Antibodies selective for the 4Ig isoform of B7-H3 and methods of use thereof

Abstract

Provided herein are antibodies and antigen-binding portions thereof that specifically bind to the 4Ig isoform of the B7 homolog 3 protein (B7-H3), various compositions of such antibodies or antigen-binding portions thereof, and recombinant nucleic acids encoding the antibodies and antigen-binding portions thereof. Also provided are methods of using the antibodies or antigen-binding portions thereof in therapies and diagnostics for cancer or inflammation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 482,201, filed January 30, 2023, the contents of which are incorporated herein by reference as if fully set forth herein.

[0002] Reference to sequence listings submitted as XML via EFS-WEB

[0001] This application contains a Sequence Listing in XML format. The Sequence Listing, created on January 30, 2024, entitled 090723-1424932-22-107PCT-SL.xml, is 49 kilobytes in size and is hereby incorporated by reference in its entirety. [Background technology]

[0003] Immunotherapies aimed at suppressing immune checkpoints on effector T cells have enabled dramatic and durable tumor responses against selected solid tumors. Antibody-based immunotherapy has prompted the identification of clinically relevant tumor antigens that can be targeted in solid tumors. Among them, the B7 ligand family is overexpressed with limited heterogeneity and high frequency on differentiated malignant and cancer-initiating cells, as well as in many different cancer types, and has limited to low expression levels in normal tissues, making it an attractive target for antibody-based immunotherapy. However, antigens such as the B7 family have different isoforms that play distinct roles, requiring antibodies with high specificity for selected isoforms. Different isoforms of the B7 family of antigens have also been identified in different inflammatory conditions. Antibodies with high specificity for selected isoforms are important for maximizing therapeutic and / or diagnostic efficacy. Therefore, novel antibodies for immunotherapeutic approaches are needed to further improve patient clinical outcomes. Summary of the Invention [Problem to be solved by the invention]

[0004] This Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended for use as an aid in limiting the scope of the claimed subject matter. [Means for solving the problem]

[0005] In one aspect, provided herein is an isolated antibody or antibody fragment that specifically binds to B7-H3. In some embodiments, the isolated antibody or antibody fragment is a humanized antibody or antibody fragment. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable region (VH) having at least 90% identity to SEQ ID NO:3, and comprising a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23, and comprising a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO:3; and a light chain variable region (VL) having at least 90% identity to SEQ ID NO:4, and comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and comprising an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4.

[0006] In some embodiments, the isolated antibody or antibody fragment thereof comprises the light chain variable sequence set forth in SEQ ID NO: 4. In some embodiments, the isolated antibody or antibody fragment thereof comprises the heavy chain variable sequence set forth in SEQ ID NO: 3. In some embodiments, the humanized monoclonal antibody or antibody fragment comprises the heavy chain variable sequence set forth in SEQ ID NO: 3 and the light chain variable sequence set forth in SEQ ID NO: 4.

[0007] In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable region (VH) having at least 95% identity to SEQ ID NO:1 or SEQ ID NO:2, and comprising a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23; and a light chain variable region (VL) having at least 95% identity to SEQ ID NO:4, and comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and having an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4. In some embodiments, the isolated antibody or antibody fragment comprises the heavy chain variable sequence set forth in SEQ ID NO:1 and the light chain variable sequence set forth in SEQ ID NO:4. In other embodiments, the isolated antibody or fragment thereof comprises the heavy chain variable sequence set forth in SEQ ID NO:2 and the light chain variable sequence set forth in SEQ ID NO:4.

[0008] In some embodiments, the isolated antibody or antibody fragment thereof is a monovalent scFv (single-chain fragment variable) antibody, a bivalent scFv, a Fab fragment, a F(ab')2 fragment, a F(ab')3 fragment, an Fv fragment, or a single-chain antibody. In some embodiments, the isolated antibody or antibody fragment thereof is a chimeric antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody, or a BiTE. In some embodiments, the isolated antibody or antibody fragment thereof is an IgG antibody, or a recombinant IgG antibody or antibody fragment.

[0009] In some embodiments, the isolated antibody or antibody fragment competes for binding to the same epitope as the humanized antibody or antibody fragment disclosed herein. In some embodiments, the isolated antibody or antibody fragment binds to the epitope on B7-H3 recognized by the isolated antibody or antibody fragment disclosed herein. In some embodiments, the isolated antibody or antibody fragment has increased binding affinity for the 4Ig isoform of B7-H3. In some embodiments, the isolated antibody or antibody fragment has increased specificity for the 4Ig isoform of B7-H3 over the 2Ig isoform of B7-H3. In certain embodiments, the isolated antibody or antibody fragment has at least 350-fold increased selectivity for the 4Ig isoform of B7-H3 over the 2Ig isoform of B7-H3.

[0010] In some embodiments, the isolated antibody or antibody fragment is conjugated or fused to an imaging agent, cytotoxic agent, metal, or radioactive moiety. In some embodiments, the imaging agent is a fluorophore. In some embodiments, the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, Tb-161, At-211, Ac-225, or Pb-212. In some embodiments, the antibody or antibody fragment is an immunoconjugate or radioimmunoconjugate. In some embodiments, the antibody or antibody fragment is conjugated to flagellin or a flagellin derivative.

[0011] In some embodiments, the isolated antibody or antibody fragment is an antibody-drug conjugate.

[0012] In another aspect, provided herein is an isolated nucleic acid encoding the antibody heavy and / or light chain variable region of the isolated antibody of any of the disclosed embodiments. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 30. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 29. In some embodiments, the nucleic acid sequence encoding the heavy chain variable region comprises SEQ ID NO: 29. In some embodiments, the nucleic acid sequence encoding the light chain variable region comprises SEQ ID NO: 30.

[0013] In some embodiments, provided herein is an expression vector comprising a nucleic acid of any one of the disclosed embodiments.

[0014] In some embodiments, provided herein are hybridoma cells or engineered cells comprising nucleic acid encoding the antibody or antibody fragment of any one of the disclosed embodiments.

[0015] In another aspect, provided herein is a method of making a humanized monoclonal antibody or antibody fragment of any one of the present embodiments, comprising culturing hybridoma cells or engineered cells of the present embodiments under conditions that allow expression of the antibody, and optionally isolating the antibody from the culture.

[0016] In a further aspect, provided herein is a chimeric antigen receptor (CAR) protein comprising an antigen-binding domain comprising a heavy chain variable region (VH) comprising the VHCDR1, VHCDR2, and VHCDR3 amino acid sequences derived from any isolated antibody or antibody fragment disclosed herein; and a light chain variable region (VL) comprising the VLCDR1, VLCDR2, and VLCDR3 amino acid sequences derived from any isolated antibody or antibody fragment disclosed herein.

[0017] In some embodiments, the antigen-binding domain comprises heavy and light chain CDR sequences such as the following: a heavy chain variable region having a heavy chain variable region sequence set forth in SEQ ID NO: 3, or a VHCDR1 amino acid sequence comprising SEQ ID NO: 9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO: 10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO: 11, 17, or 23, and comprising a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO: 3; and a light chain variable region having a light chain variable sequence set forth in SEQ ID NO: 4, or a VLCDR1 amino acid sequence comprising SEQ ID NO: 12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO: 13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO: 14, 20, or 26, and comprising an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO: 4. In some embodiments, the antigen-binding domain comprises a heavy chain variable sequence having at least 90% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and a light chain variable sequence having at least 90% identity to SEQ ID NO:4, with an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4. In some embodiments, the antigen-binding domain comprises a heavy chain variable sequence having at least 95% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and a light chain variable sequence having at least 95% identity to SEQ ID NO:4, with an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4. In some embodiments, the antigen-binding domain comprises a heavy chain variable sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and a light chain variable sequence of SEQ ID NO:4, with an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4. In some embodiments, the antigen-binding domain comprises a heavy chain variable sequence of SEQ ID NO:3, and a light chain variable sequence of SEQ ID NO:4.

[0018] In some embodiments, the antigen-binding domain specifically binds to B7-H3. In some embodiments, the antigen-binding domain specifically binds to the 4Ig isoform of B7-H3. In some embodiments, the antigen-binding domain is a humanized antigen-binding domain.

[0019] In some embodiments, the CAR protein further comprises a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR protein further comprises a hinge domain, and the hinge domain is a CD8α hinge domain or an IgG4 hinge domain. In some embodiments, the transmembrane domain is a CD8α transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ intracellular signaling domain.

[0020] In another aspect, the present disclosure provides a nucleic acid molecule encoding any of the CARs of this embodiment. In some embodiments, the sequence encoding the CAR is operably linked to an expression control sequence. In some embodiments, the nucleic acid is further defined as an expression vector.

[0021] In some embodiments, provided herein is an engineered cell comprising a nucleic acid molecule encoding any one of the CARs of the disclosed embodiments. In some embodiments, the cell is a T cell. In some embodiments, the cell is a NK cell. In some embodiments, the nucleic acid is integrated into the genome of the cell. In some embodiments, the cell is a human cell.

[0022] In another aspect, provided herein is a pharmaceutical composition comprising a population of cells according to any one of the present embodiments in a pharmaceutically acceptable carrier. In other aspects, provided herein is a pharmaceutical composition comprising an isolated antibody or antibody fragment disclosed herein in a pharmaceutically acceptable carrier. In some embodiments, the antibody or antibody fragment is conjugated or fused to a cytotoxic agent, metal, radioactive moiety, or drug.

[0023] In a further aspect, provided herein are methods of treating cancer in a human patient in need thereof. In some embodiments, the method comprises administering to the patient an anti-tumor effective amount of a cell therapy comprising one or more cells according to any one of the disclosed embodiments. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are autologous cells. In some embodiments, the cells are HLA-matched to the human subject. In other embodiments, the method comprises administering to the patient an anti-tumor effective amount of an isolated antibody or antibody fragment disclosed herein. In some embodiments, the antibody or antibody fragment is conjugated or fused to a cytotoxic agent, metal, radioactive moiety, or drug. In some embodiments, the cancer has been determined to express elevated levels of B7-H3 compared to healthy tissue. In some embodiments, the cancer is renal cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, melanoma, prostate cancer, breast cancer, glioma, lymphoma, or neuroectodermal cancer. In some embodiments, the patient has previously failed to respond to an immune checkpoint inhibitor. In some embodiments, the patient has relapsed.

[0024] In some embodiments, the method further comprises administering at least a second anticancer therapy, in some embodiments, the second anticancer therapy is chemotherapy, molecular targeted therapy, immunotherapy, radiation therapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormone therapy, epigenetic modulation, antiangiogenic therapy, or cytokine therapy.

[0025] In another aspect, provided herein are methods for diagnosing patients with cancer. In some embodiments, the methods involve detecting and / or quantifying B7-H3 using a B7-H3 antibody or antigen-binding fragment disclosed herein. In some embodiments, the B7-H3 antibody or antigen-binding fragment specifically binds to the 4Ig isoform of B7-H3. In some embodiments, the B7-H3 antibody or antigen-binding fragment is conjugated to a bioluminescent or chemiluminescent label, a metal, or a radioisotope. In some embodiments, the methods are used to select patients with cancer for treatment if the 4Ig isoform of B7-H3 is expressed by the cancer.

[0026] In yet another aspect, provided herein are methods of treating inflammation in a human patient in need thereof. In some embodiments, the method comprises treating the patient with a therapeutically effective amount of a cell therapy or antibody therapy disclosed herein. In some embodiments, the antibody is a B7-H3 antibody described herein. In some embodiments, the method further comprises administering at least a second therapeutically effective treatment for the inflammation. In some embodiments, the second therapeutically effective therapy is a molecularly targeted therapy, such as a drug, or other anti-inflammatory agent. In some embodiments, the disclosed B7-H3 antibodies and fragments thereof can be combined with corticosteroids, DMARDs, anti-cytokine therapy, or a combination thereof. In some embodiments, the disclosed B7-H3 antibodies and fragments thereof can be combined with an immunosuppressive dose of a radioisotope. For example, the radioisotope can include Zr-89, Cu-64, F-18, Y-90, Lu-177, Tb-161, At-211, Ac-225, or Pb-212. In some embodiments, an immunoinhibitory Fc domain may be coupled to the CDRs described herein. In certain embodiments, the enhanced Fc domain may enhance killing of target immune cells.

[0027] In another aspect, provided herein are methods for diagnosing patients with inflammation. In some embodiments, the methods involve detecting and / or quantifying B7-H3 using a B7-H3 antibody or antigen-binding fragment disclosed herein. In some embodiments, the B7-H3 antibody or antigen-binding fragment specifically binds to the 4Ig isoform of B7-H3. In some embodiments, the B7-H3 antibody or antigen-binding fragment is conjugated to a bioluminescent or chemiluminescent label, a metal, or a radioisotope. In some embodiments, the method is used to select patients with inflammation for treatment if the 4Ig isoform of B7-H3 is expressed by the sample.

[0028] This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the compositions and methods and to supplement any description of the compositions and methods. The drawings do not limit the scope of the compositions and methods unless the written description expressly indicates such is the case. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 is a panel of exemplary surface plasmon resonance (SPR) sensorgrams for expression, biophysical properties, and binding affinity of 11 clones preselected from a high-throughput screen for affinity (Fab-SASA) screening, along with sensorgrams for relative controls. [Figure 2] FIG. 2 is an image of a Western blot analysis of reduced and non-reduced preselected antibodies described herein. [Figure 3] FIG. 3 is a panel of exemplary sensorgrams from preselected humanized antibody clones. [Figure 4] Figure 4 is a sequence alignment showing the framework mutations present in the MIL33B-H1, MIL33B-H2, and MIL33B-H3 humanized antibodies. The sequence of the MIL33B-H3 humanized antibody is shown in a box. [Figure 5] Figure 5A shows images of Western blot analysis of 4Ig-B7-H3 and 2Ig-B7-H3 expression in non-activated leukemic monocytic cells (THP-1) in M0-, M1-, M2a-, and M2c-like states, and in phorbol myristate acetate (PMA)-activated THP-1 cells in M0-, M1-, M2a-, and M2c-like states. Figure 5B shows a panel of microscopic images of immunofluorescence staining of THP-1 cells in M0-, M1-, M2a-, and M2c-like states treated with (lower panel) or without (upper panel) PMA and further incubated with MIL33B antibody. [Figure 6] Figure 6 provides a panel of images of THP-1 cells in an undifferentiated state treated with lipopolysaccharide (LPS) and interferon-γ (INF-γ) to induce an M1 differentiated state, and THP-1 cells in an M1 differentiated state treated with PMA. [Figure 7] Figure 7A provides a panel of images of SGM3 mice intraperitoneally injected with L-012 sodium salt in saline, imaged before (day 0), and then imaged after 3 days of incubation (day 3). Figure 7B is a graph of the burst of reactive oxygen and nitrogen species (RONS) in the head in SGM3 mice intraperitoneally injected with L-012 sodium salt in saline after 5 days of treatment. M1–M4 represent values ​​for four mice. [Figure 8] Figure 8A provides a panel of SGM3 mice intraperitoneally injected with 200 μg of humanized hMIL33B-H3 in phosphate-buffered saline (PBS) twice a week for 14 days, or with PBS buffer as a control. Figure 8B provides a graph of cranial RONS bursts in SGM3 mice on days 0, 7, and 14. The slope of the line for mice treated with hMIL33B-H3 is −4823, compared with −201 for the control group. [Figure 9]Figure 9A is a graph of cranial RONS bursts in SGM3 mice imaged with L-012 after 14 days of treatment with hMIL33B-H3 antibody or PBS. Treatment was terminated on day 16, and SGM3 mice were imaged on days 21 and 28. The slope of the line for mice treated with hMIL33B-H3 is 8771, while the slope for the control group is -1043. Figure 9B is a graph of cranial RONS bursts after re-treatment with hMIL33B-H3 antibody (triangles) and PBS (circles). The slope of the line for mice treated with hMIL33B-H3 antibody is -15143, while the slope for the control group is -749. DETAILED DESCRIPTION OF THE INVENTION

[0030] The following description lists various aspects and embodiments of the present compositions and methods. Specific embodiments are not intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least within the scope of the disclosed compositions and methods. This description should be read from the perspective of a person skilled in the art; therefore, it does not necessarily include information that is known to a person skilled in the art.

[0031] I. Terminology Unless otherwise defined, all technical terms, notations, and other scientific or medical terms or terminology used herein are intended to have the meaning commonly understood by those of ordinary skill in the art. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial deviation from the definition of the term as commonly understood in the art.

[0032] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element.

[0033] As used herein, the use of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" certain elements are also contemplated as "consisting essentially of" and "consisting of those certain elements." As used herein, "and / or," when interpreted alternatively ("or"), refers to and includes any and all possible combinations, and lack of combinations, of one or more of the associated listed items.

[0034] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to encompass the recited materials or steps "and materials or steps that do not materially affect the basic and novel characteristics" of the claimed invention. See, e.g., In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in original); see also MPEP §2111.03. Thus, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising."

[0035] Unless otherwise indicated herein, the recitation of ranges of values ​​herein is intended to serve merely as a shorthand method of individually referencing each individual value falling within the range, and each individual value is incorporated herein as if it were individually recited herein. For example, if a concentration range is recited as 1% to 50%, values ​​such as 2% to 40%, 10% to 30%, or 1% to 3%, are intended to be explicitly recited herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between and including the lowest and highest recited values ​​should be considered to be explicitly recited in this disclosure.

[0036] As used herein, the terms "about" and "approximately" generally refer to an acceptable degree of error for the quantity being measured, taking into account the nature or precision of the measurement. Exemplary degrees of error are within 20% (%) of a given value or range of values; preferably within 10%; and more preferably within 5%. Any reference to "about X" or "approximately X" specifically refers to at least the values ​​X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, the expression "about X" or "approximately X" is intended to teach and provide descriptive support for a claim limitation, such as "0.98X." Numerical values ​​given herein are approximate unless otherwise specified, and the term "about" or "approximately" can be inferred if not explicitly stated. When "about" is applied to the beginning of a numerical range, it also applies to both ends of that range.

[0037] As used throughout this specification, the terms "nucleic acid," "nucleic acid sequence," "oligonucleotide," "nucleotide," or other grammatical equivalents refer to at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or their analogs, covalently linked together. A polynucleotide is a polymer of any length, including, for example, 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. The polynucleotides described herein generally contain phosphodiester bonds, but may contain at least one different linkage, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkage, as well as nucleic acid analogs that may have peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be produced; alternatively, mixtures of different polynucleotide analogs and mixtures of naturally occurring polynucleotides and analogs can be produced. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses the explicitly set forth sequence, as well as its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0038] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants, alleles, orthologs, SNPs, and complementary sequences thereof, as well as the sequence explicitly indicated.

[0039] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a single-chain polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer may contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. As used herein, the term "protein" refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single-chain polypeptides. The single-chain polypeptides of a protein may be joined by covalent bonds, e.g., disulfide bonds, or non-covalent interactions. The terms "portion" and "fragment" are used interchangeably herein to refer to a portion of a polypeptide, nucleic acid, or other molecular construct.

[0040] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of naturally occurring amino acids, unnatural amino acids, and chemically modified amino acids. Unnatural amino acids (i.e., amino acids not naturally found in proteins) are also known in the art, as shown, for example, in Zhang et al. "Protein engineering with unnatural amino acids," Curr. Opin. Struct. Biol. 23(4): 581-87 (2013); Xie et al. "Adding amino acids to the genetic repertoire," Curr. Opin. Chem. Biol. 9(6): 548-54 (2005); and the references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0041] As used herein, chemically modified amino acid refers to an amino acid whose side chain is chemically modified.For example, the side chain can be modified to include a signaling moiety such as a fluorophore or a radioactive label.The side chain can also be modified to include a new functional group such as a thiol, a carboxylic acid, or an amino group.Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0042] The term "identity" or "substantial identity," when used in the context of the polynucleotide or polypeptide sequences described herein, refers to a sequence having at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity compared to a reference sequence using the programs described herein; preferably, BLAST using standard parameters as described below. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame position, etc.

[0043] In sequence comparison, typically, one sequence serves as reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and partial sequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence with reference sequence based on program parameters.

[0044] As used herein, "comparison window" refers to any one of the segments of 20 to 600 consecutive positions, usually about 50 to about 200, more usually about 100 to about 150, where the sequence can be compared with the reference sequence of the same number of consecutive positions after the two sequences are optimally aligned.Methods for aligning sequences for comparison are well known in the art.The optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith & Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0045] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which, when aligned with words of the same length in database sequences, either match or meet a certain positive threshold score T. T is referred to as the neighborhood word score threshold [Altschul et al. (1977)]. These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops by an amount X from the maximum achieved; when the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall to zero or below; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)].

[0046] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability [P(N)], which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.01, more preferably less than about 10. -5 less than, most preferably about 10 -20 A nucleic acid is considered to be similar to a reference sequence if it is less than

[0047] As used herein, other terms used in the fields of recombinant nucleic acid technology, microbiology, immunology, antibody engineering, and molecular and cell biology will be commonly understood by those of ordinary skill in the applicable technical fields.

[0048] II. Introduction B7-H3 (also called CD276) is a co-inhibitory ligand expressed on the surface of many tumor cells and in tumor microvasculature [Suh et al., Nat. Immunol., 4:899-906, (2003); Zang et al., Proc. Natl. Acad. Sci. USA, 104:19458-63, (2007); Wang et al., Islets, 4:284-95 (2012)]. It is thought to actively inhibit the effector function of cytotoxic T lymphocytes (CTLs) or induce the generation of regulatory T cells, all of which downregulate immune responses [Pardall, Nat. Rev. Cancer, 12:252-64 (2012)]. Although both CTLA-4 and B7-H3 are members of the extended CD28 / B7 family, CTLA-4 and B7-H3 have non-redundant functions, and studies conducted in animal models suggest that the two pathways play distinct roles in immune regulation ( Zang et al., 2007 ; Wang et al., 2012 ).

[0049] B7-H3 protein is expressed in most tumor cell types and tumor-associated vasculature [Seaman et al., Cancer Cell, 31:501-515 (2017)]. For example, B7-H3 is overexpressed in renal cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, melanoma, and neuroectodermal cancer [Loo et al., Clin. Cancer Res., 18:3834-45 (2012)], as well as prostate cancer cells [Zang et al., 2007; Koenig, Medicographia, 36:285-92 (2014)], pointing to the broad applicability of targeting B7-H3 for therapy and imaging. For example, in prostatectomy specimens from 803 patients with localized disease, the majority (93%) of prostate tumors expressed B7-H3 (Zang et al., 2007). Furthermore, high levels of B7-H3 (and / or B7-H4, another co-inhibitory ligand) expression are associated with a higher risk of clinical failure (metastasis) and death within 7 years, implicating these molecules as inhibitory immune checkpoints that act to suppress antitumor immune responses [Zang et al., 2007; Zang et al., Proc. Natl. Acad. Sci. USA, 100:10388-92 (2003)]. In addition, renal carcinoma, melanoma, glioblastoma, thyroid cancer, and pancreatic cancer show up to 99% positive staining for B7-H3 by IHC (Koenig, 2014). Most importantly, B7-H3 protein is limited in normal human tissues (Koenig, 2014; Zang et al., 2003). Because B7-H3 is highly expressed on the surface of cancer cells and in cancer vasculature, but not in normal tissues, it provides an excellent target for anti-cancer immunotherapy, positron emission tomography (PET) and immuno-PET imaging, and bifunctional conjugate drug therapy.B7-H3 has also been identified on cells in inflammation [see, e.g., Chen et al., Cell. Immunol. 352:104077 (2020); Sun et al., J. Immunol. Res., 2017, Article ID 5728512 (2017); Hashimoto et al., Bone Marrow Transplantation 56:2336-54 (2021)].

[0050] B7-H3 is a type 1 membrane protein based on the presence of an extracellular N-terminal signal sequence and one predicted alpha helix, consistent with a transmembrane protein. Both the 4Ig and 2Ig isoforms of B7H3 are type 1 proteins [Zhou & Jin, Front Immunol 12:701006 (2021)]. Published localization and staining with various antibodies are consistent with a membrane protein, as are other paralogs, including PD-L1.

[0051] The extracellular domains of many type 1 membrane proteins are frequently targeted by therapeutic antibodies because they are most accessible from the blood and interstitial space. However, these domains are susceptible to further processing, including proteolytic cleavage and shedding [Tsumagari et al., iScience, 24(4): 102259 (2021); Lichtenthaler et al., EMBO J, 37(15) (2018)]. This presents two challenges: (1) antigen loss, which can be difficult to address, and (2) the shed extracellular domains can then be released into the extracellular space and enter the circulation. In the case of B7-H3, a circulating protein that most closely matches the extracellular domain of the Ig type 2 isoform has been clearly described [Zhang et al., Immunology 123(4):538-46 (2008); Baral et al., Oncol Lett 8(3):1195-1201 (2014)]. This circulating fragment increases in both inflammation and dramatically in the presence of tumors. Furthermore, it has been hypothesized that the 4Ig isoform of B7-H3, found primarily on tumor cells, may be resistant to proteolytic cleavage [Zhang et al., Bioengineered 12(2):11987-12002 (2021); Sun et al., PLoS One 6(9):e24751 (2011); Digregorio et al., Acta Neuropathol Commun, 9(1):59 (2021)].

[0052] Tumor-targeting antibodies that also target circulating proteins and protein isoforms pose significant challenges due to reduced overall efficacy and potentially increased side effects. The first challenge is delivering the antibody and antibody payload to the tumor target. Whether delivered intravenously or subcutaneously, the blood pool provides the primary route of antibody delivery to the tumor compartment. Therefore, if an antibody tightly binds to a circulating antigen, the antibody is essentially "blocked" from immediate delivery to the tumor target. If the off-rate of the tumor / circulating antigen complex is faster than the complex's excretion rate, the antibody may have some ability to target the tumor compartment. However, if the off-rate of the complex is slower than the complex's clearance rate, this effectively becomes a "sink" for the antibody, preventing it from targeting the tumor compartment. Furthermore, small antibody-antigen complexes (approximately 1:1 ratio) are cleared more rapidly than typical unconjugated antibodies due to engagement of "low affinity" FcRγ receptors in the liver and possibly the kidneys [Schlondorff & Banas, J. Am. Soc. Nephrol. 20(6):1179-87 (2009); Ganesan et al., J. Immunol., 189(10):4981-8 (2012)]. This "sink" problem is particularly relevant for low-dose, high-potency ADCs, large circulating antigen loads, and high molar activity radiotracers and radiotherapeutics (low-mass tracers). One of the first-generation antibodies targeting B7-H3, known as 8H9, preferentially binds to the extracellular domain of the 2Ig isoform of B7-H3, which is most compatible with the circulating isoform [Ahmed et al., J. Biol. Chem. 290(50):30018-29; 2015]. 8H9 failed in systemic treatment models but has been successful as a treatment for tumors in the brain or CNS via direct injection, bypassing the blood pool [Kramer et al., J. Neurooncol. 97(3):409-18 (2010)].

[0053] The second issue is the potential for increased toxicity. With antibodies injected in significant amounts, the primary consideration is the formation, tissue deposition, and reactivity of medium and larger antibody-immune complexes. This occurs when the antibody binds to the antigen at a ratio of greater than 1:1, e.g., 2:1 or higher. These larger complexes can exit the circulation and generate localized reactive immune responses, typically initiated by innate immune responses downstream of FcRγ engagement, which can damage local tissues and, in extreme cases, cause serious adverse events (Rojko et al., Toxicol. Pathol. 42(4):725-64 (2014); Mayadas et al., Circulation 120(20):2012-24 (2009)). With the advent of immune checkpoint therapies that enhance immune responses, this may become even more important. Indeed, it is interesting to note that a commercially available ADCC- or FcRγ-enhanced antibody from Macrogenics, Inc. targeting the extracellular domain of B7-H3 (the selectivity of 4Ig vs. 2Ig has not been published) demonstrated a large therapeutic index as a single agent but had serious adverse events in the context of combined PD1 therapy that also enhanced immune responses. Therefore, it is important to maximize selectivity between tumor antigens and circulating antigen isoforms, if possible.

[0054] Known anti-B7-H3 antibodies demonstrate modest nanomolar affinity for either human or mouse B7-H3 alone, with limited specificity for the 4Ig isoform compared to the 2Ig isoform. The two isoforms are structurally very similar, except that the 4Ig B7-H3 has an extra IgC-IgV immunoglobulin domain in its extracellular region. The extracellular domain of the 2Ig isoform is an excellent model for the circulating form of B7-H3 found in the blood of patients with solid tumors and, to a lesser extent, in the cerebrospinal fluid of patients with active CNS tumors / inflammation. In both radioisotope applications and antibody-drug conjugates (ADCs), antibody binding to the circulating form of B7-H3 can potentially result in less antibody payload being delivered to the tumor target due to compartment binding competition and limited immune complex clearance. Additionally, at the doses typically injected for ADC therapy, binding to soluble isoforms can result in the formation of additional medium- to large-sized immune complexes, leading to delivery of the ADC payload to cells expressing low-affinity Fc receptors in the body, particularly in the liver, kidney, and bone marrow, potentially causing general immunotoxicity due to tissue deposition of medium- to large-sized immune complexes, including type III hypersensitivity reactions and coagulopathy. Therefore, the combination of high affinity for the human 4Ig-B7-H3 target and low affinity for 2Ig-B7-H3 may be important for maximizing the therapeutic index during systemic treatment of solid tumors with this humanized antibody, antibody conjugate, or fragment thereof, especially when subsequently combined with anti-PD1 or other immune-stimulating immunotherapies. Preferred humanized monoclonal antibodies exhibit higher specificity for the 4Ig isoform over the 2Ig isoform.

[0055] MIL33B is a murine antibody that specifically binds to B7-H3 (described in PCT Publication No. WO2021 / 101991). The CDRs of MIL33B generated subnanomolar affinity for the extracellular domain of the 4Ig isoform of B7-H3, the isoform most associated with tumor cells. The initial selectivity for the extracellular domain of the 4Ig isoform over the circulating model 2Ig isoform was approximately 10-fold. This was indeed sufficiently selective to enable tumor- and antigen-specific targeting in mouse tumors. During the antibody humanization process, affinity for the initial target can be reduced, followed by an affinity maturation process. Considering that this process may involve generating random libraries in and around the CDRs, there is an opportunity to screen for additional properties beyond affinity rescue [Liang et al., Sci Rep. 11(1):22365 (2021); Fujiwara et al., Biochem. Biophys. Res. Commun. 527(2):350-357 (2020)]. Indeed, during engraftment of the previously identified CDR for MIL33B into a human IgG1 ortholog, initial affinity for human B7-H3 was lost. As discussed in the Examples, we generated a library of point mutations encompassing the framework regions surrounding the CDRs, targeting the least conserved mutations from the mouse framework to the human framework. Two additional criteria were used in selecting antibodies for further analysis and use: (1) point mutations in the framework regions that conferred the greatest affinity for the extracellular domain of the human 4Ig isoform were selected, and (2) in the case of a statistical tie, point mutations in the framework regions that maximized selectivity for human 4Ig over the human 2Ig extracellular domain were selected to minimize targeting of circulating components. Surprisingly, as shown in the Examples below, by leaving the CDR sequences unchanged but changing from a murine antibody and framework to an optimized human framework, selectivity for human 4Ig over the human 2Ig extracellular domain increased from 10-fold to over 350-fold, while maintaining subnanomolar affinity for the 4Ig isoform.Consistent with this hypothesis, improved binding data in HeLa cells expressing human B7-H3 were obtained. The humanized MIL33B-H3 antibody (also referred to herein as hMIL33B-H3) outperformed murine MIL33B in binding to live cells in binding assays, despite a modest loss of receptor binding affinity for the tumor surface 4Ig isoform from 70 pM to 120 pM.

[0056] Thus, provided herein are antibodies and antigen-binding portions thereof that specifically bind to the 4Ig isoform of B7-H3 (alternatively referred to as CD276). Also provided are various compositions of such antibodies or antigen-binding portions thereof, recombinant nucleic acids encoding the antibodies and antigen-binding portions thereof, and related methods of use. The disclosed humanized MIL33B antibody has higher affinity for human 4Ig-B7-H3 than any commercially available antibody. For moderately abundant targets, such as CD276 found on the tumor immune microenvironment, high affinity is crucial for maximizing target binding. Furthermore, this humanized antibody has first-in-class selectivity for the 4Ig-B7-H3 extracellular domain found on human tumors compared to the 2Ig-B7-H3 extracellular domain. This selectivity may be crucial for systemic therapy, whereby the antibody must first bypass circulating soluble 2Ig-B7-H3 to target solid tumors, potentially maximizing payload delivery to the tumor while minimizing potential toxicity from immune complex formation, including coagulopathy. This selectivity may also be crucial for targeting areas of inflammation in a subject.

[0057] III. Antibodies In one aspect, the present disclosure provides antibodies and antigen-binding portions thereof that specifically bind to B7-H3. As used herein, the term "antibody" encompasses, but is not limited to, all immunoglobulins (i.e., intact antibodies) of any class. Native antibodies are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of their heavy-chain constant domains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy-chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. As used herein, the term "antibody" also encompasses antibody fragments, e.g., antigen-binding fragments. An antigen-binding fragment contains at least one antigen-binding domain. An example of an antigen-binding domain is the antigen-binding domain formed by a VH-VL dimer. Antibodies and antigen-binding fragments can be represented by the antigen to which they specifically bind.

[0058] The term "variable" is used herein to refer to certain portions of antibody domains that differ in sequence among antibodies and are used to determine the binding and specificity of each particular antibody for a particular antigen. However, variability is not usually uniformly distributed throughout the variable domains of antibodies. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FRs, largely in a beta-sheet structure, connected by three CDRs, which form, and in some cases form part of, loops connecting the beta-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity. Each VH and VL generally contains three CDRs and four FRs, arranged in the following order (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and are generally thought to confer antigen specificity and binding affinity to the antibody. [Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, NIH, Bethesda, MD.] The CDR sequences on the heavy chain (VH) are sometimes designated CDRH1, 2, and 3, while the CDR sequences on the light chain (VL) are sometimes designated CDRL1, 2, and 3.

[0059] As used herein, the term "epitope" refers to a component of an antigen that can specifically bind to an antibody or its antigen-binding fragment. Such a component may include one or more contiguous amino acid residues and / or one or more noncontiguous amino acid residues. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents, but not the latter. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope bound by an antigen-binding protein can be determined using known techniques for determining epitopes, such as testing the binding of the antigen-binding protein to antigen variants with different point mutations.

[0060] As used herein, terms such as "specifically binds to," "specific for," "selectively binds to," and "selective for B7-H3 or an isoform or epitope of B7-H3 protein" refer to binding that is measurably different from nonspecific or nonselective interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. Specific binding can also be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. Specific binding is then indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.

[0061] Provided herein are antibodies and antigen-binding portions thereof that specifically bind to B7-H3. In some embodiments, the disclosed antibodies and fragments thereof specifically bind to the 4Ig isoform of B7-H3. B7-H3 antibodies and antigen-binding portions thereof are polypeptides. As used herein, the terms "antigen-binding portion" and "fragment" are used interchangeably to refer to portions of an antibody polypeptide sequence that specifically binds to B7-H3. B7-H3-specific antibodies were identified and tested as described in the Examples below. In some embodiments, the antibodies and antigen-binding portions thereof provided herein are humanized antibodies and antigen-binding portions thereof. As discussed above, the antibodies and antigen-binding portions thereof provided herein contain specific mutations to MIL33B, as described in PCT Publication No. WO2021 / 101991, that result in increased selectivity for the 4Ig isoform of B7-H3 while maintaining subnanomolar affinity.

[0062] In one aspect, provided herein is an isolated antibody or antibody fragment, wherein the antibody or antibody fragment has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:3, and comprises a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23, and wherein the VHCDR1 amino acid sequence comprises a tryptophan at position 47, a methionine at position 48, and a nucleotide at position 68 of SEQ ID NO:3. and a light chain variable region (VL) having at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:4, and comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and having an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4. The framework substitutions identified herein are shown to unexpectedly confer critical properties to the antibody.

[0063] In some embodiments, heavy chain variable region sequences and light chain variable region sequences encompassed by the present disclosure are set forth in Table 1. The CDR sequences in the variable domains listed in Table 1 are in bold and underlined text. In some embodiments, the heavy chain variable region is encoded by a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 27-29. In some embodiments, the light chain variable region is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO: 30.

[0064] Table 1. Antibody VH and VL amino acid sequences of selected clones. [Table 1]

[0065] There are various software programs that can be used to identify predicted CDRs in a given antibody polypeptide sequence, including IMGT / DomainGapAlign (described in Ehrenmann et al., Nucleic Acids Res. 38(Database issue):D301-D307 (2010); Ehrenmann & Lefranc, Cold Spring Harbor Protoc. 2011(6):737-49 (2011); Paratome (described in Kunik et al., PLoS Comput Biok, 8(2):el002388 (2012); Kunik et al., Nucleic Acids Res., 40(Web Server issue):W521-4 (2012))]; and Chothia. In certain embodiments, heavy chain CDR sequences encompassed by the present disclosure are set forth in Tables 2-4. In certain embodiments, light chain CDR sequences encompassed by the present disclosure are set forth in Tables 2-4.

[0066] Table 2. CDRs of the variable sequences of the CD276 antibody predicted by IMGT / DomainGapAlign. [Table 2]

[0067] Table 3. CDRs of the variable sequences of the CD276 antibody predicted by Paratome. [Table 3]

[0068] Table 4. CDRs of the variable sequences of the CD276 antibody predicted by Chothia. [Table 4]

[0069] As used herein, the term chimeric antibody refers to an antibody that comprises heavy and light chain variable regions from one antibody (e.g., a murine antibody) and the remaining antibody sequence from a second antibody (e.g., a human antibody). In some embodiments, the antibody or antigen-binding fragment thereof may be made from a chimeric antibody having a heavy chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 31, and a light chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 33.

[0070] As used herein, the term grafted antibody refers to a humanized antibody that contains CDR sequences from one antibody (e.g., a murine antibody) and the remaining framework and antibody sequences from a human antibody. In some embodiments, an antibody or antigen-binding fragment thereof can be made from a grafted antibody having a heavy chain variable region that contains an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 32; and a light chain variable region that contains an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 34.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1, 2, or 3; and a light chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4. In further embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:3 and comprises a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO:3; and a light chain variable region that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:4 and comprises an arginine substitution at position 45 and a tyrosine substitution at position 70 of SEQ ID NO:4.

[0072] In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising a CDRH3 comprising any one of SEQ ID NOs: 11, 17, or 23. In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising a CDRH3 comprising any one of SEQ ID NOs: 11, 17, or 23, and comprising a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO:3.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising any of the CDR1, CDR2, or CDR3 sequences set forth in Tables 2-4. In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising any of the CDR1, CDR2, or CDR3 sequences set forth in Tables 2-4, and has a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO:3. In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region comprising any of the CDR1, CDR2, or CDR3 sequences set forth in Tables 2-4. In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region comprising any of the CDR-1, CDR-2, or CDR3 sequences set forth in Tables 2-4, and has an arginine substitution corresponding to position 45 and a tyrosine substitution corresponding to position 70 of SEQ ID NO:4.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 1-3. In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and that includes a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72.

[0075] The present disclosure also provides antibodies or antigen-binding portions thereof that specifically bind to B7-H3, wherein the antibodies or antigen-binding portions thereof comprise a heavy chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 1-3, and a light chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4. Table 1 provides the sequences of SEQ ID NOs: 1-4. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 has a heavy chain variable region that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:3 and comprises an amino acid sequence that includes a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO:3; and a light chain variable region that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:4 and comprises an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:4.

[0076] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region or a light chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of the sequences in Tables 2-4, wherein the heavy chain variable region comprises a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72; and the light chain variable region comprises an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:3.

[0077] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region or a light chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of the sequences in Tables 2-4, wherein the light chain variable region comprises an arginine substitution corresponding to position 45 and a tyrosine substitution corresponding to position 70 of SEQ ID NO:4.

[0078] In each case, where a specific amino acid sequence is listed, embodiments are also provided that include sequences having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the listed sequence.

[0079] The amino acid residue sequences provided herein are indicated by the one-letter amino acid code, which may be used interchangeably with the three-letter amino acid code. Amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. The 20 naturally occurring or genetically encoded alpha-amino acids are: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). The structures of these 20 naturally occurring amino acids are shown, for example, in Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002). The term amino acid also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs.

[0080] The terms identical or percent identity, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides or amino acid residues (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or more identity over a specified region) when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0081] As with all peptides, polypeptides, and proteins (including fragments thereof), it is understood that additional modifications may be made in the amino acid sequences of the B7-H3-specific antibodies or antigen-binding fragments thereof described herein, e.g., in the heavy chain variable region and / or light chain variable region, that do not alter the properties or function of the antibody or antigen-binding fragment thereof. Such modifications include conservative amino acid substitutions, and therefore each recited sequence may contain one or more conservative amino acid substitutions. The list provided below identifies groups containing amino acids that are conservative substitutions for one another; these groups are exemplary, as other conservative substitutions will be known to those of skill in the art: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), methionine (M).

[0082] For example, if aspartic acid at a particular residue is mentioned, conservative substitutions at that residue, e.g., glutamic acid, are also contemplated. Non-conservative substitutions, e.g., substituting glycine for proline or asparagine for lysine, are also contemplated.

[0083] In some cases, the affinity of a B7-H3-specific antibody or antigen-binding fragment thereof can be optimized by mutations that increase or decrease affinity, as desired, based on one or more of the known properties of the binding interaction with B7-H3, the structure of either or both of the antibody or fragment thereof, or the B7-H3 protein. For example, in some embodiments, antibodies or antigen-binding fragments disclosed herein that contain certain conserved amino acids and certain amino acid substitutions have increased affinity and specificity for the 4Ig isoform of B7-H3 compared to the 2Ig isoform. In some cases, the mutations allow for easy elution of the purified antibody or fragment thereof under desired elution conditions during isolation and purification.

[0084] Methods for producing and screening the antibodies and antigen-binding fragments thereof provided in this disclosure are described in the Examples and are well known in the art. As described herein, methods for not only producing antigen-binding fragments but also further modifying antibodies to improve their properties (e.g., improving affinity, chimerization, humanization) are also well known in the art.

[0085] In some embodiments, the heavy and / or light chain variable regions of the isolated antibody or antibody fragment have the same sequence as the heavy and / or light chain variable regions of the antibodies produced by the methods described herein and in the Examples below. In some embodiments, the heavy and / or light chain variable regions of the isolated antibody contain one or more modifications, e.g., amino acid substitutions, deletions, or insertions.

[0086] The heavy and / or light chain variable region sequences of the antibodies described herein may be engineered to include one or more mutations in the heavy and / or light chain variable region sequences. In some embodiments, the engineered mutations improve the binding affinity of the antibody to B7-H3. In some embodiments, the engineered mutations improve the binding affinity of the antibody to the 4Ig isoform of B7-H3. In some embodiments, the engineered mutations reduce the cross-reactivity of the antibody to a second antigen.

[0087] In some embodiments, the engineered mutations are mutations in one or more CDRs, such as amino acid substitutions in the heavy chain CDRs and / or light chain CDRs described herein. In some embodiments, the engineered mutations are mutations in one or more framework regions, such as amino acid substitutions in the heavy chain framework regions and / or light chain framework regions. In some embodiments, the engineered mutations are reversions of regions of the heavy chain and / or light chain sequence to the predicted native sequence. Methods for determining predicted native immunoglobulin sequences have been described in the art. See, for example, Magnani et al., PLoS Negl Trop Dis, 2017, 11:e0005655, doi:10.1371 / journal.pntd.0005655.

[0088] In some embodiments, affinity maturation is used to engineer additional mutations that enhance the binding affinity of antibody to B7-H3 or enhance the cross-reactivity of antibody to second antigen.Methods for affinity maturation are known in the art.See, for example, Renaut et al., Methods Mol Biol, 2012, 907:451-61.

[0089] The present disclosure also encompasses antibodies or fragments thereof that bind to the same epitope of B7-H3 or the 4Ig isoform of B7-H3 as the antibodies disclosed herein. Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.

[0090] The present disclosure also provides chimeric antibodies. The term chimeric antibody refers to an antibody in which components of the heavy and / or light chain are derived from a particular source or species, while the remainder of the heavy and / or light chain are derived from a different source or species.

[0091] A human antibody is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source, a genetically modified non-human source, or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0092] Humanized forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human immunoglobulins (recipient antibodies) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, that has the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. [See Jones et al. (1986) Nature, 321:522-525; Riechmann et al. (1988) Nature, 332:323-329; and Presta, (1992) Curr Op Struct Biol., 2:593-596.] In some embodiments, the antibody or fragment thereof is a chimeric or graft antibody encoded by a nucleotide sequence set forth in Table 6.

[0093] In some embodiments, an antibody or antigen-binding fragment thereof provided herein may comprise a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, an antibody molecule comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, e.g., Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of a whole antibody or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to specifically bind to their respective antigens. Antibodies and antibody fragments can be from any class of antibody, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted, or in vitro generated. The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from either a kappa or lambda light chain.

[0094] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies. A substantially homogeneous population of antibodies contains identical or substantially similar antibodies that bind to the same epitope, excluding variations that may normally arise during monoclonal antibody production. Such variations are generally present only in minor amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from multiple clones. For example, the selection process can be the selection of a unique clone from multiple clones, such as a pool of yeast clones, phage clones, bacterial clones, mammalian cell clones, hybridoma clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, by affinity maturation, to improve its affinity for the target, to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0095] Antigen-binding fragments of antibody molecules are well known in the art and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camel or camelized variable domain; (vii) a single-chain Fv (scFv) [see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883]; and (viii) a single-domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0096] In some embodiments, a humanized monoclonal antibody comprises heavy and light chain variable region sequences derived from immunoglobulin-producing human B cells, and further comprises a kappa or lambda light chain constant region. In some embodiments, the light chain constant region (kappa or lambda) is derived from the same type of light chain (i.e., kappa or lambda) as the light chain variable region derived from immunoglobulin-producing human B cells; as a non-limiting example, if IgE-producing human B cells contain kappa light chains, the monoclonal antibody produced may comprise a light chain variable region derived from IgE-producing B cells and further comprise a kappa light chain constant region.

[0097] In some embodiments, the humanized monoclonal antibody comprises heavy and light chain variable region sequences derived from immunoglobulin-producing human B cells, and further comprises a heavy chain constant region having an IgG isotype (e.g., IgG4), an IgA isotype (e.g., IgA1), an IgM isotype, an IgD isotype, or derived from an IgG, IgA, IgM, or IgD isotype (e.g., a modified IgG4 constant region). It will be understood by those skilled in the art that different heavy chain isotypes (IgA, IgD, IgE, IgG, and IgM) have different effector functions mediated by the heavy chain constant region, and that for certain applications it may be desirable to have an antibody with the effector function of a particular isotype (e.g., IgG).

[0098] In some embodiments, the humanized monoclonal antibody comprises a native (i.e., wild-type) human IgG, IgA, IgM, or IgD constant region. In some embodiments, the monoclonal antibody comprises a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, a native human IgG4 constant region, a native human IgA1 constant region, a native human IgA2 constant region, a native human IgM constant region, or a native human IgD constant region. In some embodiments, the monoclonal antibody comprises a heavy chain constant region comprising one or more modifications. It will be understood by those skilled in the art that modifications, such as amino acid substitutions, can be made at one or more residues in the heavy chain constant region that modulate effector function. In some embodiments, the modifications reduce effector function, for example, resulting in a reduced ability to induce a certain biological function upon binding to Fc receptors expressed on effector cells that mediate effector function. In some embodiments, the modifications (e.g., amino acid substitutions) prevent Fab arm exchange in vivo, which can introduce undesirable effects and reduce the therapeutic efficacy of the antibody. See, for example, Silva et al., J Biol Chem, 2015, 280:5462-5469.

[0099] In some embodiments, a humanized monoclonal antibody comprises a native (i.e., wild-type) human IgM constant region, a human IgD constant region, a human IgG constant region derived from IgG1, IgG2, IgG3, or IgG4, or a human IgA constant region derived from IgA1 or IgA2, and comprises one or more modifications that modulate effector function. In some embodiments, a monoclonal antibody comprises a human IgM constant region, a human IgD constant region, a human IgG constant region derived from IgG1, IgG2, IgG3, or IgG4, or a human IgA constant region derived from IgA1 or IgA2. In some embodiments, a monoclonal antibody comprises a native (i.e., wild-type) human IgM constant region, a human IgD constant region, a human IgG constant region derived from IgG1, IgG2, IgG3, or IgG4, or a human IgA constant region derived from IgA1 or IgA2, and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications (e.g., amino acid substitutions). In some embodiments, the constant region comprises mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions) that affect effector function.

[0100] In some embodiments, the antibody having the identified CDRs is of an allotype other than the allotype found associated with the antibody produced by the methods described herein and in the Examples below. The antibody may comprise an allotype selected from those set forth in Table 5 below that is different from the allotype of the antibody produced by the methods described herein and in the Examples below. In some embodiments, the antibody may comprise any individual allotype selected from those set forth in Table 5, provided that the allotype is different from the corresponding allotype of the antibody produced by the methods described herein and in the Examples below.

[0101] Table 5. Human immunoglobulin allotypes. [Table 5] NB: Alphabetical designations are shown in parentheses. Source: Jefferis and Marie-Paule Lefranc, 2009, "Human immunoglobulin allotypes: Possible implications for immunogenicity" mAbs 1(4)332-338, incorporated herein by reference.

[0102] In some embodiments, the humanized monoclonal antibody comprises the CDR sequences, heavy chain variable region, and / or light chain variable region described herein (e.g., disclosed in Table 1), and further comprises heavy chain and / or light chain constant regions that are heterologous to the antibodies produced by the methods described herein and in the Examples below, and from which the CDR and / or variable region sequences are derived. For example, in some embodiments, the monoclonal antibody comprises the CDR sequences and / or variable region sequences of an antibody produced by the methods described herein and in the Examples below, and further comprises heavy chain and light chain constant regions that are heterologous to the antibodies produced by the methods described herein and in the Examples below (e.g., the heavy chain and / or light chain constant regions are wild-type or modified IgG1, IgG2, IgG3, or IgG4 constant regions), or the heavy chain and / or light chain constant regions comprise one or more modifications (e.g., amino acid substitutions) relative to the native constant regions of the antibodies produced by the methods described herein and in the Examples below.

[0103] The antibodies and fragments thereof of the present disclosure may contain modifications in the heavy chain constant region to alter the properties of the synthetic antibody relative to the corresponding naturally occurring antibody. Exemplary modifications include mutations to modulate antibody effector function (e.g., complement-based effector function or FcγR-based effector function), alter half-life, modulate antigen and FcγR co-engagement, and introduce or remove glycosylation motifs (glycoengineering). See Fonseca et al., 2018, "Boosting half-life and effector functions of therapeutic antibodies by Fc-engineering: An interaction-function review" Int J Biol Macromol. 19:306-311; Wang et al., 2018, "IgG Fc engineering to modulate antibody effector functions" Protein Cell 2018, 9(1):63-73; Schlothauer, 2016, "Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions," Protein Engineering, Design and Selection 29(10):457-466; Tam et al., 2017, "Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality" Antibodies 6, 12, each of which is incorporated herein by reference for all purposes.

[0104] The antibody molecule may also be a single-domain antibody. Single-domain antibodies may include antibodies whose complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be any of the current art or any future single-domain antibodies. Single-domain antibodies may be derived from any species, including, but not limited to, mouse, rat, guinea pig, human, camel, llama, fish, shark, goat, rabbit, and cow. Single-domain antibodies are described, for example, in International Application Publication No. WO 94 / 04678. For clarity, this variable domain derived from a heavy-chain antibody naturally lacking light chains is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies raised in Camelidae species (eg camel, llama, dromedary, alpaca and guanaco) or in other non-Camelidae species.

[0105] In some embodiments, the antigen-binding fragment may also be or include a scaffold protein, e.g., a non-antibody. These proteins are generally obtained by combinatorial chemistry-based adaptation of existing antigen-binding proteins. For example, the binding site of human transferrin to the human transferrin receptor can be diversified using the system described herein to generate a diverse library of transferrin mutants, some of which have acquired affinity for different antigens. See, e.g., Ali et al. (1999) J. Biol. Chem. 274:24066-24073. The portion of human transferrin that is not involved in binding to the receptor remains unchanged and serves as a scaffold for displaying the mutant binding site, like the framework region of an antibody. The library is then screened against the target antigen of interest according to the methods described herein for screening antibody libraries to identify mutants with optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol. 23(10):514-522.

[0106] Those skilled in the art will understand that the scaffold portion of a non-antibody scaffold protein can include, for example, all or a portion of the Z domain of S. aureus protein A, human transferrin, human fibronectin type III domain 10, the Kunitz domain of human trypsin inhibitor, human CTLA-4, ankyrin repeat proteins, human lipocalins (e.g., anticalins such as those described in International Application Publication No. WO2015 / 104406), human crystallin, human ubiquitin, or trypsin inhibitor from E. elaterium.

[0107] Any of the B7-H3-specific antibodies or antigen-binding fragments described herein may be modified by covalent and / or non-covalent modifications. Such modifications can be introduced into the antibody or antigen-binding fragment by, for example, reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be selected using any of a variety of criteria, including, for example, structural or amino acid sequence analysis of the antibody or fragment. Recombinant techniques can be used to modify the antibody or antigen-binding fragment. For example, amino acids found not to contribute to the antibody's activity, binding specificity, or affinity can be deleted without losing the respective activities. Insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues can be made, as long as the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antigen-binding fragment. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of nucleic acids encoding the antibody or fragment. [Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)]. In some cases, B7-H3-specific antibodies or antigen-binding fragments can be labeled by various means for use in diagnostic and / or pharmaceutical applications.

[0108] In some embodiments, an antibody or antigen-binding fragment thereof may be conjugated to a heterologous moiety. The heterologous moiety may be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or a detectable label, including, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag, such as biotin or streptavidin. In some embodiments, the heterologous moiety is an antibody or antigen-binding fragment thereof that specifically binds to different targets; such conjugated antibodies are referred to as bispecific antibodies. Additional suitable heterologous polypeptides include, for example, antigenic tags [e.g., FLAG (DYKDDDDK) (SEQ ID NO: 35), polyhistidine (6-His; HHHHHH (SEQ ID NO: 36) or HEHEHE (SEQ ID NO: 37)], hemagglutinin [HA; YPYDVPDYA (SEQ ID NO: 38)], glutathione-S-transferase (GST), or maltose-binding protein (MBP)] for use in purifying the antibody or fragment. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, such as luciferase, fluorescent proteins [e.g., green fluorescent protein (GFP)], or chloramphenicol acetyltransferase (CAT). Suitable radiolabels include, for example, 32 P, 33 P, 14 C. 125 I, 131 I, 35 S, and 3H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelates of diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzyme labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Another labeling technique that may result in greater sensitivity consists of coupling an antibody to a low-molecular-weight hapten. These haptens can then be specifically modified by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies. Additional acceptable heterologous moieties are described below in Section VIII.

[0109] In some cases, the B7-H3 antibody or its antigen-binding fragment can be conjugated to an imaging agent or radiotherapy agent. For example, the B7-H3 antibody or its antigen-binding fragment can be labeled for use in radionuclide imaging or radioligand therapy. In particular, the agent can be directly or indirectly labeled with a radioisotope. Examples of radioisotopes that can be used include: 225 Ac, 211 At, 128 Ba, 131 Ba, 7 Be, 204 Bi, 205 Bi, 206 Bi, 76 Br, 77 Br, 82 Br, 109 Cd, 47 Ca,11 C、 14 C、 36 Cl、 48 Cr、 51 Cr、 62 Dog, 64 Dog, 67 Dog, 165 This, 155 Yes, 18 F、 153 God, 66 Yes, 67 Yes, 68 Yes, 72 Yes, 198 Oh, 3 H、 166 Yes, 111 In, 113m In, 115m In, 123 I, 124 I, 125 I, 131 I, 189 Yes, 191m Yes, 192 Yes, 194 Yes, 52 Yes, 55 Yes, 59 Yes, 177 Hello, 15 Oh, 191m-191 If, 109 Pd, 212 Pb, 32 P、 33 P、 42 K、 226 Yes, 186 Yes, 188 Yes, 82m Rb, 153 Sm, 46 Sc, 47 Sc, 72 Yes, 75 Yes, 105 At, 22 No, 24 No, 89 Mr. 35 S、 38 S、 177 Yes, 161 Tb, 96 Tc, 99m Tc, 94m Tc, 201 Tl, 202 Tl,113 Sn, 117m Sn, 121 Sn, 166 Yb, 169 Yb, 175 Yb, 88 Y, 90 Y, 86 Zr, 89 Zr, 62 Zn, and 65 Zn. Preferably, the radioisotope is 225 Ac, 211 At, 131 I, 125 I, 124 I, 123 I, 111 I, 177 Lu, 99m Tc, 90 Y, 186 Re, 188 Re, 32 P, 153 Sm, 89 Zr, 68 Ga, 77 Br, or 18 F. In some embodiments, the imaging agent or radiotherapy agent is administered to a patient and imaged with a photoscanning device or used to treat a disease, typically cancer. In some embodiments, the imaging agent or radiotherapy agent is administered to a patient and imaged with a photoscanning device or used to treat inflammation. In certain embodiments, 18 F, 89 Zr, or 64 Cu is used for positron emission tomography (PET) imaging. In certain embodiments, 161 Tb, 177 Lu, 211 At, or 90 Y is used for radiotherapy. Procedures for labeling biological agents with radioisotopes are generally known in the art.

[0110] Two proteins (e.g., an antibody and a heterologous moiety) can be crosslinked using any of several known chemical crosslinkers. Examples of such crosslinkers are those that link two amino acid residues via a linkage containing a "hindered" disulfide bond. In these linkages, the disulfide bond within the crosslinking unit is protected (by blocking groups on both sides of the disulfide bond) from reduction, e.g., by the action of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes a terminal lysine on one side of the protein and a terminal cysteine ​​on the other to form such a linkage between two proteins. Heterobifunctional reagents that crosslink via different coupling sites on each protein can also be used. Other useful cross-linking agents include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0111] Techniques for conjugating therapeutic moieties (e.g., any of those discussed in Section VII) to the B7-H3-specific antibodies or antigen-binding fragments thereof described herein are well known and can be found, for example, in Arnon et al., Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (1985); Hellstrom et al., Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (1987); Thorpe, Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy" In: Monoclonal Antibodies For Cancer Detection And Therapy, (Baldwin et al. eds.), pp. 303-316 (1985), and Thorpe et al., Immunol. Rev. 62:119-158 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate (e.g., a bispecific antibody) as described in U.S. Pat. No. 4,676,980.

[0112] In some embodiments, the radiolabel may be conjugated directly to the amino acid backbone of the antibody. Alternatively, the radiolabel may be attached to a larger molecule (e.g., meta-[ 125 Iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS) 125Radiolabels may also be included as part of I, which bind to free amino groups to form meta-iodophenyl (mIP) derivatives of the relevant protein [see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229] or chelates (e.g., to DOTA or DTPA), which are then attached to the protein backbone. Methods for conjugating radiolabels or larger molecules / chelates containing same to the antibodies or antigen-binding fragments described herein are known in the art. Such methods include incubating the radiolabel with the protein under conditions (e.g., pH, salt concentration, and / or temperature) that promote binding of the radiolabel or chelate to the protein (see, e.g., U.S. Pat. No. 6,001,329).

[0113] Methods for conjugating fluorescent labels (sometimes referred to as fluorophores) to proteins (e.g., antibodies) are known in the field of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., of cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophore. In some embodiments, fluorophores can be conjugated to heterobifunctional crosslinker moieties such as sulfo-SMCC. A suitable conjugation method involves incubating an antibody protein or a fragment thereof with the fluorophore under conditions that promote binding of the fluorophore to the protein. See, for example, Welch and Redvanly (2003) Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0114] In some embodiments, the antibody or fragment can be modified with a moiety that improves stabilization and / or retention of the antibody in circulation, e.g., in blood, serum, or other tissues. For example, the antibody or fragment can be PEGylated or HESylated (Fresenius Kabi, Germany) as described, for example, in Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 (see, e.g., Pavisic et al. (2010) Int J Pharm 387(1-2):110-119). The stabilizing moiety can improve the stability or retention of the antibody (or fragment) by at least 1.5-fold (eg, at least 2, 5, 10, 15, 20, 25, 30, 40, or 50-fold or more).

[0115] The present disclosure also encompasses isotype modification of the antibodies described herein. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody-dependent cellular cytotoxicity, changing to class A can improve tissue distribution, and changing to class M can improve valency.

[0116] In some embodiments, an antibody or fragment thereof can be modified to include an Fc region with altered effector function, for example, by modifying C1q binding and / or FcγR binding, thereby altering CDC and / or ADCC activity. An "effector function" serves to activate or reduce biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to, C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCRs); and the like. Such effector functions may require mutating or combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0117] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be subjected to enzymatic or chemical treatment or produced from cells so that the antibodies or fragments have reduced or no glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and are described, for example, in U.S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

[0118] IV. Chimeric Antigen Receptors Also provided herein are chimeric antigen receptors comprising any of the above antibodies or antigen-binding fragments. Chimeric antigen receptors (CARs, also known as chimeric T cell receptors) are expressed in host effector cells, such as T cells or NK cells, and are designed to induce an immune response against a specific target antigen and cells expressing that antigen. Adoptive T cell immunotherapy, in which a patient's own T lymphocytes are engineered to express CARs, has shown considerable promise for the treatment of hematological malignancies. CARs can be engineered and used, for example, as described in Sadelain et al., 2013, Cancer Discov. 3:388-398. CARs typically comprise an extracellular target binding module, a transmembrane (TM) domain, and an intracellular signaling domain (ICD). The CAR domains may be joined via a flexible hinge and / or spacer region. The extracellular target binding module generally comprises an antibody or an antigen-binding fragment thereof. In some cases, multiple binding specificities may be included in the extracellular target binding module. For example, multiple antibodies or antigen-binding fragments thereof targeting different antigens can be included to create a bispecific, trispecific, or tetraspecific CAR. In some embodiments, the CAR antigen-binding domain comprises a heavy chain variable region (VH) having a VHCDR1 amino acid sequence comprising SEQ ID NO: 9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO: 10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO: 11, 17, or 23; and a light chain variable region (VL) having a VLCDR1 amino acid sequence comprising SEQ ID NO: 12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO: 13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO: 14, 20, or 26, and having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO: 4. The TM domain is primarily believed to be a structural requirement for anchoring the CAR to the cell membrane and is most commonly derived from molecules that regulate T cell function, such as CD8 and CD28. The intracellular module typically consists of the T cell receptor CD3 ζ chain and one or more costimulatory domains from either the Ig (CD28-like) or TNF receptor (TNFR) superfamily.CARs containing either the CD28 or 4-1BB costimulatory domains have been the most widely used to date, and both have produced dramatic responses in clinical trials. CAR domains are discussed in more detail below.

[0119] Provided herein is a chimeric antigen receptor comprising: (a) an extracellular target binding domain comprising a B7-H3-specific antibody or antigen-binding portion thereof; (b) a transmembrane domain; and (c) a signaling domain.

[0120] The extracellular target binding module of the CAR can comprise an antibody or antigen-binding fragment thereof that specifically binds to a target antigen (e.g., B7-H3). In certain embodiments, the extracellular target binding domain is a single-chain variable fragment (scFv) derived from an antibody, a tandem scFv, a single-domain antibody fragment (VFv), or a fusion protein. H H or sdAb), single domain bispecific antibodies (BsAb), intrabodies, nanobodies, immunokines in single chain format, and Fab, Fab', or (Fab ’)2. In other embodiments, the extracellular target binding domain can be an antibody portion comprising covalently linked multiple chains of variable fragments. In some embodiments, the extracellular target binding domain comprises any of the above antibodies or antigen-binding portions thereof. In some embodiments, the extracellular target binding domain comprises an scFv comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 1-3, and a light chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4. In some embodiments, the extracellular target binding domain comprises an scFv comprising: a heavy chain variable region (VH) having at least 90% identity to SEQ ID NO:3, and comprising a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23, and comprising a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO:3; and a light chain variable region (VL) having at least 90% identity to SEQ ID NO:4, and comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and comprising an arginine at position 45 and a tyrosine at position 70. In some embodiments, the scFv comprises a linker polypeptide between the heavy and light chain sequences (e.g., SEQ ID NO: 43 or SEQ ID NO: 44, or any of the other linkers described herein). In some embodiments, the CAR comprises an scFv comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 42.

[0121] In some embodiments, the extracellular target binding domain of a CAR provided herein further comprises one or more additional antigen-binding domains (i.e., in addition to the B7-H3-specific antibody or antigen-binding portion thereof described above). In some embodiments, the extracellular target binding domain comprises one additional antigen-binding domain. A CAR comprising such an extracellular target binding domain may be referred to as a bispecific CAR. In some embodiments, the extracellular target binding domain comprises two additional antigen-binding domains. A CAR comprising such an extracellular target binding domain may be referred to as a trispecific CAR. In some embodiments, the extracellular target binding domain comprises three additional antigen-binding domains. A CAR comprising such an extracellular target binding domain may be referred to as a tetraspecific CAR. Each of the one or more additional antigen-binding domains may comprise an antibody or antigen-binding portion thereof. In some embodiments, the one or more additional antigen-binding domains specifically bind to CD19, CD20, CD22, CD79a, CD79b, or any combination thereof.

[0122] The transmembrane domain of the CAR provided herein may be derived from natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain is derived from (i.e., comprises at least the transmembrane region of) the α, β, δ, γ, or ζ chain of the T cell receptor, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain may be selected based on, for example, the properties of various other proteins or transmembrane elements that bind to the transmembrane domain, or the cytokines induced by the transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain (e.g., a CD8α transmembrane domain). If the transmembrane domain is synthetic, it may primarily comprise hydrophobic residues such as leucine and valine. In some embodiments, a phenylalanine, tryptophan, and valine triplet can be found at each end of the synthetic transmembrane domain. In some embodiments, for example, a short oligo- or polypeptide linker having a length of about 2 to about 10 amino acids (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10) can form the link between the transmembrane domain and the intracellular signaling domain of the CAR described herein. In some embodiments, the linker is a glycine-serine doublet.

[0123] The intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell to which the CAR is or is designed to be placed. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and causes the cell to perform a specialized function. While the entire intracellular signaling domain can usually be utilized, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term "intracellular signaling sequence" is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0124] Examples of intracellular signaling domains for use in the CARs provided herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as derivatives or variants of these sequences, and any synthetic sequences that have the same functional capability.

[0125] It is known that signals generated via the TCR alone are insufficient for full activation of T cells, and secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary signaling sequences), and sequences that act antigen-independently to provide secondary or costimulatory signals (costimulatory signaling sequences).

[0126] The primary signaling sequence controls the primary activation of TCR complex either in a stimulatory or inhibitory manner.The primary signaling sequence that acts in a stimulatory manner can comprise a signaling motif known as immunoreceptor tyrosine-based activation motif or ITAM.In some embodiments, the CAR described herein comprises one or more ITAMs.

[0127] Examples of ITAMs containing primary signaling sequences of particular use in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the ITAM containing a primary signaling sequence is derived from CD3ζ.

[0128] In some embodiments, the CAR comprises a primary signaling sequence derived from CD3ζ. For example, the intracellular signaling domain of the CAR can comprise a CD3ζ intracellular signaling sequence, either alone or in combination with any other desired intracellular signaling sequence useful in the context of the CAR of the present invention. In some embodiments, the intracellular signaling domain of the CAR provided herein comprises a CD3ζ primary intracellular signaling sequence and a 4-1BB costimulatory signaling sequence (e.g., the amino acid sequence of SEQ ID NO: 41).

[0129] The CARs provided herein may include additional elements, such as a signal peptide, a leader sequence, and a hinge domain to ensure proper transport of the fusion protein to the cell surface, providing flexibility to the recognition region and enabling strong binding to the targeting moiety. In some embodiments, a spacer domain may be present between any of the domains of the CAR. The spacer domain may be any polypeptide that functions to link the two portions of the CAR. The spacer domain may contain up to about 300 amino acids, including, for example, about 10 to about 100, or about 25 to about 50 amino acids. Methods for identifying and selecting suitable spacer domains are known.

[0130] V. Antibody Expression and Purification, Nucleic Acids, Vectors, and Cells The B7-H3 antibody and its antigen-binding fragments discussed above, as well as molecules (e.g., CARs) comprising such antibodies and their antigen-binding fragments, can be produced by recombinant expression in human or non-human cells. Antibody-producing cells include non-human cells that express heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells that express heavy chains, light chains, or both heavy and light chains; and human B cells that produce heavy or light chains, but not both. The antibodies and antigen-binding fragments of the present disclosure can be heterologously expressed in vitro or in vivo in cells other than human B cells, for example, non-human cells and human cells other than B cells, optionally cells other than immune cells, and optionally cells other than cells of the B cell lineage.

[0131] The B7-H3 antibody and its antigen-binding fragments described herein, as well as molecules comprising them, can be produced using a variety of techniques known in the fields of molecular biology and protein chemistry.For example, the nucleic acid encoding the antibody or its antigen-binding fragment can be inserted into an expression vector containing transcriptional and translational regulatory sequences, including, for example, a promoter sequence, a ribosome binding site, a transcriptional start and stop sequence, a translational start and stop sequence, a transcription terminator signal, a polyadenylation signal, and an enhancer or activator sequence.Regulatory sequences include promoters and transcriptional start and stop sequences.In addition, an expression vector can contain more than one replication system, so that it can be maintained in two different organisms, for example, mammalian or insect cells for expression, and in a prokaryotic host for cloning and amplification.

[0132] Several possible vector systems are available for expressing heavy and light chain polypeptides cloned from nucleic acids in mammalian cells. One class of vectors utilizes integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be selected by co-introducing a drug resistance gene such as E. coli gpt [Mulligan & Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072] or Tn5 neo [Southern and Berg (1982) Mol. Appl. Genet. 1:327]. The selectable marker gene can be linked to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection [Wigler et al. (1979) Cell 16:77]. A second class of vectors utilizes DNA elements that confer autonomous replication capability to extrachromosomal plasmids. These vectors can be derived from animal viruses such as bovine papillomavirus [Sarver et al. (1982) Proc. Natl. Acad. Sci. USA, 79:7147], CMV, polyomavirus [Deans et al. (1984) Proc. Natl. Acad. Sci. USA 81:1292], or SV40 virus [Lusky & Botchan (1981) Nature 293:79].

[0133] The expression vector can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely determined by the target cell type, as discussed below. Exemplary methods include CaPO precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0134] Suitable host cells for expression of antibodies or antigen-binding fragments thereof include yeast, bacterial, insect, plant, and mammalian cells, of particular interest being bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human and hamster cell lines), and primary cell lines.

[0135] In some embodiments, antibodies or fragments thereof can be expressed in and purified from transgenic animals (e.g., transgenic mammals). For example, antibodies can be produced in transgenic non-human mammals (e.g., rodents) and isolated from their milk, as described, for example, in Houdebine (2002) Curr. Opin. Biotechnol. 13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res. 9(2):155-59; and Pollock et al. (1999) J. Immunol. Methods 231(1-2):147-57.

[0136] Antibodies and fragments thereof can be produced from cells transformed with an expression vector containing a nucleic acid encoding the antibody or fragment by culturing the host cell under conditions and for a time period sufficient to allow protein expression. Such conditions for protein expression vary depending on the choice of expression vector and host cell and can be easily ascertained by those skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see, e.g., Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley & Sons; and Green and Sambrook (2012) Molecular Cloning—A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001)). Codon selection, suitable expression vectors, and suitable host cells vary depending on several factors and can be easily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or other expression systems, including, but not limited to, yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220). Further description of expression vectors for use in eukaryotic cells (e.g., for treating subjects with cancer or inflammation), along with suitable delivery systems, is provided in Section VIII.A, below.

[0137] Also provided herein are nucleic acid molecules encoding a B7-H3 antibody or antigen-binding portion thereof that specifically binds to B7-H3 as described in this disclosure. In some embodiments, the nucleic acid encodes a chimeric or graft antibody disclosed herein.

[0138] In some embodiments, a nucleic acid molecule encoding a B7-H3 antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 1-3, and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid encodes an isolated antibody or antibody fragment comprising: a heavy chain variable region (VH) having at least 90% identity to SEQ ID NO:3, and comprising a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23, and comprising a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO:3; and a light chain variable region (VL) having at least 90% identity to SEQ ID NO:4, and comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and comprising an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:4. In some embodiments, a nucleic acid molecule is provided that encodes a B7-H3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable comprising an amino acid sequence at least 90% identical to any of SEQ ID NOs: 1 to 3. In some embodiments, the nucleic acid encodes a humanized monoclonal antibody or antibody fragment having at least 90% identity to any of SEQ ID NOs: 1 to 3, comprising a heavy chain variable region (VH) comprising a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO: 3. In some embodiments, a nucleic acid molecule is provided that encodes a B7-H3 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 4.In some embodiments, the nucleic acid encodes a humanized monoclonal antibody or antibody fragment having at least 90% identity to SEQ ID NO:4 and comprising a light chain variable region (VL) comprising an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:4. In some embodiments, a nucleic acid molecule encoding a B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is provided

[0139] In some embodiments, a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 is provided, wherein the nucleic acid sequence comprises a sequence that encodes an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of the sequences in Table 1. In some embodiments, the nucleic acid molecule comprises a sequence that encodes an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of the sequences in Table 1, and includes a heavy chain variable region having a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO:3; and a light chain variable region (VL) having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:4.

[0140] In some embodiments, a nucleic acid molecule is provided that comprises a nucleotide sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 27 to 30. In some embodiments, the nucleic acid molecule comprises a sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 27 to 30 and encodes an antibody or antibody fragment that comprises a heavy chain variable region having a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO: 3; and a light chain variable region having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO: 4.

[0141] In some embodiments, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is a synthetic sequence designed for expression in a host cell (e.g., a human cell). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence at least 90% identical to SEQ ID NOs: 27-30. In some embodiments, the nucleic acid molecule encodes an antibody or antibody fragment that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 27-30 and includes a heavy chain variable region having a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO: 3; and a light chain variable region having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO: 4.

[0142] In some embodiments, the nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof is operably linked to a promoter capable of directing expression in a bacterial or eukaryotic cell.

[0143] The amino acid sequences of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), AbM, and observed antigen contact ("Contact"). In some embodiments, CDRs are determined according to the IMGT definition. See Brochet et al., 2008, Nucl. Acids Rex. 36:W503-508. In some embodiments, CDRs are determined by a combination of the Kabat, Chothia, and / or Contact CDR definitions.

[0144] Also provided herein is a DNA construct comprising a promoter that drives expression in a host cell operably linked to a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a B7-H3-specific antibody or antigen-binding fragment thereof.

[0145] Also provided herein is a vector comprising a DNA construct comprising a promoter that drives expression in a host cell operably linked to a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a B7-H3-specific antibody or antigen-binding fragment thereof.

[0146] Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, such as the genomes of viruses, such as polyoma, Simian Virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably, cytomegalovirus (CMV), or heterologous mammalian promoters (e.g., β-actin promoter or EF1α promoter), or hybrid or chimeric promoters (e.g., CMV promoter fused to β-actin promoter). Of course, promoters from host cells or related species are also useful herein.

[0147] Enhancers generally refer to DNA sequences that function at any distance from the transcription start site and can be located either 5' or 3' of a transcription unit. Enhancers can also be found within introns and within the coding sequence itself. They are usually between 10 and 300 bp in length and function in cis. Enhancers typically function to increase transcription from nearby promoters. Enhancers may also contain response elements that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but enhancers from eukaryotic viruses are typically used for general expression. Preferred examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0148] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be regulated by environmental factors such as temperature and light. Optionally, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region may be cell-type-specifically active. Optionally, in certain vectors, the promoter and / or enhancer region may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the beta-actin promoter, the EF1A promoter, and retroviral long terminal repeats (LTRs).

[0149] A vector may also contain, for example, an origin of replication and / or a marker. A marker gene can confer a selectable phenotype, such as antibiotic resistance, to cells. The marker product is used to determine whether the vector has been delivered to the cell and, once delivered, whether it is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully introduced into mammalian host cells, the transformed mammalian host cells can survive if placed under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, expression vectors may contain tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus.

[0150] Also provided herein are host cells, including bacterial and eukaryotic host cells, that contain a recombinant nucleic acid molecule encoding a B7-H3 antibody or antigen-binding fragment thereof described in this disclosure. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence at least 90% identical to any of SEQ ID NOs: 27-30. In some embodiments, the nucleic acid molecule encodes an antibody or antibody fragment that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any of SEQ ID NOs: 27-30 and includes a heavy chain variable region having a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO: 3; and a light chain variable region having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO: 4.

[0151] Also provided herein are host cells engineered to express and secrete the B7-H3 antibody or antigen-binding fragment thereof described in this disclosure. In some embodiments, the cells are suitable for implantation into a patient with cancer. In some embodiments, the cells are suitable for implantation into a patient with inflammation. In some embodiments, the cells are animal or human cells and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, to reduce the likelihood of an immunological response, the cells can be encapsulated to prevent infiltration of surrounding tissues. In certain embodiments, the encapsulating material is typically a biocompatible, semipermeable polymeric enclosure or membrane that allows release of the protein product but prevents destruction of the cells by the subject's immune system or other harmful factors from surrounding tissues.

[0152] Also provided herein are immune cells (e.g., T cells) that express any of the CARs described herein. In some embodiments, the immune cells express the CAR on their surface. In some embodiments, the immune cells comprise a nucleic acid encoding a CAR, and the CAR is expressed from the nucleic acid and localized to the immune cell surface. In some embodiments, the immune cells are B lymphocytes, T lymphocytes, thymocytes, dendritic cells, natural killer (NK) cells, monocytes, macrophages, granulocytes, eosinophils, basophils, neutrophils, myelomonocytic cells, megakaryocytes, peripheral blood mononuclear cells, myeloid progenitor cells, or hematopoietic stem cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are cytotoxic T cells, helper T cells, natural killer T cells, suppressor T cells, CD8 + T cells, CD4 + T cells, CD8 + / CD4 + T cells, γδT cells, or T regulatory (T-reg) cells.

[0153] In some embodiments, immune cells expressing a CAR provided herein are obtained from a subject. When immune cells are used to treat the same subject from which they were obtained (e.g., according to the treatment methods described herein below), they are referred to as autologous cells. When they are obtained from a different subject, they are referred to as xenogeneic cells. Immune cells can be isolated from peripheral blood using techniques well known in the art, including Ficoll density gradient centrifugation followed by negative selection to remove undesired cells. In some embodiments, xenogeneic immune cells useful in the methods provided herein include allogeneic T cells, for example, as described in Bedoya et al., 2021, Front. Immunol. 12:640082.

[0154] In vitro methods are also suitable for preparing monovalent antibodies or antigen-binding fragments thereof. Digestion of antibodies to generate antibody fragments, particularly Fab fragments, can be achieved using routine techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in International Application Publication No. WO94 / 29348, U.S. Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a remaining Fc fragment. Pepsin treatment produces a fragment called the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigens.

[0155] The Fab fragment produced in antibody digestion may also contain the constant domain of the light chain and the first constant domain of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain, including one or more cysteines from the antibody hinge region. The F(ab')2 fragment is a bivalent fragment containing two Fab' fragments linked by disulfide bridges at the hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear free thiol groups.

[0156] One method for producing proteins, including the provided antibodies or fragments, is to link two or more peptides or polypeptides together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry (Applied Biosystems, Inc.; Foster City, CA). Those skilled in the art will readily understand that peptides or polypeptides corresponding to the antibodies provided herein can be synthesized, for example, by standard chemical reactions. For example, a peptide or polypeptide can be synthesized but not cleaved from its synthesis resin, whereas the other fragment of the antibody can be synthesized and then cleaved from the resin, thereby exposing a functionally blocked terminal group on the other fragment. By a peptide condensation reaction, these two fragments can be covalently linked at their carboxyl and amino termini, respectively, via peptide bonds to form an antibody or fragment thereof. [Grant GA (1992) Synthetic Peptides: A User Guide. WH Freeman and Co., NY (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer Verlag Inc., NY]. Alternatively, peptides or polypeptides can be independently synthesized in vivo. Once isolated, these independent peptides or polypeptides can be linked to form antibodies or fragments thereof via similar peptide condensation reactions.

[0157] For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to generate larger peptide fragments, polypeptides, or entire protein domains [Abrahmsen et al., Biochemistry, 30:4151 (1991)]. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct larger peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction [Dawson et al., Science, 266:776 779 (1994)]. The first step is the chemoselective reaction of the thioester of an unprotected synthetic peptide with another unprotected peptide segment containing an amino-terminal Cys residue, yielding a thioester-linked intermediate as the initial covalent product. Without changing the reaction conditions, this intermediate undergoes a spontaneous and rapid intramolecular reaction to form a native peptide bond at the ligation site. The application of this native chemical ligation method to the total synthesis of protein molecules is exemplified by the preparation of human interleukin 8 (IL-8) [Baggiolini et al., FEBS Lett. 307:97-101 (1992); Clark et al., J. Biol. Chem. 269:16075 (1994); Clark et al., Biochemistry 30:3128 (1991); Rajarathnam et al., Biochemistry 33:6623-30 (1994)].

[0158] Alternatively, unprotected peptide segments can be chemically linked, and the bond formed between the peptide segments as a result of chemical ligation is a non-natural (non-peptide) bond [Schnolzer et al., Science 256:221 (1992)]. This technique has been used to synthesize analogs of protein domains and large quantities of relatively pure proteins with full biological activity [deLisle et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)].

[0159] After expression, antibodies and their fragments can be isolated. Antibodies or their fragments can be isolated or purified in a variety of ways known in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, antibodies can be purified using a standard anti-antibody column (e.g., a protein A or protein G column). Ultrafiltration and diafiltration techniques are also useful in conjunction with protein concentration. See, for example, Scopes (1994) Protein Purification, 3rd edition, Springer-Verlag, New York City, New York. The degree of purification required depends on the desired application. In some cases, purification of the expressed antibody or its fragment is not necessary.

[0160] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, for example, Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (e.g., using a protein stain such as Coomassie blue or colloidal silver stain).

[0161] VI. Pharmaceutical Compositions and Formulations The B7-H3 antibodies and antigen-binding portions thereof described herein, as well as various molecules (e.g., CARs) comprising the antibodies and antigen-binding portions thereof, are suitable for in vitro or in vivo administration. Compositions comprising the B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure and a pharmaceutically acceptable carrier (excipient) are provided. In some embodiments, the compositions comprise a CAR comprising a B7-H3 antibody or antigen-binding fragment thereof. A pharmaceutically acceptable carrier (excipient) is a material that is not biologically or otherwise undesirable; i.e., the material is administered to a subject without causing undesired biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is included. The carrier is selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject. The compositions may further comprise diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants used in conjunction with the methods disclosed herein. Such compositions can be used, for example, in subjects with cancer or inflammation who would benefit from any of the B7-H3 antibodies or antigen-binding fragments thereof described herein.

[0162] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, Philip P. Gerbino, ed., Lippincott Williams & Wilkins (2006). In certain embodiments, acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In certain embodiments, the formulation contains an appropriate amount of a pharmaceutically acceptable salt to make the formulation isotonic. In certain embodiments, pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition.In certain embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; These include, but are not limited to, anion-forming salts (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronics, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapar, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. In certain embodiments, the optimal pharmaceutical composition can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. For example, Remington: The Science and Practice of Pharmacy, 22. ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may affect the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of a B7-H3-specific antibody or antigen-binding fragment thereof.

[0163] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier may be sterile water for injection, physiological saline, a buffered solution such as Ringer's solution, dextrose solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the physiological saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises a pH-controlled buffer, such as phosphate-buffered saline or acetate-buffered saline. In certain embodiments, compositions comprising the B7-H3-specific antibodies or antigen-binding fragments thereof disclosed herein can be prepared for storage in the form of lyophilized cakes or aqueous solutions by mixing selected compositions having the desired purity with appropriate formulations [Remington: The Science and Practice of Pharmacy, 22 ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). Furthermore, in certain embodiments, compositions comprising the B7-H3-specific antibodies or antigen-binding fragments thereof disclosed herein can be formulated as lyophilizates using appropriate excipients. In some cases, suitable excipients can include cryopreservatives, bulking agents, surfactants, or any combination thereof. Exemplary excipients include one or more polyols, disaccharides, or polysaccharides, such as, for example, mannitol, sorbitol, sucrose, trehalose, dextran 40, etc. In some cases, the cryopreservative can be sucrose or trehalose. In some cases, the bulking agent can be glycine or mannitol. In one example, the surfactant can be a polysorbate, such as, for example, polysorbate-20 or polysorbate-80.

[0164] In certain embodiments, pharmaceutical compositions may be selected for parenteral delivery (e.g., via injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intralesional routes). Preparations for parenteral administration may be in the form of pyrogen-free, parenterally acceptable aqueous solutions (i.e., water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media) containing a B7-H3-specific antibody or antigen-binding fragment thereof in a pharmaceutically acceptable vehicle. Preparations for parenteral administration may also include nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. In certain embodiments, preparations can include formulations of the desired molecule with agents such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can then provide controlled or sustained release of the product, which can be delivered via depot injection. In certain embodiments, hyaluronic acid can also be used, which may have the effect of promoting duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0165] In certain embodiments, the composition may be selected for inhalation or delivery via the digestive tract, such as orally. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be optionally desired.

[0166] In certain embodiments, compositions can be selected for topical delivery.Preparations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like are required or desirable as appropriate.

[0167] In certain embodiments, formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffering agent is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8. For example, the pH can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some cases, the pH of the pharmaceutical composition may be in the range of 6.6 to 8.5, such as 7.0 to 8.5, 6.6 to 7.2, 6.8 to 7.2, 6.8 to 7.4, 7.2 to 7.8, 7.0 to 7.5, 7.5 to 8.0, 7.2 to 8.2, 7.6 to 8.5, or 7.8 to 8.3. In some cases, the pH of the pharmaceutical composition may be in the range of 5.5 to 7.5, such as 5.5 to 5.8, 5.5 to 6.0, 5.7 to 6.2, 5.8 to 6.5, 6.0 to 6.5, 6.2 to 6.8, 6.5 to 7.0, 6.8 to 7.2, or 6.8 to 7.5. In some cases, the pH of the pharmaceutical composition may be in the range of 4.0 to 5.5, such as, for example, 4.0 to 4.3, 4.0 to 4.5, 4.2 to 4.8, 4.5 to 4.8, 4.5 to 5.0, 4.8 to 5.2, or 5.0 to 5.5.

[0168] In certain embodiments, pharmaceutical compositions may contain an effective amount of a B7-H3 antibody or antigen-binding fragment thereof in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, tablets may be prepared in unit dose form by dissolving the solution in sterile water or other suitable vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0169] Those skilled in the art can select additional pharmaceutical compositions, including formulations containing B7-H3-specific antibodies or antigen-binding fragments thereof in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. For example, see International Application Publication No. WO / 1993 / 015722, which describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained-release matrices include, for example, chemically synthesized polymers, starch-based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate [Sidman et al. (1993) Biopolymers 22:547-556], poly(2-hydroxyethyl-methacrylate) [Langer et al. (1981) J Biomed Mater Res. 15: 167-277; and Langer (1982) Chem Tech 12:98-105], ethylene vinyl acetate [Hsu & Langer (1985) J Biomed Materials Res 19(4):445-460], or polyesters, hydrogels, polylactides, including poly-D(-)-3-hydroxybutyric acid (European Patent No. EP 0133988) [e.g., U.S. Pat. Nos. 3,773,919; 5,594,091; 8,383,153; 4,767,628; International Application Publication No. WO1998043615, Calo et al. (2015) Eur. Polymer J 65:252-267, and European Patent No. EP 058,481]. In certain embodiments, sustained-release compositions may also include liposomes, which may be prepared by any of several methods known in the art.[See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent No. EP 036,676; and U.S. Patent Nos. 4,619,794 and 4,615,885].

[0170] The pharmaceutical compositions used for in vivo administration are typically sterilized.In certain embodiments, sterilization is achieved by filtration through sterile filtration membrane.In certain embodiments, when composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution.In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in solution.In certain embodiments, parenteral compositions are generally placed in a container with sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0171] In certain embodiments, once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) before administration.

[0172] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for administration to a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response discussed above in connection with its administration, i.e., appropriate route and treatment regimen. The amount administered depends on the desired effect, both according to the number of treatments and the unit dose. The actual dosage of the composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the patient's idiopathic nature, the route of administration, and the efficacy, stability, and toxicity of the particular therapeutic agent. For example, dosages can also include doses from about 1 μg / kg / body weight to about 1000 mg / kg / body weight (including such ranges) or more per administration, and any range derivable therein. Non-limiting examples of ranges derivable from the numerical values ​​described herein include about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. In any case, the physician in charge of administration will determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject. In certain examples, a B7-H3-specific antibody or antigen-binding fragment thereof may be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg, once every other day for at least four doses. Exemplary treatment regimes may include administration once daily, once weekly, twice weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. In some cases, the treatment involves administering a B7-H3-specific antibody or antigen-binding fragment thereof according to one of the above-mentioned dosing regimens for a first period, and administering another of the above-mentioned dosing regimens for a second period. In some cases, the treatment is discontinued for a period before resuming the same or a different dosing regimen. For example, a patient can continue the B7-H3-specific antibody dosing regimen for two weeks, take a week off, and continue it for another two weeks, etc.Dosing regimens for the B7-H3-specific antibodies or antigen-binding fragments thereof of the present disclosure include 0.1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, wherein the B7-H3-specific antibodies or antigen-binding fragments thereof are given using any of the following dosing schedules: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) one dose of 3 mg / kg body weight, then 1 mg / kg body weight every three weeks.

[0173] In yet another embodiment, a unit dose form is provided that includes the B7-H3-specific antibody or antigen-binding fragment thereof described in the present disclosure. The unit dose form can be formulated for administration by any of the routes described in the present disclosure. In one example, the unit dose form is formulated for intravenous or intraperitoneal administration. In yet another embodiment, a pharmaceutical package is provided that includes the unit dose form of the B7-H3-specific antibody or antigen-binding fragment thereof.

[0174] In some cases, the B7-H3 antibody or antigen-binding fragment may be an isolated B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure. When used with respect to a protein (or nucleic acid), the term "isolated" indicates that the protein (or nucleic acid) is essentially free from other cellular components with which it is naturally associated, preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high-performance liquid chromatography). In some embodiments, the isolated protein (or nucleic acid) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0175] In some cases, B7-H3 antibody or its antigen-binding fragment can be formulated into virus-like particles (VLPs).VLPs contain viral proteins derived from viral structural proteins.Methods for producing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0176] In some cases, B7-H3 antibody or its antigen-binding fragment can be formulated into subviral dense body (DB). DB transports proteins to target cells through membrane fusion. Methods for producing and using DB are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0177] VII. Kits and Packaging The B7-H3 antibodies and antigen-binding fragments thereof disclosed herein can be used to prepare kits (e.g., diagnostic test kits, radioligand therapy kits, or kits for patient treatment). In some embodiments, kits for performing any of the methods described herein are provided. The kits of the present disclosure may include a carrier container compartmentalized to receive in close contact one or more containers, such as vials, tubes, etc., each containing one of the separate elements used in the method.

[0178] In some embodiments, one of the containers may contain a B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure that is detectably labeled or capable of being labeled. The kit may also have a container containing a buffer and / or a container containing a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzyme or fluorescent label. For example, provided herein is a kit for imaging tumors in a subject with a cancer that expresses B7-H3. In another example, provided herein is a kit for radioligand therapy of tumors in a subject with a cancer that expresses B7-H3. In some embodiments, provided herein is a kit for imaging a subject with inflammation that expresses B7-H3. In some embodiments, the kit includes a container containing a labeled B7-H3 antibody or antigen-binding fragment thereof. In some embodiments, the kit includes separate containers containing the B7-H3 antibody or antigen-binding fragment thereof and a detectable label or radioisotope.

[0179] The B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure for use in treating cancer patients can be delivered to a physician, healthcare provider, treatment facility, or cancer patient in a pharmaceutical package or kit. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit or pharmaceutical package further comprises instructions for use (e.g., for administration according to the methods described herein).

[0180] In some embodiments, the B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure for use in treating a patient with inflammation can be delivered to a physician, healthcare provider, treatment facility, or patient with inflammation in a pharmaceutical package or kit (e.g., to improve patient convenience and compliance with the treatment plan). Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit or pharmaceutical package further comprises instructions for use (e.g., for administration according to the methods described herein).

[0181] In some embodiments, the pharmaceutical package or kit comprises a B7-H3 antibody or antigen-binding fragment in unit dose form, hi some embodiments, the pharmaceutical package or kit further comprises a unit dose form of one or more of a chemotherapeutic agent, a cytotoxic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

[0182] In one embodiment, the kit or pharmaceutical package contains a therapeutically effective dose of a B7-H3 antibody or antigen-binding fragment, defined as a single unit dosage form or as separate unit doses, which may be in any dosage or form described herein (e.g., pre-filled syringe, tablet, capsule, immediate release, delayed release, etc.).

[0183] In one embodiment, the kit or pharmaceutical package contains doses suitable for administration on multiple days, such as weekly, monthly, or quarterly.

[0184] In certain embodiments, the kit for producing single-dose administration unit is provided.In certain embodiments, the kit comprises the pre-filled syringe with single or multiple chambers.In certain embodiments, the kit comprises one or more containers of the formulation described in the present disclosure.

[0185] In some embodiments, the kit may further include an instruction sheet outlining the procedural steps of the methods set forth herein and following substantially the same procedures as those described herein or known to those of skill in the art. The instruction information may be a computer-readable medium containing machine-readable instructions that, when executed using a computer, display an actual or virtual procedure for delivering a therapeutically effective amount of a therapeutic agent.

[0186] VIII.How to use Provided herein are methods for treating or inhibiting diseases, conditions, or disorders associated with elevated levels of B7-H3, such as renal cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, melanoma, prostate cancer, and neuroectodermal cancer. B7-H3 function can be reduced by any suitable drug. Preferably, such a substance is an anti-B7-H3 antibody or antigen-binding fragment thereof described herein. The method includes administering to a subject a therapeutically effective amount of a composition comprising an isolated B7-H3-specific antibody or antigen-binding portion thereof described herein. Also provided are methods for prognosing and diagnosing cancer based on the detection and / or quantification of B7-H3 using the B7-H3 antibody or antigen-binding fragment described herein. Also provided are methods for detecting the presence of B7-H3 protein in a sample using the described B7-H3 antibody or antigen-binding fragment.

[0187] Provided herein are methods for treating or inhibiting inflammatory diseases, conditions, or disorders associated with elevated levels of B7-H3. B7-H3 function can be reduced by any suitable drug. Preferably, such a substance is an anti-B7-H3 antibody or antigen-binding fragment thereof described herein. The method includes administering to a subject a therapeutically effective amount of a composition comprising an isolated B7-H3-specific antibody or antigen-binding portion thereof described herein. Also provided are prognostic and diagnostic methods for inflammation based on the detection and / or quantification of B7-H3 using the B7-H3 antibody or antigen-binding fragment described herein. Also provided are methods for detecting the presence of B7-H3 protein in a sample using the described B7-H3 antibody or antigen-binding fragment thereof.

[0188] As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, or any other animal. The term does not denote a particular age or sex. Thus, it is intended to include adult and newborn subjects, regardless of male or female. As used herein, patient or subject can be used interchangeably, and the terms patient or subject include human and veterinary subjects. The B7-H3 antibodies or antigen-binding portions thereof described herein are useful for the treatment of cancer in humans, including, but not limited to, pediatric and geriatric populations, as well as animals, e.g., in veterinary applications. In one embodiment, the subject is a human. In some embodiments, the B7-H3 antibodies or antigen-binding portions thereof described herein are useful for the treatment of inflammation in humans, including, but not limited to, pediatric and geriatric populations, as well as animals, e.g., in veterinary applications. In one embodiment, the subject is a human.

[0189] As used herein, the terms "cancer" and "tumor" are used to refer to malignant tissue. Also, the term "cancer" is used to refer to a disease associated with the presence of malignant tumor cells in an individual, and the term "tumor" is used to refer to multiple cancer cells physically associated with each other. Cancer cells are malignant cells that give rise to cancer, and tumor cells are malignant cells that can form tumors and thereby give rise to cancer.

[0190] As used herein, the term "inflammation" is used to refer to a local or systemic response to cellular injury characterized by capillary dilation, leukocyte infiltration, redness, heat, pain, swelling, and often loss of function, which serves as a mechanism to initiate clearance of injurious substances and damaged tissue.

[0191] A. Treatment method Provided herein are methods of treating cancer in a subject using a B7-H3 antibody or antigen-binding fragment thereof described in this disclosure.

[0192] In another embodiment, provided herein is a method for treating inflammation in a subject using a B7-H3 antibody or antigen-binding fragment thereof described in this disclosure.

[0193] As used herein, the term "cancer" can be used to refer to a solid tumor, a metastatic cancer, or a non-metastatic cancer. In certain embodiments, the cancer can originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus.

[0194] "Inflammatory condition" or "inflammatory disorder" refers to any inflammation in an individual, which can be transient (e.g., in response to exposure to a pathogen or allergen) or chronic. Inflammation is characterized by proinflammatory cytokines such as IFN-gamma, IL-6, and TNF-alpha, which recruit and activate macrophages and other leukocytes. In some cases, inflammation can develop into a chronic, harmful condition or an autoimmune condition (e.g., multiple sclerosis, lupus, rheumatoid arthritis, Crohn's disease). Inflammation can manifest locally (e.g., at a local site of infection or exposure) or systemically (e.g., atherosclerosis, hypertension).

[0195] As used herein, "effective amount" means an amount of an agent effective to produce a desired effect in a subject. The actual dose, including the effective amount, may depend on the route of administration, the size and health of the subject, the disorder being treated (e.g., cancer or inflammation), etc.

[0196] In some embodiments, a B7-H3 antibody or antigen-binding fragment thereof can directly inhibit the growth of cancer cells and induce cell death. In some cases, a B7-H3 antibody or antigen-binding fragment thereof can inhibit tumor development, for example, by binding to B7-H3 expressed by undifferentiated leukemia cells or cancer stem cells. In some cases, a B7-H3 antibody or antigen-binding fragment thereof can sensitize cancer cells to other cancer therapies (e.g., chemotherapy). In some cases, treating a subject according to the methods described herein inhibits at least one of tumor formation, tumor cell proliferation, tumor cell growth, tumor cell survival in circulation, or tumor cell metastasis in an individual. In another embodiment, treating a subject according to the methods described herein can result in cessation of tumor growth, reduction in tumor size, and in some cases, disappearance of one or more tumors in the subject.

[0197] In some embodiments, the B7-H3 antibody or antigen-binding fragment thereof may not be therapeutic in itself, but may be used to target a therapeutic agent to cancer stem cells, as discussed further herein. In such cases, the B7-H3 antibody or antigen-binding fragment thereof only needs to specifically bind to the B7-H3 protein. Thus, in some embodiments, the B7-H3 antibody or antigen-binding fragment thereof may be conjugated to a therapeutic pharmaceutical agent, as described herein.

[0198] In some embodiments, the B7-H3 antibody or antigen-binding fragment thereof can directly inhibit inflammation in cells. In some cases, the B7-H3 antibody or antigen-binding fragment thereof can sensitize inflammation in cells to other anti-inflammatory therapies. In some cases, treating a subject according to the methods described herein inhibits inflammation in cells within an individual. In another embodiment, treating a subject according to the methods described herein can result in the cessation, reduction, and in some cases, disappearance of inflammation in the subject.

[0199] In some embodiments, the B7-H3 antibody or antigen-binding fragment thereof may not itself be therapeutic, but may be used to target a therapeutic agent to inflammatory cells, as discussed further herein. In such cases, the B7-H3 antibody or antigen-binding fragment thereof need only specifically bind to the B7-H3 protein. Thus, in some embodiments, the B7-H3 antibody or antigen-binding fragment thereof may be conjugated to a therapeutic pharmaceutical agent, as described herein.

[0200] In some embodiments, the B7-H3 antibody or its antigen-binding fragment can be conjugated to a radioisotope for use in radioligand therapy. Radiolabeled monoclonal antibodies and antibody fragments of the present disclosure can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium iodide and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. In some embodiments, the B7-H3 antibody or its antigen-binding fragment according to the present disclosure can be labeled with technetium-99m by a ligand exchange process, for example, by reducing pertechnate with a stannous solution, chelating the reduced technetium on a Sephadex column, and applying the antibody to the column. Alternatively, direct labeling techniques can be used, for example, by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediate functional groups incorporating chelators that are often used to attach radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), monomeric or dendrimeric 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), deferoxamine (DFO), or 1-hydroxy-2(1H)-pyridinone derivatives (e.g., 3,4,3-LI(1,2-HOPO) or HOPO).

[0201] Also provided is a method for treating cancer using radioligand therapy comprising the B7-H3 antibody or its antigen-binding fragment described in the present disclosure. The dosage range of the radioisotope varies widely and depends on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. Exemplary dosing regimens can be found in U.S. Patent Nos. 5,595,721 and 6,015,542, each of which is incorporated herein by reference in its entirety. For example, a radiolabeled antibody can be administered in a single dose designed to deliver a large amount of radiation. In such methods, it is contemplated that a radiometric dose of more than 200 cGy will be delivered to the patient's entire body. This "high-dose" method requires bone marrow transplantation or some other means of reconstituting hematopoietic function in the patient.

[0202] Therapeutic doses of radiolabeled antibodies can be administered, but the radiometric dose received by the patient is limited to a level that is not significant enough to cause bone marrow toxicity and does not necessitate hematopoietic reconstitution by bone marrow transplantation or other means. For example, an effective dose range for this method is one that delivers between 25 and 200 cGy, preferably 25-150 cGy, to the patient's whole body.

[0203] Also provided are methods for treating cancer using a CAR comprising a B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure. In some embodiments, these methods involve using a CAR to redirect the specificity of immune effector cells (e.g., T cells) to target cancer cells (e.g., cancer cells expressing B7-H3). Accordingly, provided herein are methods for stimulating an effector cell-mediated response (such as a T cell-mediated immune response) against a target cell population or tissue containing cancer cells in a mammal, the method comprising administering to the mammal effector cells (such as T cells) expressing a CAR described herein. In some embodiments, "stimulating" an immune cell refers to eliciting an effector cell-mediated response (such as a T cell-mediated immune response), which is distinct from activating an immune cell. Effector cells expressing a CAR described herein can be infused into a subject (e.g., a cancer patient) in need of treatment. In some embodiments, the infused cells can kill (or cause the killing of) cancer cells in the subject. Formulations and methods for generating CAR-expressing effector cells and using them in therapeutic methods are known in the art (see, e.g., Feins et al., 2019, Am. J. Hematol. 94(S1): S3-S9).

[0204] Also provided are methods of treating inflammation using a CAR comprising the B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure. In some embodiments, these methods involve using a CAR to redirect the specificity of immune effector cells to target inflammation (e.g., cells expressing B7-H3). Effector cells expressing a CAR described herein can be infused into a subject in need of treatment (e.g., a patient with or experiencing inflammation). In some embodiments, the infused cells can reduce inflammation in the subject. Formulations and methods for generating CAR-expressing effector cells and using them in therapeutic methods are known in the art (see, e.g., Feins et al., 2019, Am. J. Hematol. 94(S1):S3-S9).

[0205] A subject treated by any of the methods herein may have one of a variety of different cancers, including, for example, lymphoma, follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), leukemia, chronic lymphocytic leukemia (CLL), marginal zone lymphoma, breast cancer, ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer. In some cases, the subject may have a primary cancer. In other cases, the subject may have a metastatic cancer. In some embodiments, the cancer comprises cells that aberrantly express B7-H3 at levels above basal expression in corresponding normal / non-cancerous cells (i.e., a B7-H3-expressing cancer). In some embodiments, the subject may have chronic lymphocytic leukemia. In some embodiments, the subject may have mantle cell lymphoma. In some embodiments, the subject may have breast cancer. In some embodiments, the subject may have lung cancer.

[0206] The subject treated by any of the methods herein may have one of various different inflammatory conditions or disorders.In some embodiments, inflammatory conditions or disorders can be selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren's syndrome, dermatitis, type 1 diabetes, type 2 diabetes, thyroiditis, Addison's disease, pernicious anemia, autoimmune hepatitis, inflammatory bowel disease, multiple sclerosis, encephalitis, rheumatoid arthritis, myasthenia gravis, neuritis, primary biliary cholangitis, Goodpasture's disease, primary membranous nephropathy, cystitis, ovarian insufficiency, autoimmune orchitis, chronic obstructive pulmonary disease (COPD), asthma, pneumonia, hypertension, heart disease, myositis, myocarditis, inflammatory arteritis (Takayasu's arteritis, giant cell arteritis), lymphangitis, Parkinson's disease or graft-versus-host disease. Other such autoimmune diseases are known in the art (see, e.g., Ludwig et al. (2017, Frontiers in Immunol 8:Article 603; Hofmann et al., 2018, Frontiers in Immunol 9:Article 835). In some embodiments, the inflammatory condition is responsive to exposure to a pathogen (e.g., bacteria, virus, fungus), environmental chemical, or radiation.

[0207] In some embodiments, B7-H3 expression (e.g., in cancer cells or inflammation) can be examined by using one or more routine biochemical analyses. In some embodiments, B7-H3 expression is determined by detecting protein expression using methods such as mass spectrometry, Western blot analysis, flow cytometry, and immunohistochemical staining. In some embodiments, such methods include the use of a B7-H3 antibody or an antigen-binding portion thereof (e.g., as described in this disclosure). In some embodiments, B7-H3 expression is determined by detecting mRNA levels using methods such as RT-PCR, RNA sequencing, microarray analysis, and Northern blot analysis. In some cases, a combination of these methods may be used, or additional methods known in the art may also be used.

[0208] In one embodiment, CAR T cells comprising the B7-H3-specific antibodies provided herein induce significant lysis of cells that express human B7-H3 (e.g., B7-H3-expressing L cells and cells from B cell lymphoma cell lines including Jeko-1, sp53, and CA46), but not cells that do not express B7-H3 (e.g., parental L cells and cells from the B7-H3-negative leukemia cell line NK92).

[0209] "Treating," "treatment," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. In some embodiments, "treating" or "treatment" may refer to any indicator of success in treating or ameliorating cancer. "Treating" or "treatment" includes administering an agent to prevent cancer growth, doing one or more of the following: reducing the weight or volume of the cancer, extending the expected survival time of the subject, or extending the expected time to tumor progression. In some embodiments, "treating" or "treatment" may refer to any indicator of success in treating or ameliorating inflammation (e.g., reduction in redness, pain, warmth). The effect of treatment may be compared to an individual or pool of individuals who have not received treatment, or to the same patient at different time points before or during treatment.

[0210] The term "administering," as used herein, refers to a method of delivering an agent, compound, or composition to a desired site of biological effect. Pharmaceutical compositions (e.g., as described above) are prepared for administration in several ways, including, but not limited to, injection, ingestion, transfusion, implantation, or implantation, depending on whether local or systemic treatment is desired and the site to be treated. The preparation of such pharmaceutically acceptable compositions is within the capabilities of one of ordinary skill in the art. Compositions may be administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intraarticular, intraperitoneal, intramuscular, subcutaneous, intracavity, intralesional, transdermal, intradermal, intrahepatic, intrathecal, intracranial, rectal, transmucosal, enteral, intraocular, intraocular, otic, nasal, inhalation, or intrabronchial delivery, or any other method known in the art. In some embodiments, the B7-H3 antibody or antigen-binding fragment thereof is administered orally, intravenously, or intraperitoneally.

[0211] In one aspect, a method of treating a subject with cancer is provided, comprising administering to the patient a therapeutically effective amount of a composition comprising a B7-H3 antibody or antigen-binding portion thereof described in the present disclosure. The composition may further comprise a pharmaceutically acceptable carrier.

[0212] In another aspect, a method of treating a subject having inflammation is provided, comprising administering to the patient a therapeutically effective amount of a composition comprising a B7-H3 antibody or antigen-binding portion thereof described in the present disclosure, which composition may further comprise a pharmaceutically acceptable carrier.

[0213] In some cases, the B7-H3 antibody or antigen-binding fragment thereof may be administered via a virus-like particle, which may be formulated as described above.

[0214] In some cases, the B7-H3 antibody or antigen-binding fragment thereof may be administered via subviral dense bodies, which may be formulated as described above.

[0215] In some cases, the B7-H3 antibody or antigen-binding fragment thereof can be administered via tegument aggregates. Methods for producing and using tegument aggregates are described in International Publication No. WO2006 / 110728.

[0216] In another aspect, provided is a method of treating a subject with cancer, comprising administering to the patient cells genetically engineered to express and secrete a B7-H3 antibody, or antigen-binding portion thereof, described in this disclosure, using a method such as those described herein.

[0217] In another aspect, provided is a method of treating a subject having cancer, the method comprising administering to the patient immune cells expressing a CAR comprising a B7-H3 antibody or antigen-binding portion thereof described herein.

[0218] In another aspect, a method of treating a subject having cancer is provided, the method comprising administering to the patient a vector comprising a nucleic acid sequence encoding a B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 27-30. In some embodiments, the nucleic acid molecule is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any one of SEQ ID NOs: 27-30, and comprises a heavy chain variable region having a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO: 3; and a light chain variable region having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO: 4.

[0219] In yet another aspect, a method of treating a subject having inflammation is provided, comprising administering to the patient cells genetically engineered to express and secrete a B7-H3 antibody, or antigen-binding portion thereof, described in this disclosure, using a method such as those described herein.

[0220] In another aspect, a method of treating a subject having inflammation is provided, comprising administering to the patient immune cells expressing a CAR comprising a B7-H3 antibody or antigen-binding portion thereof described herein.

[0221] In another aspect, a method of treating a subject having inflammation is provided, the method comprising administering to the patient a vector comprising a nucleic acid sequence encoding a B7-H3 antibody or antigen-binding fragment thereof described in the present disclosure. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 27-30. In some embodiments, the nucleic acid molecule is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any one of SEQ ID NOs: 27-30, and comprises a heavy chain variable region having a tryptophan corresponding to position 47, a methionine corresponding to position 48, a valine corresponding to position 68, and an arginine corresponding to position 72 of SEQ ID NO: 3; and a light chain variable region having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO: 4.

[0222] There are several compositions and methods that can be used to deliver nucleic acid molecules and / or polypeptides to cells either in vitro or in vivo, for example, via expression vector.These methods and compositions can be broadly divided into two categories: virus-based delivery system and non-virus-based delivery system.Such methods are well known in the art and can be easily adapted to be used with the compositions and methods described herein.

[0223] As used herein, a plasmid or viral vector is an agent that transports the disclosed nucleic acid into cells without undesired degradation and contains a promoter that causes the expression of the nucleic acid molecule and / or adapter polypeptide in the cells to which it is delivered.Viral vectors are, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, Sindbis, and other RNA viruses, including those with an HIV backbone.Also preferred are any virus families that share the characteristics of these viruses and are suitable for use as vectors.Retroviral vectors are generally described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated herein by reference for vectors and methods for producing them. The construction of replication-deficient adenoviruses has been described [Berkner et al., J. Virology 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virology 57:267-74 (1986); Davidson et al., J. Virology 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)]. The advantage and utility of these viruses as vectors is that they can replicate within the initially infected cells but are unable to form new infectious viral particles, limiting their ability to spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiencies after in vivo delivery directly to respiratory epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and several other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. In some cases, nucleic acid molecules encoding the B7-H3 antibody or antigen-binding fragment thereof can be delivered via virus-like particles.

[0224] Non-viral delivery methods may include an expression vector containing a nucleic acid molecule and a nucleic acid sequence encoding an adapter polypeptide, wherein the nucleic acid is operably linked to an expression control sequence. Suitable vector backbones include those commonly used in the art, such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and introns.

[0225] In certain embodiments, the effective amount of a pharmaceutical composition comprising a B7-H3-specific antibody or antigen-binding fragment thereof to be therapeutically utilized will depend, for example, on the context and purpose of the treatment. One skilled in the art will understand that appropriate dosage levels for treatment according to certain embodiments will vary depending, in part, on the molecule being delivered, the indication for which the B7-H3-specific antibody or antigen-binding fragment thereof is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and / or condition (age and overall health). Clinicians can titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0226] Clinicians also select the frequency of administration taking into account the pharmacokinetic parameters of the B7-H3-specific antibody or antigen-binding fragment thereof in the formulation used. Such pharmacokinetic parameters are well known in the art, such as absorption rate, bioavailability, metabolism, clearance, etc. (See, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; see, e.g., Remington's, supra, most recently). In certain embodiments, clinician will administer composition until reaching the dosage that achieves desired effect.In certain embodiments, composition can be administered as a single dose or as two or more doses (which may or may not contain the same amount of desired molecule) over time, or as continuous infusion via implanted device or catheter, for example.Further refinement of suitable dosage is routinely carried out by those skilled in the art, and is within the scope of routine work carried out by those skilled in the art.In certain embodiments, suitable dosage can be confirmed by using suitable dose-response data.

[0227] In certain embodiments, the administration route of pharmaceutical composition is, for example, oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional injection; according to known methods by sustained release system or implantation device.In certain embodiments, the composition can be administered by bolus injection, or continuously by infusion, or by implantation device.In certain embodiments, the individual components of combination therapy can be administered by different routes.

[0228] In certain embodiments, the composition can be administered locally, for example, during surgery or topically.Local administration can be via implantation of a membrane, sponge, or other suitable material into which the desired molecule is absorbed or encapsulated.In certain embodiments, when an implantation device is used, the device can be implanted into any suitable tissue or organ, and the delivery of the desired molecule can be via diffusion, sustained release bolus, or continuous administration.

[0229] In certain embodiments, it may be desirable to use a pharmaceutical composition comprising a B7-H3 antibody or antigen-binding fragment thereof in an ex vivo manner. In such cases, cells removed from a subject may be exposed to a pharmaceutical composition comprising a B7-H3 antibody or antigen-binding fragment thereof, after which the cells are subsequently re-implanted into the subject.

[0230] In some embodiments, the provided methods may include administering to a subject a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to a therapeutic agent. The therapeutic agent may be at least one of a cytotoxic agent, a chemotherapeutic agent, a radiotherapeutic agent, a phototherapeutic agent, or an immunosuppressant. Such therapeutic agents are described herein.

[0231] In some embodiments, the provided method may include administering to a subject a B7-H3-specific antibody or antigen-binding fragment thereof and a second form of cancer therapy. The second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, a radiotherapeutic agent, a phototherapeutic agent, an immunosuppressant (including an immune checkpoint inhibitor), or radiation therapy. In some embodiments, the second form of cancer therapy is an antibody (e.g., a monoclonal antibody). Monoclonal antibodies that may be administered as the second form of cancer therapy include, but are not limited to, rituximab (e.g., for the treatment of B-cell lymphoma), trastuzumab (e.g., for the treatment of breast cancer), and cetuximab (e.g., for the treatment of lung cancer).

[0232] Methods and compositions involving combination therapy enhance the therapeutic or prophylactic effects and / or increase the therapeutic efficacy of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in combined amounts effective to achieve a desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. This process can include contacting cells with both an antibody or antibody fragment and a second therapy. Tissues, tumors, or cells can be contacted with one or more compositions or pharmacological formulations containing one or more of the agents (i.e., antibodies or antibody fragments or anti-cancer agents), or tissues, tumors, and / or cells can be contacted with two or more different compositions or formulations, with one composition providing 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) both an antibody or antibody fragment and an anti-cancer agent. It is also contemplated that such combination therapy can be used in conjunction with chemotherapy, radioligand therapy, external beam radiation therapy, surgery, immunotherapy, or radioimmunotherapy.

[0233] Methods and compositions involving combination therapy enhance the therapeutic or prophylactic effects and / or increase the therapeutic effect of another anti-inflammatory therapy. Therapeutic and prophylactic methods and compositions may be provided in combined amounts effective to achieve a desired effect, such as reducing inflammation. The process may include contacting cells with both an antibody or antibody fragment, or a cell-based therapy expressing an antibody fragment, and a second therapy. The tissue, tumor, or cells may be contacted with one or more compositions or pharmacological formulations containing one or more of the agents (i.e., an antibody or antibody fragment or an anti-inflammatory agent), or the tissue, tumor, and / or cells may be contacted with two or more different compositions or formulations, where one composition provides 1) an antibody or antibody fragment, 2) an anti-inflammatory agent, or 3) both an antibody or antibody fragment and an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is selected from the group consisting of a corticosteroid, a DMARD, or an anti-cytokine therapy, or a combination thereof. In some embodiments, the process may include a low dose of a radioconjugate as described herein.

[0234] The terms "contacted" and "exposed," when applied to a cell, are used herein to refer to the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell killing, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0235] The antibody may be administered before, during, or after anti-cancer drug treatment, or in various combinations. Administration may occur at intervals ranging from simultaneous administration to minutes, days, or weeks. In embodiments in which the antibody or antibody fragment is provided to the patient separately from the anti-cancer drug, it is generally ensured that no significant time elapses between deliveries, so that the two compounds can still exert their beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and anti-cancer therapy may be provided to the patient within about 12-24 hours or 72 hours of each other, more particularly within about 6-12 hours of each other. In some situations, it may be desirable to extend the treatment period significantly, with days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsed between each administration.

[0236] In certain embodiments, the treatment course lasts for 1 to 90 days or more (such ranges are inclusive). It is contemplated that one agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof, and another agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof. Within a single day (24 hours), the patient may receive one or more doses of the agent. Furthermore, it is contemplated that after the treatment course, there will be a period during which no anti-cancer treatment is administered. This period may last for 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (such ranges are inclusive), depending on the patient's condition, such as prognosis, strength, and health. It is anticipated that the treatment cycle will be repeated as necessary.

[0237] In some cases, B7-H3 antibodies or their antigen-binding fragments may be labeled, conjugated, or fused to therapeutic or diagnostic agents (such as imaging agents). The linkage may be covalent or non-covalent (e.g., ionic). Such antibodies and antibody fragments are referred to as antibody-drug conjugates (ADCs) or immunoconjugates. Antibody conjugates are useful for the local delivery of therapeutic agents, particularly cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in the treatment of cancer, allowing targeted delivery of the drug moiety to tumors and their intracellular accumulation within the tumor; systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity not only to the tumor cells being eliminated but also to normal cells. Therapeutic agents include, but are not limited to, toxins, including, but not limited to, plant and bacterial toxins, small molecules, radioligands, bifunctional chelates, peptides, polypeptides, and proteins. Also provided is a genetically engineered fusion protein in which a gene encoding an antibody, or a fragment thereof containing an Fv region, or a peptide is fused to a gene encoding a toxin, thereby enabling delivery of the toxin to a target cell. As used herein, a target cell or target cells are B7-H3-positive cells.

[0238] In some embodiments, the B7-H3 antibody or antigen-binding fragment thereof is conjugated to a moiety that specifically binds to an immune cell. In some embodiments, a bispecific antibody is provided that includes a B7-H3 antibody or antigen-binding fragment thereof described herein and an antibody or antigen-binding fragment thereof that specifically binds to an immune cell. In some embodiments, the bispecific antibody includes a B7-H3-specific antibody or antigen-binding portion thereof and an antibody portion that specifically binds to a T cell. Such molecules are referred to as bispecific T cell engagers and can induce T cell-mediated cytotoxicity of cancer cells that express B7-H3 (see, e.g., Zhou et al., 2021, Biomarker Research 9:38). In some embodiments, the bispecific antibody includes a B7-H3-specific antibody or antigen-binding portion thereof and an antibody portion that specifically binds to a natural killer cell (NK cell). Such molecules are called NK cell engagers and can induce NK cell-mediated cytotoxicity of B7-H3-expressing cancer cells [see, for example, Demaria et al., 2021, European Journal of Immunology 51(8):1934-1942].

[0239] Other examples of therapeutic agents include chemotherapeutic agents, radiotherapeutic agents, phototherapeutic agents, and immunotherapeutic agents, as well as combinations thereof. In this manner, the antibody or peptide conjugate delivered to a subject may be multifunctional in that it exerts one therapeutic effect by binding to the B7-H3 protein and exerts a second therapeutic effect by delivering an adjunctive therapeutic agent.

[0240] Therapeutic agents can act extracellularly, e.g., by initiating or affecting an immune response, or they can act intracellularly directly by translocating across the cell membrane or indirectly, e.g., by affecting transmembrane cell signaling. Therapeutic agents can be cleavable from the B7-H3 antibody or antigen-binding fragment thereof. Cleavage can be autolytic, achieved by proteolysis, or affected by contacting the cell with a cleaving agent.

[0241] In some cases, the therapeutic agent is a cytotoxic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of toxins or toxin moieties include diphtheria, ricin, streptavidin, and modifications thereof. Further examples include paclitaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicine, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), anti-cancer drugs (e.g., cyclosulfonyl 3-(2-methylpropyl)-2-hydroxybenzoates ... Antigen-binding agents include, but are not limited to, biological substances (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Cytotoxic peptides, such as the auristatin (antineoplastic) peptide auristatin E (AE), a synthetic analog of dolastatin, and monomethyl auristatin (MMAE), can also be conjugated to B7-H3-specific antibodies or antigen-binding fragments thereof. In some cases, B7-H3-specific antibodies or antigen-binding fragments thereof can be conjugated to radioactive metal ions.

[0242] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (such as TARCEVA®, Genentech / OSI Pharm.), bortezomib (such as VELCADE®, Millennium Pharm.], fulvestrant [such as FASLODEX®, AstraZeneca], Sutent (such as SU11248, Pfizer), letrozole [such as FEMARA®, Novartis], imatinib mesylate [such as GLEEVEC®, Novartis], PTK787 / ZK222584 (Novartis), oxaliplatin [such as Eloxatin®, Sanofi], 5-fluorouracil (5-FU), leucovorin, rapamycin (also known as sirolimus) [such as RAPAMUNE®, Wyeth], lapatinib [such as TYKERB®, GSK572016, GlaxoSmithKline], lonafarnib (such as SCH 66336), sorafenib (such as BAY43-9006, Bayer Labs.), capecitabine [XELODA® and the like, Roche], docetaxel [TAXOTERE® and the like], and gefitinib [IRESSA® and the like, Astrazeneca], AG1478, AG1571 (SU 5271 and the like; Sugen Inc.alkylating agents such as thiotepa and cyclosphosphamide [such as CYTOXAN®]; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictine; spongistatins; nitrogen mustards such as chlorambucil, chromafadine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1). I and calicheamicin θ1 Idynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin [morpholino-doxorubicin]. ADRIAMYCIN® including doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin], epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potyfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate and 5-fluroxin Antimetabolites such as olauracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; amino Antiadrenergics such as lutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin;Pentostatin; Fenamet; Pirarubicin; Losoxantrone; Podophyllic acid; 2-ethylhydrazide; Procarbazine; Trametes versicolor polysaccharide-K (Krestin, PSK) (JHS Natural Products, Eugene, OR); Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Cytarabine (cytosine arabinoside, "Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel [TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ, etc.], ABRAXANE™ [a cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL)], and docetaxel [TAXOTERE®, Rhoene-Poulenc Rorer, Antony, France]; chlorambucil; gemcitabine (such as GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (such as NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0243] As used herein, chemotherapeutic agents also include (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and toremifene (such as FARESTON®); (ii) anti-inflammatory drugs, such as 4(5)-isothiazolinone, 4(6)-isothiazolinone, 4(7)-isothiazolinone, 4(8)-isothiazolinone, 4(9)-isothiazolinone, 4(10)-isothiazolinone, 4(11)-isothiazolinone, 4(12)-isothiazolinone, 4(13)-isothiazolinone, 4(14)-isothiazolinone, 4(15)-isothiazolinone, 4(16)-isothiazolinone, 4(17)-isothiazolinone, 4(18)-isothiazolinone, 4(19)-isothiazolinone, 4(20)-isothiazolinone, 4(21)-isothiazolinone, 4(22)-isothiazolinone, 4(23)-isothiazolinone, 4(24)-isothiazolinone, 4(25)-isothiazolinone, 4(26)-isothiazolinone, 4(27)-isothiazolinone, 4(28)-isothiazolinone, 4(29)-isothiazolinone, 4(29)-isothiazolinone, 4(29 Aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as midazole, aminoglutethimide, megestrol acetate (such as MEGASE®), exemestane (such as AROMASIN®), formestany, fadrozole, vorozole (such as RIVISOR®), letrozole (such as FEMARA®), and anastrozole (such as ARIMIDEX®); (iii) flutamide, nilutamide, bicalutamide, leuprolide, and goserelin. and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell growth, such as PKC-alpha, Ralf, and H-Ras; (viii) VEGF receptor and angiogenesis inhibitors (including ribozymes such as ANGIOZYME®), and HER2 expression inhibitors; (ix) vaccines such as gene therapy vaccines, e.g., ALLOVECTIN-7® vaccine (a plasmid / lipid complex containing DNA sequences encoding HLA-B7 and β2 microglobulin), LEUVECTIN® vaccine (a plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE / DOPE)), and VAXID® vaccine (a patient-specific naked DNA vaccine); IL-2 or aldesleukin [such as PROLEUKIN®];Topoisomerase 1 inhibitors (such as TOPOTECAN®); gonadotropin-releasing hormone antagonists (such as ABARELIX®); (x) antiangiogenic agents such as bevacizumab (such as AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0244] In some cases, the treatment methods provided herein may further include administering an immunosuppressant, such as an immune checkpoint inhibitor, as part of the method. These treatments work by "releasing the brakes" on the immune system (which is immunosuppressive), allowing the immune system to mount a stronger and more effective attack against cancer. Currently, several different types of checkpoint inhibitors are used that target different checkpoints or "brakes" on immune cells. Immune checkpoint proteins that can be targeted by immune checkpoint blockers include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B- and T-lymphocyte attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-inducible tumor necrosis factor receptor-related protein (GITR), and HLA-DRB. These include ICOS (also known as CD278), HLA-DQAI, HLA-E, indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, OX40 (also known as CD134), programmed death 1 (PD-1), programmed death ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB 10, ST A T1, T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA, also known as C10orf54). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4. Exemplary immunosuppressants are PD-1 inhibitors (such as nivolumab and pembrolizumab), PD-L1 inhibitors (such as atezolizumab, durvalumab, and avelumab), and CTLA-4 inhibitors (such as ipilimumab). In one example, the second form of cancer therapy includes a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor.In some cases, a combination of such inhibitors may be administered. In some cases, the PD-L1 inhibitor, PD-1 inhibitor, and / or CTLA4 inhibitor may be an inhibitory antibody that specifically binds to PD-L1, PD-1, or CTLA4, respectively.

[0245] In some embodiments, the treatment methods provided herein may further include administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used to treat cancer. Radiation can be delivered by a machine outside the body (external beam radiation therapy), or radiation can come from a radioactive material placed inside the body near cancer cells (internal beam radiation therapy, also known as brachytherapy), or by radioligand therapy. Systemic radioligand therapy uses radioactive substances, such as radioisotopes, that travel into the bloodstream to kill cancer cells, as described above.

[0246] B. Diagnostic Methods In another aspect, a method for assessing the eligibility of a subject for inclusion in or exclusion from a clinical trial of a B7-H3-targeted therapy using a B7-H3 antibody or its antigen-binding fragment is provided.The method includes: (a) measuring the amount of B7-H3 in a tumor sample, serum sample, cerebrospinal fluid (CSF) sample, urine sample, tear sample, or accessible liquid sample from the subject; (b) determining whether the subject has a cancer characterized as having a high level of B7-H3 expression; and (c) indicating that if the subject's cancer is characterized as having a high level of B7-H3 expression, that is, above a predetermined threshold, the subject is eligible for the clinical trial of a B7-H3-targeted therapy, or if the subject's cancer is characterized as having a low level of B7-H3 expression, that is, below a predetermined threshold, the subject is ineligible for the clinical trial of a B7-H3-targeted therapy.In some cases, the threshold level is the median value of B7-H3 determined in a reference population of patients with the same type of cancer as the subject. In another case, the threshold level is the optimal amount of B7-H3 determined in a reference population of patients with the same type of cancer as the subject.As used herein, "optimal cutoff" refers to a predetermined measurement value in a subject that shows a certain attribute, which allows the best discrimination between two categories of attributes.For example, to determine at least one of overall survival, time to disease progression, progression-free survival, and the possibility of responding to treatment, find the optimal cutoff value that allows the best discrimination between two categories (subgroups) of patients (for example, based on clinical evaluation using RECIST criteria, as recognized in the medical field, for example, Eisenhauer, EA, et al., Eur. J. Cancer 45:228-247 (2009)).The optimal cutoff is used to separate subjects with values ​​lower or higher than the optimal cutoff to optimize a predictive model, for example, but not limited to, to maximize the specificity of the model, maximize the sensitivity of the model, maximize the difference in outcome, or minimize the p-value from the difference in hazard ratio or response.

[0247] In another embodiment, a method for assessing the responsiveness of a subject with cancer to a B7-H3 antibody or its antigen-binding fragment is provided, the method comprising: (a) measuring the amount of B7-H3 in a tumor sample from the subject; (c) determining whether the subject has a cancer characterized as having a high level of B7-H3 expression; and (d) indicating that if the subject's cancer is characterized as having a high level of B7-H3 expression, the subject is likely to respond to a B7-H3 antibody or its antigen-binding fragment. Conversely, if the subject's cancer is characterized as having a low level of B7-H3 expression, the subject is unlikely to respond to a B7-H3 antibody or its antigen-binding fragment. In some cases, the amount of B7-H3 in a tumor sample is measured using a B7-H3 antibody or its antigen-binding fragment as described herein.

[0248] In another embodiment, a method for diagnosing cancer in a subject is provided. Specifically, the diagnosis can be the diagnosis of cancer that expresses B7-H3. This method can include measuring the amount of B7-H3 in a sample from a subject, and if the amount of B7-H3 expression in the sample is high, diagnosing the subject with cancer. In some cases, this method can include: (a) using a B7-H3 antibody or its antigen-binding fragment to measure the amount of B7-H3 in a tumor sample from the subject; and (c) determining whether the subject has a cancer characterized by high levels of B7-H3 expression. Conversely, if the amount of B7-H3 expression in the sample or the subject's cancer is low, the subject cannot be diagnosed with cancer or cannot be diagnosed with cancer that expresses B7-H3.

[0249] In some embodiments, to diagnose or characterize cancer in a subject, a biopsy is typically taken from a subject with abnormal tissue growth, such as a tumor. The sample may be a formalin-fixed, paraffin-embedded tissue sample obtained from the subject's cancer (tumor). In other embodiments, such as when circulating tumor cells or exosomes or target antigens (e.g., sB7-H3) are evaluated, the sample from the subject is a blood, plasma, urine, saliva, CSF, or lymph sample. Typically, tissue or cells from the patient sample are examined under a microscope or processed to confirm the diagnosis and / or evaluate information about the tumor. In some cases, additional tests on the protein, DNA, and / or mRNA of cells in the sample may be necessary to confirm the diagnosis or characterization.

[0250] In yet another embodiment, a method for assessing the eligibility of a subject for inclusion in or exclusion from clinical trials of B7-H3 targeted therapy using B7-H3 antibody or its antigen-binding fragment is provided.This method includes: (a) measuring the amount of B7-H3 in a blood sample, serum sample, cerebrospinal fluid (CSF) sample, urine sample, tear sample, or accessible liquid sample from the subject; (b) determining whether the subject has inflammation characterized by having a high level of B7-H3 expression; and (c) indicating that if the subject's inflammation is characterized by having a high level of B7-H3 expression, that is, above a predetermined threshold, the subject is eligible for the clinical trial of B7-H3 targeted therapy, or if the subject's inflammation is characterized by having a low level of B7-H3 expression, that is, below a predetermined threshold, the subject is ineligible for the clinical trial of B7-H3 targeted therapy.In some cases, the threshold level is the median value of B7-H3 determined in a reference population of patients with the same type of inflammation as the subject. In another case, the threshold level is the optimal amount of B7-H3 determined in a reference group of patients with the same type of inflammation as the subject.As used herein, " optimal cutoff " refers to the predetermined measurement value in the subject that shows a certain attribute, which allows the best discrimination between two categories of attribute.The optimal cutoff is used to separate the subjects that have values ​​lower or higher than the optimal cutoff in order to optimize the prediction model, for example, but not limited to, to maximize the specificity of the model, maximize the sensitivity of the model, maximize the difference in outcome, or minimize the p-value from the difference in hazard ratio or response.

[0251] In another aspect, a method for assessing the responsiveness of a subject with inflammation to a B7-H3 antibody or its antigen-binding fragment is provided, the method comprising: (a) measuring the amount of B7-H3 in a sample from the subject; (c) determining whether the subject has inflammation characterized as having a high level of B7-H3 expression; and (d) indicating that if the subject's inflammation is characterized as having a high level of B7-H3 expression, the subject is likely to respond to a B7-H3 antibody or its antigen-binding fragment. Conversely, if the subject's inflammation is characterized as having a low level of B7-H3 expression, the subject is unlikely to respond to a B7-H3 antibody or its antigen-binding fragment. In some cases, the amount of B7-H3 in a sample is measured using a B7-H3 antibody or its antigen-binding fragment as described herein.

[0252] In another aspect, a method for diagnosing inflammation in a subject is provided. Specifically, the diagnosis can be the diagnosis of a patient sample expressing B7-H3. This method can include measuring the amount of B7-H3 in a sample from a subject, and diagnosing the subject with inflammation if the amount of B7-H3 expression in the sample is high. In some cases, this method can include: (a) using a B7-H3 antibody or its antigen-binding fragment to measure the amount of B7-H3 in a sample from a subject; and (c) determining whether the subject has inflammation, characterized by having a high level of B7-H3 expression. Conversely, if the amount of B7-H3 expression in a sample from a subject with inflammation is low, the subject cannot be diagnosed with inflammation or cannot be diagnosed with inflammation that expresses B7-H3.

[0253] C. How to Detect B7-H3 In another aspect, a method for detecting the presence of cells expressing B7-H3 in a biological sample is provided, the method comprising: (a) contacting the sample with a composition comprising an isolated B7-H3 antibody or antigen-binding portion thereof described herein; and (b) detecting the amount of binding of the isolated antibody or antigen-binding portion thereof as a determination of the presence of the cells expressing B7-H3. In some embodiments, the biological sample comprises a tumor sample. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0254] In some embodiments, B7-H3 expression in cancer cells or inflammation in patients can be examined by using one or more routine biochemical analyses. In some embodiments, B7-H3 expression is determined by detecting protein expression using methods such as Western blot analysis, flow cytometry, and immunohistochemistry, ELISA, or nucleic acid-labeled antibody sequencing-based detection, or staining using the B7-H3 antibody or its antigen-binding portion described in the present disclosure. In some cases, a combination of these methods may be used, or additional methods such as microarray analysis and RT-PCR may also be used.

[0255] In some cases, a threshold amount of B7-H3 protein expression is used to characterize B7-H3 expression as either high or low. A high level of B7-H3 protein expression refers to a measure of B7-H3 protein expression that exceeds a certain threshold. For example, the threshold may be the normal, average, or median amount of B7-H3 protein expression measured in a specific set of samples, referred to as a reference population. In some cases, the reference population may be a population of normal / healthy subjects. In other cases, the reference population may be a population of subjects with a specific type of cancer (the same type of cancer as the subject being evaluated has). A low level of B7-H3 expression refers to the opposite of the above. For example, the threshold can be determined by dividing samples around a mathematically determined point, such as, but not limited to, the median, to identify two distinct subgroups in the reference population, thereby creating a subgroup with high measured values ​​(i.e., higher than the median) and another subgroup with low measured values.

[0256] Also provided is a method for imaging tumors in a subject with cancer that expresses B7-H3, comprising administering to the subject an isolated antibody specific for B7-H3 or its antigen-binding portion conjugated with an imaging label, and detecting the imaging label in the subject.The imaging method can be used to evaluate tumor size and changes in tumor size during or after the course of treatment administered to the subject.This method can be useful for evaluating the response of the subject to the administered treatment.In some cases, this method can be useful for classifying the malignancy of the subject's cancer.

[0257] Also provided is a method for monitoring the response of a subject with a B7-H3-expressing cancer to cancer therapy. The method includes administering a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to an imaging label to the subject at a first time point before the subject receives the cancer therapy, detecting the imaging label in the subject to obtain a first image of the tumor, and administering a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to an imaging label to the subject at a second time point after the subject receives the cancer therapy, detecting the imaging label in the subject to obtain a second image of the tumor, and comparing the first image with the second image to determine whether a change in tumor size has occurred. In some cases, the steps of administering a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to an imaging label to the subject at a first time point after the subject receives the cancer therapy, detecting the imaging label in the subject to obtain a second image of the tumor, and comparing the first image with the second image to determine whether a change in tumor size has occurred can be repeated at a third time point (or additional time points) after the subject receives the cancer therapy.

[0258] Also provided is a method for imaging a subject with inflammation that expresses B7-H3, comprising administering to the subject an isolated antibody specific for B7-H3 or its antigen-binding portion conjugated with an imaging label, and detecting the imaging label in the subject.The imaging method can be used to evaluate the size and change of inflammation during or after the course of treatment administered to the subject.This method can be useful for evaluating the response of the subject to the administered treatment.

[0259] Also provided is a method for monitoring the response of a subject with inflammation that expresses B7-H3 to a therapy. The method includes administering a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to an imaging label to the subject at a first time point before the subject receives the therapy, detecting the imaging label in the subject to obtain a first image of the tumor, and administering a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to an imaging label to the subject at a second time point after the subject receives the therapy, detecting the imaging label in the subject to obtain a second image of the tumor, and comparing the first and second images to determine whether a change in inflammation has occurred. In some cases, the steps of administering a B7-H3-specific antibody or antigen-binding fragment thereof conjugated to an imaging label to the subject at a first time point after the subject receives the therapy, detecting the imaging label in the subject to obtain a second image of the tumor, and comparing the first and second images to determine whether a change in inflammation has occurred can be repeated at a third time point (or additional time points) after the subject receives cancer therapy. In some embodiments, the B7-H3 antibodies described herein may be used to treat inflammation in a subject.

[0260] In one embodiment, a subject is administered a labeled B7-H3 antibody or antigen-binding fragment thereof described herein conjugated to an imaging agent. The labeled B7-H3 antibody or antigen-binding fragment thereof is incubated in vivo and allowed to bind to B7-H3 in the subject's tissue. The imaging label is then localized to tumor cells or tissue, and the localized imaging label is detected using appropriate imaging equipment known to those skilled in the art.

[0261] The imaging agent may carry a bioluminescent or chemiluminescent label. Such labels include polypeptides known to be fluorescent, bioluminescent, or chemiluminescent, or polypeptides that act as enzymes on specific substrates (reagents), or polypeptides that can generate fluorescent, bioluminescent, or chemiluminescent molecules. Examples of bioluminescent or chemiluminescent labels include luciferase, aequorin, obelin, mnemiopsin, berovin, phenanthridinium ester, and variants and combinations thereof. The substrate of the bioluminescent or chemiluminescent polypeptide can also be used for imaging. For example, the chemiluminescent polypeptide can be luciferase and the reagent luciferin. The substrate of the bioluminescent or chemiluminescent label can be administered before, simultaneously (e.g., in the same formulation), or after administration of the agent.

[0262] In some embodiments, the imaging agent can carry fluorescent label.For example, the fluorescent label for use as conjugate can include but is not limited to Alexa 350, Alexa 430, Alexa 594, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0263] The imaging agent can include paramagnetic compounds such as metal-chelated polypeptides (e.g., metalloporphyrins).The paramagnetic compound can also include single-crystal nanoparticles, such as nanoparticles containing lanthanides (e.g., Gd) or iron oxide; or metal ions such as lanthanides.Examples of elements useful for magnetic resonance imaging include gadolinium, terbium, tin, iron, or their isotopes.

[0264] Whole-body imaging techniques using radioisotope-labeled agents can be used to identify the location of diseased cells and tissues (e.g., primary tumors and metastatic tumors).In some cases, labeled agents for identifying the location of tumor tissue or cells are administered intravenously.The biodistribution of the label can be monitored by scintigraphy, and the accumulation of the label is related to the presence of B7-H3 or other tumor markers.Whole-body imaging techniques are described, for example, in U.S. Patent Nos. 4,036,945 and 4,311,688.

[0265] Images according to the present disclosure can be generated by computer-assisted tomography (CAT), magnetic resonance spectroscopy (MRS) images, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence, photoacoustic, or bioluminescence imaging (BLI), or the like.

[0266] Computer-assisted tomography (CAT) and computer axial tomography (CT) systems and devices known in the art can be used to generate images. (See, e.g., U.S. Patent Nos. 6,151,377; 5,946,371; 5,446,799; 5,406,479; 5,208,581; and 5,109,97.) Imaging methods can also utilize animal imaging modalities such as MicroCAT™ (ImTek Inc.).

[0267] Magnetic resonance imaging (MRI) systems and devices known in the art can be used for imaging. For a description of MRI methods and devices, see, for example, U.S. Patent No. 6,151,377. MRI and supporting devices are commercially available from, for example, Bruker Medical GMBH; Caprius; Esaote Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; Intermagnetics General Corporation; Lunar Corp.; MagneVu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems, including, for example, imaging systems by Silicon Graphics.

[0268] Positron emission tomography (PET) systems and devices known in the art can be used for imaging. For example, the imaging method of the present disclosure can use a system designated PET VI at Brookhaven National Laboratory. For a description of PET systems and devices, see, for example, U.S. Patent No. 6,151,377. Animal imaging modalities such as microPET (Concorde Microsystems, Inc.) can also be used.

[0269] Single photon emission computed tomography (SPECT) systems and devices known in the art can be used for imaging. (See, for example, U.S. Patent Nos. 6,115,446; 6,072,177; 5,608,221; 5,600,145; 5,210,421; and 5,103,098.) Imaging methods can also be used with animal imaging modalities such as microSPECT.

[0270] Highly sensitive photon detection systems can be used to externally detect bioluminescent and fluorescent proteins; see, for example, Contag (2000), Neoplasia 2:41-52; and Zhang (1994), Clin. Exp. Metastasis, 12:87-92. The imaging method of the present disclosure can be carried out using any such photon detection device, for example, an intensified charge-coupled device (ICCD) camera coupled to an image processor. Photodetection devices are also commercially available from Xenogen / Perkin-Elmer, Hamamatsu.

[0271] Disclosed herein are materials, compositions, and methods that can be used in, in conjunction with, or in preparing the disclosed embodiments. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that although specific reference to each of the various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, when a method is disclosed and discussed, and several modifications that can be made to several molecules included in the method are discussed, each and every combination and permutation of the method and possible modifications are specifically contemplated unless otherwise indicated. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that may be performed, it is understood that each of these additional steps may be performed with any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered specifically contemplated and disclosed.

[0272] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety. The following description provides further non-limiting examples of the disclosed compositions and methods. [Example]

[0273] The following examples are offered to illustrate, but not limit, the claimed methods and compositions. [Example 1]

[0274] Analysis of MIL33B chimeric antibodies. A humanized MIL33B antibody was generated based on the mouse MIL33B antibody sequence (i.e., a mouse antibody that specifically binds to B7-H3, as described in PCT Publication No. WO2021 / 101991). DNA sequences encoding the chimeric antibody heavy and light chains listed in Table 6 were synthesized and inserted into the pcDNA3.4 vector to construct an expression plasmid for full-length IgG. Chimeric antibody expression was carried out in Expi293 cell culture, and the supernatant was purified using a Protein A affinity column. The purified antibody was buffer-exchanged into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively. Binding confirmation and affinity were assessed by surface plasmon resonance (SPR) using a Biacore™ 8K / T200.

[0275] Table 6. VH and VL nucleotide sequences of chimeric and graft antibodies. [Table 6] TIFF2026505058000008.tif205162

[0276] The affinity of the chimeric antibody for the B7-H3 antigen was determined using a surface plasmon resonance biosensor, Biacore 8K / T200 (GE Healthcare). The antibody was captured on a sensor chip using the Fc capture method. B7-H3 antigen was used as the analyte. Dissociation (kd) and association (ka) rate constant data were obtained using the Biacore 8K / T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. Binding kinetics was evaluated using SPR according to the following parameters: the system had a flow rate of 10 μl / min and a capture time of 30 seconds. The association contact time was 120 seconds, and the dissociation contact time was 360 seconds at a flow rate of 30 μl / min. The sample concentrations were 1.171875, 2.34375, 4.6875, 9.375, 18.75, 37.5, and 75 nM. The binding kinetics of the resulting chimeric antibody were as follows: ka of 6.09E05 (1 / Ms), kd of 1.21E-04 (1 / s), KD of 1.99E-10 (M), and Rmax of 65.4 (RU).

[0277] Following the testing and validation of the chimeric antibodies described in this example, grafted antibodies were also tested according to similar methods (data not shown). [Example 2]

[0278] Generation of a humanized B7-H3 antibody library. A point mutation library was created that encompassed the framework regions surrounding the CDRs and targeted the least conserved mutations from the murine framework compared to the human framework. A humanized Fast Screening for Expression Biophysical-properties and Affinity (FASEBA) screening library, including all backmutants in the inner core of the antibody structure (Fab), was designed under contract with GenScript. Library construction was performed according to GenScript's standard operating procedures (SOPs).

[0279] Specifically, a total of 48 clones were generated for sequencing for both the VH and VL regions. Sequence analysis revealed 41 VL sequences, along with 7 poor sequences, for further testing, and 38 VH sequences, along with 10 poor sequences, for further testing. Sequence alignment of the clones identified a total of 8 unique VL sequences and 14 unique VH sequences. FASEBA screening against antigen proteins was performed. Individual Fab clones were expressed in 96-well plates, and crude proteins secreted into the medium by E. coli were assayed by ELISA against bovine serum albumin (BSA) and the target antigen protein to evaluate expression and binding activity. Fifteen clones that demonstrated the best binding affinity were selected for DNA sequencing (i.e., AHF15937, AHF15938, AHF15939, AHF15940, AHF15941, AHF15942, AHF15943, AHF15944, AHF15945, AHF15946, AHF15947, AHF15948, AHF15949, AHF15950, and AHF15951). Of the 15 listed, 11 unique clones were used for binding affinity ranking.

[0280] For affinity ranking, albumin was immobilized on a sensor chip using the amine coupling method. Selected Fab-single domains fused to single-domain antibodies against serum albumin secreted into the culture medium (Fab-SASA) were injected and captured by albumin on the chip (capture phase). After equilibration, B7-H3 antigen was injected for 180 seconds (association phase), followed by 420 seconds of running buffer (dissociation phase). The surface was regenerated before injecting another Fab-SASA clone. This process was repeated until all Fab-SASA clones were analyzed. During each cycle, the response of the reference flow cell was subtracted from that of the Fab-SASA flow cell. The off-rates of Fab-SASA clones were obtained by locally fitting the experimental data to a 1:1 interaction model using Biacore 8K / T200 evaluation software. Fab-SASA clones were ranked by their off-rate constants [off-rate, kd]. Clones with binding affinities similar to or higher than those of the chimeric Fab-SASA were sequenced. Based on the sequencing results, clones with higher affinities and fewer back-mutation sites were selected. The binding affinity results for the selected 11 clones can be seen in Table 7 below, and the corresponding sensorgrams are shown in Figure 1.

[0281] Table 7. Binding affinities of 11 selected Fab-SASA clones. [Table 7] [Example 3]

[0282] Construct and production of humanized IgG. From the affinity rankings in Table 7, five clones were selected for full-length IgG1 expression, purification, and further study. DNA sequences encoding the top binder sequences were inserted into the pcDNA3.4 vector to generate full-length IgG expression plasmids. Expi293 cells were co-transfected with the heavy and light chain expression plasmids. Recombinant IgG secreted into the culture medium was purified using Protein A affinity chromatography according to the GenScript SOP. Western blot analysis was performed on the five humanized antibodies, as shown in Figure 2. The purified IgG migrated as a band of approximately 150 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and as bands of approximately 50 kDa and 25 kDa under reducing conditions.

[0283] Five humanized antibodies were purified and tested for binding affinity with human rhB7-H3. Binding confirmation was tested by surface plasmon resonance (SPR) using a Biacore 8K / T200. The binding kinetics of the humanized antibodies can be found in Table 8 below, and the corresponding sensorgrams can be found in Figure 3. Based on the binding affinity results in Table 8, three humanized monoclonal antibodies were further tested [i.e., AHF15945 (also known as MIL33B-H1), AHF15938 (also known as MIL33B-H2), and AHF15948 (also known as MIL33B-H3)].

[0284] Table 8. Binding affinities of humanized antibodies to rhB7-H3 by SPR [Table 8] [Example 4]

[0285] The humanized MIL33B antibody selectively binds to human 4Ig-B7-H3 relative to human 2Ig-B7-H3. The in vitro binding affinities of three humanized MIL33B antibodies to human 4Ig-B7-H3 and human 2Ig-B7-H3 were evaluated by biolayer interferometry. For comparison, MIL33B-mIgG2a (mouse antibody) and chimeric antibodies were run in parallel. The extracellular domains of the indicated 4Ig-B7-H3 and 2Ig-B7-H3 proteins were purchased from R&D systems. All proteins were verified by the supplier for both purity and functionality. KD values ​​were determined in "affinity" mode using capture biolayer interferometry (Octet, Molecular Devices), in which the MILB33 antibody was captured on the tip of a probe and placed in a solution containing different concentrations of the target B7-H3 extracellular domain. KD, KD error, and R 2 The values ​​are reported in Table 9. The data demonstrated that the derived human MIL33B antibodies (MIL33B-H1, MIL33B-H2, or MIL33B-H3) maintained high affinity for B7-H3, with high selectivity for 4Ig-B7-H3 over 2Ig-B7-H3, compared to the murine MIL33B antibody. MIL33B-H3 hIgG1 demonstrated the highest selectivity, with a 4Ig-B7-H3 to 2Ig-B7-H3 ratio of 360, a property that may prove crucial for the successful use of antibodies in vivo.

[0286] Table 9. Binding of various MIL33B antibodies to the extracellular domain of human 4Ig versus 2Ig B7-H3. [Table 9] [Example 5]

[0287] Humanized MIL33B-H3 binds to 4Ig pig and cynomolgus monkey proteins. The extracellular domains of the indicated 4Ig-B7-H3 and 2Ig-B7-H3 proteins were purchased from R&D systems. All proteins were verified by the supplier for both purity and functionality. KD values ​​were determined using capture biolayer interferometry (Octet, Molecular Devices) in "affinity" mode, in which the MILB33 antibody was captured on the tip of a probe and placed in a solution containing different concentrations of the target extracellular domain. KD, KD error, and R 2 The values ​​are reported in Table 10. The data demonstrated that the preferred human MIL33B antibody (MIL33B-H3) binds to porcine and cynomolgus monkey 4Ig-B7-H3 proteins comparably to the previously reported murine antibody (MIL33B-mIgG2a). In addition, these results demonstrated that the humanized MIL33B antibody binds to cynomolgus monkey and porcine 4Ig-B7-H3 with subnanomolar affinity.

[0288] Table 10. Binding of MIL33B-H3-hIgG1 and MIL33B-mIgG2a antibodies to porcine and cynomolgus 4Ig-B7-H3 proteins. [Table 10] [Example 6]

[0289] Fluorescence microscopy of tumor cell binding. Using a human HeLa tumor cell binding model and MIL33B-H3 conjugated with the fluorescent tag AlexaFluor 594, we investigated the net binding and retention of tumor cells. Live-cell fluorescence microscopy confirmed that the MIL33B-H3 antibody bound more strongly to human tumor cells than the murine MIL33B antibody (data not shown). [Example 7]

[0290] Generation and characterization of MIL33B-specific CAR T cells. To generate CAR cells, healthy donor cells can be lentivirally transduced with an anti-CD276 (4Ig-B7-H3) specific CAR, which contains the MIL33B-H3 variable region sequence (e.g., SEQ ID NO: 42), CD8α hinge, CD8α transmembrane, and 4-1BB and CD3ζ signaling domains. To assess the surface expression of anti-CD276 CARs, flow cytometry using FITC-conjugated CD276 extracellular domain protein can be used to assess transduction efficiency. These are most commonly CAR-T cells, but can also be used with CAR-NK cells, CAR-macrophages, and CAR-neutrophils.

[0291] Anti-CD276 CAR cells (e.g., CAR-T cells) can be co-cultured with CD276+ B cell lymphoma cell lines (e.g., Jeko-1, sp53, and CA46) and CD276-negative NK cell leukemia cell lines (e.g., NK92). It is understood that under co-culture conditions, anti-CD276 CAR-T cells can induce cell death (lysis) in CD276+ cells, but not in parental cells or CD276-negative cell lines. [Example 8]

[0292] M1 and M2 macrophages express human 4Ig-B7-H3. To generate M1 and M2 macrophages, 150,000 THP-1 cells were seeded in 200 μL of RPMI-1640 medium containing 10% FBS, 1% L-Glu, and 0.05 mM 2-mercaptoethanol with or without polarizing cytokines (M1-like: IFNγ 20 ng / mL; LPS 250 ng / mL) for 48 hours. After incubation at 37°C and 5% CO2, cells were activated using PMA at a final concentration of 2.5 μg / μL or 4 mM. For imaging, cells were fixed with 4% PFA for 10 minutes and washed twice with PBS. Immunofluorescence staining was performed using AF594-labeled MIL33B at 4°C for 1 hour. DAPI staining of nuclei was performed at room temperature for 10 minutes. Cells were washed three times with PBS, and epifluorescence images were captured using a Nikon TiE fluorescence microscope equipped with a Hamamatsu Orca flash 4 camera.

[0293] Western blot analysis was performed according to standard protocols to assess the molecular weight of 4Ig-B7-H3 and 2Ig-B7-H3. Figure 5A shows Western blot analysis of 4Ig-B7-H3 and 2Ig-B7-H3 expression in non-activated leukemic monocytic cells (THP-1) at M0, M1, M2a, and M2c-like states, and in phorbol myristate acetate (PMA)-activated THP-1 cells at M0, M1, M2a, and M2c-like states. Briefly, both M1 and M2 macrophages express human 4Ig-B7-H3. Without being bound by any theory, the larger than expected molecular weight is likely due to altered core glycosylation, with a weaker secondary band consistent with 4Ig-B7-H3 as found in most tumors.

[0294] Figure 5B shows a panel of microscopy images of immunofluorescence staining of THP-1 cells in M0, M1, M2a, and M2c-like states treated with PM (lower panel) or without PMA (upper panel) and then incubated with MIL33B antibody. Briefly, MIL33B still bound to its epitope in live cells in all states, with higher expression in the PMA-activated state. Additionally, the results demonstrate the feasibility of using the antibodies described herein to act as blocking antibodies targeting activated myeloid cells with antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ACDP), or in hemophagocytic lymphohistiocytosis (HLH). [Example 9]

[0295] Humanized MIL33B-H3 in the treatment of reactive oxygen and nitrogen species (RONS) bursts. To evaluate the potential for M1 and M2 macrophage generation in THP-1 cells, 150,000 THP-1 cells were seeded in 200 μL of RPMI-1640 medium containing 10% FBS, 1% L-Glu, and 0.05 mM 2-mercaptoethanol with or without polarizing cytokines (M1-like: IFNγ 20 ng / mL; LPS 250 ng / mL) for 48 h. After incubation at 37°C and 5% CO, cells were activated with PMA at a final concentration of 2.5 μg / μL and imaged with 100 μM L-012 in modified Earl's balanced salt solution (MEBSS). Bioluminescence images were captured using a supercooled CCD camera (IVIS Spectrum) with a 300-second exposure, acquiring images every 5 minutes over a 30-minute time frame.

[0296] THP-1 cells differentiated to an M1-like state with LPS and interferon-gamma treatment did not exhibit a respiratory burst, similar to the undifferentiated state, whereas THP-1 cells differentiated to an M1-like state and further treated with PMA exhibited a higher respiratory burst compared to both untreated PMA-macrophages and the undifferentiated population (Fig. 6 ). [Example 10]

[0297] Humanized MIL33B-H3 in the treatment of RONS burst in a mouse model RONS Burst Imaging Protocol Mice were pre-imaged using an intraperitoneal injection of L-012 sodium salt (20 mg kg-1) in saline. Whole-animal imaging was performed in bioluminescence and reflectance modes 10 minutes after injection of L-012 using a bioluminescence imaging system (IVIS Spectrum). Images were acquired at 19 cm FOV, F / 1.1 aperture, 4 × 4 binning, for 300 seconds or 5 minutes.

[0298] Imaging and Treatment Protocol Seventeen-week-old SGM3 mice obtained from Jackson Labs (701362) were pre-imaged for RONS before treatment initiation. Treatment commenced with 200 μL of PBS or 200 μL of H3-MIL33B (final dose 200 μg / mouse / treatment, intraperitoneal injection) twice weekly. Mice were imaged again on days 7 and 14. After this time point, treatment was stopped. During a two-week treatment break, mice were imaged on days 21 and 28. Treatment resumed for one week, followed by a final whole-animal L-012 imaging on day 35.

[0299] Figure 7A shows a panel of SGM3 mice intraperitoneally injected with L-012 sodium salt in saline, imaged beforehand (day 0) and then after 3 days of incubation (day 3). Only SGM3 mice treated with PBS showed L-012 expression above camera background. Figure 7B shows a graph of the head reactive oxygen and nitrogen species (RONS) burst determined from the mouse model in SGM3 mice intraperitoneally injected with L-012 sodium salt in saline after 5 days of treatment. Collectively, these results demonstrate that HLH occurs naturally in the brain over time in this mouse model. These findings are consistent with the literature and provide a baseline for the studies performed below.

[0300] After 3 days of incubation, mice were treated with hMIL33B-H3 antibody on days 7 and 14. Figure 8A provides a panel of SGM3 mice that were intraperitoneally injected with 200 μg of humanized hMIL33B-H3 in phosphate-buffered saline (PBS) twice a week for 14 days, or with PBS buffer as a control. Figure 8B provides a graph of head RONS bursts in SGM3 mice on days 0, 7, and 14. The slope of the line for mice treated with hMIL33B-H3 was -4823 [photons / (sec)]. * days)], whereas in the control group the slope was -201 [photons / (seconds)]. * The L-012 signal was reduced in mice treated with the hMIL33B-H3 antibody, indicating that RONS was reduced in mice treated with hMIL33B-H3. The RONS signal intensity was not reduced in mice treated with PBS as a control.

[0301] After 14 days of treatment, treatment was stopped and RONS bursts were measured on days 21 and 28 to assess whether RONS generated in the brain re-elevated. Figure 9A provides a graph of head RONS bursts in SGM3 mice after 14 days of treatment with hMIL33B-H3 antibody or PBS. Treatment was terminated on day 16, and SGM3 mice were imaged on days 21 and 28. The slope of the line for mice treated with hMIL33B-H3 was 8771 [photons / (sec)]. * days)], whereas in the control group the slope was -1043 [photons / (seconds)]. * days)]. The results indicate that treatment with hMIL33B-H3 antibody reduces RONS generation in the head, and that cessation of antibody treatment resumes RONS production in the brain. After cessation of treatment for 2 weeks (shown in Figure 9A), mice were subjected to a second round of treatment for another week. Figure 9B provides a graph of head RONS burst after retreatment with hMIL33B-H3 antibody (triangles) and PBS (circles). The slope of the line for mice treated with hMIL33B-H3 antibody was -15143 [photons / (sec *days)], whereas in the control group the slope was -749 [photons / (sec * After re-treatment, the mice's head RONS bursts were reduced, demonstrating the direct effect of the hMIL-33B-H3 antibody in lowering RONS in the brain. Together, these results demonstrate that hMIL33B-H3 acts as an effective modulator of inflammation.

[0302] Further sequences SEQ ID NO: 27: AHF15945 VH nucleotide sequence SEQ ID NO: 28: AHF15938 VH nucleotide sequence SEQ ID NO: 29: AHF15948 VH nucleotide sequence SEQ ID NO: 30: AHF15938 VL nucleotide sequence (same as AHF15945 and 15948) ATGGGCTGGTCATGTATTATTCTGTTTCTGGTCGCAACTGCTACAGGGGTCCATAGTGAGATCGTGCTGACACAGAGCCCTGCTACACTGAGCCTGAGCCCCGGCGAGCGGCCACACTCTCCTGCAGCGTGTCCAGCTCTGTCAACTACATGCACTGGTATCAGCAGAAACCTG GCCAGGCCCCTAGAAGACTGATCTACGACACCAGCAAGCTGGCCTCTGGAATCCCAGCCAGATTCAGCGGATCTGGCAGCGGCACCGATTACACCCTGACCATCAGCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGTCAGCAATGGACCTCCAACCCCCTGACCTTTGGC CAGGGCACCAAGCTGGAAATCAAGAGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCA ATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATGCTGATAA SEQ ID NO: 31 U155QGF100-chimera-VH amino acid sequence MGWSCIILFLVATATGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVRQSPGQGLEGIGYINSYSDGTKYNEKFKGKATLTSDKSSSTAYMELSGLTSEDSAVYYCAR WGGLGNGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 32 U1J155QGF100-graft-VH amino acid sequence MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINSYSDGTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR WGGLGNGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 U155QGF100-chimera-VL amino acid sequence MGWSCIILFLVATATGVHSQIVLTQSPAIMSASPGEKVTMTCSVSSSVNYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSASGSGTSYSLTISSMEAEDAATYYCQQWTSNPLTF GAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 34 U155QGF100-graft-VL amino acid sequence MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSVSSSVNYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWTSNPLTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 35 FLAG DYKDDDDK SEQ ID NO: 36 Polyhistidine (6-His) HHHHHH SEQ ID NO: 37 Alternative His tag HEHEHE SEQ ID NO: 38 Hemagglutinin (HA) YPYDVPDYA SEQ ID NO: 39: CD8 alpha hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 40: CD8α transmembrane domain IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 41:4-IBB signaling domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 42: scFv EIVLTQSPATLSLSPGERATLSCSVSSSVNYMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWTSNPLTFGQGTKLEIKGSGSGSGSGSGSGSG SQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINSYSDGTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARWGGLGNGAMDYWGQGTLVTVSS SEQ ID NO: 43: scFv linker GGGGSGGGGSGGGGS SEQ ID NO: 44: scFv linker GSGSGSGSGSGSGSGS

Claims

1. a heavy chain variable region (VH) having at least 90% identity to SEQ ID NO:3, comprising a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23, and having a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO:3; and A light chain variable region (VL) having at least 90% identity to SEQ ID NO:4, comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and having an arginine at position 45 and a tyrosine at position 70 of SEQ ID NO:

4.

1. An isolated antibody or antibody fragment comprising:

2. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a light chain variable sequence as set forth in SEQ ID NO:

4.

3. 3. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a heavy chain variable sequence as set forth in SEQ ID NO:

3.

4. 4. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a heavy chain variable sequence shown in SEQ ID NO: 3 and a light chain variable sequence shown in SEQ ID NO:

4.

5. 5. The isolated antibody or antibody fragment of any one of claims 1 to 4, wherein the antibody fragment is a monovalent scFv (single chain fragment variable) antibody, a bivalent scFv, a Fab fragment, a F(ab')2 fragment, a F(ab')3 fragment, an Fv fragment, or a single chain antibody.

6. 6. The isolated antibody or antibody fragment of any one of claims 1 to 5, wherein the antibody is a chimeric antibody, a bispecific antibody, a trispecific or other multispecific antibody, or a BiTE.

7. 7. The isolated antibody or antibody fragment of any one of claims 1 to 6, wherein the antibody is an IgG antibody, or a recombinant IgG antibody, or an antibody fragment.

8. The antibody or antibody fragment a) increased binding affinity of B7-H3 to the 4Ig isoform, and b) at least a 350-fold increased selectivity for the 4Ig isoform of B7-H3 compared to the 2Ig isoform 7. The isolated antibody or antibody fragment of any one of claims 1 to 6, having the following structure:

9. 9. The isolated antibody or antibody fragment of any one of claims 1 to 8, wherein the antibody is conjugated or fused to an imaging agent, a cytotoxic agent, a metal, or a radioactive moiety.

10. 10. The isolated antibody or antibody fragment of claim 9, wherein the imaging agent is a fluorophore.

11. 10. The isolated antibody or antibody fragment of claim 9, wherein the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, Tb-161, At-211, Ac-225, or Pb-212.

12. 9. The isolated antibody or antibody fragment of any one of claims 1 to 8, wherein the antibody is an immunoconjugate or a radioimmunoconjugate.

13. 13. The isolated antibody or antibody fragment of claim 12, wherein the antibody is conjugated to flagellin or a flagellin derivative.

14. 9. The isolated antibody or antibody fragment of any one of claims 1 to 8, wherein the antibody is an antibody-drug conjugate.

15. An isolated nucleic acid encoding the antibody heavy and / or light chain variable region of the antibody or antibody fragment of any one of claims 1 to 8.

16. An expression vector comprising the nucleic acid of claim 15.

17. A hybridoma or engineered cell comprising nucleic acid encoding the antibody or antibody fragment of any one of claims 1 to 8.

18. A hybridoma or engineered cell comprising the nucleic acid of claim 15.

19. 21. A method for producing an isolated antibody or antibody fragment of any one of claims 1 to 8, comprising culturing a hybridoma or engineered cell of claim 19 or 20 under conditions that allow expression of the antibody, and optionally isolating the antibody from the culture.

20. a heavy chain variable region (VH) having at least 90% identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and comprising a VHCDR1 amino acid sequence comprising SEQ ID NO:9, 15, or 21, a VHCDR2 amino acid sequence comprising SEQ ID NO:10, 16, or 22, and a VHCDR3 amino acid sequence comprising SEQ ID NO:11, 17, or 23; and A light chain variable region (VL) having at least 90% identity to SEQ ID NO:4, comprising a VLCDR1 amino acid sequence comprising SEQ ID NO:12, 18, or 24, a VLCDR2 amino acid sequence comprising SEQ ID NO:13, 19, or 25, and a VLCDR3 amino acid sequence comprising SEQ ID NO:14, 20, or 26, and comprising an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:

4. A chimeric antigen receptor (CAR) protein comprising an antigen-binding domain comprising:

21. The CAR of claim 20, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising VHCDR1, VHCDR2, and VHCDR3 amino acid sequences derived from SEQ ID NO: 3, and having a tryptophan at position 47, a methionine at position 48, a valine at position 68, and an arginine at position 72 of SEQ ID NO: 3; and a light chain variable region (VL) comprising VLCDR1, VLCDR2, and VLCDR3 amino acid sequences derived from SEQ ID NO: 4, and having an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:

4.

22. The CAR of claim 20, wherein the antigen-binding domain comprises: a heavy chain variable sequence having at least 95% identity with SEQ ID NO: 1; and a light chain variable sequence having at least 95% identity with SEQ ID NO: 4 and comprising an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:

4.

23. The CAR of claim 20, wherein the antigen-binding domain comprises a heavy chain variable sequence having at least 95% identity with SEQ ID NO: 2, and a light chain variable sequence having at least 95% identity with SEQ ID NO: 4 and comprising an arginine corresponding to position 45 and a tyrosine corresponding to position 70 of SEQ ID NO:

4.

24. The CAR of claim 20 or 21, wherein the antigen-binding domain comprises a heavy chain variable sequence having the sequence shown in SEQ ID NO: 3 and a light chain variable sequence having the sequence shown in SEQ ID NO:

4.

25. The CAR according to any one of claims 20 to 24, wherein the antigen-binding domain specifically binds to B7-H3.

26. The CAR of any one of claims 20 to 25, wherein the antigen-binding domain is a humanized antigen-binding domain.

27. The CAR of any one of claims 20 to 26, further comprising a hinge domain, a transmembrane domain, and an intracellular signaling domain.

28. The CAR of claim 27, wherein the hinge domain is a CD8a hinge domain or an IgG4 hinge domain.

29. The CAR of claim 27, wherein the transmembrane domain is a CD8a transmembrane domain or a CD28 transmembrane domain.

30. The CAR of claim 27, wherein the intracellular signaling domain comprises a CD3z intracellular signaling domain.

31. A nucleic acid molecule encoding the CAR of any one of claims 20 to 30.

32. 32. The nucleic acid molecule of claim 31 , wherein the sequence encoding the CAR is operably linked to an expression control sequence.

33. 32. The nucleic acid molecule of claim 31 further contained in an expression vector.

34. 31. An engineered cell comprising a nucleic acid molecule encoding the chimeric antigen receptor (CAR) of any one of claims 20 to 30.

35. 35. The cell of claim 34, which is a T cell.

36. The cell of claim 34, which is a NK cell.

37. 35. The cell of claim 34, wherein the nucleic acid is integrated into the genome of the cell.

38. 38. The cell of claim 37, which is a human cell.

39. 39. A pharmaceutical composition comprising a population of cells according to any one of claims 34 to 38 in a pharmaceutically acceptable carrier.

40. 40. A method of treating cancer in a human patient in need thereof, comprising administering to the patient an anti-tumor effective amount of a cell therapy comprising one or more cells of any one of claims 34 to 39.

41. 41. The method of claim 40, wherein the cells are allogeneic cells.

42. 41. The method of claim 40, wherein the cells are autologous cells.

43. 41. The method of claim 40, wherein the cells are HLA-matched to the human subject.

44. 41. The method of claim 40, wherein the cancer is determined to express elevated levels of B7-H3 compared to healthy tissue.

45. 41. The method of claim 40, wherein the cancer is renal cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, melanoma, prostate cancer, breast cancer, glioma, lymphoma, or neuroectodermal cancer.

46. 41. The method of claim 40, wherein the patient has previously failed to respond to an immune checkpoint inhibitor.

47. 41. The method of claim 40, wherein the patient is relapsing.

48. 41. The method of claim 40, further comprising administering at least a second anti-cancer therapy.

49. 49. The method of claim 48, wherein the second anticancer therapy is chemotherapy, molecular targeted therapy, immunotherapy, radiation therapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormone therapy, epigenetic modulation, antiangiogenic therapy, or cytokine therapy.

50. A pharmaceutical composition comprising the isolated antibody or antibody fragment of any one of claims 1 to 14.

51. 51. A method of treating cancer in a human patient in need thereof, comprising administering to the patient an anti-tumor effective amount of the pharmaceutical composition of claim 50.

52. 52. The method of claim 51, wherein the cancer is determined to express elevated levels of B7-H3 compared to healthy tissue.

53. 53. The method of claim 51 or 52, wherein the cancer is renal cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, melanoma, prostate cancer, breast cancer, glioma, lymphoma, or neuroectodermal cancer.

54. 54. The method of any one of claims 51 to 53, wherein the patient has previously failed to respond to an immune checkpoint inhibitor.

55. 55. The method of any one of claims 51 to 54, wherein the patient is relapsing.

56. 56. The method of any one of claims 51 to 55, further comprising administering at least a second anti-cancer therapy.

57. 57. The method of claim 56, wherein the second anti-cancer therapy is chemotherapy, molecular targeted therapy, immunotherapy, radiation therapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormone therapy, epigenetic modulation, anti-angiogenic therapy, or cytokine therapy.

58. 1. A method for diagnosing a patient with cancer, comprising: Detecting and optionally quantifying B7-H3 expression in cancer using the isolated antibody or antigen-binding fragment of any one of claims 1 to 14. A method comprising:

59. 59. The method of claim 58, wherein the isolated antibody or antigen-binding fragment is conjugated to a bioluminescent or chemiluminescent label, a metal, or a radioisotope.

60. 59. The method of claim 58, wherein expression of the 4Ig isoform of B7-H3 is detected.

61. 61. The method of claim 60, further comprising selecting the patient for treatment if the 4Ig isoform of B7-H3 is expressed in the cancer.

62. 40. A method of treating an inflammatory condition in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of a cell therapy comprising one or more cells of any one of claims 34 to 39.

63. 63. The method of claim 62, wherein the cells are allogeneic cells.

64. 63. The method of claim 62, wherein the cells are autologous cells.

65. 63. The method of claim 62, wherein the cells are HLA-matched to the human subject.

66. 63. The method of claim 62, wherein the inflammatory condition is determined to express elevated levels of B7-H3 compared to healthy tissue.

67. 51. A method of treating an inflammatory condition in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 50.

68. 68. The method of claim 67, wherein cells in the area of ​​inflammation are determined to express elevated levels of B7-H3 compared to healthy tissue.

69. 69. The method of any one of claims 62 to 68, wherein the inflammatory condition is selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren's syndrome, dermatitis, type 1 diabetes, type 2 diabetes, thyroiditis, Addison's disease, pernicious anemia, autoimmune hepatitis, inflammatory bowel disease, multiple sclerosis, encephalitis, rheumatoid arthritis, myasthenia gravis, neuritis, primary biliary cholangitis, Goodpasture's disease, primary membranous nephropathy, cystitis, ovarian failure, autoimmune orchitis, chronic obstructive pulmonary disease (COPD), asthma, pneumonia, hypertension, heart disease, myositis, myocarditis, inflammatory arteritis (Takayasu's arteritis, giant cell arteritis), lymphangitis, Parkinson's disease, or graft versus host disease.

70. 69. The method of any one of claims 62 to 68, wherein the inflammatory condition is caused by a pathogenic infection, an environmental chemical, or radiation.

71. 71. The method of any one of claims 62 to 70, further comprising administering at least a second anti-inflammatory therapy.

72. 72. The method of claim 71, wherein the second anti-inflammatory therapy comprises a corticosteroid, a disease-modifying antirheumatic drug (DMARD), or an anti-cytokine therapy.

Images