Patent Application
20250179186
This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office via Patent Center as an XML file entitled 0110_000693WO01 having a size of 42.5 kilobytes and created on Jan. 1, 2023. Due to the electronic filing of the Sequence Listing, 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 by reference herein.
This disclosure describes, in one aspect, an anti-B7-H3 compound. The anti-B7-H3 compound includes an anti-B7-H3 Gp2-based scaffold. The anti-B7-H3 Gp2-based scaffold includes the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:3, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.
In one or more embodiments, the anti-B7-H3 Gp2-based scaffold includes the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In one or more embodiments, the anti-B7-H3 Gp2-based scaffold includes any one of the amino acid sequences of SEQ ID NO:5 through SEQ ID NO:23.
In one or more embodiments, the anti-B7-H3 compound includes a first functional component operably coupled to the anti-B7-H3 Gp2-based scaffold. In some embodiments the first functional component includes a targeting component, an imaging component, an enzyme, or a small molecule drug. In one or more embodiments, the targeting component binds to the extracellular domain of a protein displayed on a natural killer cell or a T cell. In one or more embodiments, the imaging component includes a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label. In one or more embodiments, the enzymatic label includes sortase A. In one or more embodiments, the small molecule drug includes one or more radioisotopes.
In one or more embodiments, the anti-B7-H3 compound may include a second functional component operably couple to the anti-B7-H3 Gp2-based scaffold. In one or more embodiments, the second functional component includes a targeting component, an imaging component, an enzyme, or a small molecule drug. In one or more embodiments, the first functional component is directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional component is directly linked to the anti-B7-H3 Gp2-based scaffold. In one or more embodiments, the first functional component is directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional component is directly linked to the first functional component.
In another aspect, the present disclosure describes a composition including an anti-B7-H3 compound and a pharmaceutically acceptable carrier.
In another aspect, the present disclosure describes a method that includes administering a composition that includes an anti-B7-H3 compound to a subject. 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, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy. In one or more embodiments, the method further includes detecting the imaging component to detect the anti-B7-H3 compound bound to B7-H3 expressed by a cell. In one or more embodiments, the cell is a cancer cell.
The above summary is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the disclosure, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
This disclosure describes anti-B7-H3 Gp2-based scaffolds, compounds that include at least one of the anti-B7-H3 Gp2-based scaffolds, and methods of using such compounds. As used herein, the term “anti-B7-H3 compound” refers to a compound that includes, or is, an anti-B7-H3 Gp2-based scaffold. Exemplary platforms in which an anti-B7-H3 compound may be used include, but are not limited to, chimeric antigen receptor therapies (e.g., CAR-NK therapy, CAR-T therapy, CAR-macrophage therapy, etc.), multispecific immune cell engager technologies (e.g., bispecific killer engagers, trispecific killer engagers, bispecific T cell engagers, trispecific T cell engagers, etc.), targeted immunotherapies (e.g., targeted ADAM17 blocker (TAB) therapy), delivery of therapeutics (e.g., antibody-drug conjugates, delivery of therapeutic radioisotopes, delivery of toxins, delivery of cytokines, delivery of chemokines), imaging technologies (delivery of labeling constructs and/or labeling radioisotopes), and cell and/or ligand capture technologies (e.g., ELISA, etc.).
B7 Homolog 3 (B7-H3), also known as cluster of differentiation 276 (CD276), is a human protein encoded by the CD276 gene. The B7-H3 protein is a 316 amino acid-long type I transmembrane protein existing in two isoforms determined by its extracellular domain. B7-H3 mRNA is expressed in most normal tissues. In contrast, B7-H3 protein has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. In normal tissues, B7-H3 has a predominantly inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. As such, B7-H3 is an important immune checkpoint inhibitor of T-cell function, is a tumor vasculature biomarker, and is overexpressed in a variety of cancers, including clear cell renal cell carcinoma, cutaneous melanoma, diffuse intrinsic pontine glioma, hypopharyngeal squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, prostate cancer, and pancreatic cancer. The expression of B7-H3 is associated with tumor growth and metastasis and may ultimately lead to poor clinical prognosis.
Molecules that recognize certain targets specifically and with high affinity are useful for many clinical (e.g., diagnostic and/or therapeutic) and biotechnology applications. Typically, antibodies have been used for many of these applications, but antibodies have certain properties that may be drawbacks in certain applications. The limitations of antibodies have encouraged investigation toward alternative protein scaffolds that allow one to efficiently generate improved binding molecules. In the context of targeting solid tumors, for example, antibodies—which are typically about 150 kDa for immunoglobulin G (IgG)—can exhibit, due at least in part to their size, poor extravasation from vasculature, poor penetration through tissue, and/or long plasma clearance halftime, which can lead to poor signal-to-noise ratio, especially for diagnostic imaging. Antibodies also can exhibit thermal instability, which can lead to a loss of efficacy as a result of denaturation and/or aggregation. In addition, antibodies are typically made in mammalian cultures because many possess disulfide bonds, glycosylation, and/or multi-domain structures. This intricate structure can interfere with engineering the antibody for a particular application such as, for example, production of protein fusions for multispecific formats. Moreover, the presence of disulfide bonds in antibody molecules often precludes their intracellular use.
As a result of the limitations inherent to antibodies, alternative protein scaffolds have been developed in attempts to address many or all of these shortcomings. This disclosure describes recombinant, non-naturally occurring protein scaffolds, termed anti-B7-H3 Gp2-based scaffolds, capable of binding to the extracellular domain of B7-H3. In particular, the anti-B7-H3 Gp2-based scaffolds described herein may be used to display defined loops that are analogous to the complementarity-determining regions (“CDRs”) of an antibody variable region. The anti-B7-H3 Gp2-based scaffolds may be assembled into a multispecific compound capable of binding the extracellular domain of B7-H3 and one or more different targets. The anti-B7-H3 Gp2-based scaffolds described herein can therefore provide functional properties typically associated with antibody molecules. In particular, despite the fact that the anti-B7-H3 Gp2-based scaffold is not an immunoglobulin, its overall folding is similar in relevant respect to that of the variable region of the IgG heavy chain, making it possible for a protein scaffold to display loops in relative orientations analogous to antibody CDRs. Because of this structure, the anti-B7-H3 Gp2-based scaffolds described herein may possess ligand binding properties that are similar in nature and affinity to the binding properties of antigen and antibody.
In one aspect, this disclosure describes anti-B7-H3 compounds. The anti-B7-H3 compounds include an anti-B7-H3 Gp2-based scaffold. The anti-B7-H3 Gp2-based scaffolds of the present disclosure bind to the extracellular domain of the B7-H3 protein, hereinafter B7-H3. The anti-B7-H3 Gp2-based scaffolds may be developed from Gp2 (SEQ ID NO:1), a truncated form of T7 phage gene 2 protein.
In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds of the present disclosure include the amino acid sequence KFWXTVXXXXXXXXFEXPXYAXTXDEALXLA XXXYXXXXXXXXVXXVXP (SEQ ID NO:3), or a structurally similar sequence where each X is, independently of the amino acid at any other position, is a natural, unnatural, or modified amino acid independent of the amino acid residue at any other position in the Gp2 scaffold. In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds of the present disclosure include the amino acid sequence of SEQ ID NO:3 where X at position 4 is A or P; X at position 7 is Q, C, R, or E; X at position 8 is H, Y, or S; X at position 9 is V or S; X at position 10 is any amino acid such as Y or H, or a deletion; X at position 11 is any amino acid such as S or deletion; X at position 12 is D, E, I, or N; X at position 13 is F, H, or G; X at position 14 is G, S, or C; X at position 17 is A or V; X at position 19 can be V or I; X at position 22 is A or E; X at position 24 can be L or M; X at position 29 is Q, E, R, or L; X at position 32 is E or K; X at position 33 is V, A, or W; X at position 34 can be Q, R, or K; X at position 36 is A, V, or G; X at position 37 is N, S, P, or D; X at position 38 is S or A; X at 39 can be an optional addition such as Y; X at position 40 can be an optional addition; X at position 41 is I, G, V; X at position 42 is F or M; X at position 43 is T, M, K, D, or E; X at position 45 is T or A; X at position 46 is R or S; and X at position 49 is R or H.
In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds of the present disclosure include the amino acid sequence KFWATVQHVYSDFGFEAPVYAATLDEALQL AEVQYANSIFTVTRVRP (SEQ ID NO:4), a derivative of SEQ ID NO:3 (X at position 7 is Q; X at position 8 is H; X at position 9 is V; X at position 10 is Y; X at position 11 is S; X at position 12 is D; X at position 13 is F; X at position 14 is G; X at position 17 is A; X at position 19 is V; X at position 22 is A; X at position 24 is L; X at position 29 is Q; X at position 32 is E; X at position 33 is V; X at position 34 is Q; X at position 35 is Y; X at position 36 is A; X at position 37 is N; X at position 38 is S; X at position 39 is no amino acid; X at position 40 is no amino acid; X at position 41 is I; X at position 42 is F; X at position 43 is T; X at position 45 is T; X at position 46 Is R; and X at position 48 is R). In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds of the present disclosure include the amino acid sequence of SEQ ID NO:4 (1.4.4), or a structurally similar sequence. In one or more embodiments, the anti B7-H3 Gp2-based scaffolds of the present disclosure include an amino acid sequence that is a derivative of SEQ ID NO:4. In one or more embodiments, the anti B7-H3 Gp2-based scaffolds of the present disclosure include the amino acid SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23, or a structurally similar sequence thereof (derivatives of SEQ ID NO:4).
In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds of the present disclosure include an amino acid sequence of SEQ ID NO:2 or a structurally similar sequence.
As used herein, a polypeptide (e.g., an anti-B7-H3 Gp2-based scaffold) is “structurally similar” to a reference polypeptide (e.g., an anti-B7-H3 Gp2-based scaffold including SEQ ID NO:4) if the amino acid sequence of the polypeptide possesses a specified amount of identity compared to the reference polypeptide. Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (for example, a candidate polypeptide and the polypeptide of, for example, SEQ ID NO:4) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate polypeptide is the polypeptide being compared to the reference polypeptide. A candidate polypeptide can be isolated, for example, from an animal or other natural source, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.
A pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, polypeptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and filter on.
In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a SEQ ID NO:4 polypeptide may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free —OH is maintained; and Gln for Asn to maintain a free —NH2. Likewise, biologically active analogs of a polypeptide containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the polypeptide are also contemplated.
In one or more embodiments, an anti-B7-H3 Gp2-based scaffold as 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:3 where each X is independently selected from any amino acid. In one or more embodiments, an anti-B7-H3 Gp2-based scaffold as 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:3 where X at position 4 is A or P; X at position 7 is Q, C, R, or E; X at position 8 is H, Y, or S; X at position 9 is V or S; X at position 10 is any amino acid such as Y or H, or a deletion; X at position 11 is any amino acid such as S or a deletion; X at position 12 is D, E, I, or N; X at position 13 is F, H, or G; X at position 14 is G, S, or C; X at position 17 is A or V; X at position 19 can be V or I; X at position 22 is A or E; X at position 24 can be L or M; X at position 29 is Q, E, R, or L; X at position 32 is E or K; X at position 33 is V, A, or W; X at position 34 can be Q, R, or K; X at position 36 is A, V, or G; X at position 37 is N, S, P, or D; X at position 38 is S or A; X at position 39 can be an optional addition of any amino acid such as Y or a deletion; X at position 40 can be an optional addition of any amino acid or a deletion; X at position 41 is I, G, V; X at position 42 is F or M; X at position 43 is T, M, K, D, or E; X at position 45 is T or A; X at position 46 is R or S; and X at position 49 is R or H.
An anti-B7-H3 Gp2-based scaffold as 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; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23. When determining sequence identity, the total number of the amino acids in the sequence is multiplied by the decimal form of the percent identity in question. The resultant number is subtracted from the total number of amino acids in the sequence to reveal the total number of amino acids that can have a different identity than in the original sequence. If the total number of amino acids that can have a different identity is not a hole number, the number is rounded up (when the decimal is 0.5 or greater) or rounded down (when the decimal is less than 0.5) to give the total number of amino acids that can be different from the original sequence. For example, an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:4 can have 5 amino acids that differ from SEQ ID NO:4 (47-(0.9-47)=4.7 then round to 5). Each one of SEQ ID NO:5 through SEQ ID NO:23 have 90% or greater sequence identity to SEQ ID NO:4.
An anti-B7-H3 Gp2-based scaffold as described herein, may have with 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 SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23.
In one or more embodiments, amino acids in the loop regions of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated. The mutation may include substitution of one or more amino acid residues with a different amino acid residue, a deletion of one or more amino acid residues, or an addition of one or more residues to the amino acid sequence of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23.
For example, in one or more embodiments, there may be one or more mutations in the first loop region; that is, amino acid residues 7-14 of SEQ ID NO:3 through SEQ ID NO:23 and amino acid residues 7-12 of SEQ ID NO:2. The first loop region of the original sequence (SEQ ID NO:1) is amino acid residues of 7-12 but SEQ ID NO:3 through SEQ ID NO:23 include and insertion of two amino acids in the first loop region (see Table 1). In one or more embodiments, there may be one or more mutations in the second loop region: amino acid residues 36-43 of SEQ ID NO:3, amino acid residues 36-41 of SEQ ID NO:4 through SEQ ID NO:23, or amino acid residues of 36-40 of SEQ ID NO:2. In one or more embodiments, there may be one or more mutations in the amino acid residues of the first loop region and/or the amino acids of the second loop region.
In one or more embodiments, one or more amino acid residues may be interested into the amino acid sequence of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23. In one or more embodiments, one or more amino acid residues may be inserted into the first loop region and/or the second loop region of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 to extend to the loop region by the number of amino acid residues inserted. For example, in one or more embodiments, one or more amino acid residues may be inserted on the C-terminal side of one or more of residues of 7-14 of SEQ ID NO:3 through SEQ ID NO:23 or amino acid residues 7-12 of SEQ ID NO:2. In one or more embodiments, one or more amino acid residues may be inserted on the C-terminal side of one or more of residues 36-43 of SEQ ID NO:3, amino acid residues 36-41 of SEQ ID NO:4 through SEQ ID NO:23, or amino acid residues of 36-40 of SEQ ID NO:2. For example, in one or more embodiments, on the C-terminal side of residue 36 of SEQ ID NO:5 (0.4.1), one or more amino acid residues may be inserted prior to residue 37 (e.g., residues 36a and/or 36b in
In one or more embodiments, one or more amino acid residues may be removed from SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23. In one or more embodiments, one or more amino acid residues may be removed from the first loop region and/or the second loop region of SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 to shorten the loop region by the number of amino acids removed. For example, in one or more embodiments, one or more amino acid residues may be removed from residues 7-14 of SEQ ID NO:3 through SEQ ID NO:23 or amino acid residues 7-12 of SEQ ID NO:2. For example, in one or more embodiments, amino acid residues 10 and/or 11 of SEQ ID NO:4 are removed. In one or more embodiments, one or more amino acid residues may be removed from amino acid residues 36-43 of SEQ ID NO:3, amino acid residues 36-41 of SEQ ID NO:4 through SEQ ID NO:23, or amino acid residues of 36-40 of SEQ ID NO:2.
In one or more embodiments, one or more amino acid residues outside the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated with any amino acid. For example, in one or more embodiments, there may be one or more mutations in amino acid residues 1-6, 13-33, or 41-46 of SEQ ID NO:2; amino acid residues 1-6, 15-35, or 44-39 of SEQ ID NO:3; or amino acid residues 1-6, 15-35, or 44-47 of any one of SEQ ID NO:4 through SEQ ID NO:23. In one or more embodiments, there may be one or more mutations outside the loop regions. In one or more embodiments, there may be mutations to amino acid residues in one or more of the regions listed above (regions that are not loop regions).
In one or more embodiments, one or more amino acid residues in the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated and one or more amino acid residues outside the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated. In one or more embodiments, one or more amino acid residues within the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23, one or more amino acid residues may be removed from within the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23, and one or more amino acid residues outside the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated. In one or more embodiments, one or more amino acid residues within the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated, one or more amino acid residues may be inserted within the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23, and one or more amino acid residues outside the loop regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or any one of SEQ ID NO:6 through SEQ ID NO:23 may be mutated.
Table 1 shows that amino acid sequences of the parental Gp2 (SEQ ID NO:1) from which the anti-B7-H3 Gp2-based scaffolds are derived; the general consensus sequence (SEQ ID NO:3); and a more specific consensus sequence (SEQ ID NO:4). Table 1 also shows example identities of X for the general consensus sequence as well as example mutations for SEQ ID NO:4. In one or more embodiments, the anti-B7-H3 Gp2-based scaffolds of the present disclosure include any one of SEQ ID NO:3 or SEQ ID NO:4 having any combination of example possible mutations or identities of X shown in Table 1.
Variants of the disclosed sequences also include anti-B7-H3 Gp2-based scaffold fragments, or full-length anti-B7-H3 Gp2-based scaffolds, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original anti-B7-H3 Gp2-based scaffold (e.g., SEQ ID NO:3 and SEQ ID NO:4) over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties. Illustrative amino acid conservative substitutions include the changes of:alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate or asparagine; cysteine to serine; glutamine to asparagine or glutamate; glutamate to aspartate 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 glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine or alanine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.
In one or more embodiments, an anti-B7-H3 Gp2-based scaffold of the present disclosure may include additional sequences, such as, for example, amino acids appended to the C-terminal or N-terminal of the anti-B7-H3 Gp2-based scaffold. Such modifications can, for example, facilitate purification by trapping on columns, the use of antibodies, or facilitate recovery when expressed recombinantly in a microbe. Such tags include, for example, a histidine-rich tag that allows purification of proteins on nickel columns and/or a leader sequence that can traffic a recombinantly-expressed anti-B7-H3 Gp2-based scaffold to the membrane of the cell in which it is recombinantly expressed. Such gene modification techniques and suitable additional sequences are well known in the molecular biology arts. In one or more embodiments, the C-terminal and/or N-terminal modification may be cleaved from the anti-B7-H3 Gp2-based scaffold before being incorporated into, for example, a pharmaceutical composition. In other embodiments, retaining a C-terminal or N-terminal modification may be desired for a given application—e.g., to facilitate immobilization to a substrate.
In one or more embodiments, an anti-B7-H3 Gp2-based scaffold described herein also may include N-terminal or C-terminal functionalities other than a carboxylic acid or free amine. For example, the C-terminus, N-terminus, or both, of an anti-B7-H3 Gp2-based scaffold may be acylated, for example, acetylated. In one or more embodiments, the functionality may include a polyol (e.g., polyethylene glycol).
In one or more embodiments, an anti-B7-H3 Gp2-based scaffold described herein may include post-translational modifications 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 may be incorporated specifically or non-specifically through methods known in the biological arts.
In one or more embodiments, the anti-B7-H3 Gp2-based scaffold may be produced recombinantly in a suitable host cell and then purified. Methods for recombinant production and purification of proteins are well known in the art.
In one or more embodiments, the anti-B7-H3 Gp2-based scaffold of the present disclosure may be produced using solid phase peptide synthesis. In this method of production, the anti-B7-H3 Gp2-based scaffold may be synthesized as one complete polypeptide or in multiple polypeptide fragments that can be joined using native chemical ligation, expressed protein ligation, Staudinger ligation, or Ser/Thr ligation. Methods for solid phase peptide synthesis and ligation techniques are well known in the art.
In another aspect, this disclosure describes polynucleotides that encode any of the anti-B7-H3 Gp2-based scaffolds described herein, and the complements of such polynucleotide sequences. Given the amino acid sequence of any of the anti-B7-H3 Gp2-based scaffolds described herein, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.
The anti-B7-H3 Gp2-based scaffolds of the present disclosure may be engineered using various techniques and assays. For example, magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) may be used to select scaffolds of interest that bind to B7-H3 in a yeast display model.
The following describes a campaign that was used to engineer at least a portion of the anti-B7-H3 Gp2-based scaffolds of the present disclosure (see Example 1 and Example 2 for additional details). A Gp2 scaffold library that included Gp2 amino acid sequences derived from SEQ ID NO:1 were used to discover binders to B7-H3 via magnetic activated cell sorting and fluorescence activated cell sorting. The initial library (0.0 Gp2-based scaffold population, otherwise called a library) underwent three MACS selections (to give 0.1, 0.2, and 0.3 Gp2-based scaffold populations) using recombinant human B7-H3 extracellular domain immobilized on magnetic beads, followed by a sole FACS selection (to give a 0.4 Gp2-based population) using the same recombinant B7-H3 protein at 50 nM and gated to select for Gp2-based scaffolds with moderate affinity. DNA was isolated from the 0.4 Gp2 population (library), randomly mutated via error-prone PCR, and electroporated back into yeast. The mutated naïve population underwent a MACS selection (to give a 1.1 Gp2-based scaffold population), followed by a monovalent MACS sort (to give a 1.2 Gp2-based scaffold population) with 100 nM recombinant human B7-H3. Gp2 populations were then further enriched with two FACS selections (to give a 1.3 and 1.4 Gp2-based scaffold populations) with detergent solubilized MS1-B7-H3 lysate of increasing stringency, using estimated B7-H3 lysate concentrations of 50 nM and 1 nM. Yeast were grown and reinduced between each selection.
Single colonies of the Gp2 populations were stochastically chosen and Sanger sequenced at two selection points: after the first FACS sort (fourth sort overall; sequencing the 0.4 library sequences), as well as after all selections (two MACS selections and two FACS lysate selections; resulting in the 1.4 population) post-error-prone PCR. Sequencing results for the Gp2 variants (i.e., Gp2-based scaffolds) is shown in
Select Gp2 variants were further characterized for binding affinity (Kd). Affinity titration of Gp2 1.4.3 (SEQ ID NO:8) and Gp2 1.4.4 (SEQ ID NO:4) gave Kds of 3.4 nM (68% confidence interval:2.7-4.2 nM) and 1.0 (0.8-1.3) nM, respectively (
The 1.4 Gp2-based population was further engineered for affinity, specificity, and stability maturation (see Example 2 for additional details). DNA was isolated from the 1.4 yeast population and subjected to random mutagenesis via error-prone polymerase chain reaction (PCR) of variable regions of the affibodies. DNA was electroporated back into yeast for display and further sorted using the following sort scheme:(1) lysate MACS with 1 pmol B7-H3+ lysate sort; (2) an affinity sort (strict Kd FACS sort); (3) specificity sort (multitarget MACS depletion sort); and (4) stability sort (thermolysin at 55° C. FACS sort). After each sort, the populations were deep sequenced.
The frequencies at each sort (e.g., avidity, affinity, specificity, and stability) were determined for each sequence, as well as their subsequent enrichment. If read count was 0, a read count of 1 was assigned. The z-score for affinity, specificity, stability, and final frequency was determined and a final conditional, weighted averaged z-score was calculated (see Example 2).
Top performers were selected based on the highest score.
In addition to the anti-B7-H3 Gp2-based scaffold, in one or more embodiments, the anti-B7-H3 compound may include a first functional component operably linked to the anti-B7-H3 Gp2-based scaffold. In one or more embodiments, the first functional component may include a targeting component. In one or more embodiments, the first functional component may include an imaging component. In one or more embodiments, the first functional component may include an enzyme. In one or more embodiments, the first functional component may include a small molecule drug, a pharmacologically active derivative thereof, or an activatable inactive form of a drug (e.g., a prodrug).
In one or more embodiments, the first functional component includes a targeting component. In embodiments where the first functional component includes a targeting component, the anti-B7-H3 compound may be an immunotherapeutic compound.
Immunotherapeutic compounds can provide individualized treatment that activates or suppresses the immune system to amplify or diminish an immune response and is developing rapidly for treating various forms of cancer. Immunotherapy for cancer, such as chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, PD-1 and PD-L1 inhibitors, aim to help a subject's immune system fight cancer.
In one or more embodiments, the targeting component may bind to and recruit an effector cell to the B7-H3 displaying cell. Types of effector cells include, but are not limited to, natural killer (NK) cells, B cells, or T cells. The targeting component may bind to surface protein displayed on the effector cell. Non-limiting examples of surface proteins displayed on NK cells include, CD16, PD-1, NKp30, NKp40, NKp44, NKp46, NKG2C, or KIRs. Non-limiting examples of surface proteins displayed on B cells include CD4, CD8, or VLA-4. Non-limiting examples of surface proteins displayed on T cells include (LFA)-1, CD2, CD4, or CD8.
In one or more embodiments, the targeting component may include an antibody or fragment thereof, an affibody, a peptide, a protein, or a small molecule.
In one or more embodiments, the first functional component includes an imaging component. In embodiments where the first functional component includes an imaging component, the anti-B7-H3 compound may be used as a diagnostic tool to detect B7-H3 positive tumors.
The imaging component may be any component that can produce a detectable signal. Exemplary types of imaging components include, but are not limited to a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label. Depending on the type of imaging component, various methods may be used to detect the imaging moiety. Example 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. The imaging techniques may be accomplished in vivo or in vitro.
In one or more embodiments, the first functional component may include an enzyme. In one or more embodiments, the enzyme may modify one or more of the surface proteins displayed on the cell displaying B7-H3 to which the anti-B7-H3 Gp2-based scaffold is bound. Examples of enzymes that modifies surface proteins, include but are not limited to, a protease, a lipase, and a sortase such as sortase A.
To analyze the modularity of Gp2-based scaffolds, Gp2 1.4.4 was genetically linked to sortase A protein enzyme via a 15 amino acid glycine rich linker (SEQ ID NO:24) at the Gp2-based scaffold's C-terminus, i.e., Gp2-L15-SrtA fusion where L15 is the linker. Modularity was assessed by the retention of B7-H3 binding affinity of the Gp2-based scaffold in the presence of glycine-linked sortase A enzyme. Affinity titration of Gp2-L15-SrtA fusion gave a Kd of 1.8 nM (1.4-2.2 nM;
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 of a pharmacologically active substance (e.g., a prodrug). In embodiments where 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 where the chemotherapeutic compound includes one or more radioisotopes, the anti-B7-H3 compound may be used for radioisotope therapy (radionuclide therapy). Examples of suitable isotopes include iodine-131, iridium-192, strontium-89, samarium-153, rhenium-186, boron-10, phosphorus-32, or radium 223. The anti-B7-H3 Gp2-based scaffold may allow for localization of the small molecule drug to B7-H3 displaying cells, such as tumor cells, thereby delivering the chemotherapeutic compound to a tumor cell that expresses B7-H3. In this way, the chemotherapeutic compound may be administered to a subject systematically yet have increased local activity and reduced systemic side effects compared to administering the drug systemically without being a component of an anti-B7-H3 compound.
In one or more embodiments, the anti-B7-H3 compound may include additional functional components in addition to the first functional component and anti-B7-H3 Gp2-based scaffold. For example, the anti-B7-H3 compound may include a second functional component, a third functional component, a fourth functional component, a fifth functional component, a sixth functional component, or a seventh functional component. Each functional component may include anti-B7-H3 Gp2-based scaffold, an imaging component, an enzyme, a small molecule drug, or a targeting component (all of which are previously described herein), independent of the identity of any other functional component in the anti-B7-H3 compound.
In some embodiments where the anti-B7-H3 compound includes at least one functional component, the anti-B7-H3 Gp2-based scaffold and the functional component are operably linked. As used herein, the term “operably linked” refers to a direct or indirect covalent linking between the anti-B7-H3 Gp2-based scaffold and any additional functional component or functional components of the anti-B7-H3 compound. Thus, two functional components, or one functional component and the anti-B7-H3 Gp2-based scaffold, that are operably linked may be directly covalently coupled to one another. Conversely, two operably linked functional components, or one functional component operably linked to the anti-B7-H3 Gp2-based scaffold, may be connected by mutual covalent linking to an intervening component (e.g., a flanking sequence or linker). For example, in embodiments where the anti-B7-H3 compound includes two functional components, the first functional component and the second functional component may be separately directly linked to the anti-B7-H3 Gp2-based scaffold; or the first functional component may be directly linked to the anti-B7-H3 Gp2-based scaffold and the second functional component directly linked to the first functional component.
The anti-B7-H3 Gp2-based scaffold and the first functional component may be operably linked through one or more linkers. The term “linker” as used herein refers to any bond, small molecule, peptide sequence, or other vehicle that physically links the components. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and/or disulfide bond cleavage at conditions under which the first functional (or other functional components) component and/or the anti-B7-H3 Gp2-based scaffold remains active. Linkers are classified based on the presence of one or more chemical motifs such as, for example, including a disulfide group, a hydrazine group or peptide (cleavable), or a thioester group (non-cleavable). Linkers also include charged linkers, and hydrophilic forms thereof as known in the art.
Suitable linkers for linking the anti-B7-H3 Gp2-based scaffold and the first functional component of the present disclosure include a natural linker, an empirical linker, or a combination of natural and/or empirical linkers. Natural linkers are derived from the amino acid linking sequence of multi-domain proteins, which are naturally present between protein domains. Properties of natural linkers such as, for example, length, hydrophobicity, amino acid residues, and/or secondary structure can be exploited to confer desirable properties to a multi-domain compound that includes natural linkers connecting the components of the anti-B7-H3 compounds of the present disclosure.
The studies of linkers in natural multi-domain proteins have led to the generation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers are often classified as three types flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide a certain degree of movement or interaction at the joined components. Flexible linkers typically include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, which provide flexibility, and allow for mobility of the connected components. An example of a flexible linker useful for linking the anti-B7-H3 Gp2-based scaffold and the first functional component of the anti-B7-H3 compounds of the present disclosure is SEQ ID NO:24. Rigid linkers can successfully keep a fixed distance between the first functional component and the anti-B7-H3 Gp2-based scaffold of the anti-B7-H3 compounds to maintain their independent functions, which can provide efficient separation of the first functional component and the anti-B7-H3 Gp2-based scaffold and/or sufficiently reduce interference between the first functional component and the anti-B7-H3 Gp2-based scaffold. Cleavable linkers can allow one to control the release of the first functional component and/or the anti-B7-H3 Gp2-based scaffold in vivo. By taking advantage of unique in vivo processes, cleavable linkers can be cleaved under specific conditions such as the presence of reducing reagents or proteases. This type of linker can reduce steric hindrance, improve bioactivity, and/or achieve independent actions/metabolism of the first functional component and/or the anti-B7-H3 Gp2-based scaffold after linker cleavage.
Exemplary linker sequences include the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33; SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
In one or more embodiments, the natural linker or empirical linker is covalently attached to the anti-B7-H3 Gp2-based scaffold, the first functional component, or both, using bioconjugation chemistries. Bioconjugation chemistries are well known in the art and include, but are 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.
In one or more embodiments, the natural linker or empirical linker, the first functional component, the anti-B7-H3 Gp2-based scaffold, or combinations thereof, may include one or more unnatural amino acids that allow for bioorthogonal conjugation reactions. As used herein, “bioorthogonal conjugation” refers to a conjugation reaction that uses one or more unnatural amino acids or modified amino acids as a starting reagent. Examples of bioorthogonal conjugation reactions include, but are not limited to, Staudinger ligation, copper-catalyzed azide-alkyne cycloaddition, strain promoted [3+2]cycloadditions, tetrazine ligation, metal-catalyzed coupling reactions, or oxime-hydrazone ligations. Examples of non-natural 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, Nε-(cyclooct-2-yn-1-yloxy)carbonyl)L-lysine, Nε-2-azideoethyloxycarbonyl-L-lysine, Nε-p-azidobenzyloxycarbonyl lysine, propargyl-L-lysine, or trans-cyclooct-2-ene lysine. Such amino acids may be incorporated at any location of the anti-B7-H3 Gp2-based scaffolds of the present disclosure or at any location on any of the functional components.
In one or more embodiments, the linker is derived from a small molecule, such as a polymer. Example polymer linkers include, but are not limited to, poly-ethylene glycol, poly(N-isopropylacrylamide), and N,N′-dimethylacrylamide)-co-4-phenylazophenyl acrylate. The small molecule linkers generally include one or more reactive handles allowing conjugation to the anti-B7-H3 Gp2-based scaffold, the component, or both. In one or more embodiments, the reactive handle allows for a bioconjugation or bioorthogonal conjugation. In one or more embodiments, the reactive handle allows for any organic reaction compatible with conjugating a linker to the anti-B7-H3 Gp2-based scaffold or the first functional component.
The linker may be conjugated at any amino acid location of the anti-B7-13 Gp2-based scaffold. For example, the linker may be conjugated to the N-terminus, C-terminus, or any amino acid between.
In embodiments where the anti-B7-H3 compound includes more than one functional component, the additional functional components may be operably coupled to each other and/or the anti-B7-1-13 Gp2-based scaffold using one or more of the linkers disclosed herein.
In another aspect, this disclosure describes a host cell including any of the isolated nucleic acid sequences, anti-B7-H3 compounds, and/or anti-B7-H3 Gp2-based scaffolds described herein.
In some embodiments where the anti-B7-H3 compound includes an anti-B7-H3 Gp2-based scaffold and one or more functional components operably coupled by peptide linkers, the anti-B7-H3 compound may be produced by expression in a host cell using methods known in the art. In some embodiments where the anti-B7-H3 compound includes an anti-B7-H3 Gp2-based scaffold and one or more functional components operably coupled by peptide linkers, the anti-B7-H3 compound may be produced using solid phase peptide synthesis using methods known in the art.
In another aspect, this disclosure describes an isolated nucleic acid sequence that encodes the amino acid sequence of any embodiment of the anti-B7-H3 Gp2-based scaffolds and/or anti-B7-H3 compounds described herein or any component polypeptide fragment thereof. Given the amino acid sequence of any polypeptide, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.
In another aspect, this disclosure describes a host cell including any of the isolated nucleic acid sequences, anti-B7-H3 compounds, and/or anti-B7-H3 Gp2-based scaffolds described herein.
The nucleic acid constructs of the present disclosure may be introduced into a host cell to be altered, thus allowing expression of an anti-B7-H3 compounds and/or an anti-B7-H3 Gp2-based scaffold within the cell, thereby generating a genetically engineered cell. A variety of methods are known in the art and suitable for introduction of a nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (e.g., nanoparticles), cationic polymer mediated transfer (e.g., DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as LIPOFECTAMINE (Thermo Fisher Scientific, Inc., Waltham, MA), HILYMAX (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), JETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc USA, San Diego, CA).
The nucleic acid constructs described herein may be introduced into a host cell to be altered, thus allowing expression within the cell of the anti-B7-H3 compound and/or the anti-B7-H3 Gp2-based scaffold and/or anti-B7-H3 compounds including an anti-B7-H3 Gp2-based scaffold operably coupled to an antibody, affibody, or other polynucleotide, encoded by the nucleic acid. A variety of host cells are known in the art and suitable for protein expression. Examples of typical cell used for transfection and protein expression include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell such as, for example, 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.
In another aspect, this disclosure provides pharmaceutical compositions that include one or more anti-B7-H3 compounds described herein (including anti-B7-H3 compounds that include an anti-B7-H3 Gp2-based scaffold and no additional functional components), formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of, for example, two or more different anti-B7-H3 compounds.
As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, e.g., the material may be administered to an individual along with an anti-B7-H3 compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
A pharmaceutical composition may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. A pharmaceutical composition may include a pharmaceutically acceptable antioxidant. Those skilled in the art are generally aware of and understand how to apply the use of pharmaceutically acceptable salts and/or pharmaceutically acceptable antioxidants.
The pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier. As used herein, the term “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, hydrogel, colloid, an accessory agent, stabilizer, protein carrier, biological carrier compound, or the like. Non-limiting examples of solvents include water, ethanol, a polyol (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), a vegetable oil (e.g., olive oil), or an injectable organic ester (e.g., ethyl oleate), or combinations thereof. Non-limiting examples of a protein carrier includes keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, or the like. Non-limiting examples of a biological compound which may serve as a carrier include a glycosaminoglycan, a proteoglycan, or albumin. The carrier may include an organic solvent, (e.g., dimethyl sulfoxide), a synthetic compound, or a synthetic polymer (e.g., a polyalkyleneglycol). Ovalbumin, human serum albumin, other proteins, polyethylene glycol, or the like may be employed as the carrier. The use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient (i.e., the anti-B7-H3 compound), its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
A pharmaceutical composition may also, or alternatively, may include one or more adjuvants such as, for example, a preservative, a wetting agent, an emulsifying agent, and/or a dispersing agent. In one or more embodiments, a pharmaceutical composition can include an antibacterial agent and/or an antifungal agent such as, for example, paraben, chlorobutanol, phenol sorbic acid, or the like. It may also be desirable to include isotonic agents, such as a sugar, sodium chloride, or a polyalcohol (e.g., mannitol, sorbitol, etc.) into the compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be provided by including an agent that delays absorption such as, for example, aluminum monostearate or gelatin.
An anti-B7-H3 compound of the present disclosure may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release.
A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the active ingredient into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and/or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.
To prepare pharmaceutical compositions including an anti-B7-H3 of the present disclosure, the anti-B7-H3 compound may be mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions.
Thus, an anti-B7-H3 compound of the present disclosure may be provided in any suitable form including, but not limited to, a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle.
Nasal spray formulations include purified aqueous solutions of the active ingredient with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acids. Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye.
Topical formulations include an anti-B7-H3 compound dissolved or suspended in one or more media such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations. For example, topical formulations may include a cream, an ointment, a paste, a lotion, a powder, a solid, an aerosolized foam, or a gel. Topical formulations may contain a permeation enhancer to increase the bioavailability of the active ingredient. Topical formulations may contain preservatives and/or emulsifiers. Topical formulations may be provided in the form of a transdermal patch or bandage, wherein the formulation is incorporated into a gauze or other structure and brought into contact with the skin. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, or the like.
Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as a tablet, a troche, a capsule, a lozenge, a wafer, or a cachet, each containing a predetermined amount of the active ingredient as a powder or granules, as liposomes, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, or a draught. The tablet, troche, pill, capsule, or the like may also contain one or more of the following:a binder such as gum tragacanth, acacia, 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 sweetening agent such as sucrose, fructose, lactose, or aspartame; or a natural or artificial flavoring agent. When the unit dosage form is a capsule, it may further contain a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, a tablet, a pill, or a capsule may be coated with gelatin, wax, shellac, sugar, and/or the like. A syrup or elixir may contain one or more sweetening agent, preservative such as methyl- or propylparaben, an agent to retard crystallization of sugar, an agent to increase the solubility of any other ingredient (e.g., a polyhydric alcohol such as, glycerol or sorbitol), a dye, and/or flavoring agent. The material used in preparing any unit dosage form is substantially nontoxic in the amounts employed. The active ingredient may be incorporated into preparations or devices in formulations that may or may not be designed for sustained release.
Formulations suitable for parenteral administration can include a sterile aqueous preparation of the active ingredient, or a dispersion of a sterile powder of the active ingredient, which are preferably isotonic with the blood of the recipient. Parenteral administration of an anti-B7-H3 compound or a pharmaceutical composition containing the same of the present disclosure (e.g., through an intravenous drip) is one form of administration. Isotonic agents that may be included in the liquid preparation include a sugar, a buffer, and/or sodium chloride. Solutions of the active ingredient may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions of the active ingredient may be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), a vegetable oil, a glycerol ester, or any mixtures thereof.
In yet another aspect, this disclosure provides imaging methods and methods of treating, ameliorating, detecting, diagnosing, or monitoring a disease or a symptom or clinical sign thereof, as described herein, in a patient by administering a therapeutically effective amount of an anti-B7-H3 compound, an anti-B7-H3 Gp2-based scaffold and/or a pharmaceutical composition that includes one or more anti-B7-H3 compounds described herein. As used herein, the term “treating” and variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or clinical signs related to a condition. A “symptom” refers to any subjective evidence of disease or of a patient's condition; a “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient. A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after a condition manifests in a subject, while “prophylactic” treatment is initiated before a condition manifests in a subject. Prophylactic treatment may be administered to a subject at risk of having a condition. “At risk” refers to a subject that may or may not actually possess the described risk. In the case of a non-infectious condition, for example, a subject “at risk” for developing a specified condition is a subject that possesses one or more indicia of increased risk of having, or developing, the specified condition compared to individuals who lack the one or more indicia, regardless of whether the subject manifests any symptom or clinical sign of having or developing the condition.
Thus, in one or more embodiments, treating a subject includes a subject having, or at risk of having cancer. Generally, the method includes administering to the subject an effective amount of an anti-B7-H3 compound or a pharmaceutical composition that includes one or more anti-B7-H3 compounds described herein. As used herein, the term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) that differentiates cancer (malignant tumor) from benign tumor. As used herein, “neoplasm” or “tumor” (and grammatical variations thereof) means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, or the like.
The amount of the anti-B7-H3 compound administered can vary depending on various factors including, but not limited to, the anti-B7-H3 compound being 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 included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and/or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of anti-B7-Compound effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
For example, certain anti-B7-H3 compounds may be administered at the same dose and frequency for which the drug has received regulatory approval. In other cases, certain anti-B7-H3 compounds may be administered at the same dose and frequency at which the drug is being evaluated in clinical or preclinical studies. One can alter the dosages and/or frequency as needed to achieve a desired level of anti-B7-H3 effect. Thus, one can use standard/known dosing regimens and/or customize dosing as needed.
In one or more embodiments, the method can include administering a sufficient amount of an anti-B7-H3 compound to provide a dose of, for example, from about 100 ng/kg to about 50 mg/kg to the subject, although in some embodiments the methods may be performed by administering an anti-B7-H3 compound in a dose outside this range. In some of these embodiments, the method includes administering a sufficient amount of an anti-B7-H3 compound to provide a dose of from about 10 μg/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 μg/kg to about 1 mg/kg.
A single dose may be administered all at once, continuously for a prescribed period of time, or in multiple discrete administrations. When multiple administrations are used, the amount of each administration may be the same or different. For example, a dose of 1 mg per day may be administered as a single administration of 1 mg continuously over 24 hours, as two or more equal administrations (e.g., two 0.5 mg administrations), or as two or more unequal administrations (e.g., a first administration of 0.75 mg followed by a second administration of 0.25 mg). When multiple administrations are used to deliver a single dose, the interval between administrations may be the same or different.
In one or more embodiments, the active ingredient may be administered, for example, from a single dose to multiple doses per week, although in some embodiments the method can involve a course of treatment that includes administering doses of the active ingredient at a frequency outside this range. When a course of treatment involves administering multiple doses within a certain period, the amount of each dose may be the same or different. For example, a course of treatment can include a loading dose (e.g., an initial dose), followed by a maintenance dose that is lower than the loading dose. Also, when multiple doses are used within a certain period, the interval between doses may be the same or be different.
In certain embodiments, an anti-B7-H3 compound may be administered from about once per month to about five times per week.
An anti-B7-H3 compound or pharmaceutical composition containing the same 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 decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the anti-B7-H3 compound or pharmaceutical composition containing the same is not administered, decreasing the severity of symptoms and/or clinical signs of the condition, and/or completely resolving the condition. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the severity of symptoms and/or clinical signs of the condition compared to a subject to which the anti-B7-H3 compound or pharmaceutical composition containing the same is not administered, and/or completely resolving the condition.
The anti-B7-H3 Gp2-based scaffold may be any anti-B7-H3 Gp2-based scaffold disclosed herein. The anti-B7-H3 compound can be any embodiment of the anti-B7-H3 compound described herein having an anti-B7-H3 Gp2-based scaffold that binds to the extracellular domain of B7-H3 displayed on the target cells of a cell population. In some cases, the target cell can include a tumor cell so that the method can involve treating cancer associated with the tumor cells. Thus, in one or more embodiments, the method can include ameliorating at least one symptom or clinical sign of the tumor.
In embodiments in which the target cell includes a tumor cell, the method can further include surgically resecting the tumor and/or reducing the size of the tumor through chemical (e.g., chemotherapeutic) and/or radiation therapy. Exemplary tumors that may be treated include tumors associated with prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, and/or hematopoietic cancer.
In one or more embodiments, the anti-B7-H3 compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.
In one or more embodiments, an anti-B7-H3 compound may be administered, for example, from a single dose to multiple doses per week, although in some embodiments the method can be performed by administering an anti-B7-H3 compound at a frequency outside this range. In certain embodiments, an anti-B7-H3 compound may be administered from about once per month to about five times per week.
In one or more embodiments, the method further includes administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and/or coincident to the administration of an anti-B7-H3 compound or pharmaceutical composition containing the same. An anti-B7-H3 compound or pharmaceutical composition containing the same and the additional therapeutic agents may be co-administered. As used herein, “co-administered” refers to two or more components of a combination administered so that the therapeutic or prophylactic effects of the combination can be greater than the therapeutic or prophylactic effects of either component administered alone. Two components may be co-administered simultaneously or sequentially. Simultaneously co-administered components may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes cases in which the components are administered so that each component can be present at the treatment site at the same time. Alternatively, sequential co-administration of two components can include cases in which at least one component has been cleared from a treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a certain cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination can, in certain circumstances, include components that never exist in a chemical mixture with one another. In other embodiments, an anti-B7-H3 compound or pharmaceutical composition containing the same and the additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, the administration of an anti-B7-H3 compound, or pharmaceutical composition containing the same may allow for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic agent or agents alone, thereby decreasing the likelihood, severity, and/or extent of the toxicity observed when a higher dose of the other therapeutic agent or agents is administered.
The term “chemotherapeutic agent” as used herein refers to any therapeutic agent used to treat 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, tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumamab, Erbitux™ (cetuximab), matuzumab, IMC-JIF 8, TheraCIM hR3, denosumab, Avastin™ (bevacizumab), Humira™ (adalimumab), Herceptin™ (trastuzumab), Remicade™ (infliximab), rituximab, SynagiS™ (palivizumab), Mylotarg™ (gemtuzumab oxogamicin), Raptiva™ (efalizumab), Tysabri™ (natalizumab), Zenapax™ (dacliximab), NeutroSpec™ (Technetium (99mTc) fanolesomab), tocilizumab, ProstaScint™ (Indium-Ill labeled Capromab Pendetide), Bexxar™ (tositumomab), Zevalin™ (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), Xolair™ (omalizumab), MabThera™ (Rituximab), ReoPro™ (abciximab), MabCampath™ (alemtuzumab), Simulect™ (basiliximab), LeukoScan™ (sulesomab), CEA-Scan™ (arcitumomab), Verluma™ (nofetumomab), Panorex™ (edrecolomab), alemtuzumab, CDP 870, natalizumab, Gilotrif™ (afatinib), Lynparza™ (olaparib), Perjeta™ (pertuzumab), Otdivo™ (nivolumab), Bosulif™ (bosutinib), Cabometyx™ (cabozantinib), Ogivri™ (trastuzumab-dkst), Sutent™ (sunitinib malate), Adcetris™ (brentuximab vedotin), Alecensa™ (alectinib), Calquence™ (acalabrutinib), Yescarta™ (ciloleucel), Verzenio™ (abemaciclib), Keytruda™ (pembrolizumab), Aligopa™ (copanlisib), Nerlynx™ (neratinib), Imfinzi™ (durvalumab), Darzalex™ (daratumumab), Tecentriq™ (atezolizumab), or Tarceva™ (erlotinib). Examples of immunotherapeutic agents include, but are not limited to, an interleukin (IL-2, IL-7, IL-12, etc.), a cytokine (an interferon, G-CSF, etc.), a chemokine (CCL3, CC126, CXCL7), or an immunomodulatory imide drug (imiquimod, thalidomide, etc., or an analog thereof).
In some embodiments, of the method can include administering a sufficient amount of an anti-B7-H3 compound as described herein and administering the at least one additional therapeutic agent demonstrates therapeutic synergy. In some aspects of the methods of the present disclosure, a measurement of response to treatment observed after administering both an anti-B7-H3 compound as described herein, and the additional therapeutic agent is improved over the same measurement of response to treatment observed after administering either the anti-B7-H3 compound or the additional therapeutic agent alone.
In yet another aspect, this disclosure describes a capture assay device including any embodiment of one of the anti-B7-H3 compounds (including an anti-B7-H3 Gp2-based scaffold without an additional functional component) described herein immobilized to a substrate. For example, an anti-B7-H3 compounds described herein can be incorporated into cell and/or ligand capture technology such as, for example, an ELISA-based assay. A substrate to immobilize the anti-B7-H3 compound can include, for example, a cell culture plate or dish, a glass slide, or any other support than can be used to perform an assay requiring an immobilized anti-B7-H3 compound.
In another aspect, the present disclosure describes a method for using anti-B7-H3 compounds in molecular imaging applications including, for example, both traditional molecular imaging techniques (e.g., magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound, photoacoustic, and fluorescence) and microscopy and/or nanoscopy imaging techniques (e.g., total internal reflection fluorescence (TIRF)-microscopy, stimulated emission depletion (STRED)-nanoscopy, or atomic force microscopy (AFM).
The anti-B7-H3 compounds described herein may have in vitro and in vivo detection, diagnostic, and/or therapeutic utilities. For example, 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 as described herein, or any additional carrier or adjuvant known in the art. The method generally can include allowing an anti-B7-H3 compound that specifically binds to a target of interest with a sample that includes the target of interest, 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 radioactive isotope, a fluorescent marker, an enzyme, or a colorimetric marker.
In the preceding description and following claims, 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 “comprises,” “comprising,” and variations thereof are to be construed as open ended i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one or more embodiments,” “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The terms “administration of” and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment.
As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.
The term “polypeptide” refers to a sequence of amino acid residues without regard to the length of the sequence. Therefore, the term “polypeptide” refers to any amino acid sequence having at least two amino acids and includes full-length proteins, fragments thereof, and/or, as the case may be, polyproteins.
The term “protein” refers to any sequence of two or more amino acid residues without regard to the length of the sequence, as well as any complex of two or more separately translated amino acid sequences. Protein also refers to amino acid sequences chemically modified to include a carbohydrate, a lipid, a nucleotide sequence, or any combination of carbohydrates, lipids, and/or nucleotide sequences. As used herein, “protein,” “peptide,” and “polypeptide” are used interchangeably.
As used herein, the term “nucleic acid” or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides. A nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. A nucleic might be introduced—i.e., transfected—into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
The Gp2 library design, detailed in Kruziki et al. (ACS Comb. Sci. 20, 423-435 (2018) and Mol. Pharm. 13, 3747-3755 (2016)) comprised a truncated form of T7 phage gene 2 protein (SEQ ID NO:1), diversified at six sites in each of two loops using degenerate codons encoding for an amino acid distribution mimicking antibody CDRs (Kruziki et al., Chem. Biol. 22, 946-956 (2015)), as well designs that constrained diversity and extended the paratope (Kruziki et al., ACS Comb. Sci. 20, 423-435 (2018)).
Yeast surface display was used to display the Gp2 libraries, and B7-H3 binders were selected using both magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Populations were sorted to achieve detectable binding at 50 nM B7-H3 target. DNA was then isolated from yeast and subject to random mutagenesis via error-prone polymerase chain reaction (PCR) of paratope and entire Gp2 gene. DNA was electroporated back into yeast for display and further sorted until binding was observed to low nanomolar concentrations of B7-H3.
MACS Selections with Soluble Extracellular Domains
Magnetic bead selections were carried out using at least 15-fold oversampling of ligand diversity at all stages. Biotinylated recombinant human B7-H3 extracellular domain (available from Sino Biological, Inc., Beijing, China) for positive isolation and biotinylated Renilla renformis green fluorescent protein (rrGFP; available from Avidity, LLC, Aurora, CO) for depletion were incubated with DYNABEADS Biotin Binder (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA) to coat the beads with protein. Yeast underwent magnetic activated cell sorting, where yeast were incubated with control bare biotin binder beads for two hours at 4° C., followed by another two-hour incubation for depletion with GFP-labeled beads, to remove any non-specific and non-B7-H3 binding interactions. Yeast were then incubated two hours more with beads with immobilized recombinant human B7-H3 target protein. Bound yeast were positively selected. MACS was performed at 4° C., and yeast were washed twice between incubations. A total of three MACS selections were performed with increasing wash stringency, where yeast were grown and induced between each sort. After error-prone PCR, an additional MACS sort was performed on the large mutated naïve library, akin to the procedure described previously.
FACS Selections with Soluble Extracellular Domains
After three MACS sorts of the Gp2 libraries, library size was sampleable via FACS. Induced yeast populations were simultaneously labeled with mouse anti-c-Myc antibody (BioLegend, San Diego, CA) and 50 nM biotinylated recombinant human B7-H3 extracellular domain for 30 minutes at 4° C. Cells were washed once with PBSA, labeled with goat anti-mouse Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA) and streptavidin Alexa Fluor 488 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA) for 20 minutes at 4° C., and washed with PBSA. Cells that were Myc positive and had B7-H3 signal above background were collected.
Random mutation of the 0.4 Gp2 population (library) was performed by error-prone PCR to give the 1.4 Gp2 library using with mutagenic analogs, 8-oxo-dGTP and dPTP. Zymoprepped plasmid DNA was mutated by error-prone PCR of full Gp2 genes using primers W5/W3 and Gp2 loops using loop 1 and loop 2 mutagenesis primers (Kruziki et. at., Chem. Biol. 22, 946-956 (2015)). PCR products were purified by agarose gel electrophoresis. Gp2 loop genes were assembled into one construct using PCR assembly. Final gene inserts were amplified by PCR, concentrated by ethanol precipitation, and resuspended for electroporation. Mutated Gp2 libraries were homologously recombined with linearized pCT-Gene or into EBY100 yeast by electroporation transformation. Electroporation yielded roughly 33 million transformants for Gp2.
Monovalent MACS Sorting with Soluble Extracellular Target
After a MACS sort was performed on the large mutated naïve library (1.4 library), yeast underwent a more stringent monovalent MACS sort. Yeast were incubated with control bare biotin binder beads, washed, and then depletion by GFP-coated beads, as before. Yeast were then washed and incubated with 100 nM biotinylated recombinant human B7-H3 extracellular domain for one hour. Bare biotin binder beads were spiked in to bind to biotinylated B7-H3 and were incubated for an additional two hours at 4° C. Beads were washed three times, and yeast remaining bound were collected.
Mile Sven 1 cells stably transfected to express human B7-H3 (MS1-B7-H3) were grown at 37° C. with 5% CO2 in DMEM with 10% fetal bovine serum (v/v) and 1% penicillin and streptomycin. MS1-B7-H3 cells were grown to 70-90% confluence in 75 cm2 tissue culture-treated flasks. Cells were washed with PBS and detached with trypsin-EDTA treatment for five minutes, quenched with serum containing culture media, and pelleted at 500×g for three minutes. Pelleted cells were washed twice and resuspended in PBS with 0.5 mg/mL fresh sulfo-NHS-biotin (Thermo Fisher Scientific, Inc., Waltham, MA) for 30 minutes at room temperature. Cells were washed twice following incubation to remove excess biotin and were lysed in 250 μL lysis buffer for 15 minutes at 4° C. Cell debris was pelleted for 30 minutes at 10,000×g and removed.
FACS Selections with Detergent Solubilized Cell Lysates
Gp2 libraries underwent two rounds of flow cytometry selections with detergent solubilized cell lysate. The libraries were washed once with PBSA and incubated with cell lysate for one hour at 4° C. Following incubation, yeast were washed, incubated with chicken anti-Myc-FITC (Immunology Consultants Laboratory Inc., Portland, OR) and streptavidin, Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA) for 20 minutes at 4° C., and washed again. Yeast that were Myc positive (FITC) with the highest ratio of MS1-B7-H3 lysate binding (AF647):Myc (FITC) were collected using FACS. This sort was repeated with higher stringency by using a lower B7-H3 lysate concentration.
Enriched B7-H3-binding populations were plated on SD-CAA plates and grown for two days. Ten colonies from the Gp2 library were stochastically chosen and incubated at 100° C. for five minutes in 50 μL of distilled water. Two microliters of yeast sample were taken and underwent PCR with GeneAmp5/3 primers and DNA clean up. Amplified Gp2 genes and GeneAmp5 primer were sent to Eurofins Genomics LLC for Sanger sequencing.
Gp2 encoding regions in DNA recovered from the final B7-H3 flow cytometry sort (from the 1.4 population) were amplified by PCR, digested with NheI-IF and BamHI-HF restriction enzymes (New England Biolabs, Inc., Ipswich, MA) and ligated with T4 DNA ligase into pET-24b vector containing a C-terminal hexa-histidine tag. Plasmids were transformed into T7 Express Competent E. coli and plated on lysogeny broth (LB) plates containing 50 mg/L kanamycin. Transformants were Sanger sequenced for full-length gene and proper transformants were grown in 5 mL liquid LB with kanamycin (50 mg/L) at 37° C. at 250 rpm for 12-16 hours. Saturated cultures were added to 100 mL LB, grown, and induced. Cells were pelleted and resuspended in lysis buffer (50 mM sodium phosphate (pH 8.0), 0.5 M sodium chloride, 5% glycerol, 5 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and 25 mM imidazole), frozen and thawed five times to lyse cells, centrifuged for 10 minutes at 4° C., and 0.25 mm filtered. The resulting cell lysates were run through 0.25 mL Cobalt HisPur resin volume spin columns, washed with 30 mM imidazole, and eluted with 300 mM imidazole. Gp2 purity and concentration were analyzed via protein gel electrophoresis.
Detached MS1-B7-H3 cells were washed and individually labeled with varying concentrations of purified Gp2 and Gp2 fusions for at least 30 minutes at 4° C. Cells were pelleted at 500×g for three minutes and washed with cold PBSA prior to labeling with anti-His6 FITC conjugate (available from Abcam, Cambridge, United Kingdom) for 20 minutes at 4° C. Fluorescence was analyzed using ACCURI C6 Plus. The dissociation constant was calculated by nonlinear least-squares regression using a 1:1 binding model in PRISM software (GraphPad Software, San Diego, CA).
Gp2 gene encoding regions were amplified by PCR and assembled via NEBuilder HIFIDNA Assembly (New England Biolabs, Inc., Ipswich, MA) into pET-24b vector containing a GSGGGSGGGKGGGGT (SEQ ID NO:3) linker and amino acids 60-206 of sortase A (EC 3.4.22.70) gene with a C-terminal hexa-histidine tag. Plasmids were transformed into BL21-CodonPlus (DE3)-RIL competent E. coli and plated on LB plates containing 50 mg/L kanamycin. Transformants were Sanger sequenced for full-length gene and proper transformants were grown in 50 mg/L kanamycin and chloramphenicol for bacteria growth and protein purification. Purified proteins were affinity titrated to assess binding affinity of Gp2 in presence of conjugated protein enzyme.
Example 2 describes affinity maturation, specificity maturation, and stability maturation of the 1.4 Gp2-based scaffold population of Example 1.
DNA was isolated from the 1.5 yeast population and subjected to random mutagenesis via error-prone polymerase chain reaction (PCR) of variable regions of the affibodies. DNA was electroporated back into yeast for display and further sorted using the following sort scheme:(1) lysate MACS with 1 pmol B7-H3+ lysate sort; (2) an affinity sort (strict Kd FACS sort); (3) specificity sort (multitarget MACS depletion sort); and (4) stability sort (thermolysin at 55° C. FACS sort). After each sort, the populations were deep sequenced, filtered via USEARCH (ultra-fast sequencing analysis; Robert C. Edgar, Search and clustering orders of magnitude faster than BLAST, Bioinformatics, Volume 26, Issue 19, 1 Oct. 2010, Pages 2460-2461, doi.org/10.1093/bioinformatics/btq461), and filtered for full length Gp2-based scaffold variants.
The frequencies at each sort (e.g., avidity, affinity, specificity, and stability) were determined for each sequence, as well as their subsequent enrichment. If read count was 0, a read count of 1 was assigned. The z-score for affinity, specificity, stability, and final frequency was determined and a final conditional, weighted averaged z-score was calculated. Top performers were selected based on the highest score.
The conditional, weighted average z-score was calculated by first determining the frequency of each variant in each sort. The enrichment values for affinity, specificity, and stability sorts for each variant were then calculated by dividing the frequency of single variant at specific sort by its frequency in the previous sort. Its z-score was calculated by (sort enrichment score−average sort enrichment score of all variants) divided by (standard deviation of sort enrichment score of all variants). The z-scores were averaged based on the following weights: affinity=1; frequency=3; specificity=2; and stability=1. The final frequency and specificity z-scores were most important, as these were given the greatest weight. Additionally, the following conditions were also included: affinity enrichment >0.5 and final frequency >0. Affinity enrichment values greater than 1 indicate enrichment. A value of 0.5 was chosen to account for variants that were initially present at higher frequencies and may not have had opportunity to enrich. The final frequency condition was included because variants must be present in final sequenced population.
An avidity sort was conducted. This sort is considered an avidity sort because the B7-H3 target is expressed multivalently on avidin beads, not just monovalently in solution. The yeast are also expressing thousands of affibodies per cell, so avid binding can occur across yeast and B7-H3-coated beads.
Magnetic bead selection was performed with 20-fold oversampling to select for B7-H3 binders and easily weed out non-binders/truncated ligands resulting from error prone PCR. Yeast were incubated with bare streptavidin-coated beads for two hours at 4° C., followed by another two-hour incubation for depletion with GFP-coated beads. Yeast were incubated two hours more with 1 pmol of detergent solubilized biotinylated MS1-B7-H3 cell lysate. Usually, 33 pmol of antigen are used to coat the streptavidin beads; however, 1 pmol was chosen to be more stringent (e.g., decrease avidity). Bound yeast were selected and grown. Biotinylation of the MS1-B7-H3 cell lysate is non-specific and reacts to all proteins (non-specific N-hydoxysuccinimide (NHS) chemistry), not just B7-H3. B7-H3 is expressed at ˜1 million per cell and the Gp2-based scaffold libraries have already been primed to have binding to B7-H3.
After one round of the B7-H3 cell lysate MACS sort, library size was easily sampleable via FACS. Induced yeast populations were labeled with ˜0.2 nM biotinylated-B7-H3 cell lysate for two hours, washed, and labeled with streptavidin-AF647 and chicken anti-Myc-FITC. The top 0.4% of cells (highest affinity) was collected for the next sort (the specificity sort).
To select for more specific binders, the population isolated after the affinity sort underwent a multitarget depletion. The following proteins were biotinylated, incubated with streptavidin-coated beads, and used for negative depletion:lysozyme, bovine serum albumin, human interleukin receptor 2 gamma, tobacco etch virus (TEV) protease, human plasminogen activator urokinase receptor, human carbonic anhydrase II, and rabbit IgG-FITC. These proteins were used because they were readily available and/or already conjugated to biotin. Yeast were incubated with the aforementioned proteins coated on beads for two hours at 4° C. Yeast then underwent the same negative depletion step a second time. This was to increase confidence that non-specific binders were depleted. Non-binding yeast were selected and grown for the next sort (the stability sort).
To select for stable binders, the Gp2-based scaffold population after the specificity sort underwent a protease incubation at increased temperature. Yeast were induced to display the Gp2-based scaffold population and were incubated with 0.75 mg/mL thermolysin for 10 minutes at 55° C., then immediately put on ice. Yeast were washed and incubated with mouse anti-MYC and rabbit anti-HA-biotin for 20 minutes. Yeast were again washed and labeled with goat anti-mouse-AF647 and streptavidin-FITC for another 20 minutes. Yeast that were HA and Myc positive were sorted in a three-tiered gating approach, where the top 2%, 14%, and 19% displaying yeast were collected using FACS.
Sorted Gp2-based scaffold population were deep sequenced, filtered via USearch, and further filtered for full length Gp2-based scaffold. In the stability sort, three tiered gates were collected and sequenced. Due to lack of sequencing depth of full-length Gp2-based scaffold, the read counts from each of the stability gates were summed for a total stability read count.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, 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. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
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. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/302,296, filed on Jan. 24, 2022, which is incorporated by reference herein in its entirety.
This invention was made with government support under EB023339 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2023/011459 | 1/24/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63302296 | Jan 2022 | US |
