Anti-B7-H3 compounds and methods of use

Abstract

The anti-B7-H3 compound comprises an anti-B7-H3 Gp2-based scaffold. The anti-B7-H3 compound may comprise one or more functional moieties, such as a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug. The composition comprising anti-B7-H3 may be administered to a subject. The subject may have or be suspected of having a tumor or cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 302,296, filed January 24, 2022, which is incorporated by reference in its entirety.

[0002] government funding This invention was made with Government support under EB023339 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing This application contains a sequence listing that has been submitted electronically to the United States Patent and Trademark Office via the Patent Center as an XML file entitled 0110_000693WO01, having a size of 42.5 kilobytes and created on January 1, 2023. By filing the sequence listing in electronic format, the sequence listing submitted in electronic format serves as both the paper copy required by 37 CFR § 1.821(c) and the CRF required by § 1.821(e). The information contained in the sequence listing is incorporated herein by reference. Summary of the Invention

[0004] In one aspect, the present disclosure describes an anti-B7-H3 compound. The anti-B7-H3 compound comprises an anti-B7-H3 Gp2-based scaffold. The anti-B7-H3 Gp2-based scaffold comprises an amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:3, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

[0005] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold comprises the amino acid sequence of SEQ ID NO: 4, an amino acid sequence having at least 90% sequence similarity to SEQ ID NO: 4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4. In one or more embodiments, the anti-B7-H3 Gp2-based scaffold comprises any one of the amino acid sequences of SEQ ID NO: 5-SEQ ID NO: 23.

[0006] In one or more embodiments, the anti-B7-H3 compound comprises a first functional moiety operably linked to an anti-B7-H3 Gp2-based scaffold. In some embodiments, the first functional moiety comprises a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug. In one or more embodiments, the targeting moiety binds to an extracellular domain of a protein presented on natural killer cells or T cells. In one or more embodiments, the imaging moiety comprises a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label. In one or more embodiments, the enzymatic label comprises sortase A. In one or more embodiments, the small molecule drug comprises one or more radioisotopes.

[0007] In one or more embodiments, the anti-B7-H3 compound may include a second functional component operably linked to the anti-B7-H3 Gp2-based scaffold. In one or more embodiments, the second functional component includes a targeting component, an imaging component, an enzyme, or a small molecule drug. In one or more embodiments, the first functional component is directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional component is directly linked to the anti-B7-H3 Gp2-based scaffold. In one or more embodiments, the first functional component is directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional component is directly linked to the first functional component.

[0008] In another aspect, the disclosure describes a composition comprising an anti-B7-H3 compound and a pharma- ceutically acceptable carrier.

[0009] In another aspect, the disclosure describes a method comprising administering to a subject a composition comprising an anti-B7-H3 compound. In one or more embodiments, the subject has a tumor. In one or more embodiments, the subject has cancer. In one or more embodiments, the composition is administered prior to, concurrently with, or after chemotherapy, surgical resection of the tumor, or radiation therapy. In one or more embodiments, the method further comprises detecting the anti-B7-H3 compound bound to B7-H3 expressed by the cell by detecting an imaging moiety. In one or more embodiments, the cell is a cancer cell.

[0010] The above summary is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The following description more particularly illustrates illustrative embodiments. In several places in this disclosure, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0011] [Figure 1]Enriched Gp2 binders against B7-H3 (anti-B7-H3 Gp2-based scaffold) (A). Yeast were subjected to three rounds of magnetic activated cell sorting (MACS) selection using soluble B7-H3 extracellular domain (populations 0.1, 0.2, and 0.3 on the x-axis) to show enrichment for the B7-H3 target over negative Renilla reniformis green fluorescent protein (rrGFP) and avidin beads. The enriched Gp2 population was subjected to FACS sorting against B7-H3 (yielding population 0.4 on the x-axis). Binding to avidin beads and negative rrGFP was not analyzed in this sort, so ND indicates no data. 0.4 Gp2 library was extracted, subjected to error-prone PCR, and then electroporated back into yeast. The mutant naive library was subjected to MACS selection (population 1.1 on the x-axis) followed by monovalent MACS selection with 100 nM recombinant human B7-H3 (population 1.2 on the x-axis). The remaining selections (to obtain populations 1.3 and 1.4) were performed by fluorescence-activated cell sorting (FACS). (B) After three rounds of MACS selection, yeast were incubated with 50 nM recombinant human B7-H3 extracellular domain followed by streptavidin-Alex Fluor 647. Gp2 display was quantified using mouse anti-c-Myc antibody and goat anti-mouse-Alexa Fluor 488. Flow cytometric analysis of 10,000 random variants is shown (population 0.4). (C) The enriched 1.3 population was labeled with ~1 nM biotin-B7-H3 cell lysate and streptavidin-AF647 and chicken anti-c-Myc-FITC, and the top 0.7% was selected to obtain the 1.4 population. Boxes in (B) and (C) are cells collected during FACS sorting.

[0012] [Diagram 2]Sequences of B7-H3 binding Gp2-based scaffolds discovered and evolved (0.4.2=SEQ ID NO:1; 1.4.4=SEQ ID NO:4; 0.4.1=SEQ ID NO:5, 1.4.1=SEQ ID NO:6; 1.4.2=SEQ ID NO:7; and 1.4.3=SEQ ID NO:8). The truncated wild-type Gp2 sequence (SEQ ID NO:1) and Gp2 library design are shown for reference. Eight colonies were sequenced in the 0.4 population, with 0.4.1 (SEQ ID NO:5) observed 7 times (indicated by "x7"). Eight colonies were sequenced in the 1.4 population (1.4.1, 1.4.2, 1.4.3, 1.4.4), with 1.4.1 observed 5 times (indicated by "5x"). Amino acid diversity indicates variants with sequence families similar to the 0.4.1 sequence (SEQ ID NO:5). Circled amino acids indicate differences between variant sequences.

[0013] [Diagram 3] Affinity characterization of B7-H3 binding Gp2 scaffold and Gp2 fusion constructs. (A) Gp2 1.4.3 (SEQ ID NO: 8) and (B) Gp2 1.4.4 (SEQ ID NO: 4) were characterized for their binding affinity using flow cytometry. Gp2 1.4.3 and Gp2 1.4.4 showed high affinity binding to MS1-B7-H3 cells with Kd values ​​of 3.4 nM (2.7 nM-4.2 nM) and 1.0 nM (0.8 nM-1.3 nM), respectively. (C) Gp2 1.4.4 was genetically linked to the GSGGGGSGGGKGGGGT linker (SEQ ID NO: 24) and residues 60-206 of sortase A and produced in bacteria. Gp2 1.4.4-L15-SrtA showed comparable binding to Gp2 1.4.4 (SEQ ID NO: 4) with a Kd of 1.8 nM (1.4 nM-2.2 nM). Error bars indicate standard error and at least two replicates were performed for each concentration.

[0014] [Figure 4]Amino acid sequences of top performers after mutation of the 1.4 population by error-prone PCR, avidity selection, affinity selection, specificity selection, and stability selection. The sequence space shows a relatively constrained diversity in the 1.4Gp2-based scaffold population compared to the 1.4.4Gp2-based scaffold (see FIG. 2). Common motifs such as Q34R and T41M emerge.

[0015] [Diagram 5] Characterization of the top variants (sequences shown in Figure 4). Sequences are ranked from largest to smallest conditional weighted average z-score. Values ​​greater than 1 indicate enrichment and values ​​less than 1 indicate depletion. Color bars for affinity enrichment range from 1 to M+2SD since the mean (M) was slightly less than 1. Color bars for specificity, stability, and frequency are represented as M to M+2SD. z-scores are represented from 1 to 10.

[0016] [Figure 6] Relative frequency of top affibodies after each selection (avidity selection, affinity selection, specificity selection, and stability selection). Each line represents a single variant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure describes anti-B7-H3 Gp2-based scaffolds, compounds comprising at least one of the anti-B7-H3 Gp2-based scaffolds, and methods of using such compounds. As used herein, the term "anti-B7-H3 compound" refers to a compound that comprises or is an anti-B7-H3 Gp2-based scaffold. Exemplary platforms in which anti-B7-H3 compounds may be used include, but are not limited to, chimeric antigen receptor therapy (e.g., CAR-NK therapy, CAR-T therapy, CAR-macrophage therapy, etc.), multispecific immune cell engager technologies (e.g., bispecific killer engagers, trispecific killer engagers, bispecific T cell engagers, trispecific T cell engagers, etc.), targeted immunotherapy (e.g., targeted ADAM17 blocker (TAB) therapy), delivery of therapeutic agents (e.g., antibody-drug conjugates, delivery of therapeutic radioisotopes, delivery of toxins, delivery of cytokines, delivery of chemokines), imaging technologies (delivery of labeled constructs and / or labeled radioisotopes), and cell and / or ligand capture technologies (e.g., ELISA, etc.).

[0018] B7 homolog 3 (B7-H3), also known as cluster of differentiation 276 (CD276), is a human protein encoded by the CD276 gene. B7-H3 protein is a 316 amino acid long type I transmembrane protein that exists in two isoforms determined by its extracellular domain. B7-H3 mRNA is expressed in most normal tissues. In contrast, B7-H3 protein has very limited expression on normal tissues due to its post-transcriptional regulation by microRNAs. In normal tissues, B7-H3 plays a mainly inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. Thus, B7-H3 is a key immune checkpoint inhibitor of T cell function and a tumor vasculature biomarker, and is overexpressed in a variety of cancers, including clear cell renal cell carcinoma, cutaneous melanoma, diffuse intrinsic pontine glioma, hypopharyngeal squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, prostate cancer, and pancreatic cancer. Expression of B7-H3 is associated with tumor growth and metastasis and may ultimately lead to poor clinical prognosis.

[0019] Molecules that recognize specific targets with specificity and high affinity are useful for many clinical (e.g., diagnostic and / or therapeutic) and biotechnological applications. Typically, antibodies have been used for many of these applications, but antibodies have certain properties that may be disadvantageous in some applications. The limitations of antibodies have prompted research into alternative protein scaffolds that allow for the efficient generation of improved binding molecules. In the context of targeting solid tumors, for example, antibodies (typically about 150 kDa for immunoglobulin G (IgG)) may exhibit poor leakage from the vasculature, poor tissue penetration, and / or long plasma clearance half-life, due at least in part to their size, which may result in poor signal-to-noise ratios, especially in diagnostic imaging. Antibodies may also exhibit thermolability, which may lead to reduced efficacy as a result of denaturation and / or aggregation. In addition, antibodies are typically produced in mammalian culture, as many have disulfide bonds, glycosylation, and / or multi-domain structures. This complex structure can hinder engineering antibodies for certain applications, such as the production of protein fusions for multispecific formats. Furthermore, the presence of disulfide bonds in antibody molecules often prevents their intracellular use.

[0020] Because antibodies have inherent limitations, alternative protein scaffolds have been developed to attempt to address many or all of these shortcomings. The present disclosure describes a recombinant non-natural protein scaffold, termed the anti-B7-H3 Gp2-based scaffold, that can bind to the extracellular domain of B7-H3. In particular, the anti-B7-H3 Gp2-based scaffold described herein can be used to present defined loops that resemble the complementarity determining regions ("CDRs") of antibody variable regions. The anti-B7-H3 Gp2-based scaffold can be assembled into multispecific compounds that can bind to the extracellular domain of B7-H3 and one or more different targets. Thus, the anti-B7-H3 Gp2-based scaffold described herein can provide functional properties typically associated with antibody molecules. In particular, although the anti-B7-H3 Gp2-based scaffold is not an immunoglobulin, its overall folding is similar in that it is related to the folding of the variable region of an IgG heavy chain, allowing the protein scaffold to present loops in a relative orientation similar to that of antibody CDRs. Because of this structure, the anti-B7-H3 Gp2-based scaffold described herein can have ligand binding properties similar in nature and affinity to those of antigens and antibodies.

[0021] In one aspect, the present disclosure describes anti-B7-H3 compounds. The anti-B7-H3 compounds include anti-B7-H3 Gp2-based scaffolds. The anti-B7-H3 Gp2-based scaffolds of the present disclosure bind to the extracellular domain of B7-H3 protein (hereinafter, B7-H3). The anti-B7-H3 Gp2-based scaffolds can be developed from Gp2 (SEQ ID NO: 1), a truncated version of T7 phage gene 2 protein.

[0022] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises the amino acids KFWXTVXXXXXXXXFEXPXYAXTXDEALXLA XXXYXXXXXXXXVXXVXP (SEQ ID NO: 3), or a structurally similar sequence, where each X is a natural, non-natural, or modified amino acid, independent of the amino acids at any other position, independent of the amino acids at any other position of the Gp2 scaffold. The Gp2-based scaffold comprises the amino acid sequence of SEQ ID NO:3, wherein X at position 4 is A or P; X at position 7 is Q, C, R, or E; X at position 8 is H, Y, or S; X at position 9 is V or S; X at position 10 is any amino acid such as Y or H, or is a deletion; X at position 11 is any amino acid such as S, or is a deletion; X at position 12 is D, E, I, or N; X at position 13 is F, H, or G; X at position 14 is G, S, or C; X at position 17 is A or V; X at position 19 can be V or I; X at position 22 is A or E; X can be L or M; X at position 29 is Q, E, R, or L; X at position 32 is E or K; X at position 33 is V, A, or W; X at position 34 can be Q, R, or K; X at position 36 is A, V, or G; X at position 37 is N, S, P, or D; X at position 38 is S or A; X at position 39 can be an optional addition such as Y; X at position 40 can be an optional addition; X at position 41 is I, G, V; X at position 42 is F or M; X at position 43 is T, M, K, D, or E; X at position 45 is T or A; X at position 46 is R or S; and X at position 49 is R or H.

[0023] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises the amino acid sequence KFWATVQHVYSDFGFEAPVYAATLDEALQLAEVQYANSIFTVTRVRP (SEQ ID NO:4), which is a derivative of SEQ ID NO:3 (X at position 7 is Q; X at position 8 is H; X at position 9 is V; X at position 10 is Y; X at position 11 is S; X at position 12 is D; X at position 13 is F; X at position 14 is G; X at position 17 is A; X at position 19 is V; X at position 22 is A; X at position 24 is L; X at position 29 is Q; X at position 32 is G; X at position 33 is H; X at position 34 is H; X at position 35 is H; X at position 36 is H; X at position 37 is H; X at position 38 is H; X at position 39 is H; X at position 40 is H; X at position 41 is H; X at position 42 is H; X at position 43 is H; X at position 44 is H; X at position 45 is H; X at position 46 is H; X at position 47 is H; X at position 48 is H; X at position 49 is H; X at position 50 is H; X at position 51 is H; X at position 52 is H; X at position 53 is H; X at position 54 is H; X at position 55 is H; X at position 56 is H; X at position 57 is H; X at position 58 is H; X at position 59 is H; X at position 60 is H; X at position 61 is H; X at position 62 is H; X at position 63 is H; X at position 64 is H; X at position 65 is H; X at position 66 is H; X at (X is E; at position 33, X is V; at position 34, X is Q; at position 35, X is Y; at position 36, X is A; at position 37, X is N; at position 38, X is S; at position 39, X is no amino acid; at position 40, X is no amino acid; at position 41, X is I; at position 42, X is F; at position 43, X is T; at position 45, X is T; at position 46, X is R; and at position 48, X is R). In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises an amino acid sequence of SEQ ID NO:4 (1.4.4) or a structurally similar sequence. In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises an amino acid sequence that is a derivative of SEQ ID NO:4. In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises the amino acids of SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23, or structurally similar sequences thereof (derivatives of SEQ ID NO:4).

[0024] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises the amino acid sequence of SEQ ID NO:2, or a structurally similar sequence.

[0025] As used herein, a polypeptide (e.g., an anti-B7-H3 Gp2-based scaffold) is "structurally similar" to a reference polypeptide (e.g., an anti-B7-H3 Gp2-based scaffold comprising SEQ ID NO: 4) if the amino acid sequence of the polypeptide has a certain amount of identity compared to the reference polypeptide. The structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (e.g., a candidate polypeptide and a polypeptide such as SEQ ID NO: 4) to optimize the number of identical amino acids over the length of their sequences. In aligning to optimize the number of identical amino acids, gaps in one or both sequences are allowed, but the amino acids in each sequence must still maintain their proper order. A candidate polypeptide is a polypeptide that is compared to a reference polypeptide. A candidate polypeptide may, for example, be isolated from an animal or other natural source, or may be produced using recombinant technology, or may be chemically or enzymatically synthesized.

[0026] Pairwise comparison analysis of amino acid sequences can be performed, for example, using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, polypeptides and proteins can be compared using the Blastp program of the BLAST2 search algorithm described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)) and available at the National Center for Biotechnology Information (NCBI) website. Default values ​​can be used for all BLAST2 search parameters, including matrix=BLOSUM62; open gap penalty=11, extended gap penalty=1, gapx_dropoff=50, expectation=10, word size=3, and filter on.

[0027] In comparing two amino acid sequences, structural similarity can be referred to by percent "identity" or by percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids, but also to the presence of conservative substitutions. Conservative substitutions for an amino acid in SEQ ID NO: 4 polypeptide can be selected from other members of the class to which the amino acid belongs. For example, in the field of protein biochemistry, it is well known that an amino acid belonging to a group of amino acids with a particular size or property (e.g., charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without changing the activity of the protein, especially in regions of the protein that are not directly related to biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, substitution of Lys with Arg and vice versa to maintain a positive charge; substitution of Glu with Asp and vice versa to maintain a negative charge; substitution of Ser with Thr to maintain a free -OH; and substitution of Gln with Asn to maintain a free -NH2. Similarly, biologically active analogs of polypeptides containing one or more consecutive or non-consecutive amino acid deletions or additions that do not eliminate the functional activity of the polypeptide are also contemplated.

[0028] In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds described herein can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3, where each X is independently selected from any amino acid. The Gp2-based scaffold may have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3, where X at position 4 is A or P; X at position 7 is Q, C, R, or E; X at position 8 is H, Y, or S; X at position 9 is V or S; X at position 10 is any amino acid such as Y or H, or is a deletion; X at position 11 is any amino acid such as S, or is a deletion; X at position 12 is D, E, I, or N; X at position 13 is F, H, or G; X at position 14 is G, X at position 17 is A or V; X at position 19 can be V or I; X at position 22 is A or E; X at position 24 can be L or M; X at position 29 is Q, E, R, or L; X at position 32 is E or K; X at position 33 is V, A, or W; X at position 34 can be Q, R, or K; X at position 36 is A, V, or G; X at position 37 is N, S, P, or D; X at position 39 is S or A; X at position 39 can be the optional addition or deletion of any amino acid, such as Y; X at position 40 can be the optional addition or deletion of any amino acid; X at position 41 is I, G, V; X at position 42 is F or M; X at position 43 is T, M, K, D, or E; X at position 45 is T or A; X at position 46 is R or S; and X at position 49 is R or H.

[0029] The anti-B7-H3 Gp2-based scaffolds described herein may have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6-SEQ ID NO:23. When determining sequence identity, the total number of amino acids in the sequence is multiplied by the decimal form of the desired percent identity. The resulting number is subtracted from the total number of amino acids in the sequence to reveal the total number of amino acids that may have a different identity from the original sequence. If the total number of amino acids that may have a different identity is not an integer, the number is rounded up (if the decimal is 0.5 or more) or down (if the decimal is less than 0.5) to obtain the total number of amino acids that may differ from the original sequence. For example, an amino acid sequence having 90% or more sequence identity to SEQ ID NO:4 may have 5 amino acids that differ from SEQ ID NO:4 (47-(0.9 47)=4.7, then rounded to 5). Each of SEQ ID NOs:5 through 23 has 90% or more sequence identity to SEQ ID NO:4.

[0030] The anti-B7-H3 Gp2-based scaffolds described herein can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6-SEQ ID NO:23.

[0031] In one or more embodiments, amino acids may be mutated in the loop region of any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23. The mutation may include substitution of one or more amino acid residues with different amino acid residues, deletion of one or more amino acid residues, or addition of one or more residues to the amino acid sequence of any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23.

[0032] For example, in one or more embodiments, there may be one or more mutations in the first loop region, i.e., amino acid residues 7-14 of SEQ ID NO:3 to SEQ ID NO:23 and amino acid residues 7-12 of SEQ ID NO:2. The first loop region of the original sequence (SEQ ID NO:1) is 7-12 amino acid residues, whereas SEQ ID NO:3 to SEQ ID NO:23 contain an insertion of two amino acids in the first loop region (see Table 1). In one or more embodiments, there may be one or more mutations in the second loop region: amino acid residues 36-43 of SEQ ID NO:3, amino acid residues 36-41 of SEQ ID NO:4 to SEQ ID NO:23, or amino acid residues 36-40 of SEQ ID NO:2. In one or more embodiments, there may be one or more mutations in amino acid residues in the first loop region and / or amino acids in the second loop region.

[0033] In one or more embodiments, one or more amino acid residues may be inserted into the amino acid sequence of any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23. In one or more embodiments, one or more amino acid residues may be inserted into the first loop region and / or the second loop region of any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23, thereby extending the loop region by the number of inserted amino acid residues. For example, in one or more embodiments, one or more amino acid residues may be inserted into the C-terminal side of one or more of residues 7 to 14 of SEQ ID NO:3 to SEQ ID NO:23 or amino acid residues 7 to 12 of SEQ ID NO:2. In one or more embodiments, one or more amino acid residues may be inserted into the C-terminal side of one or more of residues 36 to 43 of SEQ ID NO:3, amino acid residues 36 to 41 of SEQ ID NO:4 to SEQ ID NO:23, or amino acid residues 36 to 40 of SEQ ID NO:2. For example, in one or more embodiments, one or more amino acid residues may be inserted before residue 37 on the C-terminal side of residue 36 of SEQ ID NO:5 (0.4.1) (e.g., residues 36a and / or 36b in FIG. 2).

[0034] In one or more embodiments, one or more amino acid residues may be removed from any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23. In one or more embodiments, one or more amino acid residues may be removed from the first loop region and / or the second loop region of any one of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23, shortening the loop region by the number of removed amino acids. For example, in one or more embodiments, one or more amino acid residues may be removed from residues 7 to 14 of SEQ ID NO:3 to SEQ ID NO:23, or from amino acid residues 7 to 12 of SEQ ID NO:2. For example, in one or more embodiments, amino acid residues 10 and / or 11 of SEQ ID NO:4 are removed. In one or more embodiments, one or more amino acid residues may be removed from amino acid residues 36 to 43 of SEQ ID NO:3, amino acid residues 36 to 41 of SEQ ID NO:4 to SEQ ID NO:23, or amino acid residues 36 to 40 of SEQ ID NO:2.

[0035] In one or more embodiments, one or more amino acid residues outside the loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23 may be mutated with any amino acid. For example, in one or more embodiments, one or more mutations may be present in amino acid residues 1 to 6, 13 to 33, or 41 to 46 of SEQ ID NO:2; amino acid residues 1 to 6, 15 to 35, or 44 to 39 of SEQ ID NO:3; or amino acid residues 1 to 6, 15 to 35, or 44 to 47 of any one of SEQ ID NO:4 to SEQ ID NO:23. In one or more embodiments, one or more mutations may be present outside the loop region. In one or more embodiments, one or more amino acid residues in the above-listed regions (regions other than the loop region) may be mutated.

[0036] In one or more embodiments, one or more amino acid residues within a loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6-23 may be mutated, and one or more amino acid residues outside the loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6-23 may be mutated. In one or more embodiments, one or more amino acid residues within a loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6-23 may be mutated, and one or more amino acid residues outside the loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6-23 may be mutated. In one or more embodiments, one or more amino acid residues within the loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23 may be mutated, one or more amino acid residues may be inserted within the loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23, and one or more amino acid residues outside the loop region of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6 to SEQ ID NO:23 may be mutated.

[0037] Table 1 shows the amino acid sequence of the parent Gp2 from which the anti-B7-H3 Gp2-based scaffold is derived (SEQ ID NO:1), the general consensus sequence (SEQ ID NO:3), and the more specific consensus sequence (SEQ ID NO:4). Table 1 also shows examples of the identities of X in the general consensus sequence and examples of mutations in SEQ ID NO:4. In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure comprises any one of SEQ ID NO:3 or SEQ ID NO:4 with any combination of the exemplary possible mutations or identities of X shown in Table 1. [Table 1]

[0038] Variants of the disclosed sequences also include anti-B7-H3 Gp2-based scaffold fragments or full-length anti-B7-H3 Gp2-based scaffolds that contain substitutions, deletions, or insertions into the protein backbone and still maintain at least about 70% homology over the corresponding portions to the original anti-B7-H3 Gp2-based scaffold (e.g., SEQ ID NO: 3 and SEQ ID NO: 4). Larger deviations from homology are permitted where similar amino acids, i.e., conservative amino acid substitutions, are not counted as changes in the sequence. Examples of conservative substitutions include amino acids with the same or similar properties. Exemplary conservative amino acid substitutions include: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid or asparagine; cysteine ​​to serine; glutamine to asparagine or glutamic acid; glutamic acid to aspartic acid or glutamine; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine or alanine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

[0039] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure may include additional sequences, such as, for example, amino acids added to the C-terminus or N-terminus of the anti-B7-H3 Gp2-based scaffold. Such modifications may, for example, facilitate purification by capture on a column, use of an antibody, or facilitate recovery when recombinantly expressed in a microorganism. Such tags include, for example, a histidine-rich tag that allows purification of the protein on a nickel column, and / or a leader sequence that may transport the recombinantly expressed anti-B7-H3 Gp2-based scaffold to the membrane of the cell in which it is recombinantly expressed. Such genetic modification techniques and suitable additional sequences are well known in the field of molecular biology. In one or more embodiments, the C-terminus and / or N-terminus modifications may be cleaved from the anti-B7-H3 Gp2-based scaffold prior to incorporation, for example, prior to incorporation into a pharmaceutical composition. In other embodiments, retaining the C-terminus or N-terminus modifications may be desirable for a given application, for example, to facilitate immobilization to a substrate.

[0040] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold described herein may also include N- or C-terminal functional groups other than carboxylic acid or free amine. For example, the C-terminus, N-terminus, or both of the anti-B7-H3 Gp2-based scaffold may be acylated, e.g., acetylated. In one or more embodiments, the functional group may include a polyol (e.g., polyethylene glycol).

[0041] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold described herein may include post-translational modifications to one or more amino acids. Examples of post-translational modifications include, but are not limited to, acetylation, methylation, glycosylation, phosphorylation, prenylation, sulfonation, palmitoylation, hydroxylation, nitration, myristoylation, formylation, or citrullination. Post-translational modifications may be incorporated specifically or non-specifically by methods known in the biological arts.

[0042] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold can be recombinantly produced in a suitable host cell and then purified. Methods for recombinant production and purification of proteins are well known in the art.

[0043] In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure can be produced using solid-phase peptide synthesis. In this production method, the anti-B7-H3 Gp2-based scaffold can be synthesized as one complete polypeptide or can be synthesized in multiple polypeptide fragments that can be linked using native chemical ligation, expressed protein ligation, Staudinger ligation, or Ser / Thr ligation. Methods for solid-phase peptide synthesis and ligation techniques are well known in the art.

[0044] In another aspect, the present disclosure describes polynucleotides encoding any of the anti-B7-H3 Gp2-based scaffolds described herein, and the complements of such polynucleotide sequences. Given the amino acid sequence of any of the anti-B7-H3 Gp2-based scaffolds described herein, one of skill in the art can use conventional routine methods to determine the full range of polynucleotides that encode that amino acid sequence.

[0045] The anti-B7-H3 Gp2-based scaffolds of the present disclosure can be engineered using a variety of techniques and assays, for example, magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) can be used to select scaffolds of interest that bind to B7-H3 in a yeast display model.

[0046] The following describes the campaign used to engineer at least some of the anti-B7-H3 Gp2-based scaffolds of the present disclosure (see Examples 1 and 2 for further details). A Gp2 scaffold library containing the Gp2 amino acid sequence from SEQ ID NO:1 was used to discover binders to B7-H3 by magnetic and fluorescence-activated cell sorting. The initial library (0.0 Gp2-based scaffold population, alternatively referred to as the library) was subjected to three rounds of MACS selection using recombinant human B7-H3 extracellular domain immobilized on magnetic beads (to obtain 0.1, 0.2, and 0.3 Gp2-based scaffold populations), followed by a single FACS sort using 50 nM of the same recombinant B7-H3 protein (to obtain a 0.4 Gp2-based population), gating to select Gp2-based scaffolds with intermediate affinity. DNA was isolated from the 0.4 Gp2 population (library), randomly mutated by error-prone PCR, and electroporated back into yeast. The mutated naive population was subjected to MACS selection (to obtain the 1.1 Gp2-based scaffold population) followed by monovalent MACS sorting with 100 nM recombinant human B7-H3 (to obtain the 1.2 Gp2-based scaffold population). The Gp2 population was then further enriched by two rounds of FACS selection using detergent-solubilized MS1-B7-H3 lysate at increasing stringency with estimated B7-H3 lysate concentrations of 50 nM and 1 nM (to obtain the 1.3 and 1.4 Gp2-based scaffold populations). Between each selection, yeast was propagated and re-induced.

[0047] FIG. 1A is a plot showing the theoretical diversity of Gp2-based scaffold populations after various sorts (0.4 is the 0.4 population). The higher the theoretical diversity, the more cells were collected for the described parameters (e.g., control, negative, or B7-H3 binders). Population 1.1 had the highest theoretical diversity of cells bound to B7-H3. FIG. 1B is a flow cytometry analysis (FACS) of 10,000 random variants of the 0.4 population. This resulted in Gp2 binders of moderate affinity to recombinant B7-H3. FIG. 1C is a flow cytometry analysis of the enriched 1.4 population resulting from mutation of the 0.4 population (indicated by the gray box) in FIG. 1B and subsequent sorting as described above. FIG. 1C shows the enriched 1.4 population resulting from mutation of the 0.4 population (indicated by the gray box) in FIG. 1B and subsequent sorting as described above. FIG. 1C shows the enriched 1.4 population resulting from mutation of the 0.4 population (indicated by the gray box) in FIG. 1B and subsequent sorting as described above. This resulted in high affinity binders of cells to B7-H3. The boxes in Figures 1B and 1C show the populations collected during FACS sorting. The collected cells express full-length Gp2 and bind B7-H3. Since the majority of the population was non-binding, a rectangle was drawn in Figure 1B to select for binders. In Figure 1C, a polygon with a diagonal line was drawn to be more stringent and collect the top binders with higher affinity.

[0048] Single colonies of the Gp2 population were stochastically selected and Sanger sequenced at two sorting points: after the first FACS sort (fourth round of sorting overall; sequencing of 0.4 library sequences) and after error-prone PCR after all selections (two rounds of MACS selection and two rounds of FACS lysate selection; yielding a 1.4 population). Sequencing results for Gp2 variants (i.e., Gp2-based scaffolds) are shown in Figure 2, where 0.4 is the 0.4 Gp2-based scaffold population and 1.4 is the 1.4 Gp2-based scaffold population (WT (wild type / parent) = SEQ ID NO: 1; 0.4.1 = SEQ ID NO: 5; 0.4.2 = SEQ ID NO: 2; 1.4.1 = SEQ ID NO: 6; 1.4.2 = SEQ ID NO: 7; 1.4.3 = SEQ ID NO: 8; 1.4.4 = SEQ ID NO: 4).

[0049] The selected Gp2 variants were analyzed by binding affinity (K d Affinity titrations of Gp2 1.4.3 (SEQ ID NO: 8) and Gp2 1.4.4 (SEQ ID NO: 4) revealed K values ​​of 3.4 nM (68% confidence interval: 2.7-4.2 nM) and 1.0 (0.8-1.3) nM, respectively. d were obtained (Figures 3A and 3B). Gp2 1.4.3 differs from Gp2 1.4.4 by the Q32R and T39M mutations (Figure 2).

[0050] The 1.4 Gp2-based population was further engineered for affinity, specificity, and stability maturation (see Example 2 for further details). DNA was isolated from the 1.4 yeast population and subjected to random mutagenesis of the variable regions of the affibodies by error-prone polymerase chain reaction (PCR). DNA was electroporated back into yeast for display and subjected to the following selection schemes: (1) lysate MACS with 1 pmol B7-H3+ lysate selection; (2) affinity selection (stringent K d (1) ELISA (FACS sorting at 1.4.4); (2) Specificity sorting (multi-targeted MACS depletion sorting); and (3) Stability sorting (thermolysin FACS sorting at 55°C). After each sort, the population was deep sequenced. Figure 4 shows the sequences of the top performers of the 1.4.4 population after all sorts were completed.

[0051] For each sequence, the frequency at each sort (e.g., avidity, affinity, specificity, and stability) and subsequent enrichment were determined. If the number of reads was 0, a read number of 1 was assigned. Z-scores for affinity, specificity, stability, and final frequency were determined and a final conditional weighted average z-score was calculated (see Example 2). Top performers were selected based on highest scores. Figure 4 shows the top performing sequences of the 1.5.1 population after all sorts were completed.

[0052] Figure 5 shows the z-scores for affinity, specificity, stability, and final frequency of the top 13 variants, as well as the conditional weighted average z-score. Values ​​greater than 1 indicate enrichment and values ​​less than 1 indicate depletion. The color bars in Figure 5 for affinity are enrichment ranges from M to M+2SD, while specificity and stability are represented as 1 to M+2SD since the mean (M) was slightly less than 1. On the bar graphs, z-scores are represented from 1 to 10 (i.e., if the z-score is 10 or greater, the bar spans the full length of the graph).

[0053] Figure 6 is a plot showing the relative frequency of a particular variant (each line) after each sort (avidity, affinity, specificity, and stability). A positive slope indicates enrichment and a negative slope indicates depletion. An ideal variant would be represented by a line with a high relative frequency across all sorts. This analysis allows the identification of notable variants with large post-sort:pre-sort differences.

[0054] In addition to the anti-B7-H3 Gp2-based scaffold, in one or more embodiments, the anti-B7-H3 compound may include a first functional component operably linked to the anti-B7-H3 Gp2-based scaffold. In one or more embodiments, the first functional component may include a targeting component. In one or more embodiments, the first functional component may include an imaging component. In one or more embodiments, the first functional component may include an enzyme. In one or more embodiments, the first functional component may include a small molecule drug, a pharmacologically active derivative thereof, or an activatable inactive form of a drug (e.g., a prodrug).

[0055] In one or more embodiments, the first functional component comprises a targeting component. In embodiments in which the first functional component comprises a targeting component, the anti-B7-H3 compound can be an immunotherapeutic compound. Immunotherapeutic compounds can provide personalized therapy to amplify or reduce immune responses by activating or suppressing the immune system, and are rapidly being developed to treat various forms of cancer. Immunotherapies for cancer, such as chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, and PD-1 and PD-L1 inhibitors, aim to help the subject's immune system fight cancer.

[0056] In one or more embodiments, the targeting moiety may bind to B7-H3 presenting cells and recruit effector cells to the B7-H3 presenting cells. Effector cell types include, but are not limited to, natural killer (NK) cells, B cells, or T cells. The targeting moiety may bind to a surface protein presented on the effector cell. Non-limiting examples of surface proteins presented on NK cells include CD16, PD-1, NKp30, NKp40, NKp44, NKp46, NKG2C, or KIR. Non-limiting examples of surface proteins presented on B cells include CD4, CD8, or VLA-4. Non-limiting examples of surface proteins presented on T cells include (LFA)-1, CD2, CD4, or CD8.

[0057] In one or more embodiments, the targeting moiety may comprise an antibody or fragment thereof, an affibody, a peptide, a protein, or a small molecule.

[0058] In one or more embodiments, the first functional component comprises an imaging moiety. In embodiments in which the first functional component comprises an imaging moiety, the anti-B7-H3 compound may be used as a diagnostic tool to detect B7-H3 positive tumors.

[0059] The imaging moiety can be any moiety that can generate a detectable signal. Exemplary types of imaging moieties include, but are not limited to, colorimetric labels, fluorescent labels, radioactive labels, magnetic labels, or enzymatic labels. Depending on the type of imaging moiety, various methods can be used to detect the imaging moiety. Exemplary detection methods include magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound, photoacoustic imaging, fluorescence microscopy, total internal reflection fluorescence (TIRF)-microscopy, or stimulated emission depletion (STRED) nanoscopy. The imaging technique can be accomplished in vivo or in vitro.

[0060] In one or more embodiments, the first functional component may include an enzyme. In one or more embodiments, the enzyme may modify one or more of the surface proteins displayed on the cells that display B7-H3 to which the anti-B7-H3 Gp2-based scaffold binds. Examples of enzymes that modify surface proteins include, but are not limited to, proteases, lipases, and sortases, such as sortase A.

[0061] To analyze the modularity of the Gp2-based scaffold, Gp2 1.4.4 was genetically linked to a sortase A protein enzyme via a 15 amino acid glycine-rich linker (SEQ ID NO: 24) at the C-terminus of the Gp2-based scaffold, i.e., Gp2-L 15 -SrtA fusion, wherein L 15 is the linker. Modularity was assessed by the retention of B7-H3 binding affinity of the Gp2-based scaffold in the presence of glycine-linked sortase A enzyme. Gp2-L 15 Affinity titration of the -SrtA fusion revealed a K of 1.8 nM (1.4–2.2 nM; Fig. 3C ). d was obtained, which is comparable to Gp2 1.4.4 alone (FIG. 3B). Thus, Gp2 1.4.4 appears to be modular and exhibits high binding affinity in linking other proteins or conjugates.

[0062] In one or more embodiments, the first functional component may include a small molecule drug. As used herein, the term "drug" is used collectively to refer to a pharmacologically active substance or an activatable inactive form (e.g., a prodrug) of a pharmacologically active substance. In embodiments in which the first functional component includes a small molecule drug, the anti-B7-H3 compound may be a chemotherapeutic compound. In one or more embodiments, the chemotherapeutic compound may include one or more radioisotopes. In embodiments in which the chemotherapeutic compound includes one or more radioisotopes, the anti-B7-H3 compound may be used for radioisotope therapy (radionuclide therapy). Examples of suitable isotopes include iodine-131, iridium-192, strontium-89, samarium-153, rhenium-186, boron-10, phosphorus-32, or radium-223. The anti-B7-H3 Gp2-based scaffold allows for the localization of small molecule drugs to B7-H3-presenting cells, such as tumor cells, thereby delivering chemotherapy compounds to tumor cells expressing B7-H3. In this way, chemotherapy compounds can be administered systemically to a subject, but with increased local activity and reduced systemic side effects compared to systemically administering a drug that is not a component of the anti-B7-H3 compound.

[0063] In one or more embodiments, the anti-B7-H3 compound may include additional functional components in addition to the first functional component and the anti-B7-H3 Gp2-based scaffold. For example, the anti-B7-H3 compound may include a second functional component, a third functional component, a fourth functional component, a fifth functional component, a sixth functional component, or a seventh functional component. Each functional component may include an anti-B7-H3 Gp2-based scaffold, an imaging component, an enzyme, a small molecule drug, or a targeting component (all of which are described herein above), regardless of the identity of any other functional components in the anti-B7-H3 compound.

[0064] In some embodiments in which the anti-B7-H3 compound comprises at least one functional component, the anti-B7-H3 Gp2-based scaffold and its functional components are operably linked. As used herein, the term "operably linked" refers to a direct or indirect covalent bond between the anti-B7-H3 Gp2-based scaffold and any additional functional component(s) of the anti-B7-H3 compound. Thus, two operably linked functional components, or one functional component and the anti-B7-H3 Gp2-based scaffold, can be directly covalently linked to each other. Conversely, two operably linked functional components, or one functional component operably linked to the anti-B7-H3 Gp2-based scaffold, can be connected by mutual covalent bonds to an intermediate component (e.g., a flanking sequence or a linker). For example, in embodiments in which the anti-B7-H3 compound comprises two functional components, the first functional component and the second functional component can be separately directly linked to the anti-B7-H3 Gp2-based scaffold, or the first functional component can be directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional component can be directly linked to the first functional component.

[0065] The anti-B7-H3 Gp2-based scaffold and the first functional component may be operably linked via one or more linkers. The term "linker" as used herein refers to any bond, small molecule, peptide sequence, or other vehicle that physically links the components. The linker may be sensitive or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and / or disulfide bond cleavage under conditions in which the first functional (or other functional component) component and / or the anti-B7-H3 Gp2-based scaffold remain active. Linkers are classified based on the presence of one or more chemical motifs, such as, for example, disulfide groups, hydrazine groups or peptides (cleavable), or thioester groups (non-cleavable). Linkers also include charged linkers and their hydrophilic forms known in the art.

[0066] Suitable linkers for linking the anti-B7-H3 Gp2-based scaffold and the first functional component of the present disclosure include natural linkers, empirical linkers, or a combination of natural and / or empirical linkers. Natural linkers are derived from the amino acid linking sequences of multidomain proteins that naturally occur between protein domains. For example, the properties of natural linkers, such as length, hydrophobicity, amino acid residues, and / or secondary structure, can be utilized to impart desirable properties to multidomain compounds that include natural linkers connecting components of the anti-B7-H3 compounds of the present disclosure.

[0067] Research on linkers in natural multi-domain proteins has led to the generation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers are often classified into three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide some degree of movement or interaction in the linked components. Flexible linkers typically contain small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids that provide flexibility and allow mobility of the connected components. An example of a flexible linker useful for linking the anti-B7-H3 Gp2-based scaffold and the first functional component of the anti-B7-H3 compound of the present disclosure is SEQ ID NO:24. The rigid linker can effectively maintain a fixed distance between the first functional component of the anti-B7-H3 compound and the anti-B7-H3 Gp2-based scaffold to maintain their independent functions, which can provide efficient separation between the first functional component and the anti-B7-H3 Gp2-based scaffold and / or sufficiently reduce the interference between the first functional component and the anti-B7-H3 Gp2-based scaffold. The cleavable linker can allow for controlled release of the first functional component and / or the anti-B7-H3 Gp2-based scaffold in vivo. By utilizing a unique in vivo process, the cleavable linker can be cleaved under certain conditions, such as the presence of a reducing agent or a protease. This type of linker can reduce steric hindrance, improve biological activity, and / or achieve independent action / metabolism of the first functional component and / or the anti-B7-H3 Gp2-based scaffold after linker cleavage.

[0068] Exemplary linker sequences include the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33; SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

[0069] In one or more embodiments, the natural or empirical linker is covalently attached to the anti-B7-H3 Gp2-based scaffold, the first functional moiety, or both, using bioconjugation chemistry, which is well known in the art and includes, but is not limited to, NHS-ester ligation, isocyanate ligation, isothiocyanate ligation, benzoyl fluoride ligation, maleimide conjugation, iodoacetamide conjugation, 2-thiopyridine disulfide exchange, 3-arylpropiononitrile conjugation, diazonium salt conjugation, 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione conjugation, and Mannich ligation.

[0070] In one or more embodiments, the natural or empirical linker, the first functional component, the anti-B7-H3 Gp2-based scaffold, or a combination thereof, may include one or more unnatural amino acids that allow for bioorthogonal conjugation reactions. As used herein, "bioorthogonal conjugation" refers to a conjugation reaction that uses one or more unnatural or modified amino acids as starting reagents. Examples of bioorthogonal conjugation reactions include, but are not limited to, Staudinger ligation, copper-catalyzed azide-alkyne cycloaddition, strain-promoted [3+2] cycloaddition, tetrazine ligation, metal-catalyzed coupling reactions, or oxime-hydrazone ligation. Examples of unnatural amino acids include azidohomoalanine, 2 homopropargylglycine, 3 homoallylglycine, 4 p-acetyl-Phe, 5 p-azido-Phe, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid, N ε -(cyclooct-2-yn-1-yloxy)carbonyl)L-lysine, N εExamples of amino acids that may be incorporated into the scaffold include, but are not limited to, -2-azidoethyloxycarbonyl-L-lysine, Nε-p-azidobenzyloxycarbonyl-L-lysine, propargyl-L-lysine, or trans-cyclooct-2-enelysine. Such amino acids may be incorporated into any position of the anti-B7-H3 Gp2-based scaffold of the present disclosure or on any of the functional moieties.

[0071] In one or more embodiments, the linker is derived from a small molecule, such as a polymer. Examples of polymer linkers include, but are not limited to, polyethylene glycol, poly(N-isopropylacrylamide), and N,N'-dimethylacrylamide)-co-4-phenylazophenylacrylate. Small molecule linkers generally include one or more reactive handles that allow conjugation to the anti-B7-H3 Gp2-based scaffold, the component, or both. In one or more embodiments, the reactive handle allows bioconjugation or bioorthogonal conjugation. In one or more embodiments, the reactive handle allows any organic reaction compatible with conjugating the linker to the anti-B7-H3 Gp2-based scaffold or the first functional component.

[0072] The linker can be conjugated at any amino acid position of the anti-B7-H3 Gp2-based scaffold. For example, the linker can be conjugated to the N-terminus, C-terminus, or any amino acid therebetween.

[0073] In embodiments in which the anti-B7-H3 compound comprises two or more functional moieties, the additional functional moieties may be operably linked to each other and / or to the anti-B7-H3 Gp2-based scaffold using one or more of the linkers disclosed herein.

[0074] In another aspect, the present disclosure describes host cells comprising any of the isolated nucleic acid sequences, anti-B7-H3 compounds, and / or anti-B7-H3 Gp2-based scaffolds described herein.

[0075] In some embodiments where the anti-B7-H3 compound comprises an anti-B7-H3 Gp2-based scaffold and one or more functional components operably linked by a peptide linker, the anti-B7-H3 compound can be produced by expression in a host cell using methods known in the art. In some embodiments where the anti-B7-H3 compound comprises an anti-B7-H3 Gp2-based scaffold and one or more functional components operably linked by a peptide linker, the anti-B7-H3 compound can be produced using solid phase peptide synthesis using methods known in the art.

[0076] In another aspect, the present disclosure describes isolated nucleic acid sequences encoding the amino acid sequence of any embodiment of a polypeptide fragment of the anti-B7-H3 Gp2-based scaffold and / or anti-B7-H3 compound described herein or any component thereof. Given the amino acid sequence of any polypeptide, one of skill in the art can determine, using conventional routine methods, the full range of polynucleotides that encode that amino acid sequence.

[0077] In another aspect, the present disclosure describes host cells comprising any of the isolated nucleic acid sequences, anti-B7-H3 compounds, and / or anti-B7-H3 Gp2-based scaffolds described herein.

[0078] The nucleic acid constructs of the present disclosure may be introduced into and modified by a host cell, thereby allowing the expression of anti-B7-H3 compounds and / or anti-B7-H3 Gp2-based scaffolds in the cells, resulting in the generation of genetically engineered cells. A variety of methods, including viral and non-viral mediated techniques, are known in the art and are suitable for the introduction of nucleic acids into cells. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated introduction, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated introduction, microinjection, microprojectile mediated introduction (e.g., nanoparticles), cationic polymer mediated introduction (e.g., DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.) or cell fusion. Other methods of transfection include proprietary transfection reagents such as Lipofectamine (Thermo Fisher Scientific, Inc., Waltham, MA), Hilymax (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), jetPEI (Polyplus Transfection, Illkirch, France), Effectene (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc USA, San Diego, CA).

[0079] The nucleic acid constructs described herein may be introduced into a host cell and modified to allow for intracellular expression of the nucleic acid-encoded anti-B7-H3 compounds, including anti-B7-H3 Gp2-based scaffolds, and / or anti-B7-H3 Gp2-based scaffolds operably linked to antibodies, affibodies, or other polynucleotides. A variety of host cells are known in the art and are suitable for protein expression. Examples of typical cells used for transfection and protein expression include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells, or plant cells, such as E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.

[0080] In another aspect, the present disclosure provides pharmaceutical compositions comprising one or more anti-B7-H3 compounds described herein (including anti-B7-H3 compounds that comprise an anti-B7-H3 Gp2-based scaffold and do not include additional functional components) formulated with a pharma- ceutical acceptable carrier. Such compositions may, for example, comprise one or a combination of two or more different anti-B7-H3 compounds.

[0081] As used herein, "pharmaceutical acceptable" refers to a substance that is not biologically or otherwise undesirable, e.g., the substance can be administered to an individual together with an anti-B7-H3 compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included.

[0082] The pharmaceutical composition may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. The pharmaceutical composition may include a pharmaceutically acceptable antioxidant. Those skilled in the art generally recognize and understand how to apply the use of pharmaceutically acceptable salts and / or pharmaceutically acceptable antioxidants.

[0083] Pharmaceutical compositions may be formulated with a pharma- ceutically acceptable carrier. As used herein, the term "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonicity agent, absorption delaying agent, buffer, carrier solution, suspension, hydrogel, colloid, adjuvant, stabilizer, protein carrier, biological carrier compound, and the like. Non-limiting examples of solvents include water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oil (e.g., olive oil), or injectable organic ester (e.g., ethyl oleate), or combinations thereof. Non-limiting examples of protein carriers include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, and the like. Non-limiting examples of biological compounds that may function as carriers include glycosaminoglycans, proteoglycans, or albumins. Carriers may include organic solvents (e.g., dimethylsulfoxide), synthetic compounds, or synthetic polymers (e.g., polyalkylene glycols). Ovalbumin, human serum albumin, other proteins, polyethylene glycol, and the like may be used as carriers. The use of such media and / or agents for pharma-ceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient (i.e., anti-B7-H3 compounds), its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0084] The pharmaceutical composition may also or alternatively contain one or more adjuvants, such as, for example, preservatives, wetting agents, emulsifying agents, and / or dispersing agents. In one or more embodiments, the pharmaceutical composition may contain antibacterial and / or antifungal agents, such as, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonicity agents in the composition, such as sugars, sodium chloride, or polyalcohols (e.g., mannitol, sorbitol, etc.). Furthermore, prolonged absorption of the injectable pharmaceutical form may be provided by including agents that delay absorption, such as aluminum monostearate or gelatin.

[0085] Thus, the anti-B7-H3 compounds of the present disclosure can be formulated into pharmaceutical compositions. The pharmaceutical compositions can be formulated in various forms adapted to the preferred route of administration. Thus, the compositions can be administered via known routes, including, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.), or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intradermally, transdermally, intrarectally, etc.). The pharmaceutical compositions can be administered to mucosal surfaces, such as, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The compositions can also be administered via sustained or delayed release.

[0086] The preparation may be provided in a convenient unit dosage form and can be prepared by a method known in the art of pharmacy.The method of preparing the composition with a pharmaceutically acceptable carrier includes the step of associating the active ingredient with the carrier that constitutes one or more accessory ingredients.In general, the preparation can be prepared by uniformly and / or intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into desired preparation.

[0087] To prepare pharmaceutical compositions comprising the anti-B7-H3 of the present disclosure, the anti-B7-H3 compounds may be mixed with a pharma- ceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents may be prepared, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension, by mixing with a physiologically acceptable carrier, excipient, or stabilizer.

[0088] Thus, the anti-B7-H3 compounds of the present disclosure may be provided in any suitable form, including, but not limited to, a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The compositions may be delivered in a formulation that includes any pharma- ceutically acceptable excipient, carrier, or vehicle.

[0089] Nasal spray formulations include purified aqueous solutions of active ingredients containing preservatives and isotonicity agents. Such formulations are preferably adjusted to a pH and isotonicity state compatible with nasal mucosa. Formulations for rectal or vaginal administration can be provided as suppositories with suitable carriers such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acids. Ophthalmic formulations are prepared in a similar manner to nasal sprays, except that pH and isotonicity factors are preferably adjusted to be compatible with those of the eye.

[0090] Topical formulations include anti-B7-H3 compounds dissolved or suspended in one or more vehicles, such as mineral oil, petroleum, polyhydroxy alcohol, or other bases used in topical pharmaceutical formulations. For example, topical formulations can include creams, ointments, pastes, lotions, powders, solids, aerosol foams, or gels. Topical formulations may contain permeation enhancers to increase the bioavailability of the active ingredient. Topical formulations may contain preservatives and / or emulsifiers. Topical formulations may be provided in the form of transdermal patches or dressings, where the formulation is incorporated into gauze or other structures and placed in contact with the skin. The formulations may further include one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, and the like.

[0091] The formulation of the present disclosure suitable for oral administration may be provided as a discrete unit such as a tablet, troche, capsule, lozenge, wafer, or cachet, each containing a predetermined amount of active ingredient as a powder or granule, as a liposome, or as a solution or suspension in an aqueous or non-aqueous liquid, such as a syrup, elixir, emulsion, or draught. Tablets, troches, pills, capsules, etc. may also contain one or more of the following: binders, such as gum tragacanth, acacia, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; sweeteners, such as sucrose, fructose, lactose, or aspartame; or natural or artificial flavors. When the unit dosage form is a capsule, it may further contain a liquid carrier, such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, and the like. Syrups or elixirs may contain one or more sweetening agents, preservatives such as methyl or propylparaben, agents that retard sugar crystallization, agents that increase the solubility of any other ingredients (e.g., polyalcohols such as glycerol or sorbitol), dyes, and / or flavorings. The materials used to prepare any unit dosage form are substantially non-toxic in the amounts employed. The active ingredient may be incorporated into the preparation or device in a formulation that may or may not be designed for sustained release.

[0092] Formulations suitable for parenteral administration may include sterile aqueous preparations of the active ingredient, or dispersions of sterile powders of the active ingredient, which are preferably isotonic with the blood of the recipient. Parenteral administration (e.g., by intravenous infusion) of the anti-B7-H3 compounds of the present disclosure or pharmaceutical compositions containing them is one form of administration. Isotonicity agents that may be included in liquid preparations include sugars, buffers, and / or sodium chloride. Solutions of the active ingredient may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions of the active ingredient may be prepared in water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, glycerol esters, or any mixture thereof.

[0093] In yet another aspect, the disclosure provides imaging methods and methods for treating, ameliorating, detecting, diagnosing, or monitoring a disease described herein or a symptom or clinical sign thereof in a patient by administering a therapeutically effective amount of an anti-B7-H3 compound described herein, an anti-B7-H3 Gp2-based scaffold, and / or a pharmaceutical composition comprising one or more anti-B7-H3 compounds. As used herein, "treat" and variations thereof refer to reducing, inhibiting the progression of, improving, or eliminating to any extent a symptom or clinical sign associated with a condition. A "symptom" refers to any subjective evidence of a disease or condition in a patient. A "sign" or "clinical sign" refers to an objective physical finding of a particular condition that can be recognized by someone other than the patient. "Treatment" can be therapeutic or prophylactic. "Therapeutic" and variations thereof refer to treatment that improves one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and variations thereof refer to treatment that limits, to any extent, the occurrence and / or appearance of a symptom or clinical sign of a condition. Generally, a "therapeutic" treatment is initiated after a condition appears in a subject, whereas a "prophylactic" treatment is initiated before a condition appears in a subject. A prophylactic treatment can be administered to a subject at risk of having a condition. As used herein, "at risk" refers to a subject who may or may not actually have the risk described. In the case of a non-infectious condition, for example, a subject at "risk" of developing a particular condition is a subject who has one or more symptoms that make him or her at high risk of having or developing a particular condition, compared to an individual who lacks one or more symptoms, regardless of whether the subject shows any symptoms or clinical signs of having or developing the condition.

[0094] Thus, in one or more embodiments, treating a subject includes a subject having or at risk of having cancer. In general, the method includes administering to the subject an effective amount of an anti-B7-H3 compound or a pharmaceutical composition comprising one or more anti-B7-H3 compounds described herein. As used herein, the term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth that begins at one site (primary site) and has the potential to invade and spread to other sites (secondary sites, metastases), which distinguishes cancer (malignant tumors) from benign tumors. As used herein, "neoplasm" or "tumor" (and grammatical variations thereof) refers to a new abnormal growth of tissue, which may be benign or cancerous. In related aspects, neoplasm refers to a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0095] The amount of anti-B7-H3 compound administered may vary depending on various factors, including but not limited to the anti-B7-H3 compound used, the weight, health condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of anti-B7-H3 compound contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and health condition of the subject, and / or the method of administration. Therefore, it is not practical to generally describe the amount that constitutes the amount of anti-B7-compound that is effective for all possible applications. However, those skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0096] For example, a certain anti-B7-H3 compound may be administered at the same dose and frequency as the drug is approved by regulatory authorities. In other cases, a certain anti-B7-H3 compound may be administered at the same dose and frequency as the drug is evaluated in clinical or preclinical studies. The dose and / or frequency can be modified as needed to achieve the desired level of anti-B7-H3 effect. Thus, standard / known dosing regimens can be used and / or dosing can be customized as needed.

[0097] In one or more embodiments, the method can include administering an anti-B7-H3 compound in an amount sufficient to provide the subject with a dose of, for example, about 100 ng / kg to about 50 mg / kg, although in some embodiments, the method can be carried out by administering an anti-B7-H3 compound at a dose outside this range. In some of these embodiments, the method includes administering an anti-B7-H3 compound in an amount sufficient to provide the subject with a dose of, for example, about 10 μg / kg to about 5 mg / kg, e.g., about 100 μg / kg to about 1 mg / kg.

[0098] A single dose may be administered all at once, continuously over a period of time, or in multiple separate doses. When multiple doses are used, the amount of each dose may be the same or different. For example, a dose of 1 mg per day may be administered as a single dose of 1 mg continuously over a 24-hour period, as two or more equal doses (e.g., two 0.5 mg doses), or as two or more unequal doses (e.g., 0.75 mg administered first, followed by 0.25 mg administered second). When multiple doses are used to deliver a single dose, the interval between doses may be the same or different.

[0099] In one or more embodiments, the active ingredient may be administered, for example, in a single dose to multiple doses per week, although in some embodiments, the method may include a course of treatment that includes administering a dose of the active ingredient at a frequency outside this range.When a course of treatment includes administering multiple doses within a specific period of time, each dose may be the same or different.For example, a course of treatment may include a loading dose (e.g., an initial dose), followed by a maintenance dose that is lower than the loading dose.Also, when multiple doses are administered within a specific period of time, the interval between administrations may be the same or different.

[0100] In certain embodiments, the anti-B7-H3 compound may be administered from about once a month to about five times a week.

[0101] The anti-B7-H3 compound or pharmaceutical composition comprising same may be administered before, during, or after the subject first exhibits symptoms or clinical signs of the condition. Treatment initiated before the subject first exhibits symptoms or clinical signs associated with the condition may result in a reduced likelihood that the subject will experience clinical evidence of the condition, a reduced severity of the symptoms and / or clinical signs of the condition, and / or a complete reversal of the condition, compared to a subject to whom the anti-B7-H3 compound or pharmaceutical composition comprising same is not administered. Treatment initiated after the subject first exhibits symptoms or clinical signs associated with the condition may result in a reduced severity of the symptoms and / or clinical signs of the condition, and / or a complete reversal of the condition, compared to a subject to whom the anti-B7-H3 compound or pharmaceutical composition comprising same is not administered.

[0102] The anti-B7-H3 Gp2-based scaffold can be any of the anti-B7-H3 Gp2-based scaffolds disclosed herein. The anti-B7-H3 compound can be any of the embodiments of the anti-B7-H3 compound described herein that has an anti-B7-H3 Gp2-based scaffold that binds to the extracellular domain of B7-H3 displayed on a target cell of a cell population. In some cases, the target cell can include a tumor cell, such that the method can include treating a cancer associated with the tumor cell. Thus, in one or more embodiments, the method can include ameliorating at least one symptom or clinical sign of a tumor.

[0103] In embodiments in which the target cells comprise tumor cells, the method may further comprise surgically removing the tumor and / or reducing the size of the tumor by chemical (e.g., chemotherapy) and / or radiation therapy. Exemplary tumors that may be treated include those associated with prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, and / or hematopoietic cancer.

[0104] In one or more embodiments, the anti-B7-H3 compound is administered prior to, concurrently with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

[0105] In one or more embodiments, the anti-B7-H3 compound may be administered, for example, in a single dose to multiple doses per week, although in some embodiments the method may be practiced by administering the anti-B7-H3 compound at a frequency outside this range. In certain embodiments, the anti-B7-H3 compound may be administered from about once a month to about five times a week.

[0106] In one or more embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and / or simultaneously with the administration of the anti-B7-H3 compound or pharmaceutical composition comprising the same. The anti-B7-H3 compound or pharmaceutical composition comprising the same and the additional therapeutic agent may be co-administered. As used herein, "co-administration" refers to two or more components of a combination administered such that the therapeutic or prophylactic effect of the combination may be greater than the therapeutic or prophylactic effect of either component administered alone. The two components may be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes when the components are administered such that each component may be present at the treatment site at the same time. Alternatively, sequential co-administration of two components may include when at least one component has been removed from the treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, co-administered combinations may, in certain circumstances, include components that are never present in a chemical mixture with each other. In other embodiments, the anti-B7-H3 compound or pharmaceutical composition comprising the same and the additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, administration of the anti-B7-H3 compound or pharmaceutical composition comprising the same may allow for the efficacy of a lower dose of the other therapeutic agent(s) compared to administration of the other therapeutic agent(s) alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when a higher dose of the other therapeutic agent(s) is administered.

[0107] The term "chemotherapeutic agent" as used herein refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Thioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, Panitumamab, Erbitux™ (Cetuximab), Matuzumab, IMC-IIF8, TheraCIM hR3, Denosumab, Avastin™ (Bevacizumab), Humira™ (Adalimumab), Herceptin™ (Trastuzumab), Remicade™ (Infliximab), Rituximab, Synagis™ (Palivizumab), Mylotarg™ (Gemtuzumab) oxogamicin), Raptiva™ (efalizumab), Tysabri™ (natalizumab), Zenapax™ (dacliximab), NeutroSpec™ (technetium (99mTc)-fanolesomab), tocilizumab, ProstaScint™ (indium-Ill-labeled capromab pendetide), Bexxar™ (tositumomab), Zevalin™ (it ibritumomab tiuxetan conjugated to lithium-90 (IDEC-Y2B8)), Xolair™ (omalizumab), MabThera™ (rituximab), ReoPro™ (abciximab), MabCampath™ (alemtuzumab), Simulect™ (basiliximab), LeukoScan™ (sulesomab), CEA-Scan™ (arcitumomab), Verluma™ (nofetumomab),Panorex™ (edrecolomab), alemtuzumab, CDP870, natalizumab, Gilotrif™ (afatinib), Lynparza™ (olaparib), Perjeta™ (pertuzumab), Otdivo™ (nivolumab), Bosulif™ (bosutinib), Cabometyx™ (cabozantinib), Ogivri™ (trastuzumab-dkst), Sutent™ (sunitinib malate), Adcetris™ (brentuximab vedotin), Alecens These include, but are not limited to, a™ (alectinib), Calquence™ (acalabrutinib), Yescarta™ (ciloleucel), Verzenio™ (abemaciclib), Keytruda™ (pembrolizumab), Aliqopa™ (copanlisib), Nerlynx™ (neratinib), Imfinzi™ (durvalumab), Darzalex™ (daratumumab), Tecentriq™ (atezolizumab), or Tarceva™ (erlotinib). Examples of immunotherapeutic agents include, but are not limited to, interleukins (IL-2, IL-7, IL-12, etc.), cytokines (interferons, G-CSF, etc.), chemokines (CCL3, CCl26, CXCL7), or immunomodulatory imidate drugs (imiquimod, thalidomide, etc. or analogs thereof).

[0108] In some embodiments, the methods can include administering a sufficient amount of an anti-B7-H3 compound described herein, where administering at least one additional therapeutic agent exhibits therapeutic synergy. In some aspects of the methods of the present disclosure, a measure of response to treatment observed after administering both an anti-B7-H3 compound described herein and an additional therapeutic agent is improved over the same measure of response to treatment observed after administering either the anti-B7-H3 compound or the additional therapeutic agent alone.

[0109] In yet another aspect, the present disclosure describes a capture assay device comprising any one of the embodiments of the anti-B7-H3 compounds described herein (including the anti-B7-H3 Gp2-based scaffold without additional functional components) immobilized on a substrate. For example, the anti-B7-H3 compounds described herein can be incorporated into cell and / or ligand capture techniques, such as ELISA-based assays. The substrate for immobilizing the anti-B7-H3 compounds can include, for example, cell culture plates or dishes, glass slides, or any other support that can be used to perform assays that require immobilized anti-B7-H3 compounds.

[0110] In another aspect, the present disclosure describes methods for using anti-B7-H3 compounds in molecular imaging applications, including, for example, both traditional molecular imaging techniques (e.g., magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound, photoacoustic, and fluorescence) and microscopy and / or nanoscopy imaging techniques (e.g., total internal reflection fluorescence (TIRF)-microscopy, stimulated emission depletion (STRED) nanoscopy, or atomic force microscopy (AFM).

[0111] The anti-B7-H3 compounds described herein may have in vitro and in vivo detection, diagnostic, and / or therapeutic utility. For example, the anti-B7-H3 compounds may be included in a detection composition for use in a detection method. The detection composition may include any carrier and / or adjuvant described herein, or any additional carrier or adjuvant known in the art. The method may generally include allowing an anti-B7-H3 compound that specifically binds to a target of interest to be combined with a sample containing the target of interest, and then detecting the formation of an anti-B7-H3 compound:target complex. Thus, the anti-B7-H3 compounds may be designed to include a detectable marker, such as a radioisotope, a fluorescent marker, an enzyme, or a colorimetric marker.

[0112] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprise", "comprising" and variations thereof are to be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably to mean one or more; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0113] In the foregoing description, certain embodiments may be described alone for clarity. Throughout this specification, references to "one or more embodiments," "an embodiment," "an embodiment," "a particular embodiment," or "some embodiments" mean that a particular feature, configuration, composition, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Moreover, certain features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment, unless the features are necessarily mutually exclusive.

[0114] In any method disclosed herein that includes separate steps, the steps may be performed in any practicable order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0115] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment.

[0116] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0117] The term "polypeptide" refers to a sequence of amino acid residues regardless of the length of the sequence. Thus, the term "polypeptide" refers to any amino acid sequence having at least two amino acids, and includes full-length proteins, fragments thereof, and / or, in some cases, polyproteins.

[0118] The term "protein" refers to any sequence of two or more amino acid residues, regardless of the length of the sequence, and any complex of two or more separately translated amino acid sequences. Protein also refers to an amino acid sequence that has been chemically modified to include carbohydrates, lipids, a nucleotide sequence, or any combination of carbohydrates, lipids, and / or nucleotide sequences. As used herein, "protein," "peptide," and "polypeptide" are used interchangeably.

[0119] As used herein, the term "nucleic acid" or "oligonucleotide" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules, such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, nucleic acid conjugates, and oligonucleotides. Nucleic acids can be single-stranded, double-stranded, linear, or covalently closed circular molecules. Nucleic acids may be isolated. The term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, such as by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, such as by cleavage and separation by gel electrophoresis, (iv) synthesized, such as by chemical synthesis, or (vi) extracted from a sample. Nucleic acids can be introduced, i.e., transfected, into a cell. When RNA is used to transfect cells, the RNA can be modified by stabilizing modifications, capping, or polyadenylation.

[0120] The present invention is illustrated in the following examples, it being understood that the specific examples, materials, amounts and procedures should be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0121] Example 1 B7-H3 Library Design The Gp2 library design detailed by Kruziki et al. (ACS Comb. Sci. 20, 423-435 (2018) and Mol. Pharm. 13, 3747-3755 (2016)) includes a truncated version of the T7 phage gene 2 protein (SEQ ID NO: 1), which has been diversified at six positions in each of the two loops using degenerate codons that encode an amino acid distribution that mimics antibody CDRs (Kruziki et al., Chem. Biol. 22, 946-956 (2015)), as well as a design that constrains diversity and extends the paratope (Kruziki et al., ACS Comb. Sci. 20, 423-435 (2018)).

[0122] Discovery and evolution strategies The Gp2 library was displayed using yeast surface display and B7-H3 binders were selected using both magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). The population was selected to achieve detectable binding at 50 nM of the B7-H3 target. DNA was then isolated from the yeast and subjected to random mutagenesis by error-prone polymerase chain reaction (PCR) of the paratope and the entire Gp2 gene. DNA was electroporated back into yeast for display and further selected until binding to low nanomolar concentrations of B7-H3 was observed.

[0123] MACS selection by soluble extracellular domains Magnetic bead sorting was performed using at least 15-fold oversampling of ligand diversity at all stages. Beads were coated with protein by incubation with DYNABEADS Biotin Binder (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA) with biotinylated recombinant human B7-H3 extracellular domain (available from Sino Biological, Inc., Beijing, China) for positive isolation and biotinylated Renilla green fluorescent protein (rrGFP; available from Avidity, LLC, Aurora, CO) for depletion. Yeast were subjected to magnetic activated cell sorting, in which yeast were incubated with control naked biotin binder beads for 2 hours at 4°C, followed by an additional 2 hours of incubation with GFP-labeled beads to remove any non-specific and non-B7-H3 binding interactions. Yeast were then incubated with beads with immobilized recombinant human B7-H3 target protein for an additional 2 hours. Yeast that bound were positively selected. MACS was performed at 4°C, with yeast washed twice between incubations. A total of three rounds of MACS selection were performed with increasing wash stringency, with yeast grown and induced between each selection. After error-prone PCR, additional MACS selections were performed on the large mutation-naive library, similar to the procedure previously described.

[0124] FACS sorting with soluble extracellular domains After three rounds of MACS sorting of the Gp2 library, the library size could be sampled by FACS. The derived yeast population was simultaneously labeled with mouse anti-c-Myc antibody (BioLegend, San Diego, CA) and 50 nM biotinylated recombinant human B7-H3 extracellular domain for 30 min at 4°C. The cells were washed once with PBSA, labeled with goat anti-mouse Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA) and streptavidin Alexa Fluor 488 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA) for 20 min at 4°C, and washed with PBSA. Cells that were Myc positive and had a B7-H3 signal above background were collected.

[0125] Error-prone PCR of Gp2: Random mutations of the 0.4 Gp2 population (library) were performed by error-prone PCR using the mutagenic analogs 8-oxo-dGTP and dPTP to obtain the 1.4 Gp2 library. Zymoprep-treated plasmid DNA was mutated by error-prone PCR of the entire Gp2 gene using primers W5 / W3 and of the Gp2 loop using loop 1 and loop 2 mutagenesis primers (Kruziki et.at., Chem. Biol. 22, 946-956 (2015)). PCR products were purified by agarose gel electrophoresis. Gp2 loop genes were assembled into one construct using PCR assembly. The final gene inserts were amplified by PCR, concentrated by ethanol precipitation, and resuspended for electroporation. The mutated Gp2 library was either homologously recombined with the linearized pCT gene or introduced into EBY100 yeast by electroporation transformation. Approximately 33 million transformants were obtained for Gp2 by electroporation.

[0126] Monovalent MACS selection with soluble extracellular targets After MACS selection against a large mutation naive library (1.4 library), yeast were subjected to a more stringent monovalent MACS selection. Yeast were incubated with control naked biotin binder beads, washed, and then removed with GFP-coated beads as before. Yeast were then washed and incubated with 100 nM biotinylated recombinant human B7-H3 extracellular domain for 1 h. Naked biotin binder beads were added to bind to the biotinylated B7-H3 and incubated for an additional 2 h at 4 °C. The beads were washed three times and yeast that remained bound were collected.

[0127] Preparation of detergent-solubilized MS1-B7-H3 cell lysates Mile Sven 1 cells stably transfected to express human B7-H3 (MS1-B7-H3) were grown in DMEM containing 10% fetal bovine serum (v / v) and 1% penicillin and streptomycin at 37°C and 5% CO2. MS1-B7-H3 cells were cultured at 75 cm 2 Cells were grown to 70–90% confluence in tissue culture-treated flasks. Cells were washed with PBS, detached by trypsin-EDTA treatment for 5 min, quenched with serum-containing culture medium, and pelleted at 500 × g for 3 min. Pelleted cells were washed twice and resuspended in PBS containing 0.5 mg / mL fresh sulfo-NHS-biotin (Thermo Fisher Scientific, Inc., Waltham, MA) for 30 min at room temperature. Cells were washed twice after incubation to remove excess biotin and lysed in 250 μL of lysis buffer for 15 min at 4 °C. Cell debris was pelleted at 10,000 × g for 30 min and removed.

[0128] FACS selection using detergent-solubilized cell lysates The Gp2 library underwent two rounds of flow cytometry selection using detergent-solubilized cell lysates. The library was washed once with PBSA and incubated with cell lysates for 1 h at 4°C. After incubation, yeast were washed and incubated with chicken anti-Myc-FITC (Immunology Consultants Laboratory Inc., Portland, OR) and streptavidin Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA) for 20 min at 4°C and washed again. Yeast that were Myc-positive (FITC) with the highest ratio of MS1-B7-H3 lysate-bound (AF647):Myc (FITC) were collected using FACS. This selection was repeated at higher stringency by using a lower B7-H3 lysate concentration.

[0129] Sanger sequencing of yeast PCR products The enriched B7-H3 binding population was plated on SD-CAA plates and grown for 2 days. Ten colonies were stochastically selected from the Gp2 library and incubated in 50 μL of distilled water at 100°C for 5 min. Two microliters of yeast sample were taken and subjected to PCR and DNA cleanup using GeneAmp5 / 3 primers. The amplified Gp2 gene and GeneAmp5 primers were sent to Eurofins Genomics LLC for Sanger sequencing.

[0130] Cloning, protein expression, and purification The Gp2 coding region in DNA recovered from the final B7-H3 flow cytometry sort (from the 1.4 population) was amplified by PCR, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Inc., Ipswich, MA), and ligated with T4 DNA ligase into a pET-24b vector containing a C-terminal hexa-histidine tag. Plasmids were transformed into T7 Express competent E. coli and plated onto lysogeny broth (LB) plates containing 50 mg / L kanamycin. Transformants were Sanger sequenced for the full-length gene, and appropriate transformants were grown in 5 mL of liquid LB containing kanamycin (50 mg / L) at 37 °C, 250 rpm for 12-16 h. Saturated cultures were added to 100 mL of LB, grown, and induced. Cells were pelleted and resuspended in lysis buffer (50 mM sodium phosphate (pH 8.0), 0.5 M sodium chloride, 5% glycerol, 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 25 mM imidazole), lysed by freeze-thawing five times, centrifuged at 4°C for 10 min, and 0.25 mm filtered. The resulting cell lysate was passed through a 0.25 mL cobalt HisPur resin spin column, washed with 30 mM imidazole, and eluted with 300 mM imidazole. Gp2 purity and concentration were analyzed by protein gel electrophoresis.

[0131] affinity titration Dissociated MS1-B7-H3 cells were washed and individually labeled with various concentrations of purified Gp2 and Gp2 fusions for at least 30 min at 4° C. Cells were pelleted at 500×g for 3 min, washed with cold PBSA, and then labeled with anti-His6 FITC conjugate (available from Abcam, Cambridge, United Kingdom) for 20 min at 4° C. Fluorescence was analyzed using ACCURI C6 Plus. Dissociation constants were calculated by nonlinear least-squares regression using a 1:1 binding model in PRISM software (GraphPad Software, San Diego, CA).

[0132] Analysis of ligand domain modularity The Gp2 gene coding region was amplified by PCR and assembled into a pET-24b vector containing the GSGGGSGGGKGGGGT (SEQ ID NO: 3) linker and amino acids 60-206 of the sortase A (EC 3.4.22.70) gene with a C-terminal hexa-histidine tag by NEBuilder HIFI DNA Assembly (New England Biolabs, Inc., Ipswich, MA). Plasmids were transformed into BL21-CodonPlus(DE3)-RIL competent E. coli and plated on LB plates containing 50 mg / L kanamycin. Transformants were Sanger sequenced for the full-length gene and appropriate transformants were grown in 50 mg / L kanamycin and chloramphenicol for bacterial growth and protein purification. The binding affinity of Gp2 in the presence of the conjugated protein enzyme was assessed by affinity titration of the purified protein.

[0133] Example 2: Example 2 describes the affinity, specificity, and stability maturation of the 1.4 Gp2-based scaffold population of Example 1.

[0134] Error-prone PCR DNA was isolated from the 1.5 yeast population and subjected to random mutagenesis by error-prone polymerase chain reaction (PCR) of the variable regions of the affibodies. The DNA was electroporated back into yeast for display using the following selection schemes: (1) lysate MACS with 1 pmol B7-H3+ lysate selection; (2) affinity selection (stringent K d(3) specificity selection (multi-targeted MACS depletion selection); and (4) stability selection (thermolysin FACS selection at 55°C). After each selection, the population was deep sequenced and filtered by USEARCH (Ultra-High-Speed ​​Sequencing Analysis; Robert C. Edgar, Search and clustering orders of magnitude faster than BLAST, Bioinformatics, Volume 26, Issue 19, 1 October 2010, Pages 2460-2461, doi.org / 10.1093 / bioinformatics / btq461) to filter for full-length Gp2-based scaffold variants.

[0135] For each sequence, the frequency at each selection (e.g., avidity, affinity, specificity, and stability) and subsequent enrichment were determined. If the number of reads was 0, a read number of 1 was assigned. Z-scores for affinity, specificity, stability, and final frequency were determined, and a final conditional weighted average z-score was calculated. Top performers were selected based on highest scores.

[0136] Conditional weighted average z-scores were calculated by first determining the frequency of each variant in each sort. The affinity, specificity, and stability sort enrichment values ​​for each variant were then calculated by dividing the frequency of a single variant in a particular sort by its frequency in the previous sort. The z-score was calculated by dividing (sort enrichment score - average sort enrichment score of all variants) by (standard deviation of sort enrichment scores of all variants). The z-scores were averaged based on the following weights: affinity = 1; frequency = 3; specificity = 2; and stability = 1. The final frequency and specificity z-scores were the most important and were given the highest weight. In addition, the following conditions were also included: affinity enrichment > 0.5 and final frequency > 0. An affinity enrichment value greater than 1 indicates enrichment. The value of 0.5 was chosen to take into account variants that were initially present at a higher frequency and may not have had a chance to be enriched. The final frequency condition was included because the variant must be present in the final sequenced population.

[0137] Figure 5 shows the z-scores for affinity, specificity, stability, and final frequency of the top 13 variants, as well as the conditional weighted average z-score. Values ​​greater than 1 indicate enrichment and values ​​less than 1 indicate depletion. The color bars in Figure 5 for affinity are enrichment ranges from M to M+2SD, while specificity and stability are represented as 1 to M+2SD since the mean (M) was slightly less than 1. On the bar graphs, z-scores are represented from 1 to 10 (i.e., if the z-score is 10 or greater, the bar spans the full length of the graph).

[0138] B7-H3 cell lysate MACS sorting (avidity sorting): An avidity selection was performed. This selection is considered an avidity selection because the B7-H3 target is expressed multivalently on the avidin beads, not simply monovalently in solution. Yeast also expresses thousands of affibodies per cell, so avid binding can occur between yeast and B7-H3 coated beads.

[0139] To select for B7-H3 binders and easily remove non-binders / truncated ligands resulting from error-prone PCR, magnetic bead selection was performed with 20-fold oversampling. Yeast were incubated with naked streptavidin-coated beads for 2 h at 4 °C, followed by removal by incubation with GFP-coated beads for an additional 2 h. Yeast were incubated with 1 pmol of detergent-solubilized biotinylated MS1-B7-H3 cell lysate for an additional 2 h. Typically, 33 pmol of antigen is used to coat streptavidin beads, but 1 pmol was chosen to be more stringent (e.g., to reduce avidity). Bound yeast were selected and grown. Biotinylation of MS1-B7-H3 cell lysate is non-specific and reacts to all proteins, not just B7-H3 (non-specific N-hydroxysuccinimide (NHS) chemistry). B7-H3 is expressed at approximately 1 million copies per cell, and the Gp2-based scaffold library has already been primed with binding to B7-H3.

[0140] Strict K d FACS selection (affinity selection) After the first round of B7-H3 cell lysate MACS sorting, the library size could be easily sampled by FACS. The induced yeast population was labeled with ~0.2 nM biotinylated B7-H3 cell lysate for 2 hours, washed, and labeled with streptavidin-AF647 and chicken anti-Myc-FITC. The top 0.4% of cells (highest affinity) were collected for the next sort (specificity sort).

[0141] Removal by multi-target MACS (specificity selection) To select for more specific binders, the population isolated after affinity selection was subjected to multi-target depletion. The following proteins were biotinylated, incubated with streptavidin-coated beads, and used for negative depletion: lysozyme, bovine serum albumin, human interleukin receptor 2 gamma, tobacco etch virus (TEV) protease, human plasminogen activator urokinase receptor, human carbonic anhydrase II, and rabbit IgG-FITC. These proteins were used because they were readily available and / or already conjugated to biotin. Yeast were incubated with the aforementioned proteins coated on the beads for 2 hours at 4°C. Yeast were then subjected to a second identical negative depletion step. This was to increase confidence that non-specific binders had been removed. Non-binding yeast were selected and grown for the next selection (stability selection).

[0142] Thermolysin Stability FACS Selection (Stability Selection) To select stable binders, the Gp2-based scaffold population after specificity selection was subjected to protease incubation at high temperature. Yeast were induced to display the Gp2-based scaffold population and incubated with 0.75 mg / mL thermolysin at 55° C. for 10 min, then immediately placed on ice. Yeast were washed and incubated with mouse anti-MYC and rabbit anti-HA-biotin for 20 min. Yeast were washed again and labeled with goat anti-mouse-AF647 and streptavidin-FITC for another 20 min. Yeast that were HA and Myc positive were sorted in a three-step gating approach, and yeast displaying the top 2%, 14%, and 19% were collected using FACS.

[0143] Deep sequencing by ISEQ (Illumina, San Diego, CA) The selected Gp2-based scaffold population was deep sequenced and filtered by USearch and further filtered for full-length Gp2-based scaffolds. For stability selection, three-stage gates were collected and sequenced. Due to the lack of sequencing depth for full-length Gp2-based scaffolds, the number of reads from each stability gate was summed to obtain the total number of stable reads.

[0144] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence deposits in GenBank and RefSeq, and amino acid sequence deposits in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be construed therefrom. The invention is not limited to the exact details shown and described, and modifications obvious to one skilled in the art will be included within the scope of the invention as defined by the claims.

[0145] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by ordinary rounding techniques.

[0146] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0147] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. Sequence Listing Free Text SEQ ID NO:1-Gp2 KFWATVESSE HSFEVPVYAE TLDEALELAE WQYVPAGFEV TRVRP SEQ ID NO:2-Gp2 0.4.2 KFWATVCYVI GCFEVPVYAA TLDEALQLAE WQYGDSYVMD VTRVRP SEQ ID NO:3 - General consensus sequence of Gp2 KFWXT VXXXX XXXXFEXPXY AXTXDEALXL AXXXYXXXXX XXXVXXVXP X4: A or P; X7: Q, C, R, or E; X8: H, Y, or S; X9: V or S; X 10 : any amino acid such as Y or H or a deletion; X 11 : any amino acid or deletion such as S; X 12 : D, E, I, or N; X 13 : F, H, or G; X 14 : G, S, or C; X 17 :A or V; X 19 :V or I; X 22 : A or E; X 24 :L or M; X 29: Q, E, R, or L; X 32 :E or K; X 33 : V, A, or W; X 34 : Q, R, or K; X 36 : A, V, or G; X 37 : N, S, P, or D; X 38 :S or A; X 39 : any amino acid or deletion; X 40 : any amino acid or deletion; X 41 :I,G,V; X 42 :F or M; X 43 : T, M, K, D, or E; X 45 :T or A; X 46 :R or S; X 49 :R or H. SEQ ID NO:4 - Second consensus sequence of Gp2 (1.4.4) KFWATVQHVY SDFGFEAPVY AATLDEALQL AEVQYANSIF TVTRVRP SEQ ID NO:5-Gp2 0.4.1 KFWATVQHVY SDFGFEVPVY AATLDEALQL AEVQYANSIF MVTRVRP SEQ ID NO:6-Gp2 1.4.1 KFWATVQHVY SDFGFEAPVY AATLDEALQL AEVQYANSIF MVTRVRP SEQ ID NO:7-Gp2 1.4.2 KFWATVQHVY SDFGFEAPVY AATLDEALQL AEVQYANSIF MVARVRP SEQ ID NO:8-Gp2 1.4.3 KFWATVQHVY SDFGFEVPVY AATLDEALQL AEVRYANSIF MVTRVRP SEQ ID NO:9 - Otu1138 KFWATVQHVH SDFGFEAPVY AATLDEALQL AEVRYANSIF MVTSVRP SEQ ID NO:10-Otu1 KFWATVQHVY SDFGFEVPVY AATLDEALQL AEVRYANSIF MVTRVRP SEQ ID NO:11 - Otu269 KFWATVQHVY SDFGFEAPVY AATLDEALQL AEVRYASSIF MVTRVRP SEQ ID NO:12 - Otu262 KFWATVQHVY SNFGFEAPVY AATLDEALQL AEVRYANSIF MVTRVRP SEQ ID NO:13 - Otu1316 KFWATVQHVY SDFGFEAPVY AATMDEALQL AEVQYASSIF MVTRVRP SEQ ID NO:14 - Otu232 KFWATVQHVY SDFGFEAPVY AATLDEALRL AEVRYANSIF MVTRVRP SEQ ID NO:15 - Otu266 KFWATVQHVY SDFGFEAPVY AATLDEALLL AEVRYANSIF MVTRVRP SEQ ID NO:16 - Otu292 KFWATVQHVY SDFGFEAPVY AATLDEALQL AEVRYANSIF MVTRVRP SEQ ID NO:17 - Otu1160 KFWPTVRHVY SDFGFEAPIYA ATLDEALQLA EVRYANSIFM VTRVRP SEQ ID NO:18 - Otu201 KFWATVRHVY SDFGFEAPIYA ATLDEALELA EVKYANSIFM VTRVRP SEQ ID NO:19 - Otu353 KFWATVQHVY SDFGFEAPVYA ATLDEALRLA EVRYANSIFM VTRVHP SEQ ID NO:20-Otu314 KFWATVQHVY SDFGFEAPVYA ATLDEALQLA EAQYANSIFK VTRVRP SEQ ID NO:21 - Otu1147 KFWATVQHVY SDFGFEAPVYA ATLDEALQLA EVRYANSIFT VTRVRP SEQ ID NO:22-Out437 KFWATVQHVH SDFGFEVPVYA ATLDEALQLA EAQYANSTF MVTRVRP SEQ ID NO:23 - Otu575 KFWATVQHVY SDFGFEAPVYA ATLDEALRLA KVQYANSIF MVTRVRP SEQ ID NO:24 - Linker GSGGGSGGGK GGGGT SEQ ID NO:25 - Linker GGGGSGGGGS GGGGS SEQ ID NO:26 - Linker GSTSGSGKPG SGEGSTKG SEQ ID NO:27 - Linker PSGQAGAAAS ESLFVSNHAY SEQ ID NO:28 - Linker EASGGPE SEQ ID NO:29 - Linker AEAAKEAAKE AAKEAAKALE AEAAKEAAKE AAKEAAKA SEQ ID NO:30 - Linker AEAAKEAAKA SEQ ID NO:31 - Linker SGGGGSGGGGS GGGGSGGGGSG SEQ ID NO:32 - Linker GGGGSGGGGS SEQ ID NO:33 - Linker GSTSGSGKPG SGEGSTKG SEQ ID NO:34 - Linker EPKSSDKTHT SPPSP SEQ ID NO:35 - Linker RATPSHNSHQ VPSAGGPTAN SGTSG SEQ ID NO:36 - Linker SSGGGGSGGG GGGSSRSSL

Claims

1. 1. An anti-B7-H3 compound comprising an anti-B7-H3 Gp2-based scaffold, the anti-B7-H3 Gp2-based scaffold comprising: SEQ ID NO:3, an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:3; or An amino acid sequence having at least 90% sequence identity to SEQ ID NO:3 4. An anti-B7-H3 compound comprising:

2. The anti-B7-H3 Gp2-based scaffold comprises an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

4.

2. The anti-B7-H3 compound of claim 1, comprising:

3. The anti-B7-H3 compound of claim 1 or claim 2, wherein the anti-B7-H3 Gp2-based scaffold comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:

24.

4. The anti-B7-H3 compound of any one of claims 1 to 3, further comprising a first functional moiety operably linked to said anti-B7-H3 Gp2-based scaffold.

5. 5. The anti-B7-H3 compound of claim 4, wherein the first functional moiety comprises a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug.

6. The anti-B7-H3 compound of claim 5, wherein the targeting moiety binds to an extracellular domain of a protein presented on natural killer cells or T cells.

7. The anti-B7-H3 compound of claim 5, wherein the imaging moiety comprises a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label.

8. The anti-B7-H3 compound of claim 5 or 7, wherein the enzyme is sortase A.

9. The anti-B7-H3 compound of claim 5, wherein the small molecule drug comprises one or more radioisotopes.

10. The anti-B7-H3 compound of any one of claims 1 to 9, further comprising a second functional moiety operably linked to the anti-B7-H3 Gp2-based scaffold.

11. 11. The anti-B7-H3 compound of claim 10, wherein the second functional moiety comprises a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug.

12. 11. The anti-B7-H3 compound of claim 10, wherein the first functional moiety is directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional moiety is directly linked to the anti-B7-H3 Gp2-based scaffold.

13. 11. The anti-B7-H3 compound of claim 10, wherein the first functional moiety is directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional moiety is directly linked to the first functional moiety.

14. An anti-B7-H3 compound according to any one of claims 1 to 13, A pharma- ceutically acceptable carrier; A composition comprising:

15. A method comprising administering an anti-B7-H3 compound according to any one of claims 1 to 13 or a composition according to claim 14 to a subject.

16. The method of claim 15 , wherein the subject has a tumor.

17. 17. The method of claim 15 or 16, wherein the subject has cancer.

18. The method of any one of claims 15 to 17, wherein the composition is administered prior to, simultaneously with, or after chemotherapy, surgical resection of a tumor, or radiation therapy.

19. The method of any one of claims 15 to 18, further comprising the imaging moiety for detecting an anti-B7-H3 compound bound to B7-H3 expressed by a cell.

20. 20. The method of claim 19, wherein the cell is a cancer cell.

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