Anti-B7-H3 Compounds and Methods of Use Thereof

Abstract

The anti-B7-H3 compound includes an anti-B7-H3 affibody. The anti-B7-H3 compound may include one or more functional moieties, such as a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug. A composition including anti-B7-H3 may be administered to a subject. The subject may have or be at risk of having a tumor or cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 302,290, 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 the invention. [Background technology]

[0003] Sequence Listing This application contains a sequence listing that has been submitted electronically to the U.S. Patent and Trademark Office via the Patent Center as an XML file entitled "0110_000692WO01.xml", created on January 22, 2023, with a size of 49.6 kilobytes. By filing the sequence listing electronically, the electronically submitted sequence listing 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 includes an anti-B7-H3 affibody. The anti-B7-H3 affibody includes an amino acid sequence of SEQ ID NO:2, an amino acid sequence having at least 90% sequence similarity with SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2.

[0005] In one or more embodiments, the anti-B7-H3 affibody comprises any one of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4, 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:22, SEQ ID NO:23, 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, and SEQ ID NO:34, an amino acid sequence having at least 90% sequence similarity to any of the listed sequences, or an amino acid sequence having at least 90% sequence identity to any of the listed sequences.

[0006] In one or more embodiments, the anti-B7-H3 compound comprises a first functional moiety operably linked to a B7-H3 affibody, hi one or more embodiments, the first functional moiety comprises a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug.

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

[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 invention. The following description more particularly illustrates preferred embodiments by way of example. In several places in this application, guidance is provided by way of listing examples, which examples can be used in various combinations. In each instance, the listing provided represents only a representative group and should not be construed as an exclusive listing. [Brief description of the drawings]

[0011] [Figure 1]Figure 1. Enriched affibody (ABY) binders for B7-H3. (A) Yeast were subjected to three rounds of magnetic activated cell sorting (MACS) selection with soluble B7-H3 extracellular domain (populations 0.1, 0.2, and 0.3 on the x-axis), which show enrichment for the B7-H3 target over negative Renilla reniformis green fluorescent protein (rrGFP) and avidin beads. The enriched ABY population was subjected to fluorescence activated cell sorting (FACS) selection against B7-H3 (generating population 0.4 on the x-axis). ND indicates no data, as binding to avidin beads and negative rrGFP was not analyzed in that sort. The ABY library from population 0.4 was extracted and subjected to error-prone PCR before being electroporated back into yeast. The mutated naive library was subjected to MACS selection (generating population 1.1 on the x-axis) followed by monovalent MACS selection with 100 nM recombinant human B7-H3 (generating population 1.2 on the x-axis). The remaining selections were performed by FACS (achieving populations 1.3 and 1.4). (B) After three rounds of MACS selection, yeast were incubated with 50 nM recombinant human B7-H3 extracellular domain and streptavidin-Alexa Fluor 647 was added. ABY display was quantified using mouse anti-c-Myc antibody and goat anti-mouse-Alexa Fluor 488. Flow cytometry analysis of 10,000 random variants is shown (population 0.4). (C) The enriched population was labeled with biotin-B7-H3 cell lysate, streptavidin-AF647, and chicken anti-c-Myc-FITC. The harvested cells were designated population 1.4. The boxes in (B) and (C) are cells collected during FACS sorting. [Diagram 2]Figure 2. Protease and thermostability of enriched ABY populations to generate population 1.5. (A) The affibody library was incubated with 1x10-6 units / μL proteinase K for 10 min at 55°C, followed by incubation with biotinylated detergent-solubilized MS1-B7-H3 cell lysate and labeling with streptavidin-Alexa Fluor 647. The top 0.5% of B7-H3 binders were selected by flow cytometry (grey box). (B) The affibody library was incubated with proteinase K (+protease) or without proteinase K (control) at 55°C, followed by incubation with biotinylated B7-H3 lysate. The slight decrease in B7-H3 binding of the +protease sample compared to the control indicates that some variants are sensitive to proteinase K and elevated temperature. [Diagram 3]Figure 3 shows the sequences of evolved B7-H3 binding affibodies in populations 0.4, 1.4, and 1.5. ABY025 refers to the affibody sequence (SEQ ID NO: 18) from which the affibodies of the present disclosure were derived (see Design of an Optimized Scaffold for Affibody Molecules, 2010 (doi:10.1016 / j.jmb.2010.03.002)). The design of the affibody library is also shown. Eight colonies were sequenced in population 0.4 (0.4.1 = SEQ ID NO: 4; 0.4.2 = SEQ ID NO: 5; 0.4.3 = SEQ ID NO: 6; 0.4.4 = SEQ ID NO: 7; 0.4.5 = SEQ ID NO: 8; 0.4.6 = SEQ ID NO: 9; 0.4.7 = SEQ ID NO: 10), of which 0.4.2 was observed twice (indicated as "x2"). The populations show diverse amino acid sequences among the variants. Ten colonies were sequenced in population 1.4 (1.4.1 = SEQ ID NO:11; 1.4.2 = SEQ ID NO:12; 1.4.3 = SEQ ID NO:13; 1.4.4 = SEQ ID NO:14; 1.4.5 = SEQ ID NO:15; 1.4.6 = SEQ ID NO:16; 1.4.7 = SEQ ID NO:17; 1.4.8 = SEQ ID NO:18), of which 1.4.1 and 1.4.2 were observed twice (indicated as "x2"). Seven colonies were sequenced in population 1.5 (1.5.1 = SEQ ID NO:19; 1.5.2 = SEQ ID NO:20), of which 1.5.1 was observed five times (indicated as "x5") and 1.5.2 was observed twice (indicated as "x2"). The amino acid diversity decreased and eventually began to indicate the predominant clones present in the enriched B7-H3 binding affibody population. [Figure 4] Figure 4 shows ABY 1.5.2 (SEQ ID NO: 20) genetically coupled to the GSG3SG3KG3GT (GSGGGSGGGKGGGGT) (SEQ ID NO: 35) linker and sortase A60-206 and produced in bacteria. The Kd of ABY 1.5.2-L15-SrtA was 4.9 nM (range 4.0-5.9 nM). Error bars indicate standard error and at least two replicates were performed for each concentration. [Diagram 5]Figure 5 shows the best candidate sequences after mutating population 1.5 and screening by avidity, affinity, specificity and stability selection. The sequence space shows variability compared to affibody 1.5.1 in population 1.5 (see Figure 3), except for the 1.5.1 point mutants Otu8 and Otu394. [Figure 6] Figure 6 shows the relative enrichment at each selection step and the weighted average z-score for the top variants (sequences shown in Figure 5). Values ​​greater than 1 indicate enrichment and values ​​less than 1 indicate depletion. Colored bars range from 1 to M+2SD for affinity enrichment, as the mean (M) was slightly below 1. Color bars for specificity, stability, and frequency range from M to M+2SD. z-scores range from 1 to 10. [Figure 7] Figure 7 is a plot showing the relative frequency of the top affibodies after each selection (avidity selection, affinity selection, specificity selection, and stability selection). Each line represents a single variant. For selected variants, the sequence names from Figure 5 are displayed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure describes anti-B7-H3 affibodies, compounds comprising at least one anti-B7-H3 affibody, 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 affibody. Examples of platforms in which anti-B7-H3 compounds may be used include, but are not limited to, chimeric antigen receptor therapy (such as, for example, CAR-NK therapy, CAR-T therapy, and CAR-macrophage therapy), multispecific immune cell engager technologies (such as, for example, bispecific killer engagers, trispecific killer engagers, bispecific T cell engagers, and trispecific T cell engagers), targeted immunotherapy (such as, for example, targeted ADAM17 blocker (TAB) therapy), delivery of therapeutic agents (such as, for example, antibody-drug conjugates, delivery of therapeutic radioisotopes, delivery of toxins, delivery of cytokines, and delivery of chemokines), imaging technologies (delivery of labeled constructs and / or labeled radioisotopes), or cell and / or ligand capture technologies (such as, for example, ELISA).

[0013] 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 with two isoforms determined by its extracellular domain. B7-H3 mRNA is expressed in most normal tissues. In contrast, B7-H3 protein is post-transcriptionally regulated by microRNAs, resulting in very limited expression in normal tissues. In normal tissues, B7-H3 plays a predominantly 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 biomarker of tumor vasculature, and is overexpressed in a variety of cancers, including clear cell renal cell carcinoma, cutaneous melanoma, diffuse pontine glioma, hypopharyngeal squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, prostate cancer, and pancreatic cancer. B7-H3 expression is associated with tumor growth and metastasis, which may ultimately lead to poor clinical prognosis.

[0014] Molecules that recognize specific targets with specificity and high affinity are useful in many clinical (e.g., diagnostic and / or therapeutic) and biotechnological applications. Antibodies have typically been used for many of these applications, but antibodies have properties that can be drawbacks in certain applications. These limitations of antibodies have prompted research into alternative protein scaffolds that can efficiently generate improved binding molecules. For example, antibodies are typically immunoglobulin G (IgG) and are approximately 150 kDa, but when targeting solid tumors, due at least in part to their size, they may have poor extravasation from the vasculature, poor tissue penetration, and / or long plasma clearance half-lives, resulting in low signal-to-noise ratios, especially in imaging diagnostics. Antibodies may also exhibit thermal instability, which may result in denaturation and / or aggregation, resulting in loss of efficacy. In addition, antibodies are typically produced in mammalian culture medium, as many of them have disulfide bonds, glycosylation, and / or multi-domain structures. This complex structure can hinder the design of antibodies for specific applications, such as the generation of protein fusions for multispecific formats, etc. Furthermore, the presence of disulfide bonds in antibody molecules often precludes their use intracellularly.

[0015] These inherent limitations of antibodies have led to the development of alternative protein scaffolds in an attempt to address many or all of these shortcomings. The present disclosure describes recombinant, non-naturally occurring protein scaffolds, called affibodies, that can bind to the extracellular domain of B7-H3. In particular, the affibodies described herein can be used to present defined helical surfaces that resemble the complementarity determining regions ("CDRs") of antibody variable regions. Affibodies can be constructed into multispecific compounds capable of binding the extracellular domain of B7-H3 and one or more different targets. Thus, the affibodies described herein can provide functional properties typically associated with antibody molecules. In particular, despite the fact that the affibody scaffold is not an immunoglobulin, it resembles in relevant respects the scaffold of the variable region of an IgG heavy chain, allowing the protein scaffold to express engineered binding paratopes that resemble antibody CDRs. Because of this structure, the ligand binding properties of the affibodies described herein can be similar in nature and affinity to those of antigens and antibodies.

[0016] In one embodiment, the present disclosure describes an anti-B7-H3 compound. The anti-B7-H3 compound is or comprises an anti-B7-H3 affibody. The term "affibody" as used herein refers to a protein that comprises at least one antigen binding site that binds to a specific antigen of interest. The anti-B7-H3 affibody of the present disclosure binds to the extracellular domain of the B7-H3 protein (hereinafter B7-H3). The anti-B7-H3 affibody of the present disclosure is developed from the affibody of SEQ ID NO:1.

[0017] An anti-B7-H3 affibody of the disclosure comprises the amino acid sequence AEAKYXKEXXXAXXXIXXLPNLXXXQXXAFXXXLXXDPSQSSELXXEAXKLNDSQAPK (SEQ ID NO:2), or a structurally similar sequence, where each X in SEQ ID NO:2 is a natural amino acid, a non-natural amino acid, or a modified amino acid, independent of the amino acid residues at other positions in the affibody. In one or more embodiments, X at position 6 is Y, N, S, T, D, or A; X at position 9 is L, R, K, W, D, I, N, or Q; X at position 10 is H, A, S, F, I, D, E, Q, T, N, or L; X at position 11 is T, Y, D, A, L, P, I, or N; X at position 13 is I, Y, L, V, T, or F; X at position 14 is Q, Y, L, V, G, F, E, A, M, or S; X at position 15 is V, S, Q, or E; X at position 17 is H, A, V, G, R, I, F, or L; X at position 18 is S, Y, L, G, T, N, M, D, R, W, E, or H; and X at position 23 is T or A. at position 24 is L, K, A, G, R, H, W, V, or E; at position 25 is D, P, T, N, V, S, H, L, R, A, W, or E; at position 27 is R, K, T, I, or A; at position 28 is A, Y, R, N, D, Q, I, H, L, S, or F; at position 31 is L, K, I, or T; at position 32 is L, G, Y, R, A, F, V, S, or Q; at position 32 is A or K; at position 35 is F, S, V, H, Y, D, E, or K; at position 36 is N or D; at position 44 is L or F; at position 46 is S or P; and at position 49 is R or K.

[0018] As used herein, a polypeptide (e.g., an affibody) is "structurally similar" to a reference polypeptide if the amino acid sequence of the polypeptide has a certain amount of identity compared to the reference polypeptide (e.g., an anti-B7-H3 affibody comprising SEQ ID NO:3). 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:3) to optimize the number of identical amino acids along the length of the sequences, and gaps may be inserted into one or both sequences when aligning to optimize the number of identical amino acids, but the order of the amino acids in each sequence must still be correct. A candidate polypeptide is a polypeptide that is compared to a reference polypeptide (e.g., SEQ ID NO:3). A candidate polypeptide can be isolated, for example, from an animal or other natural source, can be produced using recombinant techniques, or can be synthesized chemically or enzymatically.

[0019] Pairwise comparative analysis of amino acid sequences can be performed using the BESTFIT algorithm of the GCG package (version 10.2, Madison, Wis.). Alternatively, polypeptides can be compared using the Blastp program of the BLAST2 search algorithm available on the National Center for Biotechnology Information (NCBI) website as described in Tatiana et al. (FEMS Microbiol Lett, 174, 247-250 (1999)). Default values ​​for all BLAST2 search parameters can be used, including "matrix=BLOSUM62", "open gap penalty=11", "extension gap penalty=1", "gap x_dropoff=50", "expect=10", "wordsize=3", and "filter on".

[0020] In comparing two amino acid sequences, structural similarity can be expressed as a percentage of "identity" or a percentage of "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to identical amino acids, but also to the presence of conservative substitutions. Conservative substitutions of amino acids in a polypeptide can be selected from other members of the class to which the amino acid belongs. For example, it is well known in the field of protein biochemistry that amino acids belonging to a certain group of amino acids with a particular size or characteristics (such as charge, hydrophobicity, and hydrophilicity) can be substituted with other amino acids 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. Neutral polar 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. Examples of conservative substitutions include substitution of Arg with Lys or vice versa to maintain a positive charge; substitution of Asp with Glu or vice versa to maintain a negative charge; substitution of Thr with Ser to maintain a free -OH; and substitution of Asn with Gln to maintain a free -NH2. Similarly, biologically active analogs of polypeptides that include 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.

[0021] The anti-B7-H3 affibodies 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 SEQ ID NO:2, where each X is independently selected from any amino acid. The anti-B7-H3 affibodies 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 SEQ ID NO:2. X at position 6 is Y, N, S, T, D, or A; X at position 9 is L, R, K, W, D, I, N, or Q; X at position 10 is H, A, S, F, I, D, E, Q, T, N, or L; X at position 11 is T, Y, D, A, L, P, I, or N; X at position 13 is I, Y, L, V, T, or F; X at position 14 is Q, Y, L, V, G, F, E, A, M, or S; and X at position 15 is V, X at position 17 is H, A, V, G, R, I, F, or L; X at position 18 is S, Y, L, G, T, N, M, D, R, W, E, or H; X at position 23 is T or A; X at position 24 is L, K, A, G, R, H, W, V, or E; X at position 25 is D, P, T, N, V, S, H, L, R, A, W, or E; X at position 27 is R, K, T, I, or A; X is A, Y, R, N, D, Q, I, H, L, S, or F; X at position 31 is L, K, I, or T; X at position 32 is L, G, Y, R, A, F, V, S, or Q; X at position 32 is A or K; X at position 35 is F, S, V, H, Y, D, E, or K; X at position 36 is N or D; X at position 44 is L or F; X at position 46 is S or P; and X at position 49 is R or K.

[0022] The anti-B7-H3 affibodies 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:3-SEQ ID NO:20 or SEQ ID NO:22-SEQ ID NO:34.

[0023] The anti-B7-H3 affibodies 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 similarity to any one of SEQ ID NO:3-SEQ ID NO:20 or SEQ ID NO:22-SEQ ID NO:34.

[0024] This disclosure does not disclose SEQ ID NO: 21. Thus, a description of a range of SEQ ID NOs that includes SEQ ID NO: 21 does not include SEQ ID NO: 21. For example, the description "any one of SEQ ID NOs: 2 to 34" refers to any one of SEQ ID NOs: 3 to 20, and any one of SEQ ID NOs: 22 to 34.

[0025] In one or more embodiments, amino acids in the helical surface region of any one of SEQ ID NOs: 2 to 34 may be mutated. The mutations may include substitution of one or more amino acid residues with different amino acid residues, deletion of one or more amino acid residues, addition of one or more amino acid residues, or any combination thereof, in any one of SEQ ID NOs: 2 to 34.

[0026] For example, in one or more embodiments, there may be one or more mutations at amino acid residues 6-18 in any one of SEQ ID NO:2-34. In some of these embodiments, there may be a mutation at residue 6 in any one of SEQ ID NO:2-34. In one or more embodiments, there may be one or more mutations at amino acid residues 9-11 in any one of SEQ ID NO:2-34. In one or more embodiments, there may be one or more mutations at amino acid residues 13-15 in any one of SEQ ID NO:2-34. In one or more embodiments, there may be a mutation at amino acid residues 17 and / or 18 in any one of SEQ ID NO:2-34. In one or more embodiments, there may be one or more mutations at amino acid residues 24-36 of SEQ ID NO:19. In some of these embodiments, there may be a mutation at residues 24 and / or 25 in any one of SEQ ID NO:2-34. In one or more embodiments, there may be a mutation at residues 27 and / or 28 in any one of SEQ ID NO:2-34. In one or more embodiments, there may be one or more mutations at amino acid residues 31-33 in any one of SEQ ID NOs: 2-34. In one or more embodiments, there may be a mutation at residues 35 and / or 36 in any one of SEQ ID NOs: 2-34. In one or more embodiments, there may be one or more mutations in any of the amino acid residue regions described herein.

[0027] In one or more embodiments, one or more amino acid mutations may be present outside the helical surface region in any one of SEQ ID NOs: 2 to 34. For example, in one or more embodiments, one or more mutations may be present at amino acid residues 1 to 5 in any one of SEQ ID NOs: 2 to 34. In one or more embodiments, one or more mutations may be present at amino acid residues 19 to 23 in any one of SEQ ID NOs: 2 to 34. In one or more embodiments, one or more mutations may be present at amino acid residues 37 to 58 in any one of SEQ ID NOs: 2 to 34.

[0028] In one or more embodiments, any one of SEQ ID NO:2 to SEQ ID NO:34 may have one or more mutations within one or more helical surface regions, and may have one or more amino acid mutations outside the helical surface region.

[0029] Table 1 shows the amino acid sequence of the parent affibody (SEQ ID NO:1) from which the anti-B7-H3 affibody of the disclosure is derived; the general consensus sequence (SEQ ID NO:2); and the second consensus sequence (SEQ ID NO:3) of SEQ ID NOs:4-18. Table 1 also shows example identities of X relative to the general consensus sequence (SEQ ID NO:2) for SEQ ID NO:4 or any one of SEQ ID NOs:3-34, as well as example mutations. In one or more embodiments, an anti-B7-H3 affibody scaffold of the disclosure comprises any one of SEQ ID NOs:2-34 in any combination of the examples of possible mutations or possible identities of X shown in Table 1. [Table 1-1] [Table 1-2]

[0030] Variants of the disclosed sequences also include affibody fragments or full-length affibodies that contain substitutions, deletions, or insertions to the protein backbone, but that retain at least about 70% homology with the original affibody over the corresponding portions. Larger deviations in homology are also permitted, where similar amino acids, i.e., conservative amino acid substitutions, are not counted as sequence changes. Examples of conservative substitutions involve amino acids with identical or similar properties. Examples of conservative amino acid substitutions include the following changes: 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 to methionine; serine to threonine or alanine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

[0031] In one or more embodiments, the anti-B7-H3 affibody 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 affibody. Such modifications may, for example, facilitate purification by trapping on a column, using antibodies, or facilitate recovery when recombinantly expressed in a microorganism. Such tags include, for example, histidine-rich tags that allow purification of the protein on a nickel column, and / or leader sequences that allow transport of the recombinantly expressed anti-B7-H3 affibody to the membrane of the cell in which it was recombinantly expressed. Such genetic engineering techniques and suitable additional sequences are well known in the art of molecular biology. In one or more embodiments, the C-terminus and / or N-terminus modifications may be cleaved from the anti-B7-H3 affibody prior to incorporation, such as in a pharmaceutical composition. In other embodiments, retention of the C-terminus or N-terminus modifications may be desirable for certain applications, such as to facilitate immobilization to a substrate.

[0032] In one or more embodiments, the anti-B7-H3 affibodies described herein may also include N- or C-terminal functional groups other than carboxylic acids or free amines. For example, the C-terminus, the N-terminus, or both of the affibody may be acylated, e.g., acetylated. In one or more embodiments, the functional group may include a polyol (e.g., polyethylene glycol).

[0033] In one or more embodiments, the anti-B7-H3 affibodies described herein may comprise a post-translational modification on 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 can be incorporated specifically or non-specifically by methods known in the biological arts.

[0034] In one or more embodiments, the anti-B7-H3 affibodies may 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.

[0035] In one or more embodiments, the anti-B7-H3 affibodies of the present disclosure may be produced using solid phase peptide synthesis. In this production method, the anti-B7-H3 affibodies may be synthesized as a single complete polypeptide or in the form of multiple polypeptide fragments that can be joined using native chemical ligation, expressed protein ligation, Staudinger ligation, or Ser / Thr ligation. Solid phase peptide synthesis and ligation techniques are well known in the art.

[0036] In another aspect, the disclosure describes polynucleotides encoding any of the anti-B7-H3 affibodies described herein, and the complement of such polynucleotide sequences. Given the amino acid sequence of any anti-B7-H3 affibody described herein, one of skill in the art can determine, using routine conventional methods, the entire polynucleotide that encodes that amino acid sequence.

[0037] Anti-B7-H3 affibodies of the present disclosure may be designed using a variety of techniques and assays, for example, magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) may be used to select affibodies of interest that bind to B7-H3 in a yeast display model.

[0038] The following describes the campaign used to design at least some of the anti-B7-H3 affibodies of the present disclosure (see Examples 1 and 2 for further details). An affibody scaffold library comprising affibody amino acid sequences from SEQ ID NO:1 was used to discover binders to B7-H3 by yeast display, magnetic activated cell sorting, and fluorescence activated cell sorting. The library (Affibody population 0.0; also referred to as Library) was subjected to three rounds of MACS selection with recombinant human B7-H3 extracellular domain immobilized on magnetic beads (generating Affibody populations 0.1, 0.2, and 0.3), followed by a single FACS selection with the same recombinant B7-H3 protein at 50 nM, and affibodies with intermediate affinity were selected by gating into population 0.4. DNA was isolated from the entire Affibody population 0.4, randomly mutated by error-prone PCR, and electroporated back into yeast. The mutated naive population was subjected to MACS selection (generating affibody population 1.1) followed by monovalent MACS sorting with 100 nM recombinant human B7-H3 (generating affibody population 1.2). Affibody population 1.2 was then further enriched by two rounds of FACS selection with detergent-solubilized MS1-B7-H3 lysate of increasing stringency, using estimated B7-H3 lysate concentrations of 50 nM and 1 nM (generating affibody populations 1.3 and 1.4). Yeast were grown and re-induced between each selection. Affibody population 1.4 was subsequently subjected to another round of selection selecting for protease and thermostable affibodies that bind to B7-H3, resulting in population 1.5 (Figure 2).

[0039] FIG. 1A is a plot showing the theoretical diversity of affibody populations after various sorts (0.4 is population 0.4). Populations 0.2, 0.3, and 1.2 showed specific binding to B7-H3 and no binding to the negative control. The higher the theoretical diversity, the more cells were collected for a given parameter (e.g., control, negative, or B7-H3 binders). Population 1.1 had the highest theoretical diversity of cells that bound B7-H3. FIG. 1B is a flow cytometric analysis (FACS) of 10,000 random variants of population 0.4, resulting in intermediate affinity binders to recombinant B7-H3. FIG. 1C is a flow cytometric analysis of enriched population 1.4, resulting from mutation (indicated by gray box) of population 0.4 from FIG. 1B and subsequent sorting, as described above. Figure 1C shows labeling of ~1 nM biotin-B7-H3 cell lysate, streptavidin-AF647, and chicken anti-c-Myc-FITC. The result was high affinity binders to cellular B7-H3. The boxes in Figure 1B and 1C show the populations collected during FACS sorting. The collected cells express full-length affibodies and bind B7-H3. In Figure 1B, a rectangle was drawn to select binders since the majority of the population was non-binding. In Figure 1C, a shaded polygon was drawn to indicate higher stringency, resulting in the collection of top binders with higher affinity.

[0040] Single colonies from the affibody population were randomly picked and Sanger sequenced at two selection points (see Figure 3): (1) after the first FACS sort (fourth overall round of sorting; generating population 0.4) and after additional selection after error-prone PCR (two rounds of MACS selection and two rounds of FACS lysate selection; generating population 1.4). Affibody variants were subjected to a third round of Sanger sequencing following protease and thermostable B7-H3 binding selection (generating population 1.5). The sequencing results of the affibodies are shown in Figure 3 (ABY025, the affibody sequence from which the anti-B7-H3 affibody was derived = SEQ ID NO:1; 0.4.1 = SEQ ID NO:4; 0.4.2 = SEQ ID NO:5; 0.4.3 = SEQ ID NO:6; 0.4.4 = SEQ ID NO:7; 0.4.5 = SEQ ID NO:8; 0.4.6 = SEQ ID NO:9; 0.4.7 = SEQ ID NO:10; 1.4.1 = SEQ ID NO:11; 1.4.2 = SEQ ID NO:12; 1.4.3 = SEQ ID NO:13; 1.4.4 = SEQ ID NO:14; 1.4.5 = SEQ ID NO:15; 1.4.6 = SEQ ID NO:16; 1.4.7 = SEQ ID NO:17; 1.4.8 = SEQ ID NO:18; 1.5.1 = SEQ ID NO:19; 1.5.2 = SEQ ID NO:20).

[0041] Affibody population 1.5 was further engineered for affinity, specificity, and stability maturation (see Example 2 for further details). DNA was isolated from yeast population 1.5 and random mutagenesis of the affibody variable regions was performed by error-prone polymerase chain reaction (PCR). DNA was electroporated back into yeast for display and further selection using the following selection schemes: (1) lysate MACS using 1 pmol of B7-H3+ lysate (avidity selection); (2) affinity selection (strict K d (3) specificity selection (multi-target MACS depletion selection); and (4) stability selection (thermolysin FACS selection at 55° C.). After each selection, the populations were subjected to deep sequencing.

[0042] For each sequence, the frequency was determined for each sort (e.g., avidity, affinity, specificity, and stability) and for subsequent enrichments. If the read count was 0, a read count of 1 was assigned. The z-scores of affinity, specificity, stability, and final frequency of the top variants were determined, and a final conditional weighted average z-score was calculated (see Example 2). The best candidates were selected based on the highest scores. Figure 5 shows the sequences of the best candidates in population 1.5.1 after completing all sorts.

[0043] Figure 6 shows z-scores for affinity, specificity, stability, and final frequency of the top variants, as well as the conditional weighted average z-score. Values ​​greater than 1 indicate enrichment, and values ​​less than 1 indicate depletion. In Figure 6, the colored bars represent enrichment ranges of M to M+2SD for affinity, and 1 to M+2SD for specificity and stability, since the mean (M) was slightly below 1. In the bar graphs, the z-scores are a representation of 1 to 10 (i.e., for z-scores 10 or greater, the bar spans the entire length of the figure).

[0044] Figure 7 is a plot showing the relative frequency of a particular variant (each line) after each selection (avidity, affinity, specificity, and stability). An ideal variant would be represented by a line with high relative frequency in all selections.

[0045] In one or more embodiments, the anti-B7-H3 compound may include, in addition to an anti-B7-H3 affibody, a first functional moiety operably linked to the anti-B7-H3 affibody. In one or more embodiments, the first functional moiety may include a targeting moiety. In one or more embodiments, the first functional moiety may include an imaging moiety. In one or more embodiments, the first functional moiety may include an enzyme. In one or more embodiments, the first functional moiety may include a small molecule drug, a pharmacologically active derivative thereof, or an activatable inactive form of a drug (e.g., a prodrug).

[0046] 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 may be an immunotherapeutic compound. The immunotherapeutic compound may provide personalized therapy that amplifies or attenuates the immune response by activating or suppressing the immune system and is rapidly being developed for the treatment of various forms of cancer. Immunotherapies for cancer, such as those with 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.

[0047] In one or more embodiments, the targeting moiety may bind to cells presenting B7-H3 and recruit effector cells thereto. The type of effector cell may be, but is not limited to, a natural killer (NK) cell, a B cell, or a T cell. The targeting moiety may bind to a surface protein presented on an 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.

[0048] 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.

[0049] In one or more embodiments, the first functional moiety includes an imaging moiety. In embodiments in which the first functional moiety includes an imaging moiety, the anti-B7-H3 compound may be used as a diagnostic tool to detect B7-H3 positive tumors.

[0050] The imaging moiety may be any moiety capable of generating a detectable signal. Examples of 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 may be used to detect the imaging moiety. Examples of 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) nanoscopic. Imaging techniques may be accomplished in vivo or in vitro.

[0051] In one or more embodiments, the first functional component may comprise an enzyme. In one or more embodiments, the enzyme may modify one or more surface proteins displayed on a cell that displays B7-H3 to which an anti-B7-H3 affibody is bound. Examples of enzymes that modify surface proteins include, but are not limited to, proteases; lipases; or sortases, such as sortase A.

[0052] For example, to analyze the modularity of affibodies, affibody 1.5.2 (SEQ ID NO: 20) was genetically linked to a sortase A protein enzyme via a 15 amino acid glycine-rich linker (SEQ ID NO: 35) at the affibody C-terminus (i.e., affibody 1.5.2-L). 15 -SrtA fusion (see Example 1). Modularity was assessed by retention of B7-H3 binding affinity of the affibody in the presence of glycine-linked sortase A enzyme. Binding affinity of affibody 1.5.2 alone was not assessed, but affibody 1.5.2-L was. 15 -SrtA is K d was 4.9 nM (4.0-5.9 nM), indicating low nanomolar affinity in the presence of ligated sortase A (Figure 4).

[0053] In one or more embodiments, the first functional component may include a small molecule drug. As used herein, the term "drug" is used to collectively 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 in 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. Anti-B7-H3 affibodies may enable localization of small molecule drugs to B7-H3-presenting cells, such as tumor cells, thereby delivering chemotherapeutic compounds to tumor cells expressing B7-H3. Thus, the chemotherapeutic compounds may have enhanced local activity and reduced systemic side effects while being administered systemically to a subject, as compared to systemic administration of a drug that is not a component of an anti-B7-H3 compound.

[0054] In one or more embodiments, an anti-B7-H3 compound may include additional functional moieties in addition to the first functional moiety and the anti-B7-H3 affibody. For example, an anti-B7-H3 compound may include a second functional moiety, a third functional moiety, a fourth functional moiety, a fifth functional moiety, a sixth functional moiety, or a seventh functional moiety. Each functional moiety may include an anti-B7-H3 affibody, and may also include an imaging moiety, an enzyme, a small molecule drug, or a targeting moiety (all of which are described herein above), regardless of the identity of any other moiety in the anti-B7-H3 compound.

[0055] In some embodiments where the anti-B7-H3 compound comprises at least one functional component, the anti-B7-H3 affibody and the functional component are operably linked. As used herein, the term "operably linked" refers to a direct or indirect covalent bond between the anti-B7-H3 affibody and any additional functional component or components of the anti-B7-H3 compound. Thus, two operably linked functional components, or a functional component and an anti-B7-H3 affibody, may be directly covalently linked to each other. Conversely, two operably linked functional components, or a functional component operably linked to an anti-B7-H3 affibody, may be linked by covalently bonding to each other to an intervening component (e.g., a flanking sequence or a linker). For example, in embodiments in which the anti-B7-H3 compound comprises two functional moieties, the first and second functional moieties may be separately linked directly to the anti-B7-H3 affibody; or, the first functional moiety may be directly linked to the anti-B7-H3 affibody and the second functional moiety may be directly linked to the first functional moiety.

[0056] The anti-B7-H3 affibody 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 functional components of the anti-B7-H3 compound. 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 that maintain the activity of the first functional component (or other functional components) and / or the anti-B7-H3 affibody. Linkers are classified based on the presence of one or more chemical motifs, such as, for example, including disulfide groups, hydrazine groups, or peptides (cleavable), or thioester groups (non-cleavable). Linkers also include charged linkers and hydrophilic forms thereof known in the art.

[0057] Linkers suitable for linking the anti-B7-H3 affibody and the first functional component of the anti-B7-H3 compound 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 and naturally occur between protein domains. 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 linking the components of the anti-B7-H3 compound of the present disclosure.

[0058] Research into linkers in natural multidomain proteins has produced a large number of empirical linkers with various sequences and conformations for constructing 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 to the attached 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 to the attached components. One example of a flexible linker useful for linking an anti-B7-H3 affibody to a first functional component of an anti-B7-H3 compound of the present disclosure is SEQ ID NO: 35. Rigid linkers can successfully keep the distance between the first functional component of an anti-B7-H3 compound and an anti-B7-H3 affibody constant and maintain their independent functions. This provides efficient separation of the first functional component and the anti-B7-H3 affibody and / or sufficiently reduces interference between the first functional component and the anti-B7-H3 affibody. The cleavable linker may allow for controlled detachment of the first functional component and / or the anti-B7-H3 affibody in vivo. By utilizing inherent in vivo processes, the cleavable linker can be cleaved under specific conditions, such as in the presence of reducing agents or proteases. 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 affibody after linker cleavage.

[0059] Examples of suitable linker sequences include the amino acid sequences of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47.

[0060] In one or more embodiments, the natural or empirical linker is covalently attached to the anti-B7-H3 affibody, 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-arylpropiolonitrile conjugation, diazonium salt conjugation, 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione conjugation, and Mannich ligation.

[0061] In one or more embodiments, the natural or empirical linker, the first functional component, the anti-B7-H3 affibody, or a combination thereof, may include one or more unnatural amino acids that enable a bioorthogonal conjugation reaction. 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, but are not limited to, 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 ε-2-azidoethyloxycarbonyl-L-lysine, Nε-p-azidobenzyloxycarbonyl-L-lysine, propargyl-L-lysine, or trans-cyclooct-2-enelysine. Such amino acids may be incorporated at any position in the anti-B7-H3 affibody of the disclosure or at any position in the functional moiety.

[0062] 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 for conjugation to the anti-B7-H3 affibody, domain, or both. In one or more embodiments, the reactive handles allow for bioconjugation or bioorthogonal conjugation. In one or more embodiments, the reactive handles allow for any organic reaction compatible with conjugating the linker to the anti-B7-H3 affibody or first functional moiety.

[0063] The linker may be conjugated to any amino acid position of the anti-B7-H3 affibody, for example, the linker may be conjugated to the N-terminus, the C-terminus, or any amino acid in between.

[0064] In embodiments in which the anti-B7-H3 compound comprises multiple functional moieties, those additional functional moieties may be operably linked to each other and / or to the anti-B7-H3 affibody using one or more of the linkers disclosed herein.

[0065] In some embodiments, where an anti-B7-H3 compound comprises an anti-B7-H3 affibody and one or more functional moieties operably linked by a peptide linker, the anti-B7-H3 compound may be produced by expression in a host cell using methods known in the art. In some embodiments, where an anti-B7-H3 compound comprises an anti-B7-H3 affibody and one or more functional moieties operably linked by a peptide linker, the anti-B7-H3 compound may be produced by use of solid phase peptide synthesis using methods known in the art.

[0066] In another aspect, the disclosure describes isolated nucleic acid sequences that encode the amino acid sequence of any embodiment of the anti-B7-H3 compounds described herein, or any component polypeptide fragment thereof. Given the amino acid sequence of any polypeptide, one of skill in the art can determine, using routine conventional methods, the entire polynucleotide that encodes that amino acid sequence.

[0067] In another aspect, the disclosure describes host cells comprising any of the isolated nucleic acid sequences, anti-B7-H3 compounds, and / or anti-B7-H3 affibodies described herein.

[0068] The nucleic acid constructs of the present disclosure may be introduced into a host cell to be modified to allow expression of anti-B7-H3 compounds and / or anti-B7-H3 affibodies in the cell, thereby generating a genetically engineered cell. A variety of suitable methods are known for introducing nucleic acids into cells, including viral and non-viral mediated techniques. Exemplary non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated introduction, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated introduction, microinjection, biolistic mediated introduction (e.g., nanoparticles), cationic polymer mediated introduction (e.g., DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), and the like), or cell fusion. Other transfection methods include proprietary transfection reagents such as LIPOFECTAMINE (Thermo Fisher Scientific, Waltham, MA), HILYMAX (Dojindo Molecular Technologies, Rockville, MD), FUGENE (Promega, Madison, WI), JETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany), and DreamFect (OZ Biosciences, San Diego, CA, USA).

[0069] The nucleic acid constructs described herein may be introduced into a host cell to be modified to allow expression of the anti-B7-H3 compound and / or anti-B7-H3 affibody encoded by the nucleic acid in the cell. A variety of suitable host cells for protein expression are known in the art. Exemplary 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.

[0070] In another aspect, the disclosure provides pharmaceutical compositions containing one or more anti-B7-H3 compounds described herein (including anti-B7-H3 compounds that include an anti-B7-H3 affibody and do not include additional functional moieties) formulated with a pharma- ceutically acceptable carrier. Such compositions may contain, for example, a single anti-B7-H3 compound, or a combination, for example, two or more different, anti-B7-H3 compounds.

[0071] As used herein, "pharmaceutical acceptable" refers to a material that is not biologically or otherwise undesirable, e.g., the material may be administered to an individual together with an anti-B7-H3 compound without causing adverse biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included.

[0072] The pharmaceutical composition may include one or more pharma- ceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. The pharmaceutical composition may also or alternatively include a pharma- ceutically acceptable antioxidant. Those skilled in the art generally recognize and understand how to apply the use of pharma- ceutically acceptable salts and / or pharma-ceutically acceptable antioxidants.

[0073] Pharmaceutical compositions may be formulated with a pharma- ceutically acceptable carrier. The term "carrier" as used herein includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonicity agent, absorption delaying agent, buffer, carrier solution, suspension, hydrogel, colloid, accessory agent, stabilizer, protein carrier, biological carrier compound, or the like. Non-limiting examples of solvents include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (e.g., olive oil), or injectable organic esters (e.g., ethyl oleate), or combinations thereof. Non-limiting examples of protein carriers include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, or 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). Carriers may also include ovalbumin, human serum albumin, other proteins, polyethylene glycol, or the like. The use of such media and / or agents for pharma- ceutical active substances is well known in the art. Use of any conventional media or agent is contemplated in the therapeutic compositions, except insofar as it is incompatible with the active ingredient (i.e., anti-B7-H3 compounds). Supplementary active ingredients may also be incorporated into the compositions.

[0074] The pharmaceutical composition may additionally or instead 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, or the like. It may also be desirable for the pharmaceutical composition to contain isotonicity agents, such as sugars, sodium chloride, or polyalcohols, such as mannitol or sorbitol. Furthermore, prolonged absorption of the injectable pharmaceutical form may be provided by including an agent that delays absorption, such as, for example, aluminum monostearate or gelatin.

[0075] Thus, the anti-B7-H3 compounds of the present disclosure can be formulated into pharmaceutical compositions. The pharmaceutical compositions may be formulated in various forms adapted to the preferred route of administration. Thus, the compositions can be administered via known routes, such as, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.), or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intradermally, transdermally, rectally, etc.). The pharmaceutical compositions can be administered to mucosal surfaces, such as by administration (e.g., by spray or aerosol) to the nasal or respiratory mucosa. The compositions can also be administered by sustained or delayed release.

[0076] The formulation may be conveniently provided in unit dosage form and may be prepared by methods well known in the pharmaceutical field.The method of preparing the composition with pharmaceutically acceptable carrier comprises combining the anti-B7-H3 compound with the carrier which constitutes one or more accessory ingredients.The formulation can generally be prepared by uniformly and / or intimately combining the active ingredient with liquid carrier, finely divided solid carrier, or both, and then shaping the product into desired formulation as necessary.

[0077] To prepare pharmaceutical compositions containing the anti-B7-H3 compounds of the present disclosure, the anti-B7-H3 compounds can be mixed with a pharma- ceutical acceptable carrier or excipient. By mixing with a physiologically acceptable carrier, excipient, or stabilizer, the therapeutic and diagnostic formulations can be prepared in the form of, for example, a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension.

[0078] 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 in the form of any mixture, etc. The compositions may be formulated and delivered with a pharma- ceutically acceptable excipient, carrier, or vehicle.

[0079] 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 the nasal mucosa. Formulations for rectal or vaginal administration may be provided as suppositories containing 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 the pH and isotonicity factors are preferably adapted to those of the eye.

[0080] Topical formulations include anti-B7-H3 compounds dissolved or suspended in one or more vehicles, such as mineral oil, petroleum, polyhydric alcohol, or other bases used in topical pharmaceutical formulations. For example, topical formulations include creams, ointments, pastes, lotions, powders, solids, aerosolized foams, or gels. Topical formulations may include penetration enhancers to enhance the bioavailability of the active ingredient. Topical formulations may include preservatives and / or emulsifiers. Topical formulations may be provided in the form of transdermal patches or dressings, in which case the formulation may be incorporated into gauze or other structures and contacted with the skin. The formulation may further include one or more additives, such as, for example, adjuvants, skin penetration enhancers, colorants, flavors, fragrances, moisturizers, thickeners, and the like.

[0081] Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the active ingredient as a powder or granules, as liposomes, or as a solution or suspension in aqueous or non-aqueous liquid form such as a syrup, elixir, emulsion, or draught. Tablets, troches, pills, capsules, and the like may also contain one or more of the following: a binder such as gum tragacanth, gum arabic, corn starch, or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like; a lubricant such as magnesium stearate; a sweetener such as sucrose, fructose, lactose, or aspartame; or a natural or artificial flavor. When the unit dosage form is a capsule, it may further comprise a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to 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 / or the like. Syrups or elixirs may contain one or more sweeteners; preservatives such as methylparaben or propylparaben; agents that retard sugar crystallization; agents that increase the solubility of other optional ingredients (e.g., polyhydric alcohols 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 used. The active ingredient may be incorporated into a formulation or device in a formulation that may or may not be designed for sustained release.

[0082] Preparations 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., intravenous drip) of the anti-B7-H3 compounds of the present disclosure or pharmaceutical compositions containing the same 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 (such as glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, glycerol esters, or any mixture thereof.

[0083] 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 and / or a pharmaceutical composition comprising one or more anti-B7-H3 compounds described herein. As used herein, the term "treat" and variations thereof refer to any degree of alleviation, retardation, improvement, or elimination of a symptom or clinical sign associated with a condition. A "symptom" refers to any subjective evidence of a disease or patient's condition, and a "sign" or "clinical sign" refers to an objective physical finding associated with a particular condition that is detectable by a person other than the patient. A "treatment" may be therapeutic or prophylactic. The term "therapeutic" and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. The term "prophylactic" and its variations refer to a treatment that suppresses the onset and / or appearance of symptoms or clinical signs of a condition to any degree. In general, a "therapeutic" treatment is initiated after the condition appears in a subject, whereas a "prophylactic" treatment is initiated before the condition appears in a subject. Prophylactic treatment may be performed on a subject at risk of having a condition. "At risk" refers to a subject who may or may not actually have the risk described. In the case of non-infectious conditions, for example, a subject "at risk" of developing a particular condition is a subject who has one or more pieces of evidence indicating an increased risk of having or developing a particular condition, when compared to an individual who lacks the one or more pieces of evidence, regardless of whether the subject shows any symptoms or clinical signs of having or developing the condition.

[0084] Thus, in one or more embodiments, the subject to be treated 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. 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 may invade and spread to other sites (secondary sites, metastatic sites), a feature that 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, head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, or the like.

[0085] 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, physical 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 physical condition of the subject, and / or the method of administration. Therefore, it is not practical to generally prescribe the amount that constitutes an effective amount of anti-B7 compound for all possible applications. However, a person skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0086] In one or more embodiments, the method can include administering to the subject an amount of an anti-B7-H3 compound sufficient to provide, for example, a dose of about 100 ng / kg to about 50 mg / kg. However, in some embodiments, the method can be practiced by administering a dose of an anti-B7-H3 compound outside of this range. In some of these embodiments, the method includes administering to the subject an amount of an anti-B7-H3 compound sufficient to provide a dose of about 10 μg / kg to about 5 mg / kg, for example, a dose of about 100 μg / kg to about 1 mg / kg.

[0087] A dose may be administered all at once, continuously for a given period of time, or in multiple doses. When multiple doses are used, the doses may be the same or different. For example, a daily dose of 1 mg may be administered as a single dose of 1 mg administered continuously over a 24-hour period, or as two or more equal doses (e.g., 0.5 mg administered twice), or as two or more unequal doses (e.g., 0.75 mg administered once, followed by 0.25 mg administered twice). When multiple doses are used to deliver a dose, the intervals between doses may be the same or different.

[0088] In one or more embodiments, the active ingredient may be administered in a range of, for example, one dose to multiple doses per week. However, 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 period of time, the doses 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 less than the loading dose. Also, when multiple doses are used within a period of time, the intervals between doses may be the same or different.

[0089] In certain embodiments, the anti-B7-H3 compound may be administered in a range from about once per month to about 5 times per week.

[0090] The anti-B7-H3 compound may be administered before, during, or after the subject first exhibits a symptom or clinical sign of the condition. Treatment initiated before the subject first exhibits a symptom or clinical sign 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 complete elimination of the condition, compared to a subject to whom the anti-B7-H3 compound is not administered. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in a reduced severity of the symptoms and / or clinical signs of the condition, and / or complete elimination of the condition, compared to a subject to whom the composition is not administered.

[0091] The anti-B7-H3 compound can be any embodiment of an anti-B7-H3 compound described herein that has an anti-B7-H3 affibody 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 treat 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.

[0092] 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 chemotherapy (e.g., chemotherapy) and / or radiation therapy. Examples of tumors that may be treated include tumors 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.

[0093] 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.

[0094] In one or more embodiments, the anti-B7-H3 compound may be administered, for example, in a range of one dose to multiple doses per week, while 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 in a range of about once per month to about five times per week.

[0095] In one or more embodiments, the method further comprises administering one or more additional therapeutic agents. The administration of the one or more additional therapeutic agents may occur before, after, and / or simultaneously with the administration of the anti-B7-H3 compound. The anti-B7-H3 compound and the additional therapeutic agent may be administered in combination. The term "co-administered" as used herein refers to administering two or more components in combination such that the therapeutic or prophylactic effect of the combination may be greater than the therapeutic or prophylactic effect of any of the components administered alone. The two components may be co-administered simultaneously or sequentially. The components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes administering the components such that each component is simultaneously present at the treatment site. Alternatively, sequential co-administration of two or more components may include administering at least one cellular effect (e.g., cytokine production, activation of a specific cell population, etc.) of at least one component removed from the treatment site, but persisting at the treatment site until one or more additional components are administered to the treatment site. Thus, in some circumstances, a combination administered in combination may include components that are not present in a chemical mixture with each other. In other embodiments, the anti-B7-H3 compound and the additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, the administration of the anti-B7-H3 compound may allow the efficacy of the other therapeutic modality to be achieved at a lower dose than when the other therapeutic agent or agent is administered alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when the other therapeutic agent or agent is administered at a higher dose.

[0096] The term "chemotherapeutic agent" as used herein refers to any therapeutic agent used in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, 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 ozogamicin), Raptiva™ (efalizumab), Tysabri™ (natalizumab), Zenapax™ (dacliximab), NeutroSpec™ (technetium (99mTc)-fanolesomab), tocilizumab, Pr ostaScint™ (Indium-Ill labeled capromab pendetide), Bexxar™ (tositumomab), Zevalin™ (ibritumomab tiuxetan conjugated to yttrium-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), Al These include ecensa™ (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 imid drugs (imiquimod, thalidomide, etc., or analogs thereof).

[0097] In one or more embodiments, the methods can include administering a sufficient amount of an anti-B7-H3 compound described herein and administering at least one additional therapeutic agent, which exhibits therapeutic synergy. In some aspects of the methods of the present disclosure, a measure of response to treatment observed after administration of both an anti-B7-H3 compound described herein and an additional therapeutic agent is improved over a similar measure of response to treatment observed after administration of either the anti-B7-H3 compound or the additional therapeutic agent alone.

[0098] 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 anti-B7-H3 affibodies without additional functional moieties) 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. Substrates 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.

[0099] In another aspect, the disclosure describes methods for using anti-B7-H3 compounds in molecular imaging applications, including, for example, both traditional molecular imaging techniques (such as magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound, photoacoustic imaging, and fluorescence) and microscopic and / or nanoscopic imaging techniques (such as total internal reflection fluorescence (TIRF) microscopy, stimulated emission depletion (STRED) nanoscopy, or atomic force microscopy (AFM)).

[0100] The anti-B7-H3 compounds described herein may have utility in in vitro and in vivo detection, diagnosis, and / or treatment. 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 applying an anti-B7-H3 compound that specifically binds to a target of interest to 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, for example, a radioisotope, a fluorescent marker, an enzyme, a magnetic marker, or a colorimetric marker.

[0101] 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 should be interpreted as open-ended, i.e., that additional elements or steps are optional and may or may not be present; unless otherwise noted, "a", "an", "the", and "at least one" are used interchangeably and mean one or more than one; 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.).

[0102] In the above description, certain embodiments may be described separately for clarity. Throughout this specification, references to "one embodiment," "one or more embodiments," "embodiments," "particular embodiments," or "some embodiments" mean that a particular feature, configuration, composition, or characteristic described in connection with an 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. Furthermore, 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 and features described in the context of a different embodiment may be combined, unless those features are necessarily mutually exclusive.

[0103] For methods disclosed herein that include separate steps, those steps may be performed in any suitable order, and, where appropriate, any combination of two or more steps may be performed simultaneously.

[0104] 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.

[0105] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain advantages, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, if one or more preferred embodiments are described, this does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0106] 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, including full-length proteins, fragments thereof, and / or, in some cases, polyproteins.

[0107] The term "protein" refers to any sequence of two or more amino acid residues, regardless of length, 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, nucleotide sequences, or any combination of carbohydrates, lipids, and / or nucleotide sequences. As used herein, "protein," "peptide," and "polypeptide" are used interchangeably.

[0108] The term "nucleic acid" or "oligonucleotide" as used herein 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 or 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 can be isolated. The term "isolated nucleic acid" refers to (i) nucleic acids amplified in vitro, such as by polymerase chain reaction (PCR), (ii) nucleic acids recombinantly produced by cloning, (iii) nucleic acids purified, such as by cleavage and separation by gel electrophoresis, (iv) nucleic acids synthesized, such as by chemical synthesis, or (vi) nucleic acids extracted from a sample. Nucleic acids can be introduced, i.e., transfected, into a cell. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.

[0109] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0110] Example 1 Library Design The affibody library was designed following Woldring's gradient site-wise library design, in which amino acid diversity at potential hotspot positions was guided by the prevalence of amino acids in natural antibody interfaces and previously evolved affibodies (Woldring et al., Biochemistry 56, 1656-1671 (2017)).

[0111] Discovery and evolution strategies An affibody 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 the paratope and entire affibody genes were randomly mutagenized by error-prone polymerase chain reaction (PCR). DNA was electroporated back into yeast and further selection was performed until binding to low nanomolar concentrations of B7-H3 was observed upon display.

[0112] MACS selection with soluble extracellular domains Magnetic bead selection was performed with at least 15-fold oversampling of affibody diversity at all stages. Biotinylated recombinant human B7-H3 extracellular domain (available from Sino Biological, Beijing, China) for positive isolation and biotinylated Renilla green fluorescent protein (rrGFP; available from Avidity, Aurora, CO) for depletion were incubated with DYNABEADS Biotin Binder (Invitrogen, Thermo Fisher Scientific, Waltham, MA) to coat the beads with protein. Magnetic activated cell sorting was performed on yeast. For sorting, yeast were incubated with control bare biotin binder beads for 2 hours at 4°C, followed by incubation with GFP-labeled beads for an additional 2 hours for depletion to eliminate any non-specific and non-B7-H3 binding interactions. Yeast were then incubated with beads immobilized with recombinant human B7-H3 target protein for an additional 2 hours. Yeast that bound were selected as positives. MACS was performed at 4°C, with yeast washed twice between incubations. MACS selections were performed a total of three times with increasing wash stringency, with yeast grown and induced between each selection. After error-prone PCR, the large mutant naïve library was subjected to additional MACS selections as previously described.

[0113] FACS selection with soluble extracellular domains After three rounds of MACS sorting of the affibody library, the library size was easily sampled by FACS. The induced 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. Cells were washed once with PBSA, labeled with goat anti-mouse Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Waltham, MA) and streptavidin Alexa Fluor 488 conjugate (Thermo Fisher Scientific, Waltham, MA) for 20 min at 4°C, and washed with PBSA. Cells positive for Myc and with B7-H3 signal above background were harvested.

[0114] Error-prone PCR of affibodies Random mutagenesis of affibodies was performed by error-prone PCR using the mutagenic analogs 8-oxo-dGTP and dPTP. Zymoprep-treated plasmid DNA was mutated by error-prone PCR using primers W5 / W3 for the full-length affibody genes and primers ABY1F-b / ABY1R and ABY2F / ABY2R-b19 for the affibody helix. PCR products were purified by agarose gel electrophoresis. The affibody helix 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 affibody library was homologously recombined with linearized pCT-40-Helix for the affibody helix and transformed into EBY100 yeast by electroporation. Approximately 200 million transformants for the affibody library were obtained by electroporation.

[0115] Monovalent MACS selection using soluble extracellular targets Following MACS selection with the large mutant naive library, yeast were subjected to a more stringent monovalent MACS selection. As before, yeast were incubated with control naked biotin binder beads, washed, and depleted with GFP-coated beads. Yeast were then washed and incubated with 100 nM biotinylated recombinant human B7-H3 extracellular domain for 1 h. Naked biotin binder beads were spiked in to bind biotinylated B7-H3 and incubated for an additional 2 h at 4 °C. The beads were washed three times, and yeast that still bound were harvested.

[0116] 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 supplemented with 10% (v / v) fetal bovine serum and 1% penicillin and streptomycin at 37°C and 5% CO2. MS1-B7-H3 cells were cultured at 75 cm 2 Cells were grown in 1000 x g tissue culture treated flasks until 70-90% confluent. Cells were washed with PBS, detached by trypsin-EDTA treatment for 5 min, quenched with serum-containing medium, and pelleted at 500 x g for 3 min. Pelleted cells were washed twice and resuspended in PBS supplemented with 0.5 mg / mL fresh sulfo-NHS-biotin (Thermo Fisher Scientific, Waltham, MA) for 30 min at room temperature. After incubation, cells were washed twice to remove excess biotin and lysed in 250 μL lysis buffer for 15 min at 4°C. Cell debris was pelleted at 10,000 x g for 30 min and removed.

[0117] FACS selection using detergent-solubilized cell lysates The affibody library was subjected to two rounds of flow cytometry selection with detergent-solubilized cell lysate. The library was washed once with PBSA and incubated with cell lysate for 1 h at 4°C. After incubation, yeast were washed and incubated with chicken anti-Myc-FITC (Immunology Consultants Laboratory, Portland, OR) and streptavidin-Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Waltham, MA) for 20 min at 4°C, and washed again. Yeast that were Myc positive (FITC) and had the highest ratio of MS1-B7-H3 lysate bound (AF647):Myc (FITC) were collected using FACS. This selection was repeated at higher stringency using a lower B7-H3 lysate concentration.

[0118] FACS selection with protease and thermostability assays Yeast induced to display affibodies were washed and incubated with proteinase K (1 × 10 -6 Yeast were incubated with AF647 (units / μL; New England Biolabs, Ipswich, MA) for 10 min at 55°C and then immediately placed on ice. Yeast were then incubated with MS1-B7-H3 cell lysate for 1 h at 4°C. After incubation, yeast were washed and incubated with chicken anti-Myc-FITC and streptavidin-AlexaFluor 647 conjugate for 20 min at 4°C and washed again. Yeast that were Myc positive (FITC) and had the highest ratio of MS1-B7-H3 lysate bound (AF647):Myc (FITC) were harvested using FACS.

[0119] 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 randomly picked from the affibody library and incubated in 50 μL of distilled water at 100°C for 5 min. Two microliters of yeast sample was taken for PCR with GeneAmp5 / 3 primers and DNA cleanup. The amplified affibody genes and GeneAmp5 primers were sent to Eurofins Genomics for Sanger sequencing.

[0120] Cloning, protein expression, and purification The affibody coding region in DNA recovered from the final B7-H3 flow cytometry sort was amplified by PCR, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated with T4 DNA ligase into a pET-24b vector containing a C-terminal hexahistidine tag. Plasmids were transformed into T7 Express Competent E. coli and plated on lysogeny broth (LB) plates containing 50 mg / L kanamycin. Transformants were sequenced for the full length of the gene by Sanger sequencing, and suitable transformants were grown in 5 mL of liquid LB supplemented with kanamycin (50 mg / L) at 37 °C and 250 rpm for 12-16 h. Saturated cultures were added to 100 mL of LB and cultured 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 for 10 min at 4°C, and filtered through a 0.25 mm filter. The resulting cell lysate was passed through 0.25 mL Cobalt HisPur resin volume spin columns, washed with 30 mM imidazole, and eluted with 300 mM imidazole. Purity and concentration of the affibodies were analyzed by protein gel electrophoresis.

[0121] Affinity titration Detached MS1-B7-H3 cells were washed and individually labeled with various concentrations of purified affibody and affibody 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, UK) 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).

[0122] Analysis of affibody modularity Affibody gene coding regions were amplified by PCR and assembled into pET-24b vectors containing the GSGGGSGGGKGGGGT (SEQ ID NO: 35) linker and amino acids 60-206 of the sortase A (EC 3.4.22.70) gene with a C-terminal hexahistidine tag using NEBuilder HiFi DNA Assembly (New England Biolabs, Ipswich, MA). Plasmids were transformed into BL21-CodonPlus(DE3)-RIL Competent E. coli and plated on LB plates supplemented with 50 mg / L kanamycin. Full-length gene sequences of transformants were determined by Sanger sequencing, and suitable transformants were grown in 50 mg / L kanamycin and chloramphenicol for bacterial growth and protein purification. Affinity titrations were performed on the purified proteins to assess the binding affinity of selected affibodies in the presence of conjugated protein enzymes.

[0123] Example 2 Example 2 describes the affinity, specificity, and stability maturation of affibody population 1.5 from Example 1.

[0124] Error-prone PCR DNA was isolated from yeast population 1.5 and subjected to random mutagenesis of the variable regions of the affibodies by error-prone polymerase chain reaction (PCR). The DNA was electroporated back into yeast for display and further selection was performed using the following selection schemes: (1) avidity selection (lysate MACS with 1 pmol of B7-H3+ lysate); (2) affinity selection (strict K d (3) specificity selection (multi-target MACS depletion selection); and (4) stability selection (thermolysin FACS selection at 55° C.). After each selection, the populations were deep sequenced and filtered with USEARCH (ultra-fast 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 affibody variants.

[0125] For each sequence, the frequency was determined for each selection (e.g., avidity, affinity, specificity, and stability) and for subsequent enrichments. If the read count was 0, a read count of 1 was assigned. Z-scores were determined for affinity, specificity, stability, and final frequency, and a final conditional weighted average z-score was calculated. The best candidates were selected based on the highest scores.

[0126] In calculating the conditional weighted average z-score, the frequency of each variant in each sort was first determined. The enrichment values ​​for each variant in the affinity, specificity, and stability sorts 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 weighting: 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 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 but may not have had a chance to be enriched. The condition for final frequency was added because the variant must be present in the final sequenced population.

[0127] Figure 6 shows z-scores for affinity, specificity, stability, and final frequency, as well as the conditional weighted mean z-score, for the top 13 variants. Values ​​greater than 1 indicate enrichment, and values ​​less than 1 indicate depletion. The colored bars in Figure 6 are a representation of 1 to M+2SD enrichment for affinity, and 1 to M+2SD for specificity and stability, as the mean (M) was slightly below 1. In the bar graphs in Figure 6, the z-scores are a representation of 1 to 10 (i.e., for z-scores 10 or greater, the bar spans the entire length of the figure).

[0128] MACS sorting of B7-H3 cell lysate (avidity sorting): We performed an avidity selection, which we believe to be an avidity selection because the B7-H3 target is not only expressed monovalently in solution, but also multivalently on the avidin beads. Yeast also expresses thousands of affibodies per cell, so avidity-based binding between yeast and the B7-H3-coated beads is possible.

[0129] Magnetic bead selection was performed with 20-fold oversampling to select for B7-H3 binders as well as to easily remove non-binders and truncated affibodies resulting from error-prone PCR. Yeast were incubated with naked streptavidin-coated beads for 2 h at 4°C, followed by depletion 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 the 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 the reaction occurs with 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 affibody library has already been primed to bind to B7-H3.

[0130] Strict K d FACS sorting (affinity selection) After one round of MACS sorting of B7-H3 cell lysate, a library size was obtained that could be easily sampled by FACS. The induced yeast population was labeled with ~0.2 nM biotinylated B7-H3 cell lysate for 2 h, washed, and then labeled with streptavidin-AF647 and chicken anti-Myc-FITC. The top 0.4% (highest affinity) cells were collected for the next sort (specificity sort).

[0131] Multi-target MACS depletion (specificity selection) To select for more specific binders, multi-target depletion was performed on the population isolated after affinity selection. The following proteins were biotinylated and 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 pre-conjugated with biotin. Yeast was incubated with the aforementioned proteins coated on beads for 2 hours at 4°C. The yeast was then subjected to a second similar negative depletion step to more reliably deplete non-specific binders. Non-binding yeast was selected and grown for the next selection (stability selection).

[0132] Thermolysin Stability FACS Selection (Stability Selection) To select stable binders, the specificity-selected affibody population was subjected to protease incubation at elevated temperature. Yeast were induced to display the affibody population and incubated with 0.75 mg / mL thermolysin at 55°C for 10 min and 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 an additional 20 min. HA- and Myc-positive yeast were sorted using a three-step gating approach and the top 2%, 14%, and 19% displaying yeast were collected using FACS.

[0133] Deep sequencing by ISEQ (Illumina, San Diego, CA) The selected affibody population was subjected to deep sequencing, filtered with USearch, and further filtered for full-length affibodies. For stability selection, three gates were collected and sequenced. Due to lack of sequencing depth for full-length affibodies, the read counts from each stability gate were summed to obtain the total stability read count. Clones recapitulating the previously designed B7-H3 affibody were removed for sequencing.

[0134] The complete disclosures of all patents, patent applications, and publications described herein, as well as electronically available materials (e.g., nucleotide sequences submitted to GenBank and RefSeq, amino acid sequences submitted to SwissProt, PIR, PRF, and PDB, and translations from annotated coding regions in GenBank and RefSeq, etc.) 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 prevail. The detailed description and examples set forth above are provided solely for clarity of understanding. Unnecessary limitations should not be construed therefrom. The present invention is not limited to the exact details shown and described, and variations that would be obvious to one skilled in the art are encompassed within the invention as defined by the claims.

[0135] Unless otherwise indicated, all numerical values ​​expressing ingredients, molecular weights, and other quantities used in the specification and claims should be understood to be modified in all instances by the term "about." Thus, unless otherwise indicated to the contrary, 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. Without intending to limit the doctrine of equivalents to the scope of the claims, at the very least, each numerical parameter should be construed in light of at least the number of significant digits in the reported values ​​and by applying ordinary rounding techniques.

[0136] 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, every numerical value contains inherent ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0137] 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 specifically stated.

[0138] Sequence Listing Free Text SEQ ID NO:1 - ABY [ka] SEQ ID NO:2 - General consensus sequence [ka] X6: Y, N, S, T, D, or A; X9: L, R, K, W, D, I, N, or Q; X 10 : H, A, S, F, I, D, E, Q, T, N, or L; X 11 : T, Y, D, A, L, P, I, or N; X 13 : I, Y, L, V, T, or F; X 14 : Q, Y, L, V, G, F, E, A, M, or S; X 15 : V, S, Q, or E; X 17 : H, A, V, G, R, I, F, or L; X 18 : S, Y, L, G, T, N, M, D, R, W, E, or H; X 23 : T or A; X 24 : L, K, A, G, R, H, W, V, or E; X 25 : D, P, T, N, V, S, H, L, R, A, W, or E; X27 : R, K, T, I, or A; X 28 : A, Y, R, N, D, Q, I, H, L, S, or F; X 31 : L, K, I, or T; X 32 : L, G, Y, R, A, F, V, S, or Q; X 33 : A or K; X 35 : F, S, V, H, Y, D, E, or K; X 36 : N or D; X 44 : L or F; X 46 : S or P; X 49 : R or K. SEQ ID NO:3 - SEQ ID NO:4 to SEQ ID NO:18 second consensus sequence [ka] SEQ ID NO:4 - ABY 0.4.1 [ka] SEQ ID NO:5 - ABY 0.4.2 [ka] SEQ ID NO:6 - ABY 0.4.3 [ka] SEQ ID NO:7 - ABY 0.4.4 [ka] SEQ ID NO:8 - ABY 0.4.5 [ka] SEQ ID NO:9 - ABY 0.4.6 [ka] SEQ ID NO:10 - ABY 0.4.7 [ka] SEQ ID NO:11 - ABY 1.4.1 [ka] SEQ ID NO:12 - ABY 1.4.2 [ka] SEQ ID NO:13 - ABY 1.4.3 [ka] SEQ ID NO:14 - ABY 1.4.4 [ka] SEQ ID NO:15 - ABY 1.4.5 [ka] SEQ ID NO:16 - ABY 1.4.6 [ka] SEQ ID NO:17 - ABY 1.4.7 [ka] SEQ ID NO:18 - ABY 1.4.8 [ka] SEQ ID NO:19 - ABY 1.5.1 [ka] SEQ ID NO:20 - ABY 1.5.2 [ka] SEQ ID NO:22 - Otu13 [ka] SEQ ID NO:23 - Otu8 [ka] SEQ ID NO:24 - Otu12 [ka] SEQ ID NO:25 - Otu15 [ka] SEQ ID NO:26 - Otu11 [ka] SEQ ID NO:27 - Otu89 [ka] SEQ ID NO:28 - Otu227 [ka] SEQ ID NO:29 - Otu1607 [ka] SEQ ID NO:30 - Otu225 [ka] SEQ ID NO:31 - Otu394 [ka] SEQ ID NO:32 - Otu249 [ka] SEQ ID NO:33 - Otu252 [ka] SEQ ID NO:34 - Otu265 [ka] SEQ ID NO:35 - linker GSGGGSGGGK GGGGT SEQ ID NO:36 - linker GGGGSGGGGS GGGGS SEQ ID NO:37 - linker GSTSGSGKPG SGEGSTKG SEQ ID NO:38 - linker [ka] SEQ ID NO:39 - linker EASGGPE SEQ ID NO:40 - linker [ka] SEQ ID NO:41 - linker AEAAKEAAKA SEQ ID NO:42 - linker [ka] SEQ ID NO:43 - linker GGGGSGGGGS SEQ ID NO:44 - linker GSTSGSGKPG SGEGSTKG SEQ ID NO:45 - linker EPKSSDKTHT SPPSP SEQ ID NO:46 - linker [ka] SEQ ID NO:47 - linker SSGGGGSGGG GGGSSRSSL

Claims

1. 1. An anti-B7-H3 compound comprising an anti-B7-H3 affibody, said anti-B7-H3 affibody comprising: SEQ ID NO:2; an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:2; or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2; 4. An anti-B7-H3 compound comprising:

2. the anti-B7-H3 affibody Any one of SEQ ID NOs:3 to 20 or SEQ ID NOs:22 to 34; an amino acid sequence having at least 90% sequence similarity to any one of SEQ ID NO:3 to SEQ ID NO:20 or SEQ ID NO:22 to SEQ ID NO:34; or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:3 to 20 or SEQ ID NOs:22 to 34; 2. The anti-B7-H3 compound of claim 1, comprising:

3. The anti-B7-H3 compound of claim 1 or 2, further comprising a first functional moiety operably linked to said anti-B7-H3 affibody.

4. The anti-B7-H3 compound of claim 3, wherein the first functional moiety comprises a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug.

5. The anti-B7-H3 compound of claim 4, wherein the targeting moiety binds to an extracellular domain of a protein displayed on natural killer cells or T cells.

6. The anti-B7-H3 compound of claim 4, wherein the imaging moiety comprises a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label.

7. The anti-B7-H3 compound of claim 4, wherein the enzyme comprises sortase A.

8. The anti-B7-H3 compound of claim 4, wherein the small molecule drug comprises one or more radioisotopes.

9. The anti-B7-H3 compound of any of claims 1 to 8, further comprising a second functional moiety operably linked to the anti-B7-H3 affibody.

10. 10. The anti-B7-H3 compound of claim 9, wherein the second functional moiety comprises a targeting moiety, an imaging moiety, an enzyme, or a small molecule drug.

11. 10. The anti-B7-H3 compound of claim 9, wherein said first functional moiety is directly linked to said anti-B7-H3 affibody and said second functional moiety is directly linked to said anti-B7-H3 affibody.

12. 10. The anti-B7-H3 compound of claim 9, wherein the first functional moiety is directly linked to the anti-B7-H3 affibody and the second functional moiety is directly linked to the first functional moiety.

13. An anti-B7-H3 compound according to any one of claims 1 to 12; and a pharma-ceutically acceptable carrier; A composition comprising:

14. A method comprising administering to a subject the composition of claim 13.

15. 15. The method of claim 14, wherein the subject has or is at risk of having a tumor.

16. 15. The method of claim 14, wherein the subject has or is at risk of having cancer.

17. 15. The method of claim 14, wherein the composition is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

18. 15. The method of claim 14, further comprising detecting an anti-B7-H3 compound bound to cell-expressed B7-H3 by detecting the imaging moiety.

19. The method of claim 18, wherein the cell is a cancer cell that expresses B7-H3.

20. 20. The method of claim 18 or 19, wherein the imaging moiety is detectable by positron emission tomography.

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