B7-H3 antigen binding molecules

Abstract

The invention provides an antigen binding molecule, which comprises a single domain antibody sequence bound with B7-H3. The invention also provides a chimeric antigen receptor (CAR) construct comprising the antigen binding molecule, a coding nucleic acid / expression vector and a T cell expressing the CAR, and uses of the T cell expressing the CAR in methods of medical treatment, prevention and cancer treatment.

Description

Technical Field

[0001] This application relates to the field of molecular biology, and more particularly to antigen-binding molecule technology. The invention also relates to methods of medical treatment and prevention.

[0002] Background

[0003] Anti-B7-H3 drugs have been developed, including blocking antibodies, antibody-drug conjugates, CD16 affinity-engineered antibodies, and CD3-binding bispecific antibodies. Some candidate drugs have entered clinical trials (1,4). Early data from these clinical trials indicate that, although the extent of the therapeutic response remains to be verified, they have good safety and limited toxicity (1,4). Meanwhile, several groups have started developing chimeric antigen receptor (CAR) T cells targeting B7-H3. These efforts have shown good anti-tumor activity against a variety of solid tumors, leukemias, and lymphomas in preclinical studies (9-15). CART cells targeting B7-H3 are highly active in indications where current treatment regimens have proven ineffective (9-11).

[0004] In recent years, it has been recognized that scFv aggregation can induce tonic signal transduction, which in turn reduces CAR-T activity and persistence (17-19). scFv aggregation or misfolding may be due to the low folding stability of the VH or VL domains or due to the exposure of hydrophobic residues at the VH-VL interface after deletion of the constant domains. In addition, the scFv linker can spatially restrict VH-VL domain interactions and lead to oligomerization (20). Summary of the Invention

[0005] The present invention provides an antigen-binding molecule, optionally isolated, which binds to B7 homolog 3 (B7-H3).

[0006] In certain aspects and embodiments, the antigen-binding molecule contains a single-domain antibody sequence comprising the following CDRs:

[0007] CDR1 having the amino acid sequence of SEQ ID NO: 1

[0008] CDR2 having the amino acid sequence of SEQ ID NO: 2

[0009] CDR3 having the amino acid sequence of SEQ ID NO: 3.

[0010] In some embodiments, the antigen-binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0011] In some embodiments, the antigen-binding molecule contains a single-domain antibody sequence comprising the following FRs:

[0012] FR1 having the amino acid sequence of SEQ ID NO: 4

[0013] FR2 having the amino acid sequence of SEQ ID NO: 5

[0014] FR3 having the amino acid sequence of SEQ ID NO: 6

[0015] FR4 having the amino acid sequence of SEQ ID NO: 7.

[0016] In some embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule, wherein the antigen-binding molecule further comprises an antigen-binding domain that binds to an antigen other than B7-H3.

[0017] The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding molecule as described in the present invention.

[0018] 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.

[0019] The present invention also provides a nucleic acid or a plurality of nucleic acids, optionally isolated, encoding the antigen-binding molecule as described in the present invention or the CAR as described in the present invention.

[0020] The present invention also provides an expression vector or a plurality of expression vectors comprising the nucleic acid or the plurality of nucleic acids of the present invention.

[0021] The present invention also provides a cell comprising the antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, or expression vector or plurality of expression vectors of the present invention.

[0022] In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell.

[0023] In some embodiments, the cell is a virus-specific T cell. In some embodiments, the cell is an Epstein-Barr virus (EBV)-specific T cell.

[0024] The present invention also provides a method comprising culturing the cell as described in the present invention under conditions suitable for the cell to express the antigen-binding molecule or the CAR.

[0025] The present invention also provides a composition comprising the antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors or cell of the present invention, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0026] The present invention also provides methods for the medical treatment or prevention using the antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition of the present invention.

[0027] The present invention also provides the antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition of the present invention for treating or preventing cancer. In some embodiments, for treating or preventing chronic infections.

[0028] In some embodiments, the cancer is selected from the group consisting of: B7-H3 positive cancers, 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, colorectal tumor, colon cancer, colon tumor, kidney cancer, clear cell renal cell carcinoma, Wilms' tumor, prostate cancer, ovarian cancer, ovarian tumor, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, gastric tumor, 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, oropharyngeal cancer, oropharyngeal tumor, nasopharyngeal cancer, esophageal cancer, bladder cancer, urothelial carcinoma, brain cancer, medulloblastoma, ependymoblastoma, glioma, diffuse pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineal tumor, neuroblastoma, central nervous system tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brainstem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, peritoneal cancer, desmoplastic small round cell tumor, and mesothelioma.

[0029] In some embodiments, the antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition of the present invention is used to target myeloid-derived suppressor cells (MDSC). MDSC is a heterogeneous population of immune cells from the myeloid lineage (a family of cells derived from bone marrow stem cells) that expands under pathological conditions such as chronic infections or cancer. Tumors with high levels of MDSC infiltration are associated with poor patient prognosis and resistance to treatment. The MDSC may be B7-H3 positive MDSC. The treatment can eliminate the immunosuppressive effect of MDSC.

[0030] In some embodiments, the treatment is not associated with cytokine release syndrome or is less associated with cytokine release syndrome. In other words, compared to other CAR cancer treatments, the B7-H3 CARs treatment of the present invention is associated with a reduced risk of CRS. For example, the treatment is associated with no change in the levels of IL-6, IL-8, IL10, IFN-y, TNFa, and / or IL-2, neutrophils, monocytes, and / or dendritic cells, or the levels of these cytokines are lower after treatment with the B7-H3 CARs of the present invention than after treatment with other CARs (such as CD19-CAR).

[0031] The present invention also provides for using the antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition of the present invention to deplete cells expressing B7-H3 or to increase killing of cells expressing B7-H3.

[0032] The present invention also provides an in vitro complex, optionally isolated, comprising the antigen-binding molecule or CAR of the present invention that binds to B7-H3.

[0033] The present invention also provides a method for detecting B7-H3 in a sample, comprising contacting a sample containing or suspected of containing B7-H3 with the antigen-binding molecule of the present invention and detecting the formation of a complex of the antigen-binding molecule and B7-H3.

[0034] The present invention also provides a method for selecting or stratifying a subject to be treated with a B7-H3-targeted drug, the method comprising: in vitro, contacting a sample from the subject with the antigen-binding molecule of the present invention and detecting the formation of a complex of the antigen-binding molecule and B7-H3.

[0035] The present invention also provides the use of the antigen-binding molecule of the present invention as an in vitro or in vivo diagnostic or prognostic reagent. Detailed Description

[0036] The present invention provides an antigen-binding molecule that binds to B7-H3 and has novel biophysical and / or functional properties compared to the antigen-binding molecules disclosed in the prior art.

[0037] The present invention also provides a novel chimeric antigen receptor (CAR) construct having a B7-H3 binding domain that comprises the novel B7-H3-specific antigen-binding molecule of the present invention.

[0038] In a preferred embodiment, the antigen-binding molecule is a single-domain antibody (i.e., VHH). Compared to a CAR comprising a scFv antigen-binding domain, a CAR comprising a VHH antigen-binding domain has technical advantages. The size of VHH is significantly smaller than that of scFv (12 - 15 kDa versus 30 kDa), and it has more favorable in vivo immunogenicity, solubility, and stability, while retaining the ability to bind its target antigen with high affinity (21). VHH also avoids potential disruption of interactions between variable and constant domains and exposure of hydrophobic regions, both of which can severely affect solubility and stability (22).

[0039] B7-H3

[0040] B7 homolog 3 (B7-H3, CD276) is the protein identified as UniProt Q5ZPR3. B7-H3 is an immune checkpoint member belonging to the B7 and CD28 protein families and mainly exerts T cell inhibitory effects by inhibiting activation and proliferation (1).

[0041] B7-H3 subtype 1 (UniProt: Q5ZPR3-1; SEQ ID NO: 14) includes an N-terminal signal peptide (SEQ ID NO: 15), an extracellular domain of 438 amino acids (SEQ ID NO: 16), a transmembrane domain (SEQ ID NO: 17), and a short intracellular domain (SEQ ID NO: 18). B7-H3 subtype 2 (UniProt: Q5ZPR3-2; SEQ ID NO: 19) differs from B7-H3 subtype 1 in that it lacks residues 159 - 376 of SEQ ID NO: 14. B7-H3 subtype 3 (UniProt: Q5ZPR3-3; SEQ ID NO: 21) differs from B7-H3 subtype 1 in that it lacks residues 494 - 534 of SEQ ID NO: 14 and residues 465 - 493 are replaced by "GPASSAVPLSPAHPPHGSMCWSHWFSRGL". B7-H3 subtype 4 (UniProt: Q5ZPR3-4; SEQ ID NO: 22) differs from B7-H3 subtype 1 in that residues 528 - 534 are replaced by "GKDTWA". The mature sequences of human B7-H3 subtypes 1 to 4 after processing to remove the N-terminal signal peptide are shown in SEQ ID NOs: 28 - 31.

[0042] B7-H3 subtype 1, also known as 4Ig-B7-H3, refers to the four immunoglobulin (Ig)-like domains within its extracellular domain. The V-type and C2-type Ig-like domains of B7-H3 subtype 1 are shown in SEQ ID NOs: 24-27 and are also included in subtypes 3 and 4. B7-H3 subtype 2, also known as 2Ig-B7-H3, contains a set of V-type and C2-type Ig-like domains, as shown in SEQ ID NOs: 24 and 27.

[0043] High levels of B7-H3 mRNA have been detected in a wide range of normal tissues. In contrast, the expression of B7-H3 protein is limited in these tissues, indicating that the transcriptional level of B7-H3 is strictly regulated 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 has been detected in cancer cells, tumor-infiltrating blood vessels, and tumor stroma (5). Related studies have shown that high expression of B7-H3 protein is associated with poor prognosis and adverse clinical outcomes. These clinical observations are supported by studies showing that B7-H3 has a pro-cancer role in multiple types of cancer, which is independent of its immune function. B7-H3 acts upstream of multiple signal transduction pathways, including the JAK / STAT, Ras / Raf / MEK / MAPK, and PI3K / Akt / mTOR pathways. Elimination of B7-H3 expression reduces the adhesion, migration, invasion, and metastasis of many types of cancer cells in vitro and in vivo (6-8). Collectively, these studies point to the pleiotropic pro-cancer role of B7-H3 and its differential expression in tumors and healthy tissues, making B7-H3 an attractive target for cancer therapy.

[0044] As used herein, "B7-H3" refers to B7-H3 from any species, including subtypes, fragments, variants, or homologs from any species. In some embodiments, B7-H3 is B7-H3 from a mammal (e.g., Theria, Placentalia, Eutheria, Prototheria, Euarchontoglires, Primates (Rhesus macaque, Cynomolgus macaque, non-human primates, or humans)). In some embodiments, the B7-H3 is human B7-H3 or mouse B7-H3.

[0045] As used herein, a subtype, fragment, variant, or homolog of a given reference protein can be characterized as having at least 70% sequence identity, preferably having an amino acid sequence identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of the reference protein.

[0046] "Fragment" generally refers to a part of a reference protein. "Variant" generally refers to a protein with an amino acid sequence having one or more amino acid substitutions, insertions, deletions or other modifications compared to the amino acid sequence of a reference protein, but retaining a substantial degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. The minimum length of a B7-H3 fragment is one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 530 amino acids, and the maximum length is one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 530 amino acids.

[0047] "Subtype" generally refers to a variant of a reference protein expressed by the same species as the reference protein. Subtypes of B7-H3 include, of course, subtype 1 (UniProt: Q5ZPR3-1), subtype 2 (Q5ZPR3-2), subtype 3 (Q5ZPR3-3) and subtype 4 (Q5ZPR3-3).

[0048] "Homolog" generally refers to a variant of a reference protein produced by a species different from the species of the reference protein. Homologs include orthologs.

[0049] Subtypes, fragments, variants or homologs of B7-H3 can optionally be characterized as having an amino acid sequence identity of at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence of an immature or mature B7-H3 subtype from a given species (e.g., human).

[0050] In some embodiments, the B7-H3 is human B7-H3. In some embodiments, the B7-H3 is murine B7-H3 (Q8VE98-1, SEQ ID NO: 32).

[0051] Subtypes, fragments, variants or homologs can optionally be functional subtypes, fragments, variants or homologs, e.g., having the functional properties / activities of the reference B7-H3, as determined by analysis using appropriate assays for the functional properties / activities. For example, subtypes, fragments, variants or homologs of B7-H3 can induce B7-H3-mediated signal transduction.

[0052] In some embodiments, the B7-H3 comprises or consists of an amino acid sequence having an amino acid sequence identity of at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO: 14, 19, 20, 21, 22, 28, 29, 30, 31, 32 or 39.

[0053] In some embodiments, the 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% sequence identity with SEQ ID NO: 22, 16, 31 or 28. In some embodiments, the 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% sequence identity with SEQ ID NO: 16 or 20.

[0054] In some embodiments, the fragment of the 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% sequence identity with SEQ ID NO: 22, 16, 31 or 28. In some embodiments, the fragment of the 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% sequence identity with SEQ ID NO: 16 or 20.

[0055] Antigen-binding molecule

[0056] The present invention provides an antigen-binding molecule capable of binding to B7-H3 (i.e., binding). The present invention provides an antigen-binding molecule that specifically binds to B7-H3. The antigen-binding molecule according to the present invention can be provided in a purified or isolated form, i.e., provided from other naturally occurring biological materials.

[0057] As used herein, "antigen-binding molecule" refers to a molecule capable of binding to a target antigen. The term "antigen-binding molecule" includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies) and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab') 2 2, Fab 2 , diabodies, triabodies, scFv-Fc, microantibodies, single-domain antibodies (VHH), etc.) and aptamers.

[0058] More specifically, the antigen-binding molecule according to the present invention comprises an antigen-binding polypeptide portion, which may be referred to as an "antigen-binding domain". In a preferred embodiment, the antigen-binding molecule of the present invention comprises or consists of a single-domain antibody that specifically binds to B7-H3.

[0059] Single-domain antibodies (sdAbs) – also known in the art as "heavy-chain single variable domain antibodies", "VHHs", "nanobodies", and "heavy-chain only antibodies (HcAbs)" – are described, for example, in Henry and MacKenzie, Frontiers in Immunology (2018) 9:41 and Bever et al., Analytical and Bioanalytical Chemistry (2016) 408(22):5985–6002, both of which are hereby incorporated by reference in their entirety.

[0060] 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 possess heavy-chain antibodies.

[0061] The single-domain antibodies according to the present invention generally include three complementarity-determining regions CDRs: CDR1, CDR2, and CDR3. These three CDRs together define the binding site of the molecule, which is the part that binds to the target antigen.

[0062] Single-domain antibodies also include framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDRs. From the N-terminus to the C-terminus, the single-domain antibody includes the following structure: N-terminus - [FR1] - [CDR1] - [FR2] - [CDR2] - [FR3] - [CDR3] - [FR4] - C-terminus.

[0063] There are several different conventions for defining antibody CDRs and FRs, such as those of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), the Chothia system (Chothia et al., Journal of Molecular Biology 196:901-917 (1987)), the IMGT information system (the international IMGT (ImMunoGeneTics) information system (in LeFranc et al., Nucleic Acids Research (2015) 43 (Database issue):D 413-22), which uses the IMGT V-DOMAIN numbering rules described by Lefranc et al. in Developmental and Comparative Immunology (2003) 27:55-77) and VBASE2 (Retter et al., Nucleic Acids Research (2005) 33 (Suppl. 1):D671-D674). The CDRs and FRs of the antigen-binding molecules / single-domain antibodies described herein are defined according to VBASE2.

[0064] In some embodiments, the antigen-binding molecule comprises the CDRs of the B7-H3-binding single-domain antibody described herein, or comprises CDRs derived from the B7-H3-binding single-domain antibody described herein. In some embodiments, the antigen-binding molecule comprises the FRs of the B7-H3-binding single-domain antibody described herein, or comprises FRs derived from the B7-H3-binding single-domain antibody described herein. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of the B7-H3-binding single-domain antibody described herein, or comprises CDRs and FRs derived from the B7-H3-binding single-domain antibody described herein. That is, in some embodiments, the antigen-binding molecule comprises the amino acid sequence of the B7-H3-binding single-domain antibody described herein, or comprises an amino acid sequence derived from the B7-H3-binding single-domain antibody described herein.

[0065] In some embodiments, in the amino acid sequence of SEQ ID NO: 8: FR1 is formed by the amino acid sequence from position 1 to 25; CDR1 is formed by the amino acid sequence from position 26 to 33; FR2 is formed by the amino acid sequence from position 34 to 50; CDR2 is formed by the amino acid sequence from position 51 to 57; FR3 is formed by the amino acid sequence from position 58 to 96; CDR3 is formed by the amino acid sequence from position 97 to 111; FR4 is formed by the amino acid sequence from position 112 to 122.

[0066] As used herein, an amino acid sequence / domain “derived from” a reference amino acid sequence / domain comprises an amino acid sequence having at least 60%, 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 with the reference amino acid sequence.

[0067] 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.

[0068] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs of a B7-H3-binding single-domain antibody and / or the complete amino acid sequence of a B7-H3-binding single-domain antibody having the amino acid sequence shown in 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, and the antibody has the amino acid sequence shown in 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, and the antibody has the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the antigen-binding molecule comprises the CDRs (i.e., CDRs1, 2, and 3) and FRs (i.e., FRs1, 2, 3, and 4) of a B7-H3-binding single-domain antibody, and the antibody has the amino acid sequence according to SEQ ID NO: 8.

[0069] In some embodiments, the antigen-binding molecule comprises or consists of the single-domain antibody sequence shown in (1):

[0070] (1) (P2A5) single-domain antibody sequence comprising the following CDRs:

[0071] CDR1 having the amino acid sequence of SEQ ID NO: 1

[0072] CDR2 having the amino acid sequence of SEQ ID NO: 2

[0073] CDR3 having the amino acid sequence of SEQ ID NO: 3,

[0074] or a variant thereof, wherein 1 or 2 or 3 amino acids in CDR1, and / or 1 or 2 or 3 amino acids in CDR2, and / or 1 or 2 or 3 amino acids in CDR3 are replaced by another amino acid.

[0075] In some embodiments, the antigen-binding molecule comprises or consists of the single-domain antibody sequence shown in (2):

[0076] (2) (P2A5) single-domain antibody sequence comprising the following FRs:

[0077] FR1 having the amino acid sequence of SEQ ID NO: 4

[0078] FR2 having the amino acid sequence of SEQ ID NO: 5

[0079] FR3 having the amino acid sequence of SEQ ID NO: 6

[0080] FR4 having the amino acid sequence of SEQ ID NO: 7,

[0081] or a variant thereof, wherein 1, 2, or 3 amino acids in FR1, and / or 1, 2, or 3 amino acids in FR2, and / or 1, 2, or 3 amino acids in FR3, and / or 1, 2, or 3 amino acids in FR4 are replaced by another amino acid.

[0082] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence comprising the CDRs shown in (1) and the FRs shown in (2).

[0083] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence such as (3):

[0084] (3) The (P2A5) single-domain antibody sequence comprises an amino acid sequence having at least 70%, 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 shown in SEQ ID NO: 8.

[0085] In embodiments of the present invention, one or more amino acids are replaced by another amino acid. The replacement includes replacing an amino acid residue with a non-identical "substitute" amino acid residue. The substitute amino acid residues of the present invention can be naturally occurring amino acid residues (i.e., encoded by the genetic code) that are not identical to the amino acid residues at the relevant positions in the equivalent, unsubstituted amino acid sequence, 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 substitute amino acid can be a non-naturally occurring amino acid residue - i.e., an amino acid residue that is not one of those cited in the previous sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs as described in Ellman et al., Methods in Enzymology 202 (1991) 301-336.

[0086] In some embodiments, the substitution can be biochemically conservative. In some embodiments, where the amino acid to be substituted is provided in one of rows 1 to 5 of the following table, the replacement amino acid for the substitution is another non-identical amino acid provided in the same row:

[0087]

[0088] Exemplarily, in some embodiments, where the Met residue is substituted, the replacement amino acid can be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and Norleucine.

[0089] In some embodiments, the replacement amino acid for the substitution can have the same side-chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid for the substitution can have the same side-chain charge (at pH 7.4) as the amino acid residue it replaces:

[0090]

[0091]

[0092] That is, in some embodiments, one non-polar amino acid is replaced by another non-identical non-polar amino acid. In some embodiments, one polar amino acid is replaced by another non-identical polar amino acid. In some embodiments, an acidic polar amino acid is replaced by another non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is replaced by another non-identical basic polar amino acid. In some embodiments, a neutral amino acid is replaced by another non-identical neutral amino acid. In some embodiments, a positive amino acid is replaced by another non-identical positive amino acid. In some embodiments, a negative amino acid is replaced by another non-identical negative amino acid.

[0093] In some embodiments, the substitution can be functionally conservative. That is, in some embodiments, compared to an equivalent unsubstituted molecule, the substitution may not affect (or may hardly affect) one or more functional properties (such as target binding) of the antigen-binding molecule comprising the substitution.

[0094] In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin heavy chain constant region sequence (such as CH1, CH2, and / or CH3). In some embodiments, the immunoglobulin heavy chain constant region sequence is or derived from the heavy chain constant region sequence of IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE or IgM, such as human IgG (such as hIgG1, hIgG2, hIgG3, hIgG4), hIgA (such as hIgA1, hIgA2), hIgD, hIgE or hIgM. In some embodiments, the immunoglobulin heavy chain constant region sequence is or derived from the heavy chain constant region sequence of a human IgG1 allotype (such as G1m1, G1m2, G1m3 or G1m17).

[0095] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO: 40, 45 or 48.

[0096] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO: 49, 50 or 51.

[0097] In some embodiments, the antigen-binding molecule comprises a CH1 region, the CH1 region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 75%, 80%, 85%, 86%, 88%, 88%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 41 or 46. In some embodiments, the antigen-binding molecule comprises a hinge region, the hinge region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 75%, 80%, 85%, 86%, 88%, 88%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the antigen-binding molecule comprises a CH2 region, the CH2 region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 75%, 80%, 85%, 86%, 88%, 88%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 43. In some embodiments, the antigen-binding molecule comprises a CH3 region, the CH3 region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 75%, 80%, 85%, 86%, 88%, 88%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 44 or 47.

[0098] It will be appreciated that the CH2 and / or CH3 regions may provide further substitutions in the manner of modification of the Fc region of the antigen-binding molecule as described herein.

[0099] In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin light chain constant region sequence. In some embodiments, the immunoglobulin light chain constant region sequence is a human immunoglobulin kappa constant region (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant region sequence is a human immunoglobulin lambda constant region (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6 or IGLC7.

[0100] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70%, more preferably at least 75%, 75%, 80%, 85%, 86%, 88%, 88%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 52, 53, 54, 55, 56 or 57.

[0101] In some embodiments, the antigen-binding molecule of the invention comprises an Fc region.

[0102] An Fc region is composed of CH2 and CH3 regions from one polypeptide and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.

[0103] Fc-mediated functions include Fc receptor binding, antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation. Modifications to the Fc region of antibodies that affect Fc-mediated functions are known in the art, for example, as described in Wang et al., Protein & Cell (2018) 9(1): 63-73, which is hereby incorporated by reference in its entirety. Table 1 in Wang et al., Protein Cell (2018) 9(1): 63-73 summarizes exemplary Fc region modifications known to affect antibody effector functions. In some embodiments, the antigen-binding molecule of the invention comprises an Fc region that contains modifications to increase or decrease Fc-mediated functions as compared to an antigen-binding molecule that contains the corresponding unmodified Fc region.

[0104] If the Fc region / CH2 / CH3 is described as containing a modification "corresponding" to a reference substitution, equivalent substitutions in homologous Fc / CH2 / CH3 are considered. For example, the L234A / L235A substitution in human IgG1 (numbered according to the EU numbering system 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 in the C region of the mouse Igγ-2A chain (UniProtKB: P01863-1, v1).

[0105] When the Fc region is described as containing a modification, the modification can be present in one or both polypeptide chains that together form the Fc region.

[0106] In some embodiments, the antigen-binding molecule of the present invention includes a modified Fc region. In some embodiments, the antigen-binding molecule of the present invention includes an Fc region that contains one or more modifications in the CH2 and / or CH3 region.

[0107] In some embodiments, the Fc region is modified to increase Fc-mediated functions. In some embodiments, the Fc region contains modifications that increase ADCC. In some embodiments, the Fc region contains modifications that increase ADCP. In some embodiments, the Fc region contains modifications to increase CDC. Compared with an antigen-binding molecule comprising an unmodified corresponding Fc region, an antigen-binding molecule comprising modifications to increase Fc-mediated functions (such as ADCC, ADCP, CDC) induces an elevated level of the relevant effector function.

[0108] In some embodiments, the Fc region contains modifications that increase binding to an Fc receptor. In some embodiments, the Fc region contains modifications that increase binding to an Fcγ receptor. In some embodiments, the Fc region contains modifications that increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region contains modifications that increase binding to FcγRIIIa. In some embodiments, the Fc region contains modifications that increase binding to FcγRIIa. In some embodiments, the Fc region contains modifications that increase binding to FcγRIIb. In some embodiments, the Fc region contains modifications that increase binding to FcRn. In some embodiments, the Fc region contains modifications that increase binding to a complement protein. In some embodiments, the Fc region contains modifications that increase binding to C1q. In some embodiments, the Fc region contains modifications that promote hexamerization of the antigen-binding molecule. In some embodiments, the Fc region contains modifications that increase the half-life of the antigen-binding molecule. In some embodiments, the Fc region contains modifications that increase co-conjugation.

[0109] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination F243L / R292P / Y300L / V305I / P396L described in Stavenhagen et al., Cancer Research (2007) 67:8882–8890. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S239D / I332E or S239D / I332E / A330L described in Lazar et al., Proceedings of the National Academy of Sciences of the United States of America (2006) 103:4005–4010. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S298A / E333A / K334A described in Shields et al., The Journal of Biological Chemistry (2001) 276:6591–6604. In some embodiments, the Fc region comprises modifications to one heavy chain polypeptide corresponding to the substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and modifications to another heavy chain polypeptide corresponding to the substitution combination D270E / K326D / A330M / K334E, as described in Mimot et al., mAbs (2013):5:229–236. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination G236A / S239D / I332E described in Richards et al., Molecular Cancer Therapeutics. (2008) 7:2517–2527.

[0110] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination K326W / E333S described in Idusogie et al., The Journal of Immunology (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination 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 substitution combination described in Natsume et al., Cancer Research (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination E345R / E430G / S440Y described in Diebolder et al., Science (2014) 343(6176):1260-3.

[0111] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination M252Y / S254T / T256E described in Dall'Acqua et al., Journal of Immunology (2002) 169:5171–5180. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination M428L / N434S described in Zalevsky et al., Nature Biotechnology (2010) 28:157-159.

[0112] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination S267E / L328F described in Chu et al., Molecular Immunology (2008) 45:3926–3933. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination N325S / L328F described in Shang et al., Biochemistry (2014) 289:15309–15318.

[0113] In some embodiments, the Fc region comprises a modification that reduces / prevents Fc-mediated functions. In some embodiments, the Fc region comprises a modification that reduces / prevents ADCC. In some embodiments, the Fc region comprises a modification that reduces / prevents ADCP. In some embodiments, the Fc region comprises a modification that reduces / prevents CDC. An antigen-binding molecule comprising an Fc region with a modification that reduces / prevents Fc-mediated functions (e.g., ADCC, ADCP, CDC) induces a reduced level of the associated effector function compared to an antigen-binding molecule comprising an unmodified corresponding Fc region.

[0114] In some embodiments, the Fc region comprises a modification that reduces / prevents binding to an Fc receptor. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIa. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIb. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to a complement protein. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to C1q. In some embodiments, the Fc region comprises a modification that reduces / prevents glycosylation of the amino acid residue corresponding to N297.

[0115] In some embodiments, the Fc region is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to trigger Fc-mediated related functions). Thus, an antigen-binding molecule comprising such an Fc region also lacks the ability to induce the related functions. Such an antigen-binding molecule can be described as lacking the related functions.

[0116] 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 is unable to induce ADCP and / or is unable to induce CDC.

[0117] 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 a complement protein. 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.

[0118] In some embodiments, the Fc region comprises a modification corresponding to N297A or N297Q or N297G as described in Leabman et al., The Journal of Antibodies (2013) 5:896–903. In some embodiments, the Fc region comprises a modification corresponding to L235E as described in Alegre et al., The Journal of Immunology (1992) 148:3461–3468. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A or F234A / L235A as described in Xu et al., Cellular Immunology (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 & Selection (2016), 29(10):457–466. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G as described in Lo et al., J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination as described in Rother et al., Nature Biotechnology (2007) 25:1256–1264. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination S228P / L235E as described in Newman et al., Clinical Immunology (2001) 98:164–174. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination H268Q / V309L / A330S / P331S as described in An et al., The Journal of Antibodies (2009) 1:572–579. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination V234A / G237A / P238S / H268A / V309L / A330S / P331S as described in Vafa et al., Methods (2014) 65:114-126. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S as described in US2015 / 0044231A1.

[0119] It is known that substituting "L234A / L235A" and corresponding substitutions (such as F234A / L235A in human IgG4) disrupt the binding of Fc to Fcγ receptors and inhibit ADCC and ADCP, and also reduce C1q binding, thereby reducing CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10): 457–466, which is hereby incorporated by reference in its entirety). Substitutions "P329G" and "P329A" reduce C1q binding (thereby reducing CDC). It is known that substituting "N297" with "A", "G", or "Q" can eliminate glycosylation, thereby reducing the binding of Fc to C1q and Fcγ receptors, thereby reducing CDC and ADCC. Lo et al., Journal of Biological Chemistry (2017) 292(9): 3900-3908 (which is hereby incorporated by reference in its entirety) describes that the substitution combination L234A / L235A / P329G eliminates complement binding and fixation and Fcγ receptor-dependent, antibody-dependent, cell-mediated cytotoxicity in murine IgG2a and human IgG1.

[0120] The substitution combination L234A / L235E / G237A / A330S / P331S in the IgG1 Fc was disclosed in US2015 / 0044231 A1 to eliminate the induction of phagocytosis, ADCC, and CDC.

[0121] In some embodiments, the Fc region comprises a modification corresponding to substitution S228P as described in Silva et al., Journal of Biological Chemistry (2015) 290(9): 5462-5469. Substitution S228P in IgG4 Fc reduces Fab arm exchange (Fab arm exchange is undesirable).

[0122] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A. In some embodiments, the Fc region comprises a modification corresponding to the P329G substitution. In some embodiments, the Fc region comprises a modification corresponding to the N297Q substitution.

[0123] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G.

[0124] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G / N297Q.

[0125] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S.

[0126] In some embodiments, the Fc region comprises a modification corresponding to substitution S228P, such as in IgG4.

[0127] In some embodiments, particularly embodiments in which the antigen-binding molecule is a multispecific (e.g., bispecific) antigen-binding molecule, the antigen-binding molecule comprises an Fc region having modifications in one or more of the CH2 and CH3 regions that promote binding of the Fc region. Recombinant co-expression of the antigen-binding molecule constituent polypeptides and subsequent binding results in several possible combinations. To increase the yield of the desired polypeptide combinations in the antigen-binding molecule in recombinant production, it is advantageous to introduce modifications in the Fc region to promote binding of the desired heavy-chain polypeptide combinations. The modifications may promote 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., Frontiers in Immunology (2016) 7:394, which is hereby incorporated by reference in its entirety.

[0128] In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising a paired substitution in the CH3 region of the Fc region in one of the following forms, as shown in Table 1 of Ha et al., Frontiers in Immunology (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT s-s , SEED or A107.

[0129] Multispecific antigen-binding molecules are also contemplated. "Multispecific" refers to the specific binding of an antigen-binding molecule to multiple targets. 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.

[0130] 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 is capable of specifically binding to at least two different antigenic determinants.

[0131] It will be appreciated that an antigen-binding molecule according to the present invention (e.g., a multispecific antigen-binding molecule) can comprise an antigen-binding molecule capable of binding to a target specific for the antigen-binding molecule. For example, an antigen-binding molecule that binds to an antigen other than B7-H3 and B7-H3 can include: (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, the component antigen-binding molecules of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) can be referred to as, for example, the "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0132] It will also be appreciated that an antigen-binding molecule according to the present invention (e.g., a multispecific antigen-binding molecule) can comprise an antigen-binding polypeptide or an antigen-binding polypeptide complex capable of binding to a target specific for the antigen-binding molecule.

[0133] 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, such as a cell surface receptor. In some embodiments, the antigen is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen is a growth factor or a hormone.

[0134] Cancer cell antigens are antigens expressed or overexpressed by cancer cells. Cancer cell antigens can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of cancer cell antigens can be associated with cancer. Cancer cell antigens may be abnormally expressed by cancer cells (e.g., cancer cell antigens may be expressed at abnormal localizations), or may be expressed by cancer cells with abnormal structures. Cancer cell antigens may be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the antigenic portion that binds to the antigen-binding molecules described herein is displayed on the outer surface of cancer cells (i.e., is extracellular). Cancer cell antigens may be cancer-associated antigens. In some embodiments, cancer cell antigens are antigens whose expression is associated with the development, progression, or severity of cancer symptoms. Cancer-associated antigens may be related to the etiology or pathology of cancer, or may be abnormally expressed due to cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated by cancer cells (e.g., at the RNA and / or protein levels), e.g., compared to the expression levels of comparable non-cancer cells (e.g., non-cancer cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not by comparable non-cancer cells (e.g., non-cancer cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be the products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens may be overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, oncofetal antigens, or products of cell surface glycolipids or glycoproteins.

[0135] "Review of Tumor-Associated Antigens" was written by scholars such as Zarour HM, Deleo A, Finn OJ, etc., and is included in the chapter "Classification of Tumor Antigens". This chapter can be found in "Holland-Frei Cancer Medicine" (6th Edition), edited by Kufe DW, Pollock RE, Weichselbaum RR, etc., Hamilton (Ontario): BC Decker Publishing; 2003. Cancer-related antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; oncoviral antigens such as 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; lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; mutant antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; post-translationally modified antigens: MUC1, idiotypic antigens: Ig, TCR. Other cancer-related antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetal acinar protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments, the cancer-related antigen is the cancer-related antigen described by Zhao and Cao in "Frontiers in Immunology", 2019; 10: 2250, which is hereby incorporated by reference in its entirety. In some embodiments, the cancer-related 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-related antigen is an antigen expressed by cells of hematological malignancies. In some embodiments, the cancer-related antigen is selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, and BCMA. In some embodiments, the cancer-related antigen is an antigen expressed by cells of solid tumors.In some embodiments, the cancer - related antigen is selected from mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0136] The immune cell surface molecule can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on the cell surface or 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 invention is located on the outer surface of the immune cell (i.e., extracellular). The immune cell surface molecule can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell can be a hematopoietic - derived cell, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, B cell, natural killer (NK) cell, NKT cell, or innate lymphoid cell (ILC), or a precursor thereof (e.g., thymocyte or pre - B cell). In some embodiments, the antigen is a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ).

[0137] In some embodiments, the multispecific antigen - binding molecule described herein exhibits at least monovalent binding with respect to B7 - H3 and also exhibits at least monovalent binding with respect to an antigen other than B7 - H3. The binding valence refers to the number of binding sites for a given antigenic determinant in the antigen - binding molecule.

[0138] In some embodiments, the antigen - binding molecule comprises a single - domain antibody capable of binding to B7 - H3 (e.g., as described herein) and an antigen - binding region capable of binding to an antigen other than B7 - H3 (e.g., a polypeptide (e.g., a single - domain antibody), Fv, Fab, or antibody).

[0139] In some embodiments, the antigen - binding molecule comprises an immune cell - engaging portion. In some embodiments, the antigen - binding molecule is an immune cell engager. Immune cell engagers are described in Goebeler and Bargou, Nature Reviews Clinical Oncology (2020) 17:418 - 434 and Ellerman, Methods (2019) 154:102 - 117, both of which are hereby incorporated by reference in their entirety.

[0140] The immune cell engager molecule comprises an antigen - binding region for targeting a target antigen and an antigen - binding region for recruiting / engaging a target immune cell. The immune cell engager recruits / engages immune cells through an antigen - binding region specific for an immune cell surface molecule.

[0141] In some embodiments, the antigen-binding molecule comprises a CD3 polypeptide-binding portion (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide). The most intensively studied immune cell engager is the bispecific T cell engager (BiTE), which comprises a target antigen-binding domain and a CD3 polypeptide (usually CD3ε) - binding domain, through which BiTE recruits T cells. Binding of BiTE to its target antigen and the CD3 polypeptide expressed on T cells leads to T cell activation and ultimately directs T cell effector activity against cells expressing the target antigen. Other types of immune cell engagers are well known in the art, including natural killer cell engagers, such as bispecific killer cell engagers (BiKE), which can recruit and activate NK cells.

[0142] 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. The multispecific antigen-binding molecules according to the invention can be provided in any suitable format, such as those described in Brinkmann and Kontermann, mAbs (2017) 9(2):182 - 212, which are hereby incorporated by reference in their entirety.

[0143] Functional properties of antigen-binding molecules

[0144] The antigen-binding molecules described herein can be characterized by reference to certain functional properties. In some embodiments, the antigen-binding molecules described herein can have one or more of the following properties:

[0145] Bind to B7-H3 (e.g., human B7-H3 (e.g., human B7-H3 subtype 1 and / or human B7-H3 subtype 2) and / or murine B7-H3);

[0146] Bind to cells expressing B7-H3;

[0147] Do not bind to cells that do not express B7-H3;

[0148] Increase killing of cells expressing B7-H3;

[0149] Do not increase killing of cells that do not express B7-H3;

[0150] Increase ADCC of cells expressing B7-H3;

[0151] Do not increase ADCC of cells that do not express B7-H3;

[0152] Inhibit tumor growth, such as cancers expressing B7-H3; and / or

[0153] Improve the survival rate of subjects with cancer (such as cancer expressing B7-H3).

[0154] It will be understood that a given antigen-binding molecule may exhibit more than one of the properties cited in the previous paragraph. The properties cited in the previous paragraph of a given antigen-binding molecule can be evaluated using suitable detection methods. These assays can be, for example, in vitro assays, which can be cell-free or cell-based assays. Alternatively, these assays can be, for example, in vivo assays, i.e., conducted in non-human animals. The detection can use a species labeled with a detectable entity to facilitate its detection.

[0155] If the detection is a cell-based detection, they may include treating cells with a given antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more known properties. The detection can use a species labeled with a detectable entity to facilitate its detection. The assay can include evaluating the cited properties after treating the cells with a series of amounts / concentrations (such as a dilution series) of the given antigen-binding molecule, respectively. It will be understood that the cells preferably express the target antigen of the antigen-binding molecule (i.e., B7-H3).

[0156] Analysis of the results of such assays may include determining the concentration that achieves 50% of the maximum level of the relevant activity. The concentration of the antigen-binding molecule that achieves 50% of the maximum level of the relevant activity can be referred to as the "half-maximal effective concentration" of the antigen-binding molecule relative to the relevant activity, and can also be referred to as "EC 50 ". By way of example, the EC 50 of a given antigen-binding molecule binding to B7-H3 may be the concentration that achieves 50% of the maximum binding level.

[0157] Depending on the property, EC 50 can also be referred to as the "half-maximal inhibitory concentration" or "IC 50 ", which is the concentration at which the antigen-binding molecule achieves 50% of the maximum inhibitory level of the given property.

[0158] 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 an antigen and can be distinguished from non-specific binding to non-target antigens. The antigen-binding molecule / domain that specifically binds to the target molecule preferably binds to the target with greater affinity and / or for a longer duration than other non-target molecules.

[0159] The ability of a given polypeptide to specifically bind to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see Hearty et al., Methods in Molecular Biology (2012) 907:411-442), biolayer interferometry (see, for example, Lad et al. (2015) Journal of Biomolecular Screening 20(4):498-507), flow cytometry, or by a radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. By these assays, binding to the given molecule can be measured and quantified. In some embodiments, the binding can be a response detected in a given assay.

[0160] In some embodiments, the degree 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, e.g., measured by ELISA, SPR, biolayer interferometry, or by RIA. Alternatively, binding specificity can be reflected in terms of binding affinity, wherein the equilibrium dissociation constant (K D ) of the antigen-binding molecule for the antigen molecule is at least 0.1 order of magnitude greater (i.e., 0.1x10 n , where n is an integer representing the order of magnitude) than the K D of the antigen-binding molecule for the non-target molecule. This can 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.

[0161] Binding to B7-H3 can be determined by surface plasmon resonance, e.g., as described in Example 1.5 of the present invention.

[0162] In some embodiments, the antigen-binding molecules described herein bind B7-H3 with a submicromolar affinity, i.e., K D <1×10 -6 M. In some embodiments, the antigen-binding molecules described herein bind B7-H3 with an affinity in the nanomolar range, i.e., K D = 9.9×10 -7 to 1×10 -9 M. In some embodiments, the antigen-binding molecules described herein bind B7-H3 with a subnanomolar affinity, i.e., K D <1×10 -9 M. In some embodiments, the antigen-binding molecules described herein bind B7-H3 with an affinity in the picomolar range, i.e., K D = 9.9×10 -10 to 1×10 -12 M. In some embodiments, the antigen-binding molecules described herein bind B7-H3 with a subpicomolar affinity, i.e., K D <1×10-12 M.

[0163] In some embodiments, the antigen-binding molecules described herein bind to B7-H3 (e.g., human B7-H3) with a K D of 5 μM or less, preferably one of ≤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.

[0164] In some embodiments, the antigen-binding molecules described herein bind to human B7-H3 subtype 1 with a K D of 5 μM or less, preferably one of ≤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. In some embodiments, the antigen-binding molecule binds to human B7-H3 subtype 1 with a K D ≤1 μM and ≥1 nM, such as 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 ≤1 μM and ≥1 nM to human B7-H3 subtype 1, such as 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.

[0165] In some embodiments, the antigen-binding molecules described herein bind to human B7-H3 subtype 2 with a K D of 5 μM or less, preferably one of ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤450 nM, ≤400 nM, ≤350 nM. In some embodiments, the antigen-binding molecule binds to human B7-H3 subtype 2 with a K D ≤10 μM and ≥10 nM, such as 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 ≤10 μM and ≥10 nM to human B7-H3 subtype 2, such as 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.

[0166] In some embodiments, the antigen-binding molecules described herein bind to murine B7-H3 with a K D of 5 μM or less, preferably one of ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM. In some embodiments, the antigen-binding molecule binds to murine B7-H3 with a K D ≤10 μM and ≥10 nM, such as 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. DBinds to murine B7-H3 at ≤10 μM and ≥10 nM, such as 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.

[0167] The antigen-binding molecule of the present invention can bind to a particular region of interest of B7-H3. The antigen-binding molecule according to the present invention can bind to a linear epitope of B7-H3, which consists of a continuous amino acid sequence (i.e., amino acid primary sequence). In some embodiments, the antigen-binding molecule can bind to a conformational epitope of B7-H3, which consists of a discontinuous amino acid sequence of an amino acid sequence.

[0168] The region of a given target molecule to which the antigen-binding molecule binds can be determined by those skilled in the art using various methods well-known in the art, including X-ray co-crystallography of the antibody-antigen complex, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based "protection" methods. For example, these methods are described in Gershoni et al., BioDrugs 2007, 21(3): 145-156, which is hereby incorporated by reference in its entirety. The ability of the antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods familiar to those skilled in the art, including ELISA, immunoblotting (such as western blot), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[0169] In some embodiments, the antigen-binding molecule (individually) binds to human B7-H3 (such as human B7-H3 subtype 1) and murine B7-H3. In some embodiments, the antigen-binding molecule (individually) binds to human B7-H3 subtype 1 and human B7-H3 subtype 2. In some embodiments, the antigen-binding molecule (individually) binds to human B7-H3 subtype 1, human B7-H3 subtype 2, human B7-H3 subtype 3, and human B7-H3 subtype 4. In some embodiments, the antigen-binding molecule (individually) binds to human B7-H3 subtype 1, human B7-H3 subtype 2, and murine B7-H3. In some embodiments, the antigen-binding molecule (individually) binds to human B7-H3 subtype 1, human B7-H3 subtype 2, human B7-H3 subtype 3, human B7-H3 subtype 4, and murine B7-H3.

[0170] In some embodiments, the antigen-binding molecule has cross-reactivity with one or more subtypes or homologs of B7-H3 (e.g., human B7-H3 subtype 1). In some embodiments, the antigen-binding molecule has cross-reactivity with human B7-H3 (e.g., human B7-H3 subtype 1) and murine B7-H3. In some embodiments, the antigen-binding molecule has cross-reactivity with human B7-H3 subtype 1 and human B7-H3 subtype 2. In some embodiments, the antigen-binding molecule has cross-reactivity with human B7-H3 subtype 1, human B7-H3 subtype 2, human B7-H3 subtype 3, and human B7-H3 subtype 4. In some embodiments, the antigen-binding molecule has cross-reactivity with human B7-H3 subtype 1, human B7-H3 subtype 2, and murine B7-H3. In some embodiments, the antigen-binding molecule has cross-reactivity with human B7-H3 subtype 1, human B7-H3 subtype 2, human B7-H3 subtype 3, human B7-H3 subtype 4, and murine B7-H3.

[0171] 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 murine B7-H3 binds to human B7-H3 and is also capable of binding to murine B7-H3. A cross-reactive antigen-binding molecule / domain / polypeptide may exhibit specific binding to each target antigen.

[0172] In some embodiments, the antigen-binding molecule of the present invention binds to the extracellular domain of B7-H3. In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 16. In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 20. In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 30. In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 34.

[0173] In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 34.

[0174] In some embodiments, the antigen-binding molecule of the present invention binds to the Ig-like C2 domain of B7-H3. In some embodiments, the antigen-binding molecule contacts the Ig-like C2 domain of B7-H3. In some embodiments, the antigen-binding molecule binds to B7-H3 by contacting one or more amino acids of the Ig-like C2 domain of B7-H3.

[0175] In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 25. In some embodiments, the antigen-binding molecule contacts the B7-H3 region shown in SEQ ID NO: 25. In some embodiments, the antigen-binding molecule binds to B7-H3 by contacting one or more amino acids of the region shown in SEQ ID NO: 25. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25.

[0176] In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule contacts the B7-H3 region shown in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule binds to B7-H3 by contacting one or more amino acids of the region shown in SEQ ID NO: 27. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 27.

[0177] In some embodiments, the antigen-binding molecule binds to the B7-H3 region shown in SEQ ID NO: 38. In some embodiments, the antigen-binding molecule contacts the B7-H3 region shown in SEQ ID NO: 38. In some embodiments, the antigen-binding molecule binds to B7-H3 by contacting one or more amino acids of the region shown in SEQ ID NO: 38. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 38.

[0178] In some embodiments, the antigen-binding molecule according to the invention binds to the same region of B7-H3 or to an overlapping region of B7-H3, the region being bound by the antigen-binding molecule, which antigen-binding molecule comprises the CDRs, FRs and / or the complete amino acid sequence of a B7-H3-binding single-domain antibody as described herein, such as P2A5.

[0179] It can be evaluated whether a test antigen-binding molecule binds to the same or an overlapping region of a given target of a reference antigen-binding molecule, for example, by analyzing (i) the interaction between the test antigen-binding molecule and the target in the absence of the reference binding molecule, and (ii) the interaction between test antigen-binding molecules in the presence of the reference antigen-binding molecule or after incubation of the target with the reference antigen-binding molecule. After analysis according to (ii), a determination of a reduced level of interaction between the test antigen-binding molecule and the target compared to (i) may support the inference that the test and reference antigen-binding molecules bind to the same or an overlapping region of the target. Methods suitable for such analysis include, for example, competitive ELISA assays and epitope mapping assays.

[0180] In some embodiments, the antigen-binding molecule according to the invention binds to the same region of B7-H3 or to an overlapping region of B7-H3, the region binding to a polypeptide consisting of the amino acid sequence of SEQ ID NO:8.

[0181] In some embodiments, the antigen-binding molecule according to the invention can enhance (i.e., upregulate, increase) cell killing of cells comprising / expressing B7-H3. In some embodiments, the antigen-binding molecule does not enhance (i.e., substantially does not enhance) killing of cells that do not express B7-H3 on their surface.

[0182] In some embodiments, the antigen-binding molecule according to the invention can inhibit the growth of cancer or reduce cancer metastasis in cells comprising / expressing B7-H3. In some embodiments, the antigen-binding molecule can enhance (i.e., upregulate, increase) cell killing of cells against cancer cells comprising / expressing B7-H3. In some embodiments, the antigen-binding molecule can inhibit the growth of cancer or reduce cancer metastasis in cells comprising / expressing B7-H3.

[0183] For example, any method described in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616 can be used to study cell killing, which is hereby incorporated by reference in its entirety. In vitro assays for cytotoxicity / cell killing assays include release assays, such as 51Cr 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 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 / ratio of live / dead (e.g., lysed) test cells after an appropriate time. Other suitable assays include the xCELLigence real-time cytotoxicity in vitro potency assay described in Cerignoli et al., PLoS ONE, (2018) 13(3):e0193498 (hereby incorporated by reference in its entirety). Relative to a reference level of cell killing (e.g., for that cell type), an increase in resistance to cell killing and / or a decrease in sensitivity to such cell killing cells of cells expressing granzyme B (e.g., effector immune cells) can be determined by detection of a decrease in the number / ratio of dead (e.g., lysed) test cells and / or an increase in the number / ratio of live (e.g., active, unlysed) test cells after a given time.

[0184] In some embodiments, the antigen-binding molecule according to the invention is capable of reducing the number / ratio of cells expressing B7-H3. In some embodiments, the antigen-binding molecule according to the invention is capable of reducing the number / ratio of cells expressing B7-H3. In some embodiments, the antigen-binding molecule according to the invention is capable of depleting / enhancing the depletion of such cells.

[0185] The antigen-binding molecule according to the invention may comprise one or more moieties for enhancing the reduction in the number / ratio of cells expressing B7-H3. For example, the antigen-binding molecule according to the invention may, for example, comprise an Fc region and / or a drug moiety.

[0186] The Fc region provides interactions with Fc receptors and other molecules of the immune system to bring about functional effects. For example, IgG Fc-mediated effector functions are described in Jefferis et al., Immunological Reviews 1998 163:59-76 (which is hereby incorporated by reference in its entirety), and are achieved by Fc-mediated recruitment and activation of immune cells such as macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells, through interactions between the Fc region and Fc receptors expressed on immune cells, by binding of the Fc region to the complement protein C1q to recruit complement pathway components, and subsequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation.

[0187] In some embodiments, the antigen-binding molecule according to the invention comprises an Fc region capable of enhancing / guiding ADCC, ADCP, CDC, and / or enhancing the formation of MAC or cell degranulation on cells expressing B7-H3 (e.g., cells expressing B7-H3 on the cell surface).

[0188] In some embodiments, the antigen-binding molecule according to the invention is capable of enhancing / guiding ADCC against cells expressing B7-H3.

[0189] The ability and extent to which a given antigen-binding molecule can induce ADCC in a given target cell type can be analyzed, for example, according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (which is hereby incorporated by reference in its entirety), or by 51 a Cr release assay, such as that described in Jedema et al., Blood (2004) 103:2677–82 (which is hereby incorporated by reference in its entirety). The ability and extent to which a given antigen-binding molecule can induce ADCP can be analyzed, for example, according to the method described in Kamen et al., Journal of Immunology (2017) 198(1 Suppl) 157.17 (which is hereby incorporated by reference in its entirety). The ability and extent to which a given antigen-binding molecule can induce CDC can be analyzed, for example, using a C1q binding assay, such as that described in Schlothauer et al., Protein Engineering, Design & Selection (2016), 29(10):457–466 (which is hereby incorporated by reference in its entirety).

[0190] In some embodiments, the antigen-binding molecule according to the present invention comprises a drug moiety. The antigen-binding molecule may be conjugated to the drug moiety. Antibody-drug conjugates are described in Parslow et al., Biomedicine 2016 September; 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.

[0191] In some embodiments, the antigen-binding molecule according to the present invention comprises an immunocyte engaging moiety. In some embodiments, the antigen-binding molecule comprises a CD3 polypeptide binding moiety (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide).

[0192] In some embodiments, the antigen-binding molecule according to the present invention is capable of enhancing / guiding T cell-mediated cytolytic activity against cells expressing B7-H3.

[0193] In some embodiments, the antigen-binding molecule of the present invention exhibits anti-cancer activity. In some embodiments, the antigen-binding molecule of the present invention increases the killing of cancer cells. In some embodiments, the antigen-binding molecule of the present invention results in a reduction in the number of cancer cells in vivo, e.g., compared to appropriate control conditions. The cancer may be a cancer expressing B7-H3.

[0194] In some embodiments, the antigen-binding molecule according to the present invention reduces / inhibits the growth of cancer and / or cancer tumors. In some embodiments, the antigen-binding molecule reduces the tissue invasion of cancer cells. In some embodiments, the antigen-binding molecule reduces the metastasis of cancer. In some embodiments, the antigen-binding molecule exhibits anti-cancer activity. In some embodiments, the antigen-binding molecule reduces the growth / proliferation of cancer cells. In some embodiments, the antigen-binding molecule reduces the survival of cancer cells. In some embodiments, the antigen-binding molecule increases the killing of cancer cells. In some embodiments, the antigen-binding molecule of the present invention results in a reduction in the number of cancer cells, e.g., in vivo. The cancer may be a cancer composed of cells expressing B7-H3.

[0195] The antigen-binding molecule of the present invention can be analyzed for the properties described in the previous paragraph in appropriate assays. Such assays include, for example, in vivo models.

[0196] In some embodiments, administration of an antigen-binding molecule according to the invention can result in one or more of the following: inhibition of cancer development / progression, delay / prevention of cancer onset, reduction / delay / prevention of tumor growth, reduction / delay / prevention of tissue invasion, reduction / delay / prevention of metastasis, reduction in cancer symptom severity, reduction in the number of cancer cells, reduction in tumor size / volume, and / or increase in survival rate (e.g., progression-free survival or overall survival), as determined in a suitable model, for example.

[0197] In some embodiments, in a given assay, compared to tumor growth observed after treatment without an antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect tumor growth), the antigen-binding molecule of the invention is capable of reducing / inhibiting tumor growth (e.g., in an in vivo model, such as a cancer expressing B7-H3) to 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.

[0198] In some embodiments, in a given assay, compared to the level of metastasis observed after treatment without an antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect tumor growth), the antigen-binding molecule of the invention is capable of reducing / inhibiting metastasis (e.g., in an in vivo model, such as a cancer expressing B7-H3) to 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.

[0199] In some embodiments, in a given assay, the antigen-binding molecules of the present invention are capable of increasing the survival rate of a subject with cancer (e.g., in an in vivo model, such as a cancer expressing B7-H3) to more than 1-fold, e.g., ≥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, compared to the survival rate observed after treatment without antigen-binding molecules (or after treatment with a suitable control antigen-binding molecule known not to affect tumor growth).

[0200] Chimeric antigen receptors (CARs)

[0201] In some aspects and embodiments according to the present invention, the antigen-binding molecule is a chimeric antigen receptor (CAR). In some aspects and embodiments, the present invention provides a chimeric antigen receptor comprising the antigen-binding molecule or polypeptide of the present invention.

[0202] A CAR is a recombinant receptor that provides antigen-binding and T cell activation functions. CAR structure and engineering are reviewed, for example, in Dotti et al., Immunological Reviews (2014) 257(1), which is hereby incorporated by reference in its entirety. A CAR comprises an antigen-binding domain linked to a signal transduction domain via a transmembrane domain. An optional hinge or spacer domain may provide separation between the antigen-binding domain and the transmembrane domain and may act as a flexible linker. When expressed by a cell, the antigen-binding domain is extracellular and the signal domain is intracellular.

[0203] The antigen-binding domain mediates binding to the CAR-specific target antigen. The antigen-binding domain of a CAR may be based on the antigen-binding region of an antigen-binding molecule that is specific for the antigen targeted by the CAR. For example, the antigen-binding domain of the CAR may comprise the amino acid sequence of the complementarity-determining regions (CDRs) of an antibody that specifically binds 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 the target antigen. The antigen-binding domain may be provided in the form of a single-chain variable fragment (scFv), which comprises the light and heavy chain variable region amino acid sequences of an antibody. The antigen-binding domain of a CAR may be based on other protein:protein interactions targeting an antigen, such as ligand:receptor binding; for example, a CAR targeting IL-13Rα2 has been developed using an IL-13-based antigen-binding domain (see Kahlon et al., Cancer Research 64(24):9160-9166, 2004).

[0204] The CAR of the present invention comprises an antigen-binding domain, which comprises or consists of the antigen-binding molecule of the present invention, or comprises or consists of a polypeptide according to the present invention.

[0205] An optional spacer domain can provide separation between the antigen-binding domain and the transmembrane domain and can act as a flexible linker. The domain can be or comprise a flexible region, allowing the binding moiety to face different directions. The spacer domain can be derived from IgG.

[0206] The transmembrane domain is located between the antigen-binding domain and the signal transduction domain of the CAR. The transmembrane domain is used to anchor the CAR to the cell membrane of the cell expressing the CAR, such that the antigen-binding domain is located in the extracellular space and the signal domain is located intracellularly. The transmembrane domain of the CAR may be derived from the transmembrane region sequence of a cell membrane-binding protein (such as CD28, CD8, CD4, CD3-ζ, etc.).

[0207] The signal transduction domain comprises an amino acid sequence required for activating the functions of immune cells. The CAR signal domain can comprise the amino acid sequence of the intracellular domain of CD3-ζ, which provides phosphorylation and an immunoreceptor tyrosine-based activation motif (ITAM) for activating the cell expressing the CAR. Signal domains comprising other ITAM-containing protein sequences have also been used for CARs, such as a domain comprising the ITAM-containing region of FcγRI (Haynes et al. 2001 Journal of Immunology 166(1): 182-187). A CAR comprising a signal transduction domain derived from the intracellular domain of CD3-ζ is generally referred to as a first-generation CAR.

[0208] The signal transduction domain of the CAR generally further comprises the signal transduction domain of a costimulatory protein (such as CD28, 4-1BB, etc.) for providing a costimulatory signal required for enhancing immune cell activation and effector functions. A CAR having a signal transduction domain comprising an additional costimulatory sequence is generally referred to as a second-generation CAR. In some cases, the CAR is designed to provide costimulation of different intracellular signaling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB costimulation triggers signal transduction through a TNF receptor-associated factor (TRAF) adaptor protein. Thus, the signal transduction domain of the CAR sometimes comprises the costimulatory sequences of the signal transduction domains of multiple costimulatory molecules. A CAR comprising a signal transduction domain having multiple costimulatory sequences is generally referred to as a third-generation CAR.

[0209] Throughout the specification, a polypeptide, domain, and amino acid sequence "derived from" a reference polypeptide / domain / amino acid sequence has at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of the reference polypeptide / domain / amino acid sequence. The polypeptide, domain, and amino acid sequence "derived from" the reference polypeptide / domain / amino acid sequence preferably retain the functional and / or structural characteristics of the reference polypeptide / domain / amino acid sequence.

[0210] By way of example, an amino acid sequence derived from the intracellular domain of CD28 can have 60% amino acid sequence identity with the intracellular domain of CD28, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, such as shown in SEQ ID NO: 12. In addition, the amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional characteristics of the amino acid sequence of SEQ ID NO: 12, namely the ability to activate CD28-mediated signal transduction.

[0211] The amino acid sequence of a given polypeptide or its domain can be retrieved from databases known to those skilled in the art or determined from the retrieved nucleic acid sequences. Such databases include GenBank, EMBL, and UniProt.

[0212] By engineering to express a CAR specific for a particular target antigen, immune cells (usually T cells, but also including 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 triggers intracellular signal transduction, thereby triggering activation of the T cell. The stimulated CAR-T cells are activated to divide and produce factors that cause the cells expressing the target antigen to be killed.

[0213] Antigen-binding domain

[0214] The antigen-binding domain of the CAR according to the present invention comprises or consists of an antigen-binding molecule that binds to B7-H3 as described herein. Thus, the CAR according to the present invention comprises the antigen-binding molecule according to the present invention.

[0215] It will be understood that the antigen-binding molecule formed according to the present invention composes or is included in the antigen-binding domain of the CAR. Thus, in some embodiments, the antigen-binding molecule of the present invention is included in the CAR.

[0216] It can also be understood that the antigen-binding molecule of the present invention can be a CAR. A CAR having an antigen-binding domain of the antigen-binding domain contains or consists of the antigen-binding molecule of the present invention (such as a B7-H3-binding single-domain antibody) and is an antigen-binding molecule. The antigen-binding domain of the CAR of the present invention can be provided in any suitable form, such as scFv, scFab, etc.

[0217] In some embodiments, the antigen-binding domain comprises or consists of a B7-H3-binding single-domain antibody as described herein.

[0218] Spacer domain

[0219] In some embodiments, the CAR comprises a spacer domain. The spacer domain may be located 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 linkage between the CAR antigen-binding domain and the transmembrane domain.

[0220] The presence, absence, and length of the spacer domain have been shown to affect CAR function (reviewed, for example, in Dotti et al., Immunological Reviews (2014) 257(1) and Jayaraman et al., EBioMedicine (2020) 58:102931). The spacer region length can be altered to control the synaptic cleft distance, which in turn may regulate signaling. A flexible spacer can access sterically hindered epitopes on the target antigen. Multimerization of the spacer domain (such as by homotypic association) results in increased signal strength and activation stimuli.

[0221] In some embodiments, the spacer domain according to the present invention comprises or consists of an amino acid sequence that is or is derived from: the CH2-CH3 region of human IgG1 (such as, as shown in SEQ ID NO: 59), the CH2-CH3 region of human IgG2 (such as, as shown in SEQ ID NO: 61), the CH1-CH2 hinge region of human IgG1, a spacer domain derived from CD8α, such as described in WO2012 / 031744 A1, or a spacer domain derived from CD28, such as described in WO 2011 / 041093 A1. A variant CH2-CH3 region described in Hombach et al., Gene Therapy (2010) 4 17:1206-1213, for reducing activation of cells expressing FcγR (such as monocytes and NK cells). The amino acid sequence of the variant CH2-CH3 region is as shown in SEQ ID NO: 60.

[0222] 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% identity to the amino acid sequence shown in 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% identity to the amino acid sequence shown in 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%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98% or 100% identity to the amino acid sequence shown in 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%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in 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%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in 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%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in 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%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98% or 100% identity to the amino acid sequence shown in SEQ ID NO:10.

[0223] Transmembrane domain

[0224] The CAR of the present invention comprises a transmembrane domain. The transmembrane domain refers to any three-dimensional structure formed by an amino acid sequence that is thermodynamically stable in a biological membrane (such as a cell membrane). In the context of the present invention, the transmembrane domain can be an amino acid sequence that spans the cell membrane expressing the CAR.

[0225] The transmembrane domain may comprise or consist of an amino acid sequence that forms a hydrophobic α-helix or β-barrel. The amino acid sequence of the transmembrane domain of the CAR of the present invention may be or may be derived from the amino acid sequence of the transmembrane domain of a protein comprising 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 may be identified / predicted using amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001 Journal of Molecular Biology 305: 567-580).

[0226] In some embodiments, the amino acid sequence of the transmembrane domain of the CAR of the present invention 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, e.g., an immune receptor or ligand. In some embodiments, the amino acid sequence of the transmembrane domain may be or may be derived from the amino acid sequence of the transmembrane domain of one of ICOS, ICOS-L, CD86, CTLA-4, CD28, CD80, MHC class I α, MHC class II α, MHC class II β, CD3ε, CD3δ, CD3γ, CD3-ζ, TCRαTCRβ, CD4, CD8α, CD8β, CD40, CD40L, PD-1, PD-L1, PD-L2, 4-1BB, 4-1BBL, OX40, OX40L, GITR, GITRL, TIM-3, galectin-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 is or is derived from the amino acid sequence of the transmembrane domain of CD28, CD3-ζ, CD8α, CD8β, or CD4. In some embodiments, the transmembrane is or is derived from the amino acid sequence of the transmembrane domain of CD28.

[0227] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:11.

[0228] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:65.

[0229] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:66.

[0230] Signal transduction domain

[0231] The chimeric antigen receptor of the present invention comprises a signal transduction domain. The signal transduction domain provides a sequence that initiates intracellular signal transduction in cells expressing the CAR.

[0232] ITAM-containing sequence:

[0233] The signal transduction domain comprises an ITAM-containing sequence. The ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). An ITAM comprises the amino acid sequence YXXL / I (SEQ ID NO:69), where "X" represents any amino acid. In ITAM-containing proteins, the sequences shown in SEQ ID NO:69 are typically separated by 6 to 8 amino acids; YXXL / I(X) 6-8 YXXL / I (SEQ ID NO:70). When tyrosine kinase adds a phosphate group to the tyrosine residue of the ITAM, a signal cascade is initiated intracellularly.

[0234] In some embodiments, the signal transduction domain comprises one or more copies of the amino acid sequence shown in SEQ ID NO:69 or SEQ ID NO:70. In some embodiments, the signal transduction domain comprises at least 1, 2, 3, 4, 5 or 6 copies of the amino acid sequence of SEQ ID NO:69. In some embodiments, the signal transduction domain comprises at least 1, 2 or 3 copies of the amino acid sequence of SEQ ID NO:70.

[0235] In some embodiments, the signal transduction domain comprises a sequence containing an ITAM, which is or is derived from an amino acid sequence of a protein having an amino acid sequence containing an ITAM. In some embodiments, the signal transduction domain comprises a sequence containing an ITAM, which is or is derived from the amino acid sequence of the intracellular domain of one of CD3-ζ, FcγRI, CD3ε, CD3δ, CD3γ, CD79α, CD79β, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIV or DAP12. In some embodiments, the signal transduction domain contains a sequence containing an ITAM, which is or is derived from the amino acid sequence of the intracellular domain of CD3-ζ.

[0236] In some embodiments, the signal transduction domain comprises a sequence containing an ITAM, which 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% identity to the amino acid sequence shown in SEQ ID NO:13.

[0237] Co-stimulatory sequence:

[0238] The signal transduction domain may further comprise one or more co-stimulatory sequences. A co-stimulatory sequence is an amino acid sequence that provides co-stimulation to cells expressing the CAR of the present invention. After binding to the target antigen, co-stimulation promotes the proliferation and survival of CAR-expressing cells, and may also promote cytokine production, differentiation, cytotoxic function and memory formation of CAR-expressing cells. The molecular mechanisms of T cell co-stimulation are reviewed in Chen and Flies (2013) Nature Reviews Immunology 13(4):227-242.

[0239] The co-stimulatory sequence may be or be derived from the amino acid sequence of a co-stimulatory protein. In some embodiments, the co-stimulatory sequence is or is derived from the amino acid sequence of the intracellular domain of a co-stimulatory protein.

[0240] After the CAR binds to the target antigen, the co-stimulatory sequence provides co-stimulation to the cells expressing the CAR, and this co-stimulation can be provided by a co-stimulatory protein. The co-stimulatory sequence is derived from the co-stimulatory sequence when linked through its cognate ligand. For example, in the case where the CAR comprises a signal transduction domain containing a co-stimulatory sequence derived from CD28, binding to the target antigen triggers signal transduction in the cell triggered by the binding of CD80 and / or CD86 to CD28. Thus, the co-stimulatory sequence is capable of transmitting a co-stimulatory signal derived from the co-stimulatory protein.

[0241] In some embodiments, the co-stimulatory protein can be a member of the B7-CD28 superfamily (such as CD28, ICOS), or a member of the TNF receptor superfamily (such as 4-1BB, OX40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the co-stimulatory sequence is or 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 co-stimulatory sequence is or derived from the intracellular domain of CD28.

[0242] In some embodiments, the signal transduction domain comprises more than one co-stimulatory sequence. In some embodiments, the signal transduction domain includes 1, 2, 3, 4, 5 or 6 co-stimulatory sequences. Multiple co-stimulatory sequences can be provided in tandem.

[0243] Whether a given amino acid sequence can initiate signal transduction mediated by a given co-stimulatory protein can be studied, for example, by analyzing the correlation of signal transduction mediated by the co-stimulatory protein (e.g., upregulation or downregulation of the expression / activity of factors due to signal transduction mediated by the co-stimulatory protein).

[0244] Co-stimulatory proteins upregulate the expression of genes that promote cell growth, effector function, and survival through multiple transduction 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, activates ERK1 / 2 through RAS, and ICOS activates C-MAF. 4-1BB, OX40, and CD27 recruit TNF receptor-associated factors (TRAF) and signal through the MAPK pathway as well as PI3K.

[0245] In some embodiments, the signal transduction domain includes a co-stimulatory sequence that is or derived from CD28.

[0246] Kofler et al., "Molecular Therapy" (2011) 19:760-767 describes a variant CD28 intracellular domain in which the lck kinase binding site is mutated to reduce the induction of IL-2 production upon CAR ligation to minimize the inhibition of CAR-T cell activity mediated by regulatory T cells. The amino acid sequence of the variant CD28 intracellular domain is shown in SEQ ID NO: 67.

[0247] In some embodiments, the signal transduction domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:12. In some embodiments, the signal transduction domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the signal transduction domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98% or 100% identity to the amino acid sequence shown in SEQ ID NO:68.

[0248] In some embodiments, the signal transduction domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:74.

[0249] In some embodiments, the amino acid sequence comprised by the CAR has 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 shown in SEQ ID NO:75.

[0250] Linkers and additional sequences

[0251] The antigen-binding molecules, polypeptides and CARs of the present invention further additionally comprise further amino acids or amino acid sequences.

[0252] In addition to the amino acid sequence required for binding to the target antigen, the antigen-binding molecule comprises additional amino acids / amino acid sequences. In some embodiments, additional amino acids / amino acid sequences are provided at the N-terminus of the single-domain antibody sequence of the present invention. In some embodiments, additional amino acids / amino acid sequences are provided at the C-terminus of the single-domain antibody sequence of the present invention. In some embodiments, additional amino acids / amino acid sequences are provided at both the N-terminus and the C-terminus of the single-domain antibody sequence of the present invention.

[0253] The antigen-binding molecules, polypeptides, and CARs of the present invention may include one or more linker sequences located between amino acid sequences. For example, linker sequences are provided between 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 signal transduction domain). Further by way of example, linker sequences may be provided between subsequences of domains of the CAR (e.g., between the co-stimulatory sequence and the sequence of the signal transduction domain and the sequence containing ITAM).

[0254] Linker sequences are known to those skilled in the art and are described in Chen et al., Advanced Drug Delivery Reviews (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, the indicated linker sequences may be flexible linker sequences. Flexible linker sequences allow relative movement of the amino acid sequences linked by the linker sequence. Flexible linkers are well known to those skilled in the art, and several flexible linker sequences are described in Chen et al., Advanced Drug Research Revised Edition (2013) 65(10): 1357-1369, and generally contain a high proportion of glycine and / or serine residues.

[0255] 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 consists of or comprises glycine and serine residues. In some embodiments, the linker sequence has the following 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 G 4 S motif sequence. In some embodiments, the linker sequence comprises or consists of (G 4 S) 4 or (G 4 S) 6 In some embodiments, the length of the linker sequence is 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

[0256] The antigen-binding molecules, polypeptides, and CARs of the present invention may include amino acid sequences that facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecules and polypeptides of the present invention may further comprise amino acid sequences that form a detectable moiety, as described below, for example.

[0257] The antigen-binding molecules, polypeptides, and CARs of the present invention may also include a signal peptide (also referred to as a leader sequence or signal sequence). Signal peptides typically consist of a 5-30 amino acid sequence of hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed on the cell surface typically contain signal peptides. Signal peptides for many proteins are known 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).

[0258] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide / CAR or may be present in the newly synthesized antigen-binding molecule / polypeptide / CAR. The signal peptide provides efficient transport of the antigen-binding molecule / polypeptide / CAR. The signal peptide is typically removed by cleavage and thus is not included in the mature antigen-binding molecule / polypeptide / CAR.

[0259] Signal peptides for many proteins are known 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 SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0260] Labels and conjugates

[0261] In some embodiments, the antigen-binding molecules, polypeptides, or CARs of the present invention further include a detectable moiety.

[0262] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immuno-detectable label (such as an epitope tag), a radioactive label, a chemical, nucleic acid, or enzyme label. The antigen-binding molecule, polypeptide, or CAR can be labeled covalently or non-covalently with the detectable moiety.

[0263] 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. Radioisotopes include radioisotopes such as 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 , copper 67 , fluorine 18 , yttrium 90 , palladium 100 , bismuth 217 and antimony 211 . Luminescent labels include radioluminescence, chemiluminescence (e.g., acridinium ester, luminol, isoluminol), and bioluminescence labels. Immuno-detectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxin. Nucleic acid labels include aptamers.

[0264] 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 haptens (e.g., biotin, digoxin, dinitrophenol), optionally located at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide / CAR.

[0265] In some embodiments, the antigen-binding molecule / polypeptide / CAR comprises a moiety having detectable activity, such as an enzyme moiety. Enzyme moieties include, for example, luciferase, glucose oxidase, galactosidase (such as β-galactosidase), glucosidase, phosphatase (such as alkaline phosphatase), peroxidase (such as horseradish peroxidase), and cholinesterase.

[0266] In some embodiments, the antigen-binding molecule / polypeptide of the present invention is conjugated to a chemical moiety. The chemical moiety can be a moiety that provides a therapeutic effect, i.e., a drug moiety. The drug moiety can be a small molecule (e.g., a low molecular weight (<1000 daltons, typically between ~300 - 700 daltons) organic compound). For example, the drug moiety is described in Parslow et al., Biomedicine 2016 Sep;4(3):14 (which is hereby incorporated by reference in its entirety). In some embodiments, the drug moiety can be or comprise a cytotoxic agent. In some embodiments, the drug moiety can 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.

[0267] Specific exemplary polypeptides, antigen-binding molecules, and CARs

[0268] In some embodiments, the antigen-binding molecule / polypeptide of the present invention comprises or consists of an amino acid sequence comprising the CDR of P2A5.

[0269] In some embodiments, the antigen-binding molecule / polypeptide of the present invention comprises or consists of an amino acid sequence comprising the FR of P2A5.

[0270] In some embodiments, the antigen-binding molecule / polypeptide of the present invention 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% sequence identity to the amino acid sequence shown in SEQ ID NO:8.

[0271] In some embodiments of the present invention, the CAR comprises or consists of:

[0272] An antigen-binding domain comprising or consisting of an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 8;

[0273] A spacer domain comprising or consisting of an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 10;

[0274] A transmembrane domain comprising or consisting of an amino acid sequence having 60%, 65%, 70%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 75%, 80%, 75%, 85%, 89%, 89%, 90%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 11;

[0275] A co-stimulatory domain comprising or consisting of an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 12; and

[0276] A signal transduction domain comprising or consisting of an amino acid sequence having 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 shown in SEQ ID NO: 13.

[0277] In some embodiments of the present invention, the indicated 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 shown in SEQ ID NO: 9.

[0278] Nucleic acids and vectors

[0279] The present invention provides a nucleic acid or multiple nucleic acids encoding the antigen-binding molecule, polypeptide, or CAR of the present invention. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0280] In some embodiments, the nucleic acid may be or may be comprised in a vector or multiple vectors. That is, the nucleotide sequence of the nucleic acid may be comprised in a vector. The antigen-binding molecule, polypeptide, or CAR of the present invention can be produced by transcribing the vector encoding the antigen-binding molecule, polypeptide, or CAR in a cell and translation after transcribing the RNA.

[0281] Accordingly, the present invention also provides one or more vectors comprising a nucleic acid or multiple nucleic acids according to the present invention. The vectors of the present invention can facilitate the delivery of the nucleic acids encoding the antigen-binding molecule, polypeptide, or CAR. The vector may be an expression vector of the elements required for expressing the nucleic acid, which nucleic acids comprise / encode the antigen-binding molecule, polypeptide, or CAR of the present invention.

[0282] The nucleic acids and vectors of the present invention can be provided in a purified or isolated form, i.e., from other nucleic acids or naturally occurring biological materials.

[0283] The nucleotide sequence may be comprised in a vector, such as an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer an exogenous nucleic acid into a cell. The vector may be a vector for nucleic acid expression in a cell. Such a vector may include a promoter sequence that is operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. According to the present invention, any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from the vector.

[0284] The term "operably linked" may include the situation where the selected nucleic acid sequence and the regulatory nucleic acid sequence (such as a promoter and / or enhancer) are covalently linked such that the expression of the nucleic acid sequence is affected or controlled by the regulatory sequence (thus forming an expression cassette). Accordingly, the regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence can affect the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[0285] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (such as retroviral vectors, such as gamma-retroviral vectors (such as murine leukemia virus (MLV)-derived vectors, 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 (such as yeast artificial chromosomes), as described, for example, in Maus et al., Annual Review of Immunology (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines (2016) 4:9, which are hereby incorporated by reference in their entirety.

[0286] In some embodiments, the vector can be a eukaryotic vector; for example, the vector contains elements necessary for protein expression from vectors in eukaryotic cells. In some embodiments, the vector can be a mammalian vector, such as containing a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0287] The constituent polypeptides of the antigen-binding molecule / CAR of the present invention can be encoded by different nucleic acids of multiple nucleic acids or encoded by multiple different vectors.

[0288] In some embodiments, the nucleic acid encodes an antigen-binding molecule or CAR as described herein. In some examples, the vector is polycistronic (such as bicistronic, tricistronic, etc.); that is, in some embodiments, the vector encodes an mRNA having multiple protein-coding regions. In some examples, the vector is bicistronic. The constituent polypeptides of the antigen-binding molecule or CAR of the present invention can be encoded by different nucleic acids or different vectors.

[0289] The present invention provides a retroviral vector containing a nucleic acid encoding an antigen-binding molecule or CAR. In some embodiments, the retroviral vector contains a nucleic acid encoding an antigen-binding molecule that binds to B7-H3. In some embodiments, the retroviral vector contains a nucleic acid encoding a B7-H3-specific CAR.

[0290] In some embodiments, the CAR or antigen-binding molecule of the present invention can be encoded by a plasmid. The plasmid can be based on plasmid pSFG (such as described in Hakre et al., Molecular Cell, October 20, 2006, 24(2):301-8, which is incorporated herein by reference in its entirety).

[0291] Production of antigen-binding molecules and polypeptides

[0292] The antigen-binding molecules, polypeptides, and CARs of the present invention can be prepared according to methods for producing polypeptides known to those skilled in the art.

[0293] Antigen-binding molecules, polypeptides, and CARs can be prepared by chemical synthesis, such as liquid-phase or solid-phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in Chandrudu et al., Molecules (2013), 18:4373 - 4388, which is hereby incorporated by reference in its entirety.

[0294] Alternatively, antigen-binding molecules, polypeptides, and CARs can be produced by recombinant expression. Molecular biology techniques suitable for the recombinant production of polypeptides are well known in the art, such as those presented 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 hereby incorporated by reference in their entirety. Frenzel et al. also described methods for the recombinant production of antigen-binding molecules in Front Immunol (2013); 4:217 and Kunert and Reinhart, Appl Microbiol Biotechnol (2016) 100:3451–3461, both of which are hereby incorporated by reference in their entirety.

[0295] In some cases, the antigen-binding molecules and CARs of the present invention are composed of multiple polypeptide chains. In such cases, the production of the antigen-binding molecule / CAR may include the transcription and translation of multiple polypeptides, and subsequent binding of the polypeptide chains to form the antigen-binding molecule / CAR.

[0296] For recombinant production according to the present invention, any cell suitable for expressing polypeptides can be used. The cells can be prokaryotic or eukaryotic. In some embodiments, the cells are prokaryotic cells, such as cells of archaea or bacteria. In some embodiments, the bacteria can be Gram-negative bacteria, such as bacteria of the Enterobacteriaceae family, such as Escherichia coli. In some embodiments, the cells are eukaryotic cells, such as yeast cells, plant cells, insect cells, or mammalian cells, such as the cells described above.

[0297] In some cases, the cells are not prokaryotic because some prokaryotic cells do not have the same folding or post-translational modifications as eukaryotic cells. Additionally, the expression levels in eukaryotes can be very high, and it may be easier to purify proteins from eukaryotes using suitable tags. Specific plasmids can also be used to enhance protein secretion into the culture medium.

[0298] In some embodiments, polypeptides can be prepared by cell-free protein synthesis (CFPS), such as according to the system described in Zemella et al., Biochemistry (2015) 16(17):2420 - 2431, which is hereby incorporated by reference in its entirety.

[0299] Production may involve the culture or fermentation of eukaryotic cells modified to express a target polypeptide. The culture or fermentation can be carried out in a bioreactor that provides appropriate nutrients, air / oxygen, and / or growth factors. The secreted protein is collected by separating the culture medium / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate 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 by reference supra).

[0300] A bioreactor includes one or more containers in which cells can be cultured. The culture in the bioreactor can be carried out continuously, with reactants flowing continuously into the reactor and the cultured cells flowing continuously out of the reactor. Alternatively, the culture can be carried out batchwise. The bioreactor monitors and controls environmental conditions such as pH, oxygen, inflow and outflow rates, and agitation within the container to provide optimal conditions for the cells being cultured.

[0301] After culturing cells that express a polypeptide, the target polypeptide is isolated. Any suitable method known in the art can be used to isolate the protein from the cells. To isolate the polypeptide, the cells need to be separated from the nutrient medium. If the polypeptide is secreted from the cells, the cells can be separated from the medium containing the secreted target polypeptide by centrifugation. If the target polypeptide aggregates within the cells, protein isolation may include centrifugation to separate the cells from the cell medium, treatment of the cell pellet with a lysis buffer, and cell disruption, for example, by sonication, rapid freeze-thaw, or osmotic lysis.

[0302] Then, the target polypeptide needs to be isolated from the supernatant or medium, which may contain other proteins and non-protein components. A common method for separating protein components from the supernatant or medium is precipitation. Proteins with different solubilities precipitate at different concentrations of a precipitating agent such as ammonium sulfate. For example, at low concentrations of the precipitating agent, water-soluble proteins can be extracted. Thus, by adding increasing concentrations of the precipitating agent, proteins with different solubilities can be distinguished. Subsequently, ammonium sulfate can be removed from the isolated proteins using dialysis.

[0303] Other methods known in the art for distinguishing different proteins, such as ion exchange chromatography and size chromatography. These can be used as alternatives to precipitation or can be carried out after precipitation.

[0304] Once the target polypeptide is isolated from the culture, it may be necessary or desirable to concentrate the polypeptide. Many methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0305] Cells comprising / expressing antigen-binding molecules and polypeptides

[0306] The present invention also provides a cell comprising or expressing the antigen-binding molecule, polypeptide or CAR of the present invention. Also provided is a cell comprising or expressing the nucleic acid, plurality of nucleic acids, vector or plurality of vectors of the present invention.

[0307] It will be understood that when a cell is referred to herein in the singular form (i.e., “a / the cell”), a plurality / population of such cells is also contemplated.

[0308] The cell may be a eukaryotic cell, such as 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 Rodentia), cat, dog, pig, sheep, goat, cow (including dairy cows, e.g., Holstein cows or any animal in the genus Bos), horse (including any animal in the family Equidae), donkey and non-human primate).

[0309] In some embodiments, the cell is or is derived from a cell type commonly used for expressing polypeptides for human therapy. Example cells are described in Kunert and Reinhart, Applied Microbiology and Biotechnology (2016) 100:3451–3461 (hereby incorporated by reference in its entirety) and include CHO, HEK293, PER.C6, NS0 and BHK cells. In a preferred embodiment, the cell is or is derived from a CHO cell.

[0310] The present invention also provides a method of producing a cell comprising one or more nucleic acids of the present invention, comprising introducing one or more nucleic acids, one or more vectors of the present invention into the cell. In some embodiments, introducing the isolated nucleic acid or vector into the cell comprises transformation, transfection, electroporation or transduction (e.g., retroviral transduction).

[0311] The present invention also provides a method of producing a cell expressing / comprising the antigen-binding molecule, polypeptide or CAR of the present invention, comprising introducing one or more nucleic acids, one or more vectors of the present invention into the cell. In some embodiments, the method further comprises culturing the cell under conditions suitable for the cell to express the nucleic acid or vector. In some embodiments, the method is performed in vitro.

[0312] The present invention also provides a cell obtainable or obtained by the method of the present invention.

[0313] Cells of the invention expressing CARs

[0314] In some aspects and embodiments, the present invention provides a cell comprising a CAR of the present invention. The CARs of the present invention can be used to generate cells expressing the CAR, such as immune cells expressing the CAR (e.g., CAR-T or CAR-NK cells).

[0315] The cells of the present invention expressing the CAR can comprise or express a nucleic acid encoding the CAR. It will be understood that a cell expressing the CAR comprises the CAR it expresses. In addition, a cell expressing a nucleic acid encoding the CAR also expresses and comprises the CAR encoded by the nucleic acid.

[0316] The cells expressing the CAR are preferably immune cells. The immune cells can be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes or monocytes. The lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells or innate lymphoid cells (ILCs), or precursors thereof. The immune cells can express, for example, CD3 polypeptides (e.g., CD3γCD3εCD3ζ or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4 or CD8. In some embodiments, the immune cells are T cells, such as CD3+ T cells. In some embodiments, the T cells are CD3+ and CD4+ T cells. In some embodiments, the T cells are CD3+ and CD8+ T cells. In some embodiments, the T cells are helper T cells (T H cells). In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)).

[0317] Aspects and embodiments of the present invention particularly relate to T cells comprising / expressing a B7-H3 specific CAR.

[0318] The immune cells useful in the methods of the present invention can be obtained from any suitable source. The source can be an animal or a human. The source is preferably a human, more preferably a non-human mammal. The source can be of any gender. The source can be a patient to be treated with adoptive cell therapy (autologous cells). Thus, the source may have been diagnosed with a disease / condition that requires treatment, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition. In certain cases, the source is a different individual from the patient to be treated (allogeneic cells). In such cases, the source is typically a healthy individual, or an individual not known to have a disease / condition or at risk of developing / contracting such a disease / condition.

[0319] In some aspects and embodiments, the immune cells can be virus-specific immune cells. As used herein, "virus-specific immune cells" refers to immune cells that are specific for a virus. Virus-specific immune cells express / contain receptors (preferably T cell receptors) capable of recognizing viral antigenic peptides (e.g., when presented by MHC molecules). Virus-specific immune cells can express / contain such receptors as a result of the expression of endogenous nucleic acids encoding such antigen receptors, or as a result of being engineered to express such receptors. The virus-specific immune cells preferably express / contain TCRs specific for peptides of viral antigens. Virus-specific T cells may exhibit certain functional properties of T cells in response to virus antigens specific for T cells, or in response to cells that contain / express the virus / antigen. In some embodiments, the properties are functional properties associated with effector T cells, such as cytotoxic T cells.

[0320] In some embodiments, virus-specific T cells can exhibit one or more of the following properties: cytotoxicity against cells that contain / express virus / T cell-specific viral antigens; 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 by virus / viral antigens specific for T cells, or in response to exposure to cells that contain / express virus / T cell-specific viral antigens.

[0321] Virus-specific T cells express / contain TCRs capable of recognizing viral antigenic peptides specific for T cells presented by appropriate MHC molecules. Virus-specific T cells can be CD4+ T cells and / or CD8+ T cells.

[0322] The virus specific to the virus-specific immune cells can be any virus. For example, the virus can be a dsDNA virus (such as adenovirus, herpesvirus, poxvirus), ssRNA virus (such as parvovirus), dsRNA virus (such as reovirus), (+)ssRNA virus (such as picornavirus, togavirus), (-)ssRNA virus (such as orthomyxovirus, rhabdovirus), ssRNA-RT virus (such as retrovirus) or dsDNA-RT virus (such as hepadnavirus). In particular, the present invention contemplates viruses of the families Adenoviridae, Herpesviridae, Poxviridae, Polyomaviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepadnaviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae. In some embodiments, the virus is selected from Epstein-Barr virus, adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, 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, hantavirus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coliphage virus and Banna virus.

[0323] 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).

[0324] In some embodiments, the virus-specific immune cells can be specific to peptides / polypeptides of the virus, such as 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).

[0325] T cells that are specific for viral antigens are referred to herein as virus-specific T cells (VSTs). T cells that are specific for the antigens of a particular virus can be described as being specific for the relevant virus; for example, T cells that are specific for EBV antigens may be referred to as EBV-specific T cells or "EBVSTs".

[0326] Thus, in some embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVSTs), adenovirus-specific T cells (AdVSTs), cytomegalovirus-specific T cells (CMVSTs), human papillomavirus (HPVSTs), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B virus-specific T cells (HBVSTs), hepatitis C virus-specific T cells (HCVSTs), human immunodeficiency virus-specific T cells (HIVSTs), lymphocytic choriomeningitis virus-specific T cells (LCMVSTs), or herpes simplex virus-specific T cells (HSVSTs).

[0327] In some preferred embodiments, the virus-specific immune cells are specific for peptides / polypeptides of EBV antigens. In a preferred embodiment, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVSTs).

[0328] EBV virology is described in Stanfield and Luftiq, *F1000Research* (2017) 6:386 and Odumade et al., *Clinical Microbiology Reviews* (2011) 24(1):193-209, both of which are hereby incorporated by reference in their entirety.

[0329] 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, and then the viral gp42 interacts with MHC class II. These interactions trigger the fusion of the viral envelope with the cell membrane, enabling the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.

[0330] EBV has two replication modes; latent and lytic. The latent cycle does not result in the production of virions and can occur in B cells and epithelial cells. The EBV genomic circular DNA exists as an episome in the nucleus and is replicated by the host cell DNA polymerase. During latency, only a subset of EBV genes are expressed in one of three different patterns (termed latency programs), giving rise to different viral proteins and RNAs. For example, the latent cycle described in Amon and Farrell, Reviews in Medical Virology (2004) 15(3):149-56 is hereby incorporated by reference in its entirety.

[0331] The EBNA1 protein and the non-coding RNA EBER are expressed in all latency programs I - III. Latency programs II and III also involve the expression of the EBNALP, LMP1, LMP2A, and LMP2B proteins, and latency program III also involves the expression of EBNA2, EBNA3A, EBNA3B, and EBNA3C.

[0332] EBNA1 is multifunctional and plays roles in gene regulation, episomal replication, and maintenance of the EBV episomal genome through positive and negative regulation of viral promoters (Duellman et al., Journal of General Virology (2009);90(Pt 9):2251–2259). EBNA2 is involved in the regulation of latent viral transcription and contributes to the immortalization of EBV-infected cells (Kempkes and Ling, Current Topics in Microbiology and Immunology (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 to affect gene expression and contribute to the survival and growth 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., Journal of Biomedical Science (2003) 10(5):490–504). LMP2A and LMP2B inhibit normal B cell signal transduction by mimicking the activated B cell receptor (Portis and Longnecker, Oncogene (2004) 23(53):8619–8628). EBER forms ribonucleoprotein complexes with host cell proteins and is thought to play a role in cell transformation.

[0333] The latency cycle can proceed according to any of the latency programs I to III in B cells and typically goes from III to II and then to I. After infecting resting naive B cells, EBV enters the latency program III. The expression of latency III genes activates the B cells, which become proliferating blasts. Then, EBV typically progresses to latency II by restricting the expression of a subset of genes, which causes the primitive cells to differentiate into memory B cells. Further restriction of gene expression leads to EBV entering latency I. EBNA1 expression allows EBV to replicate when memory B cells divide. In epithelial cells, only latency II occurs.

[0334] In 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, subsequent replication in epithelial cells, and release of infectious virus into saliva. EBV latency III and II infections of B lymphocytes, latency II infection of oral epithelial cells, and latency II infection of NK or T cells can lead to malignancies characterized by the presence and consistent gene expression of the EBV genome.

[0335] Latent EBV in B cells can be reactivated to switch to lytic replication. The lytic cycle results in the production of infectious virus particles and can occur in in situ B cells and epithelial cells, as described by Kenney in chapter 25 of Arvin et al., Human Herpesviruses: Biology, Therapy, and Immunoprevention, Cambridge University Press (2007), which is hereby incorporated by reference in its entirety.

[0336] Lytic replication requires the EBV genome to be linear. The latent EBV genome is episomal and thus must be linearized for lytic reactivation. In B cells, lytic replication typically occurs only after latency reactivation.

[0337] Immediate-early lytic gene products (such as BZFL1 and BRLF1) act as transactivators, enhancing their own expression and the expression of late lytic cycle genes.

[0338] Early lytic gene products play roles in viral replication (e.g., EBV DNA polymerase catalyzes BALF5; DNA polymerase processes BMRF1 factor, DNA-binding BALF2 protein, BBLF4 helicase, BSLF1 primase, and primase-associated protein BBLF2 / 3) and deoxynucleotide metabolism (e.g., thymidine kinase BXLF1, dUTPase BORF2). Other early lytic gene products act as transcription factors (e.g., BMRF1, BRRF1), play roles in RNA stability and processing (e.g., BMLF1), or are involved in immune evasion (e.g., BHRF1, which inhibits apoptosis).

[0339] Late lytic gene products are traditionally classified as products expressed after the onset of viral replication. They typically encode structural components of the virion, such as nucleocapsid proteins, as well as glycoproteins that mediate EBV binding and fusion (e.g., gp350 / 220, gp85, gp42, gp25). Other late lytic gene products play a role in immune evasion; BCLF1 encodes the viral homolog of IL-10, and BALF1 encodes a protein homologous to the anti-apoptotic protein Bcl2.

[0340] As used herein, "EBV-specific immune cells" refers to immune cells that are specific for Epstein-Barr virus (EBV). EBV-specific immune cells express / contain receptors (preferably T cell receptors) capable of recognizing EBV antigen peptides (e.g., when presented by MHC molecules). EBV-specific immune cells preferably express / contain TCRs specific for EBV antigen peptides presented by MHC class I.

[0341] In some embodiments, the EBV-specific immune cells are T cells, such as 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 helper T cells (T H cells). In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)).

[0342] EBV-specific T cells preferably express / contain TCRs capable of recognizing the EBV antigen peptides specific to them, when presented by appropriate MHC molecules. EBV-specific T cells can be CD4+ T cells and / or CD8+ T cells.

[0343] EBV-specific immune cells can be specific for any EBV antigen, such as the EBV antigens described herein. A population of EBV-specific immune cells, or a composition comprising multiple EBV-specific immune cells, can comprise immune cells specific for one or more EBV antigens.

[0344] 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).

[0345] In some embodiments, according to various aspects of the present invention, a cell can comprise / express multiple (e.g., 2, 3, 4, etc.) CARs.

[0346] In some embodiments, the cell can comprise / express multiple different CARs. A cell that comprises / expresses multiple different CARs can comprise / express CARs that are specific for different target antigens. In some embodiments, each different target antigen is independently a cancer cell antigen as described herein.

[0347] Functional properties of cells expressing the CARs of the invention

[0348] A cell expressing a CAR according to the present invention (e.g., an immune cell, such as a T cell) can exhibit certain functional properties in response to B7-H3 or in response to a cell that comprises / expresses B7-H3. In some embodiments, the functional properties are functional properties associated with effector T cells, such as cytotoxic T cells.

[0349] A cell comprising a CAR / nucleic acid encoding a CAR according to the present invention can display one or more of the following properties:

[0350] Express one or more cytotoxic / effector factors (e.g., IFNγ, TNFα, GM-CSF), proliferation / population growth and / or growth factors (e.g., IL-2) in response to a cell expressing B7-H3;

[0351] Be cytotoxic to a cell expressing B7-H3;

[0352] It has no cytotoxicity to cells that do not express B7-H3 (i.e., above baseline); and / or

[0353] It has anti-cancer activity against cancers containing cells that express B7-H3 (such as cytotoxicity to cancer cells, tumor growth inhibition, reduction of tumor burden, reduction of metastasis, etc.).

[0354] In some embodiments, T cells expressing a B7-H3-specific CAR can exhibit one or more of the following characteristics: cytotoxicity to 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 B7-H3 stimulation, or in response to exposure to cells containing / expressing B7-H3;;; proliferation / population growth in response to B7-H3 stimulation, or in response to exposure to cells containing / expressing B7-H3; cytotoxicity to cancer cells expressing B7-H3; inhibition of tumor growth of cancers containing cells expressing B7-H3; reduction of tumor burden in cancer subjects containing cells expressing B7-H3; reduction of metastasis of cancers containing cells expressing B7-H3.

[0355] In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show cytotoxicity to A549 cells, MDA-MB-231 cells, and / or THP-1 cells. In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show cytotoxicity to colorectal cancer cells (such as DLD-1 cells, HT29 cells, and / or SW480 cells). In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show cytotoxicity to gastric cancer cells (such as NCI-N87 cells, MKN7 cells, and / or MKN45 cells). In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show cytotoxicity to breast cancer cells (such as MDA-MB-231 cells and / or MDA-MB-468 cells). In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show cytotoxicity to lung cancer cells (such as A549 cells, H1299 cells, H23 cells, and / or H5967 cells).

[0356] In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show continuous killing of cells expressing B7-H3, such as cancer cells expressing B7-H3. In some embodiments, cells containing the CAR / nucleic acid encoding the CAR according to the present invention show continuous killing of THP-1 cells, DLD-1 cells, HT29 cells, and / or NCI-N87 cells.

[0357] Cell proliferation / population growth can be studied by analyzing cell division or the number of cells over a period of time. Cell division can be analyzed, for example, by in vitro analysis through incorporation of 3 ³H-thymidine or by CFSE dilution assays, as described, for example, in Fulcher and Wong, Immunology and Cell Biology (1999) 77(6):559-564, which is hereby incorporated by reference in its entirety. Proliferating cells can also be identified by analyzing the incorporation of 5-ethynyl-2′-deoxyuridine (EdU), as described in Buck et al., Biotechnology, June 2008; 44(7):927-9 and Sali and Mitchison, PNAS USA, February 19, 2008; 105(7):2415–2420, which is hereby incorporated by reference in its entirety.

[0358] As used herein, "expression" can be gene or protein expression. Gene expression includes transcription of DNA to RNA and can be measured by various methods known to those skilled in the art, e.g., by measuring mRNA levels by quantitative real-time PCR (qRT-PCR) or reporter gene-based methods. Similarly, protein expression can be measured by various methods known in the art, e.g., by antibody-based methods, e.g., by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISA immunospot, or reporter gene-based methods.

[0359] Cytotoxicity and cell killing can be studied by, for example, using any method described in Zaritskaya et al., Expert Review of Vaccines (2011), 9(6):601-616, which is hereby incorporated by reference in its entirety. In vitro assays for cytotoxicity / cell killing assays include release assays, e.g., 51 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 of a given cell type can be analyzed, for example, by co-culturing test cells with a given cell type and measuring the number / ratio of live / dead test cells after an appropriate time.

[0360] In some embodiments, the killing of cells expressing B7-H3 by cells expressing CAR can be evaluated by flow cytometry as described in Example 1.13 herein or by the xCELLigence assay as described in Example 1.14. The killing of CAR-expressing cells can also be evaluated in vivo, for example, by assessing the number / proportion of cells expressing the CAR target antigen and inferring the extent to which they are killed / depleted by the CAR-expressing cells.

[0361] The anti-cancer activity of the cells (such as cytotoxicity to cancer cells, tumor growth inhibition, reduction of tumor burden, reduction of metastasis, etc.) can be evaluated in an appropriate in vitro assay or in vivo model of the relevant cancer. For example, the cells can be evaluated in a xenograft model derived from a cancer cell line, and such an analysis can be performed as described, for example, in Example 1.16 herein. By way of illustration, the examples of the present invention show that after administration of the immune cells expressing B7-H3-specific CAR of the present invention, tumor growth is inhibited and tumor burden is reduced in xenograft models derived from the HT29, SW-480, and N-87 cell lines of colorectal cancer and gastric cancer.

[0362] Production of cells expressing the CARs of the invention

[0363] Methods for producing cells expressing CAR are well known to those skilled in the art. They generally involve modifying cells (such as immune cells, such as T cells or NK cells) to express / comprise CAR, for example, by transferring a nucleic acid encoding CAR into the immune cells.

[0364] Immune cells can be modified to contain CAR or to express a nucleic acid encoding the CAR described herein according to methods well known to those skilled in the art. The methods generally include nucleic acid transfer for permanent (stable) or transient expression of the transferred nucleic acid.

[0365] Any suitable genetic engineering platform can be used to modify the cells of the present invention. Suitable methods for modifying cells include using genetic engineering platforms such as γ-retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, as described, for example, in Maus et al., Annual Review of Immunology (2014) 32:189-225, which is hereby incorporated by reference in its entirety.

[0366] The methods also include the methods described in Wang and Rivière, Molecular Therapy - Oncolytics (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0367] Methods for the in vitro / in ex vivo production / expansion of populations of immune cells expressing CARs are well known to the person skilled in the art. Suitable culture conditions (i.e., cell culture medium, additives, stimulation, temperature, gas environment), cell number, culture period, and methods for introducing nucleic acids encoding CARs into cells can be determined by reference to Hombach et al., Journal of Immunology (2001) 167: 6123-6131, Ramos et al., Journal of Clinical Investigation (2017) 127(9): 3462-3471, and WO2015 / 028444A1, all of which are incorporated herein by reference in their entirety.

[0368] Conveniently, cell cultures according to the present invention can be maintained in a humid environment containing 5% CO 2 at 37°C. The cells of the cell culture can be established and / or maintained at any suitable density, as can be readily determined by the person skilled in the art.

[0369] Cultivation can be carried out in any container suitable for the volume of the culture, for example, in the wells of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, the cells are cultured in a bioreactor, such as the bioreactor described in Somerville and Dudley, Cancer Immunology (2012) 1(8): 1435-1437, which is hereby incorporated by reference in its entirety. In some embodiments, the cells are cultured in a GRex cell culture container, such as a GRex culture flask or a GRex100 bioreactor.

[0370] Immune cells (such as T cells) may be activated prior to the introduction of nucleic acids encoding CARs. For example, in the presence of IL-2, T cells in a population of PBMCs can be non-specifically activated by stimulation with agonist anti-CD3 and agonist anti-CD28 antibodies in vitro.

[0371] The introduction of nucleic acids / vectors into cells may include transduction, such as retroviral transduction. Thus, in some embodiments, the nucleic acid is contained in a viral vector, or the vector is a viral vector. For example, the transduction of immune cells with viral vectors is described in Simmons and Alberola-Ila, Methods in Molecular Biology (2016) 1323: 99-108, which is hereby incorporated by reference in its entirety.

[0372] Reagents can be used to enhance transduction efficiency. Hexamethyl-phenanthridinium bromide (polyisoprene) is a cationic polymer commonly used to improve transduction by neutralizing the charge repulsion between viral particles and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), as well as SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0373] In some embodiments, the method includes centrifuging the cells to be introduced with the CAR-encoding nucleic acid (referred to in the art as "spin infection") in the presence of a cell culture medium containing a viral vector carrying the nucleic acid.

[0374] In some embodiments, the method includes introducing the nucleic acid or vector of the present invention into immune cells by electroporation, such as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety.

[0375] The method generally includes introducing a nucleic acid encoding a CAR into cells and culturing the cells under conditions suitable for the cells to express the nucleic acid / CAR. In some embodiments, the method includes culturing immune cells that have been introduced with a nucleic acid encoding a CAR to expand. In some embodiments, the method includes culturing immune cells introduced with a nucleic acid encoding a CAR in the presence of IL-7 and / or IL-15 (such as recombinant IL-7 and / or IL-15).

[0376] In some embodiments, the method further includes purifying / isolating the cells expressing the CAR, for example, from other cells (e.g., cells that do not express the CAR). Methods for purifying / isolating immune cells from a heterogeneous cell population are well known in the art and can employ, for example, FACS- or MACS-based methods to classify the cell population based on the expression of immune cell markers. In some embodiments, the method is used to purify / isolating a specific type of cell, such as CAR-expressing CD8+ T cells, CAR-expressing CTLs.

[0377] In a preferred embodiment, T cells expressing a B7-H3-specific CAR can be generated from T cells within a PBMC population by the following process: stimulating the PBMCs with antagonist anti-CD3 and anti-CD28 antibodies, transducing the cells with a viral vector (such as a γ-retroviral vector) encoding the B7-H3-specific CAR, and subsequently culturing the cells in the presence of IL-7 and IL-15.

[0378] Aspects and embodiments of the present invention relate particularly to EBV - specific immune cells. Methods for generating / amplifying a population of EBV - specific immune cells are described, for example, in WO2013 / 088114A1, Lapteva and Vera, International Stem Cells (2011): 434392, Straathof et al., Blood (2005) 105(5): 1898–1904, WO2017 / 202478A1, WO2018 / 052947A1, and WO2020 / 214479A1, which are hereby incorporated by reference in their entirety. Typical steps of the methods include stimulating virus / virus - antigen - specific immune cells by contacting a population of immune cells with EBV - antigen - corresponding peptide segments or antigen - presenting cells (APCs) presenting virus - antigen - corresponding peptide segments.

[0379] The present invention also provides cells and populations thereof obtained or obtainable by the methods described herein.

[0380] Compositions

[0381] The present invention also provides compositions comprising the antigen - binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[0382] The antigen - binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein can be formulated into pharmaceutical compositions or drugs for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

[0383] The compositions of the present invention may include one or more pharmaceutically acceptable carriers (such as liposomes, micelles, microspheres, nanoparticles), diluents / excipients (such as starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (such as vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), antioxidants (such as vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (such as magnesium stearate, talc, silica, stearic acid, vegetable stearic acid), binders (such as sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (such as wetting agents), masking agents, or colorants (such as titanium oxide).

[0384] As used herein, the term "pharmaceutically acceptable" means a compound, ingredient, material, composition, dosage form, etc. that is suitable for contact with the tissues of a relevant subject (e.g., a human subject) within the scope of reasonable medical judgment, without producing excessive toxicity, irritation, allergic reactions or other problems or complications, and is commensurate with a reasonable benefit / risk ratio. According to the present invention, each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, coloring agent, flavoring agent or sweetening agent of the composition must also 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, coloring agents, flavoring agents or sweetening agents can be found in standard pharmaceutical texts, e.g., Remington: The Science and Practice of Pharmacy (edited by A. Adejare), 23rd Edition (2020), Academic Press.

[0385] The composition can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intracorneal, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, the pharmaceutical composition / drug can be formulated for administration by injection or infusion, or by ingestion.

[0386] Suitable formulations can include the relevant articles in a sterile or isotonic medium. The drug and pharmaceutical composition can be formulated as a liquid, including in gel form. The liquid formulation can be formulated for administration by injection or infusion (e.g., through a catheter) to a selected area of the human or animal body.

[0387] In some embodiments, the composition is formulated for injection or infusion, e.g., into a blood vessel, a tissue / organ of interest or a tumor.

[0388] The present invention also provides a method for producing a composition having pharmaceutical efficacy. Such a production method can include one or more steps selected from the group consisting of: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or multiple thereof), expression vector (or multiple thereof) or cell of the present invention; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid (or multiple thereof), expression vector (or multiple thereof) or cell of the present invention; and / or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or multiple thereof), expression vector (or multiple thereof) or cell of the present invention with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0389] For example, another aspect of the present invention relates to a method of formulating or producing a drug or pharmaceutical composition for treating a disease / condition (such as cancer), the method comprising formulating a pharmaceutical composition or drug by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or pluralities thereof), expression vector (or pluralities thereof), or cell of the present invention with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0390] Therapeutic and prophylactic applications

[0391] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions of the present invention can be used in therapeutic and prophylactic methods.

[0392] The present invention provides antigen-binding molecules, polypeptides, CARs, nucleic acids (or pluralities thereof), expression vectors (or pluralities thereof), cells, or compositions for use in methods of medical treatment or prophylaxis. Also provided are the antigen-binding molecules, polypeptides, CARs, nucleic acids (or pluralities thereof), expression vectors (or pluralities thereof), cells, or compositions of the present invention for use in methods of treating or preventing a disease or condition described herein. Also provided is the use of the antigen-binding molecule, polypeptide, chimeric antigen receptor (CAR), nucleic acid (or pluralities thereof), expression vector (or pluralities thereof), cell, or composition of the present invention in the preparation of a medicament for treating or preventing a disease or condition of the present invention. Furthermore, provided is a method of treating or preventing a disease or condition of the present invention, the method comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the antigen-binding molecule, polypeptide, chimeric antigen receptor (CAR), nucleic acid (or pluralities thereof), expression vector (or pluralities thereof), cell, or composition of the present invention.

[0393] The method may be effective in reducing the development or progression of the disease / condition, alleviating the symptoms of the disease / condition, or reducing the pathology of the disease / condition. The method may be effective in preventing the progression of the disease / condition, for example, preventing the disease / condition from worsening or slowing the rate of development of the disease / condition. In some embodiments, the method may result in an improvement of the disease / condition, such as a reduction in the symptoms of the disease / condition or a reduction in other relevant factors of the severity / activity of the disease / condition. In some embodiments, the method may prevent the development of the disease / condition at a later stage (e.g., chronic stage or metastasis).

[0394] It is understood that the articles of the present invention can be used to treat and prevent any disease / condition that derives a therapeutic or prophylactic benefit from a reduction in B7-H3 level / activity, or a reduction in the number or activity of cells that contain / express B7-H3.

[0395] For example, the disease / condition may be a disease / condition that is pathologically associated with B7-H3 or cells containing / expressing B7-H3, such as a disease / condition in which an increase in B7-H3 level / activity, or an increase in the number / proportion of cells containing / expressing B7-H3, is positively correlated 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, an increase in the level / activity of B7-H3, or an increase in the number / proportion of cells containing / expressing B7-H3, may be a risk factor for the occurrence, development, or progression of the disease / condition.

[0396] In some embodiments, the disease / condition to be treated / prevented according to the present invention is a disease / condition characterized by an increased level of expression or activity of B7-H3, e.g., compared to the expression / activity level in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterized by an increase in the 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 equivalent non-diseased tissue). If the disease / condition is cancer, the level of expression or activity of B7-H3 may be greater than the level of expression or activity of B7-H3 in equivalent non-cancerous cells / non-tumor tissue. The cancer / its cells may contain one or more mutations (e.g., relative to equivalent non-cancerous cells / non-tumor tissue) that result in upregulation of the expression or activity of B7-H3.

[0397] Treatment according to the methods of the present invention can achieve one or more of the following in a subject (compared to an equivalent untreated subject, or compared to a subject receiving appropriate control treatment): a decrease in B7-H3 level; a decrease in B7-H3 activity; and / or a decrease in the number / proportion of cells containing / expressing B7-H3.

[0398] In aspects and embodiments according to the present invention, there is provided the use of cells (in particular immune cells, more specifically T cells) containing / expressing a CAR according to the present invention for therapeutic and prophylactic use. It will be understood that the methods generally comprise administering to a subject a population of immune cells expressing a CAR according to the present invention. In some embodiments, the immune cells expressing a CAR according to the present invention can be administered in the form of a pharmaceutical composition containing such cells.

[0399] In particular, the immune cells expressing a CAR of the present invention are used in a method for treating / preventing a disease / condition by adoptive cell transfer (ACT).

[0400] Adoptive cell transfer generally refers to the process of obtaining cells (such as immune cells) from a subject, typically by drawing a blood sample and isolating the cells therefrom. The cells are then typically modified and / or expanded and then administered to the same subject (in the case of autologous / self-gene cell adoptive transfer) or a different subject (in the case of allogeneic cell adoptive transfer). This treatment generally aims to provide the subject with a population of cells having certain desired characteristics or to increase the frequency of such cells having such characteristics in the subject. The purpose of performing adoptive transfer is to introduce cells or a population of cells into the subject and / or to increase the frequency of cells or a population of cells in the subject.

[0401] For example, the adoptive transfer of immune cells is described 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 hereby incorporated by reference in their entirety. Those skilled in the art are able to determine the appropriate reagents and procedures for adoptive cell transfer according to the present invention, for example, referring to Dai et al., Journal of the National Cancer Institute 108(7):djv439 in 2016, which is incorporated by reference in its entirety.

[0402] The immune cells expressing CAR according to the present invention can be used for treating / preventing diseases / conditions by allogeneic transplantation or autologous transplantation.

[0403] As used herein, "allogeneic transplantation" refers to the transplantation of cells, tissues or organs that are genetically different from the recipient subject into the recipient subject. The cells, tissues or organs may be from or may be derived from cells, tissues or organs of a donor subject that is not genetically identical to the recipient subject. Allogeneic transplantation is different from autologous transplantation, which refers to the transplantation of tissue from / derived from a donor subject that is genetically identical to the recipient subject (i.e., autologous material). It is understood that adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation, while adoptive transfer of autologous immune cells is a form of autologous transplantation.

[0404] The methods provided by the present invention include administering immune cells comprising / expressing CAR according to the present invention, or immune cells comprising / expressing a nucleic acid encoding CAR according to the present invention.

[0405] In some embodiments, the method includes modifying the immune cells of the present invention to comprise / express CAR. In some embodiments, the method includes modifying immune cells specific to a virus to comprise / express a nucleic acid encoding CAR according to the present disclosure.

[0406] In some embodiments, the method includes:

[0407] (a) Modifying immune cells to express or comprise a CAR according to the present invention, or to express or comprise a nucleic acid encoding a CAR according to the present invention, and

[0408] (b) Administering to a subject virus-specific immune cells modified to express or comprise a CAR as described in the present invention, or virus-specific immune cells modified to express or comprise a nucleic acid encoding a CAR as described in the present invention.

[0409] In some embodiments, the method comprises:

[0410] (a) Isolating or obtaining immune cells;

[0411] (b) Modifying immune cells to express or comprise a CAR according to the present invention, or to express or comprise a nucleic acid encoding a CAR of the present invention, and

[0412] (c) Administering to a subject immune cells modified to express or comprise a CAR as described in the present invention, or virus-specific immune cells modified to express or comprise a nucleic acid encoding a CAR as described in the present invention.

[0413] In some embodiments, the method comprises:

[0414] (a) Isolating immune cells (e.g., PBMCs) from a subject;

[0415] (b) Generating / amplifying a population of virus-specific immune cells;

[0416] (c) Modifying immune cells to express or comprise a CAR according to the present invention, or to express or comprise a nucleic acid encoding a CAR of the present invention, and

[0417] (d) Administering to a subject virus-specific immune cells modified to express or comprise a CAR as described in the present invention, or virus-specific immune cells modified to express or comprise a nucleic acid encoding a CAR as described in the present invention.

[0418] In some embodiments, the method comprises administering to a subject EBV-specific immune cells modified to express or comprise a B7-H3-specific CAR of the present invention, or expressing or comprising a nucleic acid encoding a B7-H3-specific CAR of the present invention.

[0419] In some embodiments, the subject from whom the immune cells (e.g., PBMCs) are isolated is the same as the subject to whom the cells are administered (i.e., adoptive transfer can be autologous / self-genetic cells). In some embodiments, the subject from whom the immune cells (e.g., PBMCs) are isolated is different from the subject to whom the cells are administered (i.e., adoptive transfer can be allogeneic cells).

[0420] In some embodiments, the method may comprise one or more of the following:

[0421] Obtain a blood sample from a subject;

[0422] Isolate immune cells (e.g., PBMC) from the blood sample obtained from the subject;

[0423] Produce / amplify a population of immune cells;

[0424] Culture the immune cells in vitro or in an ex vivo cell culture;

[0425] Modify the immune cells to express or comprise the CAR of the present invention, or to express or comprise a nucleic acid encoding the CAR of the present invention (e.g., by transduction with a viral vector encoding such CAR or a viral vector comprising such nucleic acid);

[0426] Culture the immune cells expressing / comprising the CAR according to the present invention, or the nucleic acid expressing / comprising the encoding CAR according to the present invention, in vitro or in an ex vivo cell culture;

[0427] Collect / isolate the immune cells expressing / comprising the CAR of the present invention, or the nucleic acid expressing / comprising the encoding CAR of the present invention;

[0428] Formulate the immune cells expressing / comprising the CAR of the present invention, or the nucleic acid encoding the CAR of the present invention, into a pharmaceutical composition, e.g., by mixing the cells with a pharmaceutically acceptable adjuvant, diluent or carrier;

[0429] Administer the immune cells expressing / comprising the CAR of the present invention, or the nucleic acid expressing / comprising the encoding CAR according to the present invention, or a pharmaceutical composition comprising such cells, to a subject.

[0430] In some embodiments, the method further comprises treating the cells or inducing / enhancing the expression of the CAR and / or inducing / enhancing the proliferation or survival of virus-specific immune cells comprising / expressing the CAR.

[0431] Cancer

[0432] In some embodiments, the disease to be treated / prevented according to the present invention is cancer.

[0433] B7-H3 expression and B7-H3-mediated signal transduction are associated with the pathogenesis of various cancers. B7-H3 promotes cancer cell migration and invasion, thereby facilitating metastasis. B7-H3 expression and signaling and their roles in diseases are described, for example, in Wu-Tong ZHOU and Wei-Lin Jin, Frontiers in Immunology (2021) 12:701006, Dong et al., Frontiers in Oncology (2018) 8:264, and Yang et al., International Journal of Biosciences (2020) 16(11):1767–1773, which are hereby incorporated herein by reference in their entireties.

[0434] Cancer may refer to any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm, or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and may be located in any tissue. Cancer may be or include solid cancer (such as a tumor) or may be a hematological cancer. The cancer may originate from the following tissues / cells: for example, adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or not including the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), 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, tonsil, trachea, uterus, vulva, and / or white blood cells.

[0435] Tumors can be neural or non-neural system tumors. Neural system tumors may originate from the central nervous system or the peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-neural system cancers / tumors may originate from any other non-neural tissue, such as melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0436] In some embodiments, the cancer is a cancer that is pathologically associated with B7-H3. That is, in some embodiments, the cancer is a cancer caused or exacerbated by the expression of B7-H3, a cancer in which the expression of B7-H3 is a risk factor, and / or a cancer in which the expression of B7-H3 is positively correlated with the onset, development, progression, severity, or metastasis of the cancer. The cancer may be characterized by the expression of B7-H3, for example, the cancer may include cells that express B7-H3. Such cancers may be referred to as B7-H3 positive. A B7-H3 "positive" cancer may be a cancer composed of cells that express B7-H3 (e.g., on the cell surface). A B7-H3 "positive" cancer may overexpress B7-H3.

[0437] Cancers pathologically related to B7-H3 are described in Yang et al., International Journal of Biology 2020;16(11):1767–1773 and Dong et al., Frontiers in Oncology (2018) 8:264 and 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 (such as triple-negative breast cancer), head and neck cancer (such as head and neck squamous cell carcinoma), oral cancer (such as oral squamous cell carcinoma), esophageal cancer, bladder cancer, urothelial cancer, brain cancer (such as medulloblastoma (e.g., ependymoblastoma medulloblastoma), glioma (e.g., diffuse pontine glioma, diffuse midline glioma), choroid plexus carcinoma, pineoblastoma), neuroblastoma, central nervous system tumors (such as primitive neuroectodermal tumors, atypical teratoid / rhabdoid tumors), brainstem glioma, sarcoma (e.g., rhabdomyosarcoma, osteosarcoma, Ewing sarcoma), peritoneal cancer, desmoplastic small round cell tumor, and mesothelioma.

[0438] In some embodiments, the cancer to be treated / prevented 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, renal cancer, clear cell renal cell carcinoma, nephroblastoma, prostate 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, oropharyngeal cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer, urothelial carcinoma, brain cancer, medulloblastoma, ependymoblastoma, glioma, diffuse pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineoblastoma, neuroblastoma, central nervous system tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brainstem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, peritoneal cancer, desmoplastic small round cell tumor, and mesothelioma.

[0439] In some embodiments, the cancer to be treated / prevented in the present invention is selected from the group consisting of: B7-H3 positive cancer, lung cancer, non-small cell lung cancer, skin cancer, cutaneous squamous cell carcinoma, melanoma, pancreatic cancer, liver cancer, hepatocellular carcinoma, intrahepatic cholangiocarcinoma, colorectal cancer, colon cancer, renal cancer, clear cell renal cell carcinoma, nephroblastoma, 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 squamous cell carcinoma, esophageal cancer, bladder cancer, urothelial carcinoma, brain cancer, medulloblastoma, ependymoblastoma, glioma, diffuse pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineoblastoma, neuroblastoma, CNS tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brainstem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, peritoneal cancer, desmoplastic small round cell tumor, and mesothelioma.

[0440] In some embodiments, the cancer may be a recurrent cancer. As used herein, a "recurrent" cancer refers to a cancer that responds to treatment (e.g., first-line treatment of the cancer), but then reappears / progresses, e.g., after a remission period. For example, a recurrent cancer may be a cancer whose growth / progression is inhibited by treatment (e.g., first-line treatment of the cancer) and then grows / progresses.

[0441] In some embodiments, the cancer can be a refractory cancer. As used herein, a "refractory" cancer refers to a cancer that does not respond to treatment (e.g., first-line treatment of cancer). For example, a refractory cancer may be a cancer in which treatment (e.g., first-line treatment of cancer) does not inhibit its growth / progression. In some embodiments, a refractory cancer can be a cancer in which a subject receiving cancer treatment does not exhibit a partial or complete response to the treatment.

[0442] Treatment of cancer according to the methods of the present invention can achieve one or more of the following therapeutic effects: 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 / lesions 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 development / progression of cancer (e.g., to an advanced stage or metastasis), reducing the severity of cancer symptoms in a subject, increasing the survival rate of a subject (e.g., progression-free survival or overall survival), reducing the correlation of the number or activity of cancer cells in a subject, and / or reducing the cancer burden of a subject.

[0443] Subjects can be evaluated to determine their response to treatment according to the Revised Response Evaluation Criteria: Lugano Classification (e.g., as described in Cheson et al., Journal of Clinical Oncology (2014) 32:3059-3068, incorporated herein by reference). In some embodiments, treatment of a subject according to the methods of the present invention results in one of the following: complete response, partial response, or stable disease.

[0444] Administration

[0445] In the present invention, administration is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to demonstrate a therapeutic or prophylactic benefit to the subject. The actual amount administered, the rate of administration, and the time course will depend on the nature and severity of the disease / condition and the particular article being administered. It is the responsibility of general practitioners and other physicians to determine the treatment prescription, e.g., decisions regarding dosage, etc., and generally take into account the disease / condition to be treated, which is known to the practitioner, the condition of the individual subject, the site of administration, the method of administration, and other factors. Examples of the above techniques and protocols can be found in Remington: The Science and Practice of Pharmacy (edited by A. Adejare), 23rd Edition (2020), Academic Press.

[0446] Administration in the present invention can be local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intracorneal, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, or transdermal. Administration can be by injection or infusion. Administration in the present invention can be intratumoral administration.

[0447] In certain aspects and embodiments of the present invention, the compositions of the present invention can be administered in a targeted manner, i.e., such that the concentration of the relevant agent in certain parts of the body is increased relative to other parts of the body. In some embodiments, the method includes intravenous, intraarterial, intramuscular, or subcutaneous administration, wherein the relevant composition is formulated in 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 silica rods, etc. The system can include a magnetic element for guiding the agent to the desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to those skilled in the art.

[0448] In some cases, the compositions of the present invention are formulated for targeted delivery to specific cells, tissues, organs, and / or tumors.

[0449] Further interventions

[0450] The administration can be administered alone or in combination with other therapies, depending on the disease / condition to be treated, simultaneously or sequentially. The antigen-binding molecules, CARs, cells or compositions, and another prophylactic / therapeutic agent described herein can be administered simultaneously or sequentially.

[0451] In some embodiments, the method includes additional therapeutic or prophylactic interventions, e.g., for treating / preventing cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination, and / or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention includes leukapheresis. In some embodiments, the therapeutic or prophylactic intervention includes stem cell transplantation.

[0452] Simultaneous administration refers to administering the antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell or composition, and the therapeutic agent together, e.g., as a pharmaceutical composition (combination product) containing both drugs, or immediately following one another, optionally by the same route of administration, e.g., the same artery, vein, or other blood vessel. Sequential administration refers to administering one of another antigen-binding molecule / composition or therapeutic agent after a given time interval. It is not required that the two formulations be administered by the same route, although this may be the case in certain embodiments. The time interval can be any time interval.

[0453] In some embodiments, the treatment of cancer also includes chemotherapy and / or radiotherapy. Chemotherapy and radiotherapy respectively refer to the use of drugs or ionizing radiation to treat cancer (e.g., radiotherapy using X-rays or gamma rays). The drugs can be chemical entities, such as small molecule drugs, antibiotics, DNA intercalators, protein inhibitors (e.g., kinase inhibitors), or biological agents, such as antibodies, antibody fragments, aptamers, nucleic acids (e.g., DNA, RNA), peptides, polypeptides, or proteins. The drugs can be formulated into pharmaceutical compositions or medicaments. The formulations can include one or more drugs (e.g., one or more active agents) and one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0454] Chemotherapy may involve the administration of more than one drug. The drugs can be administered alone or in combination with other treatments, depending on the disease to be treated, either simultaneously or sequentially.

[0455] Chemotherapy can be administered by one or more routes of administration, such as parenterally, intravenously, orally, subcutaneously, intradermally, or intratumorally.

[0456] Chemotherapy can be carried out according to a treatment regimen. The treatment regimen can be a pre-determined chemotherapy schedule, plan, protocol, or timetable, which can be prepared by a doctor or a practicing physician and can be customized according to the patient to be treated. The treatment regimen may indicate one or more of the following: the type of chemotherapy to be administered to the patient; the dose of each drug or radiation; the time interval between administrations; the duration of each treatment; the number and nature of any treatment holidays (if any), etc. For combination therapies, a single treatment regimen may be provided, specifying how each drug is to be administered.

[0457] Chemotherapeutic drugs can be selected from: abemaciclib, abiraterone acetate, abiquimod (methotrexate), albumin-bound paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, doxorubicin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), aprepitant / fosaprepitant combination (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, alectinib, alectinib, alemtuzumab, pemetrexed (pemetrexed disodium), copanlisib (copanlisib hydrochloride for injection), chlorambucil for injection (melphalan hydrochloride), chlorambucil tablets (melphalan), palonosetron (palonosetron hydrochloride), Alunbrig (brigatinib), chlorambucil, chlorambucil, amifostine, aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), nelarabine, arsenic trioxide, ofatumumab, asparaginase erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, acalabrutinib, acitinib, azacitidine, Bavencio (avelumab), BEACOPP, BiCNU (carmustine), Belinostat, belinostat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, blinatumomab, bortezomib, bosutinib, Bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib S-malate), cabozantinib S-malate, CAF, cabozantinib (acalabrutinib), Campath (alemtuzumab), capecitabine (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil - topical), carboplatin, carboplatin-paclitaxel, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, clofarabine (clofarabine), cladribine (clofarabine), CMF, cobimetinib,Cometinib (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Actinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Liposomal Cytarabine, Cytarabine (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dakogen (Decitabine), Actinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Liposomal Cytarabine, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Liposomal Cytarabine), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Liposomal Doxorubicin Hydrochloride), Doxorubicin Hydrochloride, Liposomal Doxorubicin Hydrochloride, Dox-SL (Liposomal Doxorubicin Hydrochloride), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil - Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eculizumab, Eloxatin (Oxaliplatin), Eltrombopag, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Vismodegib (Vismodegib), Erlotinib Hydrochloride, Irutinase (Asparaginase from Erwinia chrysanthemi), Ethiofos (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Liposomal Doxorubicin Hydrochloride), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil - Topical), Fareston (Toremifene), Faridac (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluorouracil (Fluorouracil - Topical), Fluorouracil Injection, Fluorouracil - Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, Fluorouracil, Calcium Folinate, Irinotecan and Bevacizumab Combination Chemotherapy, Fluorouracil, Calcium Folinate, Irinotecan and Cetuximab Combination Chemotherapy, folfirinox, folfox, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride,Gemcitabine-cisplatin, Gemcitabine-oxaliplatin, Gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel Wafer (carmustine implant), Glucuronidase, Goserelin acetate, Halaven (eribulin mesylate), Inderal (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV 9-valent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibrutinib-matuzumab, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), Idarubicin hydrochloride, Idelalisib, Idiopathic (enasidenib mesylate), Ifex (ifosfamide), Ifosfamide, Ifosfamide (ifosfamide), IL-2 (aldesleukin), Imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), Imiquimod, Immulytic (talimogene laherparepvec), Inlyta (axitinib), Inotuzumab ozogamicin, Interferon Alfa-2b, recombinant, Interleukin-2 (Aldesleukin), Intron A (recombinant interferon Alfa-2b), Iodine I131 Tositumomab and tositumomab, Ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Liposomal irinotecan hydrochloride, Istodax (romidepsin), Isphosphamide, Isatuximab citrate, Isphosphamide (isophosphamide), Jakafi (ruxolitinib phosphate), 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), Lenalidine, Levatinib mesylate, Lenvima (levatinib mesylate), Letrozole, Calcium folinate, Leukeran (chlorambucil), Leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Lobrizer (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-ped (leuprolide acetate)Lynparza (olaparib), Marqibo (vincristine sulfate liposome), Matulan (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), Metozolv (temozolomide), methotrexate, Methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mutamycin (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Melphalan (busulfan), Milosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), Abraxane (nab-paclitaxel), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Novantrone (mitoxantrone hydrochloride), Neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib), Nilandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olamaprelimab, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Trastuzumab - Idarucizumab (Denileukin Diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, nab-paclitaxel, PAD, palbociclib, palifermin, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, Pegaspargase, pegfilgrastim, pegylated interferon alfa-2b, PEG-Intron (pegylated interferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag ethanolamine), propranolol hydrochloride, Provenge (Sipuleucel-T)Purine ethanol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), Ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and hyaluronidase human), rituximab, rituximab and human hyaluronidase, rolapitant hydrochloride, romidepsin, romiplostim, Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib), rucaparib, ruxolitinib phosphate, Rydapt (midostaurin), sclerosing intrathoracic aerosol (talc), cetuximab, cixutumumab, Sandostatin LAR (lanreotide acetate), sonidegib, sorafenib mesylate, Sprycel (dasatinib), Stanford V regimen, sterile talc (talc), steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (cetuximab), Synribo (omacetaxine mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, cytarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, thalidomide (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Torak (fluorouracil - topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine-131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), triacetyluridine, VAC, valrubicin, Valstar (valrubicin), vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP,Velbalan (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), Venetoclax, abemaciclib, Vantas (leuprolide acetate sustained release implant), Vidaza (Azacitidine), vinblastine sulfate, vinca PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, vismodegib, Vistogard (triacetyluridine), Voraxaze (glucarpidase), volanesorsen, Votrient (pazopanib hydrochloride), Vyxeos (liposomal daunorubicin and cytarabine hydrochloride), Wellcovorin (calcium folinate), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (Axicabtagene Ciloleucel), Yondelis (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (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).

[0458] In some embodiments, the treatment can include administration of corticosteroids, e.g., dexamethasone and / or prednisone.

[0459] In some embodiments, the subject is administered lymphodepleting chemotherapy prior to administration of the immune cells expressing / comprising the CAR (or expressing / comprising the nucleic acid encoding the CAR) described herein.

[0460] That is, in some embodiments, the methods of treating / preventing a disease / condition of the invention comprise: (i) subjecting the subject to lymphodepleting chemotherapy, and (ii) subsequently administering the immune cells of the invention expressing / comprising the CAR, or the nucleic acid expressing / comprising the encoding CAR.

[0461] As used herein, "lymphodepleting chemotherapy" refers to the treatment of a subject being treated with a chemotherapeutic agent that results in lymphopenia (e.g., T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors). A "lymphodepleting chemotherapeutic agent" refers to a chemotherapeutic agent that results in lymphopenia.

[0462] Lymphodepleting chemotherapy and its use in adoptive cell transfer therapy methods, etc. are described in Klebanoff et al., Trends in Immunology (2005) 26(2):111 - 7 and Muranski et al., Nature Clinical Practice Oncology (2006) (12):668 - 81, both of which are hereby incorporated by reference in their entirety. The purpose of lymphodepleting chemotherapy is to deplete the endogenous lymphocyte population of the recipient subject.

[0463] In the case of treating a disease by adoptive transfer of immune cells, lymphodepleting chemotherapy is typically performed prior to adoptive cell transfer to enable the recipient subject to receive the adoptively transferred cells. Lymphodepleting chemotherapy is thought to promote the persistence and activity of the adoptively transferred cells by creating a favorable environment, e.g., by eliminating cells expressing immunosuppressive cytokines and creating the "lymphoid space" required for the expansion and activity of the adoptively transferred lymphocytes.

[0464] Chemotherapeutic drugs commonly used in lymphodepleting chemotherapy include fludarabine, cyclophosphamide, bendamustine, and pentostatin.

[0465] Multiple doses of antigen - binding molecules, polypeptides, CARs, nucleic acids (or multiples thereof), expression vectors (or multiples thereof), cells, or compositions can be provided. One or more or each dose may be accompanied by the simultaneous or sequential administration of another therapeutic agent.

[0466] Multiple doses can be spaced at predetermined time intervals, which can be selected from 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 one of 1, 2, 3, 4, 5, or 6 months. For example, the doses can be administered once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0467] According to various aspects of the present invention, methods of treating and / or preventing a disease / condition can include one or more of the following: reducing the number / proportion of cells expressing B7 - H3; inhibiting tumor growth (e.g., B7 - H3+ tumors); reducing cancer metastasis (e.g., B7 - H3+ cancers); increasing the survival rate of a subject with cancer (e.g., B7 - H3+ cancers).

[0468] Detection methods

[0469] The present invention also provides a method for using the articles of the present invention to detect, localize, or image B7-H3 or cells expressing B7-H3.

[0470] The antigen-binding molecules of the present invention can be used in methods involving detecting the binding of the antigen-binding molecule to B7-H3. Such methods may involve detecting a binding complex of the antigen-binding molecule and B7-H3. It is understood that B7-H3 may be B7-H3 expressed by a cell, such as B7-H3 expressed in or on the surface of a cell.

[0471] Accordingly, a method is provided that includes contacting a sample containing or suspected of containing B7-H3 and detecting the formation of a complex of the antigen-binding molecule and B7-H3. A method is also provided that includes contacting a sample containing or suspected of containing cells expressing B7-H3 and detecting the formation of a complex of the antigen-binding molecule and the cells expressing B7-H3.

[0472] Suitable method formats are well recognized in the art and include immunoassays, such as sandwich assays, such as ELISA. Such methods may involve labeling the antigen-binding molecule or the target, or a combination thereof, with a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immuno-detectable label, a radioactive label, a chemical, nucleic acid, or enzyme label as described herein. Detection techniques are well known to those skilled in the art and can be selected according to the corresponding labeling agent.

[0473] Methods for detecting B7-H3 or cells expressing B7-H3 include methods for diagnosing / predicting the diseases / conditions described herein.

[0474] Such methods can be performed on patient samples in vitro or after processing patient samples. Once the sample is collected, the patient does not need to be present for the in vitro method, and thus the method may be a method that is not used on humans or animals. In some embodiments, the method is performed in vivo.

[0475] Such methods may involve detecting or quantifying B7-H3 and / or cells expressing B7-H3, such as in a patient sample. When the method includes quantifying a relevant factor, the method may further include comparing the measured amount to a standard or reference value as part of a diagnostic or prognostic assessment. Other diagnostic / prognostic tests can be used in combination with the tests described herein to improve the accuracy of diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0476] Detection in a sample can be used to diagnose a disease / condition (such as cancer), susceptibility to a disease / condition, or prognosis (prognosis) of a disease / condition, such as the diseases / conditions described herein. The diagnosis or prognosis may be related to an existing (previously diagnosed) disease / condition.

[0477] Samples can be collected from any tissue or body fluid. The sample can include or may be derived from: a quantity of blood, a quantity of serum extracted from an individual's blood, which may include the liquid portion of the blood after removal of fibrin clots and blood cells, a tissue sample or biopsy, pleural effusion, cerebrospinal fluid (CSF), or cells isolated from the individual. In some embodiments, the sample can be taken from or derived from one or more tissues affected by a disease / condition (e.g., a tissue exhibiting disease symptoms or a tissue involved in the pathogenesis of the disease / condition).

[0478] Subjects can be selected for diagnostic / prognostic evaluation based on symptoms indicative of the disease / condition described herein, or based on the subject being considered at risk of having the disease / condition of the present invention.

[0479] The present invention also provides a method of selecting / stratifying subjects for treatment with a B7-H3 targeting agent. In some embodiments, subjects are selected for treatment / prevention according to the methods of the present invention, or based on the detection / quantification of B7-H3 or cells expressing B7-H3, e.g., in a sample obtained from an individual, and the subject is identified as a subject who will benefit from such treatment / prevention.

[0480] Subjects

[0481] According to various aspects of the present invention, the subject can be any animal or human. Treatment and prevention can be used for humans or animals (veterinary use).

[0482] Subjects to whom the articles of the present invention are administered (e.g., according to a therapeutic or prophylactic intervention) may be subjects in need of such an intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, more preferably a human. The subject can be male or female. The subject can be a patient.

[0483] The subject may have (e.g., may have been diagnosed with) the disease or condition described in the present invention, may be suspected of having such a disease / condition, or is at risk of developing / contracting such a disease / condition. In an embodiment according to the present invention, subjects can be selected for treatment according to a method based on the characteristics of one or more markers of such a disease / condition.

[0484] In some embodiments, subjects can be selected for the treatment or prophylactic intervention described in the present invention based on the detection of cells / tissues expressing B7-H3 or cells / tissues overexpressing B7-H3, e.g., in a sample obtained from the subject.

[0485] According to the present invention, the subject can be an allogeneic subject with respect to the intervention. The subject to be treated / prevented according to the present invention may be genetically different from the subject from which the CAR-expressing immune cells are derived. According to the present invention, the subject to be treated / prevented may be HLA mismatched with the subject from which the CAR-expressing immune cells are derived. According to the present invention, the subject to be treated / prevented can be HLA matched with the subject from which the immune cells expressing CAR are derived.

[0486] According to the present invention, the subject to whom the cells are administered can be allogeneic / non-autologous with respect to the cell source. The subject to whom the cells are administered may be different from the subject from whom the cells are obtained for producing the cells to be administered. The subject to whom the cells are administered may be genetically different from the subject from whom the cells are obtained / acquired for producing the cells to be administered.

[0487] The subject to whom the cells are administered can comprise MHC / HLA genes encoding MHC / HLA molecules that are not the same as the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from whom the cells are obtained for producing the cells to be administered. The subject to whom the cells are administered can comprise MHC / HLA genes encoding MHC / HLA molecules that are the same as the MHC / HL molecules encoded by the MHC / HLA genes of the subject from whom the cells are obtained for producing the cells to be administered.

[0488] In some embodiments, the subject to whom the cells are administered is HLA matched with respect to the subject from whom the cells are obtained for producing the cells to be administered. In some embodiments, the subject to whom the cells are administered is nearly or fully matched with respect to the subject from whom the cells are obtained for producing the cells to be administered.

[0489] In some embodiments, the matching degree of the subject on HLA-A, -B, -C and -DRB1 is ≥4 / 8 (i.e., 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8). In some embodiments, the matching degree of the subject on HLA-A, -B, -C, -DRB1 and -DQB1 is ≥5 / 10 (i.e., 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10). In some embodiments, the matching degree of the subject on HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 is ≥6 / 12 (i.e., 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, 11 / 12 or 12 / 12). In some embodiments, the subject is 8 / 8 matched on HLA-A, -B, -C and -DRB1. In some embodiments, the subject is 10 / 10 matched on HLA-A, -B, -C, -DRB1 and -DQB1. In some embodiments, the subject is 12 / 12 matched on HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1.

[0490] Kits

[0491] In certain aspects of the present invention, a kit is provided. In some embodiments, the kit can have at least one container having a predetermined amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition.

[0492] In some embodiments, the kit can include materials for generating the antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition of the present invention.

[0493] The kit can provide an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition and administration instructions for administering to a patient to treat a specific disease / condition.

[0494] In some embodiments, the kit can further include at least one container having a predetermined amount of another therapeutic agent (e.g., as described in the present invention). In such embodiments, the kit can further include a second drug or pharmaceutical composition such that the two drugs or pharmaceutical compositions can be administered simultaneously or separately so that they provide combination therapy for a specific disease or condition.

[0495] The kit according to the present invention can include instructions for use, e.g., in the form of a manual or leaflet. The instructions may include protocols for performing any one or more of the methods described in the present invention.

[0496] Sequence identity

[0497] As used herein, "sequence identity" refers to the percentage of nucleotide / amino acid residues in a target sequence that are identical to the nucleotide / 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 alignments for determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in a variety of ways known to those of skill in the art, e.g., using publicly available computer software such as ClustalOmega( J. 2005, Bioinformatics 21, 951 - 960), T - coffee (Notredame et al., 2000 Journal of Molecular Biology (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) software. When using such software, it is best to use the default parameters, for example, gap penalty and extension penalty.

[0498] Sequence

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505] ***

[0506] In the features described above, or in the present claims, or in the drawings, those represented in their specific forms or by means for performing the disclosed functions or by methods or processes for obtaining the disclosed results can be used alone or in any combination of these features to implement the present invention in various forms.

[0507] Although the present invention has been described together with the above - mentioned exemplary embodiments, many equivalent modifications and variations will be obvious to those skilled in the art when the present invention is given. Therefore, the above - mentioned exemplary embodiments of the invention are considered illustrative rather than restrictive. Various changes can be made to the described embodiments without departing from the spirit and scope of the present invention.

[0508] To avoid any doubt, any theoretical explanations provided herein are for enhancing the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0509] Any chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0510] In this specification, including the claims which follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprises", "comprising", and "including" will be understood to mean including the stated integer or step or integer or step group but not excluding any other integer or step or integer or step group.

[0511] It must be noted that, as used in the 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" 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" associated with a numerical value is optional and is meant to represent, for example, + / - 10%.

[0512] The present invention includes combinations of the described aspects and preferred features, unless such combinations are clearly impermissible or expressly avoided.

[0513] If the present invention discloses a nucleic acid sequence, its reverse complement is also expressly contemplated.

[0514] The methods described herein may preferably be carried out in vitro. The term "in vitro" is intended to include procedures carried out on cultured cells, while the term "in vivo" is intended to include procedures carried out on / with intact multicellular organisms.

[0515] Aspects and embodiments of the present invention will now be illustrated by way of example, in conjunction with the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are hereby incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0516] Figures 1A to 1D (FIGS. A1A to A1D): Binding of VHH P2A5 to B7-H3. (A) Results of flow cytometry analysis of the binding of purified P2A5 VHH to HepG2 cells expressing B7-H3. (B) to (E) Sensorgrams and binding kinetic parameters for the binding of P2A5 VHH to (B) human B7-H3 subtype 1 (also known as 4Ig-B7-H3), (C) human B7-H3 subtype 2 (also known as 2Ig-B7-H3), and (D) mouse B7-H3 determined by multi-cycle kinetic analysis and surface plasmon resonance analysis.

[0517] Figures 2A to 2D(Figures A2A to A2D): Cross-reactivity of P2A5 with murine and human B7-H3 molecules expressed on murine and human-derived cells. Murine CT26 cells and human MKN7 cells with endogenous B7-H3 gene knockout were transfected to express murine B7-H3 (A and B) and human 4Ig-B7-H3 (C and D), and stained with positive control anti-murine or anti-human B7H3 antibodies (upper row), P2A5 as a VHH-Fc fusion molecule (middle row), or isotype control (lower row).

[0518] Figure 3 (Figure A3): Generation and function of B7-H3 CAR T cells. (A) Schematic diagram of B7-H3 CAR composed of a VHH targeting B7-H3, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signal transduction domains. (B) Detection of B7-H3 CAR on activated T cells after transduction. (C) Transduction efficiency and expansion fold of untransduced and B7-H3 CAR T cells were tracked within 11 days after transduction. Data are mean ± SD of 2 independent donors. (D) Cytolysis of A549 and MDA-MB-231 cells by 2 donors was evaluated at E:T ratios of 1:1 and 5:1 using the xCELLigence real-time cell analysis system. (E) Serial cytolysis of THP-1 and CMK cells during multiple rounds of co-culture of 2 donors.

[0519] Figure 4 (Figure A4): Generation and characterization of allogeneic B7-H3 CAR EBVSTs. (A) Schematic diagram of B7-H3 CAR EBVST manufacturing. (B) Expansion fold of EBVSTs after transduction with blank (UT) or P2A5 CAR. Percentage of CAR+ cells (C) and CD8 / CD4 cell ratio (D) 4 and 11 days after transduction. (E) Expression of B7-H3 on total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all 3 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 medium, HIV, or EBV mixed peptides. Data shown in (B-D) are from 6 independent donors. Each row represents data from each donor. Marker expression data of UT or CAR EBVST final products are shown in (E-G). Data are from 3 independent donors.

[0520] Figure 5 (Figure A5): Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Cytolytic kinetics 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, effector cell:target ratio of 1:1.

[0521] Figure 6 (Figure A6): Serial killing by B7-H3 CAR EBVST. (A) Serial cytotoxicity of EBVST after each serial contact with target AML cell lines THP-1 and CMK. (B) Expansion of EBVST after each serial contact with THP-1. Cytolytic kinetics of EBVST (C) and expansion of CAR-T cells (D) after each serial contact with target cell lines DLD-1, HT29 and NCI-N87 at an effector cell:target ratio of 1:2.

[0522] Figure 7 (Figure A7). In vivo persistence and efficacy of B7-H3 CAR EBVST in colorectal cancer expressing B7-H3. (A) Experimental protocol for the HT-29 model. (B) Body weight monitoring twice a week. (C) Changes in HT-29 tumor volume and growth fold after treatment. (D) HT-29 tumor count and B7-H3 expression at the endpoint quantified by flow cytometry. (E) T cell counts in blood, liver, lung, spleen and tumor at the endpoint quantified by flow cytometry. (F) Experimental protocol for the SW-480 model. (G) Body weight monitoring twice a week. (H) Changes in SW-480 tumor volume and growth fold after treatment. (I) SW-480 tumor count and B7-H3 expression at the endpoint quantified by flow cytometry. (J) T cell counts in blood, liver, lung, spleen and tumor at the endpoint quantified by flow cytometry.

[0523] Figure 8 (Figure A8): In vivo persistence and efficacy of B7-H3 CAR EBVSTs in gastric cancer expressing B7-H3. (A) Experimental protocol for the N-87 model. (B) Body weight monitoring twice a week. (C) Changes in HT-29 tumor volume and growth fold after treatment. (D) N-87 tumor count and B7-H3 expression at the endpoint quantified by flow cytometry. (E) T cell counts in blood, liver, lung, spleen and tumor at the endpoint quantified by flow cytometry.

[0524] Figure 9 (Figure B1): Identification and characterization of B7H3 VHH lead antibodies in a phage display llama immune library. (A) B7H3-binding screening ELISA of phage eluted in the 2nd (top) and 3rd (bottom) rounds of biopanning. (B) Flow cytometry analysis of the binding of purified lead VHH candidates to HepG2 cells expressing B7H3. (C) Surface plasmon resonance screening of lead VHH candidates. Multicycle kinetic analysis of the binding of lead candidate P2A5 to (D) human B7H3 4Ig isoform, (E) human B7H3 2Ig isoform, and (F) mouse B7H3. (G) Binding kinetic parameters of P2A5 to 4Ig and 2Ig isoforms of human B7H3 and mouse B7H3.

[0525] Figure 10 (Figure B2): Cross-reactivity of P2A5 with mouse and human B7H3 molecules expressed on mouse- and human-derived cells. Mouse CT26 and human MKN7 cells transfected to knockout endogenous B7H3 to express mouse B7H3 molecules (A and C) and human 4Ig B7H3 molecules (B and D) were stained with a positive control anti-mouse or anti-human B7H3 antibody (top), P2A5 as a VHH-Fc fusion molecule (middle), or isotype control (bottom).

[0526] Figure 11 (Figure B3): Generation and function of B7H3.CAR T cells. (A) Schematic diagram of the structure of B7H3.CAR, consisting of a VHH targeting B7-H3, a 4-1BB-derived spacer, a CD28 transmembrane domain, and CD28 and CD3ζ signal transduction domains. (B) Detection of B7H3.CAR on transduced activated T cells. (C) Tracking of the transduction efficiency and fold expansion of untransduced and B7H3.CAR T cells within 11 days of transduction. Data are shown as the mean ± SD of 2 independent donors. (D)

[0527] Cytolysis of A549 and MDA-MB-231 cells by 2 donors was evaluated at E:T ratios of 1:1 and 5:1 using the xCELLigence real-time cell analysis system. (E) Serial cytolysis of THP-1 and CMK cells during multiple rounds of co-culture of 2 donors.

[0528] Figure 12 (Figure B4): Generation and characterization of allogeneic B7H3.CAR EBVSTs. (A) Schematic illustration of the generation of B7H3.CAR EBVSTs. (B) Fold expansion of EBVSTs after transduction with empty (UT) or P2A5 CAR. (C) Percentage of CAR+ cells and (D) CD8 / CD4 cell ratio at 4 and 11 days post-transduction. (E) Expression of B7-H3 on total cells. (F) Percentage of CD4 or CD8 cells expressing 0, 1, 2, or all 3 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 medium, HIV, or EBV-mixed peptides. (B–D) Data provided are from 6 independent donors. Each row represents data from each donor. (E–G) Data provided are from 3 independent donors.

[0529] Figure 13 (Figure B5): Cytotoxicity against B7-H3+ and B7-H3KO tumor cell lines. Cytolytic kinetics of B7H3.CAR EBVSTs 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, effector cell:target ratio of 1:1. (E) Quantification of IFNγ levels in the supernatant after co-incubation of untransduced or B7H3.CAR EBVST cells with tumor cell lines HT29, HT29-B7H3KO, or N87, N87-B7H3KO. (F) Cytotoxicity of EBVSTs expressing full-length or non-truncated B7H3.CAR or no CAR against B7-H3+ MKN-45 tumor cells.

[0530] Figure 14 (Figure B6): Sustained killing by B7H3.CAR EBVSTs. (A) Serial cytotoxicity of EBVSTs after each consecutive exposure to target AML cell lines THP-1 and CMK. (B) Expansion of EBVSTs after each consecutive exposure to THP-1. Serial (C) cytolytic kinetics of EBVSTs and (D) expansion of CAR-T cells after each consecutive exposure to target cell lines DLD-1, HT29, and NCI-N87, at an effector cell:target ratio of 1:2.

[0531] Figure 15 (Figure B7): B7H3.CAR EBVST in vitro targeting of allogeneic MDSCs. (A) Flow cytometry histograms showing the expression of IL-10, TGF-β, iNOS, and B7-H3 in MDSCs generated from 2 independent healthy donors. (B) Cytotoxicity of CD30.CAR EBVST against the KM-H2 cell line in the presence or absence of MDSCs, with MDSC:CAR:KM-H2 at 4:1:1 or 10:1:1. (C) Cytotoxicity of untransduced (UT) or B7H3.CAR EBVST (CAR) against allogeneic MDSCs. Data from co-cultures of effector EBVSTs generated from 2 donors and target MDSCs generated from different 2 donors. (D) Proliferation indices of untransduced and B7H3.CAR EBVSTs after anti-CD3 / CD28 stimulation in the presence of allogeneic MDSCs. Each data point represents the proliferation of each effector cell:target donor pair. The proliferation index was calculated by normalizing the percentage of proliferating cells to the percentage in the MDSC-free control condition.

[0532] Figure 16 (Figure B8): In vivo persistence and efficacy of B7H3.CAR EBVST against colorectal cancer expressing B7-H3. (A) Experimental protocol for the HT-29 model. (B) Body weight monitoring twice a week. (C) Changes in HT-29 tumor volume and growth fold after treatment. (D) Endpoint HT-29 tumor counts and B7-H3 expression quantified by flow cytometry (E) T cell counts in blood, liver, lung, spleen, and tumor at the endpoint quantified by flow cytometry. (F) Experimental protocol for the SW-480 model. (G) Body weight monitoring twice a week. (H) Changes in SW-480 tumor volume and growth fold after treatment. (I) Endpoint SW-480 tumor counts and B7-H3 expression quantified by flow cytometry (J) T cell counts in blood, liver, lung, spleen, and tumor at the endpoint quantified by flow cytometry.

[0533] Figure 17 (Figure B9): In vivo persistence and efficacy of B7H3.CAR EBVST against gastric cancer expressing B7-H3. (A) Experimental protocol for the NCI-N87 model. (B) Body weight monitoring twice a week. (C) Changes in NCI-N87 tumor volume and growth fold after treatment. (D) Endpoint NCI-N87 tumor counts and B7-H3 expression quantified by flow cytometry (E) T cell counts in blood, liver, lung, spleen, and tumor at the endpoint quantified by flow cytometry.

[0534] Figure 18 (Figure B10): In vivo persistence and efficacy of B7H3.CAR EBVST against lung cancer expressing B7-H3. (A) Experimental protocol for the NCI-H1299 model. (B) Body weight monitoring twice a week. (C) Changes in NCI-H1299 tumor volume and growth fold after treatment. (D) Endpoint NCI-H1299 tumor counts and B7-H3 expression quantified by flow cytometry.

[0535] Figure 19 (Figure B11): In vivo persistence and efficacy of B7H3.CAR EBVST against triple-negative breast cancer expressing B7-H3. (A) Experimental protocol for the MDA-MB-468 model. (B) Body weight monitoring twice a week. (C) Changes in MDA-MB-468 tumor volume measurements and growth fold after treatment; and tumor counts at the endpoint quantified by flow cytometry.

[0536] Figure 20 (Figure B12): (A and B) B7-H3 staining on breast cancer PDX by IHC. Brown represents positive B7-H3 staining. Blue represents the cell nucleus.

[0537] Figure 21 (Figure B13): In vivo persistence and efficacy of B7H3.CAR EBVST against breast cancer PDX expressing B7-H3. (A) Experimental protocol for the PDX model. (B) Body weight monitoring twice a week. (C) Changes in PDX tumor volume measurements and growth fold after treatment; (D) Tumor counts at the endpoint quantified by flow cytometry.

[0538] Figure 22 (Figure B14): Stimulation and competition of soluble B7-H3 with B7H3.CAR EBVST. (A) Cellular staining of IFNγ, TNFα, and CD25 on UT and B7H3.CAR EBVST after treatment with different concentrations of 2-Ig or 4-Ig forms of coated or soluble B7-H3. (B) Cytolysis of NCI-N87 or NCI-H1299 by B7H3.CAR EBVST in the presence of different concentrations of 2-Ig or 4-Ig forms of soluble B7-H3.

[0539] Figure 23 (Figure B15). Safety of B7H3.CAR EBVST on hematopoietic stem and progenitor cells (HSPC). (A) Flow plots showing HSPC populations expressing B7-H3 after stimulation with Flt3L, TPO, and SCF for the indicated number of days. (B) Comparison of cell surface expression of B7-H3 on HSPC and cancer cell line NCI-N87 by flow cytometry. (C) Cytotoxicity of unstimulated and stimulated HSPC after co-culture with B7H3 CAREBVST. (D) Proportion of HSPC cell populations after co-culture with untransduced or B7H3.CAR EBVST. (E) Erythroid and myeloid developmental potential of HSPC after co-culture with untransduced or B7H3.CAR EBVST.

[0540] Figure 24 (Figure B16): Safety of B7H3.CAR EBVST on peripheral blood mononuclear cells (PBMC). (A) Cell surface expression of B7-H3 on PBMC subsets after stimulation with inflammatory cytokines and cancer cell line NCI-N87. (B) Fold change in cell counts of PBMC subsets after 2 days of culture of B7H3.CAR EBVST and cytokine-stimulated PBMC. Fold change was obtained by normalizing the cell counts of PBMC subsets co-cultured with control untransduced 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 are also presented as fold change in cytotoxicity of B7H3.CAR EBVST compared to untransduced control.

[0541] Figure 25 (Figure B17): Safety of B7H3.CAR EBVST against antigen-experienced T cells. (A) Proliferation of EBVST and expression of TNFα and IFNγ, and (B) cell surface expression of B7-H3 after stimulation of antigen-presenting cells with HIV or EBV mixed peptides.

[0542] 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 murine B7-H3 (B16F10-mB7H3). (B) Experimental protocol for the immunocompetent mouse model. (C) Tumor volume measurements and fold change in growth after treatment; and tumor weight at study endpoint. (D) Body weight monitored twice a week. (E) Quantification of the total number of host blood cell subsets in mouse blood, bone marrow, spleen, and liver after treatment by flow cytometry. (F) Quantification of cytokine and chemokine levels in mouse serum after treatment by Luminex.

[0543] Figure 27 (Figure B19): CRS model in humanized mice. (A) Experimental protocol of the humanized mouse model. (B) Tumor tracking in mice. Left panel: IVIS monitoring of tumor burden in NALM-6 transplanted mice. Right panel: Fold change in tumor volume of HT29 transplanted mice. (C) Body weight monitoring of mice. (D) Cytokine and chemokine levels in serum 3 days after treatment measured by multiplex bead immunoassay. (E) Total number of myeloid cell subsets in the blood, bone marrow, spleen, and liver of humanized mice quantified by flow cytometry. (F) B7H3 expression of myeloid cell subsets in the blood, bone marrow, spleen, and liver of humanized mice quantified by flow cytometry.

[0544] Example

[0545] Compared with the limited protein expression of B7-H3 in normal human tissues and lymphoid organs, B7-H3 is aberrantly expressed in a high proportion of human malignancies. The following examples describe nanobodies targeting B7-H3, as well as related CAR constructs / EBVSTs according to the present invention.

[0546] The following examples describe the identification and utilization of a novel B7-H3 targeting nanobody as the antigen-specific domain in a CAR construct, which has superior biophysical properties compared to scFv. T cells expressing this nanobody-based B7-H3 CAR and Epstein-Barr virus-specific T cells (EBVSTs) exhibit good in vitro and in vivo activities in B7-H3 positive malignancies. Specifically, EBVSTs expressing B7-H3 CAR show excellent anti-tumor efficacy, providing an approach for allogeneic off-the-shelf CAR T cell therapy for patients with B7-H3 positive malignancies.

[0547] Example 1: Materials and methods

[0548] 1.1 Generation of a llama immune library

[0549] Native male llamas were used to generate an immune library. His-tagged recombinant human B7-H3 (Sino Biological) was immunized at a dose of 100 μg per injection, for a total of 6 injections. Four days after the last injection, 100 ml of anticoagulated blood was collected to prepare peripheral blood lymphocytes (PBL). Total RNA was isolated from the PBL and used as a template for synthesizing the first strand cDNA using an oligodT primer. Then the VHH coding sequence was amplified from the cDNA library and cloned between the PstI and NotI restriction enzyme sites of the pMECS phagemid vector, upstream of the linker, hemagglutinin (HA), and His 6 tag. Then the pMHCs vector was electrotransformed into competent Escherichia coli TG1 cells to generate a library containing 109 A VHH library of individual transformants. PCR analysis was performed on 95 randomly selected individual transformants to evaluate the percentage of transformants with the correct insert size. Then, sequence analysis was performed on 102 randomly selected colonies to evaluate the diversity and correctness of the VHH sequences. Then, M13K07 helper phage was transfected into TG1 transformants, and the VHH library was amplified and repackaged into phage particles for subsequent biopanning.

[0550] 1.2 Biopanning of a B7H3-specific VHH phage library

[0551] Several consecutive rounds of biopanning were performed on the phage-displayed VHH library to identify B7-H3 specific binders. Briefly, first, the phage library was incubated with streptavidin-coated magnetic beads to remove streptavidin binders. Then, the phage after removal was incubated with streptavidin beads pre-coated with biotinylated B7-H3. After rotating incubation at room temperature for 1 hour, the magnetic beads were separated from the unbound phage, washed multiple times with 0.1% PBST, and then eluted with 0.1 M triethylamine. The eluted phage was neutralized in 1 M Tris at pH 8.0 and used to infect TG1 cells at 37 °C for 1 hour. 2xYT containing 100 μg / mL ampicillin and 2% (v / v) glucose was added. Subsequently, the infected TG1 cells were infected with M13K07 helper phage that helped amplify and produce phage particles. Then, the TG1 cells were centrifuged at 2000 rpm for 10 minutes, and the cell pellet was resuspended in 300 mL of 2xTY containing 100 μg / mL ampicillin and 50 μg / mL kanamycin to screen for TG1 co-infected with the binder and the helper phage. Amplification was carried out overnight at 37 °C. Phage particles were precipitated from the TG1 culture supernatant using PEG / NaCl. The precipitated phage was collected and quantified by OD 260 and used for the next round of biopanning. The stringency of subsequent rounds of biopanning was increased by reducing the concentration of biotinylated B7-H3 protein coated on the streptavidin beads and increasing the number of PBST washes.

[0552] 1.3 Phage screening ELISA assay

[0553] 1.3.1 Periplasm preparation

[0554] The eluted phage was serially diluted 10-fold in PBS (10 1 -10 7Dilute (by serial dilution) for infecting TG1 cells. Then plate the infected TG1 cells on a 90 mm LB agar plate containing 100 μg / mL ampicillin and 2% (v / v) glucose to obtain single colonies. Incubate the plate overnight at 37 °C. The next day, pick single colonies and culture them in 96-well deep-well plates with 1 ml of super broth (TB) medium containing 100 μg / mL ampicillin at 37 °C for 5 - 7 hours until the OD 600 is approximately 0.6 - 0.9. Then add IPTG to each well (final concentration 1 mM) and induce overnight at 37 °C. After overnight culture, centrifuge the TG1 culture to pellet the cells and remove the supernatant. Add Tris / EDTA / sucrose (TES) buffer to the cell pellet and incubate at 4 °C for 1 hour. Further add 4-fold diluted TES and incubate the cell suspension at 4 °C for 1 hour. Then centrifuge the cell suspension at 2,500 x g for 15 minutes at 4 °C, and the supernatant (periplasmic extract) is used for binding ELISA.

[0555] 1.3.2 Binding ELISA

[0556] Coat ELISA plates with 5 μg / mL neutravidin at room temperature for at least 1 hour or overnight at 4 °C. Then wash the wells with 0.05% PBST and block with casein at room temperature for 2 hours or overnight at 4 °C. After blocking, add 0.2 μg / mL biotinylated B7H3 or control protein to each well and incubate at room temperature for 1 hour. After washing with 0.05% PBST, add the periplasmic extract to each well and bind at room temperature for 1 hour. Then wash the wells with 0.05% PBST, add anti-HA antibody (diluted 1:2000 in casein) and incubate at room temperature for 1 hour. Wash the wells again and add goat anti-mouse HRP secondary antibody (1:3000) to each well. After incubating at room temperature for 1 hour, add TMB to detect the conjugate and terminate the reaction with 1 M HCl. Measure the absorbance readings at OD450 and OD570 nm using a spectrophotometer.

[0557] 1.4 Expression and purification of recombinant VHH

[0558] For large-scale production of purified VHH, expression was carried out using the pHEN6c vector, where the intact His-tag can be used for subsequent purification and detection. The VHH was cloned between the PsteI and BstEII restriction enzyme sites of the pHEN6c vector. Then, VHH expression was performed using WK6 competent Escherichia coli cells transformed with the pHEN6c vector. Briefly, freshly transformed WK6 cell colonies were used as the starting culture and grown overnight at 37 °C in TB medium supplemented with 100 μg / mL ampicillin and 0.1% glucose. Then, 1 mL of the starting culture was further added to 150 mL of TB+Amp / Glu and grown at 37 °C until the OD600 reached 0.6 - 0.9. Induction was carried out overnight (about 16 - 18 hours) at 28 °C using 1 mM IPTG (final concentration). The next day, after centrifuging the culture, the cell pellet was harvested and resuspended in TES buffer. After incubating on ice for 1 hour, diluted TES was added, and the suspension was further incubated on ice. Then, the cell suspension was centrifuged at 8000 rpm for 30 minutes at 4 °C to obtain the supernatant (periplasmic extract containing VHH) for His-tag purification using nickel beads. The purified VHH was buffer-exchanged and stored in PBS.

[0559] To generate the VHH-Fc construct, the VHH sequence was cloned upstream of IgG1-CH2CH3 in the pTT5 expression vector. Subsequently, HEK2936E cells were transiently transfected, and recombinant VHH-Fc was purified from the culture supernatant using Protein G magnetic beads. Then, the pooled fractions were concentrated and the buffer was changed to PBS at pH 7.4 or MES at pH 6.0.

[0560] 1.5 Analysis of binding kinetics by surface plasmon resonance

[0561] The binding kinetics of VHH molecules to recombinant B7-H3 protein were evaluated by surface plasmon resonance. Human 4Ig-B7-H3 was purchased from Sino Biological (Cat. No. 11188-H08H; C-terminal His-tagged ECD (i.e., residues 1 to 461 of UniProt: Q5ZPR3-1)), and human 2Ig-B7-H3 was obtained from Acrobiosystems (Cat. No. B73-H5253; C-terminal Fc tag containing residues 29 to 245 of UniProt: Q5ZPR3-2 and residues 100 to 330 of UniProt: P01857).

[0562] All kinetic experiments were performed on a Biacore T200 (Cytiva, Uppsala, Sweden). HBS-P+ supplemented with 1% BSA w / v (Cytiva, Marlborough, USA) was used as the running buffer and dilution buffer for both the analyte and the ligand. Briefly, rabbit anti-VHH antibody (Genscript, Shanghai) was immobilized on a CM5 chip. Then, purified VHH was injected at a concentration of 100 mM for 60 seconds to be captured by the anti-VHH antibody. For the preliminary screening of VHHs, 100 nM recombinant B7-H3 was injected after stabilization. For cross-reactivity assessment, 100 nM mouse B7-H3 was injected after stabilization. The reference channel without captured VHH was used to correct for volume effects and non-specific binding, while blank runs (no antigen flow) were used to correct for surface stability. The double-reference sensorgrams were fitted with a Langmuir (1:1) binding model to obtain the association k a dissociation k d and the equilibrium dissociation constant KD, and the goodness of fit was evaluated using the chi-square value.

[0563] For multi-cycle kinetic analysis, recombinant B7-H3 at concentrations between 6.25 nM and 200 nM was injected in a six-point, two-fold dilution series. For each concentration, recombinant B7-H3 was allowed to bind for 180 seconds and dissociate for 180 seconds. Regeneration was performed with 10 mM glycine (pH 1.5). The reference channel without captured scFv-mFc was used to correct for bulk effects and non-specific binding, while blank runs (no antigen flow) were used to correct for surface stability. The double-reference sensorgrams were fitted with a Langmuir (1:1) binding model to obtain the association k a dissociation k d and the equilibrium dissociation constant KD, and the goodness of fit was evaluated using the chi-square value.

[0564] 1.6 Cross-reactivity study with transfected cell lines

[0565] Human MKN7 cells were engineered using CRISPR-Cas9 to knockout the expression of B7-H3 as described in Example 1.12. Then, mouse CT26 cells and MKN7 CD276 k / o cells were transfected with the corresponding pCMV3 vectors (Sino Biological) to express mouse B7-H3-GFPSpark and human 4Ig-B7-H3-GFPSpark fusion proteins. Briefly, cells were seeded in 6-well plates to 70% confluence and allowed to adhere overnight. Then, the plasmid was pre-mixed with 6 transfection reagent (Promega) and added to the cells. Transfection was then evaluated by flow cytometry and fluorescence microscopy.

[0566] 1.7 CAR construct targeting B7-H3 and retrovirus production

[0567] The VHH targeting B7-H3 was cloned upstream of the 4-1BB-derived spacer of the pSFG retroviral vector, followed by the CD28 transmembrane domain, the CD28 and CD3ζ signal transduction domains. A truncated form of the B7-H3-specific CAR consisting only of the extracellular domain was also cloned into the pSFG retroviral vector. The pSFG vector was transiently transfected using the PEIpro transfection reagent (Polyplus, Illkirch, France), and retroviruses carrying the full-length or truncated B7-H3 CAR were produced in the RD114 packaging cell line (BioVec Pharma, Quebec, Canada). The medium containing retroviruses was harvested at 48 and 72 hours post-transfection and concentrated 10 to 50-fold using a RetroX concentrator (TakaraBio, Kusatsu, Shiga, Japan). The retroviruses were either used immediately or snap-frozen and stored at -80 °C.

[0568] A stable RD114 retroviral packaging cell line that produces high-titer GFP-Firefly luciferase (GFP-FFluc) viral particles was used.

[0569] 1.8 Donors

[0570] By Spectra Leukapheresis products enriched in ACD-A anticoagulant collected from consented healthy donors by the Spectra apheresis system CMNC collection protocol were purchased from HemaCare (Northridge, California, USA). The frozen leukocyte bags were thawed, and PBMCs were extracted by gradient centrifugation using Ficoll-Paque PLUS (Cytiva, MA, USA). The PBMCs were either used immediately for experiments or frozen at 30 - 50 x 10 6 cells per cryotube in small aliquots in cryotubes containing CS10 cell freezing medium (STEMCELL Technologies, Cambridge, Massachusetts, USA).

[0571] 1.9 Generation of B7-H3-targeted CAR T cells

[0572] The PBMCs were thawed and seeded on cell culture plates pre-coated with anti-CD3 / CD28, at 37 °C and 5% CO 2Below, it is cultured in a medium containing 10% FBS, 45% Advanced RPMI, and 45% Click's medium to generate activated T cells (ATC). Two days after culturing, IL-7 and IL-15 are added to the cell culture. On the 3rd day of culturing, the retrovirus containing the B7-H3 specific CAR is transduced into ATC by spin transfection. The retrovirus is washed away after 24 hours, and the medium is occasionally changed to supplement IL-7 and IL-15 for culturing ATC. On the 11th day after transduction, use Freeze the ATC and store it in liquid nitrogen for further research needs.

[0573] Before cell counting and evaluation in in vitro or in vivo studies, all effector T cells are thawed and allowed to stand overnight in IL-7 and IL-15.

[0574] 1.10 Generation of B7-H3 CAR EBVST

[0575] Thaw PBMCs from healthy donors and perform CD45RA depletion using negative selection with CD45RA MACS beads (Miltenyi Biotec, Germany). PBMCs depleted of CD45RA are co-cultured with viral peptides from an overlapping Epstein-Barr virus (EBV) peptide library (15-mers, overlapping by 11 amino acids) from JPT Technologies (Berlin, Germany), covering EBV antigens BMRF2, BALF2, BNLF2b, BNLF2a, LMP1, LMP2, EBNA1, BZLF1, BRLF1, BMRF1, BMLF1, and BARF1. The culture is performed using VST medium [47.5% advanced RPMI 1640 (Gibco), 47.5% Clicks' medium (FUJIFILM Irvine Scientific), 5% human platelet lysate (Sexton Biotechnologies), and 2 mM GlutaMAX (Gibco)], supplemented with 10 ng / ml IL-7 and IL-15 (R&D Systems). Five days after culturing, the B7-H3 specific CAR construct is transduced into the cells using RetroNectin (Takara Bio, Japan). On the fourth day after transduction, the T cells are stimulated with irradiated co-stimulatory cells expressing markers such as CD80, CD86, 4-1BB, etc. After culturing for 7 to 8 days, harvest the VST and use Freeze it or use it directly for cell experiments.

[0576] Before cell counting and evaluation in in vitro or in vivo studies, all effector T cells are thawed and allowed to stand overnight in IL-7 and IL-15.

[0577] 1.11 Detection of transduction efficiency and phenotype by flow cytometry and antibody staining

[0578] Flow cytometry was performed using an Aurora flow cytometer (Cytek Biosciences) or a FACSymphony A3 cell analyzer (BD Biosciences). Up to 200,000 T cells were stained with a live / dead TM NIR viability dye (Thermo Fisher), and surface epitope presentation was detected using fluorescently labeled anti-CD3 (clone SK7, Biolegend), CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BioLegend), CD56 (clone B159, BD Biosciences), CD19 (clone SJ25C1, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), and LAG3 (clone 11C3C65, BioLegend) monoclonal antibodies. B7-H3 CAR expression was measured using an anti-camel VHH antibody (clone 96A3F5, Genscript). Data analysis and gating were performed in FlowJo v10.8.1 for Windows.

[0579] 1.12 Gene knockout of B7-H3 in cell lines

[0580] The B7-H3 gene was knocked out in the cell line using the CRISPR-Cas9 system. The sequences of single-guide RNA targeting sequences 5'-CTGGTGCACAGCTTTGCTGA-3', 5'-GTGCCCACCAGTGCCACCAC-3', and 5'-TGCCCACCAGTGCCACCACT-3' (Integrated DNA Technologies, Inc and Synthego) were incubated with Cas9 (Integrated DNA Technologies, Inc) for 10 minutes to form an RNP complex. The cell line washed with PBS was briefly incubated with the RNP complex and then electroporated using a 4D-Nucleofector (Lonza). Then the cells were allowed to recover in complete medium and then seeded onto tissue culture plates. The cells were stained with an antibody against B7-H3 (clone 7-517, BD Biosciences), and the knockout efficiency was evaluated using flow cytometry. B7-H3 knockout cells were selected by FACS using a BD InfluxTM cell sorter (BD Biosciences).

[0581] 1.13 Real-time cytotoxicity detection

[0582] Prior to assessing CAR potency by cytotoxicity assay, the cell culture medium was changed to 2% assay medium containing RPMI and 2% FBS. Using a real-time cell analysis system, cytotoxicity assays were performed at 37 °C with 5% CO 2 (Agilent). Target tumor cells were added to the PET plates according to the manufacturer's manual (Agilent). After 24 hours, CAR T cells were seeded into the PET plates at a CAR T:target cell ratio of 0.5:1, 1:1, or 5:1. The PET plates were then returned to the system, and cytotoxicity was monitored for 48 hours.

[0583] 1.14 Continuous killing potency detection using the xCelligence real-time cell analysis system

[0584] Using a real-time cell analysis system, serial killing was evaluated at 37 °C with 5% CO 2 2 (Agilent). Target cells DLD-1, HT-29, and NCI-N87 cells were added to the PET plates according to the manufacturer's instructions (Agilent). After the target cells had adhered for 24 hours, CAR T cells were seeded onto the PET plates at a CAR T:target cell ratio of 1:2. The PET plates were returned to the system, and cytotoxicity was monitored for 48 hours. After 48 hours, the CAR T cell suspension was harvested, counted, and added to the target cell plates seeded 24 hours earlier to establish a second round of contact at a CAR T:target cell ratio of 1:2. After another 48 hours, the cells were harvested and added to a new set of target cells per well for a third round of contact. At 48 hours after each contact, the cells were stained with a live / dead TM NIR viability dye (Thermo Fisher) and antibodies against anti-camel VHH antibody (clone 96A3F5, Genscript), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), and LAG3 (clone 11C3C65, BioLegend), along with CountBright TM absolute counting beads (Invitrogen TM)Count the effector cell population present after each round of target contact together. Data analysis and gating were performed in FlowJo v10.8.1 on Windows. Cytolysis was calculated as (cell count in target-only control wells - cell count in test wells) / cell count in target-only control.

[0585] 1.15 Determination of continuous killing potency by flow cytometry analysis

[0586] THP-1 and CMK cells were used as target cell lines, with an initial E:T ratio of 1:2. Each well of the 96-well plate for the first round of contact had 50,000 effector cells and 100,000 target cells. Three parallel experiments were set up. After 48 hours, two groups of cells were harvested and added to two new groups of 100,000 target cells per well to establish the second round of contact. After another 48 hours, one group of cells was harvested and added to a new group of 100,000 target cells per well for the third round of contact. To distinguish target cells of different rounds of contact, THP-1 and CMK cells for the second and third rounds of contact were labeled with the lipophilic membrane dyes PKH67 and PKH26 (Sigma-Aldrich), respectively. Forty-eight hours after each round of contact, live / dead TM NIR viability dye (Thermo Fisher) and anti-camel VHH antibody (clone 96A3F5, Genscript), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), LAG3 (clone 11C3C65, BioLegend) antibodies were used for staining, and CountBright TM Absolute Counting Beads (Invitrogen TM ) were used to count the remaining target cell population and effector cell population in each 48-hour culture. Data were analyzed and gated in FlowJo v10.8.1 on Windows. Cytolysis was calculated as (target count in target-only control wells - target count in analyzed wells) / target count in target-only control wells.

[0587] 1.16 Colorectal and gastric cancer mouse models

[0588] In a colorectal cancer model, 5x10 6 HT-29 or 2x10 6 SW-480 cells were subcutaneously injected into NOD-scidIL2Rgammanull Kb Dbnull I-A null The right abdominal cavity of (NSG-MHC I / IIDKO) mice. In the gastric cancer model, 2x10 6 NCI-N87 cells were subcutaneously injected into the right abdominal cavity of NOD-scid IL2Rgammanull Kb Db null I-A null (NSG-MHC I / IIDKO) mice. On day 7, the mice were randomly assigned to treatment groups based on tumor volume. 5x10 6 untransduced or P2A5-B7-H3 CAR EBVST were intravenously injected into the mice. Untreated mice served as controls. Physical examinations, tumor, and body weight measurements were performed twice a week until the endpoint. When the mice were sacrificed on day 21, blood, spleen, liver, lung, and tumors were collected for endpoint flow cytometry analysis.

[0589] Endpoint antibody staining was performed using a live / dead TM NIR viability dye (Thermo Fisher) and fluorescently labeled anti-camelid VHH antibodies (clone 96A3F5, Genscript), mouse CD45 (clone 30-F11, BD Biosciences), human CD45 (clone H130, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BDBiosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), LAG3 (clone 11C3C65, BioLegend) antibodies, along with CountBright TM Absolute Counting Beads (Invitrogen TM ) to count cell populations. Data analysis and gating were performed using FlowJo v10.8.1 for Windows.

[0590] 1.17 EBV reactivity assay

[0591] To measure the reactivity of EBVSTs against EBV antigens, cells were stimulated with HIV or EBV peptide pools (JPTPeptideTechnologies) in the presence of anti-CD49d (clone L25, BD Biosciences) and anti-CD28 (clone L293, BD Biosciences). One hour after stimulation, cells were treated with GolgiSTOP and GolgiPlug solutions (BD Biosciences) to accumulate cytokines in the Golgi complex. After overnight incubation, cells were surface stained with fluorescently labeled antibodies against CD3 (clone SK7, BioLegend), CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BioLegend), and camel VHH (clone 96A3F5, Genscript). Cells were fixed and permeabilized and then stained with anti-TNF (clone Mab11, BD Biosciences) and IFN-γ (clone B27, BD Bioscienes) antibodies. Flow cytometry was performed using a FACSymphony A3 cell analyzer (BD Biosciences), and data and gating were analyzed in FlowJo v10.8.1 for Windows.

[0592] 1.18 In vivo evaluation of B7H3.CAR EBVST in colorectal cancer (CRC), non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), and gastric cancer (GC) mouse models 1.19 Breast cancer patient-derived xenograft model

[0593] In the CRC model, 5x10 6 HT-29 or 2x10 6 SW480 cells were subcutaneously injected into the right flank of NOD-scidIL2Rgammanull Kb Dbnull I-Anull (NSG-MHC I / IIDKO) mice. In the NSCLC, TNBC, and GC cancer models, 2x10 6 NCI-H1299, 5x106 MDA-MB-468, or 2x106 NCI-N87 cells were subcutaneously injected into the right flank of NSG-MHC I / IIDKO mice, respectively. When tumors became palpable (100 - 200 mm 3 ) between days 7 and 16, mice were randomly assigned to treatment groups and stratified by tumor volume. 5x10 6 untransduced or P2A5-B7H3.CAR EBVSTs were intravenously injected into the mice, and untreated mice served as controls. Physical examinations, tumor, and body weight measurements were performed twice a week until the tumors in the control mice reached the endpoint size of 1000 mm 3 When mice were sacrificed between days 21 and 42, blood, spleen, liver, lung, and tumors were collected for endpoint flow cytometry analysis. Using live / deadTM Aqua reactive dyes (ThermoFisher) and fluorescently labeled anti-camel VHH (clone 96A3F5, Genscript), mouse CD45 (clone 30-F11, BD Biosciences), human CD45 (clone H130, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), LAG3 (clone 11C3C65, BioLegend) antibodies were used for endpoint antibody staining, and CountBright TM Absolute Counting Beads (Invitrogen TM ) were used to count cell populations by flow cytometry.

[0594] 1.20 B7H3 CAR activation and soluble B7-H3 competition assay

[0595] Patient-derived xenografts (PDX) from breast cancer patients were subcutaneously injected into the right flank of NOD-scid IL2Rgammanull (NSG) mice. When the PDX became palpable on day 32, the mice were randomly assigned to treatment groups, stratified by tumor volume. 5x10 6 Untransduced or GFP-luciferase-expressing P2A5-B7H3.CAR EBVSTs were intravenously injected into the mice. Untreated mice served as controls. Physical examinations, tumor and body weight measurements, and imaging were performed twice a week until the study endpoint at 22 days post-treatment.

[0596] 1.21 In vitro safety assay of hematopoietic stem and progenitor cells (HSPCs)

[0597] Untransduced and B7H3.CAR EBVST were stimulated with recombinant soluble B7-H3 (AcroBiosystems) in 4-Ig or 2-Ig form. For controls, untransduced and B7H3.CAR EBVST were seeded onto microtiter plates coated with recombinant B7-H3 in 4-Ig or 2-Ig form. Cytokine expression and activation were then detected by interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and CD25 cell surface staining (ICS). For CD25 staining, cells were harvested 2 days after stimulation and stained with a live / dead TM NIR viability dye (Thermo Fisher) and fluorescently labeled anti-camel VHH antibody (clone 96A3F5, Genscript), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), and CD25 (clone 2A3, BD Biosciences) antibodies. For ICS, GolgiSTOP and GolgiPlug (BD Biosciences) were added to the cells for 1 hour of stimulation, and after overnight incubation, the cells were stained with a live / dead TM NIR viability dye (Thermo Fisher) and fluorescently labeled anti-camel VHH (clone 96A3F5, Genscript), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences) monoclonal antibodies, fixed and permeabilized with the Cytofix / Cytoperm kit (BD Biosciences), and stained with fluorescently labeled anti-IFN-γ (clone B27, BioLegend) and TNF-α (clone Mab11, BD Biosciences) monoclonal antibodies.

[0598] For the competition assay, cytotoxicity of B7H3.CAR EBVST against NCI-N87 or NCI-H1299 was measured using a real-time cell analysis system (Agilent) in the presence of different concentrations of soluble B7-H3 for 48 hours.

[0599] 1.22 In vitro safety assay of peripheral blood mononuclear cells (PBMCs)

[0600] CD34+ HSPCs were stimulated with 10 ng / mL of Flt3 ligand (FLT3L), stem cell factor (SCF), and thrombopoietin (TPO) (all from Miltenyi Biotec) at 5,000–20,000 cells per well for a specific time.

[0601] In cytotoxic co-cultures of CD34+ HSPCs and T cells, the stimulated HSPCs were seeded with untransduced or B7H3.CAR EBVSTs at an E:T ratio of 1:1 for 24 h. Using live / dead TM NIR viability dye (Thermo Fisher) and fluorescently labeled anti-CD34 (clone 561, BD Biosciences), CD133 (clone 7, BioLegend), CD45RA (clone HI100, BD Biosciences), CD38 (clone HIT2, BD Biosciences), CD10 (clone HI10a, BD Biosciences) monoclonal antibodies, HSPC subsets were analyzed by flow cytometry. Using CountBright TM Absolute Counting Microspheres (Invitrogen TM ) the cell population was counted by flow cytometry.

[0602] The StemMACS TM HSC-CFU assay kit (Miltenyi Biotec) was used to evaluate erythroid and myeloid developmental potential. Cells were labeled with fluorescently conjugated antibodies against CD14, CD15, and CD235a as part of the StemMACS kit antibody mixture and colony types were identified according to the manufacturer's protocol. Colony types included colony-forming units of granulocytes (CFUG) and macrophages (CFU-M), and burst-forming units of erythrocytes (BFU-E). For more primitive progenitors, CFU-GM gave rise to granulocytes and macrophages, while CFU-GEMM differentiated into all three cell populations.

[0603] 1.23 In vitro safety assay using antigen-experienced T cells

[0604] PBMCs were stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF), IFNγ, TNFα, lipopolysaccharide (LPS), or medium alone at the indicated concentrations and then stained with live / dead TM NIR viability dye (Thermo Fisher) and a fluorescently labeled anti-B7-H3 monoclonal antibody (clone 7-517, BD Biosciences).

[0605] In the cytotoxic co-culture experiment, PBMCs were stimulated with GM-CSF, IFNγ, TNFα, LPS, or medium at the indicated concentration alone for one day, then the cytokines were washed away, and the PBMCs were further cultured with untransduced or B7H3.CAR EBVST labeled with CellTrace TM Violet for 2 days. The cells were stained with a live / dead TM NIR viability dye (Thermo Fisher) and fluorescently labeled anti-human CD14 (clone M5E2, BD Biosciences), human CD56 (clone HCD56, BD Biosciences), human CD16 (clone 3G8, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), and camel VHH (clone 96A3F5, Genscript) antibodies, and CountBright TM Absolute Counting Beads (Invitrogen TM ) were added, and the cell population was counted by flow cytometry.

[0606] Similarly, in the monocyte co-culture experiment, monocytes purified with CD14 beads (Miltenyi Biotec) were incubated with allogeneic EBVST or B7H3.CAR EBVST labeled with CellTrace TM Violet for 2 days. The cells were stained with a live / dead TM NIR viability dye (Thermo Fisher) and monoclonal antibodies against human CD14 (clone M5E2, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), and camel VHH (clone 96A3F5, Genscript), and CountBright TM Absolute Counting Beads (Invitrogen TM ) were added, and the cell population was counted by flow cytometry.

[0607] 1.24 In vitro MDSC generation and co-culture assay

[0608] PBMC plates were coated with anti-CD3 / CD28 antibodies and activated for 7 days to generate activated T cells (ATC), which were then stimulated with a mixture of HIV or EBV peptides. The stimulated ATC were then irradiated and co-cultured with untransduced or B7H3.CAR EBVST labeled with CellTraceTM Violet. GolgiSTOP and GolgiPlug (BD Biosciences) were added to the cells for 1 hour of stimulation. After overnight incubation, the cells were stained with a live / dead TM NIR viability dye (Thermo Fisher) and monoclonal antibodies against camel VHH (clone 96A3F5, Genscript), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences). The cells were then fixed and permeabilized with the Cytofix / Cytoperm kit (BD Biosciences) and stained with monoclonal antibodies against IFNγ (clone B27, BioLegend) and TNFα (clone Mab11, BD Biosciences) labeled with fluorescence. To analyze cell proliferation and B7-H3 expression, cells were collected on days 2 and 5 of culture and stained with a live / dead TM NIR viability dye (Thermo Fisher) and monoclonal antibodies against camel VHH (clone 96A3F5, Genscript), CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), and B7-H3 (clone 7-517, BD Biosciences).

[0609] 1.25 In vivo safety model in immunocompetent mice

[0610] To generate MDSC, monocytes were isolated from PBMC using CD14 microbeads (Miltenyi Biotec) and cultured in IL-6 and GM-CSF for 7 days. Then, MDSC were harvested on day 7 using Accutase cell dissociation medium (Thermo Fisher). Stained with a live / dead TMCells were stained with NIR viability dye (Thermo Fisher) and monoclonal antibodies conjugated with fluorescent labels against CD14 (clone M5E2, BD Biosciences), CD11b (clone ICRF44, BD Biosciences), CD33 (clone HIM3-4, BD Biosciences), CD15 (clone W6D3, BioLegend), CD66b (clone 6 / 40c, BioLegend), HLA-DR (clone G46-6, BD Biosciences), and B7-H3 (clone 7-517, BD Biosciences) to verify the expression of their surface markers. Cells were also fixed and permeabilized prior to staining with monoclonal antibodies conjugated with fluorescent labels against IL-10 (clone JES3-9D7, BD Biosciences), TGF-β1 (clone S200006A, BioLegend), and iNOS (clone CXNFT, Thermo Fisher) to detect the expression of inhibitory molecules.

[0611] For the MDSC co-culture experiment, untransduced and B7H3.CAR EBVST were first labeled with CellTrace TM Violet (Thermo Fisher) and then co-cultured with allogeneic MDSCs overnight at a defined effector cell:target ratio. To evaluate proliferation, CellTrace TM Violet-labeled untransduced and B7H3.CAR EBVST were cultured with allogeneic MDSCs on anti-CD3 / CD28-coated plates for 6 days. Cells were harvested and stained with fluorescent-labeled monoclonal antibodies as described above to distinguish effector cells and target cells. CountBright TM Absolute Counting Beads (Invitrogen) were added to count cell populations by flow cytometry. Proliferation was evaluated based on the percentage of cells with CellTrace TM Violet dilution. The proliferation index was calculated by normalizing the percentage of proliferating cells to the percentage of proliferating cells under the MDSC-free control condition.

[0612] 1.26 In vivo cytokine release syndrome (CRS) model in humanized mice

[0613] B16F10-WT cells were transduced with a retrovirus containing the human B7-H3 construct to generate the B16F10-hB7H3 murine tumor cell line expressing human B7-H3. 5x10 5B16F10-hB7H3 cells were subcutaneously injected into the right lower abdomen of wild-type C57BL6 / J mice. When the tumors became palpable, the mice were irradiated with 5 Gy to create a lymphocytopenic environment. Three days after irradiation, the mice were randomly assigned to treatment groups and were injected intravenously with 10x10 6 unstimulated or P2A5.mCAR-T cells, which were generated from activated T cells isolated from the spleens of B6SJL-Ptprca Pepc b / BoyJ mice (Jackson Laboratory). Untreated mice served as controls. Physical examinations, tumor, and body weight measurements were performed twice weekly until the tumors in the control mice reached the endpoint size of 1000 mm 3 . Mouse sera were collected by cheek bleeds 3 days after treatment. Serum cytokine levels were quantified using bead-based multiplex assays with Luminex technology (Merck-Millipore). When the mice were sacrificed 10 days after treatment, blood, spleen, liver, lung, tumor, brain, and bone marrow were collected for endpoint flow cytometry and pathology evaluations. Endpoint antibody staining was performed using a monoclonal antibody against the live / dead NIR viability dye (ThermoFisher TM ), fluorescein-labeled anti-camel VHH (clone 96A3F5, Genscript), mouse CD45.1 (clone A20, BD Biosciences), CD45.2 (clone 104, BD Biosciences), CD11b (clone M1 / 70, BD Biosciences), CD11c (clone N418, BioLegend), Gr1 (clone RB6-8C5, BD Biosciences), CD19 (clone ID3, BD Biosciences), NK1.1 (clone PK136, BD Biosciences), CD3 (clone 145-2C11, BD Biosciences), CD4 (clone RM4-5, BD Biosciences), CD8 (clone 53-6.7, BD Biosciences), B7-H3 (clone EPNCIR122, Abcam), and CountBright TM Absolute Count Beads (Invitrogen TM ) were added, and cell populations were counted by flow cytometry.

[0614] 1.27 Flow cytometry analysis

[0615] To generate humanized mice, 1x10 5CD34+ cord blood cells were intravenously injected into sub-lethally irradiated triple transgenic NSG (NSG-SGM3) mice (Jackson Laboratory) expressing human IL3, GM-CSF, and stem cell factor (SCF). Four weeks after humanization, successful humanization of the mice was verified by positive staining of human CD45 on peripheral blood cells. For the B7H3 CAR test group, 5x10 6 HT-29 cells were subcutaneously injected into the right abdominal cavity of humanized mice. When the tumors became palpable, the mice were randomly assigned to treatment groups and stratified by tumor volume. 5x10 6 untransduced or B7H3.CAR EBVST were intravenously injected into the mice, and untreated mice served as controls. For the CRS positive control group, 2x10 6 CD19-expressing NALM-6 cells were intravenously injected into humanized mice. Tumor burden in NALM-6 transplanted mice was tracked using IVIS imaging. Eighteen days after NALM-6 tumor implantation, the mice were randomly grouped and received CD19 CAR-T treatment or not. Physical examinations, tumor, and body weight measurements were performed twice a week until the endpoint was reached 7 days after treatment. Mouse serum was collected by cheek bleeding 3 days after treatment. Serum cytokine levels were quantified using a LEGENDplex multiplex bead-based immunoassay (BioLegend). When the mice were sacrificed, blood, spleen, liver, and bone marrow were collected for endpoint flow cytometry analysis.

[0616] Endpoint antibody staining used Live / Dead TMCells were stained with monoclonal antibodies against Aqua reactive dyes (Thermo Fisher) and fluorescently labeled anti-camel VHH (clone 96A3F5, Genscript), mouse CD45 (clone 30-F11, BD Biosciences), human CD45 (clone H130, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD14 (clone M5E2, BioLegend), CD19 (clone SJ25C1, BioLegend), CD11b (clone ICRF44, BD Biosciences), CD66b (clone G10F5, BioLegend), CD11c (clone B-ly6, BD Biosciences), CD56 (clone HCD56, BioLegend), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), HLA-A3 (clone GAP.A3, BD Biosciences), B7-H3 (clone 7-517, BD Biosciences), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), LAG3 (clone 11C3C65, BioLegend), and CountBright TM Absolute Counting Beads (Invitrogen TM ) were added, and cell populations were counted using flow cytometry.

[0617] 1.28 Statistical analysis

[0618] Cell flow cytometry was performed on a FACSymphony A3 cell analyzer (BD Biosciences), and the data were analyzed in FlowJo v10.8.1 for Windows.

[0619] Example 2: Results

[0620] Statistical analysis and visualization were performed using Prism 9 software for Windows (Graphpad Software Inc.). For comparisons between two groups, a two-tailed unpaired t-test was used where appropriate. To compare over time or between three or more groups, one-way or two-way analysis of variance (ANOVA) with Tukey or Dunnett or Sidak's post hoc was used where appropriate.

[0621] 2.1 B7-H3-specific VHH

[0622] Figure 1A

[0623] After multiple rounds of biopanning with recombinant human 4Ig-B7-H3, B7H3-specific VHHs were isolated from an immunized llama library. The accuracy was increased by reducing the concentration of the target protein from 100 nM in the first round to 20 nM in the second round and finally to 5 nM in the third round. Phage eluted in the second and third rounds was screened by ELISA for B7-H3 binding. After sequencing to identify specific VHH sequences, 20 candidate lead antibodies were expressed and purified to further screen for binding to B7-H3 expressed on the surface of HepG2 cells expressing B7-H3, as well as the binding kinetics to recombinant 4Ig B7H3 by surface plasmon resonance. Although most of the lead antibodies bound very tightly to the recombinant B7-H3 protein, only 9 lead antibodies were able to bind strongly to HepG2 cells expressing B7-H3.

[0624] Clone P2A5 had a strong binding affinity for HepG2 cells expressing human B7-H3 ( Figure 1B ), and had a moderate dissociation rate ( Figure 1B ).

[0625] The affinity of the CAR for its target antigen affects not only its efficacy but also the safety of CAR T cell therapy. The higher the affinity binding to a target such as B7-H3, the greater the risk of off-target, non-oncotoxic effects, as B7-H3 is expressed at low levels in non-malignant cells and tissues. Therefore, the balance between efficacy and specificity for B7-H3-high expressing tumor cells can be regulated by selecting medium affinity and avoiding high affinity VHHs as the binding domain of the CAR. Therefore, the inventors decided to focus on the medium affinity clone P2A5.

[0626] The binding affinity of P2A5 for human 4Ig-B7-H3 was evaluated by multi-cycle kinetic analysis by SPR ( Figure 1C ).

[0627] In humans, B7-H3 exists in two major subtypes with similar structures, consisting of a pair of extracellular immunoglobulin domains IgV-IgC (in the shorter 2Ig form (2Ig-B7-H3)), or by a tandem pair IgV-IgC-IgV-IgC (for the larger 4Ig form (4Ig-B7-H3)). Mouse B7-H3 contains a pair of IgV-IgC domains and has 87% sequence identity with human 2Ig-B7-H3. The inventors further investigated the binding of P2A5 to 2Ig-B7-H3 and mouse B7-H3 ( Figure 1B and 1D)。Although the binding of P2A5 to 4Ig-B7-H3 is relatively strong, with an equilibrium dissociation constant K D of ~30.9 nM( Figure 1C ), its binding affinity to 2Ig-B7-H3 and murine B7-H3 is ~10-fold weaker, with K D values of 340 nM and 473 nM, respectively( Figure 1C and 1D ).

[0628] 2.2 Cross-reactivity of B7H3-specific P2A5 VHH with murine and human B7H3 expressed by cells

[0629] B7-H3 is a highly glycosylated protein, and the glycosylation patterns may vary between human and murine cells. Therefore, to determine that the binding epitope of P2A5 on B7-H3 is not affected by glycosylation in different cells, the B7-H3-negative murine colon cancer cell line CT26 and the gastric adenocarcinoma cell line MKN7, in which B7-H3 expression had been knocked out by CRISPR / Cas9, were transfected to express human (4Ig) and murine B7-H3 molecules. Then, a commercially available anti-murine B7-H3 antibody (clone ab134161, Abcam, Cat. No. EPNCIR122) or anti-human B7-H3 antibody (clone DCN.70, Biolegend, Cat. No. 331606) was used as a positive control, and P2A5 expressed in the VHH-Fc form and an isotype control (VHH to CD19) were used to stain the transfected cells. The staining of the transfected cells was then observed by fluorescence microscopy (Figure 2). It was observed that P2A5 could bind not only to murine B7-H3 expressed on murine CT26 cells( Figure 2A ) and human B7-H3 expressed on human MKN7 cells( Figure 2D ), but also to murine B7H3 expressed on MKN7 cells( Figure 2B ) and human B7-H3 expressed on CT26 cells( Figure 2C ). Therefore, the cross-reactivity of P2A5 with murine and human B7-H3 is not affected by the cells from different sources and thus not by the glycosylation patterns of these cells.

[0630] 2.3 Expression and functional cytotoxicity of B7-H3 CAR T cells

[0631] The lead anti-B7-H3 VHH candidate P2A5 was cloned into the retroviral vector pSFG as a CAR with a 4-1BB spacer domain, a CD28-derived transmembrane and intracellular domain, and a CD3ζ intracellular signaling domain( Figure 3A)。Primary PBMCs from 2 healthy donors were activated on CD3- and CD28-coated plates and transduced with retroviral particles carrying the B7-H3 CAR transgene. Transduction efficiency was evaluated by flow cytometry 6 days after transduction using labeled anti-camel VHH antibodies ( Figure 3B )。B7-H3 CAR T cells from both donors showed good transduction efficiency (>60%) and fold expansion (more than 20-fold) ( Figure 3C )。To detect the cytotoxic efficiency of B7-H3 CAR T cells against B7-H3-expressing A549 non-small cell lung cancer and MDA-MB-231 triple-negative breast cancer cell lines, a real-time cytotoxicity assay was established, consisting of effector cells with effector-to-target ratios of 1:1 and 5:1 effector T cells. B7-H3 CAR T cells showed good and specific killing effects against A549 and MDA-MB-231 cells at E:T ratios of 1:1 and 5:1 ( Figure 3D )。The cytotoxicity of B7-H3 CAR T cells was further evaluated in multiple rounds of co-culture with THP-1 acute myeloid leukemia cells expressing B7-H3, with an effector cell:target ratio of 1:2. B7-H3 low CMK leukemia cells were used as a negative control. B7-H3 CAR T cells completely eliminated THP-1 cells through 3 consecutive rounds of target contact. In contrast, CMK cells were not lysed in the presence of B7-H3 CAR T cells ( Figure 3E )。

[0632] 2.4 Generation and characterization of CAR EBVST expressing specific B7-H3

[0633] To create an off-the-shelf allogeneic T cell therapy for cancer, a virus-specific T cell (VSTs) platform was adopted to manufacture our allogeneic B7-H3 CAR-T therapy. EBVSTs were generated by pulsing CD45RA-depleted donor PBMCs with a mixture of E8V proteins and maintained in a medium containing human platelet lysate (hPL), human IL-7, and IL-15. On day 5, EBVSTs were transduced with retroviral particles containing the above B7-H3 CAR. On day 9, cells were restimulated with irradiated MHC I / II knockout universal LCL (uLCL) at an EBVST:uLCL ratio of 1:4. CAR EBVSTs were harvested, analyzed, and cryopreserved on day 16 of culture ( Figure 4A )。B7-H3 CAR EBVSTs generated from 6 donors expanded approximately 100- to 270-fold after transduction (Figure 48). B7-H3 CAR EBVSTs from all donors had good CAR+ cell enrichment in the final product, with more than 80% of EBVSTs expressing CAR ( Figure 4C)。In the final B7-H3 CAR EBVST product, most donor cells showed enrichment of CD4+ cells (Figure 40).

[0634] Since B7-H3 is known to be expressed on T cells and is hypothesized to be an immune checkpoint molecule, the B7-H3 expression of the final product was examined and found that CAR EBVST expressed lower levels of B7-H3 than untransduced EBVST( Figure 4F )。Staining for checkpoint molecules PD-1, Tim-3, and LAG-3 showed that the number of markers expressed by B7-H3 CAR EBVST was higher than that of untransduced EBVST, and the difference was greater in CD8+ cells than in CD4+ cells( Figure 4F )。

[0635] To evaluate whether B7-H3 CAR EBVSTs respond to EBV antigens, EBVSTs were stimulated with a mixture of EBV protein mixed peptides. B7-H3 CAR EBVSTs upregulated the expression of TNFα and / or IFNγ upon stimulation with EBV mixed peptides, although the levels were lower than those of untransduced EBVSTs( Figure 4G )。

[0636] 2.5 Cytotoxicity of B7-H3 CAR EBVST against B7-H3+ cancer cell lines in vitro

[0637] To evaluate the specific cytolysis of B7-H3 CAR EBVST, EBVST was co-incubated with B7-H3+ cell lines and their B7-H3KO counterparts at an effector cell:target ratio of 1:1 using xCELLigence. B7-H3 CAR EBVST showed rapid killing of DLD-1, SW480, NCI-N87, and MDA-MB-468, and all target cells were lysed within 24 hours after co-incubation. Although the cytolysis of anti-HT29, MKN7, MKN45, MDA-MB-231, and A549 was slower, good cytolysis was still achieved at the end point. The cytolysis of anti-B7-H3KO cell lines was delayed and attenuated, showing a level similar to background killing in some donors, indicating that the killing of these cell lines was mediated by the B7-H3 CAR activation system( Figures 5A to 5D )。

[0638] To further evaluate the continuous killing ability of B7-H3 CAR EBVST, multiple rounds of co-culture were performed with target cells at an effector cell:target ratio of 1:2. Flow cytometry analysis showed that B7-H3 CAR EBVSTs maintained 100% cytolysis of the target up to 3 times against the B7-H3 high AML cell line THP-1, while the cytotoxicity against B7-H3 low CMK was negligible( Figure 6A). At the same time, the number of B7-H3 CAR also increased, but the number of non-transduced EBVST effector cells did not increase ( Figure 6B ).

[0639] Serial killing efficacy against solid tumor cell lines was also evaluated using the xCELLigence system. After each exposure, effector cells were counted and transferred to freshly inoculated tumor cells at an effector cell:target ratio of 1:2. B7-H3 CAREBVST showed similar or equal killing in 2 rounds of serial target exposure. Subsequently, CAR EBVST showed reduced killing efficacy ( Figure 6C ). After the first two rounds of continuous target contact, the number of effector cells also mostly increased ( Figure 6D ).

[0640] 2.6 In vivo activity of B7-H3 CAR EBVST against B7-H3+ tumors

[0641] 2.6.1 In vivo efficacy and safety of B7-H3 CAR T cells in B7-H3 positive colorectal cancer

[0642] To evaluate the antitumor activity of B7-H3 CAR EBVST against B7-H3-expressing colorectal cancer, immunodeficient NSG-MHC I / IIDKO mice were implanted with HT-29 cells. After tumor implantation, mice were randomized to receive no treatment or to receive untransduced or B7-H3 CAR EBVST treatment ( Figure 7A Within 14 days after treatment, the body weight of mice was similar and stable between treatment groups ( Figure 7B ).

[0643] While tumor growth continued uncontrolled in untreated or non-transduced EBVST mice, treatment with B7-H3CAR EBVST induced significant tumor reduction ( Figure 7C Similarly, endpoint flow cytometry analysis showed a significant decrease in the population of viable HT-29 cells in tumors from mice treated with B7-H3 CAR EBVST compared to untreated and non-transduced mice ( Figure 7D The small population of HT-29 cells remaining in the tumors of B7-H3 CAR EBVST-treated mice retained high B7-H3 expression, indicating the absence of tumor antigen downregulation ( Figure 7D ).

[0644] Further evaluation showed that the number of B7-H3 CAR EBVSTs detected in the blood, liver, lungs, and spleen of mice was significantly increased compared with that of non-transduced EBVSTs. More importantly, the number of B7-H3 CAR EBVSTs observed in tumors far exceeded that of non-transduced EBVSTs ( Figure 7E ). Altogether, this suggests that B7-H3 CAR EBVST can effectively migrate to tumor sites to exert its anti-tumor activity.

[0645] To evaluate the anti-tumor activity of B7-H3 CAR EBVST against a second B7-H3-expressing colorectal cancer cell line, in vivo studies were conducted. Immunodeficient NSG-MHC I / IIDKO mice were implanted with SW-480 cells. After tumor implantation, the mice were randomly assigned to receive no treatment or treatment with untransduced or B7-H3 CAR EBVST ( Figure 7F ). Within 15 days after treatment, the body weights of the mice in the treatment groups remained stable ( Figure 7G ).

[0646] Tumor control was significantly improved in mice treated with B7-H3 CAR EBVST compared to no treatment or treatment with untransduced EBVST ( Figure 7H ), consistent with endpoint analysis, in which a significantly reduced population of viable SW-480 tumor cells was observed in mice treated with B7-H3 CAR EBVST ( Figure 7I ). SW-480 tumor cells in mice treated with B7-H3 CAR EBVST retained high B7-H3 expression, indicating no tumor antigen downregulation ( Figure 7I ).

[0647] Tests showed that more B7-H3 CAR EBVST could be detected in the liver, lungs, and spleens of mice compared to untransduced EBVST. Additionally, B7-H3 CAR EBVST was more abundant in tumors compared to untransduced EBVST, and they also exceeded the amount of B7-H3 CAR EBVST in other organs. Overall, this indicates that B7-H3 CAR EBVST is capable of effective migration and accumulation at the tumor site to exert its anti-tumor activity ( Figure 7J ).

[0648] 2.6.2 In vivo efficacy and safety of B7-H3 CAR T cells in B7-H3 positive gastric cancer

[0649] To evaluate the anti-tumor activity of B7-H3 CAR EBVST against B7-H3-expressing gastric cancer, immunodeficient NSG-MHC I / IIDKO mice were implanted with NCI-N87 cells. After tumor implantation, the mice were randomly assigned to receive no treatment or treatment with untransduced or B7-H3 CAR EBVST ( Figure 8A ). Between 14 days after treatment, the body weights of the mice among the treatment groups were similar and stable ( Figure 8B ).

[0650] While tumor growth continued to increase in mice that received no treatment or untransduced EBVST, treatment with B7-H3 CAREBVST induced significant tumor regression, with almost undetectable tumor masses at the endpoint ( Figure 8C) Flow cytometry analysis at the end point showed that the viable N-87 cell population was significantly reduced in mice treated with B7-H3 CAR EBVST compared to untreated and untransduced mice ( Figure 7D )。A small residual N-87 cell population in tumors of mice treated with B7-H3 CAR EBVST retained high B7-H3 expression, indicating no downregulation of tumor antigens ( Figure 8D )。

[0651] Further evaluation showed that significantly increased numbers of B7-H3 CAR EBVST were detected in the blood, liver, lung, and spleen of mice compared to untransduced EBVST. More importantly, the number of B7-H3 CAR EBVST in tumors was observed to far exceed that of untransduced EBVST ( Figure 8E )。Taken together, this indicates that B7-H3 CAR EBVST can effectively migrate to the tumor site to exert its anti-tumor activity.

[0652] Example 3

[0653] 3.1 Biopanning of B7H3-specific VHH

[0654] After multiple rounds of biopanning with recombinant 4Ig B7H3 protein, VHHs specific for B7H3 were isolated from an immunized llama library. The accuracy was increased by reducing the concentration of the target protein from 100 nM in the first round to 20 nM in the second round and finally to 5 nM in the third round. Phage eluted from the second and third rounds was screened by ELISA for B7H3 binding (Figure B1A( Figure 9A ))。After sequencing to identify the specific VHH sequences, 20 shortlisted lead antibodies were expressed and purified for further screening by binding to B7H3 expressed on the surface of HepG2 cells expressing B7H3 (Figure B1B( Figure 9B )) and by binding kinetics to recombinant 4Ig B7H3 by surface plasmon resonance (Figure B1C( Figure 9C ))。Only 9 lead antibodies were able to strongly bind to HepG2 cells expressing B7H3, although most lead antibodies bound very strongly to recombinant B7H3. Notably, clone P2A5 showed strong binding to HepG2 cells with a moderate dissociation rate (Figure B1B & C( Figure 9B&C)). The binding affinity of the CAR not only affects its efficacy but also the safety of CAR T cells (23). The risk of on-target, off-tumor toxicity increases with increasing affinity because targets such as B7H3 can be expressed at low levels in non-malignant tissues. Therefore, the balance between efficacy and specificity for B7H3 highly expressed on tumor cells can be adjusted by selecting medium affinity and avoiding high affinity VHHs as the binding domain of the CAR. Therefore, clone P2A5 was selected for further study. The binding affinity of P2A5 to the 4Ig form of B7H3 was further measured by multi-cycle kinetic analysis of SPR (Figure B1D( Figure 9D ). B7H3 exists in two forms with similar structures, consisting of a pair of extracellular immunoglobulin domains IgV-IgC (the shorter 2Ig form) or a tandem pair IgV-IgC-IgV-IgC (the larger 4Ig form). Mouse B7H3 consists of a pair of IgV-IgC domains and has 87% sequence identity with the human 2Ig form. Therefore, the binding of P2A5 to the 2Ig subtypes of human B7H3 and mouse B7H3 was studied (Figure B1E and F( Figure 9E and F). We saw that while P2A5 could bind relatively strongly to 4Ig B7H3 with a dissociation constant K D of approximately 30.9 nM, its binding to the 2Ig subtype and mouse B7H3 was approximately 10-fold weaker, with K D of 340 nM and 473 nM, respectively (Figure B1G( Figure 9G ).

[0655] 3.2 Cross-reactivity of B7H3-specific P2A5 VHH with cell-expressed murine and human B7H3.

[0656] B7H3 is a highly glycosylated protein, and the glycosylation patterns may differ between human and mouse cells. Therefore, to determine that the binding epitope of P2A5 on B7H3 is not affected by glycosylation in different cells, the B7H3-negative mouse colon cancer cell line CT26 and the gastric adenocarcinoma cell line MKN7, in which B7H3 expression had been knocked out by CRISPR / Cas9, were transfected to express human (4Ig) and mouse B7H3 molecules. Then, the transfected cells were stained with commercially available anti-mouse or anti-human B7H3 antibodies as positive controls, P2A5 expressed in the VHH-Fc format, and an isotype control. Then, the staining of the transfected cells was observed by fluorescence microscopy (Figure B2 (Figure 10)). It was observed that P2A5 could bind not only to mouse B7H3 expressed on mouse CT26 cells (Figure B2A( Figure 10A )) and human B7H3 expressed on human MKN7 cells (Figure B2D( Figure 10D )) but also to mouse B7H3 expressed on MKN7 cells (Figure B2B( Figure 10B)) and human B7H3 expressed on CT26 cells (Figure B2C( Figure 10C ). Thus, the cross-reactivity of P2A5 with murine and human B7H3 is not affected by the expression of cells from different sources, nor by the glycosylation patterns of these cells.

[0657] 3.3 Expression and functional cytotoxicity of B7H3.CAR T cells

[0658] The lead anti-B7-H3 VHH candidate P2A5 was cloned into the retroviral vector pSFG as a CAR with a 4-1BB spacer domain, a CD28-derived transmembrane and intracellular domain, and a CD3ζ intracellular signaling domain (Figure B3A( Figure 11A ). Primary PBMCs from 2 healthy donors were activated and transduced with retroviral particles carrying the B7H3.CAR transgene. The transduction efficiency was evaluated by flow cytometry using a labeled anti-camel VHH antibody 6 days after transduction (Figure B3B( Figure 11B ). B7H3 CART cells from both donors showed good transduction efficiency (>60%) and fold expansion (more than 20-fold) (Figure B3C( Figure 11C ). A real-time cytotoxicity assay was established to detect the cytotoxic efficiency of B7H3.CART cells against B7-H3 of non-small cell lung cancer expressing A549 and MDA-MB-231 triple-negative breast cancer cell lines, consisting of effector cell to target cell ratios of 1:1 and 5:1. B7H3.CART cells showed good and specific killing effects on A549 and MDA-MB-231 cells at E:T ratios of 1:1 and 5:1 of effector T cells to target cells (Figure B3D( Figure 11D ). The cytotoxic activity of B7H3.CART cells was further evaluated by co-culturing with THP-1 acute myeloid leukemia cells expressing B7-H3 in multiple rounds at an effector cell:target ratio of 1:2. B7-H3-low CMK leukemia cells were used as a negative control. B7H3.CART cells completely eliminated THP-1 cells through 3 consecutive rounds of target contact. In contrast, CMK CAR cells were not lysed in the presence of B7H3 (Figure B3E( Figure 11E ).

[0659] 3.4 Generation and characterization of B7H3.CAR EBVST

[0660] To develop an off-the-shelf third-party T cell therapy for cancer, a virus-specific T cell (VST) platform was used to manufacture our allogeneic B7H3.CAR-T therapy. EBVSTs were generated by pulsing CD45RA-depleted donor PBMCs with a mix of EBV proteins mixed peptides and maintained in media containing human platelet lysate (hPL), human IL-7, and IL-15. On day 5, EBVSTs were transduced with a retrovirus carrying the B7H3 CAR transgene. On day 9, irradiated feeder cells were used to re-stimulate the cells at an EBVST:feeder cell ratio of 1:4. CAR EBVSTs were harvested, analyzed, and cryopreserved on day 16 of culture (Figure B4A( Figure 12A ))). B7H3.CAR EBVSTs generated from 6 donors expanded approximately 100 to 270-fold after transduction (Figure B4B( Figure 12B ))). B7H3.CAR EBVSTs from all donors had good enrichment of CAR-posi...

Claims

1. An antigen-binding molecule, optionally isolated, which binds to B7 homolog 3 (B7-H3), wherein the antigen-binding molecule comprises a single-domain antibody sequence containing 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.

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

8.

3. The antigen-binding molecule according to claim 1 or claim 2, wherein, the antigen-binding molecule contains a single-domain antibody sequence containing the following FRs: 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.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein, the antigen-binding molecule is a multispecific antigen-binding molecule, wherein the antigen-binding molecule 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 according to any one of claims 1 to 4.

6. The CAR according to claim 5, wherein, the CAR comprises or consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

9.

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

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

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

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

11. The cell according to claim 9 or claim 10, wherein, the cell is a virus-specific T cell, optionally an Epstein-Barr virus (EBV)-specific T cell.

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

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

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

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

16. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 15, wherein, 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, colorectal tumor, colon cancer, colon tumor, kidney cancer, clear cell renal cell carcinoma, nephroblastoma, prostate cancer, ovarian cancer, ovarian tumor, cervical cancer, endometrial cancer, germ cell tumor, gastric cancer, gastric tumor, 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, oropharyngeal cancer, oropharyngeal tumor, nasopharyngeal cancer, esophageal cancer, bladder cancer, urothelial carcinoma, brain cancer, medulloblastoma, ependymoblastoma, glioma, diffuse pontine glioma, diffuse midline glioma, choroid plexus carcinoma, pineal tumor, neuroblastoma, central nervous system tumor, primitive neuroectodermal tumor, atypical teratoid / rhabdoid tumor, brainstem glioma, sarcoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, peritoneal cancer, desmoplastic small round cell tumor and mesothelioma.

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

18. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of claims 1 to 4 that binds to B7-H3 or a CAR according to claim 5 or 6.

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

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

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

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