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MULTIMERIC PROTEIN COMPLEXES AS ANTIBODY SUBSTITUTES FOR NEUTRALIZATION OF VIRAL PATHOGENS IN PROPHYLACTIC AND THERAPEUTIC APPLICATIONS

20230382977 · 2023-11-30

    Inventors

    Cpc classification

    International classification

    Abstract

    The present patent consists of an engineered multimeric protein complex as antibody substitute composed of human proteins, with an m-fold symmetry, with each m-fold element containing a modified monomeric protein derived from a symmetric human multimeric protein complex fused to a module containing n fused, modified human beta solenoid proteins (mBSP), and that fused to a human derived pathogen binding domain (PBD), as well as a separate antibody substitute composed of P human PBD complexes. The invention may find application in prophylactic and therapeutic treatments for viral infections, especially for COVID19 by neutralizing the SARS-CoV-2 virus.

    Claims

    1. A multimeric protein complex as antibody substitute complex comprising a plurality (e.g., m≥2) of monomeric proteins with modular protein domains of the form (α-β.sub.n-γ.sub.p).sub.m or (γ.sub.p-β.sub.n-α).sub.m wherein the monomeric proteins α-β.sub.n-γ.sub.p or γ.sub.p-β.sub.n-α comprise fused protein domains wherein: α is a monomeric protein from a symmetric human multimeric protein complex of point group symmetry C.sub.m or D.sub.m, β is a fused domain of n modified beta solenoid proteins (mBSPs) with n≥0, and γ.sub.p is a complex of p pathogen binding domains (PBDs) either fused or bound to each other by intermolecular forces and wherein p≥1.

    2. The multimeric protein complex as antibody substitute of claim 1, wherein the multimeric protein complex as antibody substitute is symmetrical.

    3. The multimeric protein complex as antibody substitute of claim 2, wherein the multimeric protein complex as antibody substitute has two-fold symmetry, three-fold symmetry, four-fold symmetry, five-fold symmetry, six-fold symmetry, or twelve-fold symmetry.

    4-8. (canceled)

    9. The multimeric protein complex as antibody substitute of claim 1, wherein the modular protein domain a is a monomeric protein from a wild type symmetric human multimeric protein complex α.sub.m.

    10. (canceled)

    11. The multimeric protein complex as antibody substitute of claim 1, wherein α is a monomeric protein from: (a) the m=3 human collagen trimerization domain which is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 9. or (b) the m=3 human growth factor EDA-Al which is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 10; or (c) the m=3 human Langerin which is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 11; or (d) the m=4 human tetrameric diubiquitin which is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 12.

    12-14. (canceled)

    15. The multimeric protein complex as antibody substitute of claim 1, where n=0, p=1 and the protein binding domain (PBD) γ is at least 90%, 95%, 98% or 99% identical to the N-terminus domain (residues 19-85 or residues 19-91) of the human ACE2 receptor protein of SEQ ID NO: 6 or SEQ ID NO: 7.

    16. The multimeric protein complex as antibody substitute of claims 1, wherein the monomeric protein sequence is at least 90%, 95%, 98% or 99% identical to SEQ ID NO:1, 2 or 3.

    17. (canceled)

    18. (canceled)

    19. The multimeric protein complex as antibody substitute of claim 1, wherein the modified human beta solenoid (mBSP) is: (a) at least 80%, 90%, 95%, 98%, or 99% identical to the dynactin p27 domain (3VTO) of SEQ ID NO: 8. or (b) modified to be at least 80%, 90%, 95%, 98% or 99% identical to the human Retinitis Pigmentosa Protein 2 (RP2) (2BX6).

    20. The multimeric protein complex as antibody substitute of claim 1, wherein the monomeric sequence is at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 4 and n=1 and p=1, or to SEQ ID NO: 5, and n=4 and p=1.

    21. (canceled)

    22. The multimeric protein complex as antibody substitute of claim 1, wherein the sequence is of the form y2 and y is at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 6 or SEQ ID NO: 7.

    23. (canceled)

    24. The multimeric protein complex as antibody substitute of claim 1, wherein the pathogen binding domain is at least 90%, 95%, 98% or 99% identical to the helix-turn-helix (HTH) complex from the N-terminus of the ACE2 receptor protein of SEQ ID NO: 6 or SEQ ID NO: 7.

    25. The multimeric protein complex as antibody substitute of claim 1, wherein at least one (e.g., 1, 2, 3, 4, 5, or more) amino acid of the a or modified human beta solenoid domain is modified to allow attachment to a nanoparticle, a solid support, or other biological molecule.

    26. The multimeric protein complex as antibody substitute of claim 1, wherein the multimeric protein complex is attached to a nanoparticle, a solid support, or other biological molecule.

    27. The multimeric protein complex as antibody substitute of claim 1, wherein the multimeric protein complex is attached to human serum albumin.

    28. The pathogen binding domain of the multimeric protein complex as antibody substitute of claim 1, wherein at least one (e.g., 1, 2, 3, 4, or more) amino acid of one or more of the module domains is modified to allow attachment to a nanoparticle, a solid support, or other biological molecule.

    29. A multimeric protein complex as antibody substitute comprising a plurality of pathogen binding domains (e.g., 2, 3, 4, or more).

    30. The multimeric protein complex as antibody substitute of claim 29, wherein the pathogen binding domain is modified to be at least 90%, 95%, 98% or 99% identical to the HTH domain (residues 19-85 or 19-91) of the N-terminus of the ACE2 receptor protein of SEQ ID NO: 6 or SEQ ID NO: 7.

    31. The multimeric protein complex as antibody substitute of claim 30, wherein at least one (e.g., 1, 2, 3, 4, or more) amino acid of either or all pathogen binding domains are modified to allow attachment to a nanoparticle, a solid support, or other biological molecule.

    32. A method for neutralizing a pathogen comprising contacting said pathogen with the multimeric protein complex as antibody substitute of claim 1, wherein one or more pathogen binding domains binds to one or more sites on the pathogen.

    33. A method for immobilizing a pathogen comprising contacting said pathogen with the multimeric protein complex as antibody substitute of claim 1, wherein one or more pathogen binding domains binds to one or more sites on the pathogen.

    34. (canceled)

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0068] FIG. 1. Schematic drawing of a multimeric protein complex as antibody substitute .

    [0069] In this case the multimeric protein complex is a trimer (m=3) comprised of a monomeric protein from a symmetry human trimer such as the human collagen trimerization domain (PDB ID 3N3F), fused to a modified beta solenoid (mBSP) domain in which n copies of a single mBSP such as the p27 domain from dynactin (PDB ID 3VT0) are fused together, and p copies of a pathogen binding domain (PBD) such as residues 19-91 of the human ACE2 receptor protein. If the sequence begins with the α-monomer, it will have the form (α-β.sub.n-γ.sub.p).sub.m with α-β.sub.n-γ.sub.p the fused (by peptide bond) monomeric protein of the α, β.sub.n, and γ.sub.p domains. If the sequence begins with the PBD, it will have the form (γ.sub.p-β.sub.n-α).sub.m. The multimeric value m is chosen to match the symmetry of the corresponding viral envelope protein (VEP).sub.m where, e.g., VEP can be a monomeric protein of the Spike trimer from the SARS-CoV-2 virus.

    [0070] FIG. 2A-C Sample trimer neutralizing structure (γ-α).sub.3 where α is a monomeric protein from the A) human collagen trimerization domain (PDB Record 3N3F) and γ comes from N-terminus residues (19-85) of the human ACE2 protein with mutations per SEQ ID NO: 1. B) Full Trimer. C) This is designed to neutralize a single spike trimer complex of SARS-CoV-2 virions.

    [0071] FIG. 3A-C. A) Positive Control of binding of ACE2 protein to SARS-CoV-2 RBD protein measured in a biolayer interferometry (BLI) experiment, with an inferred 3 nM equilibrium dissociation constant, K.sub.D. B) Binding of HTH fused with red fluorescent protein (RFP) in a BLI experiment with inferred dissociation constant K.sub.D=5 nM. C) Binding of trimer synthesized from SEQ ID NO: 1 to monomeric RBD in a BLI experiment. The inferred dissociation constant K.sub.D=5 nM showing that the monomeric binding is comparable to the ACE2.

    [0072] FIG. 4A-D. Results from Simulations of (γ-α).sub.3 where α=monomeric protein from the human collagen trimerization domain, γ=PBD from residues (19-91) from human ACE2 protein with mutations per SEQ ID NO: 3. A) 10 nanosecond YASARA molecular dynamics simulations together show interfacial hydrogen bond counts for the ACE2 derived PBD binding to SARS-CoV-2 Spike RBD variants (wild type, alpha, beta, gamma) show little variation. As a corollary a combined analysis from HawkDock MM/GBSA correlated with Deep Mutational Scan measurements of binding energy changes from point mutations on RBD allows predictions of variant dissociation constant with wildtype, and the changes are small. B) Structure of SEQ ID NO: 3 simulated binding to single Spike RBD. C) Interfacial hydrogen bond counts from 12 nanosecond YASARA simulations of monomeric RBD bound to single HTH, and trimeric form of SEQ ID NO: 3 bound to three RBDS held by constraining potential in positions found experimentally for all 3 spike RBDs out. Avidity is demonstrated by >3X enhancement of interfacial hydrogen bound count for SEQ ID NO: 3, which correlates well with binding energy. D) Simulated structure of trimeric RBDs constrained to positions of full spike trimer.

    [0073] FIG. 5 SDS PAGE confirming trimer expression from SEQUENCE ID 2 and SEQUENCE ID 3 from P. pastoris. Lane 1—mass standard for SDS-PAGE, protein mass markers (approximate) in kilodaltons (KDa). Lane 10—Cytochrome C Oxidase standard. Monomers at ˜KDa. Lane 2, 3: expression from one P. pastoris culture of the protein of SEQUENCE ID 2 in nonreducing (2) and reducing (3) conditions. Same for different P. pastoris culture in Lanes 6,7. Note monomeric protein bands near 14 KDa (arrows) and identified (red ovals) trimer bands near 38 KDa. Lanes 5,9: Expression from P. pastoris culture of SEQUENCE ID 3 in nonreducing (5) and reducing (9) conditions. Again, note monomeric protein bands near 14 KDa and trimer bands near 38 KDa. The lighter apparent mass of the trimer per unit is attributable to excess negative charge. Lanes 4,8: Failed expression of modified version of SEQUENCE ID 1.

    [0074] FIG. 6. Sample trimeric neutralizing structure (α-β.sub.1-γ).sub.3 where a is a monomeric protein of the trimerization domain (PDB Record 1RJ7), β is an mBSP from the dynactin p27 BSP (PDB record 3VT0), and γ is the PBD taken from the N-terminus residues (19-83) of the human ACE2 protein. This is designed to neutralize a single spike trimer complex of SARS-CoV-2 virions.

    [0075] FIG. 7. Sample trimeric neutralizing structure (γ-β.sub.4-α).sub.3 where α is a monomeric protein of the trimerization domain (1RJ7), β is an mBSP from the dynactin p27 BSP (3VT0) with four copies fused together in a single unit, and γ=HTH comes from N-terminus residues (19-83) of the human ACE2 protein. This structure is designed to neutralize up to three spike 25 complexes of SARS-CoV-2 virions.

    [0076] FIG. 8. Sample model γ.sub.2 dimer constructed from γ=HTH from residues (19-83) of the ACE2 human receptor protein . The two HTH complexes (one red, one blue) bind together because of hydrophobic interactions on the inner surface of the peptides.

    DETAILED DESCRIPTION OF THE INVENTION

    [0077] The inventors have discovered that engineered protein trimers and dimers composed of linked modular sequences from human proteins can neutralize viral pathogens in a prophylactic or therapeutic context to form a multimeric protein complex as antibody substitute. By fusing a purely human multimeric protein complex comprised of m monomeric proteins α.sub.m to mBSPs and PBDs to match the m-fold symmetry and geometry of the viral envelope protein, it is possible to neutralize the multimeric VEP protein with a multimeric protein as an antibody substitute properly attuned to the VEP symmetry. In contrast monoclonal antibodies which at best exhibit dimeric (m=2) protein binding, but without a geometry tuned to the VEP.sub.m geometry they typically exhibit monomeric protein binding. The multimeric protein binding increases the net binding affinity to the VEP.sub.m complex. The inventors have previously demonstrated, for example, that a similar binding domain to that of the PBD from the ACE2 of SEQ ID NOS:1-6 attached to mBSP polymers can successfully bind vascular endothelial growth factor (VEGF) protein. The symmetric human multimeric protein complexes expressible in E. coli can be, for example, the human collagen trimerization domain (PDB code 3N3F) or other multimeric human protein complexes including but not limited to trimeric EDA-1 (PDB code 1RJ7), trimeric Langerin (3KQG), and tetrameric diubiquitin (2XEW), as detailed in SEQ ID NO: 9, 10, 11, and 12. FIG. 2 shows the design from SEQ ID NO: 1 using the human collagen trimerization domain and a single modified HTH per collagen monomer.

    [0078] The inventors have discovered that in the case of the PBD in the embodiment of the N-terminus sequence (see, e.g., SEQ ID NO: 6 and SEQ ID NO: 7) from the ACE2 protein that the binding is effective and essentially unchanged from the ACE2 dimer itself (FIG. 3). We denote this PBD as the HTH complex since it has an alpha helix (H)-turn(T)-alpha helix (H) structure. This indicates that the construct itself is sufficient for neutralizing a single RBD. Note that the trimer synthesized in E. coli from SEQ. ID NO:1 binds to the monomeric RBD with the same affinity as the RFP-HTH construct. Previous computer simulations suggest that the HTH binds RBD..sup.62

    [0079] The inventors have discovered through extensive simulations using the YASARA molecular dynamics program.sup.63 that mutations associated with extensive variants of the SARS-CoV-2 virus do not significantly alter the binding of the PBD from residues 19-91 of the ACE2 to the RBD of the SARS-CoV-2 spike protein (FIG. 4a) and that the multimeric protein complex as antibody substitute trimer construct of SEQ ID NO 3 binds to the full spike protein with all RBDs out with >3X the interfacial hydrogen bond count than the monomer, which will lead to up to a million fold greater binding affinity to the full spike protein with all RBDs out (FIG. 4c) confirming the avidity of the trimer per the definition.

    [0080] The inventors have discovered that the multimeric protein complex as antibody substitute designs of SEQ ID NO: 2 and SEQ ID NO: 3 are expressible in P. pastoris with the trimers secreted from the cells and needing no post-translation modification, confirming the advantage of the invention over monoclonal antibodies. (FIG. 5). Note that a modification of SEQ ID NO: 1 does not express well from the P. Pastoris.

    [0081] The inventors have discovered that by inserting a human β.sub.n=mBSP.sub.n domain consisting of n fused copies of a single mBSP domain between a monomeric proteins and the γ=PBD element of the multimeric protein complex as antibody substitute that they can adapt the size of the multimeric protein complex as antibody substitute to match the size of the VEPm to bind to more than one VEP element at once (FIG. 2 for the SARS-CoV-2 virus, with n=0, FIG. 6 with n=1), or to have sufficient length to bind to more than one VEPm complex at a time (FIG. 7 for the SARS-CoV-2 virus, with n=4). The human mBSP expressible in E. coli, for example, is shown in SEQ ID NO: 8 modified from the dynactin p27 protein (PDB ID 3TV0) or can be obtained from modifying the Retinitis Pigmentosa 2 protein (RP2)(PDB ID 2BX6).

    [0082] The inventors have discovered that in the case of the N-terminus ACE2 HTH example of SEQ ID NO: 6 or SEQ ID NO: 7 for a PBD, that there is a tendency to dimerize when detached from the ACE2 protein (FIG. 8). The source of this dimerization is a relatively large patch of hydrophobic residues which tend to associate together. Given that this dimer is a small construct (mass of approximately 15 KDa) this discovery allows the dimer itself to be a potent, easily dispersed neutralizer of spike proteins via binding to RBD.

    [0083] By making use of purely or nearly (see the following paragraph) human proteins in each of the modular domains of (α-β.sub.n-γ.sub.p) or (γ.sub.p-β.sub.n-α) for the multimeric protein complex as antibody substitute, the invention avoids immunogenic response of the human host in prophylactic or therapeutic applications.

    [0084] Immunogenic tolerance of the host to these multimeric protein complexes as antibody substitutes can be maintained by modifying up to 5 residues of the PBD to either (a) increase the innate binding strength to the VEP complex, or (b) to inhibit the dimerization of a PBD such as the HTH PBD construct from the ACE2 protein. For example, the substitutions of lysines or arginines at positions 62,69 of the HTH PBD in SEQ ID NO: 2 or 3 helps to significantly diminish the hydrophobic dimerization tendency.

    [0085] The probability for attachment of N-linked glycans to the multimeric proteins as antibody substitutes can be substantially reduced by modifying NIT sequences to QIT, as for example in SEQ ID NO:2 or 3.

    [0086] Exemplary multimeric protein complex as antibody substitute sequences of the form (α-β.sub.n-γ.sub.p).sub.m or (γ.sub.p-β.sub.n-α).sub.m include but are not limited to polypeptides comprising an amino acid sequence at least 90%, 95%, 98%, 99% or 100% identical any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, or 7 as provided below (numbers at right are amino acid count, beginning with N-terminal).

    TABLE-US-00001 ((γ-α).sub.m, m = 3, n = 0, p = 1, α is a monomeric protein of the human collagen trimerization domain 3N3F, γ is residues 19-85 of the human ACE2 receptor with the mutations S19G, T20C, P84A, L85C) This is the monomeric sequence γ-α. SEQ ID NO: 1 GCIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQST      (60) LAQMYACGGGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELI     (120) PIPA                                                             (124) ((γ-α).sub.m, m = 3, n = 0, p = 1, α is a monomeric protein of the human collagen trimerization domain 3N3F, γ is residues 19-91 of the human ACE2 receptor with the mutations N52Q, M62K, M69K) This is the monomeric sequence γ-α. SEQ ID NO: 2 STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTQITEENVQNKNNAGDKKSAFLKEQST      (60) LAQMYPLQEIQNLGGGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKL     (120) QLGELIPIPA                                                       (130) ((γ-α).sub.m, m = 3, n = 0, p = 1, α is a monomeric protein of the human collagen trimerization domain 3N3F, γ is residues 19-91 of the human ACE2 receptor with the mutations N52Q, M62R, M69R) This is the monomeric sequence γ-α. SEQ ID NO: 3 STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTQITEENVQNRNNAGDKRSAFLKEQST      (60) LAQMYPLQEIQNLGGGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKL.    (120) QLGELIPIPA                                                       (130) ((α-β-γ).sub.3, m = 3, n = 1, m = 1, α is a monomeric protein of the 1RJ7 human trimer, β = mBSP is from the 3VT0, γ is residues 19-83 from the ACE2 with the mutations S19G. This is the monomeric sequence α-β-γ. SEQ ID NO: 4 QPAVVHLQGQGSAIQVKNDLSGGVLNDWSRITMNPKVFKLHPRSGELEVLVDGTYFIYSQ      (60) VEVYYINFTDFASYEVVVDEKPFLQCTRSIETGKTNYNTCYTAGVCLLKARQKIAVKMVH     (120) ADISINMSKHTTFFGAIRLGEAPAPGAVVCVESEIRGDVTIGPRTVIHPKARIIAEAGPI     (180) VIGEGNLIEEQALIINAYPDNIKPMIIGTNNVFEVGCYSQAMKMGDNNVIESKAYVGRNV     (240) ILTSGCIIGACCNLNTFEVIPSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEE     (300) NVQNMNNAGDKWSAFLKEQSTLAQMYG                                      (327) ((γ-β.sub.4-α).sub.3, m = 3, n = 4, m = 1, α is a monomeric protein of the 1RJ7 human trimer, β = mBSP is the 3VT0, γ from residues 19-83 of the ACE2. This is the monomeric sequence γ-β.sub.4-α. SEQ ID NO: 5 STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQST      (60) LAQMYGGYPDNIKPMIIGTNNVFEVGCYSQAMKMGDNNVIESKAYVGRNVILTSGCIIGA     (120) CCNLNTFEVIPENRTVIHPKARIIAEAGPIVIGEGNLIEEQALIINAYPDNIKPMIIGTN     (180) NVFEVGCYSQAMKMGDNNVIESKAYVGRNVILTSGCIIGACCNLNTFEVIPENRTVIHPK     (240) ARIIAEAGPIVIGEGNLIEEQALIINAYPDNIKPMIIGTNNVFEVGCYSQAMKMGDNNVI     (300) ESKAYVGRNVILTSGCIIGACCNLNTFEVIPENRTVIHPKARIIAEAGPIVIGEGNLIEE     (360) QALIINAYPDNIKPMIIGTNNVFEVGCYSQAMKMGDNNVIESKAYVGRNVILTSGCIIGA     (420) CCNLNTFEVIPENRTVIHPKARIIAEAGPIVIGEGNLIEEQALIINAYPDNQPAVVHLQG     (480) QGSAIQVKNDLSGGVLNDWSRITMNPKVFKLHPRSGELEVLVDGTYFIYSQVEVYYINFT     (540) DFASYEVVVDEKPFLQCTRSIETGKTNYNTCYTAGVCLLKARQKIAVKMVHADISINMSK     (600) HTTFFGAIRLGEAP                                                   (614) EXEMPLARY PBD SEQUENCES (One PBD in γ.sub.p, p = 2, from ACE2 N-terminus HTH, residues 19-83 of ACE2) SEQ ID NO: 6 STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQST      (60) LAQMYP                                                            (66) (One PBD in γ.sub.p, p = 1, from ACE2 N-terminus HTH, residues 19-91 of ACE2) SEQ ID NO: 7 STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQST      (60) LAQMYPLQEIQNLG                                                    (74) EXEMPLARY mBSP SEQUENCES (mBSP from dynactin p27 BSP, 3VT0) SEQ ID NO: 8 GAVVCVESEIRGDVTIGPRTVIHPKARIIAEAGPIVIGEGNLIEEQALIINAYPDNIKPM      (60) IIGTNNVFEVGCYSQAMKMGDNNVIESKAYVGRNVILTSGCIIGACCNLNTFEVIPEP       (118)

    EXEMPLARY SYMMETRIC MULTIMERIC PROTEIN SEQUENCES

    [0087]

    TABLE-US-00002 (α monomeric protein from om human collagen trimerization domain, m = 3, 3N3F) SEQ ID NO: 9 NLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA              (54) (α monomeric protein from α.sub.m human growth factor EDA-A1, m = 3, 1RJ7) SEQ ID NO: 10 GSHMGPSGAADKAGTRENQPAVVHLQGQGSAIQVKNDLSGGVLNDWSRITMNPKVFKLHP        (60) RSGELEVLVDGTYFIYSQVEVYYINFTDFASYEVVVDEKPFLQCTRSIETGKTNYNTCYT.      (120) AGVCLLKARQKIAVKMVHADISINMSKHTTFFGAIRLGEAPAS                        (163) (α monomeric protein from α.sub.m human langerin, m = 3, 3KQG) SEQ ID NO: 11 ASTLNAQIPELKSDLEKASALNTKIRALQGSLENMSKLLKRQNDILQVVSQGWKYFKGNF        (60) YYFSLIPKTWYSAEQFCVSRNSHLTSVTSESEQEFLYKTAGGLIYWIGLTKAGMEGDWSW       (120) VDDTPFNKVQSARFWIPGEPNNAGNNEHCGNIKAPSLQAWNDAPCDKTFLFICKRPYVPS       (180) EP                                                                 (182) (α monomeric protein from α.sub.m human tetrameric diubiquitin, m = 4, 2XEW) SEQ ID NO: 12 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN        (60) IQKESTLHLVLRLRGG                                                    (76)

    EXAMPLES

    Example 1

    [0088] We have developed antibody replacements for viral neutralization from modular domain designs of proteins into multimeric proteins as antibody substitutes, extending work the inventors have previously demonstrated skill in developing for basic science.sup.64-70 and for applications.sup.53. The invention is to identify human symmetric multimeric protein complexes (α.sub.m) such as the human collagen trimerization domain (SEQ ID NO: 9 and PDB code 3N3F, M=3), with an example shown in FIG. 2, modify these in a genetic construct by fusion to a number of n fused copies of human modified beta solenoid proteins (mBSP) such as arising from the dynactin p27 domain (SEQ ID NO: 8 modified from 3VT0) to provide length as needed for matching the geometry of the (VEP).sub.m complexes to optimize binding, and at the end attach a Pathogen binding domain (PBD) such as the N-terminus helix-turn-helix motif from the ACE2 receptor (SEQ ID NO: 6 and SEQ ID NO: 7) that the SARS-CoV-2 complex binds to (residues 19-83). The data attached herein speaks to the potency of the HTH binding (see FIG. 3).

    [0089] As example 1, we consider the multimeric protein complex as antibody substitute design of SEQ ID 1 and FIG. 2, in which the human collagen trimerization domain of SEQ ID NO: 9 is modified by fusion to the n-terminal HTH domain of ACE2. As illustrated in FIG. 6, this domain can in principle bind to three RBDs of a single spike complex in the up configuration, thus providing potent neutralization capability. Because a single domain can bind with dissociation constant in the nanomolar range, per. FIG. 3, and because the dissociation constant

    [00001] K D ∼ exp ⁡ ( m ⁢ Δ ⁢ G B k B ⁢ T ) ,

    where m is the number of binders (identical to the multimeric number m in this example) and ΔG.sub.B is the (negative) binding free energy associated with a single binding event, the dissociation constant of the trimeric antibody substitute of SEQ ID NOS: 1-4 is likely to be in the femtomolar regime when all three RBDs are simultaneously bound by a single trimer. This avidity concept is borne out by considerable theoretical evidence and argument.sup.71-75 and by recent evidence of engineered nanobody binding to the down domain of the spike protein where trimers have likely femtomolar affinity.sup.76. FIG. 4 illustrates several relevant points from simulations supporting this. First, FIG. 4A shows that several of the most concerning variants (alpha, beta, gamma) have little variation in interfacial hydrogen bound count measured in >10 ns of YASARA.sup.63 all atom molecular dynamics simulations of the PBD from the N-terminus (SEQ ID NO: 6 or SEQ ID NO: 7) binding to the RBD. The mutations produce small variation of interfacial hydrogen bond number on average. We find an excellent correlation of the hydrogen bond count at the protein-protein interface with the binding energy measured in the MM/GBSA program HawkDock.sup.77. A separate correlation of binding energy point mutations of the RBD binding to the HTH shows that the relation between the estimated HawkDock change in binding energy with mutation and the measured Deep Mutational Scan energies is given by (all in kcal/mole)


    ΔΔG.sub.Mut−DMS=0.03437*(ΔΔG.sub.Mut−HawkDock−3.8)

    from which we can predict, with the theoretical ΔΔG.sub.Mut−HawkDock, the dissociation constants relative to wildtype (WT) as

    [00002] K D K D , WT = exp ⁡ ( 1 ⁢ 0 ⁢ 0 ⁢ 0 * 4 . 1 ⁢ 8 * Δ ⁢ Δ ⁢ G M ⁢ u ⁢ t - D ⁢ M ⁢ S R ⁢ T )

    where T is taken at room temperature and R=8.29 J/mole-K is the ideal gas constant. This is the third column of FIG. 4A. These results support the independence of the PBD binding strength with respect to Spike RBD variant sequence since they vary less than a factor of 2 from wild type. FIG. 4B shows a monomeric protein of SEQ ID NO: 3 bound to the RBD. FIG. 4C shows simulation results for 12 ns of simulation time of a trimer of SEQ ID NO: 3 bound to the RBDs of the 7KMS PDB file, constrained to the positions of 7KMS to mimic the full spike trimer. We find >3X the number of interfacial hydrogen bonds on average compared to the simulation of a single monomeric protein of HTH bound to a single RBD. This strongly supports the avidity concept above, and (i) the linear relationship of binding energy with number of bound interfaces, and (ii) the linear relationship of binding energy with interfacial hydrogen bound counts we have observed, we expect an affinity of the trimer of as low as femtomolar.

    [0090] By working with human constructs for the multimeric proteins as antibody substitutes containing a minor number (≤4-5) of point mutations, it is anticipated that our modular protein designs will be resistant to immunogenic response/rejection, and proteolytic degradation inside the body. Moreover, by using common domains from ubiquitous human proteins, the potential for autoimmune response is avoided.

    [0091] By choosing protein domains for our multimeric proteins as antibody substitutes which have been expressed from well proven, scalable, industrialized prokaryotic or single celled eukaryotic processes, we save the expense and unpredictability of monoclonal antibody production. FIG. 5 shows successful expression of trimers from SEQ ID NO: 2 and SEQ ID NO: 3.

    [0092] The molecular weight of each monomeric protein in our multimeric protein complex as antibody substitute designs is relatively small compared to antibodies. The design of SEQ ID 1 and FIG. 2 has a mass of approximately 14 KDa per binding unit, compared with 150KDa typical of monoclonal antibodies (mABs). The significance of this is that less protein mass is needed per prophylactic or therapeutic unit to achieve the same efficacy as for mABs.

    Example 2

    [0093] In example 2, we consider the somewhat larger construct of FIG. 6, and SEQ ID NO: 4 where the modified multimeric protein am is taken from the trimeric EDA-1 complex (SEQ ID NO: 10 and PDB Code 1RJ7), and we add a single β=mBSP element from the dynactin p27 complex (SEQ ID NO:8 and PDB Code 3VT0), and the γ=HTH domain from the ACE2 protein as mentioned above. This complex has a mass of approximately 42 KDa.

    [0094] This design can access the RBD domain even when it is flipped out away from the spike complex, and thus is amenable to a wider range of binding conformations than the smaller design of FIG. 2, because the center to center spacing of the HTH regions is about 7-8 nm, comparable to the RBD separation when all are fully out of the spike complex.

    Example 3

    [0095] The much larger multimeric protein complex as antibody substitute design of FIG. 7 and SEQ ID NO: 5 is made from the modified am EDA-1 complex (SEQ ID NO: 10) as per EXAMPLE 2, and this is fused to a fused 4-fold repeat of the β=mBSP complex modified from the human dynactin p27 protein (SEQ ID NO: 8), which is attached at the end to γ=HTH PBD from the ACE2 protein (SEQ ID NO: 7). The choice of four mBSP repeats provides an HTH-to-HTH separation of approximately 24 nm. The spike complexes on the surface of the SARS-CoV-2 virus are separated on average about 22 nm.sup.76, so this flexible trimer could potentially neutralize three spike proteins at once. The mass per fused monomeric protein is approximately 72 KDa, but if successful in neutralizing three spike proteins at once, the mass per RBD is like that of FIG. 2 and SEQ ID NO: 1.

    Example 4

    [0096] The HTH complex itself, in dimer form, can be a potent multimeric protein complex as antibody substitute neutralizing agent. The dimer construct of FIG. 8 and SEQ ID NO: 6 and SEQ ID NO: 7 has been demonstrated to have negligible affinity difference from the full length ACE2 per the inventors' data of FIG. 3. The mass per binding unit here is the smallest (about 7 KDa) of any of the other constructs, and we estimate the dimer self-affinity to be K.sub.D≈250 nM, weaker than the affinity of the HTH to the RBD domain.

    [0097] This multimeric protein complex as antibody substitute complex can be mutated by up to 4 amino acids to achieve higher affinity binding with the RBD without inducing immunogenic response, and such mutations can enhance the affinity per HTH to sub-nanomolar KD values.sup.78.

    [0098] If necessary, particularly in attachment to the complexes of EXAMPLES 1-3, we can modify the hydrophobic “underside” of the HTH complex by 1-4 residues to prevent self-association.

    Example 5

    [0099] By fusing a specific human serum albumin binding sequence to the side of the trimers in the multimeric protein complex as antibody substitutes discussed in EXAMPLES 1-3 opposite the binding face to the spike proteins, we can attach to albumin in the blood. For example, the SA2 peptide invented by Genentech.sup.79-82, has specific binding to serum albumin. This provides steric hindrance to viral binding in addition to the explicit blocking of the spike proteins and engenders enhanced lifetimes in vivo.

    Example 6

    [0100] The multimeric protein complex as antibody substitute inventions herein, while using specific examples of binding to the SARS-CoV-2 virus, are not solely restricted to this application. For example, the general multimeric protein complex as antibody substitute schema (α-β.sub.n-γ.sub.p).sub.m or (γ.sub.p-β.sub.n-α).sub.m can be extended to develop neutralization agents for the trimeric haemagglutinin VEPs on the surface of influenza virions, the tetrameric neuraminidase complexes on influenza virions, or the trimeric gp120 VEPs on the surface of HIV virions.

    [0101] Additionally, the general (α-β.sub.n-γ.sub.p).sub.m or (γ.sub.p-β.sub.n-α).sub.m scheme can be extended to binding to multimeric fusion complexes on microorganisms or tumor cells.

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    [0185] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.