B7-H3 antigen-binding molecules

Abstract

The present disclosure provides an antigen-binding molecule comprising a single domain antibody sequence that binds to B7-H3. Also disclosed are a chimeric antigen receptor (CAR) construct comprising the antigen-binding molecule, a nucleic acid / expression vector encoding the CAR, and a T cell expressing the CAR, as well as the use of the CAR-expressing T cell in methods for cancer drug treatment, prevention, and treatment.

Description

[Technical Field]

[0001] The present disclosure relates to the field of molecular biology, and more particularly to the field of antigen-binding molecule technology. The present invention also relates to methods of medical therapy and prevention. [Background technology]

[0002] Anti-B7-H3 agents are being developed, including blocking antibodies, antibody-drug conjugates, CD16 affinity-engineered antibodies, and CD3-engaging bispecific antibodies. Some candidates have advanced to clinical trials (1, 4). Early data from these clinical trials suggest a favorable safety profile with limited toxicity, although the extent of therapeutic response remains to be established (1, 4). In parallel, several groups have embarked on the development of B7-H3-targeted chimeric antigen receptor (CAR) T cells. These efforts have demonstrated excellent antitumor activity against multiple solid tumors, leukemias, and lymphomas in preclinical studies (9-15). B7-H3-targeted CAR T cells have been highly active in indications for which current treatment options have proven ineffective (9-11). Summary of the Invention [Problem to be solved by the invention]

[0003] Recently, it has been recognized that scFv aggregation can induce persistent signaling, which in turn reduces the activity and persistence of CAR-T (17-19). Aggregation or misfolding of scFvs may be caused by poor folding stability of the VH or VL domains or exposure of hydrophobic residues at the VH-VL interface after constant domain deletion. Furthermore, the scFv linker may constrain the VH-VL domain interactions in terms of spatial arrangement, leading to oligomerization (20). [Means for solving the problem]

[0004] The present disclosure provides antigen-binding molecules, optionally isolated, that bind to B7 homolog 3 (B7-H3). In some aspects and embodiments, the antigen binding molecule has the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 1; CDR2 having the amino acid sequence of SEQ ID NO: 2; CDR3 having the amino acid sequence of SEQ ID NO: 3 The present invention includes a single domain antibody sequence incorporating:

[0005] In some embodiments, the antigen-binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antigen-binding molecule has the following FR: FR1 having the amino acid sequence of SEQ ID NO: 4, FR2 having the amino acid sequence of SEQ ID NO: 5; FR3 having the amino acid sequence of SEQ ID NO: 6; FR4 having the amino acid sequence of SEQ ID NO:7 The present invention includes a single domain antibody sequence incorporating:

[0006] In some embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule, and the antigen-binding molecule further comprises an antigen-binding domain that binds to an antigen other than B7-H3. The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present disclosure.

[0007] In some embodiments, the CAR comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9. The present disclosure also provides a nucleic acid or nucleic acids, optionally isolated, encoding an antigen-binding molecule or CAR according to the present disclosure.

[0008] The present disclosure also provides an expression vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The present disclosure also provides a cell comprising an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, or an expression vector or expression vectors according to the present disclosure.

[0009] In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the cells are virus-specific T cells. In some embodiments, the cells are Epstein-Barr virus (EBV)-specific T cells.

[0010] The present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for expression of the antigen binding molecule or CAR by the cell. The present disclosure also provides compositions comprising an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or vectors, or a cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0011] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, a cell or a composition according to the present disclosure for use in a method of medical therapy or prophylaxis.

[0012] The present disclosure also provides an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, a cell, or a composition according to the present disclosure for use in treating or preventing cancer. In some embodiments, the use is for treating or preventing chronic infection.

[0013] In some embodiments, the cancer is B7-H3 positive cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin cancer, squamous cell carcinoma of the skin, melanoma, pancreatic cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, colorectal cancer, colorectal cancer, colon cancer, colon carcinoma, kidney cancer, renal clear cell carcinoma, Wilms' tumor, prostate cancer, ovarian cancer, ovarian cancer, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma, breast cancer, triple negative breast cancer, head and neck cancer, head and neck squamous cell carcinoma carcinoma, oral cavity cancer, oral squamous cell carcinoma, laryngeal cancer, oropharyngeal cancer, oropharyngeal cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer, urothelial cancer, brain cancer, medulloblastoma, ependymoma, medulloblastoma, glioma, diffuse intrinsic pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineoblastoma, neuroblastoma, CNS tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brain stem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, peritoneal carcinoma, desmoplastic small cell tumor, and mesothelioma.

[0014] In some embodiments, the antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell, or composition according to the present disclosure is used to target myeloid-derived suppressor cells (MDSCs). MSDCs are a heterogeneous group of immune cells derived from the myeloid lineage (a family of cells originating from bone marrow stem cells) and expand under pathological conditions such as chronic infection or cancer. Tumors infiltrated with high levels of MDSCs have been shown to be associated with poor patient outcomes and resistance to therapy. The MDSCs may be B7-H3-positive MDSCs. Treatment can prevent the immunosuppressive effects of MDSCs.

[0015] In some embodiments, treatment is not associated with cytokine release syndrome or only minimally associated with cytokine release syndrome.In other words, compared with other CAR treatments for cancer, treatment with the B7-H3 CAR disclosed herein is associated with a lower risk of CRS.For example, treatment is associated with no change in the levels of IL-6, IL-8, IL10, IFN-γ, TNFα and / or IL-2, neutrophils, monocytes and / or dendritic cells, or the levels of these cytokines in cells are lower after treatment with the B7-H3 CAR described herein than after treatment with other CARs, such as CD19-CAR.

[0016] The present disclosure also provides the use of an antigen-binding molecule, a CAR, a nucleic acid or nucleic acids, an expression vector or expression vectors, a cell or a composition according to the present disclosure to deplete or increase killing of cells that express B7-H3.

[0017] The present disclosure also provides an in vitro complex, optionally isolated, comprising an antigen-binding molecule or CAR according to the present disclosure bound to B7-H3. The present disclosure also provides a method for detecting B7-H3 in a sample, the method comprising contacting a sample containing or suspected of containing B7-H3 with an antigen-binding molecule according to the present disclosure, and detecting the formation of a complex between the antigen-binding molecule and B7-H3.

[0018] The present disclosure also provides a method of selecting or stratifying a subject for treatment with a B7-H3 targeting agent, the method comprising contacting a sample from the subject in vitro with an antigen-binding molecule according to the present disclosure, and detecting formation of a complex between the antigen-binding molecule and B7-H3.

[0019] The present disclosure also provides the use of an antigen-binding molecule according to the present disclosure as an in vitro or in vivo diagnostic or prognostic agent. DETAILED DESCRIPTION OF THE INVENTION

[0020] explanation The present disclosure provides antigen-binding molecules that bind to B7-H3 and have novel biophysical and / or functional properties compared to antigen-binding molecules disclosed in the prior art.

[0021] The present disclosure also provides novel chimeric antigen receptor (CAR) constructs having a B7-H3-binding domain that comprise the novel B7-H3-specific antigen binding molecules of the present disclosure. In a preferred embodiment, the antigen-binding molecule is a single-domain antibody (i.e., VHH). CARs containing VHH antigen-binding domains offer technical advantages over CARs containing scFv antigen-binding domains. VHHs are significantly smaller in size than scFvs (30 kDa versus 12-15 kDa) and possess more favorable in vivo immunogenicity, solubility, and stability qualities, while retaining the ability to bind their target antigens with high affinity (21). VHHs also avoid potential disruption of interactions between variable and constant domains and exposure of hydrophobic patches, both of which can significantly affect solubility and stability (22). B7-H3 B7 homolog 3 (B7-H3, CD276) is a protein identified by UniProt Q5ZPR3. B7-H3 is a member of the immune checkpoint family of B7 and CD28 proteins that has a predominantly T cell inhibitory role by suppressing activation and proliferation (1).

[0022] B7-H3 isoform 1 (UniProt: Q5ZPR3-1; SEQ ID NO: 14) contains an N-terminal signal peptide (SEQ ID NO: 15), a 438-amino acid extracellular domain (SEQ ID NO: 16), a transmembrane domain (SEQ ID NO: 17), and a short intracellular domain (SEQ ID NO: 18). B7-H3 isoform 2 (UniProt: Q5ZPR3-2; SEQ ID NO: 19) differs from B7-H3 isoform 1 in that it lacks positions 159-376 of SEQ ID NO: 14. B7-H3 isoform 3 (UniProt: Q5ZPR3-3; SEQ ID NO: 21) differs from B7-H3 isoform 1 in that it lacks positions 494-534 of SEQ ID NO: 14 and that positions 465-493 are replaced with "GPASSAVPLSPAHPPHGSMCWSHWFSRGL." B7-H3 isoform 4 (UniProt: Q5ZPR3-4; SEQ ID NO: 22) differs from B7-H3 isoform 1 by the substitution of "GKDTWA" at positions 528-534. SEQ ID NOs: 28-31 show the mature sequences of human B7-H3 isoforms 1-4 after processing to remove the N-terminal signal peptide.

[0023] B7-H3 isoform 1 is also known as 4Ig-B7-H3, referring to the presence of four immunoglobulin (Ig)-like domains within its extracellular domain. SEQ ID NOS: 24-27 show the V- and C2-type Ig-like domains of B7-H3 isoform 1, which are also contained in isoforms 3 and 4. B7-H3 isoform 2 is also known as 2Ig-B7-H3, and contains a single set of V- and C2-type Ig-like domains as shown in SEQ ID NOS: 24 and 27.

[0024] The detection of high levels of B7-H3 mRNA in various normal tissues contrasts with the limited expression of B7-H3 protein in these tissues, suggesting tight transcriptional regulation of B7-H3 in healthy tissues (2, 3). In contrast, B7-H3 is overexpressed at both the mRNA and protein levels in multiple types of human tumors (4), and B7-H3 protein is detected in cancer cells, tumor-infiltrating vessels, and tumor stroma (5). Association studies have revealed that high B7-H3 protein expression correlates with poor prognosis and clinical outcome. These clinical observations are supported by studies demonstrating a pro-tumorigenic role for B7-H3 in various types of cancer, independent of its immune function. B7-H3 acts upstream of multiple signaling pathways, including the JAK / STAT, Ras / Raf / MEK / MAPK, and PI3K / Akt / mTOR pathways. Interfering with B7-H3 expression reduces the adhesion, migration, invasion, and metastasis of many types of cancer cells in vitro and in vivo. (6-8) Taken together, these studies point to the differential expression of B7-H3 in tumors versus healthy tissues, in addition to its pleiotropic pro-tumorigenic role, making it an attractive target for cancer therapy.

[0025] As used herein, "B7-H3" refers to B7-H3 from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, the B7-H3 is B7-H3 from a mammal (e.g., a Therian, a placental mammal, a Supertherian, a preptotheria, an archontan, a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human)). In some embodiments, the B7-H3 is human B7-H3 or mouse B7-H3.

[0026] As used herein, an isoform, fragment, variant, or homologue of a given reference protein may be characterized as having at least 70% sequence identity, and preferably one having one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of the reference protein.

[0027] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but that retains a substantial degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. A fragment of B7-H3 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 530 amino acids, and a maximum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 530 amino acids.

[0028] "Isoform" generally refers to the variant of a reference protein that is expressed by the same species as the reference protein. Of course, the isoforms of B7-H3 include isoform 1 (UniProt: Q5ZPR3-1), isoform 2 (Q5ZPR3-2), isoform 3 (Q5ZPR3-3) and isoform 4 (Q5ZPR3-3).

[0029] A "homologue" generally refers to a variant of a reference protein produced by a species different from that of the reference protein. Homologues include orthologs. An isoform, fragment, variant, or homolog of B7-H3 may optionally be characterized as having at least 70% amino acid sequence identity, and preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, with the amino acid sequence of an immature or mature B7-H3 isoform from a given species, e.g., human.

[0030] In some embodiments, the B7-H3 is human B7-H3. In some embodiments, the B7-H3 is mouse B7-H3 (Q8VE98-1, SEQ ID NO: 32). An isoform, fragment, variant, or homolog may optionally be a functional isoform, fragment, variant, or homolog, e.g., have a functional property / activity of a reference B7-H3, as determined by analysis with a suitable assay for that functional property / activity. For example, an isoform, fragment, variant, or homolog of B7-H3 can induce signaling mediated by B7-H3.

[0031] In some embodiments, B7-H3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 14, 19, 20, 21, 22, 28, 29, 30, 31, 32 or 39.

[0032] In some embodiments, B7-H3 comprises an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 22, 16, 31 or 28. In some embodiments, B7-H3 comprises an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 16 or 20.

[0033] In some embodiments, a fragment of B7-H3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 22, 16, 31, or 28. In some embodiments, a fragment of B7-H3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 16 or 20. antigen binding molecule The present disclosure provides antigen-binding molecules capable of binding to (i.e., bind to) B7-H3. The present disclosure provides antigen-binding molecules that specifically bind to B7-H3. The antigen-binding molecules of the present disclosure may be provided in purified or isolated form, i.e., purified or isolated from other naturally occurring biological materials.

[0034] As used herein, "antigen-binding molecule" refers to a molecule capable of binding to a target antigen. The term "antigen-binding molecule" encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (VHH), etc.), and aptamers.

[0035] More specifically, antigen-binding molecules according to the present disclosure comprise an antigen-binding polypeptide portion, which may also be referred to as an "antigen-binding domain." In a preferred embodiment, an antigen-binding molecule according to the present disclosure comprises or consists of a single-domain antibody that specifically binds to B7-H3.

[0036] Single domain antibodies (sdAbs), also referred to variously in the art as "single variable domains on heavy chain antibodies," "VHHs," "nanobodies," and "heavy chain-only antibodies (HcAbs)," are described, for example, in Henry and MacKenzie, Front Immunol. (2018) 9:41 and Bever et al., Anal Bioanal Chem. (2016) 408(22):5985-6002, both of which are incorporated by reference in their entireties.

[0037] Single domain antibodies are formed from a single monomeric antibody variable domain. The first single domain antibodies were engineered from heavy chain antibodies found in camelids, and cartilaginous fish also have heavy chain antibodies.

[0038] A single domain antibody according to the present disclosure generally comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The three CDRs together define the paratope of the molecule, which is the portion through which it binds to its target antigen.

[0039] Single domain antibodies further comprise framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From N- to C-terminus, single domain antibodies comprise the following structure: N-terminus-[FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4]-C-terminus.

[0040] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), the Clothia system (Chothia et al., J. Mol. Biol. 196:901-917 (1987)), the IMGT information system (International IMGT (Immunogenetics) Information System (described in LeFranc et al., Nucleic Acids Res. (2015) 43 (Database Edition):D413-22), which uses the IMGT V-domain numbering rules described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77), and Retter et al., Nucleic Acids Res. (2003) 27:55-77. Res. (2005) 33 (Supplement 1): D671 to D674. The CDRs and FRs of the antigen-binding molecules / single domain antibodies described herein are defined according to VBASE2.

[0041] In some embodiments, the antigen-binding molecule comprises the CDRs of the B7-H3-binding single-domain antibodies described herein or comprises CDRs derived from the B7-H3-binding single-domain antibodies described herein. In some embodiments, the antigen-binding molecule comprises the FRs of the B7-H3-binding single-domain antibodies described herein or comprises FRs derived from the B7-H3-binding single-domain antibodies described herein. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of the B7-H3-binding single-domain antibodies described herein or comprises CDRs and FRs derived from the B7-H3-binding single-domain antibodies described herein. That is, in some embodiments, the antigen-binding molecule comprises the amino acid sequence of the B7-H3-binding single-domain antibodies described herein or comprises an amino acid sequence derived from the B7-H3-binding single-domain antibodies described herein.

[0042] In some embodiments, in the amino acid sequence of SEQ ID NO: 8, FR1 is formed by the amino acid sequence at positions 1 to 25; CDR1 is formed by the amino acid sequence at positions 26 to 33; FR2 is formed by the amino acid sequence at positions 34 to 50; CDR2 is formed by the amino acid sequence at positions 51 to 57; FR3 is formed by the amino acid sequence at positions 58 to 96; CDR3 is formed by the amino acid sequence at positions 97 to 111; and FR4 is formed by the amino acid sequence at positions 112 to 122.

[0043] As used herein, an amino acid sequence / domain "derived from" a reference amino acid sequence / domain includes an amino acid sequence that has at least 60% sequence identity, such as at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the reference sequence.

[0044] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and / or the complete amino acid sequence of the B7-H3-binding single domain antibody P2A5. In some embodiments, the antigen binding molecule comprises the CDRs, FRs and / or the complete amino acid sequence of a B7-H3 binding single domain antibody having the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the antigen binding molecule comprises the CDRs (i.e., CDR1, 2 and 3) of a B7-H3 binding single domain antibody having the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the antigen binding molecule comprises the FRs (i.e., FR1, 2, 3 and 4) of a B7-H3 binding single domain antibody having the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the antigen binding molecule comprises the CDRs (i.e., CDR1, 2 and 3) and FRs (i.e., FR1, 2, 3 and 4) of a B7-H3 binding single domain antibody having the amino acid sequence according to SEQ ID NO: 8.

[0045] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to (1): (1)(P2A5) has the following CDR: CDR1 having the amino acid sequence of SEQ ID NO: 1; CDR2 having the amino acid sequence of SEQ ID NO: 2; CDR3 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein one, two or three amino acids in CDR1, and / or one, two or three amino acids in CDR2, and / or one, two or three amino acids in CDR3 are substituted with another amino acid.

[0046] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to (2): (2) (P2A5) is a single domain antibody sequence containing the following FR: FR1 having the amino acid sequence of SEQ ID NO: 4, FR2 having the amino acid sequence of SEQ ID NO: 5; FR3 having the amino acid sequence of SEQ ID NO: 6; FR4 having the amino acid sequence of SEQ ID NO: 7; or a variant thereof, in which one, two or three amino acids in FR1, and / or one, two or three amino acids in FR2, and / or one, two or three amino acids in FR3, and / or one, two or three amino acids in FR4 are substituted with another amino acid.

[0047] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence comprising a CDR according to (1) above and a FR according to (2). In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to (3): (3) (P2A5) is a single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO:8.

[0048] In embodiments according to the present disclosure, one or more amino acids are substituted with another amino acid. Substitutions include replacing an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue of a substitution according to the present disclosure may be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is not identical to the amino acid residue at the relevant position of the equivalent unsubstituted amino acid sequence, and is selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the replacement amino acid may be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than those listed in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. 202 (1991) 301-336.

[0049] In some embodiments, substitutions may be biochemically conservative. In some embodiments, when a substituted amino acid is provided in one of rows 1-5 of the table below, the replacement amino acid for the substitution is another non-identical amino acid provided in the same row.

[0050] [Table 1]

[0051] By way of example, in some embodiments where the substitution is for a Met residue, the replacement amino acid may be selected from Ala, Val, Leu, lie, Trp, Tyr, Phe, and norleucine.

[0052] In some embodiments, the replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces, or the same side chain charge (at pH 7.4) as the amino acid residue it replaces.

[0053] [Table 2]

[0054] That is, in some embodiments, a non-polar amino acid is substituted with another non-identical non-polar amino acid. In some embodiments, a polar amino acid is substituted with another non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another non-identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another non-identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another non-identical negative amino acid.

[0055] In some embodiments, substitutions may be functionally conservative, i.e., the substitution may not affect (or not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule comprising the substitution compared to an equivalent unsubstituted molecule.

[0056] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more regions (e.g., CH1, CH2, and / or CH3) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0057] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 40, 45 or 48.

[0058] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 49, 50, or 51.

[0059] In some embodiments, the antigen-binding molecule comprises a CH1 region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 41 or 46. In some embodiments, the antigen-binding molecule comprises a hinge region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antigen-binding molecule comprises a CH2 region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antigen-binding molecule comprises a CH3 region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 44 or 47.

[0060] It will be appreciated that the CH2 and / or CH3 regions may be provided with additional substitutions in accordance with the modifications to the Fc region of the antigen binding molecules described herein. In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin kappa constant (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0061] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 52, 53, 54, 55, 56, or 57.

[0062] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region. An Fc region is composed of the CH2 and CH3 regions from one polypeptide and the CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.

[0063] Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), membrane attack complex (MAC) formation, cellular degranulation, cytokine and / or chemokine production, and antigen processing and presentation. Modifications to antibody Fc regions that affect Fc-mediated functions are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated herein by reference in its entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region containing modifications to increase or decrease an Fc-mediated function compared to an antigen-binding molecule containing a corresponding unmodified Fc region.

[0064] When an Fc region / CH2 / CH3 is described as containing an alteration "corresponding to" a referenced substitution, the equivalent substitution in the homologous Fc / CH2 / CH3 is assumed. By way of illustration, the L234A / L235A substitution in human IgG1 (numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991) corresponds to the L to A substitution at positions 117 and 118 of the mouse Ig gamma-2 A chain C region (UniProtKB:P01863-1, v1).

[0065] When an Fc region is described as comprising a modification, the modification may be present in one or both of the polypeptide chains that together form the Fc region. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that comprises a modification. In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region that comprises a modification in one or more of the CH2 and / or CH3 regions.

[0066] In some embodiments, the Fc region comprises a modification to increase an Fc-mediated function. In some embodiments, the Fc region comprises a modification to increase ADCC. In some embodiments, the Fc region comprises a modification to increase ADCP. In some embodiments, the Fc region comprises a modification to increase CDC. Antigen-binding molecules comprising an Fc region comprising a modification to increase an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce increased levels of the relevant effector function compared to a corresponding antigen-binding molecule comprising an unmodified Fc region.

[0067] In some embodiments, the Fc region comprises a modification to increase binding to an Fc receptor. In some embodiments, the Fc region comprises a modification to increase binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification to increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification to increase binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification to increase binding to FcγRIIa. In some embodiments, the Fc region comprises a modification to increase binding to FcγRIIb. In some embodiments, the Fc region comprises a modification to increase binding to FcRn. In some embodiments, the Fc region comprises a modification to increase binding to complement proteins. In some embodiments, the Fc region comprises a modification to increase binding to C1q. In some embodiments, the Fc region comprises a modification to promote hexamerization of the antigen-binding molecule. In some embodiments, the Fc region comprises a modification to increase the half-life of the antigen-binding molecule, hi some embodiments, the Fc region comprises a modification to increase co-engagement.

[0068] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions F243L / R292P / Y300L / V305I / P396L described in Stavenhagen et al., Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S239D / I332E or S239D / I332E / A330L described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005-4010. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S298A / E333A / K334A described in Shields et al., J Biol Chem. (2001) 276:6591-6604. In some embodiments, the Fc region comprises modifications to one of the heavy chain polypeptides corresponding to the combination of substitutions L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and modifications to the other heavy chain polypeptide corresponding to the combination of substitutions D270E / K326D / A330M / K334E, as described in Mimoto et al., MAbs. (2013):5:229-236. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions G236A / S239D / I332E, as described in Richards et al., Mol Cancer Ther. (2008)7:2517-2527.

[0069] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions K326W / E333S described in Idusogie et al., J Immunol. (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S267E / H268F / S324T described in Moore et al., MAbs. (2010) 2(2):181-9. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions E345R / E430G / S440Y described in Diebolder et al., Science (2014) 343(6176):1260-3.

[0070] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions M252Y / S254T / T256E described in Dall'Acqua et al., J Immunol. (2002) 169:5171-5180. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions M428L / N434S described in Zalevsky et al., Nat Biotechnol. (2010) 28:157-159.

[0071] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S267E / L328F described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions N325S / L328F described in Shang et al., Biol Chem. (2014) 289:15309-15318.

[0072] In some embodiments, the Fc region comprises a modification to reduce / prevent an Fc-mediated function. In some embodiments, the Fc region comprises a modification to reduce / prevent ADCC. In some embodiments, the Fc region comprises a modification to reduce / prevent ADCP. In some embodiments, the Fc region comprises a modification to reduce / prevent CDC. Antigen-binding molecules comprising an Fc region comprising a modification to reduce / prevent an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce a reduced level of the relevant effector function compared to an antigen-binding molecule comprising a corresponding unmodified Fc region.

[0073] In some embodiments, the Fc region comprises a modification to reduce / prevent binding to an Fc receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcγRIIa. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcγRIIb. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to complement proteins. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to C1q. In some embodiments, the Fc region comprises a modification to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0074] In some embodiments, the Fc region is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to trigger relevant Fc-mediated functions). Thus, antigen-binding molecules comprising such Fc regions also lack the ability to induce relevant functions. Such antigen-binding molecules may also be described as lacking the relevant functions.

[0075] In some embodiments, the Fc region is unable to induce ADCC. In some embodiments, the Fc region is unable to induce ADCP. In some embodiments, the Fc region is unable to induce CDC. In some embodiments, the Fc region is unable to induce ADCC and / or unable to induce ADCP and / or unable to induce CDC.

[0076] In some embodiments, the Fc region is unable to bind to an Fc receptor. In some embodiments, the Fc region is unable to bind to an Fcγ receptor. In some embodiments, the Fc region is unable to bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region is unable to bind to FcγRIIIa. In some embodiments, the Fc region is unable to bind to FcγRIIa. In some embodiments, the Fc region is unable to bind to FcγRIIb. In some embodiments, the Fc region is unable to bind to FcRn. In some embodiments, the Fc region is unable to bind to complement proteins. In some embodiments, the Fc region is unable to bind to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0077] In some embodiments, the Fc region comprises a modification corresponding to N297A or N297Q or N297G as described in Leabman et al., MAbs. (2013) 5:896-903. In some embodiments, the Fc region comprises a modification corresponding to L235E as described in Alegre et al., J Immunol. (1992) 148:3461-3468. In some embodiments, the Fc region comprises a modification corresponding to the combination of substitutions L234A / L235A or F234A / L235A as described in Xu et al., Cell Immunol. (2000) 200:16-26. In some embodiments, the Fc region comprises a modification corresponding to P329A or P329G as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235A / P329G described in Lo et al., J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions described in Rother et al., Nat Biotechnol. (2007) 25:1256-1264. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions S228P / L235E described in Newman et al., Clin. Immunol. (2001) 98:164-174. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions H268Q / V309L / A330S / P331S described in An et al., MAbs. (2009) 1:572-579. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions V234A / G237A / P238S / H268A / V309L / A330S / P331S described in Vafa et al., Methods. (2014) 65:114-126. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235E / G237A / A330S / P331S described in US2015 / 0044231A1.

[0078] The substitution combination "L234A / L235A" and corresponding substitutions (such as F234A / L235A in human IgG4) are known to disrupt Fc binding to Fcγ receptors, inhibit ADCC, ADCP, and also reduce C1q binding and therefore CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, incorporated herein by reference in its entirety). The substitutions "P329G" and "P329A" reduce C1q binding (thereby reducing CDC). Substitution of "N297" with "A," "G," or "Q" is known to eliminate glycosylation, thereby reducing Fc binding to C1q and Fcγ receptors, and therefore reducing CDC and ADCC. Lo et al., J. Biol. Chem (2017) 292(9):3900-3908 (incorporated herein by reference in its entirety) report that the substitution combination L234A / L235A / P329G eliminated complement binding and fixation, as well as Fcγ receptor-dependent, antibody-dependent, cell-mediated cytotoxicity in both mouse IgG2a and human IgG1.

[0079] The combination of substitutions L234A / L235E / G237A / A330S / P331S in IgG1 Fc to abolish the induction of phagocytosis, ADCC and CDC is disclosed in US2015 / 0044231A1.

[0080] In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The substitution S228P in an IgG4 Fc reduces Fab-arm exchange, which may be undesirable.

[0081] In some embodiments, the Fc region comprises a modification corresponding to the combination of substitutions L234A / L235A. In some embodiments, the Fc region comprises a modification corresponding to substitution P329G. In some embodiments, the Fc region comprises a modification corresponding to substitution N297Q.

[0082] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235A / P329G. In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235A / P329G / N297Q.

[0083] In some embodiments, the Fc region comprises modifications corresponding to the combination of substitutions L234A / L235E / G237A / A330S / P331S. In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P, for example, in IgG4.

[0084] In some embodiments, particularly those in which the antigen-binding molecule is a multispecific (e.g., bispecific) antigen-binding molecule, the antigen-binding molecule comprises an Fc region containing modifications in one or more of the CH2 and CH3 regions that promote assembly of the Fc regions. Recombinant coexpression and subsequent assembly of the constituent polypeptides of an antigen-binding molecule results in numerous possible combinations. To improve the yield of the desired combination of polypeptides in an antigen-binding molecule during recombinant production, it is advantageous to introduce modifications into the Fc region that promote assembly of the desired combination of heavy chain polypeptides. The modifications can promote, for example, hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described, for example, in Ha et al., Front. Immunol (2016) 7:394, the entire contents of which are incorporated herein by reference.

[0085] In some embodiments, the antigen-binding molecules of the present disclosure are in the following formats as shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT s-s , SEED, or A107.

[0086] Multispecific antigen-binding molecules are also envisioned. "Multispecific" means that an antigen-binding molecule exhibits specific binding to more than one target. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains.

[0087] In some embodiments, the antigen-binding molecule binds to B7-H3 and another target (e.g., an antigen other than B7-H3), and is thus at least bispecific. The term "bispecific" means that the antigen-binding molecule can specifically bind to at least two distinct antigenic determinants.

[0088] It will be understood that antigen-binding molecules (e.g., multispecific antigen-binding molecules) according to the present disclosure may comprise antigen-binding molecules capable of binding to a target for which the antigen-binding molecule is specific. For example, an antigen-binding molecule that binds to B7-H3 and an antigen other than B7-H3 may comprise (i) an antigen-binding molecule that binds to B7-H3 and (ii) an antigen-binding molecule that binds to an antigen other than B7-H3. In some embodiments, an antigen-binding molecule that is a component of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to, for example, as the "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0089] It will also be understood that an antigen-binding molecule (e.g., a multispecific antigen-binding molecule) according to the present disclosure may comprise an antigen-binding polypeptide or antigen-binding polypeptide complex capable of binding to a target for which the antigen-binding molecule is specific.

[0090] In some embodiments, the antigen other than B7-H3 in the multispecific antigen-binding molecule is an immune cell surface molecule. In some embodiments, the antigen is a cancer cell antigen. In some embodiments, the antigen is a receptor molecule, for example, a cell surface receptor. In some embodiments, the antigen is a cell signaling molecule, for example, a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen is a growth factor or hormone.

[0091] A cancer cell antigen is an antigen expressed or overexpressed by a cancer cell. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen may be aberrantly expressed by a cancer cell (e.g., the cancer cell antigen may be expressed with abnormal localization) or may be expressed in an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of a cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen that binds to the antigen-binding molecule described herein is displayed on the external surface of a cancer cell (i.e., is extracellular). A cancer cell antigen may be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of a cancer symptom. Cancer-associated antigens may be associated with the cause or pathology of cancer, or may be aberrantly expressed as a result of cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, for example, compared to the level of expression by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer-associated antigens may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer-associated antigens may be the product of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens may be the product of overexpressed cellular proteins, cancer antigens produced by tumor viruses, oncofetal antigens, or cell surface glycolipids or glycoproteins.

[0092] Cancer-associated antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al., Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors, Holland-Frei Cancer Medicine. 6th ed. Hamilton (ON): BC Decker; 2003. Cancer-associated antigens include oncofetal antigens: CEA, immature laminin receptor, Tag-72; tumor virus antigens, e.g., HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; and lineage-restricted antigens. Other cancer-associated antigens include MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, and prostate-specific antigen; mutated antigens include β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, and TGF-βRII; posttranslationally modified antigens include MUC1; idiotypic antigens include Ig and TCR. Other cancer-associated antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placenta-like 2 (ALPPL-2), Siglec-5, stress-inducible phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments, the cancer-associated antigen is a cancer-associated antigen described in Zhao and Cao, Front Immunol. 2019;10:2250, which is incorporated by reference in its entirety.In some embodiments, the cancer-associated antigen is selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA. In some embodiments, the cancer-associated antigen is an antigen expressed by cells of hematological malignancies. In some embodiments, the cancer-associated antigen is selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, and BCMA. In some embodiments, the cancer-associated antigen is an antigen expressed by cells of solid tumors. In some embodiments, the cancer-associated antigen is selected from mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0093] The immune cell surface molecule may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on or at the cell surface of an immune cell. In some embodiments, the portion of the immune cell surface molecule that binds to the antigen-binding molecule of the present disclosure is on the external surface of the immune cell (i.e., extracellular). The immune cell surface molecule may be expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, for example, a T cell, a B cell, a natural killer (NK) cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor thereof (e.g., a thymocyte or a pre-B cell). In some embodiments, the antigen is a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ).

[0094] In some embodiments, the multispecific antigen-binding molecules described herein exhibit at least monovalent binding to B7-H3 and also exhibit at least monovalent binding to antigens other than B7-H3. Binding valency refers to the number of binding sites in an antigen-binding molecule for a given antigenic determinant.

[0095] In some embodiments, the antigen-binding molecule comprises a single domain antibody (e.g., as described herein) capable of binding to B7-H3, and an antigen-binding region (e.g., a polypeptide (e.g., a single domain antibody), Fv, Fab, or antibody) capable of binding to an antigen other than B7-H3.

[0096] In some embodiments, the antigen-binding molecule comprises an immune cell-engaging moiety. In some embodiments, the antigen-binding molecule is an immune cell engager. Immune cell engagers are reviewed, for example, in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418-434 and Ellerman, Methods (2019) 154:102-117, both of which are incorporated herein by reference in their entireties.

[0097] Immune cell engager molecules contain an antigen-binding region for a target antigen of interest and an antigen-binding region for recruiting / engaging immune cells of interest. Immune cell engagers recruit / engage immune cells via the antigen-binding region specific for an immune cell surface molecule.

[0098] In some embodiments, the antigen-binding molecule comprises a portion that binds to a CD3 polypeptide (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide). The most well-studied immune cell engager is the bispecific T cell engager (BiTE), which comprises a target antigen-binding domain and a CD3 polypeptide (typically CD3ε)-binding domain, through which the BiTE recruits T cells. Binding of the BiTE to its target antigen and further to the CD3 polypeptide expressed by the T cell results in T cell activation, ultimately directing T cell effector activity against cells expressing the target antigen. Other types of immune cell engagers are known in the art, including natural killer cell engagers, such as bispecific killer engagers (BiKEs), which recruit and activate NK cells.

[0099] In some embodiments, the immune cells engaged by the immune cell engager are T cells or NK cells. In some embodiments, the immune cell engager is a T cell engager. Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable manner, for example, as described in Brinkmann and Kontermann, MAbs (2017) 9(2):182-212, which is incorporated herein by reference in its entirety. Functional properties of antigen-binding molecules The antigen-binding molecules described herein can be characterized by certain functional properties. In some embodiments, the antigen-binding molecules described herein may have one or more of the following properties: binding to B7-H3 (e.g., human B7-H3 (e.g., human B7-H3 isoform 1 and / or human B7-H3 isoform 2) and / or mouse B7-H3); binding to cells expressing B7-H3; not binding to cells that do not express B7-H3; increasing the killing of cells that express B7-H3; not increasing the killing of cells that do not express B7-H3; increasing ADCC of cells that express B7-H3; not increasing ADCC of cells that do not express B7-H3; inhibiting tumor growth, for example, tumor growth of cancers that express B7-H3; and / or increasing the survival of subjects with cancer, for example, cancers that express B7-H3.

[0100] It will be understood that a given antigen-binding molecule may exhibit more than one of the properties listed in the previous paragraph. A given antigen-binding molecule can be evaluated for the properties listed in the previous paragraph using a suitable assay. The assay may be, for example, an in vitro assay, and such an assay may be a cell-free or cell-based assay. Alternatively, the assay may be, for example, an in vivo assay, i.e., performed in a non-human animal. The assay may employ species labeled with a detectable entity to facilitate their detection.

[0101] If the assay is a cell-based assay, the assay may involve treating cells with a given antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the listed properties. The assay may employ a species labeled with a detectable entity to facilitate its detection. The assay may involve treating cells separately with various amounts / concentrations of the given antigen-binding molecule (e.g., a dilution series) and then evaluating the listed properties. It will be understood that preferably, the cells express the target antigen (i.e., B7-H3) of the antigen-binding molecule.

[0102] Analysis of the results of such assays may include determining the concentration at which 50% of the maximal level of the relevant activity is achieved. The concentration of the antigen-binding molecule at which 50% of the maximal level of the relevant activity is achieved may also be referred to as the "half-maximal effective concentration" of the antigen-binding molecule for the relevant activity, which is also referred to as the "EC 50 By way of example, the EC of a given antigen-binding molecule for binding to B7-H3 may be 50 may be the concentration at which 50% of the maximal level of binding is achieved.

[0103] Depending on the characteristics, EC 50 is also known as the "half maximal inhibitory concentration" or "IC 50 ", which is the concentration of an antigen-binding molecule at which 50% of the maximal level of inhibition of a given property is observed.

[0104] The antigen-binding molecules and antigen-binding domains described herein preferably exhibit specific binding to B7-H3. As used herein, "specific binding" refers to binding that is selective for the antigen and can be distinguished from non-specific binding to non-target antigens. An antigen-binding molecule / domain that specifically binds to a target molecule preferably binds to the target with greater affinity and / or for a longer duration than it binds to other non-target molecules.

[0105] The ability of a given polypeptide to specifically bind to a given molecule may be determined by assays according to methods known in the art, such as ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or by radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. Through such assays, binding to a given molecule can be measured or quantified. In some embodiments, binding can be a response detected in a given assay.

[0106] In some embodiments, the extent of binding of the antigen-binding molecule to a non-target molecule is less than about 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, binding specificity is measured in terms of binding affinity, where the antigen-binding molecule exhibits at least 0.1 orders of magnitude (i.e., 0.1 x 10 n , where n is an integer representing the number of digits), D ) and the K D If it is greater, it may be reflected, which may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0107] Binding to B7-H3 can be determined by surface plasmon resonance, for example, by surface plasmon resonance as described in Example 1.5 of the present disclosure. In some embodiments, the antigen-binding molecules described herein bind to antigens with an affinity of less than 1 micromolar, i.e., 1×10 -6 K less than M D In some embodiments, the antigen-binding molecules described herein bind to B7-H3 with affinities in the nanomolar range, i.e., 9.9 x 10 -7 ~1×10 -9 K of M D In some embodiments, the antigen-binding molecules described herein bind to B7-H3 with an affinity of less than 1 nanomolar, i.e., 1 x 10 -9 K less than M D In some embodiments, the antigen-binding molecules described herein bind to B7-H3 with affinities in the picomolar range, i.e., 9.9 x 10 -10 ~1×10 -12 K of M D In some embodiments, the antigen-binding molecules described herein bind to B7-H3 with sub-picomolar affinity, i.e., 1 x 10 -12 K less than M D binds to B7-H3.

[0108] In some embodiments, the antigen-binding molecules described herein have a K D and preferably one of the following K values: ≦5 μM, ≦2 μM, ≦1 μM, ≦500 nM, ≦450 nM, ≦400 nM, ≦350 nM, ≦300 nM, ≦250 nM, ≦200 nM, ≦150 nM, ≦100 nM, ≦75 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, or ≦10 nM. D and binds to B7-H3 (e.g., human B7-H3).

[0109] In some embodiments, the antigen-binding molecules described herein have a K Dand preferably one of the following K values: ≦5 μM, ≦2 μM, ≦1 μM, ≦500 nM, ≦450 nM, ≦400 nM, ≦350 nM, ≦300 nM, ≦250 nM, ≦200 nM, ≦150 nM, ≦100 nM, ≦75 nM, ≦50 nM, ≦40 nM, or ≦30 nM. D In some embodiments, the antigen-binding molecule binds to human B7-H3 isoform 1 with a K of ≦1 μM and ≧1 nM. D and, for example, one of ≦500 nM and ≧5 nM, ≦200 nM and ≧10 nM, ≦150 nM and ≧15 nM, ≦100 nM and ≧20 nM, or ≦50 nM and ≧25 nM. D and binds to human B7-H3 isoform 1.

[0110] In some embodiments, the antigen-binding molecules described herein have a K D and preferably one of the following K values: ≦5 μM, ≦2 μM, ≦1 μM, ≦500 nM, ≦450 nM, ≦400 nM, ≦350 nM. D In some embodiments, the antigen-binding molecule binds to human B7-H3 isoform 2 with a K of ≦10 μM and ≧10 nM. D and a K of, for example, one of ≦5 μM and ≧50 nM, ≦2 μM and ≧100 nM, ≦1 μM and ≧150 nM, ≦750 nM and ≧200 nM, or ≦500 nM and ≧250 nM. D and binds to human B7-H3 isoform 2.

[0111] In some embodiments, the antigen-binding molecules described herein have a K D and preferably one of K ≦5 μM, ≦2 μM, ≦1 μM, ≦500 nM. D In some embodiments, the antigen-binding molecule binds to mouse B7-H3 with a K of ≦10 μM and ≧10 nM. D and, for example, one of K ≦5 μM and ≧50 nM, ≦2 μM and ≧100 nM, ≦1 μM and ≧150 nM, ≦750 nM and ≧200 nM, or ≦500 nM and ≧250 nM. Dand binds to mouse B7-H3.

[0112] The antigen-binding molecules of the present disclosure can bind to specific target regions of B7-H3. The antigen-binding molecules of the present disclosure can bind to a linear epitope of B7-H3 consisting of a continuous sequence of amino acids (i.e., a primary amino acid sequence). In some embodiments, the antigen-binding molecules can bind to a conformational epitope of B7-H3 consisting of a discontinuous sequence of amino acids in the amino acid sequence.

[0113] The region of a given target molecule to which an antigen-binding molecule binds can be determined by those skilled in the art using various methods well known in the art, such as X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometric hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, the entire contents of which are incorporated herein by reference. The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, such as ELISA, immunoblotting (e.g., Western blot), immunoprecipitation, surface plasmon resonance, and biolayer interferometry analysis.

[0114] In some embodiments, the antigen-binding molecule binds (independently) to human B7-H3 (e.g., human B7-H3 isoform 1) and mouse B7-H3. In some embodiments, the antigen-binding molecule binds (independently) to human B7-H3 isoform 1 and human B7-H3 isoform 2. In some embodiments, the antigen-binding molecule binds (independently) to human B7-H3 isoform 1, human B7-H3 isoform 2, human B7-H3 isoform 3, and human B7-H3 isoform 4. In some embodiments, the antigen-binding molecule binds (independently) to human B7-H3 isoform 1, human B7-H3 isoform 2, and mouse B7-H3. In some embodiments, the antigen-binding molecule binds (independently) to human B7-H3 isoform 1, human B7-H3 isoform 2, human B7-H3 isoform 3, human B7-H3 isoform 4, and mouse B7-H3.

[0115] In some embodiments, the antigen-binding molecule is cross-reactive with one or more isoforms or homologs of B7-H3 (e.g., human B7-H3 isoform 1). In some embodiments, the antigen-binding molecule is cross-reactive with human B7-H3 (e.g., human B7-H3 isoform 1) and mouse B7-H3. In some embodiments, the antigen-binding molecule is cross-reactive with human B7-H3 isoform 1 and human B7-H3 isoform 2. In some embodiments, the antigen-binding molecule is cross-reactive with human B7-H3 isoform 1, human B7-H3 isoform 2, human B7-H3 isoform 3, and human B7-H3 isoform 4. In some embodiments, the antigen-binding molecule is cross-reactive with human B7-H3 isoform 1, human B7-H3 isoform 2, and mouse B7-H3. In some embodiments, the antigen-binding molecule is cross-reactive with human B7-H3 isoform 1, human B7-H3 isoform 2, human B7-H3 isoform 3, human B7-H3 isoform 4, and mouse B7-H3.

[0116] As used herein, a "cross-reactive" antigen-binding molecule / domain / polypeptide binds to a target antigen with which the antigen-binding molecule / domain has cross-reactivity. For example, an antigen-binding molecule / domain / polypeptide that is cross-reactive with human B7-H3 and mouse B7-H3 can bind to both human B7-H3 and mouse B7-H3. The cross-reactive antigen-binding molecule / domain / polypeptide can exhibit specific binding to each of the target antigens.

[0117] In some embodiments, the antigen-binding molecule of the present disclosure binds to the extracellular domain of B7-H3. In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 16. In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 20. In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 30. In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 34.

[0118] In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34.

[0119] In some embodiments, the antigen-binding molecules of the present disclosure bind to the Ig-like C2-type domain of B7-H3. In some embodiments, the antigen-binding molecules contact the Ig-like C2-type domain of B7-H3. In some embodiments, the antigen-binding molecules bind to B7-H3 through contact with one or more amino acids in the Ig-like C2-type domain of B7-H3.

[0120] In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 25. In some embodiments, the antigen-binding molecule contacts the region of B7-H3 set forth in SEQ ID NO: 25. In some embodiments, the antigen-binding molecule binds to B7-H3 through contact with one or more amino acids in the region set forth in SEQ ID NO: 25. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25.

[0121] In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule contacts the region of B7-H3 set forth in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule binds to B7-H3 through contact with one or more amino acids in the region set forth in SEQ ID NO: 27. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 27.

[0122] In some embodiments, the antigen-binding molecule binds to the region of B7-H3 set forth in SEQ ID NO: 38. In some embodiments, the antigen-binding molecule contacts the region of B7-H3 set forth in SEQ ID NO: 38. In some embodiments, the antigen-binding molecule binds to B7-H3 through contact with one or more amino acids in the region set forth in SEQ ID NO: 38. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 38.

[0123] In some embodiments, antigen-binding molecules according to the present disclosure bind to a region of B7-H3 that is the same as or overlaps with a region of B7-H3 that binds to an antigen-binding molecule comprising the CDRs, FRs and / or the complete amino acid sequence of P2A5 of a B7-H3-binding single domain antibody described herein, e.g., P2A5.

[0124] Whether a test antigen-binding molecule binds to the same or overlapping region of a given target as a reference antigen-binding molecule can be evaluated, for example, by analyzing (i) the interaction between the test antigen-binding molecule and the target in the absence of the reference antigen-binding molecule, and (ii) the interaction between the test antigen-binding molecule in the presence of the reference antigen-binding molecule or after incubation of the target with the reference antigen-binding molecule. Determining a reduced level of interaction between the test antigen-binding molecule and the target after analysis by (ii) compared to (i) may support the inference that the test and reference antigen-binding molecules bind to the same or overlapping region of the target. Suitable assays for such analysis include, for example, competitive ELISA assays and epitope binning assays.

[0125] In some embodiments, an antigen-binding molecule according to the present disclosure binds to a region of B7-H3 that is the same as or overlaps with the region of B7-H3 that binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:8.

[0126] In some embodiments, antigen binding molecules according to the present disclosure can enhance (i.e., upregulate, potentiate) cell killing of cells containing / expressing B7-H3. In some embodiments, antigen binding molecules do not enhance (i.e., do not substantially enhance) cell killing of cells that lack surface expression of B7-H3.

[0127] In some embodiments, antigen-binding molecules according to the present disclosure can inhibit the growth or reduce metastasis of cancers comprising cells that contain / express B7-H3. In some embodiments, antigen-binding molecules can enhance (i.e., upregulate, strengthen) cell killing of cancer cells that contain / express B7-H3. In some embodiments, antigen-binding molecules can inhibit the growth or reduce metastasis of cancers comprising cells that contain / express B7-H3.

[0128] Cell killing can be assessed using, for example, any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include: 51Examples of suitable release assays include Cr release assays, lactate dehydrogenase (LDH) release assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assays, and calcein-acetoxymethyl (calcein-AM) release assays. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed, for example, by co-culturing the test cells with the effector immune cells and measuring the number / proportion of viable / dead (e.g., lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3): e0193498 (incorporated herein by reference in its entirety). Increased resistance to cell killing by cells expressing granzyme B (e.g., effector immune cells) and / or decreased susceptibility to cell killing by such cells compared to a reference level of cell killing (e.g., by cell type) can be determined by detecting a decrease in the number / proportion of dead (e.g., lysed) test cells and / or an increase in the number / proportion of live (e.g., viable, non-lysed) test cells after a given period of time.

[0129] In some embodiments, antigen-binding molecules according to the present disclosure are capable of reducing the number / proportion of cells expressing B7-H3. In some embodiments, antigen-binding molecules according to the present disclosure are capable of reducing the number / proportion of cells expressing B7-H3. In some embodiments, antigen-binding molecules according to the present disclosure are capable of depleting / enhancing the depletion of such cells.

[0130] The antigen-binding molecules of the present disclosure may comprise one or more moieties for enhancing the reduction of the number / proportion of cells expressing B7-H3. For example, the antigen-binding molecules of the present disclosure may comprise, for example, an Fc region and / or a drug moiety.

[0131] The Fc region interacts with Fc receptors and other molecules of the immune system to produce functional effects. The effector functions mediated by IgG Fc are reviewed, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein by reference in its entirety), and include Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through interactions between the Fc region and Fc receptors expressed by immune cells, recruitment of components of the complement pathway via binding of the Fc region to the complement protein C1q, and resulting in activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0132] In some embodiments, an antigen-binding molecule according to the present disclosure comprises an Fc region capable of enhancing / directing one or more of ADCC, ADCP, CDC against cells expressing B7-H3 (e.g., cells expressing B7-H3 on the cell surface), and / or an Fc region capable of enhancing MAC formation on or cellular degranulation of cells expressing B7-H3.

[0133] In some embodiments, antigen binding molecules according to the present disclosure are capable of enhancing / directing ADCC against cells expressing B7-H3. The ability and extent to which a given antigen-binding molecule can induce ADCC of a given target cell type can be determined, for example, according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (incorporated herein by reference in its entirety), or as described, for example, in Jedema et al., Blood (2004) 103:2677-82 (incorporated herein by reference in its entirety). 51 The ability and extent of a given antigen-binding molecule to induce ADCP can be analyzed by a Cr release assay. The ability and extent of a given antigen-binding molecule to induce CDC can be analyzed, for example, according to the method described in Kamen et al., J Immunol (2017) 198 (Supplement 1) 157.17 (incorporated herein by reference in its entirety). The ability and extent of a given antigen-binding molecule to induce CDC can be analyzed, for example, using a C1q binding assay, such as the assay described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (incorporated herein by reference in its entirety).

[0134] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a drug moiety. The antigen-binding molecule may be conjugated to a drug moiety. Antibody-drug conjugates are generally discussed, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14 (incorporated herein by reference in its entirety). In some embodiments, the drug moiety is or comprises a cytotoxic agent, such that the antigen-binding molecule exhibits cytotoxicity against cells expressing B7-H3 (e.g., cells expressing B7-H3 on the cell surface). In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0135] In some embodiments, an antigen-binding molecule according to the present disclosure comprises an immune cell-engaging portion. In some embodiments, an antigen-binding molecule comprises a portion that binds to a CD3 polypeptide (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide).

[0136] In some embodiments, antigen-binding molecules according to the present disclosure are capable of enhancing / directing T cell-mediated cytolytic activity against cells expressing B7-H3.

[0137] In some embodiments, the antigen-binding molecules of the present disclosure exhibit anti-cancer activity. In some embodiments, the antigen-binding molecules of the present disclosure increase the killing of cancer cells. In some embodiments, the antigen-binding molecules of the present disclosure reduce the number of cancer cells in vivo, for example, compared to an appropriate control condition. The cancer may be a cancer that expresses B7-H3.

[0138] In some embodiments, antigen-binding molecules according to the present disclosure reduce / inhibit cancer and / or cancer tumor growth. In some embodiments, antigen-binding molecules reduce tissue infiltration by cancer cells. In some embodiments, antigen-binding molecules reduce cancer metastasis. In some embodiments, antigen-binding molecules exhibit anti-cancer activity. In some embodiments, antigen-binding molecules reduce cancer cell growth / proliferation. In some embodiments, antigen-binding molecules reduce cancer cell survival. In some embodiments, antigen-binding molecules increase cancer cell killing. In some embodiments, antigen-binding molecules of the present disclosure result in a reduction in the number of cancer cells, for example in vivo. The cancer may be a cancer comprising cells that express B7-H3.

[0139] Antigen-binding molecules of the present disclosure can be analyzed for the properties described in the previous paragraph in suitable assays, including, for example, in vivo models.

[0140] In some embodiments, administration of an antigen-binding molecule according to the present disclosure may result in one or more of: inhibiting cancer development / progression; delaying / preventing cancer onset; reducing / delaying / preventing tumor growth; reducing / delaying / preventing tissue invasion; reducing / delaying / preventing metastasis; reducing the severity of cancer symptoms; reducing cancer cell number; reducing tumor size / volume; and / or increasing survival (e.g., progression-free survival or overall survival), e.g., as determined in a suitable model.

[0141] In some embodiments, the antigen-binding molecules of the disclosure are capable of reducing / inhibiting tumor growth (e.g., in an in vivo model, e.g., of a cancer that expresses B7-H3) in a given assay by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, compared to the tumor growth observed in the absence of treatment with the antigen-binding molecule (or a suitable control following treatment with the antigen-binding molecule that is known to have no effect on tumor growth).

[0142] In some embodiments, the antigen-binding molecules of the disclosure are capable of reducing / inhibiting metastasis in a given assay (e.g., in an in vivo model, e.g., of a cancer that expresses B7-H3) to a level of less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of metastasis observed in the absence of treatment with the antigen-binding molecule (or a suitable control following treatment with an antigen-binding molecule known to have no effect on metastasis).

[0143] In some embodiments, an antigen-binding molecule of the disclosure is capable of increasing survival of a subject having cancer (e.g., in an in vivo model, e.g., a cancer that expresses B7-H3) in a given assay by more than 1-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold, the level of survival observed in the absence of treatment with the antigen-binding molecule (or a suitable control following treatment with the antigen-binding molecule that is known not to affect survival). Chimeric antigen receptor (CAR) In some aspects and embodiments according to the present disclosure, the antigen-binding molecule is a chimeric antigen receptor (CAR). In some aspects and embodiments, the present disclosure provides a chimeric antigen receptor comprising an antigen-binding molecule or polypeptide according to the present disclosure.

[0144] CAR is a recombinant receptor that provides both antigen binding and T cell activation functions. The structure and operation of CAR are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257 (1), the entire contents of which are incorporated herein by reference. CAR comprises an antigen binding domain linked to a signaling domain via a transmembrane domain. An optional hinge or spacer domain can separate the antigen binding domain and the transmembrane domain and act as a flexible linker. When expressed by a cell, the antigen binding domain is provided in the extracellular space, and the signaling domain is intracellular.

[0145] The antigen-binding domain mediates binding of the CAR to its specific target antigen. The antigen-binding domain of the CAR may be based on the antigen-binding region of an antigen-binding molecule specific for the antigen to which the CAR is targeted. For example, the antigen-binding domain of the CAR may comprise the amino acid sequence of the complementarity-determining region (CDR) of an antibody that specifically binds to the target antigen. The antigen-binding domain of the CAR may comprise or consist of the light and heavy chain variable region amino acid sequences of an antibody that specifically binds to the target antigen. The antigen-binding domain may be provided as a single-chain variable fragment (scFv) comprising the light and heavy chain variable region amino acid sequences of the antibody. The antigen-binding domain of the CAR may also target antigens based on other protein:protein interactions, such as ligand:receptor binding; for example, an IL-13Rα2-targeting CAR was developed using an antigen-binding domain based on IL-13 (see, e.g., Kahlon et al., 2004 Cancer Res 64(24):9160-9166).

[0146] A CAR of the present disclosure comprises an antigen-binding domain that comprises or consists of an antigen-binding molecule of the present disclosure, or that comprises or consists of a polypeptide according to the present disclosure. The optional spacer domain can separate the antigen-binding domain and the transmembrane domain and act as a flexible linker. Such a domain can be or include a flexible region, allowing the binding moiety to be oriented in various directions. The spacer domain can be derived from IgG.

[0147] The transmembrane domain is provided between the antigen binding domain and the signal transduction domain of CAR.The transmembrane domain, together with the antigen binding domain in the extracellular space and the signal transduction domain inside the cell, causes the CAR to be fixed to the cell membrane of the cell expressing the CAR.The transmembrane domain of CAR can be derived from the transmembrane region sequence for cell membrane-associated proteins (such as CD28, CD8, CD4, CD3-ζ, etc.).

[0148] The signaling domain contains an amino acid sequence required for activation of immune cell function. The CAR signaling domain may contain the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of cells expressing the CAR. Signaling domains containing sequences from other ITAM-containing proteins have also been employed in CARs, such as the ITAM-containing domain containing the FcγRI region (Haynes et al., 2001 J Immunol 166(1):182-187). CARs containing a signaling domain derived from the intracellular domain of CD3-ζ are often referred to as first-generation CARs.

[0149] The signaling domain of a CAR also typically includes the signaling domain of a costimulatory protein (e.g., CD28, 4-1BB, etc.) to provide the costimulatory signal necessary to enhance immune cell activation and effector function. CARs with signaling domains that include additional costimulatory sequences are often referred to as second-generation CARs. In some cases, CARs are engineered to generate costimulation of different intracellular signaling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1BB costimulation initiates signaling via TNF receptor-associated factor (TRAF) adaptor proteins. Therefore, the signaling domain of a CAR sometimes contains costimulatory sequences from the signaling domains of more than one costimulatory molecule. CARs that include signaling domains with multiple costimulatory sequences are often referred to as third-generation CARs.

[0150] Throughout this specification, polypeptides, domains and amino acid sequences "derived from" a reference polypeptide / domain / amino acid sequence have at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of the reference polypeptide / domain / amino acid sequence. Polypeptides, domains and amino acid sequences "derived from" a reference polypeptide / domain / amino acid sequence preferably retain functional and / or structural properties of the reference polypeptide / domain / amino acid sequence.

[0151] By way of example, an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the intracellular domain of CD28, for example as set forth in SEQ ID NO: 12. Furthermore, the amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional property of the amino acid sequence of SEQ ID NO: 12, i.e., the ability to activate signaling mediated by CD28.

[0152] The amino acid sequence of a given polypeptide or domain thereof may be retrieved from, or determined from, nucleic acid sequences retrieved from, databases known to those of skill in the art, such as GenBank, EMBL, and UniProt.

[0153] By engineering them to express CARs specific for a particular target antigen, immune cells (typically T cells, but also other immune cells, such as NK cells) can be directed to kill cells expressing the target antigen. Binding of a CAR-expressing T cell (CAR-T cell) to its specific target antigen initiates intracellular signaling, resulting in T cell activation. Activated CAR-T cells are stimulated to divide and produce factors that result in the killing of cells expressing the target antigen. antigen-binding domain The antigen-binding domain of a CAR according to the present disclosure comprises or consists of an antigen-binding molecule that binds to B7-H3 as described herein. Thus, a CAR according to the present disclosure comprises an antigen-binding molecule as described herein.

[0154] It will be understood that the antigen-binding molecule according to the present disclosure constitutes or is included in the antigen-binding domain of a CAR. Thus, in some embodiments, the antigen-binding molecule of the present disclosure is included in a CAR.

[0155] It will also be understood that the antigen-binding molecule of the present disclosure may be a CAR. A CAR having an antigen-binding domain comprising or consisting of an antigen-binding molecule of the present disclosure (e.g., a B7-H3-binding single-domain antibody) is an antigen-binding molecule. The antigen-binding domain of the CAR of the present disclosure may be provided in any suitable format, for example, in the form of scFv, scFab, etc.

[0156] In some embodiments, the antigen-binding domain comprises or consists of a B7-H3-binding single-domain antibody described herein. spacer domain In some embodiments, CAR comprises a spacer domain. The spacer domain may be provided between the antigen binding domain and the transmembrane domain. The spacer domain may also be referred to as a hinge domain. The spacer domain is an amino acid sequence that provides a flexible connection between the antigen binding domain and the transmembrane domain of CAR.

[0157] The presence, absence, and length of a spacer domain have been shown to affect CAR function (e.g., Dotti et al., Immunol Rev (2014) 257(1) and Jayaraman et al., EBioMedicine (2020) 58:102931, reviewed above). Spacer length can be altered to control synaptic cleft distance, thereby modulating signal transduction. Flexible spacers can allow access to sterically hindered epitopes on target antigens. Multimerization of spacer domains (e.g., via homotypic association) results in increased signal strength and activation stimulation.

[0158] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence that is or is derived from the CH2-CH3 region of human IgG1 (e.g., as set forth in SEQ ID NO: 59), the CH2-CH3 region of human IgG2 (e.g., as set forth in SEQ ID NO: 61), the CH1-CH2 hinge region of human IgG1, a spacer domain derived from CD8α, e.g., as described in WO 2012 / 031744 A1, or a spacer domain derived from CD28, e.g., as described in WO 2011 / 041093 A1. Hombach et al., Gene Therapy (2010) 17:1206-1213, describe variant CH2-CH3 regions for reduced activation of cells expressing FcγR, such as monocytes and NK cells. The amino acid sequence of the variant CH2-CH3 region is set forth in SEQ ID NO: 60.

[0159] In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the spacer domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. Transmembrane domain The CAR of the present disclosure comprises a transmembrane domain.Transmembrane domain refers to any three-dimensional structure formed by the sequence of thermodynamically stable amino acids in biological membrane, for example, cell membrane.In the context of the present disclosure, transmembrane domain can be the amino acid sequence that spans the cell membrane of the cell that expresses CAR.

[0160] The transmembrane domain may comprise or consist of a sequence of amino acids that form a hydrophobic alpha helix or beta barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be or be derived from the amino acid sequence of the transmembrane domain of a protein that contains a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001 J Mol Biol 305:567-580).

[0161] In some embodiments, the amino acid sequence of the transmembrane domain of a CAR of the present disclosure may be or may be derived from the amino acid sequence of the transmembrane domain of a protein expressed on the cell surface. In some embodiments, the protein expressed on the cell surface is a receptor or ligand, for example, an immunoreceptor or ligand. In some embodiments, the amino acid sequence of the transmembrane domain is selected from the group consisting of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I alpha, MHC class II alpha, MHC class II beta, CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, TCR alpha, TCR beta, CD4, CD8 alpha, CD8 beta, CD40, CD40L, PD-1, PD-L1, PD-L2, 4-1BB, 4-1BBL, OX40, OX40L, GITR, GITRL, TIM-3, and Gale. The transmembrane domain may be or may be derived from the amino acid sequence of one of the following: cutin-9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1, ICAM1, LFA-1, LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1, CD2, CD48, 2B4, SLAM, CD30, CD30L, DR3, TL1A, CD226, CD155, CD112, and CD276. In some embodiments, the transmembrane domain is or is derived from the amino acid sequence of the transmembrane domain of CD28, CD3-zeta, CD8α, CD8β, or CD4. In some embodiments, the transmembrane domain is or is derived from the amino acid sequence of the transmembrane domain of CD28.

[0162] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:11.

[0163] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:65.

[0164] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:66. Signaling domains The chimeric antigen receptors of the present disclosure comprise a signaling domain that provides a sequence for initiating intracellular signaling in cells expressing the CAR. ITAM-containing sequences: Signaling domains include ITAM-containing sequences. ITAM-containing sequences contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs include the amino acid sequence YXXL / I (SEQ ID NO: 69), where "X" represents any amino acid. In ITAM-containing proteins, sequences according to SEQ ID NO: 69 are often separated by 6 to 8 amino acids; YXXL / I(X) 6-8 YXXL / I (SEQ ID NO: 70). When a phosphate group is added to the tyrosine residue of an ITAM by a tyrosine kinase, a signal transduction cascade within the cell is initiated.

[0165] In some embodiments, the signaling domain comprises one or more copies of the amino acid sequence according to SEQ ID NO: 69 or SEQ ID NO: 70. In some embodiments, the signaling domain comprises at least 1, 2, 3, 4, 5, or 6 copies of the amino acid sequence according to SEQ ID NO: 69. In some embodiments, the signaling domain comprises at least 1, 2, or 3 copies of the amino acid sequence according to SEQ ID NO: 70.

[0166] In some embodiments, the signaling domain comprises an ITAM-containing sequence that is or is derived from the amino acid sequence of an ITAM-containing sequence of a protein having an ITAM-containing amino acid sequence. In some embodiments, the signaling domain comprises an ITAM-containing sequence that is or is derived from the amino acid sequence of the intracellular domain of one of CD3-zeta, FcγRI, CD3ε, CD3δ, CD3γ, CD79α, CD79β, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIV, or DAP12. In some embodiments, the signaling domain comprises an ITAM-containing sequence that is or is derived from the amino acid sequence of the intracellular domain of CD3-zeta.

[0167] In some embodiments, the signaling domain comprises an ITAM-containing sequence that comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13. Costimulatory sequence: The signaling domain may additionally comprise one or more costimulatory sequences. A costimulatory sequence is an amino acid sequence that provides costimulation of cells expressing a CAR of the present disclosure. Costimulation promotes the proliferation and survival of cells expressing a CAR upon binding to a target antigen, and can also promote cytokine production, differentiation, cytotoxic function, and memory formation by cells expressing a CAR. The molecular mechanisms of T cell costimulation are reviewed in Chen and Flies (2013) Nat Rev Immunol 13(4):227-242.

[0168] The costimulatory sequence may be or may be derived from the amino acid sequence of a costimulatory protein, hi some embodiments, the costimulatory sequence is or is derived from the amino acid sequence of the intracellular domain of a costimulatory protein.

[0169] When a CAR binds to a target antigen, the costimulatory sequence, when ligated with its cognate ligand, provides costimulation to a cell expressing the CAR of the type predicted to be provided by the costimulatory protein from which the costimulatory sequence is derived. As an example, in the case of a CAR that includes a signaling domain that includes a costimulatory sequence derived from CD28, binding to the target antigen initiates signaling in a cell expressing the CAR of the type predicted to be initiated by binding of CD80 and / or CD86 to CD28. Thus, the costimulatory sequence is capable of delivering the costimulatory signal of the costimulatory protein from which the costimulatory sequence is derived.

[0170] In some embodiments, the costimulatory protein domain may be a member of the B7-CD28 superfamily (e.g., CD28, ICOS) or a member of the TNF receptor superfamily (e.g., 4-1BB, OX40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the costimulatory sequence is or is derived from the intracellular domain of one of CD28, 4-1BB, ICOS, CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226, and LIGHT. In some embodiments, the costimulatory sequence is or is derived from the intracellular domain of CD28.

[0171] In some embodiments, the signaling domain comprises more than one costimulatory sequence. In some embodiments, the signaling domain comprises 1, 2, 3, 4, 5, or 6 costimulatory sequences. Multiple costimulatory sequences may be provided in tandem.

[0172] Whether a given amino acid sequence is capable of initiating signal transduction mediated by a given costimulatory protein can be investigated, for example, by analyzing correlations in costimulatory protein-mediated signal transduction (e.g., expression / activity of factors whose expression / activity is up-regulated or down-regulated as a result of costimulatory protein-mediated signal transduction).

[0173] Costimulatory proteins upregulate the expression of genes that promote cell growth, effector function, and survival through numerous pathways. For example, CD28 and ICOS signal through phosphatidylinositol 3-kinase (PI3K) and AKT, and upregulate the expression of genes that promote cell growth, effector function, and survival through NF-κB, mTOR, NFAT, and AP1 / 2. CD28 also activates AP1 / 2 through CDC42 / RAC1 and ERK1 / 2 through RAS, and ICOS activates C-MAF. 4-1BB, OX40, and CD27 recruit TNF receptor-associated factors (TRAFs) and signal through PI3K in addition to the MAPK pathway.

[0174] In some embodiments, the signaling domain comprises a costimulatory sequence that is or is derived from CD28. Kofler et al., Mol. Ther. (2011) 19:760-767, describe a variant CD28 intracellular domain in which the lck kinase binding site is mutated to reduce induction of IL-2 production upon CAR ligation in order to minimize regulatory T cell-mediated suppression of CAR-T cell activity. The amino acid sequence of the variant CD28 intracellular domain is set forth in SEQ ID NO: 67.

[0175] In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67. In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:68.

[0176] In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:74.

[0177] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 75. Linkers and additional sequences The antigen-binding molecules, polypeptides and CARs of the present disclosure may additionally comprise additional amino acids or sequences of amino acids.

[0178] The antigen-binding molecule may contain additional amino acids / amino acid sequences in addition to the amino acid sequence required for binding to the target antigen. In some embodiments, such additional amino acids / amino acid sequences are provided at the N-terminus of the single domain antibody sequence according to the present disclosure. In some embodiments, such additional amino acids / amino acid sequences are provided at the C-terminus of the single domain antibody sequence according to the present disclosure. In some embodiments, such additional amino acids / amino acid sequences are provided at the N-terminus and C-terminus of the single domain antibody sequence according to the present disclosure.

[0179] The antigen-binding molecules, polypeptides, and CARs of the present disclosure may contain one or more linker sequences between amino acid sequences. For example, a linker sequence may be provided between the domains of the CAR (e.g., between the antigen-binding domain and the spacer domain, and / or between the spacer domain and the transmembrane domain, and / or between the transmembrane domain and the signaling domain). In a further example, a linker sequence may be provided between subsequences of the domains of the CAR (e.g., between the costimulatory sequence and the ITAM-containing sequence of the signaling domain).

[0180] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0181] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm, where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5 or 6) copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0182] The antigen-binding molecules, polypeptides, and CARs of the present disclosure may comprise an amino acid sequence to facilitate expression, folding, trafficking, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids that forms a detectable moiety, for example, as described herein below.

[0183] The antigen-binding molecules, polypeptides, and CARs of the present disclosure may additionally contain a signal peptide (also known as a leader sequence or signal sequence). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed on the cell surface often contain signal peptides. Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0184] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide / CAR or may be present in a newly synthesized antigen-binding molecule / polypeptide / CAR. The signal peptide provides efficient trafficking of the antigen-binding molecule / polypeptide / CAR. The signal peptide is often removed by cleavage and is therefore not included in the mature antigen-binding molecule / polypeptide / CAR.

[0185] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176). Labels and conjugates In some embodiments, the antigen-binding molecule, polypeptide, or CAR of the present disclosure additionally comprises a detectable moiety.

[0186] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radiolabel, a chemical label, a nucleic acid label, or an enzyme label. The antigen-binding molecule, polypeptide, or CAR may be covalently or non-covalently labeled with a detectable moiety.

[0187] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radiolabels include hydrogen 3 ,sulfur 35 ,carbon 14 , Phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 ,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper67 , fluorine 18 ,yttrium 90 ,palladium 100 , bismuth 217 and antimony 211 Examples of the label include radioisotopes such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 13

[0188] In some embodiments, the antigen-binding molecule / polypeptide / CAR comprises an epitope tag, such as His (e.g., 6xHis), FLAG, c-Myc, StrepTag, hemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and a hapten (e.g., biotin, digoxigenin, dinitrophenol), optionally at the N- or C-terminus of the antigen-binding molecule / polypeptide / CAR.

[0189] In some embodiments, the antigen-binding molecule / polypeptide / CAR comprises a moiety with detectable activity, such as an enzymatic moiety, including luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucuronidases, phosphatases (e.g., alkaline phosphatase), peroxidases (e.g., horseradish peroxidase), and cholinesterases.

[0190] In some embodiments, the antigen-binding molecule / polypeptide / CAR of the present disclosure is conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect, i.e., a drug moiety. The drug moiety may be a small molecule (e.g., an organic compound with a low molecular weight (<1000 daltons, typically about 300-700 daltons)). Drug moieties are described, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14 (incorporated herein by reference in its entirety). In some embodiments, the drug moiety may be or comprise a cytotoxic agent. In some embodiments, the drug moiety may be or comprise a chemotherapeutic agent. Drug moieties include, for example, calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD. Certain exemplary polypeptides, antigen-binding molecules and CARs In some embodiments, the antigen-binding molecules / polypeptides of the present disclosure comprise or consist of an amino acid sequence comprising the CDRs of P2A5.

[0191] In some embodiments, the antigen-binding molecules / polypeptides of the present disclosure comprise or consist of an amino acid sequence comprising the FR of P2A5. In some embodiments, an antigen-binding molecule / polypeptide of the present disclosure comprises or consists of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:8.

[0192] In some embodiments of the present disclosure, the CAR comprises: an antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8; a spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10; a transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11; a costimulatory domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12; and a signaling domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13. It comprises or consists of:

[0193] In some embodiments of the present disclosure, the CAR comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9. Nucleic acids and vectors The present disclosure provides a nucleic acid or multiple nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0194] In some embodiments, the nucleic acid may be or be included in a vector or vectors. That is, the nucleotide sequence of the nucleic acid may be contained in a vector. The antigen-binding molecule, polypeptide, or CAR according to the present disclosure may be produced intracellularly by transcription from a vector encoding the antigen-binding molecule, polypeptide, or CAR, followed by translation of the transcribed RNA.

[0195] Therefore, the present disclosure also provides a vector or vectors containing a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate the delivery of a nucleic acid encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. The vector may be an expression vector containing the elements necessary to express a nucleic acid comprising / encoding an antigen-binding molecule, polypeptide, or CAR according to the present disclosure.

[0196] The nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e., purified or isolated from other nucleic acids or naturally occurring biological materials.

[0197] The nucleotide sequence may be contained in a vector, e.g., an expression vector. As used herein, a "vector" refers to a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acids into cells. The vector may be a vector for expressing a nucleic acid in a cell. Such a vector may include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0198] The term "operably linked" can include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked such that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript can then be translated into a desired peptide / polypeptide.

[0199] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., retroviral vectors, such as gammaretroviral vectors (e.g., vectors derived from murine leukemia viruses (MLV) such as SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated by reference in their entireties.

[0200] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector that includes elements necessary for expression of a protein from the vector in a eukaryotic cell, hi some embodiments, the vector may be a mammalian vector, e.g., a vector that includes a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0201] The component polypeptides of an antigen binding molecule / CAR according to the present disclosure may be encoded by different nucleic acids of a plurality of nucleic acids or by different vectors of a plurality of vectors.

[0202] In some embodiments, the nucleic acid encodes the antigen-binding molecule or CAR described herein. In some embodiments, the vector is polycistronic (e.g., bicistronic, tricistronic, etc.); that is, in some embodiments, the vector encodes an mRNA with multiple protein-coding regions. In some embodiments, the vector is bicistronic. The component polypeptides of the antigen-binding molecule or CAR according to the present disclosure may be encoded by different nucleic acids or different vectors.

[0203] The present disclosure provides a retroviral vector comprising a nucleic acid encoding an antigen-binding molecule or CAR according to the present disclosure. In some embodiments, the retroviral vector comprises a nucleic acid encoding an antigen-binding molecule that binds to B7-H3. In some embodiments, the retroviral vector comprises a nucleic acid encoding a CAR specific to B7-H3.

[0204] In some embodiments, a CAR or antigen-binding molecule according to the present disclosure may be encoded by a plasmid. The plasmid may be based on the plasmid pSFG (e.g., as described in Hakre et al., Mol Cell. 2006 Oct. 20;24(2):301-8, which is incorporated herein by reference in its entirety). Production of antigen-binding molecules and polypeptides The antigen-binding molecules, polypeptides and CARs according to the present disclosure can be prepared according to methods for producing polypeptides known to those skilled in the art.

[0205] Antigen-binding molecules, polypeptides, and CARs may be prepared by chemical synthesis, for example, by liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in, for example, Chandrudu et al., Molecules (2013), 18:4373-4388, the entire contents of which are incorporated herein by reference.

[0206] Alternatively, antigen-binding molecules, polypeptides, and CARs may be produced by recombinant expression. Molecular biology techniques suitable for the recombinant production of polypeptides are well known in the art, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and Nat Methods. (2008); 5(2): 135-146, both of which are incorporated herein by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461, both of which are incorporated herein by reference in their entirety.

[0207] In some cases, the antigen-binding molecules and CARs of the present disclosure are composed of more than one polypeptide chain. In such cases, production of the antigen-binding molecule / CAR may involve transcribing and translating more than one polypeptide, and then associating the polypeptide chains to form the antigen-binding molecule / CAR.

[0208] For recombinant production according to the present disclosure, any cell suitable for expressing a polypeptide can be used. The cell may be a prokaryotic or eukaryotic organism. In some embodiments, the cell is a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium may be a gram-negative bacterium, such as a bacterium belonging to the Enterobacteriaceae family, such as Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, such as those described above.

[0209] In some cases, the cells are not prokaryotic because some prokaryotic cells do not tolerate the same folding or post-translational modifications as eukaryotic cells. In addition, much higher expression levels are possible in eukaryotes, and proteins can be more easily purified from eukaryotes using appropriate tags. Specific plasmids are also available that enhance the secretion of proteins into the culture medium.

[0210] In some embodiments, the polypeptides may be prepared by cell-free protein synthesis (CFPS), for example, according to the system described in Zemella et al., Chembiochem (2015) 16(17):2420-2431, the entire contents of which are incorporated herein by reference.

[0211] Production may involve the culture or fermentation of eukaryotic cells engineered to express the polypeptide of interest. The culture or fermentation can be carried out in a bioreactor with an appropriate supply of nutrients, air / oxygen, and / or growth factors. The secreted protein can be collected by removing the culture medium / fermentation broth from the cells, extracting the protein content, separating the individual proteins, and isolating the secreted polypeptide. Culture, fermentation, and separation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.; incorporated herein by reference above).

[0212] A bioreactor contains one or more vessels in which cells can be cultured. Cultivation in a bioreactor can be continuous, with a continuous flow of reactants into the reactor and a continuous flow of cultured cells out of the reactor. Alternatively, cultivation can be performed in batches. Bioreactors are monitored and controlled for environmental conditions such as pH, oxygen, flow rates into and out of the vessel, and agitation within the vessel to provide optimal conditions while culturing the cells.

[0213] After culturing the cells expressing the polypeptide, the polypeptide of interest can be isolated. Any suitable method for isolating proteins from cells known in the art can be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide is secreted from the cells, the cells can be separated from the culture medium containing the secreted polypeptide of interest by centrifugation. If the polypeptide of interest is collected intracellularly, protein isolation may include centrifugation to separate the cells from the cell culture medium, treating the cell pellet with a lysis buffer, and lysing the cells, for example, by sonication, rapid freeze-thawing, or osmotic lysis.

[0214] It may then be desirable to isolate the polypeptide of interest from the supernatant or culture medium, which may contain other proteins and non-protein components. A common approach to separating protein components from the supernatant or culture medium is precipitation. Proteins with different solubilities are precipitated with different concentrations of a precipitant, such as ammonium sulfate. For example, low concentrations of the precipitant extract water-soluble proteins. Therefore, by adding different increasing concentrations of the precipitant, proteins with different solubilities can be differentiated. Then, dialysis may be used to remove ammonium sulfate from the separated proteins.

[0215] Other methods for distinguishing different proteins are known in the art, such as ion exchange chromatography and size chromatography, which may be used as an alternative to precipitation or may be performed after precipitation.

[0216] Once the polypeptide of interest has been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide. Numerous methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization. Antigen-binding molecules and cells containing / expressing polypeptides The present disclosure also provides cells comprising or expressing an antigen-binding molecule, polypeptide, or CAR according to the present disclosure. Also provided are cells comprising or expressing a nucleic acid, nucleic acids, vector, or vectors according to the present disclosure.

[0217] It will be understood that where a cell is referred to in the singular herein (ie, "a / the cell"), a plural / population of such cells is also contemplated. The cell may be a eukaryotic cell, for example a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human), or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodents), cat, dog, pig, sheep, goat, cow (including cattle, e.g., dairy cows, or any animal in the order Bos), horse (including any animal in the family Equidae), donkey, and non-human primate).

[0218] In some embodiments, the cells are or are derived from a cell type commonly used for the expression of polypeptides for therapeutic use in humans. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (incorporated herein by reference in its entirety), including, for example, CHO, HEK293, PER.C6, NS0, and BHK cells. In a preferred embodiment, the cells are or are derived from CHO cells.

[0219] The present disclosure also provides a method for producing a cell containing a nucleic acid or vector according to the present disclosure, the method comprising introducing a nucleic acid, multiple nucleic acids, one vector, or multiple vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0220] The present disclosure also provides a method for producing a cell that expresses / contains an antigen-binding molecule, polypeptide, or CAR according to the present disclosure, the method comprising introducing a nucleic acid, multiple nucleic acids, one vector, or multiple vectors according to the present disclosure into the cell. In some embodiments, the method additionally comprises culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the method is carried out in vitro.

[0221] The present disclosure also provides cells obtained or obtainable by a method according to the present disclosure. Cells expressing the CAR of the present disclosure In some aspects and embodiments, the present disclosure provides cells comprising a CAR according to the present disclosure. The CAR according to the present disclosure can be used to generate cells that express the CAR, such as CAR-expressing immune cells (e.g., CAR-T or CAR-NK cells).

[0222] A cell that expresses a CAR may contain or express a nucleic acid encoding a CAR according to the present disclosure. It will be understood that a cell that expresses a CAR contains the CAR that it expresses. It will also be understood that a cell that expresses a nucleic acid encoding a CAR also expresses and contains the CAR encoded by the nucleic acid.

[0223] The cell expressing the CAR is preferably an immune cell. The immune cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, for example, a T cell, a B cell, a NK cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor thereof. The immune cell may express, for example, a CD3 polypeptide (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), a TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the immune cell is a T cell, such as a CD3+ T cell. In some embodiments, the T cell is a CD3+ and a CD4+ T cell. In some embodiments, the T cell is a CD3+ and a CD8+ T cell. In some embodiments, the T cell is a T helper cell (T H In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0224] Aspects and embodiments of the present disclosure particularly relate to T cells comprising / expressing a CAR specific for B7-H3 according to the present disclosure. Immune cells useful in the methods described herein can be obtained from any suitable source. The source may be an animal or a human. The source may be a non-human mammal, but is more preferably a human. The source may be of any gender. The source may be a patient expected to be treated with adoptive cell therapy (autologous cells). Thus, the source may be one diagnosed with a disease / condition requiring treatment, one suspected of having such a disease / condition, or one at risk of developing / obtaining such a disease / condition. In some cases, the source is an individual different from the patient expected to be treated (allogeneic cells). In such cases, the source is typically expected to be a healthy individual or an individual not known to have or be at risk of developing / obtaining such a disease / condition.

[0225] In some aspects and embodiments, the immune cells may be virus-specific immune cells. "Virus-specific immune cells," as used herein, refer to immune cells specific to a virus. Virus-specific immune cells express / contain a receptor (preferably a T cell receptor) capable of recognizing a viral antigen peptide (e.g., when presented by an MHC molecule). Virus-specific immune cells can express / contain such a receptor as a result of expression of endogenous nucleic acid encoding such an antigen receptor or as a result of being engineered to express such a receptor. Virus-specific immune cells preferably express / contain a TCR specific for a viral antigen peptide. Virus-specific T cells can display certain functional properties of T cells in response to the viral antigen for which the T cell is specific or in response to cells containing / expressing the virus / antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g., cytotoxic T cells.

[0226] In some embodiments, virus-specific T cells may exhibit one or more of the following properties: cytotoxicity towards cells containing / expressing the virus / viral antigen to which the T cell is specific; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression in response to stimulation with the virus / viral antigen to which the T cell is specific or in response to exposure to cells containing / expressing the virus / viral antigen to which the T cell is specific.

[0227] Virus-specific T cells express / contain a TCR that, when presented by the appropriate MHC molecule, is capable of recognizing a peptide of the viral antigen for which the T cell is specific. Virus-specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0228] The virus that virus-specific immune cell is specific for can be any virus.For example, virus can be dsDNA virus (for example, adenovirus, herpesvirus, poxvirus), ssRNA virus (for example, parvovirus), dsRNA virus (for example, reovirus), (+)ssRNA virus (for example, picornavirus, togavirus), (-)ssRNA virus (for example, orthomyxovirus, rhabdovirus), ssRNA-RT virus (for example, retrovirus) or dsDNA-RT virus (for example, hepadnavirus). Specifically, the present disclosure contemplates viruses belonging to the families Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae. In some embodiments, the virus is selected from the group consisting of Epstein-Barr virus, adenovirus, herpes simplex 1 virus, herpes simplex 2 virus, varicella zoster virus, human cytomegalovirus, human herpesvirus 8, human papillomavirus, BK virus, JC virus, smallpox virus, hepatitis B virus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, The virus is selected from yellow fever virus, dengue virus, West Nile virus, TBE virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coltivirus, and bannavirus.

[0229] In some embodiments, the virus is selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).

[0230] In some embodiments, the virus-specific immune cells may be specific for a viral peptide / polypeptide selected from, for example, Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).

[0231] T cells specific for viral antigens are sometimes referred to herein as virus-specific T cells (VSTs). T cells specific for a particular viral antigen may also be described as specific for the related virus, for example, T cells specific for EBV antigens may also be referred to as EBV-specific T cells, or "EBVSTs."

[0232] Thus, in some embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVST), adenovirus-specific T cells (AdVST), cytomegalovirus-specific T cells (CMVST), human papillomavirus (HPVST), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B virus-specific T cells (HBVST), hepatitis C virus-specific T cells (HCVST), human immunodeficiency virus-specific T cells (HIVST), lymphocytic choriomeningitis virus-specific T cells (LCMVST), or herpes simplex virus-specific T cells (HSVST).

[0233] In some preferred embodiments, the virus-specific immune cells are specific for an EBV antigenic peptide / polypeptide. In preferred embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVST).

[0234] EBV virology is described, for example, in Stanfield and Luftiq, F1000 Res. (2017) 6:386 and Odumade et al., Clin Microbiol Rev (2011) 24(1):193-209, both of which are incorporated by reference in their entireties.

[0235] EBV infects epithelial cells through the binding of the viral protein BMFR2 to β1 integrin and the binding of the viral proteins gH / gL to integrins avβ6 and avβ8. EBV infects B cells through the interaction of the viral glycoprotein gp350 with CD21 and / or CD35, followed by the interaction of viral gp42 with MHC class II. These interactions initiate fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.

[0236] EBV has two replication modes: latent and lytic. The latent cycle does not result in the production of virions and can occur in niches, i.e., B cells and epithelial cells. The circular DNA of the EBV genome exists as an episome in the cell nucleus and is copied by the host cell's DNA polymerase. During latency, only a fraction of EBV's genes are expressed in one of three distinct patterns known as the latency program, which produces distinct sets of viral proteins and RNA. The latent cycle is described, for example, in Amon and Farrell, Reviews in Medical Virology (2004) 15(3):149-56, the entire contents of which are incorporated herein by reference.

[0237] The EBNA1 protein and the non-coding RNA EBER are expressed in each of the latency programs I to III. Latency programs II and III further include the expression of EBNALP, LMP1, LMP2A, and LMP2B proteins, while latency program III further includes the expression of EBNA2, EBNA3A, EBNA3B, and EBNA3C.

[0238] EBNA1 is multifunctional and plays a role in gene regulation, extrachromosomal replication, and genomic maintenance of EBV episomes through positive and negative regulation of viral promoters (Duellman et al., J Gen Virol. (2009); 90(Pt9):2251-2259). EBNA2 is involved in regulating latent viral transcription and contributes to the immortalization of EBV-infected cells (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391:35-59). EBNA-LP is required for the transformation of naive B cells and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog. (2018); 14(2):e1006890). EBNA3A, 3B, and 3C interact with RBPJ and influence gene expression, contributing to the survival and proliferation of infected cells (Wang et al., J Virol. (2016) 90(6):2906-2919). LMP1 regulates the expression of genes involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5):490-504). LMP2A and LMP2B inhibit normal B cell signaling by mimicking activated B cell receptors (Portis and Longnecker, Oncogene (2004) 23(53):8619-8628). EBERs form ribonucleoprotein complexes with host cell proteins and are proposed to play a role in cellular transformation.

[0239] The latent cycle can progress through either latency program I or III in B cells, usually progressing from III to II and from II to I. EBV enters latency program III when it infects resting naive B cells. Expression of latency III genes activates the B cell, which becomes a proliferating blast. EBV then typically progresses to latency II by restricting expression to a subset of genes, which causes the blast to differentiate into a memory B cell. Further restriction of gene expression causes EBV to enter latency I. When memory B cells divide, EBNA1 expression allows EBV to replicate. In epithelial cells, only latency II occurs.

[0240] During primary infection, EBV replicates in oropharyngeal epithelial cells and establishes latency III, II, and I infections in B lymphocytes. EBV latent infection of B lymphocytes is required for viral persistence and subsequent replication in epithelial cells and for the release of infectious virus into saliva. EBV latency III and II infections of B lymphocytes, latency II infections of oral epithelial cells, and latency II infections of NK or T cells can result in malignancies characterized by the presence and gene expression of the normal EBV genome.

[0241] Latent EBV in B cells can be reactivated and switch to lytic replication. The lytic cycle leads to the production of infectious virions and can occur in niches, i.e., B cells and epithelial cells, as reviewed, for example, by Kenney in Chapter 25 of Arvin et al., Human Herpesviruses: Biology, Therapy and Immunoprophylaxis; Cambridge University Press (2007), which is incorporated herein by reference in its entirety.

[0242] Lytic replication requires that the EBV genome be linear. The latent EBV genome is episomal and must be linearized for lytic reactivation. In B cells, lytic replication usually occurs only after reactivation from latency.

[0243] Immediate-early lytic gene products, such as BZFL1 and BRLF1, act as transactivators, enhancing their own expression and that of late lytic cycle genes. Early lytic gene products have roles in viral replication (e.g., EBV DNA polymerase catalytic component BALF5; DNA polymerase processivity factor BMRF1, DNA-binding protein BALF2, helicase BBLF4, primase BSLF1, and primase-related protein BBLF2 / 3) and deoxyribonucleotide metabolism (e.g., thymidine kinase BXLF1, dUTPase BORF2). Other early lytic gene products act as transcription factors (e.g., BMRF1, BRRF1), have roles in RNA stability and processing (e.g., BMLF1), or are involved in immune evasion (e.g., BHRF1, which inhibits apoptosis).

[0244] Late lytic gene products are traditionally classified as those expressed after the onset of viral replication. They generally encode structural components of the virion, such as nucleocapsid proteins, as well as glycoproteins (e.g., gp350 / 220, gp85, gp42, gp25) that mediate EBV binding and fusion. Other late lytic gene products have roles in immune evasion; BCLF1 encodes the viral homolog of IL-10, and BALF1 encodes a protein with homology to the anti-apoptotic protein Bcl2.

[0245] "EBV-specific immune cells," as used herein, refer to immune cells specific for Epstein-Barr virus (EBV). EBV-specific immune cells express / contain a receptor (preferably a T cell receptor) capable of recognizing an EBV antigenic peptide (e.g., when presented by an MHC molecule). EBV-specific immune cells preferably express / contain a TCR specific for an EBV antigenic peptide presented by MHC class I.

[0246] In some embodiments, the EBV-specific immune cells are T cells, e.g., CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (T H In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0247] EBV-specific T cells preferably express / contain a TCR that is capable of recognizing a peptide of an EBV antigen for which the T cell is specific when presented by an appropriate MHC molecule. EBV-specific T cells may be CD4+ and / or CD8+ T cells.

[0248] The EBV-specific immune cells may be specific for any EBV antigen, such as those described herein. A population of EBV-specific immune cells, or a composition comprising a plurality of EBV-specific immune cells, may comprise immune cells specific for one or more EBV antigens.

[0249] In some embodiments, the EBV antigen is an EBV latent antigen, such as a type III latent antigen (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latent antigen (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B or BARF1), or a type I latent antigen (e.g., EBNA1 or BARF1). In some embodiments, the EBV antigen is an EBV lytic antigen, such as an immediate early lytic antigen (e.g., BZLF1, BRLF1 or BMRF1), an early lytic antigen (e.g., BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU or EBNA1-FUK), or a late lytic antigen (e.g., BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3 or gp350).

[0250] In some embodiments according to various aspects of the present disclosure, the cells may contain / express more than one (e.g., two, three, four, etc.) CARs. In some embodiments, a cell may contain / express more than one non-identical CAR. A cell containing / expressing more than one non-identical CAR may contain / express CARs specific for non-identical target antigens. In some embodiments, each non-identical target antigen is independently a cancer cell antigen described herein. Functional properties of cells expressing the CAR of the present disclosure A cell (e.g., an immune cell, e.g., a T cell) expressing a CAR according to the present disclosure can display certain functional properties in response to B7-H3 or in response to cells containing / expressing B7-H3. In some embodiments, the property is a functional property associated with an effector T cell, e.g., a cytotoxic T cell.

[0251] Cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure may exhibit one or more of the following properties: expression of one or more cytotoxic / effector factors (e.g., IFNγ, TNFα, GM-CSF), proliferation / population expansion, and / or expression of a growth factor (e.g., IL-2) in response to cells expressing B7-H3; cytotoxicity towards cells expressing B7-H3; lack of cytotoxicity (i.e., above baseline) towards cells that do not express B7-H3; and / or anti-cancer activity against cancers comprising cells expressing B7-H3 (e.g., cytotoxicity to cancer cells, tumor growth inhibition, reduced tumor burden, reduced metastasis, etc.).

[0252] In some embodiments, T cells expressing a B7-H3-specific CAR may exhibit one or more of the following properties: cytotoxicity towards cells containing / expressing B7-H3; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression in response to stimulation with B7-H3 or exposure to cells containing / expressing B7-H3; proliferation / population expansion in response to stimulation with B7-H3 or exposure to cells containing / expressing B7-H3; cytotoxicity towards cells of a cancer expressing B7-H3; inhibition of tumor growth of a cancer comprising cells expressing B7-H3; reduction of tumor burden in a subject with a cancer comprising cells expressing B7-H3; reduction of metastasis of a cancer comprising cells expressing B7-H3.

[0253] In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit cytotoxicity to A549 cells, MDA-MB-231 cells, and / or THP-1 cells. In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit cytotoxicity to colorectal cancer cells (e.g., DLD-1 cells, HT29 cells, and / or SW480 cells). In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit cytotoxicity to gastric cancer cells (e.g., NCI-N87 cells, MKN7 cells, and / or MKN45 cells). In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit cytotoxicity to breast cancer cells (e.g., MDA-MB-231 cells and / or MDA-MB-468 cells). In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit cytotoxicity to lung cancer cells (e.g., A549 cells, H1299 cells, H23 cells, and / or H5967 cells).

[0254] In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit sequential killing of cells expressing B7-H3, e.g., cancer cells expressing B7-H3, hi some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure exhibit sequential killing of THP-1 cells, DLD-1 cells, HT29 cells and / or NCI-N87 cells.

[0255] Cell proliferation / population expansion can be studied by analyzing cell division or cell number over a period of time. Cell division can be measured, for example, by: 3Proliferating cells may be analyzed by in vitro analysis of H-thymidine incorporation, or by the CFSE dilution assay described, for example, in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, which are incorporated herein by reference in their entireties. Proliferating cells can also be identified by analysis of 5-ethynyl-2'-deoxyuridine (EdU) incorporation by a suitable assay, for example, as described in Buck et al., Biotechniques. 2008 January;44(7):927-9, and Sali and Mitchison, PNAS USA 2008 February 19;105(7):2415-2420, both of which are incorporated herein by reference in their entireties.

[0256] "Expression" as used herein may refer to gene or protein expression. Gene expression encompasses the transcription of DNA into RNA and can be measured by various means known to those skilled in the art, for example, by measuring mRNA levels with quantitative real-time PCR (qRT-PCR) or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, for example, by antibody-based methods, such as Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0257] Cytotoxicity and cell killing can be investigated using, for example, any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include: 51Examples of such release assays include Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing by a given cell type can be analyzed, for example, by co-culturing test cells with the given cell type and measuring the number / ratio of viable / dead test cells after a suitable period of time.

[0258] In some embodiments, cell killing of cells expressing B7-H3 by cells expressing a CAR can be assessed by flow cytometry, as described herein in Example 1.13, or by the xCELLigence assay, as described herein in Example 1.14. Cell killing by cells expressing a CAR can also be assessed in vivo, for example, by assessing the number / proportion of cells expressing the target antigen for the CAR and inferring their killing / depletion by cells expressing the CAR.

[0259] The cells can be evaluated for anti-cancer activity (e.g., cytotoxicity to cancer cells, tumor growth inhibition, reduction in tumor burden, reduction in metastasis, etc.) by appropriate in vitro assays or analysis in in vivo models of the relevant cancer. For example, the cells can be evaluated in xenograft models derived from cancer cell lines, and such analysis can be performed, for example, as described in Example 1.16 herein. By way of illustration, the Examples of the present disclosure demonstrate inhibition of tumor growth and reduction in tumor burden following administration of immune cells expressing a CAR specific for B7-H3 according to the present disclosure in xenograft models derived from HT29, SW-480, and N-87 cell lines of colorectal cancer and gastric cancer. Producing cells expressing the CAR of the present disclosure Methods for producing cells that express a CAR are well known to those of skill in the art. They generally involve modifying a cell (e.g., an immune cell, such as a T cell or an NK cell) to express / contain a CAR, e.g., introducing a nucleic acid encoding a CAR into the immune cell.

[0260] Immune cells may be modified to contain / express a CAR or a nucleic acid encoding a CAR described herein by methods well known to those of skill in the art. The methods generally involve transfer of a nucleic acid for permanent (stable) or transient expression of the transferred nucleic acid.

[0261] Any suitable genetic engineering platform can be used to modify cells according to the present disclosure.Suitable methods for modifying cells include using genetic engineering platforms, such as gammaretroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery and RNA transfection, as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, the entire contents of which are incorporated herein by reference.

[0262] Methods also include, for example, those described in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is incorporated herein by reference in its entirety. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0263] Methods for generating / expanding a population of immune cells expressing a CAR in vitro / ex vivo are well known to those skilled in the art. Suitable culture conditions (i.e., cell culture medium, additives, stimuli, temperature, gas atmosphere), cell number, culture period, and method for introducing a nucleic acid encoding a CAR into cells can be determined by reference to, for example, Hombach et al., J Immunol (2001) 167:6123-6131, Ramos et al., J. Clin. Invest. (2017) 127(9):3462-3471, and WO2015 / 028444A1, all of which are incorporated herein by reference in their entirety.

[0264] Conveniently, cultures of cells according to the present disclosure may be maintained in a humidified atmosphere containing 5% CO at 37° C. Cells in cell culture may be established and / or maintained at any suitable density, as can be readily determined by one of skill in the art.

[0265] Culturing can be carried out in any vessel suitable for the volume of culture, such as a well of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, the cells are cultured in a bioreactor, such as those described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, which is incorporated herein by reference in its entirety. In some embodiments, the cells are cultured in a GRex cell culture vessel, such as a GRex flask or a GRex100 bioreactor.

[0266] Immune cells (e.g., T cells) may be activated prior to introduction of a nucleic acid encoding a CAR. For example, T cells within a population of PBMCs may be nonspecifically activated in vitro by stimulation with agonistic anti-CD3 and anti-CD28 antibodies in the presence of IL-2.

[0267] Introducing nucleic acid / vector into cell can include transduction, for example, retroviral transduction.Therefore, in some embodiments, nucleic acid is contained in a viral vector, or vector is a viral vector.The transduction of immune cells with viral vector is described, for example, in Simmons and Alberola-Ila, Methods Mol Biol.(2016)1323:99-108, which is incorporated herein by reference in its entirety.

[0268] Drugs may be employed to enhance transduction efficiency. Hexadimethrine bromide (polybrene) is a cationic polymer commonly used to improve transduction by neutralizing charge repulsion between virions and sialic acid residues expressed on the cell surface. Other drugs commonly used to enhance transduction include, for example, poloxamer-based drugs such as LentiBOOST (Sirion Biotech), Retronectin (Takara), and Vectofusin (Miltenyi Biotech), as well as SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0269] In some embodiments, the method comprises centrifuging cells into which it is desired to introduce a nucleic acid encoding a CAR in the presence of cell culture medium containing a viral vector comprising the nucleic acid (referred to in the art as "spinfection").

[0270] In some embodiments, the method comprises introducing a nucleic acid or vector according to the present disclosure into an immune cell by electroporation, e.g., as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is incorporated herein by reference in its entirety.

[0271] The methods generally include introducing a nucleic acid encoding a CAR into a cell and culturing the cell under conditions suitable for expression of the nucleic acid / CAR by the cell. In some embodiments, the methods include culturing the immune cells into which the nucleic acid encoding the CAR has been introduced to expand their numbers. In some embodiments, the methods include culturing the immune cells into which the nucleic acid encoding the CAR has been introduced in the presence of IL-7 and / or IL-15 (e.g., recombinant IL-7 and / or IL-15).

[0272] In some embodiments, the method further comprises purifying / isolating cells that express a CAR, e.g., from other cells (e.g., cells that do not express a CAR). Methods for purifying / isolating immune cells from a heterogeneous population of cells are well known in the art, and for example, FACS- or MACS-based methods can be employed to sort a population of cells based on the expression of immune cell markers. In some embodiments, the method purifies / isolates a specific type of cell, e.g., CD8+ T cells that express a CAR, CTLs that express a CAR.

[0273] In a preferred embodiment, T cells expressing a B7-H3-specific CAR may be generated from T cells within a population of PBMCs by a process comprising stimulating the PBMCs with antagonist anti-CD3 and anti-CD28 antibodies, transducing the cells with a viral vector (e.g., a gamma-retroviral vector) encoding a B7-H3-specific CAR, and then culturing the cells in the presence of IL-7 and IL-15.

[0274] Aspects and embodiments of the present disclosure relate specifically to EBV-specific immune cells. Methods for generating / expanding a population of EBV-specific immune cells are described, for example, in WO2013 / 088114A1, Lapteva and Vera, Stem Cells Int. (2011): 434392, Straathof et al., Blood (2005) 105(5): 1898-1904, WO2017 / 202478A1, WO2018 / 052947A1, and WO2020 / 214479A1, all of which are incorporated herein by reference in their entirety. The methods typically include stimulating virus / viral antigen-specific immune cells by contacting a population of immune cells with APCs presenting peptides corresponding to EBV antigens or viral antigens.

[0275] The present disclosure also provides cells, and populations thereof, obtained or obtainable by the methods described herein. composition The present disclosure also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[0276] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may include a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0277] The compositions of the present disclosure may be formulated with one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants, or the like. The formulation may contain an agent (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), a lubricant (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), a binder (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), a stabilizer, a solubilizer, a surfactant (e.g., a wetting agent), a masking agent, or a colorant (e.g., titanium dioxide).

[0278] The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of a subject of interest (e.g., a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of a composition according to the present disclosure must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings or sweeteners can be found in standard pharmaceutical textbooks, for example, Remington's "The Science and Practice of Pharmacy" (A. Adejare, ed.), 23rd Edition (2020), Academic Press.

[0279] The composition may be formulated for local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration. In some embodiments, the pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or by ingestion.

[0280] Suitable formulations may include the relevant items in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid form, such as gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to a selected region of the human or animal body.

[0281] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel, a tissue / organ of interest, or a tumor. The present disclosure also provides methods for producing a pharmaceutically useful composition, which may include one or more steps selected from: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein; and / or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0282] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., cancer), comprising the step of formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein together with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent. Therapeutic and prophylactic uses The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.

[0283] The present disclosure provides an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein for use in a method of medical therapy or prevention. Also provided are an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided are methods of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein.

[0284] The method may be effective in reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. The method may be effective in preventing the progression of a disease / condition, for example, preventing the worsening of a disease / condition or slowing the rate of its onset. In some embodiments, the method may result in an improvement in a disease / condition, for example, a reduction in the symptoms of a disease / condition or a reduction in some other correlates of the severity / activity of a disease / condition. In some embodiments, the method may prevent the disease / condition from progressing to a later stage (e.g., a chronic stage or metastasis).

[0285] It will be understood that the articles of the present disclosure can be used to treat / prevent any disease / condition in which a therapeutic or prophylactic benefit is expected to result from a reduction in the level / activity of B7-H3 or a reduction in the number or activity of cells containing / expressing B7-H3.

[0286] For example, the disease / condition may be one in which B7-H3 or cells containing / expressing B7-H3 are pathologically associated, e.g., a disease / condition in which increased levels / activity of B7-H3 or an increased number / proportion of cells containing / expressing B7-H3 are positively associated with the onset, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition. In some embodiments, increased levels / activity of B7-H3 or an increased number / proportion of cells containing / expressing B7-H3 may be a risk factor for the onset, development, or progression of the disease / condition.

[0287] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is one characterized by an increased level of B7-H3 expression or activity, e.g., compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is one characterized by an increased number / proportion / activity of cells expressing B7-H3, e.g., compared to the level / number / proportion / activity in the absence of the disease / condition (e.g., in a healthy subject or in an equivalent non-diseased tissue). If the disease / condition is cancer, the level of B7-H3 expression or activity may be greater than the level of B7-H3 expression or activity in an equivalent non-cancerous cell / non-tumor tissue. The cancer / its cells may contain one or more mutations that result in upregulation of B7-H3 expression or activity (e.g., compared to an equivalent non-cancerous cell / non-tumor tissue).

[0288] Treatment with the methods of the present disclosure can achieve one or more of the following in a subject (compared to an equivalent untreated subject, or a subject treated with an appropriate control): a reduction in the level of B7-H3; a reduction in the activity of B7-H3; and / or a reduction in the number / proportion of cells containing / expressing B7-H3.

[0289] In aspects and embodiments according to the present disclosure, cells (particularly immune cells, more particularly T cells) comprising / expressing a CAR according to the present disclosure are provided for therapeutic and prophylactic use. It will be understood that the methods generally include administering to a subject a population of immune cells expressing a CAR according to the present disclosure. In some embodiments, immune cells expressing a CAR according to the present disclosure may be administered in the form of a pharmaceutical composition comprising such cells.

[0290] In particular, the use of immune cells expressing a CAR according to the present disclosure in methods for treating / preventing diseases / conditions by adoptive cell transfer (ACT) is envisioned. Adoptive cell transfer generally refers to the process of obtaining cells (e.g., immune cells) from a subject, typically by taking a blood sample and isolating the cells therefrom. The cells are then typically modified and / or expanded, and then administered to either the same subject (in the case of adoptive transfer of autologous / autologous cells) or a different subject (in the case of adoptive transfer of allogeneic cells). The treatment typically aims to provide a population of cells with certain desired characteristics to the subject, or to increase the frequency of such cells with such characteristics in the subject. Adoptive transfer may be performed with the aim of introducing a cell or a population of cells into the subject and / or increasing the frequency of a cell or a population of cells in the subject.

[0291] Adoptive transfer of immune cells is described, for example, in Kalos and June (2013), Immunity 39(1):49-60, and Davis et al. (2015), Cancer J. 21(6):486-491, both of which are incorporated by reference in their entireties. One of skill in the art can determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure by referring, for example, to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, which is incorporated by reference in its entirety.

[0292] Immune cells expressing a CAR according to the present disclosure can be employed in the treatment / prevention of diseases / conditions by allogeneic or autologous transplantation. "Allogeneic transplantation," as used herein, refers to the transplantation of cells, tissues, or organs into a recipient subject that are genetically non-identical to the recipient subject. The cells, tissues, or organs may be of or derived from the cells, tissues, or organs of a donor subject that is genetically non-identical to the recipient subject. Allogeneic transplantation differs from autologous transplantation in that it refers to the transplantation of cells, tissues, or organs that are / are from a donor subject that is genetically identical to the recipient subject (i.e., autologous material). It will be understood that adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation, and adoptive transfer of autologous immune cells is a form of autologous transplantation.

[0293] The present disclosure provides methods that include administering to a subject immune cells that contain / express a CAR according to the disclosure, or immune cells that contain / express a nucleic acid encoding a CAR according to the disclosure.

[0294] In some embodiments, the method comprises modifying an immune cell to contain / express a CAR according to the present disclosure. In some embodiments, the method comprises modifying a virus-specific immune cell to contain / express a nucleic acid encoding a CAR according to the present disclosure.

[0295] In some embodiments, the method comprises: (a) modifying an immune cell to express or contain a CAR according to the present disclosure, or to express or contain a nucleic acid encoding a CAR according to the present disclosure, and (b) administering to the subject virus-specific immune cells that have been modified to express or contain a CAR according to the disclosure, or that have been modified to express or contain a nucleic acid encoding a CAR according to the disclosure. Includes:

[0296] In some embodiments, the method comprises: (a) isolating or obtaining immune cells; (b) modifying an immune cell to express or contain a CAR according to the disclosure, or to express or contain a nucleic acid encoding a CAR according to the disclosure, and (c) administering to the subject immune cells that have been modified to express or contain a CAR according to the disclosure, or that have been modified to express or contain a nucleic acid encoding a CAR according to the disclosure. Includes:

[0297] In some embodiments, the method comprises: (a) isolating immune cells (e.g., PBMCs) from a subject; (b) generating / expanding a population of virus-specific immune cells; (c) modifying virus-specific immune cells to express or contain a CAR according to the present disclosure, or to express or contain a nucleic acid encoding a CAR according to the present disclosure; and (d) administering to the subject virus-specific immune cells that have been modified to express or contain a CAR according to the disclosure, or that have been modified to express or contain a nucleic acid encoding a CAR according to the disclosure. Includes:

[0298] In some embodiments, the method includes administering to a subject EBV-specific immune cells that have been modified to express or contain a B7-H3-specific CAR according to the present disclosure, or that have been modified to express or contain a nucleic acid encoding a B7-H3-specific CAR according to the present disclosure.

[0299] In some embodiments, the subject from which the immune cells (e.g., PBMCs) are isolated is the same subject to which the cells are administered (i.e., adoptive transfer can be transfer of autologous / autologous cells). In some embodiments, the subject from which the immune cells (e.g., PBMCs) are isolated is a different subject from the subject to which the cells are administered (i.e., adoptive transfer can be transfer of allogeneic cells).

[0300] In some embodiments, the method may include one or more of the following: obtaining a blood sample from the subject; isolating immune cells (e.g., PBMCs) from a blood sample obtained from the subject; generating / expanding a population of immune cells; Culturing immune cells in in vitro or ex vivo cell culture; modifying immune cells to express or contain a CAR according to the disclosure, or to express or contain a nucleic acid encoding a CAR according to the disclosure (e.g., by transduction with a viral vector encoding such a CAR or containing such a nucleic acid); culturing immune cells that express / comprise a CAR according to the present disclosure or that express / comprise a nucleic acid encoding a CAR according to the present disclosure in an in vitro or ex vivo cell culture; collecting / isolating immune cells that express / comprise a CAR according to the present disclosure or that express / comprise a nucleic acid encoding a CAR according to the present disclosure; formulating the immune cells expressing / comprising a CAR according to the disclosure, or a nucleic acid encoding a CAR according to the disclosure, into a pharmaceutical composition, e.g., by mixing the cells with a pharmaceutically acceptable adjuvant, diluent, or carrier; Administering immune cells that express / comprise a CAR according to the disclosure, or that express / comprise a nucleic acid encoding a CAR according to the disclosure, or a pharmaceutical composition comprising such cells to the subject.

[0301] In some embodiments, the method may additionally include treating a cell or subject to induce / enhance expression of a CAR and / or to induce / enhance proliferation or survival of virus-specific immune cells that contain / express a CAR.

[0302] cancer In some embodiments, the disease to be treated / prevented in accordance with the present disclosure is cancer. B7-H3 expression and B7-H3-mediated signaling are involved in the pathogenesis of various cancers. B7-H3 promotes cancer cell migration and invasion, and therefore metastasis. B7-H3 expression and signaling, as well as its role in disease, are reviewed, for example, in Wu-Tong Zhou and Wei-Lin Jin, Front. Immunol. (2021) 12:701006; Dong et al., Front Oncol. (2018) 8:264; and Yang et al., Int J Biol Sci. (2020) 16(11):1767-1773, all of which are incorporated herein by reference in their entireties.

[0303] Cancer can also refer to any unwanted cell proliferation (or any disease that manifests itself through unwanted cell proliferation), neoplasm, or tumor. Cancer can be benign or malignant, primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. Cancer can be or include a solid cancer (e.g., a tumor), or can be a blood cancer. The cancer may be, for example, a cancer of the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, and / or tissue / cells derived from white blood cells.

[0304] The tumor may be a nervous system tumor or a non-nervous system tumor. Nervous system tumors may originate from either the central or peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-nervous system cancers / tumors may originate from any other non-nervous tissue, such as melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0305] In some embodiments, the cancer is a cancer in which B7-H3 is pathologically associated. That is, in some embodiments, the cancer is a cancer caused or exacerbated by B7-H3 expression, a cancer in which B7-H3 expression is a risk factor, and / or a cancer in which B7-H3 expression is positively associated with the onset, development, progression, severity, or metastasis of the cancer. The cancer may be characterized by B7-H3 expression; for example, the cancer may contain cells that express B7-H3. Such cancers may also be referred to as being positive for B7-H3. A cancer that is "positive" for B7-H3 may be a cancer that contains cells that express B7-H3 (e.g., on the cell surface). A cancer that is "positive" for B7-H3 may overexpress B7-H3.

[0306] Cancers in which B7-H3 is pathologically associated are described, for example, in Yang et al., Int J Biol Sci. 2020; 16(11): 1767-1773 and Dong et al., Front Oncol. (2018) 8: 264, and examples thereof include lung cancer (e.g., non-small cell lung cancer), skin cancer (e.g., cutaneous squamous cell carcinoma, melanoma), pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma), colorectal cancer (e.g., colorectal carcinoma), kidney cancer (e.g., renal clear cell carcinoma, Wilms' tumor), prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, breast cancer (e.g., triple-negative breast cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), and oral cancer. (e.g., oral squamous cell carcinoma), esophageal cancer, bladder cancer, urothelial cancer, brain cancer (e.g., medulloblastoma (e.g., ependymoma medulloblastoma), glioma (e.g., diffuse intrinsic pontine glioma, diffuse midline glioma), choroid plexus carcinoma, pineoblastoma), neuroblastoma, CNS tumors (e.g., primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor), brain stem glioma, sarcoma (e.g., rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), peritoneal cancer, desmoplastic small cell tumor, and mesothelioma.

[0307] In some embodiments, the cancer to be treated / prevented in accordance with the present disclosure is B7-H3 positive cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin cancer, squamous cell carcinoma of the skin, melanoma, pancreatic cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, colorectal cancer, colorectal cancer, colon cancer, colon cancer, kidney cancer, renal clear cell carcinoma, Wilms' tumor, prostate cancer, ovarian cancer, ovarian cancer, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma, breast cancer, triple negative breast cancer, head and neck cancer, The cancer is selected from the group consisting of: head and neck squamous cell carcinoma, oral cavity cancer, oral squamous cell carcinoma, laryngeal cancer, oropharyngeal cancer, oropharyngeal cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer, urothelial cancer, brain cancer, medulloblastoma, ependymoma, medulloblastoma, glioma, diffuse intrinsic pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineoblastoma, neuroblastoma, CNS tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brain stem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, peritoneal carcinoma, desmoplastic small cell tumor, and mesothelioma.

[0308] In some embodiments, the cancer to be treated / prevented in accordance with the present disclosure is a B7-H3 positive cancer, lung cancer, non-small cell lung cancer, skin cancer, squamous cell carcinoma of the skin, melanoma, pancreatic cancer, liver cancer, hepatocellular carcinoma, intrahepatic cholangiocarcinoma, colorectal cancer, colorectal cancer, kidney cancer, renal clear cell carcinoma, Wilms' tumor, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, breast cancer, triple negative breast cancer, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, The cancer is selected from the group consisting of: pulmonary squamous cell carcinoma, esophageal cancer, bladder cancer, urothelial carcinoma, brain cancer, medulloblastoma, ependymoma, medulloblastoma, glioma, diffuse intrinsic pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineoblastoma, neuroblastoma, CNS tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brain stem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, peritoneal carcinoma, desmoplastic small cell tumor, and mesothelioma.

[0309] In some embodiments, cancer may be a recurrent cancer. As used herein, "recurrent" cancer refers to a cancer that has responded to treatment (e.g., a first-line therapy for cancer), but then reappears / progresses, for example, after a period of remission. For example, a recurrent cancer may be a cancer whose growth / progression has been inhibited by treatment (e.g., a first-line therapy for cancer), and then grows / progresses.

[0310] In some embodiments, the cancer may be a refractory cancer. As used herein, "refractory" cancer refers to a cancer that has not responded to treatment (e.g., a first-line therapy for cancer). For example, a refractory cancer may be a cancer whose growth / progression has not been inhibited by treatment (e.g., a first-line therapy for cancer). In some embodiments, a refractory cancer may be a cancer in which a subject receiving treatment for cancer has not shown a partial or complete response to the treatment.

[0311] Treating cancer with the methods of the present disclosure achieves one or more of the following effects of treatment: reducing the number of cancer cells in a subject; reducing the size of a cancerous tumor / lesion in a subject; inhibiting (e.g., preventing or slowing) the growth of cancer cells in a subject; inhibiting (e.g., preventing or slowing) the growth of a cancerous tumor / lesion in a subject; inhibiting (e.g., preventing or slowing) the onset / progression of cancer (e.g., to a later stage or to metastasis); reducing the severity of cancer symptoms in a subject; prolonging the survival (e.g., progression-free survival or overall survival) of a subject; reducing the number or activity correlates of cancer cells in a subject; and / or reducing the cancer burden in a subject.

[0312] The subject can be evaluated according to the Revised Criteria for Response Assessment: The Lugano Classification (e.g., as described in Cheson et al., J Clin Oncol (2014) 32:3059-3068, incorporated by reference above) to determine their response to treatment. In some embodiments, treatment of a subject with the methods of the present disclosure achieves one of the following: a complete response, a partial response, or stable disease. Administration Administration of the articles of the present disclosure is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is an amount sufficient to provide a therapeutic or prophylactic benefit to the subject. The actual amount administered, and the rate and time course of administration, will depend on the nature and severity of the disease / condition and the particular article being administered. Prescribing treatment, such as determining dosage, is within the responsibility of a general practitioner or other physician, and typically takes into account the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and protocols described above can be found in Remington's "The Science and Practice of Pharmacy" (A. Adejare, ed.), 23rd Edition (2020), Academic Press.

[0313] Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, intranasal, or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral.

[0314] In some aspects and embodiments of the present disclosure, the article of the present disclosure is delivered in a targeted manner, i.e., the concentration of the relevant drug in a subject is increased in some parts of the body compared to other parts of the body. In some embodiments, the method includes intravenous, intraarterial, intramuscular, or subcutaneous administration, in which case the relevant article is formulated in the form of a targeted drug delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes, or micro-sized silica rods. Such systems may also include magnetic elements that direct the drug to the desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to those skilled in the art.

[0315] In some cases, the articles of the present disclosure are formulated for targeted delivery to specific cells, tissues, organs and / or tumors. Further interventions Administration can be alone or in combination with other treatments, either simultaneously or sequentially, depending on the disease / condition to be treated. The antigen-binding molecules, CARs, cells or compositions described herein and another prophylactic / therapeutic agent can be administered simultaneously or sequentially.

[0316] In some embodiments, the method includes additional therapeutic or preventive intervention, for example, for the treatment / prevention of cancer. In some embodiments, the therapeutic or preventive intervention is selected from chemotherapeutic agents, immunotherapy, radiation therapy, surgery, vaccination, and / or hormone therapy. In some embodiments, the therapeutic or preventive intervention includes leukapheresis. In some embodiments, the therapeutic or preventive intervention includes stem cell transplantation.

[0317] Simultaneous administration refers to the administration of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), cell, or composition and a therapeutic agent together, for example, as a pharmaceutical composition (combined preparation) containing both agents, or administered immediately after each other, optionally via the same administration route, for example, into the same artery, vein, or other blood vessel. Sequential administration refers to the administration of one of the antigen-binding molecule / composition or therapeutic agent, followed by separate administration of the other agent after a given time interval. The two agents do not necessarily have to be administered by the same route, although in some embodiments they are. The time interval may be any time interval.

[0318] In some embodiments, the cancer treatment further includes chemotherapy and / or radiation therapy. Chemotherapy and radiation therapy refer to the treatment of cancer with drugs or ionizing radiation (e.g., radiation therapy using X-rays or gamma rays), respectively. The drug may be a chemical entity, such as a small molecule drug, an antibiotic, a DNA intercalator, a protein inhibitor (e.g., a kinase inhibitor), or a biological substance, such as an antibody, an antibody fragment, an aptamer, a nucleic acid (e.g., DNA, RNA), a peptide, a polypeptide, or a protein. The drug may be formulated as a pharmaceutical composition or medicament. The formulation may include one or more drugs (e.g., one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0319] Chemotherapeutic agents may involve the administration of more than one drug, which may be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated.

[0320] The chemotherapeutic agent may be administered by one or more routes of administration, for example, parenterally, intravenously, orally, subcutaneously, intradermally, or intratumorally. Chemotherapeutic agents may be administered according to a treatment plan. A treatment plan may be a predetermined timetable, plan, scheme, or schedule for administering chemotherapy, which can be created by a doctor or medical professional and can be tailored to the patient who needs treatment. A treatment plan may indicate one or more of the following: the type of chemotherapy agent to be administered to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; and, if there is a treatment break, the number and nature of any treatment breaks. In the case of simultaneous therapy, a single treatment plan may be provided that indicates how each drug should be administered.

[0321] Chemotherapy drugs include abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (albumin-stabilized nanoparticle formulation of paclitaxel), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta ( Pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran (melphalan hydrochloride) for injection, Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), Arsenic trioxide, Arzera (ofatumumab), Asparaginase from Erwinia blackleg chrysanthemi), atezolizumab, Avastin (bevacizumab), avelumab, axiconib-ciloreucel, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodak (belinostat), belinamustine hydrochloride, BEP, Besponsa (inotuzumab ozolomide) gamycin), bevacizumab, bexarotene, Bexar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Bilincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan),Cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Calquence (acalabrutinib), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (topical fluorouracil), carboplatin, carboplatin-taxol, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide) ), CEM, ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), chloral (clofarabine), CMF, cobimetinib, Cometrik (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-AB V, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride Salt and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, DepoCyt (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab,Efudex (topical fluorouracil), Elitek (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, Epoch, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase derived from blackleg disease bacteria), Ethio (amifostine), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical), Fairston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), Filgra Stim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (topical fluorouracil), fluorouracil injection, topical fluorouracil, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, Folfox, Folotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil Lu9 (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Giotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemandiol (propranolol hydrochloride),Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nonavalent vaccine, recombinant HPV quadrivalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), Ifo Sufamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene laherparepvec), Inlyta (Axitinib), Inotuzumab ozogamicin, Interferon alfa-2b, Recombinant Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon alfa-2b), Iodine I131 Tositumomab and Tositumomab Mab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah ( Tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukelan (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levran (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposomal),Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marquibo (vincristine sulfate liposomal), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), metazolastone (Methazolamide) olastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), mutamycin (mitomycin C), Myleran (busulfan), Mirosar Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (albumin-stabilized nanoparticle formulation of paclitaxel), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexa Bar (sorafenib tosylate), Nilandrone (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal),Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, albumin-stabilized nanoparticle formulation of paclitaxel, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate dihydrate, Tritium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst ( pomalidomide), ponatinib hydrochloride, Portraza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human Human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, Relistol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycera (rituximab and human-derived hyaluronidase), rituximab, rituximab and human-derived hyaluronan Nidase, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparibe camsylate), rucaparibe camsylate, ruxolitinib phosphate, Rydapt (midostaurin), Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc), Steritalc (talc),Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxine mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (venom) Xarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (topical fluorouracil), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I-131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, tra Bectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, valrubicin, Valstar (valrubicin), vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib) vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate),Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xelori, Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicabtagene cilolucel), Yondelis (trabectedin), Zaltrap (Z iv-aflibercept), Zarxio (filgrastim), Zejula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zynecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0322] In some embodiments, treatment may include administration of corticosteroids, such as dexamethasone and / or prednisone. In some embodiments, the subject is administered lymphodepleting chemotherapy prior to administration of immune cells expressing / comprising a CAR described herein (or expressing / comprising a nucleic acid encoding such a CAR).

[0323] That is, in some embodiments, a method of treating / preventing a disease / condition in accordance with the present disclosure comprises (i) administering lymphodepleting chemotherapy to a subject, and (ii) thereafter administering immune cells that express / comprise a CAR according to the present disclosure or that express / comprise a nucleic acid encoding a CAR according to the present disclosure.

[0324] "Lymphodepleting chemotherapy," as used herein, refers to treatment with a chemotherapeutic agent that results in the depletion of lymphocytes (e.g., T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors) in the subject to whom the treatment is administered. "Lymphodepleting chemotherapeutic agent" refers to a chemotherapeutic agent that results in lymphocyte depletion.

[0325] Lymphodepleting chemotherapy and its use in methods of treatment by adoptive cell transfer are described, for example, in Klebanoff et al., Trends Immunol. (2005) 26(2):111-7 and Muranski et al., Nat Clin Pract Oncol. (2006)(12):668-81, both of which are incorporated herein by reference in their entireties. The goal of lymphodepleting chemotherapy is to deplete the recipient subject's endogenous lymphocyte populations.

[0326] In the context of disease treatment by adoptive transfer of immune cells, lymphodepleting chemotherapy is typically administered before adoptive cell transfer to prepare recipient subject to receive adoptively transferred cells.Lymphodepleting chemotherapy is thought to promote the persistence and activity of adoptively transferred cells by, for example, creating a permissive environment through the elimination of cells that express immunosuppressive cytokines, and creating the "lymphocyte space" that is necessary for the expansion and activity of adoptively transferred lymphoid cells.

[0327] Chemotherapeutic agents commonly used in lymphodepleting chemotherapy include, for example, fludarabine, cyclophosphamide, bedamustine, and pentostatin.

[0328] Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), cell, or composition may be provided, and one or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0329] The multiple doses may be separated by predetermined time intervals, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. As an example, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0330] According to various aspects of the present disclosure, methods of treating and / or preventing a disease / condition may include one or more of the following: reducing the number / proportion of cells expressing B7-H3; inhibiting tumor growth (e.g., of a B7-H3+ tumor); reducing metastasis of cancer (e.g., a B7-H3+ cancer); or prolonging survival of a subject with cancer (e.g., a B7-H3+ cancer). Methods of detection The present disclosure also provides articles of the present disclosure for use in methods for detecting, localizing, or imaging B7-H3, or cells expressing B7-H3.

[0331] The antigen-binding molecules described herein can be used in methods comprising detecting binding of the antigen-binding molecule to B7-H3. Such methods may include detecting a bound complex of the antigen-binding molecule and B7-H3. It will be understood that the B7-H3 may be B7-H3 expressed by a cell, for example, in or on the surface of a B7-H3-expressing cell.

[0332] Thus, methods are provided that include contacting a sample containing or suspected of containing B7-H3 and detecting the formation of a complex between an antigen-binding molecule and B7-H3. Also provided are methods that include contacting a sample containing or suspected of containing cells that express B7-H3 and detecting the formation of a complex between the antigen-binding molecule and the cells that express B7-H3.

[0333] Suitable method formats, including sandwich assays, immunoassays such as ELISA, are well known in the art. The method may include labeling the antigen-binding molecule, or the target, or both, with a detectable moiety as described herein, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label, a radiolabel, a chemical label, a nucleic acid label, or an enzyme label. Detection techniques are well known to those skilled in the art and can be selected to be compatible with the labeling agent.

[0334] Methods that include detecting B7-H3 or cells that express B7-H3 include methods for diagnosing / predicting the diseases / conditions described herein. This type of method may be performed on a patient sample in vitro, or may be performed after processing of the patient sample. Once the sample is collected, patient participation is not required for the in vitro method to be performed, and therefore the method may not be performed on a human or animal body. In some embodiments, the method is performed in vivo.

[0335] Such methods may include, for example, detecting or quantifying B7-H3 and / or cells expressing B7-H3 in a patient sample. If the method includes quantifying a related factor, the method may further include comparing the determined amount with a standard or reference value as part of a diagnostic or prognostic assessment. Other diagnostic / prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0336] Detection in a sample can be used for diagnostic purposes of a disease / condition (e.g., cancer), a predisposition to a disease / condition, or to provide a prognosis (predict a prognosis) with respect to a disease / condition, such as a disease / condition described herein. The diagnosis or prognosis may be with respect to an existing (previously diagnosed) disease / condition.

[0337] The sample can be taken from any tissue or body fluid. The sample can include or be derived from a predetermined amount of blood; a predetermined amount of serum from an individual's blood, which may include the liquid portion of blood obtained after fibrin clot and blood cell removal; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual. In some embodiments, the sample domain can be obtained from or derived from one or more tissues affected by a disease / condition (e.g., one or more tissues that exhibit symptoms of the disease or are involved in the pathogenesis of the disease / condition).

[0338] Subjects can be selected for diagnostic / prognostic evaluation based on the presence of symptoms indicative of a disease / condition described herein, or based on the subject being considered at risk for developing a disease / condition described herein.

[0339] The present disclosure also provides methods for selecting / stratifying subjects for treatment with a B7-H3 targeting agent. In some embodiments, a subject is selected for treatment / prevention by a method of the present disclosure, or identified as a subject who would benefit from such treatment / prevention, based, for example, on detection / quantification of B7-H3 or cells expressing B7-H3 in a sample obtained from the individual. subject The subject according to various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in humans or animals (veterinary use).

[0340] The subject to which the article of the present disclosure is to be administered (e.g., following a therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient.

[0341] The subject may have (e.g., been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / obtaining such a disease / condition. In embodiments according to the present disclosure, subjects may be selected for treatment by the present methods based on their characterization with respect to one or more markers for such a disease / condition.

[0342] In some embodiments, a subject can be selected for a therapeutic or prophylactic intervention as described herein based on the detection of cells / tissues that express B7-H3 or that overexpress B7-H3, e.g., in a sample obtained from the subject.

[0343] The subject may be an allogeneic subject for intervention according to the present disclosure. The subject to be treated / prevented according to the present disclosure may be genetically non-identical to the subject from which the CAR-expressing immune cells are derived. The subject to be treated / prevented according to the present disclosure may be HLA-mismatched to the subject from which the CAR-expressing immune cells are derived. The subject to be treated / prevented according to the present disclosure may be HLA-matched to the subject from which the CAR-expressing immune cells are derived.

[0344] The subject to whom cells are administered in accordance with the present disclosure may be allogeneic / non-autologous with respect to the source from which the cells are derived. The subject to whom cells are administered may be a different subject from the subject from which the cells were obtained / derived for the production of the cells to be administered. The subject to whom cells are administered may be genetically non-identical to the subject from which the cells were obtained / derived for the production of the cells to be administered.

[0345] The subject to whom the cells are administered may contain MHC / HLA genes encoding MHC / HLA molecules that are not identical to the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from which the cells were / are obtained for the production of the cells to be administered.The subject to whom the cells are administered may contain MHC / HLA genes encoding MHC / HLA molecules that are identical to the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from which the cells were / are obtained for the production of the cells to be administered.

[0346] In some embodiments, the subject to whom the cells are administered is HLA-matched to the subject from whom the cells were obtained / derived for the production of the cells to be administered. In some embodiments, the subject to whom the cells are administered is nearly or perfectly HLA-matched to the subject from whom the cells were obtained / derived for the production of the cells to be administered.

[0347] In some embodiments, the subject is ≧4 / 8 (i.e., 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8) matched across HLA-A, -B, -C, and -DRB1. In some embodiments, the subject is ≧5 / 10 (i.e., 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10) matched across HLA-A, -B, -C, -DRB1, and -DQB1. In some embodiments, the subject is ≧6 / 12 (i.e., 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, 11 / 12, or 12 / 12) matched across HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1. In some embodiments, the subject is 8 / 8 matched across HLA-A, -B, -C, and -DRB1. In some embodiments, the subject is 10 / 10 matched across HLA-A, -B, -C, -DRB1 and -DQB1, hi some embodiments, the subject is 12 / 12 matched across HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1. kit In some aspects of the present disclosure, a kit of parts is provided. In some embodiments, the kit may include at least one container containing a predetermined amount of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), cell, or composition described herein.

[0348] In some embodiments, the kit may include materials for producing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein.

[0349] The kit can provide an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cells or composition along with instructions for administration to a patient to treat a specified disease / condition.

[0350] In some embodiments, the kit may further include at least one container with a predetermined amount of another therapeutic agent (e.g., as described herein). In such embodiments, the kit may also include a second medicament or pharmaceutical composition, such that the two medicaments or pharmaceutical compositions can be administered simultaneously or separately to provide a combined treatment for a particular disease or condition.

[0351] Kits according to the present disclosure may include instructions, for example in the form of an instruction booklet or leaflet, which may include protocols for carrying out any one or more of the methods described herein. Sequence identity "Sequence identity," as used herein, refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780). When using such software, default parameters are preferably used, for example, with respect to gap penalties and extension penalties. array

[0352] [Table 3-1]

[0353] [Table 3-2]

[0354] [Table 3-3]

[0355] [Table 3-4]

[0356] [Table 3-5]

[0357] [Table 3-6]

[0358] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, while expressed in their specific form, or in terms of means for performing a disclosed function, or methods or processes for obtaining a disclosed result, can, where appropriate, be utilized separately or in any combination of such features to realize the invention in diverse forms thereof.

[0359] While the present invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will be apparent to those skilled in the art in light of this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments can be made without departing from the spirit and scope of the invention.

[0360] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of improving the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0361] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the claims set out below, and unless the context requires otherwise, the terms "comprise" and "include," and variations such as "comprises," "comprising," and "including," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0362] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" with respect to numerical values is optional and means, for example, + / - 10%.

[0363] The present disclosure includes combinations of the described aspects and preferred features except where such combinations are clearly unacceptable or explicitly avoided. When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0364] The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed with cultured cells, whereas the term "in vivo" is intended to encompass procedures with / on intact multicellular organisms.

[0365] Aspects and embodiments of the present disclosure will now be illustrated by way of example with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this document are incorporated herein by reference. [Brief explanation of the drawings]

[0366] [Figure 1A] 1A-1D (Figures A1A-A1D). Binding of VHH P2A5 to B7-H3. (A) Flow cytometry analysis of the binding of purified P2A5 VHH to HepG2 cells expressing B7-H3. (B)-(E) Sensorgrams and binding kinetic parameters determined by multi-cycle kinetic analysis of the binding of P2A5 VHH to (B) human B7-H3 isoform 1 (also known as 4Ig-B7-H3), (C) human B7-H3 isoform 2 (also known as 2Ig-B7-H3), and (D) mouse B7-H3, as determined by surface plasmon resonance analysis. [Figure 1B] 1A-1D (Figures A1A-A1D). Binding of VHH P2A5 to B7-H3. (A) Flow cytometry analysis of the binding of purified P2A5 VHH to HepG2 cells expressing B7-H3. (B)-(E) Sensorgrams and binding kinetic parameters determined by multi-cycle kinetic analysis of the binding of P2A5 VHH to (B) human B7-H3 isoform 1 (also known as 4Ig-B7-H3), (C) human B7-H3 isoform 2 (also known as 2Ig-B7-H3), and (D) mouse B7-H3, as determined by surface plasmon resonance analysis. [Figure 1C]1A-1D (Figures A1A-A1D). Binding of VHH P2A5 to B7-H3. (A) Flow cytometry analysis of the binding of purified P2A5 VHH to HepG2 cells expressing B7-H3. (B)-(E) Sensorgrams and binding kinetic parameters determined by multi-cycle kinetic analysis of the binding of P2A5 VHH to (B) human B7-H3 isoform 1 (also known as 4Ig-B7-H3), (C) human B7-H3 isoform 2 (also known as 2Ig-B7-H3), and (D) mouse B7-H3, as determined by surface plasmon resonance analysis. [Figure 1D] 1A-1D (Figures A1A-A1D). Binding of VHH P2A5 to B7-H3. (A) Flow cytometry analysis of the binding of purified P2A5 VHH to HepG2 cells expressing B7-H3. (B)-(E) Sensorgrams and binding kinetic parameters determined by multi-cycle kinetic analysis of the binding of P2A5 VHH to (B) human B7-H3 isoform 1 (also known as 4Ig-B7-H3), (C) human B7-H3 isoform 2 (also known as 2Ig-B7-H3), and (D) mouse B7-H3, as determined by surface plasmon resonance analysis. [Figure 2] Figures 2A-2D (Figures A2A-A2D) show the cross-reactivity of P2A5 with mouse and human B7-H3 molecules expressed by cells of mouse and human origin. Mouse CT26 cells engineered to knock out endogenous B7-H3 and human MKN7 cells transfected to express mouse B7-H3 (A and B) and human 4Ig-B7-H3 (C and D) were stained with positive control anti-mouse or anti-human B7H3 antibodies (top row), P2A5 as a VHH-Fc fusion molecule (middle row), or an isotype control (bottom row). [Figure 3A]Figure 3 (Figure A3). Generation and functionality of B7-H3 CAR T cells. (A) Schematic of the B7-H3 CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of the B7-H3 CAR on activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7-H3 CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 3B] Figure 3 (Figure A3). Generation and functionality of B7-H3 CAR T cells. (A) Schematic of the B7-H3 CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of the B7-H3 CAR on activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7-H3 CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 3C]Figure 3 (Figure A3). Generation and functionality of B7-H3 CAR T cells. (A) Schematic of the B7-H3 CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of the B7-H3 CAR on activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7-H3 CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 3D] Figure 3 (Figure A3). Generation and functionality of B7-H3 CAR T cells. (A) Schematic of the B7-H3 CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of the B7-H3 CAR on activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7-H3 CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 3E]Figure 3 (Figure A3). Generation and functionality of B7-H3 CAR T cells. (A) Schematic of the B7-H3 CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of the B7-H3 CAR on activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7-H3 CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 4A] Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 4B]Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 4C] Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 4D]Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 4E] Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 4F]Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 4G] Figure 4 (Figure A4). Generation and characterization of allogeneic B7-H3 CAR EBVST. (A) Schematic of B7-H3 CAR EBVST production. (B) Fold expansion of EBVST after transduction with mock (UT) or P2A5 CAR. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells at 4 and 11 days post-transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Marker expression data of UT or CAR EBVST final products. Data shown are from three independent donors. [Figure 5A]Figure 5 (Figure A5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7-H3 CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. [Figure 5B] Figure 5 (Figure A5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7-H3 CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. [Figure 5C] Figure 5 (Figure A5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7-H3 CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. [Figure 5D]Figure 5 (Figure A5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7-H3 CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. [Figure 6A] Figure 6 (Fig. A6). Sequential killing of B7-H3 CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) CAR-T cell expansion after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 6B] Figure 6 (Fig. A6). Sequential killing of B7-H3 CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) CAR-T cell expansion after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 6C] Figure 6 (Fig. A6). Sequential killing of B7-H3 CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) CAR-T cell expansion after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 6D]Figure 6 (Fig. A6). Sequential killing of B7-H3 CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) CAR-T cell expansion after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 7A] Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7B]Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7C] Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7D]Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7E] Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7F]Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7G] Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7H]Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7I] Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 7J]Figure 7 (Figure A7). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing colorectal cancer. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 8A] Figure 8 (Figure A8). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing gastric cancer. (A) Experimental scheme for the N-87 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint N-87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 8B] Figure 8 (Figure A8). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing gastric cancer. (A) Experimental scheme for the N-87 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint N-87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 8C]Figure 8 (Figure A8). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing gastric cancer. (A) Experimental scheme for the N-87 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint N-87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 8D] Figure 8 (Figure A8). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing gastric cancer. (A) Experimental scheme for the N-87 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint N-87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 8E] Figure 8 (Figure A8). Durability and efficacy of B7-H3 CAR EBVST in vivo in B7-H3-expressing gastric cancer. (A) Experimental scheme for the N-87 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint N-87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 9A]Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 9B] Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 9C]Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 9D] Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 9E]Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 9F] Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 9G]Figure 9 (Figure B1). Identification and characterization of lead B7H3 VHHs from an immunized llama library by phage display. (A) B7H3 binding screening ELISA of eluted phage from round 2 (top) and round 3 (bottom) of biopanning. (B) Flow cytometry analysis of binding of purified lead VHH candidates to B7H3-expressing HepG2 cells. (C) Surface plasmon resonance screening of lead VHH candidates. Multi-cycle kinetic analysis of binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Measured binding kinetic parameters of P2A5 binding to the 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3. [Figure 10A] Figure 10 (Figure B2) shows the cross-reactivity of P2A5 to mouse and human B7H3 molecules expressed by cells of mouse and human origin. Mouse CT26 and human MKN7 cells, in which endogenous B7H3 was knocked out, were transfected to express mouse B7H3 molecules (A and C) and human 4Ig B7H3 molecules (B and D), and stained with positive control anti-mouse or anti-human B7H3 antibodies (top), P2A5 as a VHH-Fc fusion molecule (middle), or an isotype control (bottom). [Figure 10B] Figure 10 (Figure B2) shows the cross-reactivity of P2A5 to mouse and human B7H3 molecules expressed by cells of mouse and human origin. Mouse CT26 and human MKN7 cells, in which endogenous B7H3 was knocked out, were transfected to express mouse B7H3 molecules (A and C) and human 4Ig B7H3 molecules (B and D), and stained with positive control anti-mouse or anti-human B7H3 antibodies (top), P2A5 as a VHH-Fc fusion molecule (middle), or an isotype control (bottom). [Figure 10C]Figure 10 (Figure B2) shows the cross-reactivity of P2A5 to mouse and human B7H3 molecules expressed by cells of mouse and human origin. Mouse CT26 and human MKN7 cells, in which endogenous B7H3 was knocked out, were transfected to express mouse B7H3 molecules (A and C) and human 4Ig B7H3 molecules (B and D), and stained with positive control anti-mouse or anti-human B7H3 antibodies (top), P2A5 as a VHH-Fc fusion molecule (middle), or an isotype control (bottom). [Figure 10D] Figure 10 (Figure B2) shows the cross-reactivity of P2A5 to mouse and human B7H3 molecules expressed by cells of mouse and human origin. Mouse CT26 and human MKN7 cells, in which endogenous B7H3 was knocked out, were transfected to express mouse B7H3 molecules (A and C) and human 4Ig B7H3 molecules (B and D), and stained with positive control anti-mouse or anti-human B7H3 antibodies (top), P2A5 as a VHH-Fc fusion molecule (middle), or an isotype control (bottom). [Figure 11A] Figure 11 (Figure B3). Generation and functionality of B7H3.CAR T cells. (A) Schematic of B7H3.CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of B7H3.CAR in activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7H3.CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 11B]Figure 11 (Figure B3). Generation and functionality of B7H3.CAR T cells. (A) Schematic of B7H3.CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of B7H3.CAR in activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7H3.CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 11C] Figure 11 (Figure B3). Generation and functionality of B7H3.CAR T cells. (A) Schematic of B7H3.CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of B7H3.CAR in activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7H3.CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 11D]Figure 11 (Figure B3). Generation and functionality of B7H3.CAR T cells. (A) Schematic of B7H3.CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of B7H3.CAR in activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7H3.CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 11E] Figure 11 (Figure B3). Generation and functionality of B7H3.CAR T cells. (A) Schematic of B7H3.CAR, consisting of a B7-H3-targeting VHH, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signaling domains. (B) Detection of B7H3.CAR in activated T cells after transduction. (C) Transduction efficiency and fold expansion of untransduced and B7H3.CAR T cells followed for 11 days after transduction. Data shown are the mean ± SD of two independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells was assessed at E:T ratios of 1:1 and 5:1 for two donors using the xCELLigence real-time cell analysis system. (E) Sequential cytolysis of THP-1 and CMK cells during multiple rounds of coculture for two donors. [Figure 12A]Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 12B] Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 12C]Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 12D] Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 12E]Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 12F] Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 12G]Figure 12 (Figure B4). Generation and characterization of allogeneic B7H3.CAR EBVST. (A) Schematic of B7H3.CAR EBVST production. (B) Fold expansion of EBVST after mock (UT) or P2A5 CAR transduction. (C) CAR+ percentage and (D) CD8 / CD4 ratio of cells 4 and 11 days after transduction. (E) B7-H3 expression among total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all three of the exhaustion markers PD-1, Tim-3, and LAG-3. (G) Percentage of cells expressing TNF-α and IFN-γ after overnight stimulation of cells with media, HIV, or EBV pepmix. (B-D) Data shown are from six independent donors. Each line represents data from each donor. (E-G) Data shown are from three independent donors. [Figure 13A] Figure 13 (Figure B5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7H3.CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. (E) Quantification of IFNγ levels in cell supernatants of untransduced or B7H3.CAR EBVST after co-incubation with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVST expressing full-length or non-truncated B7H3, with or without CAR, against B7-H3+MKN-45 tumor cells. [Figure 13B]Figure 13 (Figure B5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7H3.CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. (E) Quantification of IFNγ levels in cell supernatants of untransduced or B7H3.CAR EBVST after co-incubation with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVST expressing full-length or non-truncated B7H3, with or without CAR, against B7-H3+MKN-45 tumor cells. [Figure 13C] Figure 13 (Figure B5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7H3.CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. (E) Quantification of IFNγ levels in cell supernatants of untransduced or B7H3.CAR EBVST after co-incubation with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVST expressing full-length or non-truncated B7H3, with or without CAR, against B7-H3+MKN-45 tumor cells. [Figure 13D]Figure 13 (Figure B5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7H3.CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. (E) Quantification of IFNγ levels in cell supernatants of untransduced or B7H3.CAR EBVST after co-incubation with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVST expressing full-length or non-truncated B7H3, with or without CAR, against B7-H3+MKN-45 tumor cells. [Figure 13E] Figure 13 (Figure B5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7H3.CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. (E) Quantification of IFNγ levels in cell supernatants of untransduced or B7H3.CAR EBVST after co-incubation with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVST expressing full-length or non-truncated B7H3, with or without CAR, against B7-H3+MKN-45 tumor cells. [Figure 13F]Figure 13 (Figure B5). Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Kinetics of cytolysis of B7H3.CAR EBVST against (A) colorectal cancer cell lines DLD-1, HT29, SW480; (B) gastric cancer cell lines NCI-N87, MKN7, MKN45; (C) breast cancer cell lines MDA-MB-231, MDA-MB-468; (D) lung cancer cell lines A549, H1299, H23, and H596, and their B7-H3 knockout counterparts, at an effector:target ratio of 1:1. (E) Quantification of IFNγ levels in cell supernatants of untransduced or B7H3.CAR EBVST after co-incubation with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVST expressing full-length or non-truncated B7H3, with or without CAR, against B7-H3+MKN-45 tumor cells. [Figure 14A] Figure 14 (Fig. B6). Sequential killing of B7H3.CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) expansion of CAR-T cells after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 14B] Figure 14 (Fig. B6). Sequential killing of B7H3.CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) expansion of CAR-T cells after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 14C]Figure 14 (Fig. B6). Sequential killing of B7H3.CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) expansion of CAR-T cells after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 14D] Figure 14 (Fig. B6). Sequential killing of B7H3.CAR EBVST. (A) Sequential cytotoxicity of EBVST after each successive encounter with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each successive encounter with THP-1. (C) Kinetics of EBVST cytolysis and (D) expansion of CAR-T cells after each successive encounter with target cell lines DLD-1, HT29, and NCI-N87 at an effector:target ratio of 1:2. [Figure 15A] Figure 15 (Figure B7). In vitro targeting of allogeneic MDSCs by B7H3.CAR EBVST. (A) Flow cytometry histograms showing IL-10, TGF-β, iNOS, and B7-H3 expression in MDSCs generated from two independent healthy donors. (B) Cytotoxicity of CD30.CAR EBVST against the KM-H2 cell line in the presence or absence of MDSCs at MDSC:CAR:KM-H2 ratios of 4:1:1 or 10:1:1. (C) Cytotoxicity of untransduced (UT) or B7H3.CAR EBVST (CAR) against allogeneic MDSCs. Data compiled from co-cultures of effector EBVST generated from two donors and target MDSCs generated from two different donors. (D) Proliferation index of untransduced and B7H3.CAR EBVST after anti-CD3 / CD28 stimulation in the presence of allogeneic MDSCs. Each data point represents proliferation from each effector:target donor pair. The proliferation index was calculated by normalizing the percentage of proliferated cells to that of the control condition without MDSCs. [Figure 15B]Figure 15 (Figure B7). In vitro targeting of allogeneic MDSCs by B7H3.CAR EBVST. (A) Flow cytometry histograms showing IL-10, TGF-β, iNOS, and B7-H3 expression in MDSCs generated from two independent healthy donors. (B) Cytotoxicity of CD30.CAR EBVST against the KM-H2 cell line in the presence or absence of MDSCs at MDSC:CAR:KM-H2 ratios of 4:1:1 or 10:1:1. (C) Cytotoxicity of untransduced (UT) or B7H3.CAR EBVST (CAR) against allogeneic MDSCs. Data compiled from co-cultures of effector EBVST generated from two donors and target MDSCs generated from two different donors. (D) Proliferation index of untransduced and B7H3.CAR EBVST after anti-CD3 / CD28 stimulation in the presence of allogeneic MDSCs. Each data point represents proliferation from each effector:target donor pair. The proliferation index was calculated by normalizing the percentage of proliferated cells to that of the control condition without MDSCs. [Figure 15C]Figure 15 (Figure B7). In vitro targeting of allogeneic MDSCs by B7H3.CAR EBVST. (A) Flow cytometry histograms showing IL-10, TGF-β, iNOS, and B7-H3 expression in MDSCs generated from two independent healthy donors. (B) Cytotoxicity of CD30.CAR EBVST against the KM-H2 cell line in the presence or absence of MDSCs at MDSC:CAR:KM-H2 ratios of 4:1:1 or 10:1:1. (C) Cytotoxicity of untransduced (UT) or B7H3.CAR EBVST (CAR) against allogeneic MDSCs. Data compiled from co-cultures of effector EBVST generated from two donors and target MDSCs generated from two different donors. (D) Proliferation index of untransduced and B7H3.CAR EBVST after anti-CD3 / CD28 stimulation in the presence of allogeneic MDSCs. Each data point represents proliferation from each effector:target donor pair. The proliferation index was calculated by normalizing the percentage of proliferated cells to that of the control condition without MDSCs. [Figure 15D]Figure 15 (Figure B7). In vitro targeting of allogeneic MDSCs by B7H3.CAR EBVST. (A) Flow cytometry histograms showing IL-10, TGF-β, iNOS, and B7-H3 expression in MDSCs generated from two independent healthy donors. (B) Cytotoxicity of CD30.CAR EBVST against the KM-H2 cell line in the presence or absence of MDSCs at MDSC:CAR:KM-H2 ratios of 4:1:1 or 10:1:1. (C) Cytotoxicity of untransduced (UT) or B7H3.CAR EBVST (CAR) against allogeneic MDSCs. Data compiled from co-cultures of effector EBVST generated from two donors and target MDSCs generated from two different donors. (D) Proliferation index of untransduced and B7H3.CAR EBVST after anti-CD3 / CD28 stimulation in the presence of allogeneic MDSCs. Each data point represents proliferation from each effector:target donor pair. The proliferation index was calculated by normalizing the percentage of proliferated cells to that of the control condition without MDSCs. [Figure 16A] Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16B]Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16C] Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16D]Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16E] Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16F]Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16G] Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16H]Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16I] Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 16J]Figure 16 (Figure B8). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing colorectal cancer in vivo. (A) Experimental scheme for the HT-29 model. (B) Body weight monitoring twice weekly. (C) Fold change in HT-29 tumor volume and growth after treatment. (D) Endpoint HT-29 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. (F) Experimental scheme for the SW-480 model. (G) Body weight monitoring twice weekly. (H) Fold change in SW-480 tumor volume and growth after treatment. (I) Endpoint SW-480 tumor count and B7-H3 expression quantified by flow cytometry. (J) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 17A] Figure 17 (Figure B9). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing gastric cancer in vivo. (A) Experimental scheme for the NCI-N87 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-N87 tumor volume and growth after treatment. (D) Endpoint NCI-N87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 17B] Figure 17 (Figure B9). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing gastric cancer in vivo. (A) Experimental scheme for the NCI-N87 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-N87 tumor volume and growth after treatment. (D) Endpoint NCI-N87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 17C] Figure 17 (Figure B9). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing gastric cancer in vivo. (A) Experimental scheme for the NCI-N87 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-N87 tumor volume and growth after treatment. (D) Endpoint NCI-N87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 17D] Figure 17 (Figure B9). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing gastric cancer in vivo. (A) Experimental scheme for the NCI-N87 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-N87 tumor volume and growth after treatment. (D) Endpoint NCI-N87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 17E] Figure 17 (Figure B9). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing gastric cancer in vivo. (A) Experimental scheme for the NCI-N87 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-N87 tumor volume and growth after treatment. (D) Endpoint NCI-N87 tumor count and B7-H3 expression quantified by flow cytometry. (E) Endpoint T cell count in blood, liver, lung, spleen, and tumor quantified by flow cytometry. [Figure 18A]Figure 18 (Figure B10). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing lung cancer in vivo. (A) Experimental scheme for the NCI-H1299 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-H1299 tumor volume and growth after treatment. (D) Endpoint NCI-H1299 tumor count and B7-H3 expression quantified by flow cytometry. [Figure 18B] Figure 18 (Figure B10). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing lung cancer in vivo. (A) Experimental scheme for the NCI-H1299 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-H1299 tumor volume and growth after treatment. (D) Endpoint NCI-H1299 tumor count and B7-H3 expression quantified by flow cytometry. [Figure 18C] Figure 18 (Figure B10). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing lung cancer in vivo. (A) Experimental scheme for the NCI-H1299 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-H1299 tumor volume and growth after treatment. (D) Endpoint NCI-H1299 tumor count and B7-H3 expression quantified by flow cytometry. [Figure 18D] Figure 18 (Figure B10). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing lung cancer in vivo. (A) Experimental scheme for the NCI-H1299 model. (B) Body weight monitoring twice weekly. (C) Fold change in NCI-H1299 tumor volume and growth after treatment. (D) Endpoint NCI-H1299 tumor count and B7-H3 expression quantified by flow cytometry. [Figure 19A]Figure 19 (Figure B11). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing triple-negative breast cancer in vivo. (A) Experimental scheme for the MDA-MB-468 model. (B) Body weight monitoring twice weekly. (C) Measurement of MDA-MB-468 tumor volume and fold change in proliferation after treatment; and tumor count at study endpoint quantified by flow cytometry. [Figure 19B] Figure 19 (Figure B11). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing triple-negative breast cancer in vivo. (A) Experimental scheme for the MDA-MB-468 model. (B) Body weight monitoring twice weekly. (C) Measurement of MDA-MB-468 tumor volume and fold change in proliferation after treatment; and tumor count at study endpoint quantified by flow cytometry. [Figure 19C] Figure 19 (Figure B11). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing triple-negative breast cancer in vivo. (A) Experimental scheme for the MDA-MB-468 model. (B) Body weight monitoring twice weekly. (C) Measurement of MDA-MB-468 tumor volume and fold change in proliferation after treatment; and tumor count at study endpoint quantified by flow cytometry. [Figure 19D] Figure 19 (Figure B11). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing triple-negative breast cancer in vivo. (A) Experimental scheme for the MDA-MB-468 model. (B) Body weight monitoring twice weekly. (C) Measurement of MDA-MB-468 tumor volume and fold change in proliferation after treatment; and tumor count at study endpoint quantified by flow cytometry. [Figure 20A] Figure 20 (Figure B12). (A and B) Staining of B7-H3 in breast cancer PDX by IHC. Brown represents positive B7-H3 staining. Blue represents cell nuclei. [Figure 20B]Figure 20 (Figure B12). (A and B) Staining of B7-H3 in breast cancer PDX by IHC. Brown represents positive B7-H3 staining. Blue represents cell nuclei. [Figure 21A] Figure 21 (Figure B13). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing breast cancer PDX in vivo. (A) Experimental scheme for PDX model. (B) Body weight monitoring twice weekly. (C) Measurement of PDX tumor volume and fold change in proliferation after treatment; and (D) Tumor count at study endpoint quantified by flow cytometry. [Figure 21B] Figure 21 (Figure B13). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing breast cancer PDX in vivo. (A) Experimental scheme for PDX model. (B) Body weight monitoring twice weekly. (C) Measurement of PDX tumor volume and fold change in proliferation after treatment; and (D) Tumor count at study endpoint quantified by flow cytometry. [Figure 21C] Figure 21 (Figure B13). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing breast cancer PDX in vivo. (A) Experimental scheme for PDX model. (B) Body weight monitoring twice weekly. (C) Measurement of PDX tumor volume and fold change in proliferation after treatment; and (D) Tumor count at study endpoint quantified by flow cytometry. [Figure 21D] Figure 21 (Figure B13). Durability and efficacy of B7H3.CAR EBVST against B7-H3-expressing breast cancer PDX in vivo. (A) Experimental scheme for PDX model. (B) Body weight monitoring twice weekly. (C) Measurement of PDX tumor volume and fold change in proliferation after treatment; and (D) Tumor count at study endpoint quantified by flow cytometry. [Figure 22A]Figure 22 (Figure B14). Stimulation and competition of soluble B7-H3 on B7H3.CAR EBVST. (A) Cell staining for IFNγ, TNFα, and CD25 in UT and B7H3.CAR EBVST after treatment with various concentrations of coated or soluble B7-H3 in the form of 2-Ig or 4-Ig. (B) Cell lysis of B7H3.CAR EBVST against NCI-N87 or NCI-H1299 in the presence of various concentrations of soluble B7-H3 in the form of 2-Ig or 4-Ig. [Figure 22B] Figure 22 (Figure B14). Stimulation and competition of soluble B7-H3 on B7H3.CAR EBVST. (A) Cell staining for IFNγ, TNFα, and CD25 in UT and B7H3.CAR EBVST after treatment with various concentrations of coated or soluble B7-H3 in the form of 2-Ig or 4-Ig. (B) Cell lysis of B7H3.CAR EBVST against NCI-N87 or NCI-H1299 in the presence of various concentrations of soluble B7-H3 in the form of 2-Ig or 4-Ig. [Figure 23A] Figure 23 (Fig. B15). Safety of B7H3.CAR EBVST on hematopoietic stem and progenitor cells (HSPCs). (A) Flow plot showing the B7-H3-expressing HSPC population after stimulation with Flt3L, TPO, and SCF for the indicated days. (B) Comparison of cell surface expression of B7-H3 on HSPCs and the cancer cell line NCI-N87 by flow cytometry. (C) Cytotoxicity of unstimulated and stimulated HSPCs after co-culture with B7H3.CAR EBVST. (D) Percentage of HSPC populations after co-culture with untransduced or B7H3.CAR EBVST. (E) Erythroid and myeloid developmental potential of HSPCs after co-culture with untransduced or B7H3.CAR EBVST. [Figure 23B]Figure 23 (Fig. B15). Safety of B7H3.CAR EBVST on hematopoietic stem and progenitor cells (HSPCs). (A) Flow plot showing the B7-H3-expressing HSPC population after stimulation with Flt3L, TPO, and SCF for the indicated days. (B) Comparison of cell surface expression of B7-H3 on HSPCs and the cancer cell line NCI-N87 by flow cytometry. (C) Cytotoxicity of unstimulated and stimulated HSPCs after co-culture with B7H3.CAR EBVST. (D) Percentage of HSPC populations after co-culture with untransduced or B7H3.CAR EBVST. (E) Erythroid and myeloid developmental potential of HSPCs after co-culture with untransduced or B7H3.CAR EBVST. [Figure 23C] Figure 23 (Fig. B15). Safety of B7H3.CAR EBVST on hematopoietic stem and progenitor cells (HSPCs). (A) Flow plot showing the B7-H3-expressing HSPC population after stimulation with Flt3L, TPO, and SCF for the indicated days. (B) Comparison of cell surface expression of B7-H3 on HSPCs and the cancer cell line NCI-N87 by flow cytometry. (C) Cytotoxicity of unstimulated and stimulated HSPCs after co-culture with B7H3.CAR EBVST. (D) Percentage of HSPC populations after co-culture with untransduced or B7H3.CAR EBVST. (E) Erythroid and myeloid developmental potential of HSPCs after co-culture with untransduced or B7H3.CAR EBVST. [Figure 23D]Figure 23 (Fig. B15). Safety of B7H3.CAR EBVST on hematopoietic stem and progenitor cells (HSPCs). (A) Flow plot showing the B7-H3-expressing HSPC population after stimulation with Flt3L, TPO, and SCF for the indicated days. (B) Comparison of cell surface expression of B7-H3 on HSPCs and the cancer cell line NCI-N87 by flow cytometry. (C) Cytotoxicity of unstimulated and stimulated HSPCs after co-culture with B7H3.CAR EBVST. (D) Percentage of HSPC populations after co-culture with untransduced or B7H3.CAR EBVST. (E) Erythroid and myeloid developmental potential of HSPCs after co-culture with untransduced or B7H3.CAR EBVST. [Figure 23E] Figure 23 (Fig. B15). Safety of B7H3.CAR EBVST on hematopoietic stem and progenitor cells (HSPCs). (A) Flow plot showing the B7-H3-expressing HSPC population after stimulation with Flt3L, TPO, and SCF for the indicated days. (B) Comparison of cell surface expression of B7-H3 on HSPCs and the cancer cell line NCI-N87 by flow cytometry. (C) Cytotoxicity of unstimulated and stimulated HSPCs after co-culture with B7H3.CAR EBVST. (D) Percentage of HSPC populations after co-culture with untransduced or B7H3.CAR EBVST. (E) Erythroid and myeloid developmental potential of HSPCs after co-culture with untransduced or B7H3.CAR EBVST. [Figure 24A]Figure 24 (Figure B16). Safety of B7H3.CAR EBVST on peripheral blood mononuclear cells (PBMCs). (A) Cell surface expression of B7-H3 on PBMC subsets after stimulation with inflammatory cytokines and the cancer cell line NCI-N87. (B) Fold change in cell count of PBMC subsets after 2 days of culturing cytokine-stimulated PBMCs with B7H3.CAR EBVST. Fold change is obtained by normalizing the cell count of PBMC subsets co-cultured with untransduced control EBVST. (C) Cell surface expression of B7-H3 on monocytes after co-culture with allogeneic EBVST. (D) Cytotoxicity of monocytes after co-culture with untransduced or B7H3.CAR EBVST. Data also expressed as fold change in cytotoxicity of B7H3.CAR EBVST relative to the untransduced control. [Figure 24B] Figure 24 (Figure B16). Safety of B7H3.CAR EBVST on peripheral blood mononuclear cells (PBMCs). (A) Cell surface expression of B7-H3 on PBMC subsets after stimulation with inflammatory cytokines and the cancer cell line NCI-N87. (B) Fold change in cell count of PBMC subsets after 2 days of culturing cytokine-stimulated PBMCs with B7H3.CAR EBVST. Fold change is obtained by normalizing the cell count of PBMC subsets co-cultured with untransduced control EBVST. (C) Cell surface expression of B7-H3 on monocytes after co-culture with allogeneic EBVST. (D) Cytotoxicity of monocytes after co-culture with untransduced or B7H3.CAR EBVST. Data also expressed as fold change in cytotoxicity of B7H3.CAR EBVST relative to the untransduced control. [Figure 24C]Figure 24 (Figure B16). Safety of B7H3.CAR EBVST on peripheral blood mononuclear cells (PBMCs). (A) Cell surface expression of B7-H3 on PBMC subsets after stimulation with inflammatory cytokines and the cancer cell line NCI-N87. (B) Fold change in cell count of PBMC subsets after 2 days of culturing cytokine-stimulated PBMCs with B7H3.CAR EBVST. Fold change is obtained by normalizing the cell count of PBMC subsets co-cultured with untransduced control EBVST. (C) Cell surface expression of B7-H3 on monocytes after co-culture with allogeneic EBVST. (D) Cytotoxicity of monocytes after co-culture with untransduced or B7H3.CAR EBVST. Data also expressed as fold change in cytotoxicity of B7H3.CAR EBVST relative to the untransduced control. [Figure 24D] Figure 24 (Figure B16). Safety of B7H3.CAR EBVST on peripheral blood mononuclear cells (PBMCs). (A) Cell surface expression of B7-H3 on PBMC subsets after stimulation with inflammatory cytokines and the cancer cell line NCI-N87. (B) Fold change in cell count of PBMC subsets after 2 days of culturing cytokine-stimulated PBMCs with B7H3.CAR EBVST. Fold change is obtained by normalizing the cell count of PBMC subsets co-cultured with untransduced control EBVST. (C) Cell surface expression of B7-H3 on monocytes after co-culture with allogeneic EBVST. (D) Cytotoxicity of monocytes after co-culture with untransduced or B7H3.CAR EBVST. Data also expressed as fold change in cytotoxicity of B7H3.CAR EBVST relative to the untransduced control. [Figure 25A] Figure 25 (Fig. B17). Safety of B7H3.CAR EBVST on antigen-experienced T cells. (A) Proliferation and expression of TNFα and IFNγ in EBVST and (B) cell surface expression of B7-H3 after stimulation with antigen-presenting cells pulsed with HIV or EBV pepmix. [Figure 25B]Figure 25 (Fig. B17). Safety of B7H3.CAR EBVST on antigen-experienced T cells. (A) Proliferation and expression of TNFα and IFNγ in EBVST and (B) cell surface expression of B7-H3 after stimulation with antigen-presenting cells pulsed with HIV or EBV pepmix. [Figure 26A] Figure 26 (Figure B18). Safety of P2A5-mCAR in an immunocompetent mouse model. (A) Cytotoxicity of P2A5-mCAR-T against B16F10-WT, B16F10 engineered to express human B7-H3 (B16F10-hB7H3), and B16F10 transfected with mouse B7-H3 (B16F10-mB7H3). (B) Experimental scheme for the immunocompetent mouse model. (C) Measurement of tumor volume and fold change in growth after treatment; and tumor weight at study endpoints. (D) Body weight monitoring twice weekly. (E) Total counts of host hematological cell subsets in the blood, bone marrow, spleen, and liver of treated mice, quantified by flow cytometry. (F) Serum levels of mouse cytokines and chemokines after treatment, quantified by Luminex. [Figure 26B] Figure 26 (Figure B18). Safety of P2A5-mCAR in an immunocompetent m...

Claims

1. 1. An antigen-binding molecule, optionally isolated, that binds to B7 homolog 3 (B7-H3), comprising the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, CDR3 having the amino acid sequence of SEQ ID NO: 3 a single domain antibody sequence incorporating The antigen-binding molecule.

2. The antigen-binding molecule of claim 1, comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

8.

3. The following FR: FR1 having the amino acid sequence of SEQ ID NO: 4, FR2 having the amino acid sequence of SEQ ID NO: 5, FR3 having the amino acid sequence of SEQ ID NO: 6, FR4 having the amino acid sequence of SEQ ID NO:7 The antigen-binding molecule of claim 1 or 2, comprising a single domain antibody sequence incorporating:

4. The antigen-binding molecule of any one of claims 1 to 3, which is a multispecific antigen-binding molecule and further comprises an antigen-binding domain that binds to an antigen other than B7-H3.

5. A chimeric antigen receptor (CAR) comprising the antigen-binding molecule of any one of claims 1 to 4.

6. The CAR of claim 5, comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

9.

7. 7. A nucleic acid or nucleic acids, optionally isolated, encoding the antigen-binding molecule of any one of claims 1 to 4, or the CAR of claim 5 or 6.

8. An expression vector or vectors comprising the nucleic acid or nucleic acids of claim 7.

9. A cell comprising the antigen-binding molecule of any one of claims 1 to 10, the CAR of claim 5 or 6, the nucleic acid or nucleic acids of claim 7, or the expression vector or expression vectors of claim 8.

10. 10. The cell of claim 9, wherein the cell is an immune cell, optionally wherein the immune cell is a T cell.

11. The cell of claim 9 or 10, which is a virus-specific T cell, optionally an Epstein-Barr virus (EBV)-specific T cell.

12. 12. A method comprising culturing the cell of any one of claims 9 to 11 under conditions suitable for expression of the antigen-binding molecule or CAR by the cell.

13. A composition comprising the antigen-binding molecule of any one of claims 1 to 4, the CAR of claim 5 or 6, the nucleic acid or nucleic acids of claim 7, the expression vector or expression vectors of claim 8, or the cell of any one of claims 9 to 11, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

14. 14. The antigen-binding molecule of any one of claims 1 to 4, the CAR of claim 5 or 6, the nucleic acid or nucleic acids of claim 7, the expression vector or expression vectors of claim 8, the cell of any one of claims 9 to 11, or the composition of claim 13, for use in a method of drug therapy or prevention.

15. 14. The antigen-binding molecule of any one of claims 1 to 4, the CAR of claim 5 or 6, the nucleic acid or nucleic acids of claim 7, the expression vector or expression vectors of claim 8, the cell of any one of claims 9 to 11, or the composition of claim 13, for use in the treatment or prevention of cancer.

16. The cancer is selected from the group consisting of B7-H3 positive cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin cancer, cutaneous squamous cell carcinoma, melanoma, pancreatic cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, colorectal cancer, colon cancer, kidney cancer, renal clear cell carcinoma, Wilms' tumor, prostate cancer, ovarian cancer, ovarian cancer, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma, breast cancer, triple negative breast cancer, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, oral squamous cell carcinoma, laryngeal cancer, oropharynx cancer, oropharynx cancer, nasopharyngeal cancer, esophageal cancer, esophageal cancer, esophageal ulcer ...

16. The antigen-binding molecule, CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell, or composition for use according to claim 15, wherein the cancer is selected from the group consisting of urinary tract cancer, bladder cancer, urothelial cancer, brain cancer, medulloblastoma, ependymoma, medulloblastoma, glioma, diffuse intrinsic pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineoblastoma, neuroblastoma, CNS tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brain stem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, peritoneal carcinoma, desmoplastic small cell tumor, and mesothelioma.

17. Use of the antigen-binding molecule of any one of claims 1 to 4, the CAR of claim 5 or 6, the nucleic acid or nucleic acids of claim 7, the expression vector or expression vectors of claim 8, the cell of any one of claims 9 to 11, or the composition of claim 13, to deplete or increase killing of cells expressing B7-H3.

18. 7. An in vitro complex, optionally isolated, comprising the antigen-binding molecule of any one of claims 1 to 4 or the CAR of claim 5 or 6 bound to B7-H3.

19. A method for detecting B7-H3 in a sample, the method comprising the steps of contacting a sample containing or suspected of containing B7-H3 with an antigen-binding molecule of any one of claims 1 to 4, and detecting the formation of a complex between the antigen-binding molecule and B7-H3.

20. 10. A method of selecting or stratifying a subject for treatment with a B7-H3 targeting agent, comprising contacting a sample from the subject in vitro with an antigen-binding molecule of any one of claims 1 to 4, and detecting formation of a complex between the antigen-binding molecule and B7-H3.

21. Use of the antigen-binding molecule of any one of claims 1 to 4 as an in vitro or in vivo diagnostic or prognostic agent.

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