 transmembrane domain.

[0310] Embodiment 42. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44.

[0311] Embodiment 43. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the activating domain comprises a CD3ζ-activating polypeptide.

[0312] Embodiment 44. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD3ζ-activating polypeptide comprises the amino acid sequence of SEQ ID NO: 52.

[0313] Embodiment 45. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein at least one co-stimulatory domain comprises at least one 4-1BB co-stimulatory polypeptide.

[0314] Embodiment 46. An anti-CD99 CAR polypeptide of any one of the prior embodiments, wherein at least one 4-1BB co-stimulatory polypeptide comprises the amino acid sequence of SEQ ID NO: 50.

[0315] Embodiment 47. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein at least one co-stimulatory domain comprises at least one CD28 co-stimulatory polypeptide.

[0316] Embodiment 48. An anti-CD99 CAR polypeptide of any one of the prior embodiments, wherein at least one CD28 co-stimulatory polypeptide comprises the amino acid sequence of SEQ ID NO: 48.

[0317] Embodiment 49. An anti-CD99 CAR polypeptide of any one of the prior embodiments, wherein the signal peptide comprises a CD8 signal peptide.

[0318] Embodiment 50. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 56.

[0319] Embodiment 51. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the signal peptide comprises a GMCSF signal peptide.

[0320] Embodiment 52. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the GMCSF signal peptide comprises the amino acid sequence of SEQ ID NO: 58.

[0321] Embodiment 53. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the hinge domain comprises a CD8 hinge domain.

[0322] Embodiment 54. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 42.

[0323] Embodiment 55. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the hinge domain comprises a CD28 hinge domain.

[0324] Embodiment 56. An anti-CD99 CAR polypeptide from any one of the prior embodiments, wherein the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 40.

[0325] Embodiment 57. An anti-CD99 CAR polypeptide from any one of the prior embodiments, comprising, from N-terminus to C-terminus, a signal peptide containing a CD8 signal peptide, an anti-CD99 antigen-binding domain containing anti-CD276 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and an activation domain containing a CD3ζ activating polypeptide.

[0326] Embodiment 58. An anti-CD99 CAR polypeptide of any one of the prior embodiments, comprising the amino acid sequence of SEQ ID NO: 65.

[0327] Embodiment 59. An anti-CD99 CAR polypeptide from any one of the prior embodiments, comprising, from N-terminus to C-terminus, a signal peptide containing a CD8 signal peptide, an anti-CD99 antigen-binding domain containing an anti-CD99 scFv, a hinge domain containing a CD28 hinge polypeptide, a transmembrane domain containing a CD28 transmembrane polypeptide, and at least one costimulatory domain containing at least one CD28 costimulatory polypeptide.

[0328] Embodiment 60. An anti-CD99 CAR polypeptide of any one of the prior embodiments, comprising the amino acid sequence of SEQ ID NO: 61.

[0329] Embodiment 61. An anti-CD99 CAR polypeptide from any one of the prior embodiments, comprising, from N-terminus to C-terminus, a signal peptide containing a GMCSF signal peptide, an anti-CD99 antigen-binding domain containing an anti-CD99 scFv, a hinge domain containing a CD8 hinge polypeptide, a transmembrane domain containing a CD8 transmembrane polypeptide, and at least one costimulatory domain containing at least one 4-1BB costimulatory polypeptide.

[0330] Embodiment 62. An anti-CD99 CAR polypeptide of any one of the prior embodiments, comprising the amino acid sequence of SEQ ID NO: 60.

[0331] Embodiment 63. A logical pair of CAR polypeptides comprising (i) an anti-CD276 CAR polypeptide from any one of the prior embodiments; and (ii) an anti-CD99 CAR polypeptide from any one of the prior embodiments.

[0332] Embodiment 64. A nucleic acid molecule comprising one or more nucleic acid sequences encoding an anti-CD276 CAR polypeptide of any one of the prior embodiments.

[0333] Embodiment 65. A nucleic acid molecule comprising one or more nucleic acid sequences encoding an anti-CD99 CAR polypeptide of any one of the prior embodiments.

[0334] Embodiment 66. A nucleic acid molecule comprising (i) one or more nucleic acid sequences encoding an anti-CD276 CAR polypeptide of any one of the prior embodiments; and (ii) one or more nucleic acid sequences encoding an anti-CD99 CAR polypeptide of any one of the prior embodiments.

[0335] Embodiment 67. A nucleic acid molecule from any one of the prior embodiments, wherein one or more nucleic acid sequences encoding an anti-CD276 CAR polypeptide and one or more nucleic acid sequences encoding an anti-CD99 CAR polypeptide are separated by a nucleic acid sequence encoding a self-cleaving peptide.

[0336] Embodiment 68. A nucleic acid molecule from any one of the prior embodiments, wherein the self-cleaving peptide is a P2A peptide.

[0337] Embodiment 69. A nucleic acid molecule from any one of the prior embodiments, comprising the nucleic acid sequence of SEQ ID NO: 67.

[0338] Embodiment 70. A nucleic acid molecule from any one of the prior embodiments, comprising the nucleic acid sequence of SEQ ID NO: 69.

[0339] Embodiment 71. A nucleic acid molecule from any one of the prior embodiments, comprising the nucleic acid sequence of SEQ ID NO: 71.

[0340] Embodiment 72. A nucleic acid molecule from any one of the prior embodiments, comprising the nucleic acid sequence of Sequence ID No. 73.

[0341] Embodiment 73. A vector comprising a nucleic acid molecule from any one of the prior embodiments.

[0342] Embodiment 74. A viral vector, which is one of the vectors of the prior embodiments.

[0343] Embodiment 75. A vector according to any one of the prior embodiments, wherein the viral vector is an AAV vector or a lentiviral vector.

[0344] Embodiment 76. Cells expressing an anti-CD276 CAR polypeptide from any one of the prior embodiments and an anti-CD99 CAR polypeptide from any one of the prior embodiments.

[0345] Embodiment 77. A cell expressing a logical pair of CAR polypeptides from any one of the prior embodiments.

[0346] Embodiment 78. A cell from any one of the prior embodiments, which is an immune cell.

[0347] Embodiment 79. A cell from any one of the prior embodiments, wherein the immune cell is a T cell, NK cell, NK-like cell, NKT cell, or cytokine-induced killer (CIK) cell.

[0348] Embodiment 80. A cell from any one of the prior embodiments, wherein the immune cell is a T cell.

[0349] Embodiment 81. A population of cells from any one of the prior embodiments.

[0350] Embodiment 82a. A method for treating cancer in a subject, comprising the step of administering to a subject one or more amounts of a cell population from any one of the prior embodiments.

[0351] Embodiment 82b. A method for preventing cancer metastasis in a subject, comprising the step of administering to the subject one or more amounts of a cell population from any one of the prior embodiments.

[0352] Embodiment 83. Any one of the prior embodiments, further comprising the step of administering at least one additional therapy to a subject.

[0353] Embodiment 84. Any one of the prior embodiments, wherein at least one additional therapy includes radiotherapy.

[0354] Embodiment 85. Any one of the methods of the prior embodiments, wherein at least one additional therapy includes chemotherapy.

[0355] Embodiment 86. Any one of the prior embodiments, wherein at least one additional therapy includes surgical intervention.

[0356] Embodiment 87. A method according to any one of the prior embodiments, wherein cancer is characterized by the expression of both CD99 and CD276 in cancer cells.

[0357] Embodiment 88. A method according to any one of the prior embodiments, wherein the cancer is a diffuse pontine glioma.

[0358] Embodiment 89. A method according to any one of the prior embodiments, wherein the cancer is acute myeloid leukemia.

[0359] Embodiment 90. A method according to any one of the prior embodiments, wherein the cancer is Ewing's sarcoma.

[0360] Embodiment 91. A method according to any one of the prior embodiments, wherein the cancer is a posterior fossa type A (PFA) ependymoma.

[0361] Embodiment 92. A method according to any one of the prior embodiments, wherein the cancer is an atypical teratomatoid rhabdoid tumor (ATRT).

[0362] Embodiment 93. A method according to any one of the prior embodiments, wherein the cancer is NK / T-cell lymphoma.

[0363] Embodiment 94. A method according to any one of the prior embodiments, wherein the cancer is large granular lymphocytic leukemia.

[0364] Embodiment 95. A method according to any one of the prior embodiments, wherein the cancer is chronic myeloid leukemia.

[0365] Embodiment 96. A method according to any one of the prior embodiments, wherein the cancer is glioblastoma.

[0366] Embodiment 97. A method according to any one of the prior embodiments, wherein the cancer is a glioma.

[0367] Embodiment 98. A method according to any one of the prior embodiments, wherein the cancer is an ependymoma.

[0368] Embodiment 99. A method according to any one of the prior embodiments, wherein the cancer is neuroblastoma.

[0369] Embodiment 100. A method according to any one of the prior embodiments, wherein the cancer is glioblastoma multiforme.

[0370] Embodiment 101. A method according to any one of the prior embodiments, wherein the cancer is recurrent cancer.

[0371] Embodiment 102. A method according to any one of the prior embodiments, wherein the cancer is refractory cancer.

[0372] Embodiment 103. A method according to any one of the prior embodiments for treating metastatic cancer.

[0373] Embodiment 104. One of the methods of the prior embodiments, wherein the subject has previously been administered at least one prior therapy.

[0374] Embodiment 105. A method according to any one of the prior embodiments, wherein at least one prior therapy includes radiotherapy.

[0375] Embodiment 106. A method according to any one of the prior embodiments, wherein at least one prior therapy includes chemotherapy.

[0376] Embodiment 107. A method according to any one of the prior embodiments, wherein at least one prior therapy includes surgical intervention.

[0377] Embodiment 108. Any one of the prior embodiments, wherein the surgical procedure involves the amputation of one or more body parts. [Examples]

[0378] Example 1. Genetic engineering of dual antigen CARs to improve efficacy and safety The following experimental example tests the genetic manipulation of a biantigen chimeric antigen receptor (CAR) by designing CAR-T cells such that only binding to both antigens on the same cell leads to complete activation of the CAR (Figure 1A). Using anti-CD99 and anti-CD276 scFv described herein, two logical pairs of CARs were designed: "AND CAR-T-1" (incorporating the 4-1BB costimulatory domain) and "AND CAR-T-2" (incorporating the CD28 costimulatory domain) (see Figure 1B).

[0379] More specifically, the logical AND CAR T-1 included an anti-CD99 CAR corresponding to sequence number 60 and an anti-CD276 CAR corresponding to sequence number 62. The logical AND CAR T-2 included an anti-CD99 CAR corresponding to sequence number 61 and an anti-CD276 CAR corresponding to sequence number 62.

[0380] The applicants tested the specificity of CD99 / CD276 logic gate type CARs in targeting only cells expressing both CD99 and CD276 on their surface, and found that both constructs of the CD99 / CD276 logic gate type CAR were effective in targeting single antigen-expressing T cells (CD99). + We found that it protected (only) and inhibited sibling killing of T cells. Cytokine secretion was present in CD99-expressing T cells only when treated with CD99-BBz CAR-T cells in a 1:5 (tumor cells:CAR-T cells) ratio, and was not present when treated with mock CAR-T cells, anti-CD276 CAR-T cells, or CD99 / CD276 logic gate type CAR-T cells (Figure 2). T cells transduced by either AND CAR T-1 logic pair or AND CAR T-2 logic pair showed increased viability compared to T cells transduced by CD99-BBz CAR, indicating that CD99 / CD276 logic gate type CARs rescue T cells from sibling killing (Figure 3).

[0381] Example 2. Functionality of single antigen CD99 and CD276 CAR-T cells against DIPG. The following experimental examples test the functionality of single antigens CD99 and CD276 CAR-T cells against DIPG. The applicants tested the protein expression of CD99 and CD276 in DIPG patient tumors and cell lines (testED) and found that CD99 and CD276 are highly expressed on DIPG cells (Figures 3A-3B).

[0382] The applicants tested the functionality of single-antigen CD99 CAR-T cells or CD276 CAR-T cells against DIPG007 or DIPG BT245 cells. DIPG007 cells were treated with single-antigen anti-CD99 CAR-T cells or single-antigen anti-CD19 CAR-T cells (mock) in a 1:1 (tumor cells:CAR-T cells) ratio, or single-antigen anti-CD276 CAR-T cells in a 1:2 (tumor cells:CAR-T cells) ratio, and changes in growth were measured using the XCELLigence RTCA system to monitor complete tumor lysis. This analysis is shown in Figure 4A. Compared to DIPG007 cells treated with single-antigen CD19 CAR-T cells, cytokine production increased when DIPG007 cells were treated with single-antigen anti-CD99 CAR-T cells in a 1:1 ratio and single-antigen anti-CD276 CAR-T cells in a 1:2 ratio (Figure 4B). Compared to BT245 cells treated with single-antigen anti-CD19 CAR-T cells, DIPG BT245 tumor cells were also treated with single-antigen anti-CD99 CAR-T cells in a 1:1 ratio and single-antigen anti-CD276 CAR-T cells in a 1:2 ratio, resulting in increased cytokine production (Figure 4C).

[0383] In subsequent experiments, a DIPG xenograft model was created by transplanting BT245 tumor cells into mouse bridges. These mice were then administered a single dose (20 million cells) of the aforementioned single-antigen anti-CD99 CAR-T cells or anti-CD19 CAR-T cells. In vivo bioluminescence imaging (IVIS) of the xenografts revealed that anti-CD99 CAR-T cells were the first to eliminate the DIPG tumor (up to 9 days after treatment), but the tumor eventually recurred (up to 42 days after treatment).

[0384] While we do not wish to dwell on theory, the results presented in this example demonstrate that DIPG tumor cells express both CD99 and CD276 at higher levels than other normal cells, but neither is a tumor-specific antigen individually. Furthermore, targeting CD99 alone using single-antigen CAR-T cell therapy leads to the eventual relapse of DIPG.

[0385] Example 3. Functionality and specificity of CD99 / CD276 logic gate type CAR-T cells against DIPG. The following experimental examples test the functionality and specificity of the anti-CD99 / anti-CD276 CAR logic pair of this disclosure for use in the treatment of DIPG.

[0386] The applicants tested the antitumor efficacy of the AND CAR T-1 and AND CAR T-2 logical pairs described in Example 1. DIPG007 tumor cells were treated with T cells expressing one of the logical pairs in a 1:2 ratio (tumor cells:CAR-T cells). Changes in growth were measured using XCELLigence real-time measurement. As shown in Figure 5A, this analysis revealed that both logical pairs resulted in complete tumor lysis. Furthermore, cytokine production was increased when DIPG007 cells were treated with either of the two different logical pairs in a 1:2 ratio (tumor cells:CAR-T cells) compared to DIPG007 cells treated with single-antigen anti-CD19 CAR-T cells (Figure 5B).

[0387] In subsequent experiments, RAJI cells, a Burkitt lymphoma cell line expressing only CD99 and not CD276, were treated with single-antigen anti-CD19 CAR-T cells, single-antigen anti-CD276 CAR-T cells, CAR-T cells expressing the AND CAR T-2 logical pair, and single-antigen anti-CD99 CAR-T cells. Increased cell death (as measured by the increase in caspase 3 / 7 incucyte reagent) was observed only with single-antigen anti-CD99 CAR-T cells and not with CAR-T cells expressing the logical pair of this disclosure (Figure 5C).

[0388] In another experiment, the applicants tested the antitumor efficacy of CAR-T cells expressing the logical pair of this disclosure against DIPG BT245 and DIPG007 xenograft models. Briefly, DIPG-Luc tumor cells were transplanted into the pons of NSG (immunodeficient) mice. After tumor establishment, a single dose (20 × 10⁶) of either single-antigen anti-CD19 CAR-T cells, single-antigen anti-CD99 CAR-T cells, single-antigen anti-CD276 CAR-T cells, or CAR-T cells expressing the AND CAR T-2 logical pair of Example 1 ("AND CAR T-2 CAR-T cells") was administered. 6(100 CAR-T cells) were delivered systemically. Kaplan-Meier survival analysis of xenografts from BT245 mouse models after each CAR-T treatment showed survival times of approximately 30 days in mice treated with single-antigen anti-CD19 CAR-T cells, approximately 50 days in mice treated with single-antigen anti-CD99 CAR-T cells, approximately 75 days in mice treated with single-antigen anti-CD276 CAR-T cells, and over 200 days in mice treated with AND CAR T-2 CAR-T cells (Figure 6A). Similarly, Kaplan-Meier survival analysis of xenografts from the DPIG007 mouse model after each CAR-T treatment showed survival times of approximately 60 days for mice treated with single-antigen anti-CD19 CAR-T cells, approximately 90 days for mice treated with single-antigen anti-CD99 CAR-T cells, approximately 80 days for mice treated with single-antigen anti-CD276 CAR-T cells, and over 100 days for mice treated with AND CAR T-2 CAR-T cells (Figure 6B).

[0389] While we do not wish to dwell on theory, the results presented in this embodiment demonstrate that the logical pair of CAR polypeptides of this disclosure can be used to specifically target and eliminate DIPG cells both in vitro and in vivo.

[0390] Example 4. Functionality of CD99 / CD276 logic gate type CAR-T cells in PFA ependymoma (PFA EPN). The following experimental examples test the functionality and specificity of CD99 / CD276 logic-gate CAR-T cells against posterior fossa type A (PFA) ependymoma (PFA EPN). The applicants tested the expression of CD99 and CD276 in patient tumors and cultured cell lines of PFA EPN and found that both CD99 and CD276 were highly expressed in patient tumors compared to normal cerebellum (NC), as shown by RNA-seq analysis and flow cytometry in patient tumors and PFA EPN cell lines.

[0391] The applicants investigated the antitumor efficacy of CAR-T cells expressing the AND CAR T-1 logic pair and CAR-T cells expressing the AND CAR T-2 logic pair, as described in Example 1, against PFA EPN MAF928 cells. PFA EPN MAF928 tumor cells were treated with CAR-T cells expressing the logic pair in a 1:1 ratio (tumor cells:CAR-T cells). Changes in growth were measured using XCELLigence real-time measurement, and complete tumor lysis was observed when PFA EPN cells were treated with CAR-T cells expressing the logic pair (Figure 7). Compared to PFA EPN MAF928 cells treated with single-antigen anti-CD19 CAR-T cells (mock), cytokine production also increased when PFA EPN MAF928 cells were treated with logic-gate type CAR-T cells in a 1:1 ratio (Figure 8).

[0392] The applicants also tested the antitumor efficacy of CD99 / CD276 logic-gate CAR-T cells against a PFN EPN MAF928 mouse model. Briefly, PFN EPN MAF928-luciferase tumor cells were transplanted into the fourth ventricle of NSG (immunodeficient) mice. After tumor establishment, a single dose (20 million CAR-T cells) of either single-antigen anti-CD19 CAR-T or CAR-T cells expressing the AND CAR T-2 logic pair as in Example 1 was administered. BLI imaging of the tumors showed complete tumor clearance up to day 7 post-injection in mice treated with AND CAR T-2 CAR-T cells. In another experiment, luciferase-expressing MAF293 PFA EPN tumor cells were transplanted into the fourth ventricle of mice and metastasized. Forty days after injection, the mice were treated with a single systemic dose of single-antigen anti-CD19 CAR-T or AND CAR T-2 CAR-T cells. BLI imaging of tumors demonstrated complete tumor clearance and survival in logic-gated CAR-T treated mice after 140 days.

[0393] While we do not wish to dwell on theory, the results described in this embodiment demonstrate that logical pairs of CAR polypeptides can be used to effectively target and eliminate PFA EPN cells both in viro and in vivo.

[0394] Example 5. Functionality of CD99 / CD276 logic gate type CAR-T cells in atypical teratomatoid rhabdoid tumor (ATRT) The following experimental examples test the functionality and specificity of CD99 / CD276 logic gate type CAR-T cells for ATRT. The applicants tested the expression of CD99 and CD276 in tumors of ATRT patients and found that both CD99 and CD276 were highly expressed in patient tumors compared to normal brain cells, as shown by RNA-seq analysis.

[0395] The applicants tested the antitumor efficacy of CAR-T cells expressing the AND CAR T-2 logic pair described in Example 1 against an ATRT mouse model. Briefly, ATRT-luciferase tumor cells were transplanted into the fourth ventricle of NSG (immunodeficient) mice. After tumor establishment, a single dose (20 million CAR-T cells) of either single-antigen anti-CD19 CAR-T or AND CAR T-2 logic pair-expressing CAR-T cells was delivered systemically. Tumor BLI imaging showed complete tumor clearance in CD99 / CD276 logic-gated CAR-T treated mice up to 7 days post-injection. Kaplan-Meier survival analysis of xenografts from single-antigen CD19 CAR-T treated mice showed a survival period of approximately 40 days compared to over 150 days for mice treated with CD99 / CD276 logic-gated CAR-T cells (Figure 9).

[0396] Example 6. Functionality of CD99 / CD276 logic gate type CAR-T cells in Ewing sarcoma. The following experimental examples test the functionality and specificity of CD99 / CD276 logic gate type CAR-T cells against Ewing sarcoma. The applicants tested the expression of CD99 and CD276 in Ewing sarcoma cell lines and found that both CD99 and CD276 were highly expressed, as shown by flow cytometry analysis.

[0397] The applicants investigated the antitumor efficacy of CD99 / CD276 logic-gate CAR-T cells (T cells expressing the AND CAR T-2 logic pair described in Example 1) against an orthotopic TC71 Ewing sarcoma mouse model. Kaplan-Meier survival analysis of TC71 xenografts treated with single-antigen anti-CD19 CAR-T cells, single-antigen anti-CD99 CAR-T cells, single-antigen anti-CD276 CAR-T cells, and CD99 / CD276 logic-gate CAR-T cells showed increased survival in CD99 / CD276 logic-gate CAR-T cell treated mice. CAR-T cells were administered at a cell dose of 20 million cells. Death in mice treated with CD99 / CD276 logic-gate CAR-T cells was attributed to the development of graft-versus-host disease (GVHD) and not to tumor tissue volume (Figure 10). Both single-antigen anti-CD99 CAR-T cell treatment and single-antigen anti-CD276 CAR-T cell treatment resulted in a transient reduction in tumor tissue volume, but these tumors rapidly recurred. Recurrence ultimately required amputation of the leg. Conversely, CD99 / CD276 logic-gate CAR-T cell treated mice did not experience recurrence and therefore did not require amputation of the leg.

[0398] Mice from each CAR-T cell treatment were also tested for lung metastasis using ddPCR. The presence of lung metastasis was indicated by increased detection of EWS-FLI1 DNA in plasma collected from single-antigen anti-CD99 CAR-T cell treated mice and single-antigen anti-CD276 CAR-T cell treated mice. No evidence of lung metastasis was found in mice treated with CD99 / CD276 logic-gate CAR-T cells.

[0399] While we do not wish to dwell on theory, the results described in this embodiment demonstrate that the CD99 / CD276 logic gated T cells of this disclosure can be used to effectively treat Ewing sarcoma and to effectively prevent metastasis of Ewing sarcoma.

[0400] Example 7. Fractionated irradiation increases the expression of both CD99 and CD276 in DIPG cells and Ewing sarcoma cells. The following experimental examples test the effects of fractionated radiotherapy on CD99 and CD276 expression in DIPG and Ewing sarcoma cells. The applicants subjected DIPG BT245 cells or Ewing sarcoma cells to fractionated low-dose irradiation (4 Gy) over three consecutive days and observed increased CD99 and CD276 expression after 48 hours. While we do not wish to dwell on theory, these results demonstrate that the CD99 / CD276 logic-gated T cells of this disclosure can be used in combination with radiotherapy to effectively treat cancers, including DIPG and Ewing sarcoma.

[0401] Example 8. Heterogeneity of CD276 expression in tumor cells and the functionality of CD99 / CD276 logic gate type CAR-T cells against tumor cells with low or high CD276 levels. The following experimental example tests the impact of heterogeneity in CD276 expression in tumor cells. While CD99 is expressed at high and uniform levels, significant heterogeneity in CD276 expression levels is evident in tumors from DIPG patients.

[0402] The applicants tested CD99 / CD276 logic gate CAR-T cells against CD276-high and CD276-low Ewing sarcoma cells. The applicants found that the logic gate CAR-T cells expressing the AND CAR T-1 logic pair or AND CAR T-2 logic pair of Example 1 were cytotoxic to both CD276-high and CD276-low expression cells in vitro.

[0403] The applicants tested CD99 / CD276 logic-gate CAR-T cells against the low-CD276-expressing human erythromyeloid leukemia cell line, K562. Stable mCherry-expressing K562 cells were co-cultured with CD99 / CD276 logic-gate CAR-T cells and single-antigen CD276 CAR-T cells at a 1:2 tumor:effector (T:E) ratio for 24 hours, and the cytotoxic efficacy of the CAR-T cells was determined by measuring the presence of viable mCherry-positive cells by flow cytometry. A reduction in mCherry-positive / viable cells was demonstrated in both CD99 / CD276 logic-gate CAR-T cell and single-antigen CD276 CAR-T cell treated lines compared to those treated with single-antigen CD19 CAR-T cells (mock).

[0404] The applicants tested CD99 / CD276 logic-gate CAR-T cells for their ability to mitigate the impairment of tumor evasion in low-CD276-expressing cells by conducting in vivo studies using subcutaneously established K562 mouse xenografts. Equal numbers of single-antigen CD19 CAR-T cells, single-antigen CD276 CAR-T cells, or CD99 / CD276 logic-gate CAR-T cells were injected into these tumor-bearing mice, and tumor volume was periodically measured using Caliper. CD99 / CD276 logic-gate CAR-T cells performed better than single-antigen CD276 CAR-T cells in tumor tissue reduction and, primarily, resistance to tumor recurrence (Figure 11).

[0405] While we do not wish to dwell on theory, these results presented in this embodiment demonstrate that the CD99 / CD276 logic gate type CAR-T cells of this disclosure can be used to target and eliminate cancer cells, even when they express low levels of CD276.

Claims

1. A pair of chimeric antigen receptor (CAR) polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, The anti-CD276 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD276 antigen-binding domain containing an anti-CD276 scFv containing the amino acid sequence of Sequence ID No. 12; (iii) A hinge domain containing a CD28 hinge polypeptide containing the amino acid sequence of SEQ ID NO: 40; (iv) A transmembrane domain containing the CD28 transmembrane polypeptide with the amino acid sequence of SEQ ID NO: 44; (v) A costimulatory domain containing the CD28 costimulatory polypeptide with the amino acid sequence of SEQ ID NO: 48 Including; and The anti-CD99 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD99 antigen-binding domain containing an anti-CD99 scFv containing the amino acid sequence of SEQ ID NO: 38; (iii) A hinge domain containing a CD8 hinge polypeptide with the amino acid sequence of SEQ ID NO: 42; (iv) A transmembrane domain containing a CD8 transmembrane polypeptide having the amino acid sequence of SEQ ID NO: 46; and (v) Activation domain containing CD3ζ activating polypeptide with amino acid sequence of SEQ ID NO: 52 A pair of the above CAR polypeptides, including the above.

2. The anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 63, and The anti-CD99 CAR contains the amino acid sequence of SEQ ID NO:

65. A pair of CAR polypeptides according to claim 1.

3. A pair of CAR polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, The anti-CD276 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD276 antigen-binding domain containing an anti-CD276 scFv containing the amino acid sequence of Sequence ID No. 12; (iii) A hinge domain containing a CD8 hinge polypeptide with the amino acid sequence of SEQ ID NO: 42; (iv) A transmembrane domain containing a CD8 transmembrane polypeptide with the amino acid sequence of SEQ ID NO: 46; (v) A costimulatory domain containing a 4-1BB costimulatory polypeptide with the amino acid sequence of SEQ ID NO: 50 Including; and The anti-CD99 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD99 antigen-binding domain containing an anti-CD99 scFv containing the amino acid sequence of SEQ ID NO: 38; (iii) A hinge domain containing a CD8 hinge polypeptide with the amino acid sequence of SEQ ID NO: 42; (iv) A transmembrane domain containing a CD8 transmembrane polypeptide having the amino acid sequence of SEQ ID NO: 46; and (v) Activation domain containing CD3ζ activating polypeptide with amino acid sequence of SEQ ID NO: 52 A pair of the above CAR polypeptides, including the above.

4. The anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 64, and The anti-CD99 CAR contains the amino acid sequence of SEQ ID NO:

65. A pair of CAR polypeptides according to claim 2.

5. A pair of chimeric antigen receptor (CAR) polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, The anti-CD99 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD99 antigen-binding domain containing an anti-CD99 scFv containing the amino acid sequence of SEQ ID NO: 38; (iii) A hinge domain containing a CD28 hinge polypeptide containing the amino acid sequence of SEQ ID NO: 40; (iv) A transmembrane domain containing the CD28 transmembrane polypeptide with the amino acid sequence of SEQ ID NO: 44; (v) A costimulatory domain containing the CD28 costimulatory polypeptide with the amino acid sequence of SEQ ID NO: 48 Including; and The anti-CD276 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD276 antigen-binding domain containing an anti-CD276 scFv containing the amino acid sequence of Sequence ID No. 12; (iii) A hinge domain containing a CD8 hinge polypeptide with the amino acid sequence of SEQ ID NO: 42; (iv) A transmembrane domain containing a CD8 transmembrane polypeptide having the amino acid sequence of SEQ ID NO: 46; and (v) Activation domain containing CD3ζ activating polypeptide with amino acid sequence of SEQ ID NO: 52 A pair of the above CAR polypeptides, including the above.

6. The anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 61, and The anti-CD99 CAR contains the amino acid sequence of SEQ ID NO:

62. A pair of CAR polypeptides according to claim 1.

7. A pair of CAR polypeptides comprising an anti-CD276 CAR polypeptide and an anti-CD99 CAR polypeptide, The anti-CD99 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD99 antigen-binding domain containing an anti-CD99 scFv containing the amino acid sequence of SEQ ID NO: 38; (iii) A hinge domain containing a CD8 hinge polypeptide with the amino acid sequence of SEQ ID NO: 42; (iv) A transmembrane domain containing a CD8 transmembrane polypeptide with the amino acid sequence of SEQ ID NO: 46; (v) A costimulatory domain containing a 4-1BB costimulatory polypeptide with the amino acid sequence of SEQ ID NO: 50 Including; and The anti-CD276 CAR polypeptide is structured from the N-terminus to the C-terminus. (i) signal peptides; (ii) an anti-CD276 antigen-binding domain containing an anti-CD276 scFv containing the amino acid sequence of Sequence ID No. 12; (iii) A hinge domain containing a CD8 hinge polypeptide with the amino acid sequence of SEQ ID NO: 42; (iv) A transmembrane domain containing a CD8 transmembrane polypeptide having the amino acid sequence of SEQ ID NO: 46; and (v) Activation domain containing CD3ζ activating polypeptide with amino acid sequence of SEQ ID NO: 52 A pair of the above CAR polypeptides, including the above.

8. The anti-CD276 CAR contains the amino acid sequence of SEQ ID NO: 60, and The anti-CD99 CAR contains the amino acid sequence of SEQ ID NO:

62. A pair of CAR polypeptides according to claim 2.

9. At least nucleic acid sequences encoding an anti-CD276 CAR polypeptide of a pair of CAR polypeptides according to any one of claims 1 to 8; and At least nucleic acid sequences encoding the anti-CD99 CAR polypeptide of the CAR polypeptide pair according to any one of claims 1 to 8 Nucleic acid molecules containing these molecules.

10. The nucleic acid molecule according to claim 9, wherein at least one nucleic acid sequence encoding an anti-CD276 CAR polypeptide and at least one nucleic acid sequence encoding an anti-CD99 CAR polypeptide are separated by at least one nucleic acid sequence encoding a self-cleaving peptide, preferably the self-cleaving peptide is a P2A self-cleaving peptide.

11. A vector comprising the nucleic acid molecule described in claim 9 or claim 10, preferably a viral vector, and preferably the viral vector is an AAV vector or a lentiviral vector.

12. A cell expressing a pair of CAR polypeptides according to any one of claims 1 to 8.

13. The cell according to claim 12, which is an immune cell.

14. The cell according to claim 13, wherein the immune cell is a T cell, NK cell, NK-like cell, NKT cell, or cytokine-induced killer (CIK) cell, and preferably the immune cell is a T cell.

15. A population of cells according to any one of claims 12 to 14.

16. A method for treating cancer in a subject, comprising the step of administering one or more amounts of the cell population described in claim 15 to the subject.

17. A method for preventing cancer metastasis in a subject, comprising the step of administering one or more amounts of the cell population described in claim 15 to the subject.

18. The method according to claim 16 or claim 17, further comprising the step of administering at least one additional therapy to the subject.

19. The method according to claim 18, wherein at least one additional therapy is at least one of radiotherapy, chemotherapy, and surgery.

20. The method according to any one of claims 16 to 19, wherein the cancer is diffuse pontine glioma (DIPG), acute myeloid leukemia (AML), Ewing's sarcoma, posterior fossa type A (PFA) ependymoma, or atypical teratomatous rhabdoid tumor (ATRT).

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