SYNTHETIC CD16a BINDING PROTEINS, MANUFACTURE, AND USES THEREOF

Abstract

The present disclosure provides synthetic CD16a binding proteins, compositions containing such binding proteins, and methods of making and using such binding proteins.

Description

SYNTHETIC CD16a BINDING PROTEINS, MANUFACTURE, AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This present application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 566,180, filed March 15, 2024, the entire contents of which are hereby incorporated by reference for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing XML is hereby incorporated by reference in its entirety. The XML file, created on February 19, 2025, is named AIP- 01 IWO SL.xml and is 62,542 bytes in size.FIELD

[0003] The disclosure relates generally to synthetic cluster of differentiation 16a (CD 16a) binding proteins, their manufacture and use in the treatment of various disorders, including cancer.BACKGROUND

[0004] According to the National Cancer Institute (“NCI”), in 2022 there were 18.1 million cases of cancer in the United States, over 1.9 million of which were new. See, e.g., Siegel et al. (2022) CA CANCER J. CLIN. 72:7-33. NCI estimates that direct cancer-related medical costs in the US were $183 billion in 2015 and are projected to increase to $246 billion by 2030. See. e.g., The American Cancer Society Cancer Action Network, The Costs of Cancer 2020 Edition. Cancer is caused by abnormal proliferation of cells and can occur in any tissue in the body. These abnormal cells can evade the body’s natural defenses, making it difficult for the immune system to fight cancer cells.

[0005] Natural killer (NK) cells are innate lymphoid cells that play a critical role in immunity, including, for example, by killing cancer cells and secreting cytokines and chemokines. See, e.g, Paul et al. (2017) FRONT. IMMUNOL. 8: 1124; Mandal et al. (2014) HEMATOL. ONCOL. STEM CELL THER. 8: 1 124. Unlike certain other types of immune cells, NK cells are able to detect and kill tumor cells without “pre-activation.” See, e.g., Rahman et al. (2024) Histology7, Natural Killer Cells, STATPEARLS atncbi.nlm.nih.gov / books / NBK565844 / . NK cells may also be less likely to induce a cytokine storm, making them safer (for patients) to target as compared to other immune cells (e.g, T cells, etc.). See, e.g., Page et al. (2024) CE L MOL. IMMUNOL.; Wang et al. (2020) MEDCOMM 4:e422. In addition, NK cells are believed to be less susceptible to exhaustion than other types of immune cells (e.g., T cells) and may have greater abi li ty to infiltrate tissues. See, e.g., Courtney (2023) BLOOD. 141:869-876.

[0006] One way NK cells are activated is through their receptors, such as CD 16 proteins. See, e.g., Gonzalez (2022) IMMUNOL. REV. 309:64-74. One way to leverage these receptors and their activity is through NK engagers. NK engagers are proteins that can simultaneously bind to receptors on NK cells (e.g., CD16a) and surface antigens (e.g., tumor-associated antigens) on cells such as cancer cells. See, e.g., Capuano (2021) CANCERS. 13:2500. This dual binding can trigger tumor-cell specific cytotoxicity, induced by NK cells. See, e.g, Capuano et al. (2021) supra. NK cell-mediated immunotherapy including NK engagers has shown promise due to safety and efficacy, but cost, including manufacturability, remains a concern. See, e.g., Chu et al. (2022) J. TRANSL. MED. 20:240. In addition, specificity for targets such as NK cell-specific proteins and certain cancer cell antigens remains a challenge. In particular, given the high homology between amino acid sequences (e.g., the amino acid sequences of CD 16a and CD 16b), designing binding proteins that specifically bind NK cells has been a challenge.

[0007] The CD16 family of proteins (also known as Fey receptors, or FcyR) is a group of receptor proteins that modulates immune cell responses via the antibody-dependent cell- mediated cytotoxicity (ADCC) upon binding to the Fc region of antibodies. See, e.g., Gonzalez (2022) supra. The CD 16 protein family includes CD 16a. which is predominantly expressed on NK cells, and CD 16b, which is predominantly expressed on neutrophils. See. e.g., Coenon et aZ. (2022) FRONT IMMUNOL. 13:913215; Li et al. (2016) EXP MOL PATHOL. 101: 281-289; Ravetch et aZ. (1989) J EXP MED. 170:481-497. CD16a and CD16b are known to have different binding affinities to Fc regions. See, e.g., Roberts et al. (2018) J. BIOL. CHEM. 293(51): 19899. Although CD16a and CD16b share over 97% amino acid sequence identity', specific amino acid residues in CD 16a are believed to be responsible for CD16a’s stronger binding affinity' to the Fc region than CD16b. See, e.g., Roberts et al. (2018) J. BIOL. CHEM. 293(51): 19899; see also Ravetch et al. (1989) supra. Furthermore, while engagement of NK cells via CD16a can activate immune responses, Fc binding toCD16b in neutrophils can dampen ADCC and reduce or prevent immune responses. See, e.g, Treffers et al. (2019) FRONT. IMMUNOL. 9:3124.

[0008] Binding of CD16a with the Fc region of an antibody begins an intracellular signaling cascade in the NK cells, which includes phosphorylation of intracellular domains by protein kinase C (PKC) and, consequently, production of proinfl ammatory cytokines by the cells. See, e.g., Coenon et al. (2022) FRONT. IMMUNOL. 13:913215; see also Li et al. (2012) J. IMMUNOL. 189(9): 4284. Furthermore, N-glycosylation of the Fc region is believed to be an additional variable that impacts the robustness of binding between CD 16a and Fc. See, e.g.. Dekkers et al. (2017) FRONT. IMMUNOL. 8:877. Because NK cells play an important role in antitumor and antiviral responses, as well as regulation of other immune cells, engagement of NK cells via CD16a shows promise as an immunotherapeutic candidate. See Vivier et al. (2008) NAT. IMMUNOL. 9: 503. Moreover, the high homology between CD16a and CD16b poses a major challenge in specifically targeting CD16a. See. e.g., Zhang et al. (2023) FRONT. IMMUNOL. 14: 1207276; Nikkhoi et al. (2023) FRONT. IMMUNOL. 3:1039969. Thus, despite the efforts made to date, there remains a need for immunotherapies that can specifically target CD 16a relative to CD 16b.SUMMARY

[0009] The disclosure is based, in part, upon the discovery of synthetic CD16a binding proteins that specifically and preferentially bind CD16a (relative to CD16b). When IgG antibodies bind to CD 16a, NK cells can be activated and ADCC activity triggered. Accordingly, proteins that specifically bind CD 16a can be used to "engage" NK cells. Such engagement can be accomplished, for example, by attaching a CD 16a binding protein to another protein that binds to a cell that the NK cell can target for destruction.Accordingly, engaging NK cells may allow a subject's own immune system to successfully increase target cell death (e.g.. cancer cells) by targeting NK cells and improving treatment outcomes. Currently, there is a long felt and unmet need for improvements in compositions and methods for engaging NK cells, e.g., to fight cancer cells. As provided herein, CD 16a binders with improved safety, selectivity, efficacy, potency, manufacturability, scalability, and stability can meet such needs.

[0010] Accordingly, the disclosure provides, among other things, synthetic CD 16a binding proteins, methods of making such binding proteins, and methods of using such proteins to treat a disease or disorder by NK cell-mediated cytotoxicity’ of a target cell.

[0011] In one aspect, the disclosure provides a synthetic CD 16a binding protein, comprising: (a) an amino acid sequence from 35 amino acids to 100 amino acids in length; (b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for CD 16a stronger than 1 pM; and (d) a stability profile such that the binding protein (i) retains at least 90% binding affinity to CD16a upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the binding protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to CD 16a after incubation for 16 hours at 37°C of incubation in PBS relative to the binding protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to CD16a in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the binding protein prior to chemical denaturation.

[0012] In another aspect, the disclosure provides a synthetic CD 16a binding protein, comprising: (a) an amino acid sequence from 35 amino acids to 100 amino acids in length; (b) a net negative charge in PBS; (c) a binding affinity for CD16a stronger than 1 pM; (d) at least one alpha helix; (e) at least three beta sheets; (1) at least three amino acid loops, wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first beta sheet to a terminal amino acid (e.g., aN- tenninal amino acid) of a second beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., an C-terminal amino acid) of the second beta sheet to a terminal amino acid (e.g.. an N-terminal amino acid) of a first alpha helix sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g, an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta sheet; and (g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets. In certain embodiments, the N-terminus of the first beta sheet is preceded by one or more N-terminal amino acids and / or the C-terminus of the third beta sheet is followed by one or more C-terminal amino acids.

[0013] In certain embodiments, the synthetic CD16a binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acid residues; (d) does not comprise an unpaired cysteine ammo acid residue when cysteine amino acid residues are present in the binding protein; (e) free of N-linked glycosylation sites; (f) free of protease cleavage sites (e.g., L-X-R-R sequences (wherein X represents any amino acid residue); and (g) soluble up to at least 0.5 mM in PBS at 4 °C for one month.

[0014] In certain embodiments, the synthetic CD 16a binding protein has a binding affinity from between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM, about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM. The synthetic CD 16a binding protein can have a binding affinity stronger than about 10 pM, 7.5 pM, 5 pM, about 2.5 pM, about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM. about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0015] In certain embodiments, the synthetic CD 16a binding protein comprises from 35 to95 amino acid residues in length, from 35 to 90 amino acid residues in length, from 35 to 85 amino acid residues in length, from 35 to 80 amino acid residues in length, from 35 to 75 amino acid residues in length, from 35 to 70 amino acid residues in length, from 35 to 65 amino acid residues in length, from 35 to 60 amino acid residues in length, from 35 to 55 amino acid residues in length, from 35 to 50 amino acid residues in length, from 35 to 45 amino acid residues in length, from 35 to 40 amino acid residues in length, from 40 to 95 amino acid residues in length, from 40 to 90 amino acid residues in length, from 40 to 85 amino acid residues in length, from 40 to 80 amino acid residues in length, from 40 to 75 amino acid residues in length, from 40 to 70 amino acid residues in length, from 40 to 65 amino acid residues in length, from 40 to 60 amino acid residues in length, from 40 to 55 amino acid residues in length, from 40 to 50 amino acid residues in length, from 40 to 45 amino acid residues in length, from 45 to 95 amino acid residues in length, from 45 to 90 amino acid residues in length, from 45 to 85 amino acid residues in length, from 45 to 80 amino acid residues in length, from 45 to 75 amino acid residues in length, from 45 to 70 amino acid residues in length, from 45 to 65 amino acid residues in length, from 45 to 60 amino acid residues in length, from 45 to 55 amino acid residues in length, from 45 to 50 amino acid residues in length, from 50 to 95 amino acid residues in length, from 50 to 90 amino acid residues in length, from 50 to 85 amino acid residues in length, from 50 to80 amino acid residues in length, from 50 to 75 amino acid residues in length, from 50 to70 amino acid residues in length, from 50 to 65 amino acid residues in length, from 50 to60 amino acid residues in length, or from 50 to 55 amino acid residues in length. In certain embodiments, the synthetic CD16a binding protein comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0016] In certain embodiments, the synthetic CD16a binding protein comprises one or more of the following features: (a) the alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible; (b) the first, second, and / or third beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) the alpha helix contains at least one or two solvent accessible amino acids; (d) the first, second, and / or third beta sheet contains at least one or two solvent accessible amino acids; (e) the first alpha helix contains at least one or two solvent accessible amino acids; (f) the first and / or second and / or third loop contains at least one hydrophobic amino acid. In some embodiments, a synthetic CD 16a binding protein comprises (a) the first, second, and / or third beta sheet each contains at least two hydrophobic amino acids; (b) the first, second, and third beta sheet each contains at least one solvent accessible amino acid; (c) the first, second, and third beta sheet each contains at least two hydrophobic and one solvent accessible amino acids; (d) the first alpha helix contains at least four solvent accessible amino acids; and / or (e) the first, second, and / or third loop each contains at least one hydrophobic amino acid.

[0017] In another aspect, the disclosure provides a synthetic CD16a binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3- L3-D4 (Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 20, wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of X2LX4VX6VX8X9, wherein X2 is T, E. or Y; X4 is R or L; X6 is T or I; X8 is T, Y, Q, I, or A; and X9 is H or I; (b) D2 comprises an amino acid sequence of SEQ ID NO: 25, wherein X13 is S or P; X15 is R or N; and X16 is R, I, V, L, or T; (c) D3 comprises an amino acid sequence of SEQ ID NO: 26, wherein X30 is R or Q; and X34 is R, E, H, or A; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 27. wherein X42 is H, Q, or R. In certain embodiments, the synthetic CD 16a binding protein comprises LI, which comprises an amino acid sequence of XI 0X11, wherein XI 0 is P, S, G,E, or A; and XI 1 is D, G, or F; and / or L2, which comprises an amino acid sequence of X21DD, wherein X21 is A or S; and / or L3, which comprises an amino acid sequence of X35PEGX39, wherein X35 is T, G, A, or S; and X39 is T or Q.

[0018] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 20, wherein X2 is T, E, or Y; X4 is R or L; X6 is T or I; X8 is T, Y, Q, I, or A; and X9 is H or I; X10 is P, S, G, E, or A; XI 1 is D, G, or F; X13 is S or P; X15 is R or N; and X16 is R, I, V, L, or T; X21 is A or S; X30 is R or Q; and X34 is R, E, H, or A; X35 is T, G. A, or S; X39 is T or Q; and X42 is H, Q, or R. In certain embodiments, the synthetic CD 16a binding protein further comprises one or more N- terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 20.

[0019] In certain embodiments, the synthetic CD 16a binding protein comprises one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C- terminal to D4.

[0020] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: I -19.

[0021] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3- L3-D4 (Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 21, wherein DI, D2, D3. and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 28; (b) D2 comprises an amino acid sequence of SEQ ID NO: 29, wherein XI 6 is V or I; (c) D3 comprises an amino acid sequence of SEQ ID NO: 30, wherein X34 is H, A, or E; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 31. In certain embodiments, the synthetic CD16a binding protein comprises LI, which comprises an amino acid sequence of X10D, wherein X10 is S. G, E. or A; and / or L2, which comprises an amino acid sequence of ADD; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 32, wherein X35 is A or S.

[0022] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 21, wherein X10 is S, G, E, or A ; X16 is I or V; X34 is H, A, or E; and X35 is A or S. In certain embodiments, the synthetic CD16abinding protein further comprises one or more N-terminal amino acids and / or one or more C- terminal amino acids to the amino acid sequence of SEQ ID NO: 21.

[0023] In certain embodiments, the synthetic CD 16a binding protein comprises one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C- terminal to D4.

[0024] In certain embodiments the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 4, 9, 1 1 and 12.

[0025] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3- L3-D4 (Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI. L2. and L3 are loops 1, 2, and 3. respectively; and (ii) an amino acid of SEQ ID NO: 22. wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 33, wherein X9 is H or I; (b) D2 comprises an amino acid sequence of SEQ ID NO: 34, wherein XI 3 is P or S and XI 6 is V or I; (c) D3 comprises an amino acid sequence of SEQ ID NO: 35; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 31. In certain embodiments, the synthetic CD 16a binding protein comprises LI, which comprises an amino acid sequence of PX11, wherein XI 1 is D, F, or G; and / or L2, which an amino acid sequence of ADD; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 44, wherein X35 is A or G.

[0026] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 22, wherein X9 is H or I; XI 1 is D, F, or G; X13 is P or S: X16 is V or I; and X35 is A or G. In certain embodiments, the synthetic CD 16a binding protein further comprises one or more N-terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 22.

[0027] In certain embodiments, the synthetic CD16a binding protein comprises one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C- terminal to D4. In certain embodiments the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 3, 13, 18, and 19.

[0028] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3- L3-D4 (Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO: 23,wherein DI, D2, D3, and D4 independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 36, wherein X6 is I or T and X8 is Y or T; (b) D2 comprises an amino acid sequence of SEQ ID NO: 43, wherein XI 6 is R or V;(c) D3 comprises an amino acid sequence of SEQ ID NO: 37, wherein X30 is R or Q; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 38, wherein X42 is R, H, or Q. In certain embodiments the synthetic CD 16a binding protein comprises LI, which comprises an amino acid sequence of X10D, wherein XI 0 is P or G; and / or L2, which comprises an amino acid sequence of ADD; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 45, wherein X35 is A or T.

[0029] In certain aspects, the disclosure provides a synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 23, wherein X6 is I or T; X8 is Y or T; X10 is P or G; XI 6 is R or V; X30 is R or Q; X35 is A or T; and X42 is R, H, or Q. In certain embodiments, the synthetic CD16a binding protein further comprises one or more N- terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 23.

[0030] In certain embodiments, the synthetic CD 16a binding protein comprises one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C- terminal to D4.

[0031] In certain embodiments, the synthetic CD16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1, 2, 5, and 6.

[0032] In certain aspects, the disclosure provides a synthetic CD 16a binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3- L3-D4 (Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and (ii) an amino acid of SEQ ID NO:24, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 39, wherein X2 is E, T, or Y; X4 is R or L; and X8 is T, Q, Y, I, or A; (b) D2 comprises an amino acid sequence of SEQ ID NO: 40, wherein X15 is R or N; and X16 is I, L, R, T, or V; (c) D3 comprises an amino acid sequence of SEQ ID NO: 41; and (d) D4 comprises an amino acid sequence of SEQ ID NO: 31. In certain embodiments, the synthetic CD 16a binding protein comprises LI, which comprises an amino acid sequence of PD; and / or L2, which comprises an amino acidsequence of X21DD. wherein X21 is A or S; and / or L3, which comprises an amino acid sequence of SEQ ID NO: 42.

[0033] In certain aspects, the disclosure provides a synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 24. wherein X2 is E. T, or Y; X4 is R or L; X8 is T, Q, Y, I, or A; XI 5 is R or N; XI is I, L, R, T, or V; and X21 is A or S. In certain embodiments, the synthetic CD 16a binding protein further comprises one or more N-terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 24.

[0034] Depending on circumstances, it is understood that in a CD 16a binding protein comprising DI and D4 domains, that the synthetic CD 16a binding protein can be flanked by one or more N-terminal amino acid residues, N-terminal to DI, and / or one or more C- tenninal amino acid residues, C-terminal to D4.

[0035] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 7, 8, 10, and 14-17.

[0036] In certain embodiments, synthetic CD 16a binding proteins of the present disclosure have binding affinities for CD 16a of stronger than 1 pM. Depending upon the circumstances, the synthetic CD 16a binding protein has a binding affinity from between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM: about 1 pM to about 0.75 nM: about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM. In some embodiments, the synthetic CD 16a binding protein has a binding affinity stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM. about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0037] In certain embodiments, the amino acid sequence of the synthetic CD16a binding protein has at least 70 (e.g., 75, 80, 81, 82, 83. 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97. 97.5. 98. 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9) percent identify to that of any of SEQ ID NOs: 1-19.

[0038] In certain embodiments, a synthetic CD 16a binding protein provided herein has an amino acid sequence comprising or according to one or more sequences as set forth in Table 1, with one or more amino acid substitutions as set forth in Table 4.

[0039] In certain embodiments, the amino acid sequence of the synthetic CD 16a has an amino acid sequence comprising or according to one or more sequences set forth in Table 6. In certain embodiments, the synthetic binding protein comprises a paratope represented by X28-X29-X32-X39-X41-X42, wherein X28 is V or L, X29 is D, X32 is D, X39 is T or V, X41 is I or L, and X42 is R or Q, wherein the amino acid positions correspond to those of SEQ ID NO: 5 from N-terminus to C-terminus.

[0040] In certain embodiments, the synthetic binding protein comprises a paratope defined by X28 may also be L, X39 may also be V, X41 may also be L, and X42 may be Q, provided that neither X29 nor X32 is substituted.

[0041] In certain embodiments, the synthetic CD16a binding protein comprises a paratope defined by any combination of positions as set forth in Table 5.

[0042] In another aspect, the disclosure provides a synthetic CD 16a binding protein comprising an amino acid sequence with reference to any of SEQ ID NOs. 1-19, but having one or more changes to one or more amino acid residues as set forth in Table 4.

[0043] Also provided herein is a fusion protein comprising a synthetic CD 16a binding protein and an effector (e.g. LILRB4, ROR1, CD30, CD33. CD123, EpCAM, CD79B. CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, B7-H3).

[0044] Also provided herein is a pharmaceutical compositions comprising a synthetic CD 16a binding protein and a pharmaceutically acceptable carrier. In any of the compositions, the CD16a binding protein can further comprise an effector (e.g., leukocyte immunoglobulin-like receptor B4 (LILRB4)).

[0045] In another aspect, the disclosure provides a method of targeting CD16a, the method comprising contacting a cell that expresses CD 16a on its cell surface with a composition comprising a synthetic CD 16a binding protein, or a fusion protein comprising a synthetic CD16a binding protein and an effector, or a pharmaceutical composition as provided herein. In some embodiments, the cell is an immune cell.

[0046] In another aspect, the disclosure provides a method of modulating immune cell activity comprising contacting an immune cell that expresses CD 16a on its cell surface with acomposition comprising a synthetic CD 16a binding protein, or a fusion protein comprising a synthetic CD 16a binding protein and an effector, or a pharmaceutical composition provided herein under conditions that permit the CD 16a binding protein to bind to the CD 16a on the immune cell, wherein the synthetic CD 16a binding protein further comprises an effector, wherein the effector binds to a protein on a target cell and the activity of the immune cell is modulated in that it initiates a cytotoxic response against the target cell.

[0047] In certain embodiments, the synthetic CD 16a binding protein promotes or increases a cell-mediated activity (e.g., ADCC) of a target cell expressing a target (e.g., LILRB4) relative to cell-mediated activity in the target cell in the absence of the CD 16a binding protein.

[0048] In each of the foregoing methods, the immune cell can be an NK cell. In certain embodiments, the target cell is a cancer cell. In some embodiments, the effector molecule is a molecule that binds to a tumor-associated antigen on the surface of the cancer cell or a cellsurface molecule. In certain embodiments, the tumor-associated antigen is selected from any of LILRB4, ROR1, CD30. CD33, CD123. EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, and B7-H3. In certain embodiments the tumor-associated antigen is LILRB4.

[0049] In yet another aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof a synthetic CD16a binding protein, a fusion protein comprising a synthetic CD16a binding protein and an effector, or a pharmaceutical composition as provided herein, wherein the effector targets a target cell, wherein the administration localizes a CD16a-expressing immune cell into proximity with a cancer cell expressing a tumor-associated antigen, and promoting or increasing CD16a-mediated cytotoxicity' in the cancer cell.

[0050] In yet another aspect, the present disclosure provides a method of treating one or more cancers (e.g., myeloma, lymphoma, leukemia, solid tumor, etc.) in a subject in need thereof, the method comprising administering to the subject an effective amount of a synthetic CD 16a binding protein, or a fusion protein comprising a synthetic CD 16a binding protein and an effector, or pharmaceutical composition as provided herein. In certain embodiments, the administration is before, during, or after administration or use of one or more other treatments. In certain embodiments, the one or more other treatments is or comprises a biological agent (e.g, biologies, gene therapy, peptides), a small molecule (e.g, chemotherapy, corticosteroids, antivirals, antibiotics, anti-inflammatory agents, etc.), one ormore cells (e.g., immunotherapy), and / or one or more mechanical interventions (e.g., surgery, cryotherapy). In certain embodiments, the subject is diagnosed as having or at risk of having a cancer or population of cancerous cells (e.g.. myeloma, lymphoma, leukemia, solid tumor, etc.). In certain embodiments, the subject has been diagnosed as having cancer and / or a population of cancerous cells.

[0051] In another aspect, the disclosure provides a method of targeting a population of leukocyte immunoglobulin-like receptor B4 (LILRB4)-expressing cancer cells, the method comprising contacting the population with a composition comprising a synthetic CD 16a binding protein, wherein the synthetic CD 16a binding protein further comprises an effector that binds to LILRB4, wherein after the contacting with the composition, a greater portion of the population is dead as compared to contacting without a composition that does not comprise the synthetic CD16a binding protein and / or the effector.

[0052] These and other aspects and features of the disclosure are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIGs. 1A-1C are schematic representations of various aspects of CD16a and exemplary synthetic binding proteins. FIG. 1A is a schematic representation of a CD 16a receptor in a complex with an exemplary synthetic CD 16a binding protein (denoted as a ribbon diagram and labeled as a synthetic miniprotein in FIG. 1 A). Upon binding of an exemplary7sy nthetic CD 16a binding protein to CD 16a and crosslinking with other receptors, NK cells can be activated. FIG. IB shows one schematic representation indicating certain structural differences in key binding areas in CD 16a (which has a G147 and Y158) as compared to CD16b (which has a D147 and H158), suggesting their potential importance for selective binding of synthetic CD 16a binding proteins. Exemplary synthetic CD 16a binding domains bind to CD 16a with high affinity and do not bind to CD 16b. FIG. 1C shows a schematic representation of an exemplary fusion protein comprising an exemplary CD 16a binding protein and an exemplary effector binding protein, wherein the effector binding protein binds to an effector target (e.g., a cell-surface protein, such as a tumor-associated antigen, etc.) on a target cell (e.g., cancer cell).

[0054] FIGs. 2A-2C show binding and specificity characteristics of exemplary synthetic CD 16a binding proteins. FIG. 2A is a graph that shows binding affinity of an exemplary' synthetic CD16a miniproteins Reference Miniprotein 1 (SEQ ID NO: 1) to CD16a. FIG.2B are graphs that show specific, measurable binding of Reference Miniprotein 1 (SEQ ID NO: 1) to the CD16a ectodomain protein (left graph of FIG. 2B) and not to either CD16b (right graph of FIG. 2B) or the streptavidin protein chip surface used for testing (data not show n). FIG. 2C is a graph of circular dichroism (CD) spectra showing the stability profile of Reference Miniprotein 1 (SEQ ID NO: 1) over a wavelength range of 200-260 nm during heating and cooling measured between 25°C - 95°C (not all data points shown for clarity), indicating that the protein showed proper folding, unfolding, and refolding, and was thermostable as shown by circular dichroism spectroscopy at about 25°C (open circle) going up to 95°C (solid inverted triangle) and properly folding as cooling back down to 25°C (open triangle) occurred.

[0055] FIG. 3 is a graph depicting binding affinity7of an exemplary7CD 16a binding protein of the present disclosure (Reference Miniprotein 5; SEQ ID NO: 5) to CD16a as measured by SPR at 0.781 nM. 3.125 nM, 12.5 nM and 50 nM. These data show that exemplary CD16a binding proteins bind to soluble CD16a.

[0056] FIGs. 4A-4C show data related to structural and binding differences of exemplary CD16a miniproteins between CD16a and CD16b. FIG. 4A are graphs depicting affinity7measurement by surface plasmon resonance (SPR) of an exemplary synthetic CD 16a binding domain (Reference Miniprotein 5; SEQ ID NO: 5) for two variants of CD16a (176V (left graph of FIG. 4A) and 176F (right graph of FIG. 4A). FIG. 4B are graphs depicting affinity measurement by' surface plasmon resonance (SPR) of an exemplary synthetic CD16a binding domain (Reference Miniprotein 5; SEQ ID NO: 5) for two alleles of CD16b (NA1 (left graph of FIG. 4B) and NA2 (right graph of FIG. 4B). FIG. 4C is a graph of circular dichroism (CD) spectra showing the stability profile of Reference Miniprotein 5 (SEQ ID NO: 5) over a wavelength range of 200-260 nm during heating measured at 5°C intervals between temperatures of 25°C - 95°C (not all data points shown for clarity ).

[0057] FIGs. 5A and 5B show7data and a schematic of binding localization of exemplary' CD 16a binding proteins of the present disclosure. FIG. 5A is a graph showing results from a binding assay using an exemplary synthetic CD 16a binding domain (Reference Miniprotein 5; SEQ ID NO: 5) immobilized on an SPR chip and determining binding (y- axis) of CD 16a in the presence and absence of human serum over time (x-axis). FIG. 5B is a schematic representation of the results of FIG. 5A, show ing that the binding location of the exemplary synthetic CD 16a miniprotein (at the bottom of the modeled image) does notbind to the same epitope as the Fc domain (at the top of the modeled image) and thus should not impact binding to the Fc domain of a human antibody. It is believed that the CD 16a miniproteins provided herein should not compete with antibodies present in human serum through Fc regions.

[0058] FIG. 6 shows results from a cell binding assay using primary natural killer (NK) cells. FIG. 6 is a graph showing percent cell count on the y-axis and fluorescent intensity of the detectable marker on the x-axis. As shown in the graph, cells treated with either no exemplary miniprotein are indicated by the left-side peak and cells treated with an exemplary flag-tagged CD16a miniprotein (SEQ ID NO: 5; Reference Miniprotein 5) are indicated by the right side peak, showing stronger signal and confirming that the exemplary CD16 a miniprotein can bind to surface bound CD16a on NK cells.DETAILED DESCRIPTION

[0059] The disclosure is based, in part, upon the discovery of synthetic CD16a binding proteins that specifically and preferentially bind CD16a (relative to CD16b). It is contemplated that CD 16a binding proteins provided herein have the ability to modulate NK cell activation and cytotoxic activity against target cells. Without wishing to be bound by theory, it is believed that synthetic CD16a binding proteins as provided herein can be used to “engage’’ NK cells (e.g., to target one or more target cells, e.g., cancer cells). Such engagement can be accomplished by, for example, attaching a CD 16a binding protein to another protein that binds to a cell that the NK cell targets for destruction. Accordingly, engaging NK cells may allow a subject’s own immune system to successfully initiate ADCC against certain cells (e.g., cancer cells) by targeting NK cells to cancer cells through use of a CD 16a binding protein attached to a target cell binding protein, thus, improving treatment outcomes.

[0060] Accordingly, the present disclosure provides, among other things, synthetic CD 16a binding proteins, methods of making such binding proteins, and methods of using such proteins to treat related disorders.I. DEFINITIONS

[0061] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g., polypeptide and polynucleotide chemistry and synthesis, molecular and cellularbiology, protein biology and biochemistry, immunology', etc. as described herein are those well-known and commonly used in the art.

[0062] As used herein, the singular forms “a,” "an" and “the" include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g. , a synthetic CD16a binding protein includes a single binding protein, a plurality7of synthetic binding proteins, etc.

[0063] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0064] Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations.

[0065] As used herein, the phrases “solvent accessible residue” and “solvent accessible amino acid” refer to an amino acid that, when disposed in a folded molecule (e.g., in its a tertiary conformation) and in a solvent, is characterized in that the amino acid is at least partially accessible or exposed to the solvent. Solvent accessible amino acids can be determined using a variety of approaches including, e.g., Rosetta software suite, Neighbor Count, and Neighbor vector algorithms (Durham et. al. (2009) J. MO . MODEL. 15(9): 1093-108).

[0066] As used herein, the phrase “conservative substitution” refers to a substitution with a structurally and / or functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) and Threonine (T); 2) Aspartic Acid (D) and Glutamic Acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W). Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAMsubstitution: p matrix (e.g., the PAM 250 matrix). In certain embodiments, a binding protein of the disclosure comprises 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions relative to a reference amino acid sequence.

[0067] As used herein, the phrase “corresponding to” designates a position / identity of an amino acid or a nucleic acid in a polymeric molecule such as an amino acid in an amino acid sequence or a nucleic acid in a nucleic acid sequence. It is understood by the skilled artisan that such amino acids or nucleic acids in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that, for example, an amino acid in a first polymer “corresponding to” position seven in the reference amino acid, for example, need not actually be the seventh amino acid in the first polymer. Those of ordinary skill in the art are aware of methodology to identify “corresponding” amino acids or nucleic acids between two molecules (e.g, a polymer and a reference polymer), including, such as. commercially available algorithms, databases, or other information given context regarding particular polymers.

[0068] As used herein, the term “domain” refers to a region or segment of a given synthetic binding protein disclosed herein, and can include one or more structural features (e.g., amino acid, primary7, structure or secondary structure features) and / or one or more functional features (e.g., binding properties). In the context of secondary structure, a “structural domain” can be an uninterrupted linear sequence that adopts a single type of secondary structure, for example, ten continuous amino acid residues that are all part of the same alpha helix structure or beta sheet. In the context of binding, a “binding domain” can be a discontinuous portion of the overall amino acid sequence that facilitates chemical interactions with a target molecule or indirectly stabilizes such interactions.

[0069] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g.. a synthetic CD 16a binding protein disclosed herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0070] As used herein, the term “effector” refers to a molecule or molecular entity that confers one or more particular characteristics on itself or another molecule or molecular entity with or to which it is associated. For example, an effector may include a synthetic binding protein (e.g. , a miniprotein, or something other than a miniprotein that is associatedwith a synthetic CD 16a binding protein disclosed herein (e.g., via a covalent linkage), such as a detectable label (e.g, visualizable or otherwise measurable such as by fluorescence or radiolabel detection), small molecule, nanoparticle (e.g, a lipid nanoparticle, a polymer nanoparticle, etc.), polynucleotide (e.g, an aptamer, an siRNA, an shRNA, an oligonucleotide, etc.), a radionuclide or a chelating moiety complexed with a radionuclide, etc. An effector may be a synthetic binding protein (e.g, a monovalent synthetic binding protein linked to a CD 16a binding protein disclosed herein to create a bivalent synthetic protein where one or both of the proteins causes a change, e.g, in a cellular function, e.g, in a disease state, etc.).

[0071] As used herein, the term “loop” refers to (i) a structure (e.g., polypeptide) that connects two structural domains (e.g, a loop may be disposed between two alpha helices, between an alpha helix and a beta sheet, or between two beta sheets in a given synthetic CD16a binding protein) and / or (ii) a structure (e.g, peptide) present at the N- and / or C- terminal end of a given monovalent synthetic binding protein.

[0072] As used herein, the term “linker” refers to a structure (e.g. a polypeptide linker), or a chemical crosslinker (e.g, a homobifunctional or a heterobifunctional cross linking agent) between two molecules (e.g., two synthetic CD 16a binding proteins disclosed herein) or between, e.g, a synthetic CD16a binding protein and an effector, wherein each of the entities that are linked is covalently linked to one another.

[0073] As used herein, the terms / phrases “synthetic binding protein,” “synthetic miniprotein,” and “miniprotein” are used interchangeably, and refer to a polypeptide between about 35 to about 100 amino acids in length, e.g., from about 35 to about 90 amino acids in length, from about 35 to about 85 amino acids in length, from about 35 to about 80 amino acids in length, from about 35 to about 75 amino acids in length, from about 35 to about 70 amino acids in length, from about 35 to about 65 amino acids in length, from about 35 to about 60 amino acids in length, from about 35 to about 55 amino acids in length, from about 35 to about 50 amino acids in length, from about 35 to about 45 amino acids in length, from about 35 to about 40 amino acids in length, from about 35 to about 45 amino acids in length, from about 35 to about 40 amino acids in length, from about 40 to about 90 amino acids in length, from about 40 to about 85 amino acids in length, from about 40 to about 80 amino acids in length, from about 40 to about 75 amino acids in length, from about 40 to about 70 amino acids in length, from about 40 to about 65 amino acids in length, from about 40 to about 60 amino acids in length, from about 40 to about 55amino acids in length, from about 40 to about 50 amino acids in length, from about 40 to about 45 amino acids in length, from about 45 to about 90 amino acids in length, from about 45 to about 85 amino acids in length, from about 45 to about 80 amino acids in length, from about 45 to about 75 amino acids in length, from about 45 to about 70 amino acids in length, from about 45 to about 65 amino acids in length, from about 45 to about 60 amino acids in length, from about 45 to about 55 amino acids in length, from about 45 to about 50 amino acids in length, from about 50 to about 90 amino acids in length, from about 50 to about 85 amino acids in length, from about 50 to about 80 amino acids in length, from about 50 to about 75 amino acids in length, from about 50 to about 70 amino acids in length, from about 50 to about 65 amino acids in length, from about 50 to about 60 amino acids in length, from about 50 to about 55 amino acids in length, or 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length, that are capable of binding to a given target, e.g., CD16a with a desired binding affinity (e.g, stronger than 1 pM).

[0074] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences (e.g., a polypeptide) have the same amino acid or nucleotide at the same positions in an alignment. The percent identity between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. It is contemplated that a reference sequence can be an amino acid sequence corresponding to an entire CD 16a binding protein or a portion thereof. A reference sequence may be an amino acid sequence that corresponds to a particular domain or domains (e.g, an alpha helix, a loop region) or a combination of domains (e.g., a combination of a loop and an alpha helix). Alignment for purposes of determining percent sequence identity’ (e.g, amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, MUSCLE, or BioPython software. For a discussion of basic issues in searching sequence databases, see Altschul et al. (1994) NATURE GENETICS 6: 1 19-129, which is incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0075] As used herein, the term “synthetic’' refers to a molecule that is (i) not naturally occurring, (ii) not present in nature, (iii) does not comprise entirely natural components, or (iv) a combination of any one of (i), (ii) and (iii). For example, a synthetic peptide does not exist naturally, is produced or otherwise modified by human intervention, such as techniques including recombinant or cell-free synthesis, and / or the peptide may comprise one or more non-naturally occurring amino acids.

[0076] As used herein, the phrase “pharmaceutically acceptable’' refers to those compounds, materials, compositions, and / or dosage forms which are. within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity7, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0077] As used herein, the phrase “pharmaceutically acceptable carrier” as used herein refers to an agent (e.g., excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various ty pes of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g.. Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020).

[0078] As used herein, the term “epitope” refers to a region of a protein that is specifically recognized by a binding partner, such as an antibody or another binding protein. The epitope may generally span a portion of the protein. Often, proteins may have multiple such regions where binding partners can attach. Epitopes ty pically fall into two classes: continuous epitopes (also known as linear epitopes), which are epitopes defined by linear sequences of consecutive amino acids, and discontinuous epitopes (also known asconformational epitopes), which are epitopes defined by discontinuous amino acids that are brought together into spatial proximity’ when a protein is in its folded state.

[0079] As used herein, the term "paratope" refers to the specific region of a binding molecule (e.g.. a binding protein, e.g., a CD 16a binding protein) that recognizes and binds an epitope of a target molecule (e.g., CD1 a). A paratope of a given binding molecule ty pically comprises 5-20 amino acids that are solvent accessible and in close proximity in three-dimensional space.

[0080] As used herein, the terms “subject'’ and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g, murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0081] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subj ect, e.g. , in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0082] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0083] Throughout the text, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or theelement or component can be selected from a group consisting of two or more of the recited elements or components.

[0084] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular protein, that protein can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described and depicted herein.

[0085] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0086] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0087] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0088] It should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0089] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within orencompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc.II. CLUSTER OF DIFFERENTIATION 16a (CD 16a)

[0090] Provided herein are synthetic binding proteins (also referred to as miniproteins), that bind cluster of differentiation 16a (CD16a) also referred to as the Low Affinity Immunoglobulin Gamma Fc Region Receptor III-A (FCGR3A). As depicted in the schematic representation of FIG. 1A, binding and crosslinking with another cell or receptor can induce NK cell activation and ADCC activity. CD 16a is a member of the Fc gamma receptor family that binds to IgG. CD 16a has two extracellular Ig-like domains, a transmembrane domain and a short C-terminal cytoplasmic tail. See, e.g, Coenon et al. (2022) supra.

[0091] While Fc gamma receptors are broadly expressed throughout the body, CD16a is expressed on natural killer (NK) cells and macrophages and anchors to the membrane via a transmembrane domain. See, e.g., Coenon et al. (2021) supra. A highly homologous isoform, CD 16b, is expressed on neutrophils and is glycosylphosphatidylinositol (GPI) anchored to the neutrophil cell membrane. See, e.g., Coenon et al. (2022) supra. Li et al. (2016) supra,' and Ravetch et al. (1989) supra. In most individuals, CD16a is the only Fc gamma receptor expressed on NK cells and is important for antibody-dependent cell mediated cytotoxicity (ADCC). See, e.g., Patel et al. (2020) J Bio CHEM. 296: 100183.

[0092] Without being bound by theory, binding specificity of CD 16a over CD 16b appears to involve at least two key residues, 147 and 158, with G147 and Y158 in CD16a as compared to amino acids at corresponding residues in CD 16b, D147 and Hl 58 (see FIG. IB). CD 16a expression has more recently been leveraged to target NK cells to cancer cells using antibodies that bind to surface-expressed markers (e.g., tumor-associated antigens), however specificity of binding proteins to CD 16a over CD 16b has remained a challenge. See, e.g., Whalen (2023) MABS. 15: 2208697; and Capuano (2021) supra.

[0093] The interaction of CD16a with IgG is believed to occur at least through the IgG upper CH2 and lower hinge region and is influenced by the glycan composition of the IgG Fc region. See, e.g., Coenon et al. (2022) supra. Additionally, CD 16a can be glycosylated with high mannose and N-glycan type structures. Without being limited by theory, glycan profiles are thought to impact interactions between CD16a and binding partners. Forexample, glycosylation of N45 can stabilizes CD 16a and influence IgG binding; Coenon et al. (2021) supra. Fucosylation or lack thereof may also influence the signaling through IgG-CD 16a interactions. See, e.g., Gonzalez (2022) supra; Coenon et al. (2021) supra.

[0094] CD 16a is known to lack immunoreceptor tyrosine-based activation motifs (IT AMs). Thus, CD16a has been shown to cluster in lipid rafts and interact with other signaling molecules that contain ITAMs such as CD3zeta. Coenon et al. (2021) supra. This interaction is believed to lead to subsequent phosphorylation of kinases that trigger NK cell degranulation and calcium release into the cytosol, signaling ADCC of target cells. Id. In some contexts, ADCC by NK cells can be leveraged to target certain cells for destruction by combining CD 16a expressing cells with one or more other binding molecules, so that NK cells are engaged to induce a cytotoxic response on the target cell, such as, for example a cancer cell. See, e.g., Gonzalez (2022) supra; Capuano (2021) supra.

[0095] As depicted schematically in FIG. 1C, engagement of CD 16a with a synthetic CD 16a binding protein and an effector can activate NK cell pathways to target a target cell. When paired with another molecule to “engage’’ the NK cell to target another cell, e.g., a cancer cell expressing a tumor associated antigen, NK-cell mediated cytotoxicity (e.g., ADCC) of the cancer cell is induced.

[0096] Synthetic CD16a binding proteins provided herein are designed to selectively and tightly bind to CD 16a. This specificity', selectivity', and binding strength allows targeted binding to NK cells (which express CD 16a) over other cell types (e.g., cells expressing CD 16b). Such binding characteristics will allow improvements in approaches targeting NK cells, including for use in targeting and treating cancer cells in subjects in need of such treatment.III. CD 16a BINDING PROTEINS

[0097] The disclosure provides synthetic CD 16a binding proteins, and methods of identity ing, making, characterizing, formulating, and using such CD16a binding proteins. The synthetic CD16a binding proteins have improved manufacturability, increased specificity, and affinity as compared to existing proteins that bind to CD16a. For example, other CD16a antibodies and / or binding proteins may suffer from off-target activities (e.g., binding to CD16b on non-NK cells). Similarly, large molecules such as biologies (e.g., monoclonal antibodies that bind to CD 16a) are expensive to produce, are challenging to produce in uniform batches of drug substance (including, especially, e.g.. at commercially-scalable amounts), and can be challenging to formulate, transport, store, and administer to subjects.

[0098] The synthetic CD 16a binding proteins disclosed herein avoid certain such disadvantages as they have high binding affinity to CD 16a, can be engineered to have desired pharmacodynamic and pharmacokinetic properties (e.g, a desirable circulating half-life in plasma), reduced cross-reactivity (e.g., do not bind to CD 16b), are chemically and thermally stable, and are resistant to protease degradation (e.g., via L-X-R-R sites, wherein X represents any amino acid residue), deamination and lack post-translational modification (e.g, glycosylation, e.g, N-linked glycosylation, e.g, glycosylation through N-X-S / T, wherein X represents any amino acid residue), and are stable in different redox environments.

[0099] A synthetic CD16a binding protein described herein can have mM-level solubility. In some embodiments, the CD16a binding protein can have a solubility greater than 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL. 10 mg / mL. 15 mg / mL. 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, or 80 mg / mL in an aqueous solution.

[0100] The CD 16a binding proteins described herein designed to specifically bind CD16a, and preferably bind CD16a over CD16b. Preferably, the binding proteins do not bind CD 16b. Depending upon the circumstances, the synthetic CD 16a binding proteins bind CD 16a and upon crosslinking with another receptor results in downstream NK cell activation. Synthetic CD 16a miniproteins as provided herein can, in some embodiments, bind with an affinity at least at a level of a reference CD 16a binding protein.

[0101] The synthetic CD16a binding proteins disclosed herein can be conjugated to or conjugated with (e.g., chemically conjugated or as fusion proteins) one or more effector molecules. For the example, the CD 16a binding proteins described herein can be bound to one or more other synthetic binding proteins, which can be the same or different. The resulting proteins can be monovalent, bivalent or multivalent. For example, a first CD 16a binding protein described herein can be conjugated to (e.g., by a linker) and / or produced as a fusion (e.g, a genetic fusion) that binds to a second binding protein that binds a different target of interest such as. e.g, a tumor associated antigen, e.g., L1LRB4, ROR1, CD30, CD33, CD123, EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, B7-H3,etc. The resulting molecule is bivalent and can bind CD 16a on a first cell (e.g., an NK cell) as well as the second target molecule (e.g., LILRB4, ROR1, CD30. CD33, CD123.EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, B7-H3, etc.) on a second cell e.g., a cancer cell).

[0102] The synthetic CD 16a binding proteins disclosed herein can be conjugated to or conjugated with (e.g., chemically conjugated or as fusion proteins) one or more effector molecules. For the example, the CD16a binding proteins described herein can be bound to one or more other synthetic binding proteins, which can be the same or different. The resulting proteins can be monovalent, bivalent or multivalent. For example, a first CD 16a binding protein described herein can be conjugated to a second CD 16a binding protein that can be the same as or different from the first CD 16a binding protein. The resulting molecule is bivalent. Alternatively, a first CD 16a binding protein described herein can be conjugated to a second binding protein that binds a different target of interest such as, e.g.. a cell-specific surface protein (e.g., a tumor-associated antigen, e.g.. LILRB4 to, for example, engage the CD16a-expressing cell to a cancer cell expressing LILRB4), serum albumin (e.g., to extend serum half life), etc., which may optionally be bound to a third binding protein that can be, in some embodiments, a second CD 16a binding protein or the protein to the second target. The resulting molecule is bivalent or trivalent and can bind to NK cells expressing CD16a and another cell expressing another target (e.g., a cancer cell). In addition, it is contemplated that the resulting molecule can be multivalent and contain two, three, four, five, six or seven miniproteins, which can be the same or different.

[0103] In one aspect, the disclosure provides a synthetic CD 16a binding protein that comprises:(a) an amino acid sequence from 35 amino acids to 100 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for CD 16a stronger than 1 pM; and(d) a stabi li ty profile such that the protein (i) retains at least 90% binding affinity to CD 16a upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to CD 16a after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% bindingaffinity to CD16a in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0104] In another aspect, the disclosure provides a synthetic CD 16a binding protein, the binding protein comprising:(a) an amino acid sequence from 35 amino acids to 100 amino acids in length;(b) a net negative charge in PBS;(c) a binding affinity for CD 16a stronger than 1 qM;(d) at least one alpha helix;(e) at least three beta sheets;(1) at least three amino acid loops, wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g, a C-terminal amino acid) of a first beta sheet to a terminal amino acid (e.g., a N-terminal amino acid) of a second beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g, an C-terminal amino acid) of the second beta sheet to a terminal amino acid (e g., an N-terminal amino acid) of a first alpha helix sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e g., an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta sheet; and(g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets.

[0105] In another aspect, the disclosure provides a synthetic CD 16a binding protein, wherein(a) the first alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible;(b) the first, second, and / or third beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible;(c) the first alpha helix contains at least one or two solvent accessible amino acids;(d) the first, second, and / or third beta sheet each contains at least one or two solvent accessible amino acids;(e) the first alpha helix contains at least one or two solvent accessible amino acids;(f) the first and / or second and / or third loop each contains at least one hydrophobic amino acid; or (g) the binding protein comprises any combination of elements selected from (a), (b). (c), (d), (e), and (f).

[0106] Synthetic CD16a binding proteins of the present disclosure may include naturally-occurring or non-naturally occurring amino acids. It is understood that certain amino acids may have and / or take on different characteristics (e.g., hydrophobic, hydrophilic, neutral, etc.) depending upon the context (e.g, macro and / or microenvironment including, but not limited to surrounding amino acids, environmental conditions such as solvent type. pH, etc.). Various terms and phrases may be used herein to describe, identify, and / or characterize amino acids. In addition, a single amino acid, at any given time, may have more than one characteristic or identify. Depending upon the context, a hydrophobic amino acid may be selected from alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, tryptophan, tyrosine, lysine, and arginine. A hydrophilic amino acid may be an amino acid selected from cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tryptophan, and tyrosine. A charged amino acid may be an amino acid selected from arginine, histidine, lysine, aspartic acid (aspartate), and glutamic acid (glutamate). A positively-charged amino acid may be an ammo acid selected from arginine, histidine, and lysine. A negatively- charged amino acid may be an amino acid selected from aspartic acid (aspartate) and glutamic acid (glutamate). An uncharged or neutral amino acid may be selected from alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Synthetic CD 16a binding proteins of present disclosure have a primary structure comprising certain key features. For example, synthetic CD16a binding proteins can have amino acid sequences that include various combinations of hydrophobic and solvent accessible amino acids organized into certain domains. Primary structures (i.e., amino acid sequences) of the synthetic CD16a binding proteins will have a combination of one or more types (e.g., hydrophobic, e.g., solvent accessible) of amino acids.

[0107] In each of the foregoing aspects, the synthetic CD16a binding protein comprises one or more of the following features: (a) free of try ptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the binding protein (e) free ofN-linked glycosylation sites (e.g., N-X-S / T, wherein X represents any amino acid residue); (f) free of protease cleavage sites (e.g., L-X-R-R sequences, wherein X represents any amino acid residue); and (g) soluble up to at least 0.5 mM in PBS at 4 °C for one month.

[0108] Preferably, the CD 16a binding sites are designed to be free of glycosylation sites (e.g., free of N-linked glycosylation sites), for example, free of peptide sequences that are substrates for glycosylation (e.g., N-X-S / T (wherein X represents any amino acid residue)), which can be a substrate for an oligosaccharyltransferase (OST) complex. Alternatively or in addition, the CD 16a binding sites are designed to be free of protease cleavage sites, for example, free of peptide sequences that are substrates for proteases (e.g., L-X-R-R (wherein X represents any amino acid residue), which can be a substrate for a Kexin / KEX2 protease). Similarly, the CD16a binding proteins may also be designed to be free of other protease cleavage sites for other proteolytic enzymes such as trypsin, chymotrypsin, elastase, subtilisin. etc. Some synthetic CD 16a binding proteins may be designed to avoid cleavage by certain other enzymes, including depending upon linkers and fusion protein partners.

[0109] Depending upon the circumstances, the N-terminus of the first beta sheet is preceded by one or more N-terminal amino acids and / or the C-terminus of the third beta sheet is followed by one or more C-terminal amino acids.

[0110] It is contemplated that the synthetic CD16a binding protein can comprises from 35 amino acids to 100 amino acids in length. For example, the CD 16a binding protein comprises from about 35 to about 90 amino acids in length, from about 35 to about 85 amino acids in length, from about 35 to about 80 amino acids in length, from about 35 to about 75 amino acids in length, from about 35 to about 70 amino acids in length, from about 35 to about 65 amino acids in length, from about 35 to about 60 amino acids in length, from about 35 to about 55 amino acids in length, from about 35 to about 50 amino acids in length, from about 35 to about 45 amino acids in length, from about 35 to about 40 amino acids in length, from about 35 to about 45 amino acids in length, from about 35 to about 40 amino acids in length, from about 40 to about 90 amino acids in length, from about 40 to about 85 amino acids in length, from about 40 to about 80 amino acids in length, from about 40 to about 75 amino acids in length, from about 40 to about 70 amino acids in length, from about 40 to about 65 amino acids in length, from about 40 to about 60 amino acids in length, from about 40 to about 55 amino acids in length, from about 40 to about 50 amino acids in length, from about 40 to about 45 amino acids in length, fromabout 45 to about 90 amino acids in length, from about 45 to about 85 amino acids in length, from about 45 to about 80 amino acids in length, from about 45 to about 75 amino acids in length, from about 45 to about 70 amino acids in length, from about 45 to about 65 amino acids in length, from about 45 to about 60 amino acids in length, from about 45 to about 55 amino acids in length, from about 45 to about 50 amino acids in length, from about 50 to about 90 amino acids in length, from about 50 to about 85 amino acids in length, from about 50 to about 80 amino acids in length, from about 50 to about 75 amino acids in length, from about 50 to about 70 amino acids in length, from about 50 to about 65 amino acids in length, from about 50 to about 60 amino acids in length, from about 50 to about 55 amino acids in length, or 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46. 47, 48, 49, or 50 amino acids in length, that are capable of binding to a given target, e.g, CD16a with a desired binding affinity (e.g, stronger than 1 pM).

[0111] A synthetic CD16a binding protein may have an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 81%, about 82%. about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%. about 90%. about 91%, about 92%, about 93%, about 94%. about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9% or more or 100% identity with or to a reference sequence or component thereof, wherein the reference sequence is selected from any of SEQ ID NOs: 1-19, and portions (e.g, domains) thereof. In some embodiments, a synthetic CD 16a binding protein may have an amino acid sequence comprising an amino acid sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%. about 98%, about 99%, about 99.5%, about 99.6%. about 99.7%. about 99.8%, about 99.9% or more or 100% identity with or to a reference sequence or component thereof selected from any of SEQ ID NOs: 20-45, and portions thereof.

[0112] By way of a non-limiting example, a structural arrangement in a synthetic CD16a binding protein may be depicted as N-EniLn2En3Ln4Hn5Ln6En7-C, where E is a beta sheet, H is an alpha helix, L is a loop, each of ni-n? represents an integer indicating the number of amino acids in that structural domain, N and C represent N-terminal and C-terminal domains, respectively. As disclosed herein, certain CD 16a binding proteins can berepresented according to a formula: D1-L2-D2-L2-D3-L3-D4 (Formula I). In some embodiments, DI, D2. and D4 each correspond to a beta sheet domain, and D3 corresponds to an alpha helical domain; LI, L2, and L3 represent loops connecting the beta sheets and alpha helix to one another. The components of Formula 1 can correspond to the aforementioned structural arrangement as follows: D3 to Hns and each of DI, D2, and D4 to each of Eni, En3, and En7. The number of E amino acids does not have to be the same across sheets, for example, m. ns, and may be, but do not have to be. the same numbers. Similarly, , and ne, may be, but do not have to be, the same number. For example, an exemplary formula of a synthetic CD 16a binding protein may comprise N- E8L2E9L3H11L4E6-C as depicted pictorially below denoting the amino acids in a helix domain (H) or a loop domain (L):N-EEEEEEEELLEEEEEEEEELLLHHHHHHHHHHHLLLLEEEEEE-C.A person skilled in the art can determine which amino acids of a given sequence constitute a loop, sheet, or a helix. See, e.g. Mirdita, et al. (2022) NAT. METHODS(19): 679-682.

[0113] Any given H domain (e.g.. Hns) in an alpha helix that is part of a synthetic CD16a binding protein may independently contain between about 4 amino acids and about 20 amino acids in length. An H domain may independently comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length. Any given E domain (e.g., En3) in a beta sheet that is part of a synthetic CD 16a binding protein may independently contain between about 4 and about 16 amino acids in length. An E domain may independently comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more amino acids in length.

[0114] Loops disposed between alpha helices and / or beta sheets may also be of the same or different lengths. Each loop may independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 amino acids or more in length. In some embodiments, each loop is independently between at least 2, 3, 4, 5, or 6 amino acids in length.

[0115] In a protein with more than one alpha helical structure, each alpha helix may comprise the same number of amino acids in each of its H domains or different numbers of amino acids in length in reference to the primary structure of each helical region. That is, in some synthetic CD 16a binding proteins having more than one alpha helix, each H domain in the binding protein is the same length. In some synthetic CD 16a binding proteins having one or more alpha helix, one or more H domains has a different lengthrelative to other H domains in the binding protein. In some embodiments, an H domain has zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more conserved amino acids (e.g. relative to other CD 16a binding proteins).

[0116] In a protein with more than one beta sheet, each beta sheet may comprise the same number of amino acids in each of its E domains or different numbers of amino acids in length in reference to the primary' structure of each sheet region. That is, in some synthetic CD 16a binding proteins having more than one beta sheet, each E domain in the binding protein is the same length. In some synthetic CD 16a binding proteins having one or more beta sheets, one or more E domains has a different length relative to other E domains in the binding protein. In some embodiments, an E domain has zero, one, two, three, four, five, six. seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more conserved amino acids (e.g., relative to other CD 16a binding proteins).

[0117] In an exemplary’ CD16a binding protein disclosed herein, the binding protein comprises at least one alpha helix, at least three beta sheets, and at least three loops (a first loop, a second loop, and a third), wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g. , a C-terminal amino acid) of a first beta sheet to a terminal amino acid (e.g., a N-terminal amino acid) of a second beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g. an C-terminal amino acid) of the second beta sheet to a terminal amino acid (e.g. , an N- terminal amino acid) of a first alpha helix sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g., an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g. , an N-terminal amino acid) of a third beta sheet.

[0118] In some embodiments, a synthetic CD16a binding protein may have (a) a first alpha helix containing at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible; (b) a first, second, and / or third beta sheet each containing at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) a first alpha helix containing at least one or two solvent accessible amino acids; (d) a first, second, and / or third beta sheet each containing at least one or two solvent accessible amino acids; (e) a first alpha helix containing at least one or two solvent accessible amino acids; (1) a first and / or second and / or third loop containing atleast one hydrophobic amino acid; or (g) any combination of elements selected from (a), (b). (c). (d), (e), and (f).

[0119] In some embodiments, a synthetic CD 16a binding protein may have (a) a first, second, and / or third beta sheets each containing at least two hydrophobic amino acids; (b) a first, second, and third beta sheet each containing at least one solvent accessible amino acid; (c) a first, second, and third beta sheet each containing at least two hydrophobic and one solvent accessible amino acids; (d) a first alpha helix containing at least four solvent accessible amino acids; and / or (e) a first, second, and / or third loop each containing at least one hydrophobic amino acid.

[0120] A synthetic CD 16a binding protein may have one of several consensus sequence structures. Consensus sequences will generally have certain ‘'fixed’’ amino acid positions as well as those that can be varied, such as by changing to another amino acid. Sometimes changing amino acids at certain positions can alter the function of the synthetic binding protein by increasing or decreasing affinity for the target ( / .<?., CD 16a). However, all the binding proteins disclosed herein, although having different primary structures have a minimal “threshold” binding affinity to CD 16a. In some embodiments, a threshold binding affinity may be stronger than about 10 pM, about 1 pM. about 100 nM, about 10 nM, or about 1 nM.

[0121] A synthetic CD 16a binding protein disclosure may be represented according to a formula shown as one or more domains, wherein each domain optionally has one or more conserved amino acid residues and / or a particular structure (e.g, loop, e.g., helix). For example, an exemplary' synthetic CD16a binding protein comprises an amino acid sequence arranged in a primary structure of:D1-L1-D2-L2-D3-L3-D4 (Formula I) where DI, D2, and D4 represent beta sheets, and D3 represents an alpha helix, and LI, L2, and L3 represent loops connecting the alpha helix and / or beta sheets. The amino acid sequence of the starting “parental” protein and exemplary consensus sequences for various miniproteins developed in Examples 1-2 are set forth in TABLE 1. Bold, underlined residues represent beta sheets and correspond to DI, D2, and D4, and bold, italicized residues represent helical residues and correspond to D3, in order along a given consensus sequence. Exemplary consensus sequences for DI, D2. D3, D4, LI, L2, and L3 for each miniprotein are set forth in TABLE 2A. Positions for each variable amino acid along thelength of a consensus sequence as set forth in TABLES 1 and 2A are set forth in TABLE2B

[0122] As demonstrated in the Examples that follow, a synthetic CD 16a binding protein having the amino acid sequence of a first miniprotein (referred to a Reference Miniprotein 1 ) RTLRVTVTHPDGSVRRLTVDADDVVDTVDRLDARTPEGTVTHIEEA (SEQ ID NO: 1) was developed, characterized, and optimized.TABLE 1. Exemplary CD16a Synthetic Binding Protein Consensus Sequences*Bolded and italicized text indicates amino acids present in an alpha helix (positions 24-34); and bolded, underlined text indicates amino acids present in a beta sheet (positions 2-9; 12-20; and 40-45).TABLE 2A. Exemplary CD16a Synthetic Binding Protein Domain Consensus SequencesTABLE 2B. Exemplary Miniprotein Variable Substitutions

[0123] In one aspect, the disclosure provides a synthetic CD16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4 (Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 20, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of X2LX4VX6VX8X9, wherein X2 is T, E, or Y; X4 is R or L; X6 is T or I; X8 is T, Y, Q, I, or A; and X9 is H or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 25, wherein X13 is S or P; XI 5 is R or N; and X16 is R, I, V, L, or T;(c) D3 comprises an amino acid sequence of SEQ ID NO: 26, wherein X30 is R or Q; and X34 is R, E, H, or A; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 27, wherein X42 is H, Q, or R.

[0124] Furthermore, LI can comprise an amino acid sequence of XI 0X11, wherein XI 0 is P,S, G, E, or A; and XI 1 is D, G, or F; and / or L2 comprises an amino acid sequence of X21DD, wherein X21 is A or S; and / or L3 comprises an amino acid sequence of X35PEGX39, wherein X35 is T, G, A, or S; and X39 is T or Q.

[0125] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 20 may have at least one N-terminal amino acid, N-terminal to DI, and / or at least one C- terminal amino acid, C-terminal to D4.

[0126] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence of SEQ ID NO: 20, wherein X2 is T, E, or Y; X4 is R or L; X6 is T or I; X8 isT, Y, Q, I, or A; X9 is H or I; XIO is P, S, G, E, or A; XI 1 is D, G, or F; X13 is S or P; X15 is R or N; X16 is R, I, V, L, or T; X21 is A or S; X30 is R or Q; X34 is R, E, H, or A; X35 is T, G, A, or S; X39 is T or Q; and X42 is H, Q, or R.

[0127] In certain embodiments, the CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1-19.

[0128] In another aspect, the disclosure provides a synthetic CD16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, andLI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 21, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 28;(b) D2 comprises an amino acid sequence of SEQ ID NO: 29, wherein XI 6 is V or I.(c) D3 comprises an amino acid sequence of SEQ ID NO: 30, wherein X34 is H, A, or E; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 31

[0129] Furthermore, LI can comprise an amino acid sequence of X10D, wherein X10 is S, G, E, or A; and / or L2 comprises an amino acid sequence of ADD; and / or L3 comprises an amino acid sequence of SEQ ID NO: 32, wherein X35 is A or S.

[0130] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 21 may have at least one N-terminal amino acid, N-terminal to DI, and / or at least one C- terminal amino acid, C-terminal to D4.

[0131] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence of SEQ ID NO: 21, wherein X10 is S, G, E, or A ; X16 is I or V; X34 is H, A, or E; and X35 is A or S.

[0132] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 4, 9, 11 and 12.

[0133] In one aspect, the disclosure provides a synthetic CD16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 22, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 33, wherein X9 is H or I.(b) D2 comprises an amino acid sequence of SEQ ID NO: 34, wherein XI 3 is P or S and X16 is V or l.(c) D3 comprises an amino acid sequence of SEQ ID NO: 35; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 31.

[0134] Furthermore, LI can comprises an amino acid sequence of PX11, wherein XI 1 is D, F, or G; and / or L2 comprises an amino acid sequence of ADD; and / or L3 comprises an amino acid sequence of SEQ ID NO: 44, wherein X35 is A or G.

[0135] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 22 may have at least one N-terminal amino acid, N-terminal to DI, and / or at least one C- terminal amino acid, C-terminal to D4.

[0136] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence of SEQ ID NO: 22, wherein X9 is H or I; XI 1 is D, F, or G; X13 is P or S; X16 is V or I; and X35 is A or G.

[0137] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 3, 13, 18, and 19.

[0138] In one aspect, the disclosure provides a synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 23, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 36, wherein X6 is I or T and X8 is Y or T.(b) D2 comprises an amino acid sequence of SEQ ID NO: 43, wherein X16 is R or V(c) D3 comprises an amino acid sequence of SEQ ID NO: 37, wherein X30 is R or Q.(d) D4 comprises an amino acid sequence of SEQ ID NO: 38, wherein X42 is R, H, or Q.

[0139] Furthermore, LI can comprise an amino acid sequence of X10D, wherein X10 is P or G; and / or L2 comprises an amino acid sequence of ADD; and / or L3 comprises an amino acid sequence of SEQ ID NO: 45, wherein X35 is A or T.

[0140] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 23 may have at least one N-terminal amino acid, N-terminal to DI, and / or at least one C- terminal amino acid, C-terminal to D4.

[0141] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence of SEQ ID NO: 23, wherein X6 is I or T; X8 is Y or T; X10 is P or G; X16 is R or V; X30 is R or Q; X35 is A or T; and X42 is R, H, or Q.

[0142] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1, 2, 5, and 6.

[0143] In yet another aspect, the disclosure provides a synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, andLI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 24, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 39, wherein X2 is E, T, or Y; X4 is R or L; and X8 is T, Q, Y, I, or A.(b) D2 comprises an amino acid sequence of SEQ ID NO: 40, wherein XI 5 is R or N; and X16 is l, L, R, T, or V.(c) D3 comprises an amino acid sequence of SEQ ID NO: 41; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 31.

[0144] Furthermore, LI comprises an amino acid sequence of PD; and / or L2 comprises an amino acid sequence of X21DD, wherein X21 is A or S; and / or L3 comprises an amino acid sequence of SEQ ID NO: 42.

[0145] In addition, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 24 may have at least one N-terminal amino acid, N-terminal to DI, and / or at least one C- terminal amino acid, C-terminal to D4.

[0146] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence of SEQ ID NO: 24, wherein X2 is E, T, or Y; X4 is R or L; X8 is T, Q, Y, I, or A; X15 is R or N; X16 is I, L, R, T, or V; and X21 is A or S.

[0147] In certain embodiments, the synthetic CD 16a binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 7, 8, 10, and 14-17.

[0148] The synthetic CD 16a binding proteins disclosed herein can have a binding affinity from between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM; about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM. Furthermore, the synthetic CD 16a binding protein can have a binding affinity stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0149] In some embodiments, a threshold binding affinity may be stronger than about 1 pM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM.

[0150] It is contemplated that optimization of synthetic binding proteins may be achieved using optimized designs, such as, for example, modifications of one or more amino acids by substitution at one or more positions with a different amino acid. Optimization, such as by amino acid modifications, allows tunability of certain characteristics such as changes to (e.g., increases in) binding affinity and / or avidity.

[0151] Synthetic CD16a binding proteins can be optimized by affinity maturation techniques. For example, affinity maturation may be used on a sequence of a binding protein to create another synthetic CD 16a binding protein with at least the same or better selectivity and / or affinity for CD16a as compared to the starting sequence. Affinity maturation can be accomplished using techniques known to those of ordinary skill in the art, including, for example, generating libraries using error prone PCR, degenerate codons, synthetic oligonucleotide pools, or a combination thereof. These libraries can then be transformed into yeast and improved variants may be isolated by methods such as magnetic, flow cytometric, and / or FACS-based approaches. Computational design / redesign strategies may also be used when affinity maturing proteins and computer programs for implementing such approaches are known in the art. Prior to affinity maturation, synthetic binding proteins may be characterized to determine functional and structural features, such as binding affinity (e.g., for CD16a) and conformation.

[0152] Synthetic CD 16a binding proteins provided herein are engineered to have certain characteristics (e.g., binding affinity / avidity, binding specificity, e.g., for a target, e.g., for CD 16a). Various in silica, in vitro, and in vivo characterization assays may be used to evaluate these CD 16a binding proteins. For example, binding assays can be used to determine binding specificity to a target, e.g., CD 16a as compared to binding to another molecule such as, e.g., another receptor or a ligand such as CD 16b. Other assays can be used to determine binding affinity of a binding protein, e.g., a CD 16a binding protein for its target, which can include for example, surface plasmon resonance (SPR), and flow cytometry.

[0153] The synthetic CD 16a binding proteins of the present disclosure are designed to have certain stability characteristics. For example, a binding protein is stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to CD16a upon cooling to room temperature after thermal denaturation at 95°C in a solution (e.g., phosphate buffered saline (PBS)) for at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60 or more minutes relative to the synthetic CD 16a binding protein prior to thermal denaturation.

[0154] In some embodiments, a synthetic CD16a binding protein is stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to CD 16a after incubation at about 37°C (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more hours) relative to the synthetic CD 16a binding protein prior to incubation.

[0155] Synthetic CD16a binding proteins of the present disclosure may also display stability in resistance to chemical denaturation and / or retention of stability after exposure to chemical denaturants. For example, a synthetic CD 16a binding protein may be stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to CD16a in PBS following exposure to a denaturing chemical (e.g., 4M urea) at room temperature for about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 or more hours relative to the binding affinity of the synthetic CD 16a binding protein prior to exposure to the chemical denaturant.

[0156] Synthetic CD 16a binding proteins engineered, developed, and produced herein are selected and / or specific for CD 16a. That is, in some embodiments, a synthetic CD 16a binding proteins does not bind to a non- CD16a target (e.g., CD16b). In some embodiments, a synthetic CD16a binding protein binds to another receptor, but binds to CD16a with a much greater affinity. For example, the binding affinity of a synthetic CD16a binding protein may be between 1 and 200-fold greater than that for any other binding partner (e.g., CD 16b).

[0157] Affinity of a synthetic CD 16a binding protein may be modified and may vary depending on modifications made to, for example, its primary sequence. A binding affinity may be at least 1-fold greater, at least 2-fold greater, at least 3 -fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 125-fold greater, at least 150-fold greater, at least 175-fold greater, at least 200-fold greater than theaffinity of the synthetic CD16a binding protein for an unrelated (e.g., different) target (e.g, CD 16b).

[0158] As provided herein, the synthetic CD 16a binding proteins are highly specific for CD 16a (e.g, as compared to CD 16b). It is contemplated that the specificity of the proteins developed and disclosed is due in part at least to the unique paratopic residues interacting with CD 16a.

[0159] A CD 16a binding protein in accordance with the present disclosure may have certain conserved or allowable residues in particular locations. For example, with respect to Reference Miniprotein 5 (SEQ ID NO: 5), it is contemplated that a synthetic CD16a binding protein with certain substitutions in accordance with TABLE 5 (see Example 8) will maintain strong and specific binding to CD16a. It is further contemplated that certain residues are critical to specific binding, as set forth in TABLE 5.

[0160] In certain embodiments, the synthetic binding protein of the disclosure comprises a paratope defined by a paratope represented by X28 - X29 - X32 - X39 - X41 - X42, wherein X28 is V or L, X29 is D, X32 is D, X39 is T or V, X41 is I or L, and X42 is R or Q, wherein the amino acid positions correspond to those of SEQ ID NO: 5 from N-terminus to C-terminus. A synthetic binding protein of the disclosure comprises a paratope defined by any combination of positions as set forth in Table 5.IV. SYNTHESIS OF CD16a BINDING PROTEINS

[0161] The synthetic CD 16a binding proteins described herein may be produced by methods known to those of ordinary skill in the art. Methods may include, for example, biological approaches, such as recombinant approaches and / or chemical approaches, such as solid phase and / or liquid phase chemical synthesis, etc., or combinations thereof.

[0162] With regard to recombinant approaches, a variety of methodologies can be implemented to produce the binding proteins disclosed herein. For example, DNA molecules encoding the binding proteins can be synthesized chemically and / or cloned / produced using recombinant DNA methodologies. The resulting DNA molecules encoding binding proteins of interest can be ligated to other nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct (i.e., expression vector). Thereafter,the resulting expression vectors are introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include E. coll cells, Bacillus subtilis cells, Pichia Pastor is cells, Saccharomyces cerevisiae cells, Kluyveromyces lactis cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). The transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the binding proteins.

[0163] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, c. ., T7, lac, Trp or Tac, and, in some contexts, a prokaryotic signal sequence or fusion to a protein such as, e.g., Trx, MBP, SUMO, or OsmY. The expressed protein may be secreted. The expressed protein can be harvested after disruption of the cells by French press or sonication (e.g., in the presence of 4-6 M urea). Alternatively, or in addition, the binding proteins can be harvested and purified or isolated from cell extracts using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags. Protease cleavage with SUMO Protease (Ulp), thrombin, enterokinase, TEV protease, 3C protease may be used to cleave affinity tags and fusion proteins from the miniprotein binder. The protein may be further purified with reverse phase HPLC using a C- 18 column and eluted in a solvent gradient (e.g., gradient of acetonitrile). The protein may then be lyophilized to remove solvent and may be resuspended in phosphate buffered saline. Purification by reverse phase HPLC may be used to remove endotoxin from samples expressed in E. coli.

[0164] If the engineered gene is expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers. The vector may also optionally contain fusion domains which can be used to facilitate expression and secretion. Vectors may also optionally contain enzyme (e.g., protease) cleavage sites. The gene construct can be introduced into eukaryotic host cells using conventional transfection (e.g., for mammalian) and transformation (e.g., for yeast).

[0165] In addition, the synthetic binding proteins may be produced in cell-free systems. For example, chemical synthesis such as organic chemical synthesis using liquid and / or solid phase chemical processes may be used. Such processes and tools for performing such processes, such as various automatic synthesizers, are well known to those of ordinary skill in the art and such tools are widely commercially available. More specifically, methods of chemically synthesizing polypeptides are well known in the art and include, but are not limited to, solidphase peptide synthesis, liquid-phase peptide synthesis, and organic synthesis methods. In some synthetic approaches, an amino group of one amino acid (or amino acid derivative) is linked to a carboxyl group of another amino acid (or amino acid derivative) that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group attacks the activated carboxyl group, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the a-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) may be blocked (“protected”) from participating in the chemical reaction. Thus, only particular active groups react such that the desired product is formed. Blocking groups useful for this purpose include, without limitation, tertbutoxy carbonyl groups (t-Boc) and benzoyloxy carbonyl groups to protect amine groups; and simple esters (such as methyl and ethyl groups) and benzyl esters to protect carboxyl groups. Blocking groups can typically be subsequently removed with a treatment that leaves peptide bonds intact (for example, treatment with dilute acid). This process of protecting reacting groups that should not react, coupling to form a peptide bond, and deprotecting reactive groups may be repeated. A peptide may be synthesized by sequentially adding amino acids to a growing peptide chain.

[0166] Both liquid-phase and solid phase peptide synthesis methods can be used to make the binding proteins described herein. In solid-phase peptide synthesis, the growing peptide chain is typically linked to an insoluble matrix (such as, for example, polystyrene beads) by linking the carb oxy terminal amino acid to the matrix. At the end of synthesis, the peptide can be released from the matrix using a cleaving reagent that does not disrupt peptide bonds, such as hydrofluoric acid (HF). Protecting groups are also typically removed at this time. Automated, high throughput, and / or parallel peptide synthesis methods may also be used in accordance with the disclosure. For more information about peptide synthesis methods, see, e.g.,Merrifield (1969) ADV. ENZYMOL. RET, AT. AREAS MOT,. BIOL., 32: 221-96; Fridkin et al. (1974) ANN. REV. BIOCHEM. 43(0): 419-43; Merrifield (1997) METH. ENZYMOL. 289: 3-13; Sabatino et al. (2009) CUR . OPIN. DRUG DISCOV. DEVEL., 11(6): 762-70.

[0167] Once synthesized, the binding proteins can be purified using standard approaches including, for example, chromatographic (e.g., reverse phase HPLC) and affinity binding approaches. The resulting binding proteins can then be characterized using a variety of chemical, biological and biophysical approaches.V. CHARACTERIZATION OF CD16a BINDING PROTEINSA. Biophysical Characterization

[0168] The synthetic binding proteins described herein may be characterized using a variety of approaches to determine, e.g., secondary and tertiary conformation, binding affinity, binding selectivity, stability (e.g., thermostability, chemical stability, propensity to degrade, etc.), solubility, etc.

[0169] For example, protein conformation may be measured via circular dichroism spectroscopy, infrared spectroscopy, NMR, X-ray crystallography, cryo-electron microscopy and AlphaFold (alphafold.ebi.ac.uk / ). Binding affinity and / or selectivity may be determined using assays such as flow cytometric analyses using, e.g., yeast or mammalian cells, biolayer interferometry and / or surface plasmon resonance measurements, each of which will be able to determine different types and specificities of binding.

[0170] Binding affinity and / or avidity can be determined by measuring the equilibrium dissociation constant (KD) of a synthetic CD 16a binding protein to a target. In some embodiments, the binding affinity (KD) of synthetic CD 16a binding proteins is in the range of ICT5M or less, or ranging down to IO'10M or lower, (e.g., about 10'6, about 10'7, about 10'8, about 10’9, about 10’10M or less).

[0171] In some embodiments, the synthetic CD16a binding protein comprises a binding affinity characterized by a dissociation constant ranging from about 1 pM to about 1 pM. In some embodiments, the binding affinity is between about 0.001 nM to about 1 pM; about 0.01 nM to about 1 pM; about 0.1 nM to about 1 pM; about 1 nM to about 1 pM; about 1 nM to about 0.5 pM; about 1 nM to about 0.25 pM; about 1 nM to about 0.10 pM; about 1 nM toabout 75 nM; about 1 nM to about 50 nM; about 1 nM to about 25 nM; about 1 nM to about 10 nM; and about 1 nM to about 5 nM. In some embodiments, the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM to about 0.01 nM, and about 0.01 nM to about 0.001 nM. One of ordinary skill in the art will readily know how to calculate equilibrium dissociation constants using measured Ka (1 / sec) and Kd (1 / secM) of the synthetic CD16a binding proteins.

[0172] Other analytical techniques (some of which are also used for synthesis and purification) include, without limitation, HPLC, LCMS, quantitative thin layer chromatography and others known to those of skill in the art. Stability can be measured using assays that expose binding proteins to elevated temperatures (e.g., 37 °C, e.g., 95 °C, etc.) and / or chemical denaturants (e.g., urea, and guanidine hydrochloride) and then observe whether the protein refolds into its pre-exposure structure / conformation and / or regains binding activity to a given target molecule. Degradation can be evaluated using techniques such as reverse phase HPLC or gel electrophoresis to monitor resistance of a synthetic binding protein to degradation.B. Biochemical Characterization

[0173] The biological activity of the binding proteins can be determined via various assays, such as in vitro binding assays on chips and / or on primary cells (see, e.g., Examples 4, 5, and 6). Such assays can be used to determine whether a binding protein has agonistic (or antagonistic) properties. For example, the suitable assays can be performed to determine whether a synthetic CD 16a binding protein disclosed herein can, e.g., bind to an NK cell in a way that will allow it to be engaged with another cell (e.g., a cancer cell) when connected (e.g., fused) to another protein (e.g., a tumor-associated antigen, e.g., LILRB4).

[0174] As will be understood to those in the art, given context, various assays may be used to characterize phenotype and activity of synthetic CD16a binding proteins. For example, to evaluate efficacy of synthetic CD 16a binding proteins and determine their ability to engage CD16a-mediated signaling and cytotoxicity cell markers and functional assays may be used. To give but one example, cell surface markers associated with NK cell phenotype and differentstages of activation (e.g., CD25, CD69, and / or CD107) may be evaluated. Evaluation may include various assays and methods known to those of skill in the art including, but not limited to, immunocytochemistry including flow cytometric and flow activated cell sorting (FACS) analyses, western blotting, etc. In some embodiments, functional assays determining, for example, how many target cells are killed by a synthetic CD 16a binding protein further comprising an effector that binds to a target on the target cell.[00175J Cellular phenotype or function, e.g., through a cell receptor such as CD 16a, can be measured using any number of commercial assays used to characterize, e.g., cellular phenotype using surface markers, cell death of cancer cells (e.g., achieved via NK -induced cytotoxicity, etc.). For example, a FACS-based assay may be used to identify cell surface markers such as CD25, CD69, and / or CD 107, all of which are known NK-cell markers. CD25 and CD69 are considered early and late immune cell markers, respectively, and CD 107 is considered a marker of activated NK cells. Expression of one or more of these cell surface markers may be used to phenotype and “stage” an NK cell, e.g., to which a synthetic CD16a binding protein of the present disclosure has bound.

[0176] Assays conducted with cells, such as primary immune cells e.g., NK cells from healthy human subjects, NK cells from subjects with a disease (e.g., cancer), PBMCs from healthy human subjects, PMBCs from human subjects with a disease (e.g., cancer), etc. may be used to evaluate and characterize synthetic CD16a binding proteins. For example, the cells may have a visualizable reporter that is detectable upon CD16a-mediated binding. In some embodiments, CD 16a binding is measured by contacting a population of cells with a labeled CD 16a miniprotein (e.g., IgG (FcyRIII)) and measuring cellular activity (e.g., NK activation, etc.). Such a measurement may be compared to a measurement made after contacting a population of cells with a synthetic CD 16a binding protein as provided in accordance with the present disclosure. The amount of signaling activity can be induced or increased contacting the population of cells (before, concomitant with, or after exposure to a ligand) with a synthetic CD 16a binding protein (including, e.g., as compared to binding with a different CD16a binding protein such as, e.g., IgG (FcyRIII)).

[0177] Characterization assays may also be conducted in vivo. For example, synthetic CD 16a binding proteins may be tested for selective binding by comparing a control CD 16abinding protein (e.g., a scaffold with the same size and shape as synthetic CD16a binding proteins, but without any identity in paratope regions) to synthetic CD 16a binding proteins as provided herein (e.g, as in Table 2A).VI. CD16a-BINDING PROTEIN CONJUGATES

[0178] It is contemplated that the synthetic binding proteins may be engineered to modify certain desired properties (e.g., binding affinity, binding avidity, bi or multi-specificity, or pharmacokinetic or pharmacodynamic properties, etc.). That is, a CD 16a binding protein may also include a CD16a binding protein conjugate. Production of such conjugates can be achieved by conjugating (e.g., chemical conjugation or via a fusion protein) a synthetic binding protein to an effector molecule or to a chelating moiety which can complex with an effector molecule (e.g., a metal such as a radionuclide). For example, the synthetic binding protein can be conjugated to a second binding molecule, e.g., a second synthetic binding molecule, which can be the same or different from the first synthetic binding protein, or an antibody or antibody fragment) or a molecule that directly (e.g., bovine serum albumin (BSA), murine serum albumin (MSA), or human serum albumin (HSA)) or indirectly (e.g., an engineered binding site for BSA, MSA, or HSA) enhances the PK or PD properties of the binding molecule. By way of non-limiting example, half-life can be extended by a variety of approaches known to those of skill in the art including use of polyethylene glycol (PEG), fusion proteins (e.g., Fc fusions, albumin fusions), engineered Fc binding such as engineered binding to neonatal Fc receptor (FcRn), antibody conjugation (e.g., to an antibody or fragment thereof), e.g., other protein engineering approaches that change the stability and / or clearance of a protein from an organism. It is contemplated that certain half-life extension approaches such as PEGylation may reduce clearance and prolong circulation. Fusions, such as to Fc or albumin, and / or addition of binding domains such as engineered HSA domains may prolong half-life due to circulatory half-life properties of the molecules (e.g., longer circulatory half-life via binding to albumin in the serum). For instance, a serum albumin binding domain may facilitate longer half-life via binding to albumin in circulating blood. One mechanism that extension could occur may include via recycling of a synthetic CD 16a binding protein by binding to neonatal Fc receptor (FcRn), which could thereby extend the serum half-life of the synthetic CD 16a binding protein.

[0179] It is contemplated that a variety of effector molecules can be used to modify the properties of the synthetic CD16a binding protein disclosed herein. Furthermore, effector molecules may be, for example, binding proteins that bind to a different target on a different cell. Such effector molecules may “engage” the CD16a-positive cell (e.g., NK cell) and bring it apposed to the different cell, such as a cancer cell expressing a cell surface tumor associated antigen (e.g., LILRB4). An effector molecule may be a molecule that binds to a protein or fragment thereof on a surface of a cell, such as a cancer cell. Exemplary tumor associated antigens include, but are not limited to R0R1, CD30, CD33, CD123, EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, or B7-H3.

[0180] Furthermore, it is contemplated that the effector may be a cytotoxic molecule or detectable label (e.g., radiolabel or fluorescent tag) which can be used in a detection assay, e.g., a diagnostic assay, or, e.g., in an assay intended to target NK cells for delivery of a molecule, etc. in a condition where NK cells may themselves be targeted for destruction (e.g., transplantation, e.g., of an organ, of cells, etc.).

[0181] In some embodiments, provided synthetic binding proteins are conjugated to a chelating moiety, e.g., a chelating moiety which can complex with a metal such as a radionuclide or a non-metal radiolabel. Non-limiting examples of chelating moi eties include DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DOTMA (lR,4R,7R,10R)-a, a', a", a'"-tetramethyl- 1,4, 7, 10-tetraazacyclododecane- 1,4, 7, 10-tetraacetic acid, DOTAM (1,4,7,10- tetrakis(carbamoylmethyl)-l,4,7, 10-tetraazacyclododecane), DOTPA (1,4,7, 10- tetraazacyclododecane-l,4,7,10-tetra propionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2- amino-2-oxoethyl)-l,4,7,10-tetraazacyclododecan-l-yl)acetic acid), DOTA-GA anhydride (2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-l,4,7,10-tetraazacyclododecane-l,4,7- triyl )triacetic acid, DOTP (1,4, 7, 10-tetraazacyclododecane- 1,4, 7, 10-tetra(m ethylene phosphonic acid)), DOTMP (l,4,6,10-tetraazacyclodecane-l,4,7,10-tetramethylene phosphonic acid, DOTA- 4AMP (1,4,7, 10-tetraazacyclododecane- 1, 4,7, 10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8, 1 l-tetraazabicyclo[6.6.2]hexadecane-4,l 1-diacetic acid), NOTA (1,4,7- triazacyclononane-l,4,7-triacetic acid), NOTP (l,4,7-triazacyclononane-l,4,7-tri(methylene phosphonic acid), TETPA ( 1,4, 8, 11-tetraazacy clotetradecane- 1,4, 8,11 -tetrapropionic acid), TETA (1,4,8, 11-tetraazacy clotetradecane- 1, 4,8,11 -tetra acetic acid), HEHA (1,4,7,10,13,16- hexaazacyclohexadecane-l,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N'", N'"'-pentaacetic acid), FUOctapa (N,N'-bis(6-carboxy- 2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H2Dedpa (l,2-[[6-(carboxy)-pyridin-2- yl]-methylamino]ethane), Hephospa (N,N'-(methylenephosphonate)-N,N'-[6- (methoxycarbonyl)pyridin-2-yl]-methyl-l,2-diaminoethane), TTHA (triethyl enetetramine- N,N,N',N",N'", N"'-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA(ethylenedi aminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinones), and porphyrin.

[0182] In some embodiments, the chelating moiety is DOTA.

[0183] In some embodiments, the chelating moiety has the structure:(Formula III) wherein Y1is — CH2OCH2(L2)n-B, C=O(L2)n-B, or C=S(L2)n-B and Y2is — CH2CO2H; or wherein Y1is H and Y2is LJ-(L2)n-B.

[0184] In some embodiments, L1has the structure:(Formula IV) wherein R2is optionally substituted hydrogen or — CO2H.

[0185] Persons skilled in the art may determine the appropriate chelating moiety depending on the particular situation, e.g., depending on the metal (e.g., radionuclide) intended to be complexed with the chelating moiety.

[0186] In some embodiments, the chelating moiety is complexed with a radionuclide.

[0187] In some embodiments, the radionuclide is useful in diagnostic or imaging applications. Examples of such radionuclides, include, but are not limited to, gamma emitters, such as62Cu,64Cu,67Ga,68Ga,86Y,89Zr, andi nIn.

[0188] In some embodiments, the radionuclide is useful in therapeutic applications, such as beta or alpha emitters.

[0189] In certain embodiments, the radionuclide is an alpha-emitting radionuclide. As one of ordinary skill in the art would appreciate, the chelator can chelate an alpha-emitting radionuclide through one or more coordinate bonds. In some embodiments, the alpha-emitting radionuclide is selected from the group consisting of211At,212Bi,213Bi,223Ac,223Ra,212Pb,227Th,149Tb, and255Fm. In some embodiments, the alpha-emitting radionuclide is225Ac.225Ac (Actinium-225) is an alpha-emitting radioisotope with a 10-day half-life that is of particular interest for medical applications.225Ac decays in a series of steps that ultimately emits 4 alpha particles before reaching a stable isotope,209Bi, thereby increasing the potency.

[0190] In some embodiments, the radionuclide is a beta and / or gamma-emitting radionuclide, such as a radionuclide selected from the group consisting of32P,47Sc,67Cu,77As,89Sr,90Y, "Tc,105Rh,109Pd,mAg,131I,153Sm,159Gd,165Dy,166Ho,169Er,177Lu,186Re,188Re,194Ir,198Au, and199Au.

[0191] In some embodiments, the radioisotope is177Lu (Lutetium- 177), which emits both gamma-irradiation suitable for imaging and medium-energy beta-irradiation suitable for radiotherapy.

[0192] It is also contemplated that the effector molecule can be chemically conjugated to the synthetic binding protein or can be incorporated into the synthetic binding protein as a fusion protein. The chemical conjugation can be accomplished by including a conjugation site into the synthetic binding protein, e.g, via inclusion of a derivatizable amino acid (e.g, a lysine or cysteine amino acid). The conjugation site can then be used to link the effector molecule to the binding protein, either directly or indirectly (e.g., via a linker, such as homobifunctional or a heterobifunctional cross-linking agent). By way of non-limiting example, linkers may be or include hydrazone, PEG, bifunctional 4-(4-acetylphenoxy) butanoic acid moiety, maleimidocaproyl, maleimidom ethyl cyclohexane- 1 -carboxylate, maleimidocaproyl group with a tetrapeptide portion consisting of the amino acid sequence, glycine-glycine-phenylalanine-glycine, and / or maleimidocaproyl group with PEG. Exemplary homo- and heterobifunctional cross-linking agents can also include, for example, EDC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride), sulfo-NHS (N-hydroxysulfo succinimide), NHS (N-hydroxysuccinimide), dimethyl pimelimidate dihydrochloride, suberic acid bis(N-hydroxysuccinimide ester), DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DSP (dithiobis(succinimidyl propionate)), sulfo-SMCC (sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate), SPDP ((succinimidyl 3-(2- pyridyldithio)propionate), N-succinimdyl oxy carbonyl ethyl methanethio sulfonate, 6- Maleimidohexanoic acid N-hydroxysuccinimide ester, 6-Maleimidocaproic acid sulfo-N- succinimidyl ester, maleimidoacetic acid N-hydroxysuccinimide ester, N-succinimidyl iodoacetate, and 4-(4-maleimidophynyl) butyric acid N-hydroxysuccinimide ester.

[0193] Linkers may be used to link two or more synthetic CD 16a binding proteins to one another, and may also be used to link one or more synthetic CD 16a binding proteins to one or more effectors. A linker may be a peptide linker or a chemical linker. Linkers may be covalently bound (e.g, to an amino acid) to a synthetic CD 16a binding protein and covalently bound to a second agent (e.g., an effector, e.g., a second binding protein). The composition and / or length of the linker may be designed with a particular functionality in mind, but preferably is non-immunogenic. A linker may contain one or more glycine amino acids and / or one or more serine amino acids. Exemplary linkers can comprise one or multiples of (Gly2Ser)n, (GlyaSerjn, or (Gly4Ser)n, where n can be 1, 2, 3 / c.%

[0194] It is contemplated that the synthetic CD 16a binding proteins disclosed herein can be monovalent or multivalent. Multivalent proteins may include, but are not limited to bivalent and trivalent formats. A multivalent molecule may include two, three, four, or more monovalent synthetic CD 16a binding proteins or, for example, one synthetic CD 16a binding protein and another binding protein that binds another target (e.g. serum albumin). In any of these formats, at least one linker can connect a C-terminal amino acid of a first monovalent binding protein to an N-terminal amino acid of a second monovalent synthetic binding protein, such that the first and the second monovalent synthetic CD 16a binding proteins are linked together. For example, in some embodiments, a multivalent (e.g., bivalent) miniprotein may comprise two or three miniproteins, each independently between about 35 and 100 amino acids and associated (e.g., linked, conjugated) with one another. In some embodiments, multivalentmolecules may be fused and / or combined to another molecule, such as an effector molecule, e.g., a half-life extender (e.g., a site or a protein that binds to serum albumin to extend serum half-life, etc.) or a multivalent protein may comprise a miniprotein (e.g., a synthetic CD 16a binding protein), and a miniprotein engineered to bind to serum albumin (e.g., to extend serum half-life). In some such embodiments, a half-life extension molecule may be fused or conjugated to either a C-terminal amino acid or an N-terminal amino acid (or to both the bland C-terminal amino acids) of a synthetic CD 16a binding protein disclosed herein.

[0195] In certain embodiments, the disclosure provides a multivalent (e.g., bivalent) protein comprising a multiple synthetic CD16a binding proteins disclosed herein. The multivalent protein can comprise a first synthetic CD16a binding protein and a second synthetic CD16a binding protein linked together through at least one linker. A linker (e.g., a glycine and serine containing linker (e.g., GGS), can connect a C-terminal amino acid of the first synthetic CD 16a binding protein to an N-terminal amino acid of the second synthetic CD 16a binding protein. Depending upon the circumstances, the multivalent binding protein can have a binding affinity stronger than the binding affinity of each synthetic CD 16a binding protein alone.

[0196] In certain embodiments, the multivalent protein comprises a synthetic CD 16a binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in TABLE 6.VII. FUSION PROTEINS

[0197] Also provided herein are fusion proteins comprising a synthetic CD 16a binding protein of the present disclosure and an effector.

[0198] In certain aspects, the fusion protein of the present disclosure comprises a synthetic CD 16a binding protein and an effector that binds to a tumor-associated antigen on the surface of the cancer cell or a cell-surface molecule. In certain embodiments, the tumor-associated antigen is selected from any of LILRB4, R0R1, CD30, CD33, CD123, EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, and B7-H3. In certain embodiments the tumor-associated antigen is LILRB4.VIII. PHARMACEUTICAL COMPOSITIONS

[0199] Once produced, a synthetic CD16a binding protein disclosed herein can be formulated into a pharmaceutical composition.

[0200] For therapeutic use, a synthetic CD16a binding protein disclosed herein is combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc. used in formulating and preparing pharmaceutical compositions are known and / or readily accessible to those of skill in the art. Depending upon the circumstances, a carrier can include a liquid (e.g., a sterile liquid) or a solid. A carrier may be selected from or comprise water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically a carrier is approved by United States Food and Drug Administration and meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other International Pharmacopoeia. Suitable formulations for use in the present disclosure are found in see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). For a brief review of methods for drug delivery, see, e.g., Langer (1990) SCIENCE 249: 1527-1533. The resulting pharmaceutical compositions are suitable for administration to a subject (e.g., an animal, e.g., a mammal, e.g., a human).

[0201] A pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, betacyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chi orhexi dine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020)).

[0202] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0203] Depending upon the circumstances, a pharmaceutical composition may contain nanoparticles, or lipid droplets, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo el al. (2016) BIOENG. TRANSL. MED. 1 : 10-29).

[0204] Pharmaceutical compositions containing a synthetic CD 16a binding protein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectaladministration. In certain embodiments, the synthetic peptide is administered by subcutaneous administration.

[0205] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see e.g, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0206] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, poly ethoxylated castor oil or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0207] Pharmaceutical formulations preferably are sterile. Formulations can be sterilized, for example, by methods appropriate to retain activity and stability of the synthetic CD 16a binding protein included therein. Sterilization can be accomplished by any suitable method, e.g., fdtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0208] Depending upon the drug substance and formulation, the resulting dosage forms can be stable for extended periods of time, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more, when the dosage form is a liquid or solid. The formulations can be stable at room temperature or higher. It is contemplated that the dosage form is stable at ambient conditions in PBS. Alternatively the dosage form is frozen (e.g., a liquid or a lyophilizate) and stable under appropriate temperatures such as, e.g., -20°C, -80°C).

[0209] Depending upon the circumstances, the dosage forms can be formulated as a unit dose, which can include, for example, about 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg,1.75 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 g, 1.5 g, 2.5 g, 5 g, or 10 g of the drug substance.

[0210] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein are generally administered by parenteral administration. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion. In certain embodiments, it is contemplated that the synthetic constructs disclosed herein can be administered by systemic administration.

[0211] Generally, a therapeutically effective amount of active component, for example, a synthetic CD16a binding protein disclosed herein, is in the range of 0.01 pg / kg to 250 mg / kg, e.g., 0.1 pg / kg to 25 mg / kg, 1 pg / kg to 15 mg / kg, 10 pg / kg to 10 mg / kg, 0.1 mg / kg to 100 mg / kg, 2.5 mg / kg to 175 mg / kg, 5 mg / kg to 250 mg / kg. In certain embodiments, the effective amount is 0.01 pg / mg. In certain embodiments, the effective amount is 0.1 mg / kg. In certain embodiments, the effective amount is 1 mg / kg. In certain embodiments, the effective amount is 15 mg / kg. In certain embodiments, the effective amount is 30 mg / kg. In certain embodiments, the effective amount is 50 mg / kg. In certain embodiments, the effective amount is 100 mg / kg. A dose may also be a flat dose, for example, about 0.25 mg to 25 mg, and depending on context e.g., administration route such as intravenous vs. subcutaneous vs. oral), dose can change. In certain embodiments e.g. intravenous administration) a dose may be about 0.075 mg to about 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 0.1 mg to 50 mg or 1 mg to 50 mg. In certain embodiments (e.g. subcutaneous administration) a dose may be about 0.25 mg to about 2.5 mg (e.g., about 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, or 2.5 mg. In other embodiments, (e.g. oral administration) a dose may be about 5 mg to about 25 mg (e.g., about 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, or 25 mg). The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissuelevel. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosagemay be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the synthetic peptide, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks.IX. METHODS OF USE AND TREATMENT

[0212] The CD 16a binding proteins described herein can be used in a variety of different approaches or contexts, including, for example, therapeutic and / or diagnostic contexts. For example, the synthetic CD16a binding proteins of the present disclosure can be used, along with one ore more detectable labels (e.g., a fluorescent label), to determine if a binding protein specifically binds to CD16a as compared to CD16b. In addition, such labeled CD16a miniproteins can be used to determine if binding occurs to soluble CD 16a and / or cell-surface bound CD 16a. Such methods may be used, for example, in diagnostic contexts or in screens identifying proteins that specifically bind to CD16a as compared to, e.g., CD16b.

[0213] The synthetic CD 16a binding proteins of the present disclosure can be used in a method of targeting CD 16a to a cell expressing a different cell surface antigen, such as a method which comprises contacting a cell that expresses CD 16a on its cell surface with a composition comprising a synthetic CD 16a binding protein disclosed herein, wherein the CD 16a binding protein further comprises an effector, and the effector binds to a cell-surface antigen on another cell, thereby engaging the CD16a-expressing cell to target the cell to which the effector is bound for death.

[0214] For example, the binding proteins can be used in a method of targeting CD16a. The method comprises contacting a cell that expresses CD 16a on its cell surface with a composition comprising the synthetic CD 16a binding protein disclosed herein. In addition the CD 16a binding proteins described herein can be used to modulate CD16a-positive cell activity (e.g., NK cell activity). The method comprises contacting a cell that expresses CD16a on its cell surface with a composition comprising the synthetic CD16a binding protein disclosed herein. In each method, the CD 16a binding protein or the pharmaceutical composition comprising a CD 16a binding protein further comprises an effector molecule. In each of the foregoing methods, the synthetic CD 16a binding protein or the pharmaceutical compositioncan leverage CD16a expression to (1) bind to an NK cell and (2) engage the bound NK cell to target another cell via an effector.

[0215] It is further contemplated that CD 16a binding proteins provided herein can be used to target NK cells for delivery of a molecule, etc. in a condition where NK cells may themselves be targeted for destruction (e.g., transplantation, e.g., of an organ, of cells, etc.).

[0216] The CD 16a binding proteins provided herein may be used in treatment of a disease, disorder, or condition mediated by CD16a-positive cell activity. CD16a is expressed on NK cells that can target cancer cells, which cells may express one or more surface associated antigens (e.g., tumor associated antigens, etc.).

[0217] In certain embodiments, the disclosure provides a method of targeting CD 16a- by contacting a cell that expresses CD 16a on its cell surface with a composition comprising a synthetic CD 16a binding protein as provided herein.

[0218] In one aspect, the present disclosure provides a method of targeting CD16a, the method comprising contacting a cell that expresses CD 16a on its cell surface with a composition comprising a synthetic CD 16a binding protein or pharmaceutical composition as provided herein.

[0219] In one aspect, the present disclosure provides a method of modulating immune cell activity, the method comprising contacting an immune cell that expresses CD16a on its cell surface with a composition comprising a synthetic CD16a binding protein or a pharmaceutical composition as provided herein, and under conditions that permit the CD 16a binding protein to bind to the CD 16a on the immune cell, wherein the synthetic CD 16a binding protein further comprises an effector, wherein the effector binds to a protein on a target cell and the activity of the immune cell is modulated in that it initiates a cytotoxic response against the target cell.

[0220] In some embodiments, an immune cell is an NK cell. In some embodiments, a target cell is a cancer cell. In some embodiments, the cell is a senescent cell. In some embodiments, the cell is a healthy cell such as a cell in a tumor microenvironment that has suppressive capabilities.

[0221] In some such embodiments, an effector molecule is a molecule that binds to an antigen on the target cell. In some embodiments, the antigen is a tumor-associated antigen.Without being limited, tumor associated antigens may be selected from LILRB4, ROR1, CD30, CD33, CD123, EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, and B7-H3.

[0222] Without being bound by theory, it is contemplated that a synthetic CD 16a binding protein as provided by the present disclosure promotes or increases a cell-mediated activity (e.g., ADCC) in a target cell expressing a target (e.g., LILRB4) relative to cell-mediated activity in the target cell in the absence of the CD 16a binding protein.

[0223] In one aspect, the disclosure provides a method of treating cancer by administering to a subject in need thereof a synthetic CD16a binding protein, a fusion protein comprising a synthetic CD 16a binding protein and an effector, or a pharmaceutical composition as provided herein. In some embodiments, wherein the effector targets a target cell. In some embodiments, the administration localizes a CD16a-expressing immune cell into proximity with a cancer cell expressing a tumor-associated antigen, and promotes or increases CD 16a- mediated cytotoxicity in the cancer cell.

[0224] In one aspect, the disclosure provides a method of treating one or more cancers or populations of cancer cells (e.g., tumor) in a subject in need thereof, the method comprising administering to the subject an effective amount of a synthetic CD 16a binding protein, a fusion protein comprising a synthetic CD 16a binding protein and an effector, or pharmaceutical composition as provided herein.

[0225] Subjects that can be treated include those suspected as having, having, or at risk of having a disease, disorder, or condition that would benefit from targeted cell toxicity by CD 16a expressing cells. The methods described herein may include a step of selecting a treatment for a subject in need thereof. The method includes (a) identifying e.g., diagnosing) the subject with such a disease, disorder, or condition, and (b) selecting a synthetic CD16a binding protein as described herein, to treat the subject.

[0226] Synthetic CD 16a binding proteins (or fusion proteins comprising a synthetic CD 16a binding protein and an effector, or a pharmaceutical compositions disclosed herein) administered in an effective amount to a subject in need thereof may result in NK-cell mediated death of one or more target cells, which target cells express a cell-surface antigen targeted by an effector, which effector is attached to the CD 16a binding protein.

[0227] The present disclosure provides methods of treating a subject in need thereof by administering an effective amount of the synthetic CD 16a binding protein, a fusion protein comprising the synthetic CD 16a binding protein and an effector, or a pharmaceutical composition comprising the synthetic CD 16a binding protein to the subject.

[0228] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The phrase administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0229] It is contemplated that therapy can be accomplished using a synthetic CD16a binding as part of a combination therapy wherein the CD 16a binding protein further comprises an effector and, optionally, one ore more additional agents, which agent(s) may be administered prior, concomitant with, and / or after treatment with a synthetic CD 16a binding protein comprising an effector.

[0230] The combination therapy comprising the CD16a binding protein and effector may include one or more additional agents or therapeutic approaches known to those of skill in theart and may have been previously used, be already ongoing, or added to a treatment for a subject in need thereof.

[0231] In methods of the present disclosure, where administration occurs, administration can be before, during, or after administration or use of one or more other treatments. In some embodiments, one or more other treatments may be a biological agent (e.g., biologies, gene therapy, peptides), a small molecule e.g., chemotherapy, corticosteroids, antivirals, antibiotics, anti-inflammatory agents, etc.), one or more cells (e.g., immunotherapy), and / or one or more mechanical interventions (e.g., surgery, cryotherapy, radiation). Exemplary small molecules can include, paclitaxel, cyclophosphamide. Exemplary biologies can include, for example, immune checkpoint modulator targeting antibodies such as anti-PD-1 or anti-CTLA-4 antibodies. Exemplary other treatments include, but are not limited to, cell therapy such as ex vivo expanded and differentiated NK cell. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes.

[0232] Exemplary diseases, disorders, or conditions that may be treated with the CD16a binding proteins disclosed herein include those such as cancer or population of cancerous cells such as, for example, myeloma (e.g., multiple myeloma), leukemia (e.g., acute myeloid leukemia (AML)), lymphoma (e.g., mantle cell lymphoma), solid tumors, etc.).

[0233] Compositions of the present disclosure may be used to treat a subject diagnosed as having or at risk of having a one or more such conditions. In some embodiments, the subject has been diagnosed as having cancer and / or a cancer or population of cancerous cells.

[0234] In one aspect, the disclosure provides a method of targeting a population of leukocyte immunoglobulin-like receptor B4 (LILRB4)-expressing cancer cells, the method comprising contacting the population with a composition comprising a synthetic CD 16a binding protein, wherein the synthetic binding protein further comprising an effector that binds to LILRB4, wherein after the contacting, a greater portion of the population is dead as compared to contacting without a composition that does not comprise the synthetic CD 16a binding protein and / or the effector.

[0235] A subject may be evaluated, e.g., by a healthcare provider, before, during, and / or after treatment with a composition provided herein. Depending on the outcome of the evaluation, a treatment may be continued or ceased, treatment frequency or dosage may change, or thepatient may be treated with a different synthetic CD16a binding protein and / or effector. Subjects may be administered a composition comprising the synthetic CD16a binding protein for a discrete period of time according to dosage paradigms described herein, including, optionally, until the disease, disorder, or condition is treated.X. KITS

[0236] Synthetic CD 16a binding proteins of the present disclosure may be included as part of a kit. A kit may comprise a container comprising or consisting essentially of a unit of a pharmaceutical composition comprising a synthetic CD 16a binding protein, instructions for use, and optionally, one or more agents (e.g., a buffer or diluent, if appropriate, to dissolve the binding protein or dilute a solution containing the binding protein), and a dispenser. A kit may include a label indicating the intended use of the contents of the kit. The contents of the kit may be used for treating, monitoring and / or diagnosing a subject in need thereof.

[0237] The present disclosure is further illustrated by the following examples which should not be construed as further limiting.EXAMPLESEXAMPLE 1: INITIAL SCREENING

[0238] This Example describes an initial in vitro screen of synthetic proteins using a library screen to identify synthetic proteins capable of binding to human CD 16a. The library was designed in silico and members screened for certain characteristics, such as selectivity and specificity of binding to the target (CD16a) at particular concentrations of target (e.g., 10 pM, e.g., 1 pM, etc.). Once identified, certain synthetic binding proteins were synthesized for use in downstream screening and discovery processes.

[0239] Two unique proteins (Reference Miniprotein 1 (SEQ ID NO: 1), and Reference Miniprotein 2 (SEQ ID NO: 2)) were individually tested for CD 16a binding by surface plasmon resonance (SPR). Initially, the miniproteins encoded by each of these sequences were expressed and purified from E. coli to use in SPR testing.

[0240] Binding affinity and specificity of the miniproteins for CD 16a and CD 16b were tested by SPR (single cycle and multicycle). Both Reference Miniprotein 1 (SEQ ID NO: 1) (FIG. 2A graph) and Reference Miniprotein 2 (SEQ ID NO: 2) demonstrated binding affinitiesstronger than 1 mM. Both proteins also showed specific binding to CD16a (as determined by measurable binding to the CD 16a ectodomain protein (FIG. 2B, left panel) and not to either CD 16b or the streptavidin protein chip surface used for testing (FIG. 2B, right panel).

[0241] Reference Miniprotein 1 (SEQ ID NO: 1) was confirmed to bind to CD16a as follows: kon 3.07 e4s^M’1; koff 3.88 e^s’1; and KD 1.26 e'5M. Furthermore, Reference Miniprotein 1 showed proper folding, unfolding, and refolding, and was thermostable as shown by circular dichroism spectroscopy at about 25°C going up to 95°C and properly folding as cooling back down to 25°C occurred (FIG. 2C).

[0242] Reference Miniprotein 1 (SEQ ID NO: 1) was selected as the basis to generate a new library, which was aimed at maintaining specificity for CD 16a while improving binding affinities.EXAMPLE 2: MINIPROTEIN OPTIMIZATION

[0243] This Example describes the optimization of CD 16a miniproteins developed from Reference Miniprotein 1 (SEQ ID NO: 1). The goal of optimizing Reference Miniprotein 1 was to improve binding affinity while maintaining binding specificity for CD 16a.

[0244] Reference Miniprotein 1 (SEQ ID NO: 1) was used to create a new library having greater than 1 billion members. These members were screened for binding to human CD 16a using standard yeast surface display techniques. CD 16b was used for negative selection, in order to maintain selectivity of the CD16a-specific parent sequences. Miniproteins that bound to CD16a were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using standard labeling and selection techniques. Thousands of unique miniprotein sequences were identified by next generation sequencing (NGS).

[0245] The miniproteins with higher NGS counts (SEQ ID NOs: 3-19), which serves as a surrogate for binding affinity, were expressed and purified in soluble form from E. coli and further characterized.

[0246] Reference Miniproteins 3-19 (SEQ ID NOs: 3-19) showed improved binding relative to Reference Miniproteins 1 and 2 see FIG. 3 showing binding affinity of an exemplary CD16a binding protein (Reference Miniprotein 5; SEQ ID NO.: 5) to CD16a and TABLE 3) and kept a very high specificity for two variants of CD 16a (FIG. 4A,) as compared to twovariants of CD 16b (FIG. 4B, NA1 and NA2, which are two known allelic variants of CD16b that are 95% homologous to one another and 95-97% similar to CD 16a). The ability to specifically distinguish between CD16a and CD 16b provides a solution to the longfelt need.

[0247] Each of Reference Miniproteins 3-19 (SEQ ID NOs: 3-19) had the same secondary structural arrangement of loop amino acids, alpha helix amino acids, and beta sheet amino acids as its parent, Reference Miniprotein 1 (SEQ ID NO: 1).

[0248] The binding characteristics of exemplary Reference Miniproteins 3-19 to human CD 16a were measured and binding affinity and selectivity / specificity of each miniprotein was determined using SPR (see data in TABLE 3).

[0249] Thermostability of the CD 16a binding mini proteins was also analyzed. CD 16a binding miniproteins were exposed to 4M urea at temperatures of at least 50°C. These proteins retained their properly folded three-dimensional structure as confirmed by circular dichroism spectroscopy until between at least about 60°C - 70°C, and returned to their properly folded structure upon cooling (as shown for Reference Miniprotein 5, SEQ ID NO: 5 in FIG. 4C; data for other miniproteins not shown), and, even when heated to 95°C, although less stable by CD, returned to its properly folded and stable structure upon cooling (data not shown). FIG.4C (Reference Miniprotein 5, SEQ ID NO: 5) shows an exemplary CD spectra of an exemplary CD 16a binding proteins measured at 5°C intervals between 25°C - 95°C, demonstrating that CD 16a binding proteins with improved affinity and specificity / selectivity for CD 16a retains its folding (not all data points shown).

[0250] The amino acid sequences of Reference Miniproteins 3-19 share about 82%-95% identity with one another, and about 88-92% identity with that of Reference Miniprotein 1 (SEQ ID NO: 1).TABLE 3. Binding Characteristics of Exemplary Reference Miniproteins

[0251] Reference Miniproteins 1-19 were used to develop a consensus sequence of SEQ ID NO: 20. Additional subconsensus sequences were developed for Reference Miniproteins (i) 4, 9, 11, and 12 (SEQ ID NOs: 4, 9, 11, and 12, respectively; subconsensus sequence of SEQ ID NO: 21); (ii) Reference Miniproteins 3, 13, 18, and 19 (SEQ ID NOs: 3, 13, 18, and 19, respectively; subconsensus sequence of SEQ ID NO: 22); Reference Miniproteins 1, 2, 5, and 6 (SEQ ID NOs: 1, 2, 5, and 6, respectively; subconsensus sequence of SEQ ID NO: 23); and Reference Miniproteins 7, 8, 10, 14, 15, 16, and 17 (SEQ ID NOs: 7, 8, 10, 14, 15, 16, and 17, respectively; subconsensus sequence of SEQ ID NO: 24). Reference Miniproteins 4, 9, 11, and 12 (SEQ ID NOs: 4, 9, 11, and 12, respectively) share about 93-96 % identity with one another and about 91% identity with that of Reference Miniprotein 1 (SEQ ID NO: 1);Reference Miniproteins 3, 13, 18, and 19 (SEQ ID NOs: 3, 13, 18, and 19, respectively) share about 91-96 % identity with one another and about 91% identity with that of Reference Miniprotein 1 (SEQ ID NO: 1); Reference Miniproteins 1, 2, 5, and 6 (SEQ ID NOs: 1, 2, 5, and 6, respectively) share about 87-98 % identity with one another and about 87-100 % identity with that of Reference Miniprotein 1 (SEQ ID NO: 1); and Reference Miniproteins 7, 8, 10, 14, 15, 16, and 17 (SEQ ID NOs: 7, 8, 10, 14, 15, 16, and 17, respectively) share about 89-96 % identity with one another and about 89-91 % identity with that of Reference Miniprotein 1 (SEQ ID NO: 1).EXAMPLE 3: VARIANT MINIPROTEIN STABILITY

[0252] This Example describes in vitro chemical stability of various Reference Miniproteins 2-19 (SEQ ID NOS. 2-19) using biophysical characterization after exposure to exemplary chemical denaturants.

[0253] Each of the CD 16a miniproteins developed and characterized in Example 2 were exposed to 4M urea and showed stability (data not shown). That is, after exposure to urea and subsequent dilution into PBS, the Reference Miniproteins each folded into their expected, respective, molecular structures (as prior to urea exposure) at 25°C as measured by circular dichroism (CD) spectroscopy (data not shown).

[0254] In addition, miniproteins were thermally stable during heating and showed proper refolding during cooling. Measurements of whether these proteins had properly folded three- dimensional structure after heating to 95°C and then returning to 25°C as measured by circular dichroism (CD) spectroscopy were determined. An example is shown in Example 2, FIG. 4C, demonstrating that exemplary Reference Miniprotein 5 (SEQ ID NO: 5) was thermally- stable from 25°C - 95°C.EXAMPLE 4: VALIDATION OF SELECTIVE BINDING OF CD16a MINIPROTEINS

[0255] This Example describes assays for measuring selectivity of binding by human CD 16a miniproteins. Specificity of binding to CD16a over CD16b was confirmed via multiple measurements. Figures presented here show schematic representation (FIG. IB) and data (FIGs. 4A-4B) related to structural and binding differences between CD16a and CD16b.FIGs. 4A-4B show binding characteristics of an exemplary synthetic CD 16a binding protein, demonstrating specificity of CD16a binding proteins for CD16a over CD16b.

[0256] Specificity of binding to CD16a over CD16b was also confirmed. FIGs. 4A and 4B are line graphs showing affinity measurement by surface plasmon resonance (SPR), demonstrating selectivity of an exemplary synthetic CD 16a binding domain (Reference Miniprotein 5; SEQ ID NO: 5) for two variants of CD16a (FIG. 4A; 176V (left graph in FIG. 4A) and 176F (right graph in FIG. 4A)) over two alleles of CD 16b (FIG. 4B; NA1 (left graph in FIG. 4B) and NA2 (right graph in FIG. 4B)) as evidenced by presence of binding (RU; y- axis) over time (x-axis) in FIG. 4A and no binding in FIG. 4B. FIG. 4C is a graph of circular dichroism (CD) spectra showing the stability profile of Reference Miniprotein 5 (SEQ ID NO: 5) over a wavelength range of 200-260 nm during heating and cooling measured at 5°C intervals between temperatures of 25°C - 95°C (not all data points shown for clarity).EXAMPLE 5: COMPETITION FOR FC BINDING

[0257] This Example describes binding location of CD 16a relative to its natural ligands. CD 16a is a Fey receptor that recognizes IgG molecules found in high abundance in human serum. This study assessed whether synthetic CD 16a proteins of the present disclosure would be incompatible with in vivo use, if, for example, it bound to Fc regions of serum antibodies.

[0258] Synthetic CD 16a miniprotein binding to CD 16a in the presence of human serum was measured by SPR. An exemplary CD 16a miniprotein (Reference Miniprotein 5, SEQ ID NO: 5) was immobilized on a SPR chip and soluble CD 16a was added in the presence or absence of human serum. The binding curves in FIG. 5A show that binding of synthetic CD 16a miniproteins does not occur in a way that competes for Fc receptors, such that CD 16a miniproteins do not bind to the same epitope as the Fc domain and should not impact binding to the Fc domain of a human antibody, thus making these synthetic miniproteins compatible with use in the presence of human serum and with binding of antibodies found in human serum. That is, it is believed that the CD 16a miniproteins provided herein should not compete with antibodies present in human serum through Fc regions, including because they each bind at distinct locations.

[0259] FIGs. 5A and 5B show data and a schematic of binding localization of exemplaryCD 16a binding proteins of the present disclosure. FIG. 5A is a graph showing results from abinding assay using an exemplary synthetic CD16a binding protein (Reference Miniprotein 5; SEQ ID NO: 5) immobilized on an SPR chip and determining binding (y-axis) of CD16a in the presence and absence of human serum over time (x-axis). FIG. 5B is a schematic representation of the results of FIG. 5A, showing that binding of a synthetic CD 16a miniprotein does not bind at the Fc domain in a human antibody, indicating that CD 16a binding proteins will not compete with binding of antibodies present in human serum through antibody Fc regions.EXAMPLE 6: VALIDATION OF CELL BINDING BY CD16a MINIPROTEINS

[0260] This Example shows that synthetic CD 16a binding proteins can bind soluble CD 16a (e.g., as shown by SPR) as well as membrane-bound CD 16a (e.g., such as on the surface of an NK cell).

[0261] Synthetic miniprotein was expressed as a genetic fusion to a flag tag and incubated with NK primary cells. After washing, an anti-Flag antibody conjugated to a fluorophore was added and miniprotein binding was assessed by flow cytometry. The anti-Flag antibody was detected in a cell-based assay, which confirms that CD 16a binding proteins were detected on NK cell surfaces. FIG. 6 shows results from a cell binding assay using primary natural killer (NK) cells. The graph shows percent cell count on the y-axis and fluorescent intensity of the detectable marker on the x-axis. As shown in the graph, cells treated with either no exemplary miniprotein are indicated by the left-side peak and cells treated with an exemplary flag-tagged CD16a miniprotein (SEQ ID NO: 5; Reference Miniprotein 5) are indicated by the right side peak, showing stronger signal and confirming the exemplary CD 16a miniprotein bound to NK cell surfaces, confirming that CD 16a miniproteins of the present disclosure can bind to surface bound CD 16a.EXAMPLE 7: SUBSTITUTION TOLERANCE

[0262] This Example describes use of single-site saturation mutagenesis (SSM) for identification and interrogation binding tolerance at each of 46 amino acid positions in an exemplary CD16a Miniprotein.

[0263] Reference Miniprotein 5 (SEQ ID NO: 5) was selected according to its strong binding affinity for CD16a. To better understand the sequence requirements for binding, single aminoacid substitutions were made at each of the 46 linear amino acid positions, from N-terminus to C-terminus, of SEQ ID NO: 5.

[0264] Allowable residues in linear positions from N-to-C terminus in Formula I are set forth in TABLE 4, with reference to SEQ ID NO: 5 (Reference Miniprotein 5). The “allowable residues” represent those residues which may be changed from the reference residue at a given position in Reference Miniprotein 5 (SEQ ID NO: 5). For example, position 3 in TABLE 4 is listed as having “M” as an allowable residue, but position 3 in SEQ ID NO: 5 is L, so the allowable residues are in addition to those set forth in SEQ ID NO: 5. To give but another example, position 42 in TABLE 4 is listed as R, and Q is an allowable residue, thus, allowable residues should be considered to be those in addition to the residues present in SEQ ID NO: 5.

[0265] For example, at position 1, Arginine (R) was substituted with alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The binding affinity (KD) for CD16a was assessed using yeast surface display. This process was continued for each of the remaining 45 amino acids in SEQ ID NO: 5, and a map of substitutions tolerances was developed as shown in TABLE 4. To be considered an allowable residue, binding of the mutated miniproteins had to occur at an affinity 120 nM or stronger. As shown in TABLE 4, there were allowable residues at positions other than 29, 32, and 41, which were immutable in that they could not be varied (to another amino acid residue) and still maintain binding at an affinity of 120 nM or stronger.TABLE 4. Allowable Residues in Synthetic Miniproteins* Relative to linear positions and amino acids in SEQ ID NO: 5. Allowable residues are in addition to those already present in SEQ ID NO: 5.EXAMPLE 8: PARATOPE IDENTIFICATION

[0266] This Example describes the identification and interrogation of the paratope of CD 16a miniproteins using substitution tolerance data from Example 7.

[0267] Affinity measurements (KD) were determined using titration of soluble CD 16a used to label yeast cells expressing individual substitutions of Reference Miniprotein 5 (SEQ ID NO: 5) generated as described in Example 7. The binding signal at 0 pM, 244 pM, 488 pM, 976 pM, 1.95 nM, 3.91 nM, 7.81 nM, 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1 pM of CD16a was measured. The Hill equation (Formula II),(Formula II)where (HH) is the Hill coefficient, K0.5 is the half-saturation constant, Y is the output response, I is the input concentration. The goodness of the curve fit was assessed using the R2value of the fit equation and collected only those binding affinities with an R2greater than 0.95. In total, measurable KD values were obtained for 374 sequence variants (which variants can be envisaged according to a combination of consensus sequences and substitutions at least as in TABLES 2A, 2B, and 4) that are all one amino acid substitution different from the amino acid sequence of Reference Miniprotein 5 (SEQ ID NO: 5). The structure of Reference Miniprotein 5 (SEQ ID NO: 5) was predicted with AlphaFol d2 and used to identify solvent accessible surface residues. These residues were then analyzed within the context of single-site- mutational data to assess mutational tolerance in terms of binding affinity to CD16a.

[0268] Paratope residues were identified at positions corresponding to amino acid residues relative to SEQ ID NO: 5, which is a 46-mer. Certain paratope residues were found to be immutable in that they were completely intolerant to substitution without loss of function (see, e.g., TABLE 5)

[0269] The amino acid corresponding to position 28 of SEQ ID NO: 5 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 46 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-19 and using SSM analysis of SEQ ID NO: 5, could only tolerate substitution to leucine (L) without materially decreasing CD 16a binding potency. The amino acid corresponding to position 29 of SEQ ID NO: 5 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 46 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-19 and using SSM analysis of SEQ ID NO: 5, could only tolerate substitution to glutamic acid (E) without materially decreasing CD 16a binding potency. The amino acid corresponding to position 32 of SEQ ID NO: 5 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 46 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-19 and using SSM analysis of SEQ ID NO: 5 could not tolerate any substitutions without materially decreasing CD16a binding potency. The amino acid corresponding to position 39 of SEQ ID NO: 5 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 46 as the C-terminal amino acid) was eithera glutamine (Q) or threonine (T) in each of SEQ ID NOs: 1-19 and using SSM analysis of SEQ ID NO: 5, which has a T at position 39, could only tolerate substitution to valine (V) without materially decreasing CD 16a binding potency. The amino acid corresponding to position 41 of SEQ ID NO: 5 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 46 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-19 and using SSM analysis of SEQ ID NO: 5, could only tolerate substitution to leucine (L) without materially decreasing CD 16a binding potency. The amino acid corresponding to position 42 of SEQ ID NO: 5 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 46 as the C-terminal amino acid) was either a glutamine (Q), histidine (H), or arginine (R) in each of SEQ ID NOs: 1-19. Using SSM analysis of SEQ ID NO: 5, which is an R at position 42, the protein could only tolerate substitution to glutamine (Q) without materially decreasing CD 16a binding potency.

[0270] A paratope of a CD 16a binding protein of the present disclosure can be represented as X28-X29-X32-X39-X41-X42, where X28 is V, X29 is D, X32 is D, X39 is T, X41 is I, and X42 is R. Without losing binding potency as measured by binding at 120 nM or stronger, X28 may also be L, X39 may also be V, X41 may also be L, and X42 may be Q. Neither X29 nor X32 can be substituted with other amino acids.TABLE 5. Paratope Residues and Tolerances* Amino acid identities at positions in SEQ ID NO. 5. The Paratope was determined using SSM data with Reference Miniprotein 5 (SEQ ID NO. 5) as a starting sequence; position refers to linear, ordinal position with amino acid position 1 being the N-terminal amino acid in SEQ ID NO. 5 and amino acid 46 being the C-terminal amino acid in SEQ ID NO. 5.INCORPORATION BY REFERENCE

[0271] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entiretyfor all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.EQUIVALENTS

[0272] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.TABLE 6. Table of Sequences

Claims

CLAIMSWhat is Claimed is:

1. A synthetic CD 16a binding protein, the binding protein comprising:(a) an amino acid sequence from 35 amino acids to 100 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for CD 16a stronger than 1 pM; and(d) a stability profile such that the binding protein (i) retains at least 90% binding affinity’ to CD 16a upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the binding protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to CD16a after incubation for 16 hours at 37°C of incubation in PBS relative to the binding protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to CD 16a in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the binding protein prior to chemical denaturation.

2. A synthetic CD16a binding protein, the binding protein comprising:(a) an amino acid sequence from 35 amino acids to 100 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for CD 16a stronger than 1 pM;(d) at least one alpha helix;(e) at least three beta sheets;(1) at least three amino acid loops, wherein a first loop having a first amino acid sequence connects a terminal amino acid (e.g. , a C-terminal amino acid) of a first beta sheet to a terminal amino acid (e.g. , a N-terminal amino acid) of a second beta sheet, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., an C-terminal amino acid) of the second beta sheet to a terminal amino acid (e.g. , an N-terminal amino acid) of a first alpha helix sheet, and a third loop having a third amino acid sequence connects a third, terminal amino acid (e.g., an C-terminal amino acid) of the first alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third beta sheet; and(g) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one of the alpha helices and / or at least one of the beta sheets.

3. The synthetic CD 16a binding protein of claim 1 or 2, wherein the binding protein comprises one or more of the following features:(a) free of tryptophan amino acids;(b) free of methionine amino acids;(c) free of lysine amino acids;(d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the binding protein;(e) free of N-linked glycosylation sites;(1) free of protease cleavage sites (e.g, L-X-R-R sequences, wherein X represents any amino acid residue); and(g) soluble up to at least 0.5 rnM in PBS at 4 °C for one month.

4. The synthetic CD16a binding protein of any one of claims 1-3, wherein the binding affinity is between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM, about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0. 10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM.

5. The synthetic CD 16a binding protein of any one of claims 1-4, wherein the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM. about 0. 1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM. about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM. and about 0.001 nM.

6. The synthetic CD16a binding protein of any one of claims 2-5, wherein the N-terminus of the first beta sheet is preceded by one or more N-terminal amino acids.

7. The synthetic CD 16a binding protein of any one of claims 2-6, wherein the C-terminus of the third beta sheet is followed by one or more C-terminal amino acids.

8. The synthetic CD16a binding protein of any of the preceding claims, wherein the binding protein comprises from 35 to 95 amino acid residues in length, from 35 to 90 amino acid residues in length, from 35 to 85 amino acid residues in length, from 35 to 80 amino acid residues in length, from 35 to 75 amino acid residues in length, from 35 to 70 amino acid residues in length, from 35 to 65 amino acid residues in length, from 35 to 60 amino acid residues in length, from 35 to 55 amino acid residues in length.from 35 to 50 amino acid residues in length, from 35 to 45 amino acid residues in length, from 35 to 40 amino acid residues in length, from 40 to 95 amino acid residues in length, from 40 to 90 amino acid residues in length, from 40 to 85 amino acid residues in length, from 40 to 80 amino acid residues in length, from 40 to 75 amino acid residues in length, from 40 to 70 amino acid residues in length, from 40 to 65 amino acid residues in length, from 40 to 60 amino acid residues in length, from 40 to 55 amino acid residues in length, from 40 to 50 amino acid residues in length, from 40 to 45 amino acid residues in length, from 45 to 95 amino acid residues in length, from 45 to 90 amino acid residues in length, from 45 to 85 amino acid residues in length, from 45 to 80 amino acid residues in length, from 45 to 75 amino acid residues in length, from 45 to 70 amino acid residues in length, from 45 to 65 amino acid residues in length, from 45 to 60 amino acid residues in length, from 45 to 55 amino acid residues in length, from 45 to 50 amino acid residues in length, from 50 to 95 amino acid residues in length, from 50 to 90 amino acid residues in length, from 50 to 85 amino acid residues in length, from 50 to 80 amino acid residues in length, from 50 to 75 amino acid residues in length, from 50 to 70 amino acid residues in length, from 50 to 65 amino acid residues in length, from 50 to 60 amino acid residues in length, or from 50 to 55 amino acid residues in length.

9. The synthetic CD 16a binding protein of claim 8, wherein the binding protein comprises35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45. 46, 47, 48, 49, or 50 amino acids.

10. The synthetic CD 16a binding protein of any one of claims 2-9, wherein(a) the at least one alpha helix contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible;(b) the first, second, and / or third beta sheet each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible;(c) the at least one alpha helix contains at least one or two solvent accessible amino acids;(d) the first, second, and / or third beta sheet contains at least one or two solvent accessible amino acids;(e) the first alpha helix contains at least one or two solvent accessible amino acids;(f) the first and / or second and / or third loop contains at least one hydrophobic amino acid; or(g) the binding protein comprises any combination of elements selected from (a), (b), (c), (d). (e), and (f).

11. The synthetic CD 16a binding protein of claim 10, wherein:(a) the first, second, and / or third beta sheet each contains at least two hydrophobic amino acids:(b) the first, second, and third beta sheet each contains at least one solvent accessible amino acid;(c) the first, second, and third beta sheet each contains at least two hydrophobic and one solvent accessible amino acids;(d) the first alpha helix contains at least four solvent accessible amino acids; and / or(e) the first, second, and / or third loop each contains at least one hydrophobic amino acid.

12. A synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure ofD1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 20, wherein DI, D2, D3. and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of X2LX4VX6VX8X9, wherein X2 is T, E, or Y;X4 is R or L; X6 is T or I; X8 is T, Y, Q, I, or A; and X9 is H or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 25, wherein XI 3 is S or P;X15 is R or N; and X16 is R, I, V, L, or T;(c) D3 comprises an amino acid sequence of SEQ ID NO: 26, wherein X30 is R or Q; andX34 is R, E, H, or A; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 27, wherein X42 is H, Q, or R.

13. The synthetic CD 16a binding protein of claim 12. whereinLI comprises an amino acid sequence of X10X11. wherein X10 is P, S, G, E, or A; andXI 1 is D, G, or F; and / orL2 comprises an amino acid sequence of X21DD, wherein X21 is A or S; and / orL3 comprises an amino acid sequence of X35PEGX39, wherein X35 is T, G, A, or S: and X39 is T or Q.

14. The synthetic CD 16a binding protein of claim 12 or 13, further comprising one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C-terminal to D4.

15. The synthetic CD16a binding protein of any one of claims 12-14, comprising an amino acid sequence selected from any of SEQ ID NOs: 1-19.1 . A synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 20, wherein X2 is T, E, or Y; X4 is R or L; X6 is T or I; X8 is T, Y, Q, I, or A; X9 is H or I; X10 is P, S, G, E, or A; XI 1 is D. G, or F; X13 is S or P; X15 is R or N; X16 is R, I. V, L. or T; X21 is A or S; X30 is R or Q; X34 is R, E, H. or A; X35 is T. G, A. or S; X39 is T or Q; and X42 is H, Q, or R.

17. The synthetic CD16a binding protein of claim 16, further comprising one or more N- terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 20.

18. The synthetic CD 16a binding protein of claim 16 or 17, comprising an amino acid sequence selected from any of SEQ ID NOs: 1-19.

19. A synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure ofD1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively ; and(ii) an amino acid of SEQ ID NO:

21. wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 28;(b) D2 comprises an amino acid sequence of SEQ ID NO: 29, wherein XI 6 is V or I;(c) D3 comprises an amino acid sequence of SEQ ID NO: 30, wherein X34 is H. A, orE; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 31.

20. The synthetic CD 16a binding protein of claim 17, whereinLI comprises an amino acid sequence of X10D, wherein XI 0 is S, G, E, or A; and / orL2 comprises an amino acid sequence of ADD; and / orL3 comprises an amino acid sequence of SEQ ID NO: 32, wherein X35 is A or S.

21. The synthetic CD16a binding protein of claim 19 or 20, further comprising one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C-terminal to D4.

22. The synthetic CD 16a binding protein of any one of claims 19-21, comprising an amino acid sequence selected from any of SEQ ID NOs: 4, 9, 11 and 12.

23. A synthetic CD 16a binding protein comprising an amino acid sequence of SEQ ID NO: 21, wherein X10 is S, G, E, or A; X16 is I or V; X34 is H, A, or E; and X35 is A or S.

24. The synthetic CD16a binding protein of claim 23, further comprising one or more N- terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 21.

25. The synthetic CD 16a binding protein of claim 23 or 24, comprising an amino acid sequence selected from any of SEQ ID NOs: 4, 9, 11 and 12.

26. A synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure ofD1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2. D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 22, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 33, wherein X9 is H or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 34, wherein X13 is P or S and X16 is V or I;(c) D3 comprises an amino acid sequence of SEQ ID NO: 35; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 31.

27. The synthetic CD 16a binding protein of claim 26, whereinLI comprises an amino acid sequence of PX11, wherein Xl l is D, F, or G; and / orL2 comprises an amino acid sequence of ADD; and / orL3 comprises an amino acid sequence of SEQ ID NO: 44, wherein X35 is A or G.

28. The synthetic CD16a binding protein of claim 26 or 27, further comprising one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids. C-terminal to D4.

29. The synthetic CD 16a binding protein of any one of claims 26-28, comprising an amino acid sequence selected from any of SEQ ID NOs:

3.

13.

18. and 19.

30. A synthetic CD16a binding protein comprising an amino acid sequence of SEQ ID NO: 22, wherein X9 is H or I; XI 1 is D, F, or G; XI 3 is P or S; XI 6 is V or I; and X35 is A or G.

31. The synthetic CD 16a binding protein of claim 30, further comprising one or more N- terminal amino acids, and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 22.

32. The synthetic CD 16a binding protein of claim 30 or 31, comprising an amino acid sequence selected from any of SEQ ID NOs: 3, 13, 18, and 19.

33. A synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure ofD1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2, D3, and D4 are domains 1, 2, 3, and 4, respectively, andLI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 23, wherein DI, D2, D3. and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO:

36. wherein X6 is I or T and X8 is Y or T;(b) D2 comprises an amino acid sequence of SEQ ID NO: 43, wherein X16 is R or V;(c) D3 comprises an amino acid sequence of SEQ ID NO:

37. wherein X30 is R orQ: and(d) D4 comprises an amino acid sequence of SEQ ID NO: 38, wherein X42 is R, H, or Q.

34. The synthetic CD16a binding protein of claim 33, whereinLI comprises an amino acid sequence of X10D, wherein X10 is P or G; and / orL2 comprises an amino acid sequence of ADD; and / orL3 comprises an amino acid sequence of SEQ ID NO:

45. wherein X35 is A or T.

35. The synthetic CD 16a binding protein of claim 33 or 34, further comprising one or more N-terminal amino acid. N-terminal to DI, and / or one or more C -terminal amino acid. C- terminal to D4.

36. The synthetic CD16a binding protein of any one of claims 33-35, comprising an amino acid sequence selected from any of SEQ ID NOs: 1, 2, 5, and 6.

37. A synthetic CD16a binding protein comprising an amino acid sequence of SEQ ID NO: 23, wherein X6 is I or T; X8 is Y or T; X10 is P or G; X16 is R or V; X30 is R or Q; X35 is A or T; and X42 is R, H, or Q.

38. The synthetic CD16a binding protein of claim 37, further comprising one or more N- terminal amino acids and / or one or more C-terminal amino acids to the amino acid sequence of SEQ ID NO: 23.

39. The synthetic CD 16a binding protein of claim 37 or 38, comprising an amino acid sequence selected from any of SEQ ID NOs: 1, 2, 5, and 6.

40. A synthetic CD 16a binding protein comprising:(i) an amino acid sequence arranged in a primary structure ofD1-L1-D2-L2-D3-L3-D4(Formula I), wherein DI, D2. D3, and D4 are domains 1, 2, 3, and 4, respectively, and LI, L2, and L3 are loops 1, 2, and 3, respectively; and(ii) an amino acid of SEQ ID NO: 24, wherein DI, D2, D3, and D4 independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 39, wherein X2 is E, T, or Y; X4 is R or L; and X8 is T, Q, Y, I, or A;(b) D2 comprises an amino acid sequence of SEQ ID NO: 40, wherein XI 5 is R or N; and X16 is I, L, R, T, or V;(c) D3 comprises an amino acid sequence of SEQ ID NO: 41; and(d) D4 comprises an amino acid sequence of SEQ ID NO: 31.

41. The synthetic CD 16a binding protein of claim 40, whereinLI comprises an amino acid sequence of PD; and / orL2 comprises an amino acid sequence of X21DD, wherein X21 is A or S; and / orL3 comprises an amino acid sequence of SEQ ID NO: 42.

42. The synthetic CD 16a binding protein of claim 40 or 41, further comprising one or more N-terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C-terminal to D4.

43. The synthetic CD 16a binding protein of any one of claims 40-42 comprising an amino acid sequence selected from any of SEQ ID NOs:

7. 8, 10, and 14-17.

44. A synthetic CD16a binding protein comprising an amino acid sequence of SEQ ID NO: 24, wherein X2 is E, T, or Y; X4 is R or L; X8 is T, Q, Y. I. or A; XI 5 is R or N; XI 6 is I, L, R, T, or V; and X21 is A or S.

45. The synthetic CD16a binding protein of claim 44, further comprising one or more N- terminal amino acids, N-terminal to DI, and / or one or more C-terminal amino acids, C- terminal to D4.

46. The synthetic CD 16a binding protein of claim 44 or 45 comprising an amino acid sequence selected from any of SEQ ID NOs: 7, 8, 10, and 14-17.

47. The synthetic CD 16a binding protein of any one of claims 12-46, wherein the binding protein has a binding affinity for CD16a stronger than 1 pM.

48. The synthetic CD16a binding protein of any one of claims 12-47, wherein the binding affinity is between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM; about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 0.01 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 0.10 pM to about 0.01 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM.

49. The synthetic CD 16a binding protein of any one of claims 12-48, wherein the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0. 1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM. about 0.75 nM. about 0.5 nM, about 0.25 nM. about 0.1 nM, about 0.01 nM, and about 0.001 nM.

50. The synthetic CD 16a binding protein of any one of claims 12-49, wherein the amino acid sequence of the binding protein has at least 70 (e.g.. 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9) percent identity to that of any of SEQ ID NOs: 1-19.

51. The synthetic CD 16a binding protein of any one of claims 1-50, wherein the binding protein has an amino acid sequence comprising or according to one or more sequences set forth in Table 6.

52. The synthetic CD 16a binding protein of any one of claims 1-51, wherein the binding protein has an amino acid sequence comprising or according to one or more sequences as set forth in Table 1, with one or more amino acid substitutions as set forth in Table 4.

53. The synthetic CD16a binding protein of any one of claims 1-52, wherein the binding protein comprises a paratope defined by a paratope represented by X28 - X29 - X32 - X39 - X41 - X42, wherein X28 is V or L, X29 is D, X32 is D, X39 is T or V, X41 is I or L, and X42 is R or Q, wherein the amino acid positions correspond to those of SEQ ID NO: 5 from N-terminus to C-terminus.

54. The synthetic CD16a binding protein of any one of claims 1-53, comprising a paratope defined by any combination of positions as set forth in Table 5.

55. A synthetic CD16a binding protein comprising an amino acid sequence with reference to any of SEQ ID NOs. 1-19, but having one or more changes to one or more amino acid residues as set forth in Table 4.

56. A fusion protein comprising the synthetic CD16a binding protein of any one of claims 1-55 and an effector (e g. LILRB4, ROR1, CD30, CD33, CD123, EpCAM, CD79B, CD38, BCMA. CD 19, SLAMF7, DLL3. FcRH5, B7-H3).

57. A pharmaceutical composition comprising the synthetic CD 16a binding protein of any one of claims 1-55; and a pharmaceutically acceptable carrier.

58. The pharmaceutical composition of claim 57, wherein the synthetic CD 16a binding protein further comprises an effector (e.g., LILRB4, ROR1, CD30, CD33, CD123, EpCAM, CD79B, CD38, BCMA, CD19, SLAMF7, DLL3, FcRH5, B7-H3).

59. A method of targeting CD16a, the method comprising contacting a cell that expresses CD16a on its cell surface with a composition comprising the synthetic CD16a binding protein of any one of claims 1-55, the fusion protein of claim 56, or the pharmaceutical composition of claim 57 or 58.

60. The method of claim 59, wherein the cell is an immune cell.

61. A method of modulating immune cell activity, the method comprising contacting an immune cell that expresses CD 16a on its cell surface with a composition comprising the synthetic CD16a binding protein of any one of claims 1-55, the fusion protein of claim 56, or the pharmaceutical composition of claim 57 or 58 under conditions that permit the synthetic CD 16a binding protein to bind to the CD 16a on the immune cell, wherein the synthetic CD 16a binding protein further comprises an effector, wherein the effector binds to a protein on a target cell and the activity of the immune cell is modulated in that it initiates a cytotoxic response against the target cell.

62. The method of claim 60 or 61, wherein the immune cell is an NK cell.

63. The method of claim 61 or 62. wherein the target cell is a cancer cell.

64. The method of any one of claims 61-63, wherein the effector is a molecule that binds to a tumor-associated antigen on the surface of the cancer cell.

65. The method of claim 64. wherein the tumor-associated antigen is leukocyte immunoglobulin-like receptor B4 (LILRB4).

66. The method of any one of claims 61-65, wherein the synthetic CD 16a binding protein promotes or increases a cell-mediated activity (e.g., ADCC) in a target cell expressing a target (e.g., LILRB4) relative to cell-mediated activity in the target cell in the absence of the synthetic CD 16a binding protein.

67. A method of treating cancer by administering to a subject in need thereof a synthetic CD16a binding protein of any one of claims 1-55, a fusion protein comprising a synthetic CD 16a binding protein of any one of claims 1-55 and an effector, or the pharmaceutical composition of claims 57 or 58.

68. The method of claim 67, wherein the effector targets a target cell.

69. The method of claim 67 or 68. wherein the administration localizes a CD16a-expressing immune cell into proximity with a cancer cell expressing a tumor antigen and promotes or increases CD16a-mediated cytotoxicity7in the cancer cell.

70. A method of treating one or more cancer (e.g., tumor) -related conditions in a subject in need thereof, the method comprising administering to the subject an effective amount of the synthetic CD16a binding protein of any one of claims 1-55, the fusion protein of claim 56. or the pharmaceutical composition of claim 57 or 58.

71. The method of claim 70, wherein the administration is before, during, or after administration or use of one or more other treatments.

72. The method of claim 71, wherein the one or more other treatments is or comprises a biological agent (e.g, biologies, gene therapy, peptides), a small molecule (e.g, chemotherapy, corticosteroids, antivirals, antibiotics, anti-inflammatory agents, etc.), one or more cells (e.g., immunotherapy), and / or one or more mechanical interventions (e.g., surgery, cry otherapy).

73. The method of any one of claims 70-72, wherein the subject is diagnosed as having or at risk of having a cancer or population of cancerous cells (e.g., myeloma, lymphoma, leukemia, a solid tumor).

74. The method of claim 73, wherein the subject has been diagnosed as having cancer and / or a population of cancerous cells.

75. A method of targeting a population of leukocyte immunoglobulin-like receptor B4 (LILRB4)-expressing cancer cells, the method comprising contacting the population with a composition comprising a synthetic CD 16a binding protein, wherein the synthetic CD 16a binding protein further comprises an effector that binds to LILRB4, wherein after the contacting with the composition, a greater portion of the population is dead as compared to contacting without a composition that does not comprise the synthetic CD 16a binding protein and / or the effector.