WO2021188736A1 - MINIATURE GUIDANCE AND NAVIGATION CONTROL (miniGNC) ANTIBODY-LIKE PROTEINS AND METHODS OF MAKING AND USING THEREOF - Google Patents

Abstract

A multi-specific antibody-like protein having a first monomer comprising a first binding monomer, a CH1 domain, a first hinge, a first CH2 domain, and a first CH3 domain, a second monomer comprising a second binding monomer, a CL domain, a second hinge, a second CH2 domain, and a second CH3 domain, wherein the first binding monomer and a second binding monomer are configured to form a dimer, wherein the first monomer and the second monomer are covalently paired through at least one disulfide bond between the CH1 domain and the CL domain and at least one disulfide bond between the first hinge and the second hinge, wherein the first monomer and second monomer may comprise optionally a first binding domain (D1) and a second binding domain (D2), and a fourth binding domain (D4) and a fifth binding domain (D5), and wherein the multi-specific antibody-like protein is at least bi-specific.

Description

MINIATURE GUIDANCE AND NAVIGATION CONTROL (miniGNC) ANTIBODY-LIKE PROTEINS AND

METHODS OF MAKING AND USING THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 62/991,042 filed March 17, 2020 under 35 U.S.C. 119(e), the entire disclosures of which are incorporated by reference herein.

TECHNICAL FIELD

The present application generally relates to the technical field of multi-specific antibody for cancer immunotherapy and more particularly relates to making and using miniature Guidance and Navigation Control (miniGNC) antibodies with multiple binding activities against surface molecules of both immune cells and tumor cells.

BACKGROUND

Therapeutic antibodies have become a mainstay in treatment of several diseases, including but not limited to cancer, infection, and autoimmunity. While monoclonal, monospecific antibodies provide a straightforward mechanism to treat disease, e.g., via inhibition or activation of specific signalling pathways, multi-specific antibodies allow more complex therapeutic mechanisms to be explored. By targeting multiple distinct antigens, or multiple epitopes within a single antigen, biological responses such as cell colocalization and activation can be elicited, which enables, for example, the redirection of T cells and other immune cells to the site of specific antigen-expressing tumor cells. In this way, bispecific and multi-specific antibodies have become important platforms in the field of immunooncology.

Due to the advantages of multi-specific antibody platforms, many such frameworks have been developed. One such platform is known as Guidance and Navigation Control (GNC). The GNC proteins include the proteins linking multiple functionally independent binding moieties into a single entity that is capable of bringing both effector cells and target cells together (see Applicant's applications, WO/2019/005641, W02019191120, and PCT/US20/59230, incorporated herein in its entirety). In general, these platforms are restricted to at least two specificities, preventing the exploration of complex therapeutic mechanisms that require binding to more than two antigens. For antibody platforms with more than two specificities, the increased number of binding domains usually necessitates formation of large molecules which often have undesirable physical and biological properties. The increase in molecular weight can reduce developability, as larger and more complex molecules are more likely to have problems with aggregation and solubility. Furthermore, the ability of large macromolecules to penetrate into solid tumors may be hindered compared to proteins of smaller size. Thus, there is a need for multi-specific antibody platforms with more than two specificities that are not significantly larger than monoclonal IgG antibodies.

SUMMARY The following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

The application provides proteins with binding specificities such as multi-specific protein like antibodies including multi-specific antibodies, and the fragments of these binding proteins including without limitation scFv domain, Fab region, Fc domain, VH, VL, light chains, heavy chains, variable regions, and complementary determining region (CDR). The application further provides method of making and using the antibody-like proteins disclosed herein.

In one aspect, the application provides multi-specific antibody-like proteins. In one embodiment, the multi-specific antibody-like protein having a N-terminus and a C-terminus, including, a first monomer, including, from the N-terminus to the C-terminus, a first binding monomer, a CHI domain, a first hinge, a first CH2 domain, and a first CH3 domain, wherein the first monomer may comprise optionally a first binding domain (Dl) linked to the N-terminus, a fourth binding domain (D4) linked to the C-terminus, or both, a second monomer, including, from the N-terminus to the C-terminus, a second binding monomer, a CL domain, a second hinge, a second CH2 domain, and a second CH3 domain, wherein the second monomer may comprise optionally a second binding domain (D2) linked to the N-terminus, a fifth binding domain (D5) linked to the C-terminus, or both, wherein the first binding monomer and a second binding monomer are configured to form a dimer, wherein the first monomer and the second monomer are covalently paired through at least one disulfide bond between the CHI domain and the CL domain and at least one disulfide bond between the first hinge and the second hinge, and wherein the multi-specific antibody-like protein is at least bi-specific.

In one embodiment, the multi-specific antibody-like protein may be tri-specific, tetra- specific, or penta-specific. In one embodiment, the multi-specific antibody-like protein may be monoclonal antibodies. In one embodiment, the multi-specific antibody-like protein may be purified monoclonal antibody. In one embodiment, the multi-specific antibody-like protein may be humanized antibodies.

In one embodiment, the multi-specific antibody-like protein may further comprise a disulfide bond between the first CH3 domain and a second CH3 domain.

In one embodiment, the multi-specific antibody-like protein may further comprise a second disulfide bond between the first hinge and the second hinge.

In one embodiment, connecting linkers are used between various domains. In one embodiment, the connecting linker may comprise a (G_xS_y)_n linker, wherein n, x, and y each independently is an integer from 1 to 10. In one embodiment, Dl, D2, D4 or D5 are liked to N- or C-terminus through a linker. In one embodiment, the linker may comprise a (G_xS_y)_n linker. n, x, and y each independently may be an integer from 1 to 10. In one embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In one embodiment, x is 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In one embodiment, y is 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

In one embodiment, the first CH3 domain is configured to form a hole structure, wherein the second CH3 domain is configured to form a knob structure, and wherein the first CH2 and CH3 domains and the second CH2 and CH3 domains are configured to heterodimerize to form a complementary Fc domain.

In one embodiment, the first CH3 domain may comprise at least one 'hole' mutation at T366S, L368A or Y407V and the second CH3 domain may comprise a 'knob' mutation at T366W.

In one embodiment, the Fc domain may comprise mutations at H435R/Y436F.

In one embodiment, the Fc domain is engineered to eliminate effector cell functions selected from ADCC, ADCP, or CDC.

In one embodiment, the Fc domain may comprise at least one mutation at L234A, L235A, G237A, K322A (Eu numbering). In one embodiment, the Fc region comprise mutations at L234A/L235A/G237A/K322A. In one embodiment, the Fc region comprise mutations at L234A/L235A/K322A (Eu numbering). In one embodiment, the Fc domain comprise null mutation. In one embodiment, the lgG4 Fc domain comprises the mutation S228P (Eu numbering). In one embodiment, the lgG4 Fc domain comprises the mutations S228P/F234A/L234A (Eu numbering).

In one embodiment, the heavy chain constant sequence may be derived from IgGl or lgG4.

In one embodiment, the Fc domain may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO. 313 or 314.

In one embodiment, the first binding monomer may comprise a VH domain, a second binding monomer may comprise a VLdomain, and the VH, CHI, VL, CL domains form a Fab region as a third binding domain (D3). In one embodiment, the Fab region may comprise a disulfide bond between VH-44C and VL lOOC.

In one embodiment, the first binding monomer and the second binding monomer form a NKG2D receptor as a third binding domain (D3).

In one embodiment, the Dl, D2, D4, and D5 is independently a scFv domain, a VHH domain, a receptor, or a ligand.

In one embodiment, the scFv domain may have a VH domain linked to a VL domain. In one embodiment, the scFv domain have may have VH-VL orientation. In one embodiment, the scFv domain have a VL-VH orientation.

In one embodiment, the scFv domain may comprise a disulphide bond between VL and VH. In one embodiment, the disulfide bond is between VL100 and VH44 of the scFv domain. In one embodiment, the scFv domain may comprise the mutation R19S in the VH.

In one embodiment, the VHH domain may comprise the mutation R19S in the VH or VHH. In one embodiment, at least one, two, or three of Dl, D2, D4, and D5 may be scFv. In one embodiment, all of Dl, D2, D4, and D5 may be scFv.

In one embodiment, at least one, two, or three of Dl, D2, D4, and D5 may be a VHH domain. In one embodiment, all of Dl, D2, D4, and D5 may be VHH domains.

In one embodiment, at least one, two, or three of Dl, D2, D4, and D5 may be a receptor. In one embodiment, all of Dl, D2, D4, and D5 may be receptors.

In one embodiment, at least one, two, or three of Dl, D2, D4, and D5 may be a ligand. In one embodiment, all of Dl, D2, D4, and D5 may be ligands.

In one embodiment, the Dl, D2, D3, D4, and D5 each independently may have a binding specificity against an antigen selected from a T cell activating receptor, an immune cell binding receptor, an immune checkpoint molecule, a co-stimulation factor, a receptor of a leukocyte, a tumor antigen, a tumor associated antigen (TAA), a receptor of a tissue cell, a receptor of a cancer cell, or a combination thereof.

In one embodiment, the binding domain for T cell activating receptor is adjacent to the binding domain for the tumor associated antigen (TAA).

In one embodiment, T cell activating receptor may comprise CD3.

In one embodiment, an immune checkpoint receptor may comprise PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PD-L2, CD160, LOX-1, siglec-15, CD47, HVEM SIRPa CSF1R, CD73, Siglec-15, CD47or a combination thereof.

In one embodiment, a co-stimulating receptor may comprise 4-1BB, CD28, 0X40, GITR, CD40L, CD40, ICOS, LIGHT, CD27, CD30, or a combination thereof.

In one embodiment, a tumor associated antigen may comprise EGFR, HER2, HER3, EGRFVIII, CD19, BCMA, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D ligand, CD39, CLDN18.2, DLL3, HLA-G, FcRH5, GPRC5D, LIV-1, MUC1, CD138, CD70, uPAR, CD38 or a combination thereof.

In one embodiment, the Dl, D2, D3, D4, and D5 each independently may have a binding specificity against an antigen selected, EGFR, HER2, HER3, EGFRvlll, ROR1, CD3, CD28, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D, NKG2D ligand, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, PD-L1, PD1, 0X40, 4-1BB, GITR, TIGIT, TIM-3, LAG- 3, CTLA4, CD40, CD40L, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73, CD39, CLDN18.2, DLL3, HLA-G, FcRH5, GPRC5D, LIV-1, MUC1, CD138, CD70, CD16, uPAR, Siglec-15, CD47, CD38, , NKp46, PD-L2, CD160, LOX-1, SIRPa CD27 and the Fc domain may comprise a human IgG Fc domain.

In one embodiment, Dl may have a binding specificity to CD3, CD20, CEA, HER2, EGFR or NKG2D ligand. In one embodiment, D2 may have a binding specificity to HER3, EGFR, CD3, or CD19. In one embodiment, D3 may have a binding specificity to HER3, EGFR, CD3, or NKG2D ligands. In one embodiment, D4 may have a binding specificity to 4-1BB, or EGFR. In one embodiment, D5 may have a binding specificity to PD-L1 or HER3. In one embodiment, D1 may have a binding specificity to CD3, D2 may have a binding specificity to HER3, D3 may have a binding specificity to EGFR, D4 may have a binding specificity to 4-1BB, and D5 may have a binding specificity to PD-L1.

In one embodiment, D1 may have a binding specificity to EGFR, D2 may have a binding specificity to HER3, D3 may have a binding specificity to CD3, D4 may have a binding specificity to 4-1BB, and D5 may have a binding specificity to PD-L1.

In one embodiment, D1 may have a binding specificity to EGFR, D2 may have a binding specificity to CD3, D3 may have a binding specificity to HER3, D4 may have a binding specificity to 4-1BB, and D5 may have a binding specificity to PD-L1.

In one embodiment, D1 may have a binding specificity to CD3 or EGFR, D2 may have a binding specificity to CD19, D3 may have a binding specificity to CD3 or EGFR, D4 may have a binding specificity to 4-1BB, and D5 may have a binding specificity to PD-L1.

In one embodiment, D1 and D4 each may have a binding specificity to EGFR, D2 and D5 each may have a binding specificity to HER3, and D3 may have a binding specificity to CD3.

In one embodiment, D1 may have a binding specificity to CD20, D2 may have a binding specificity to CD19, D3 may have a binding specificity to CD3, D4 may have a binding specificity to 4-1BB, and D5 may have a binding specificity to PD-L1.

In one embodiment, D1 may have a binding specificity to NKG2D ligand, D2 may have a binding specificity to CD19, D3 may have a binding specificity to CD3, D4 may have a binding specificity to 4-1BB, and D5 may have a binding specificity to PD-L1.

In one embodiment, D1 may have a binding specificity to CD3, and D3 may comprise a NKG2D receptor. In one embodiment, the protein may further comprise D2 having a binding specificity to CD19. In one embodiment, the protein may further comprise D5 having a binding specificity to PD-L1. In one embodiment, the protein may further comprise D4 having a binding specificity to 4-1BB.

In one embodiment, the multi-specific antibody-like protein is bi-specific. In one embodiment, D2 may have a binding specificity to HER3, D3 may have a binding specificity to CD3. In one embodiment, D1 may have a binding specificity to HER2, D3 may have a binding specificity to CD3. In one embodiment, D1 may have a binding specificity to EGFR, D3 may have a binding specificity to CD3. In one embodiment, D3 may have a binding specificity to CD3, D5 may have a binding specificity to HER3. In one embodiment, D3 may have a binding specificity to CD3, D4 may have a binding specificity to EGFR.

In one embodiment, the bi-specific antibody-like protein may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO. 1 and 3; 5 and 7; 9 and 11; 13 and 15; 53 and 55; 57 and 59; 113 and 115; 117 and 119; 121 and 123; 125 and 127; 157 and 159; 297 and 299; or 199 and 201.

In one embodiment, the multi-specific antibody-like protein is tri-specific. In one embodiment, D1 may have a binding specificity to EGFR, D2 may have a binding specificity to HER3, D3 may have a binding specificity to CD3. In one embodiment, D1 may have a binding specificity to CEA, D2 may have a binding specificity to EGFR, D3 may have a binding specificity to CD3. In one embodiment, D3 may have a binding specificity to CD3, D4 may have a binding specificity to EGFR, D5 may have a binding specificity to HER3. In one embodiment, the antibody is a tri-specific antibody having the binding specificity to EGFR, HER3, and CD3. In one embodiment, the tri-specific antibody-like protein may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO. 41 and 43; 45 and 47; 49 and 51; 101 and 103; 105 and 107; 109 and 111; 195 and 197; 137 and 139; or 161 and 163.

In one embodiment, the multi-specific antibody-like protein is tetra-specific. In one embodiment, D1 may have a binding specificity to EGFR, D2 may have a binding specificity to HER3, D3 may have a binding specificity to CD3, D4 may have a binding specificity to 4-1BB. In one embodiment, D1 may have a binding specificity to EGFR, D2 may have a binding specificity to HER3, D3 may have a binding specificity to CD3, D5 may have a binding specificity to PD-L1. the antibody is a tetra-specific antibody having the binding specificity to EGFR, HER3, CD3, and 4-1BB. the antibody is a tetra-specific antibody having the binding specificity to EGFR, HER3, CD3, and PD-L1. In one embodiment, the tetra-specific antibody-like protein may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO. 33 and 35; 37 and 39; 141 and 143; 145 and 147; or 165 and 167.

In one embodiment, the antibody is penta-specific. In one embodiment, the antibodylike protein may have the binding specificity to EGFR, HER3, CD3, 4-1BB and PD-L1. In one embodiment, the penta-specific antibody-like protein may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO. 17 and 19; 21 and 23; 25 and 27; 29 and 31; 69 and 71; 73 and 75; 77 and 79; 81 and 83; 85 and 87; 89 and 91; 93 and 95; 97 and 99; 129 and 131; 133 and 135; 149 and 151; 169 and 171; 173 and 175; or 177 and 179.

In a second aspect, the application provides novel sequences for complimentary determining regions (CDRs). In one embodiment, the application provides proteins, antibodylike proteins or antibodies comprising the CDRs as disclosed herein.

In one embodiment, the CDRs may have an affinity to CEA. In one embodiment, the CDRs may have an equilibrium dissociation constant (KD) to CEA, wherein the KD is not more than 0.1 nM, 2nM, 5nM, 10 nM, 20nM, 30nM or 50nM. In one embodiment, the CDR may include an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 301, 302, 303, 304, 305, or 306.

In one embodiment, the application provides a protein having an affinity to CEA. In one embodiment, the protein may have a CDR HI having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.301. In one embodiment, the protein may have a CDR H2 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.302. In one embodiment, the protein may have a CDR H3 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.303. In one embodiment, the protein may have a CDR LI having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.314. In one embodiment, the protein may have a CDR L2 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.305. In one embodiment, the protein may have a CDR L3 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.306. In one embodiment, the protein may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO. 279, 280, 281 or 282. In one embodiment, the protein may include an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO. 279, 280, 281 or 282.

In one embodiment, the application provides a multi-specific antibody-like protein having a variable region, wherein the variable region may comprise an amino acid sequence selected from SEQ ID NO. 301, 302, 303, 304, 305, or 306.

In one embodiment, the CDRs may have an affinity to CD3. In one embodiment, the CDR may have an equilibrium dissociation constant (KD) to CD3, wherein the KD is not more than 10 nM, 20nM, 30nM or 50nM, lOOnM, 200nM, 300nM, 400nM or 500nM. In one embodiment, the CDR may include an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 307, 308, 309, 310, 311 or 312.

In one embodiment, the protein having an affinity to CD3 includes CDR HI having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.307. In one embodiment, the protein having an affinity to CD3 includes CDR H2 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.308. In one embodiment, the protein having an affinity to CD3 includes CDR H3 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.309. In one embodiment, the protein having an affinity to CD3 includes CDR LI having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.310. In one embodiment, the protein having an affinity to CD3 includes CDR L2 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.311. In one embodiment, the protein having an affinity to CD3 includes CDR L3 having an amino acid sequence having at least 80% sequence identity to SEQ ID NO.312.

In one embodiment, the protein may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO. 227-230, 231-234, 235-238, 239-242 and 291- 294. In one embodiment, the multi-specific antibody-like protein may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NO. 227-230, 231-234, 235- 238, 239-242 and 291-294. In one embodiment, the multi-specific antibody-like protein may have a variable region wherein the variable region may comprise an amino acid sequence selected from SEQ ID 307, 308, 309, 310, 311 or 312.

In a further aspect, the application provides isolated nucleic acid sequences encoding the multi-specific antibody-like proteins, fragments, domains, regions as disclosed herein.

In a further aspect, the application provides expression vectors including the isolated nucleic acid sequences as disclosed herein.

In a further aspect, the application provides host cells including the isolated nucleic acid sequences as disclosed herein. In one embodiment, the host cell includes the expression vector as disclosed herein. In one embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell.

In a further aspect, the application provides methods for producing the multi-specific antibody like proteins as disclosed herein. In one embodiment, the method includes the steps of culturing a host cell such that the DNA sequence encoding the multi-specific antibody-like protein is expressed, and purifying said multi-specific antibody. In one embodiment, the method includes the steps of culturing a host cell under conditions wherein said multi-specific antibody- like proteins are produced and recovering said antibody-like protein.

In a further aspect, the application provides immunoconjugates. In one embodiment, the immunoconjugate may include the multi-specific antibody-like proteins linked to a cytotoxic agent, an imaging agent, or both.

In a further aspect, the application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition may include the multi-specific antibody-like protein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may further include radioisotope, radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent or a combination thereof. In one embodiment, the pharmaceutical composition may include the immunoconjugate as disclosed thereof and a pharmaceutically acceptable carrier.

In a further aspect, the application provides methods for treating or preventing a cancer, an autoimmune disease, or an infectious disease in a subject. In one embodiment, the method may include the steps of administering to the subject a pharmaceutical composition including a purified multi-specific antibody-like protein, an immunoconjugate, or a pharmaceutical composition as disclosed herein. In one embodiment, the method may further include co- administering an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent may comprise an antibody, a chemotherapy agent, an enzyme, or a combination thereof.

In one embodiment, the subject is a human. In one embodiment, the subject is a mammal. In one embodiment, the subject is a chimpanzee. In one embodiment, the subject is a pet animal. In a further aspect, the application provides a solution including an effective concentration of the multi-specific antibody-like protein, immunoconjugate, or pharmaceutical composition as disclosed herein. In one embodiment, the solution is blood plasma in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:

Figure 1 depicts the heterodimeric configuration of miniGNC molecules with five binding domains (D1-D5), of which the two monomers are encoded by Chain A (N-D1-D3/VH-CH1-CH2-CH3-D4-C) and Chain B (N-D2-D3/VL-CL-CH2-CH3-D5-C);

Figure 2 shows the dimerization of engineered miniGNC Chain A and Chain B with knockout mutations (KO) on either VH or Fc: (2A) by Protein-A purification to fractionate the mixture into the homodimer (a), the desired heterodimer (b), and the Chain B monomer (c); and (2B) by SDS- PAGE analysis of Protein-A fractionated homodimer (a), heterodimer (b), Chain B monomer (c), and Chain A monomer (d), at the expected sizes under non-reducing and reducing conditions; Figure 3 shows the comparative potency of multi-specific miniGNC molecules mediated TDCC on BXPC3 tumor cells by using penta-miniGNC (SI-75P6), tetra-miniGNC (SI-75E2), tri-miniGNC (Sl- 75X3), bi-miniGNC (SI-75X2), and mono-miniGNC (SI-7502) molecules;

Figure 4 shows the comparative potency of miniGNC molecules mediated TDCC on BXPC3 tumor cell line with EC50 values in the range of 0.1 to 5.5 pM;

FIGURE 5 shows the survival curves of TDCC assays measuring comparative potency of (A) multi- specific miniGNC molecules with stapled domains, including penta-miniGNC (SI-68P1), tetra- miniGNC (SI-68E2 and SI-68E1), and tri-miniGNC (Sl-68x2), for killing pancreatic cancer cells (HPAFII) in TDCC assay, and (B) SI-68X1 against MCF-7 breast cancer cells; and Figure 6 shows the functionality of multi-specific miniGNC molecules possessing a dimeric NKG2D receptor, including tri-specific (SI-49R25), tetra-specific (SI-49P_X), penta-specific (SI-49PM1 and SI-49P8) in killing breast cancer cells (MDA-MB-231) in TDCC assay.

DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

The disclosure provides, among others, isolated antibodies, methods of making such antibodies, bispecific or multi-specific molecules, antibody-drug conjugates and/or immuno- conjugates composed from such antibodies or antigen binding fragments, pharmaceutical compositions containing the antibodies, bispecific or multi-specific molecules, antibody-drug conjugates and/or immuno-conjugates, the methods for making the molecules and compositions, and the methods for treating cancer using the molecules and compositions disclosed herein.

The present application discloses multi-specific Guidance and Navigation Control (GNC) antibodies in miniature configurations (miniGNC). A miniGNC platform molecule is an assembled heterodimer of two polypeptide chains characterized by the formation of a Fab-Hinge-Fc region core structure with one, two, three, or four additional binding domains (from D1 to D5), as elucidated in Figure 1. The two polypeptides may be configured, for Chain A (or Chain 1): N-Dl- D3(VH)-CH1-Hinge-CH2-CH3-D4-C; and for Chain B (or chain 2): N-D2-D3(VL)-CL-Hinge-CH2-CH3- D5-C. The Fc domains of the two chains are engineered to contain complementary mutations, also known as "knobs-into-holes", to enhance the formation of the heterodimer. A miniGNC platform molecule may have variable components. For example, the VH and VL of the Fab region may be replaced by a non-Fab dimer with or without binding specificity. An additional binding domain may be an antibody fragment, such as scFv or VHH, or a non-antibody structure, such as a ligand or a receptor. A miniGNC platform antibody may be bivalent, trivalent, tetravalent, or pentavalent with up to five distinct specificities, and retains the approximate size of a normal bivalent IgG antibody (tri-miniGNC Ab at 150 kD) or slightly larger (tetra-miniGNC Ab at 175 kD, and penta-miniGNC Ab at 200 kD).

The present application relates to methods of making and using miniGNC antibodies, in particular, penta-specific miniGNC antibodies (penta-miniGNC Ab). In general, GNC proteins, such as GNC antibodies, are characterized by comprising two moieties for engaging immune cells, such as activating T cells, while targeting tumor cells. Similar to GNC antibodies, miniGNC antibodies retain multiple antigen binding domains for engaging immune cells, such as anti-CD3 for T cell activation, anti-4-lBB for co-stimulation, and anti-PD-Ll for inhibiting immune checkpoint. To improve the efficacy of antibody therapy for treating cancer, miniGNC antibodies are designed to be structurally stable and compact while retaining the characteristic feature of two moieties in GNC antibodies. This improvement allows an additional binding specificity to a second tumor associated antigen on the same or different tumor cell. As compared to GNC antibodies, miniGNC contains an Fc domain that allows for FcRn-mediated recycling and half-life extension, as well as facile protein A-based purification. The Fc receptor-mediated immunity may be incorporated if desired. An Fc-containing miniGNC antibody is quite compact, which allows for better developability and increased tumor penetration. GNC antibodies are usually larger than an IgG antibody due to increased number of antigen binding domains (AgBD), which provides spatial flexibility for binding to both a T cell and a tumor cell. On the other hand, miniGNC antibodies retains the same approximate size as human IgG antibodies calculated at approximately 110-130 kD for bi-specific;120-160 for tri-specific; 130-190 kD for tetra-specific; 140-220 kD for penta-specific; as compared to 150 kD for human IgG. The incorporation of one or two scFvs onto each chain may reduce chain-pairing complications and improve stability while navigating through tumor microenvironment. With these characteristic features, GNC and miniGNC antibodies may be alterative of an efficacious antibody therapy for treating the same cancer, since the moiety for targeting tumor associated antigens remains the same, including but not limited to, EGFR, HER2, HER3, EGRFVIII, CD19, BCMA, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D ligand, CD39, CLDN18.2, DLL3, HLA-G, FcRH5, GPRC5D, LIV-1, MUC1, CD138, CD70, uPAR, CD38.

The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, polyclonal, chimeric, single chain, bispecific or bi-effective, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv and Fv fragments, including the products of aFab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above. In some embodiments, antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically bind an antigen. The immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgGl, lgG2, lgG3, lgG4, IgAl and lgA2) or subclasses of immunoglobulin molecule. In one embodiment, the antibody may be whole antibodies and any antigen-binding fragment derived from the whole antibodies. A typical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is comprised of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4from amino-terminus to carboxy-terminus. Within the variable regions of the light and heavy chains there are binding regions that interacts with the antigen.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

The monoclonal antibodies may include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

Monoclonal antibodies can be produced using various methods including mouse hybridoma or phage display (see Siegel. Transfus. Clin. Biol. 9:15-22 (2002) for a review) or from molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453- 7 (2011)). In the present disclosure antibodies were created by methods of immunizing rabbits, mice, or llama in combination with subsequent strategies like hybridoma or display. Rabbits are known to create antibodies of high affinity, diversity and specificity (Weber et al. Exp. Mol. Med. 49:e305). Besides immunization of rabbits followed by B cell culture, other common strategies for antibody generation and discovery include immunization of other animals (e.g., mice, llamas) followed by hybridoma and/or display on phage, yeast, or mammalian cells; or display using synthetic variable gene libraries. This general method of antibody discovery is similar to that described in Seeber et al. PLOS One. 9:e86184 (2014).

The term "antigen- or epitope-binding portion or fragment" refers to fragments of an antibody that are capable of binding to an antigen. These fragments may be capable of the antigen-binding function and additional functions of the intact antibody. Examples of binding fragments include, but are not limited to a single-chain Fv fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker or a Fab fragment which is a monovalent fragment consisting of the VL, constant light (CL), VH and constant heavy 1 (CHI) domains. Antibody fragments can be even smaller sub- fragments and can consist of domains as small as a single CDR domain, in particular the CDR3 regions from either the VL and/or VH domains (for example see Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods known to those skilled in the art. The antibody fragments can be screened for utility using the same techniques employed with intact antibodies.

The "antigen-or epitope-binding fragments" can be derived from an antibody of the present disclosure by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

The Fab fragment may contain the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.

"Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (l), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-l, lgG-2, lgG-3, and lgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, delta, epsilon, γ, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

A "humanized antibody" refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain "humanized antibodies" are well known to those skilled in the art. (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991)).

The terms "polypeptide", "peptide", and "protein", as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.

The terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate.

By "isolated" is meant a biological molecule free from at least some of the components with which it naturally occurs. "Isolated," when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody," refers to an antibody which is substantially free of other antibodies having different antigenic a binding specificity.

"Recombinant" means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells.

The term "antigen" refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.

Also, as used herein, the term "immunogenic" refers to substances which elicit or enhance the production of antibodies, T-cells or other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals. An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present disclosure to moderate or alleviate the disorder to be treated.

"Specific binding" or "specifically binds to" or is "specific for" a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

The term "affinity" refers to a measure of the attraction between two polypeptides, such as antibody/antigen, receptor/ligand, etc. The intrinsic attraction between two polypeptides can be expressed as the binding affinity equilibrium dissociation constant (KD) of a particular interaction. A KD binding affinity constant can be measured, e.g., by Bio-Layer Interferometry, where KD is the ratio of kdis (the dissociation rate constant) to kon (the association rate constant), as KD = kdis/kon.

Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10^-4 M, at least about 10^-5 M, at least about 10^-6 M, at least about 10^-7 M, at least about 10^-8 M, at least about 10^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 -₁₂ M or greater, where KD refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50- , 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.

"Homology" between two sequences is determined by sequence identity. If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs. The deviations appearing in the comparison between a given sequence and the above-described sequences of the disclosure may be caused for instance by addition, deletion, substitution, insertion or recombination.

The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein. Although the present disclosure has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure.

EXAMPLES

Example 1: Configuration of penta-specific miniGNC antibodies

The design of miniGNC antibodies relies on the co-expression and assembling of two polypeptide chains of a heterodimer between Chain A and Chain B (Figure 1). While Chain A may be a native heavy chain (HC), Chain B is a recombinant fusion peptide between the light chain and the Fc domain of HC. At the core of this antibody structure, there is a Fab region (VH-CH1/VL- CL) followed by the hinge and Fc region (CH2-CH3/CH2-CH3) of human IgGl. "Knobs-into-holes" mutations or other complementary sets of mutations may be introduced into the CH3 domains to destabilize homodimers and favor heterodimer formation (Ridgway JB, Presta LG, Carter P., Protein Eng 1996;9:617-21). The "hole" mutations in the CH3 domain of Chain A consist of T366S, L368A, and Y470V, while the "knob" mutation in the CH3 domain of Chain B is T366W. The "knob" created by the T366W mutation on Chain B preferentially interacts with the "hole" created by the three mutations on Chain A. This preferential pairing may further stabilize the heterodimer while the non-covalent pairing of the Fab domain components (i.e. between VH- CH1 on Chain A and VL-Ck on Chain B) creates a functional Fab region. In other words, a functional D3 binding domain, such as a functional Fab region or a non-Fab receptor dimer, can only be created in Chain A-Chain B heterodimers. As compared to GNC antibodies, miniGNC antibodies also contain antibody fragments from native IgG (Fab, Fc) and other variable-sequence based structures, such as scFv and VHH, but are engineered to preferentially configure a Fab region-Hinge-Fc region structure after the formation of a heterodimer. The characteristic feature of miniGNC antibodies is the compactness of this engineered structure.

One or more structurally diversified antigen binding domain may be added to the two N- terminals and the two C-terminals of a miniGNC molecule as D1, D2 D4, or D5, thereby creating up to five binding specificities. Each of these added binding domains may be variable-sequence- based structures, such as scFv and VHH, or non-variable sequence-based structures, such as ligand and receptor. As elucidated in Figure 1, D1 and D2 refer to the binding domains linked to the N-terminals of Chain A and Chain B, respectively, whereas D4 and D5 refer to the binding domains linked to the C-terminals of Chain A and Chain B, respectively. Thus, the two polypeptides of a penta-specific miniGNC (penta-miniGNC) antibody may be configured as Chain A: N-Dl-D3(VH)-CH1-Hinge-CH2-CH3-D4-C; and Chain B: N-D2-D3(VL)-CL-Hinge-CH2-CH3-D5-C; where VL may be either nl or VK and CL may be either C or Ck as shown in Figure 1.

Depending on the number of binding domains and their binding specificities, the structural configuration of the penta-miniGNC antibody in Figure 1 may be simplified by reducing one or more binding domains and becoming tetra-, tri-, and bi-miniGNC. For incidence, Table 1 depicts the generation and characterization of a family of anti-CD3 miniGNC antibodies for optimizing their efficacy of targeting EGFR and HER3. In this case, each binding domain exerts one binding specificity. When two of its five binding domains target the same epitope of an antigen, i.e. the same binding specificity, the penta-miniGNC antibody becomes a tetra-specific penta-miniGNC antibody.

Example 2: Engineered core structure of miniGNC antibodies

To demonstrate that the configuration of miniGNC may be translated into stable and functional heterodimeric protein, a simplified asymmetric format was designed for evaluating the effect of configuration and mutations to the heterodimer formation of miniGNC molecules. Briefly, a single scFv (Dl) is fused to the N-terminus of Chain A, whereas the Chain B does not have any added binding domain. In this way, the proportion of each species of dimerization between Chain A and Chain B may be distinguished by SDS-PAGE and SEC, as the predicted sizes for a Chain A homodimer (not observed), a Chain B homodimer, and a Chain A and Chain B heterodimer are 100 kDa, 150 kDa, and 125 kDa, respectively) (Figure 2).

To engineer a strategy for selectively purifying the heterodimeric miniGNC molecule from a mixture, two amino acid residues in the CH3 domain (H435 and Y436) of the Chain B are substituted. In particular, the histidine (H435) is critical forthe Chain B to bind to protein-A resins during the purification of therapeutic antibodies (Tustian et al., 2016), and the substitution of this amino acid or a ProA knockout mutation (ProAKO) terminates the protein-A binding. This strategy may allow separation of the heterodimer via selective elution from a protein-A column. As the protein-A column retains the heterodimer through its parental heavy chain binding site, the Chain B homodimer flows through as the Chain B lacks protein-A binding. The design of a ProAKO on the Chain B minimizes any possible issue of altering immunogenicity by amino acid substitutions of the protein-A binding site with residues taken from the homologous region in the lgG3 subclass. Although most of miniGNC molecules will predominantly incorporate the structure and sequence of IgGl subtype, lgG3 has an overall similar structure to other IgG subclasses with some specific structural perturbations in binding to protein-A, protein-G and FcRn (neonatal Fc receptor). This difference is based on Fc sequence difference as revealed earlier where the presence of R435 (H435 in other subtypes) in lgG3 ablates its Fc interaction with protein-A. Hence, in representative miniGNC molecules, amino acids critical for protein-A binding on miniGNC Chain B (H435 and F436) have been mutated to the lgG3 counterpart sequences (R435 and Y436).

In the class of multi-specific miniGNC antibodies with an addition of scFv fused to the N terminal of Chain B, the interaction to protein-A binding is still possible if the scFv comes from VH3 family. VH3 encoded antibodies are known to interact with Staphylococcal Protein A (SPA) and residues in FR1, CDR2 and FR3 have been identified to be involved in SPA binding (Roben, et al., 1995), and structural data indicated that the replacement of a single involved region with the corresponding region from the nonbinding immunoglobulin ablates protein-A binding by VH3 encoded antibodies. Substitution of the amino acid R19 in FR1 was identified to be critical in binding to protein-A and was undertaken with the amino acid sequence (S19) from equivalent region in the VH4 family.

Four sets of miniGNC molecules (SEQ ID 1-16) with four different versions of chain B mutations (SEQ ID 3,7,11 and 15) embracing different combinations of ProAKO: H435R/Y436F (Fc) and R19S (VH) were purified and analyzed with analytical size exclusion chromatography. Only protein containing Chain B with both sets of mutations allowed complete removal of Chain B contaminants (Figure 2A). This VH3 protein A knockout mutation (R19S) together with Fc ProteinA knockout mutations (H435R, F436Y) on Chain B was incorporated in most of the multi- specific antibody formats and used to produce bi-, tri-, tetra- and penta-miniGNC molecules.

To demonstrate the feasibility of generating functional miniGNC proteins with alternative antibody isotypes, an example miniGNC containing lgG4 constant regions was generated. SI- 75XM9 (SEQ ID 297-300) is analogous to SI-75X5 (SEQ ID 1-4) bispecific miniGNC, except that the IgGl CHI, hinge, and Fc were replaced with lgG4 CHI, hinge, and Fc. Notably, both proteins have the same binding domains (anti-HER3 domain in D2 and anti-CD3* domain in D3) and contain the same core structure and mutations (Knobs-into-Holes Fc mutation; R19S in anti-HER3 scFv; and H435R/Y436F mutations in Fc). As shown in Table 8, SI-75XM9 had comparable titer to SI-77X5, and had a significant 19% increase in the percent protein of interest (85.6% POI vs. 72.2% POI) after protein A purification, as assessed by analytical SEC. The reduction in high molecular weight species could be related to the shorter hinge length of lgG4 (12 amino acids instead of 15), which could reduce the propensity for association of higher-order oligomers. Thus, generation of miniGNC proteins with alternative antibody isotypes is not only possible, but can lead to more favorable properties such as decreased aggregation.

Example 3: Configuration of multi-specific miniGNC antibodies

To generate and characterize the first group of multi-specific miniGNC antibodies, a trio of moiety 1 binding domains, i.e. aCD3, aPD-Ll, and a4-lBB, and two moiety 2 binding domains against EGFR and HER3, respectively, were selected for comparison. As shown in Table 1, the group was subdivided into penta-miniGNC, tetra-miniGNC, tri-miniGNC, and bi-miniGNC groups. Of each subgroup, there was at least one miniGNC antibody having the CD3 binding from D3. The penta-miniGNC group includes SI-75P6 (SEQ ID 17, 19, with aEGFR from D3 position), SI-75P4 (SEQ ID 21, 23, with 41BBL, the ligand to 4-1BB receptor, at D4), SI-75P3 (SEQ ID 25, 27), and Sl- 75P9 (SEQ ID 29, 31, with aHER3 from D3). The tetra-miniGNC group have two antibodies, Sl- 75E1 (SEQ ID 33, 35) and SI-75E2 (SEQ ID 37, 39) comparing the effect in the presence and absence of either a4-lBB or aPD-Ll domain. The tri-miniGNC group have three antibodies, SI-75X3 (SEQ ID 41, 43), SI-75X16 (SEQ ID 45, 47), and SI-75X18 (SEQ ID 49, 51) for comparing the two moiety 2 binding domains on either N- or C-terminus of the heterodimer. The bi-miniGNC group have three antibodies, SI-75X1 (SEQ ID 53, 55), SI-75X2 (SEQ ID 57, 59), and SI-75X5 (SEQ ID 1, 3), each of which has one moiety 1 binding domain and one moiety 2 binding domain.

The DNA encoding each member of the first group of miniGNC antibodies were cloned into vector pTT5, which were expressed with acceptable titers using ExpiCHO expression systems for 9 days. The miniGNC antibodies were purified with 5mL MabSelect protein-A columns followed by Size Exclusion using a highload 16/600 200 pg preparative SEC column on either an Akta Avant or Purifier system. SEC aggregates were analyzed using a waters HPLC linked to multi angle light scattering (MALS, Wyatt systems) to identify correct molecular weight by dn/dc calculated methods.

Example 4: Octet analysis of binding affinity

To assess the functionality of miniGNC antibodies, the binding affinity of each domain was determined by using Biolayer Interferometry (Octet 384 system). Octet analysis was used to ensure that the selected miniGNC antibody retains the binding specificity and affinity to all their cognate antigens. Each miniGNC antibody was loaded onto AHC sensors for 180 seconds at 10 ug/ml, followed by a 60-second baseline step, a 180-second association step with 100 nM of commercially purchased human antigen, and a 360-second dissociation step. Samples for all steps were in Octet buffer (PBS containing 0.1% Tween 20 and 1% BSA). Fits were performed using a 1:1 binding model to extract affinity KD values. As shown in Table 1, the binding affinity for each binding domain, as measured by its KD, varies within an acceptable range. For moiety 1 binding, the KD for aCD3 domain may vary between 17.4 and 36.2 nM, for aPD-Ll between 1 and 2.72 nM, and for a4-1BB between 7.51 and 37.3 nM; and for moiety 2 binding, the KD for aEGFR varies between 5.56 and 11.1, and for aHER3 between 112.8 and 185 nM.

Example 5: Comparative potency of multi-specific miniGNC antibodies

The first group of multi-specific miniGNC antibodies was subjected to T-cell dependent cellular cytotoxicity (TDCC) assay for measuring the potency for killing cancer cells. With an aCD3 binding domain in all molecules, this group of miniGNC antibodies was equipped to engage T cells, redirect T-cell mediated cytolysis, and ultimately kill target cells.

A luminescence-based T-cell dependent cellular cytotoxicity (TDCC) assay was used to measure the extent of antibody-induced cellular cytotoxicity by quantification of cell viability via constitutive expression of luciferase. Luciferised BXPC3 human pancreatic cancer cell lines (ATCC, Manassas, VA) were cultured at 37°C, 5% C02 in RPMI (ATCC, Manassas, VA) medium with 10% heat-inactivated fetal bovine serum (Invitrogen, Waltham, MA). Cell viability was monitored with a Vi-CELL automated cell counter (Beckman Coulter, Pasadena, CA). Target cell surface expression was measured by flow cytometry. Human pancreatic cancer cells, BXPC3 cells, were co-cultured with human pan T-cells at an effector-to-target (E:T) ratio of 5:1 and antibodies were added with a 5-fold dilution series (0 - 30 nM). Using a Multidrop bulk liquid dispenser (BIOTEK, Winooski, VT), 500 cells per well of target cells (20 pL per well) and 25,00 cells per well of T-cells (20 pL/ well) were plated sequentially into 384-well, white, flat-bottom, polystyrene TC-treated microplates (Corning, Corning, NY). Antibody dilutions were added (10 pL/well) and plates were incubated for 72 hrs at 37°C, 5% C02 before luminescence-based cell viability quantification. 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of tumor cells was determined using a CLARIOstar plate reader.

Data were fit to a sigmoidal function to calculate and list EC50 values in Table 1 for comparison. Figure 3 shows the dose-dependent cell viability curves of miniGNC antibodies selected from a penta-miniGNC (SI-75P6), a tetra-miniGNC (SI-75E2), a tri-miniGNC (SI-75X3), a bi-miniGNC (SI-75X2), and a mono-miniGNC (SI-7502) molecule. While all multi-specific miniGNC antibodies were capable of killing BXPC3 cells completely, both pent- and tetra-miniGNC antibodies steadily displayed high potency in the range of EC50 in pM. In this regard, SI-75X1 was exceptionally potent. The data demonstrate the important roles of moiety 1 binding domains, in particular, the aPD-Ll domain as an immune checkpoint inhibitor when targeting BXPC3 cancer cells.

Example 6: Configuration of CD3 binding domain in miniGNC antibodies

By definition, a miniGNC antibody is capable of interfacing with at least one immune effector cell and one target cancer cell through the binding of its moiety 1 and 2 binding domains, respectively. Using the general scheme of miniGNC antibody configuration (Figure 1), it is possible that assigning two moiety 2 binding domains to D1 and D2 may lead to different efficacy from assigning them to D1 and D4 or D2 and D5 because the relative position of the targets on the cell surface is different, either on the top and bottom or on both sides. In this context, a second group of penta-miniGNC antibodies were configured and listed in Table 2. This group of minGNC antibodies share the same binding specificities to CD3, CD19, EGFR, 4-1BB, and PD-L1, and the same binding domains at D2, D4, and D5. SI-68P13 (SEQ ID 69, 71) has aCD3 D3 domain, which is comparable to SI-75P3 of the first group except the difference in D2, i.e. aCD19 vs. aHER3, and to SI-68P17 (SEQ ID 73, 75) of the second group except the switch of aCD3 domain to Dl. The rest of the second group of miniGNC antibodies share the same binding specificities from Dl (aCD3) and D3 (aEGFR). SI-68P17 (with the binding specificity to CD19, 4-1BB and PD- Ll, respectively.

Anti-CD3 antibody plays a central role in T cell activation based immune therapy. Humanized antibody is desirable for the development of a therapeutic antibody. CD3 domain was incorporated into positions Dl and D3 to generate SI-68P17 (SEQ ID 73,75) and SI-68P13 (SEQ ID 69, 71) for testing positional effect. The anti-CD3 sequences in humanized framework 1 (CD3**), 2 (CD3***) 3 (CD****) and 4 (CD*****) were cloned into an expression cassette for producing SI-68P15 (SEQ ID NO. 77, 79), SI-68P18 (SEQ ID 81, 83), SI-68P19 (SEQ ID 85, 87), and SI-68P16 (SEQ ID 89, 91) (Table 2). Each expression cassette was transfected into 25 mL of ExpiCHO and expressed for 8 days followed by protein-A affinity chromatography for harvesting and purifying each penta-miniGNC antibody. The antibodies were produced with good titer (Table 2). Octet was used to verify that the penta-miniGNC antibodies containing different CD3 domains, can bind to human CD3, respectively (Table 2). Each penta-miniGNC antibody was loaded via AHC sensors at 10 mg/ml and bound to a serial dilution (highest 200 nM, 1:2.5 dilutions) or a single 100-nM concentration of His-tagged human CD3. The resulting global fit to a 1:1 binding model demonstrated that the penta-GNC antibodies bind to CD3 with affinities in the low nanomolar range (Table 2).

To evaluate the effect of CD3-mediated T cell directed cytotoxicity on cancer cells, BXPC3 tumor cell line was used as target. Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of antibodies were added to a white 384-well plate containing luciferized target cells and activated T cells (plated immediately before drug; effector:target = 5:1) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferized tumor cells was determined using a CLARIOstar plate reader. Data were fit to a sigmoidal function to calculate EC50 values, which were in the range of 0.1 to 5.5 pM as shown by Figure 4 and listed in Table 2. The data demonstrate that there are certain degrees of flexibility in anti-CD3 positioning, configuration, and binding affinity.

Example 7. multi-specific miniGNC antibodies having VHH as binding domains

To multi-specific antibody platforms, such as GNC and miniGNC antibodies that contain multiple scFv domains, a potential issue is related to chain mispairing due to multiple VH and VL domains in the same molecule. Different VH and VL domains have different propensities to interact with each other. If binding domains of different specificity have superior interaction energy, then the wrong pairings may form when the protein assembles. As a result, none of the binding domains will bind their targeted antigens.

VHH refers to "antibodies" with single Ig domains, i.e. "heavy chain only", also known as a single-domain antibody (sdAb) or a nanobody. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids, also known as VHH fragment (VHH). VHH domains have been increasingly incorporated into antibody-based therapeutics due to several favorable properties. VHH domains may have increased stability and solubility compared to more traditionally used scFv domains. Whereas the VH/VL interface of traditional antibody variable regions is hydrophobic, the transient exposure of these surfaces can result in significant aggregation or precipitation. On the other hand, VHH domains have a naturally more hydrophilic surface and therefore can have superior solubility and stability properties. An obvious benefit of VHH domains over Fab or scFv domains is their small size. Where Fab domains are about 50 kDa and scFv domains are about 25 kDa, VHH domains are a very compact 12-15 kDa. The penta- miniGNC antibodies contain five binding domains (Figure 1). Incorporation of VHH domains instead of scFv domains may significantly decrease molecular size, and increase tumor penetration.

A third group of multi-specific miniGNC antibodies were created and configured to have VHH binding domains against EGFR and HER3 (Gottlin et al., 2009; Eliseev et al., 2018) (Table 3). Penta, Tri, and bi specific single domain variable regions (VHH) -incorporated miniGNC molecules were constructed and purified with reasonable titer. Binding activity of each domain of multi- specific miniGNCs : SI-75P5 (SEQ ID 93, 95), SI-75P8 (SEQ ID 97, 99), SI-75X4 (SEQ ID 101,103), Sl- 75X17 (SEQ ID 105, 107), SI-75X19 (SEQ ID 109, 111),, SI-75X9 (SEQ ID 113-115), SI-75X11 (SEQ ID 117,119), SI-75X15 (SEQ ID 121,123)and SI-75X13 (SEQ ID 125, 127) were verified using Octet. AHC sensors were used to capture the GNC antibody at a concentration of 10 ug/ml for 180 seconds. After a 60-second baseline step, a single concentration of antigen was used for a 180- second association step. Antigens tested include 200 nM human EGFR (purified in-house), 100 nM human CD3 (Aero CDD-H52W1), 20 nM human PD-L1 (Aero PD1-H5229), 400 nM human 4- 1BB (purified in-house), 200 nM human HER3 (Aero ER3-H5223). After association, a 420-second dissociation step was used. The tabulated KD values were calculated using a one-to-one binding model in ForteBio Data Analysis software version 11. The data demonstrated that all domains retained high affinity in the VHH-incorporated miniGNC platform.

T cell directed cytotoxicity on cancer cells, BXPC3 tumor Cell line was used as target. Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of antibodies were added to a white 384-well plate containing luciferized target cells and activated T cells (plated immediately before drug; effector: target = 5:1) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferized tumor cells was determined using a CLARIOstar plate reader. EC50 values are tabulated in Table 3.

Example 8. Engineered Fab (D3) domain for improved expression and protein quality.

A disulphide staple was optionally introduced in the VH-VL interface of the fab region (Figure 5). The purpose of this engineering was improving heterodimer pairing and stabilize the overall structure by the formation of a covalent disulphide bond (Weatherill et al., 2012) at the core-fab interface. Cysteines pair was optionally introduced to form disulphide bond at VLAIOO on Chain B and its corresponding V_HA44 on Chain A fab region.

A pair of penta-miniGNC antibodies (SI-38P11 and SI-76PM1) (SEQ ID NO. 129 and 131; 133 and 135) with identical binding specificities were created for analysing the effect of stapled variable region in the Fab. The Chain A of the two antibodies comprises aCD20 scFv at Dl, aCD3 VH at D3 (VH Fab-CHl-Fc), and a4-1BB at D4, whereas Chain B comprises aCD19 at D2, aCD3V_L at D3 (VKappa Fab CL-Fc) and aPD-Ll scFv at D5 (Table 4) according to the naming system in Figure 1. SI-76PM1 was constructed with an additional disulphide in the D3 (VH/VL Fab-CHl/CL- Fc) and SI-38P11 was constructed without any additional disulphide pairing in the VH/VL fab.

Penta-miniGNC antibody constructs with and without the disulphide staple at position D3 were expressed in ExpiCHO system. Construct with stapled D3 (SI-76PM1) was produced with significantly higher titer than SI-38P11. Both proteins were purified with 5mL MabSelect protein A columns followed by Size Exclusion using a hiload 16/600 200 pg preparative SEC column on either an Akta Avant or Akta Pure Purifier system. SEC aggregates were analyzed using a waters HPLC linked to multi angle light scattering (MALS, Wyatt systems) to identify correct molecular weight by dn/dc calculated methods. With all of the analyses conducted as shown in Table 4, the disulfide bonded, i.e. "D3 stapled Fab", penta-miniGNC antibodies displayed 5% higher protein of interest production. Both molecules exhibited comparable antigen binding kinetics (Table 4).

Example 9. Multi-specific miniGNC antibodies with stapled scFv binding domains

The core structure of multi-specific miniGNC molecules were engineered to acquire several features in order to stabilize the heterodimer, including the covalently linked hinge and non-covalent and preferential interaction of "knobs-into-holes" by the CH3 domains in Fc region. While the stability and compactness are desirable, it remains unclear whether the closer proximity of D1 and D2 or D3 and D4 may affect their stability and independent function. To maintain the stability and independence of each added binding domains, one option is to introduce a disulfide bond at VLIOO and VH44 in each scFv domain, i.e. to staple each scFv domain. A disulfide bond between VL and VH may be used for all scFv domains to stabilize the overall structure. Alternatively, a disulphide bond may be introduced into at least one selected scFv domain at any position.

Four miniGNC antibodies have their D1 and D2 targeting EGFR and HER3 were grouped for measuring comparative potency of TDCC to the pancreatic cancer cells (HPAF-II), including tri- miniGNC (SI-68X2, SEQ ID NO. 137,139), tetra-miniGNC (SI-68E1, SEQ ID NO, 141, 143, and Sl- 68E2, SEQ ID NO. 145, 147), and penta-miniGNC (SI-68P1, SEQ ID NO. 149, 151) molecules. The moiety 1 binding specificities to 4-1BB and PD-L1 were the variable, whereas CD3 was the constant as D3 in the group interms of multispecificity. The expression construct encoding each of these four miniGNC antibodies was modified such that all scFv domains carrying a disulfide bond. The constructs were expressed individually in ExpiCHO system and each antibody was purified with reasonable titer. The binding kinetics to respective antigens were verified using Octet. AHC sensors were used to capture the miniGNC antibody at a concentration of 10 ug/ml for 180 seconds. After a 60-second baseline step, a single concentration of antigen was used for a 180-second association step. Antigens tested included 200 nM human EGFR (purified in- house), 100 nM human CD3 (Aero CDD-H52W1), 20 nM human PD-L1 (Aero PD1-H5229), 400 nM human 4-1BB (purified in-house), 200 nM human HER3 (Aero ER3-H5223). After association, a 420-second dissociation step was used. The tabulated KD values were calculated using a one-to- one binding model in ForteBio Data Analysis software version 11. Analysed KD values for each miniGNC molecule with all stapled scFv domains, as shown in Table 5, were compared with KDs of monoclonal antibody controls for respective antigens as shown in Table 6. The binding kinetics data shows comparable binding affinity of each stapled scFv domain with its respective mAb or Fc-scFv control SI-1C3 (SEQ ID 181, 183) SI-1C7 (SEQ ID 185), SI-9C21 (SEQ ID 187, 189), SI-35SF11 (SEQ ID 191), SI-3SF11 (SEQ ID 193), SI-20C14 (SEQ ID 287 and 289) (Table6), suggesting that stapling one or more scFv domains seems to have little effect to the binding affinity.

To evaluate the effect of stapled scFv domains to the potency of the miniGNC antibody, T cell directed cytotoxicity (TDCC) assay was used and the target cells were HPAF -II, human a human pancreatic cancer cell line (ATCC, Manassas, VA). The surface expression of the target cells was validated by flow cytometry. Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of antibodies were added to a white 384-well plate containing target cells and activated T cells (plated immediately before drug; effector: target = 5:1) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferised tumor cells was determined using a CLARIOstar plate reader. Data were fit to a sigmoidal function to calculate EC50 values. As shown in Figure 6 and Table 5, the survival curves revealed that the potency of all four miniGNC antibodies was be within the dose range of pM and with a tendency of the penta-miniGNC antibody exerting higher TDCC potency than that of tetra-miniGNC and tri-miniGNC antibodies. This observation again shows little effect by using stapled scFv domains.

Example 10. miniGNC having NKG2D receptor dimer as a binding domain

An increased number of binding specificities allows the GNC antibodies to bind not only T cells but also subsets of T cells, natural killer cells, and other types of immune cells, collectively known as moiety 1 binding domains/specificities (see Applicant's applications, WO/2019/005641 and W02019191120, incorporated herein in its entirety). Some of moiety 1 binding specificities may replace the cellular response to or recognition of targeted cells. For example, NKG2D is a major recognition receptor for the detection and elimination of transformed and infected cells, as its ligands are induced during cellular stress, either as a result of viral infection or genomic stress, such as in cancer. In humans, NKG2D is expressed by NK cells, cells, and CD8+ T cells. The addition of NKG2D as a binding domain/specificity to the class of miniGNC antibodies may improve the cytotoxicity and efficacy of the antibody as a single multi-functional therapeutic agent.

In NK cells, NKG2D serves as an activating receptor, which itself can trigger cytotoxicity, whereas on CD8⁺ T cells the function of NKG2D is to send co-stimulatory signals to activate them. NKG2D forms a homodimer whose ectodomains serve for ligand binding. This feature qualifies NKG2D as a non-variable-sequence-based binding domain in a miniGNC format and other binding domains can be added to create a class of multi-specific NKG2D- miniGNC protein. In one miniGNC format, individual NKG2D monomer was incorporated in the D3 position on chain A and chain B which formed a dimeric NKG2D receptor on Fc dimerization. Thus, NKG2D can act as a receptor for the multi-specific miniGNC molecule to bind its ligand. In other miniGNC format, a NKG2D tandem repeat was designed by adding a (GxSy)n spacer/linker between individual NKG2D monomers which homodimerizes and forms a functional dimeric receptor. This NKG2D tandem dimeric structure can be positioned in Dl, D2, D4 or D5.

As listed in Table 7, this class includes mono-NKG2D-miniGNC (SI-49R26, SEQ ID NO. 153, 155), bi-miniGNC (SI-49R27, SEQ ID NO. 157, 159), tri-miniGNC (SI-49R25, SEQ ID NO. 161, 163), tetra-miniGNC (SI-49P_X, SEQ ID NO. 165, 167), and penta-mini-GNC molecules (SI49P8, SI-49P9, SEQ ID. NO. 169, 171, 177, and 179). A control penta-miniGNC, SI-49PM1 (SEQ ID NO. 173, 175), was created by switching the positions of Dl (aCD3) and D3 of SI-49P8, and the Octet binding affinity for either NKG2D or CD3 was not affected by this switch. While the binding affinity of other moiety 1 binding domains remained stable in this class of NKG2D-miniGNC molecules, the binding affinity of NKG2D was within 2-fold. Thus, the penta-miniGNC molecule possesses the binding function of NKG2D receptor for binding to NKG2D ligand. To assess and compare the potency of TDCC of NKG2D-miniGNC molecules, tri-, tetra-, and penta-miniGNC molecules, SI-49R25, SI-49P_X, and SI-49P8 and SI-49PM1 were used to target MDA-MB-231 breast cancer cell line. Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of the miniGNC protein were added to a white 384-well plate containing luciferised MDA-MB-231 cells 24 hours prior and grown at 37 °C. Activated T cells were plated immediately before miniGNC molecules (effector: target = 15:1) in a total volume of 50 ul. After incubated for additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and the luminescence corresponding to viability of luciferised tumor cells was determined using a CLARIOstar plate reader. Data were fit to a sigmoidal function to calculate EC50 values (Figure 7). Based on the dose-viability curves, there were two groups, the tetra- and penta-miniGNC molecules were more potent than the tri-miniGNC molecule, SI-49R25. As listed in Table 7, the difference might result from the addition of aPD-Ll. However, the EC50 value (59.3nM) of SI-49R25 should be considered as relatively potent. To the breast cancer cell line MDA-MB-231, CD19, which is a pan-B cell marker, is a non-participating tumor antigen. In this regard, SI-49R25 may be viewed as having two binding specificities to CD3 and NKG2D ligand. Thus, the data indicates that the incorporation of NKG2D receptor to different miniGNC antibody format contributes to the potency of TDCC. In a broader sense, the miniGNC antibody molecules can accommodate multiple binding specificities to modulate, cooperate, and direct an optimized immune response to targeted cells, such as cancer.

TABLES

Table 1. The configuration, production, binding affinity, and potency of multi-specific minGNC molecules when targeting EGFR and/or HER3-expressing pancreatic cancer cells (BXPC3).

Table 2. The effect of the position and binding affinity of the CD3 binding domain on the potency of penta-minGNC molecules when targeting EGFR-expressing pancreatic cancer cells (BXPC3).

Table 3. The effect of VHH single binding domain(s) on the potency of multi-specific minGNC molecules when targeting BXPC3 tumor cells

Table 4. The effect of "stapled" CD3 binding domain at D3 position on the production of multi specific miniGNC molecules.

Table 5. Comparative potency of multi-specific miniGNC molecules with "stapled" scFv at all positions when targeting tumor cells.

Table 6. Characterization of the unstapled versions of each domain (D1-D5) in mAb orscFvformat to respective antigens.

Table 7. The characterization of multi-specific minGNC molecules with NKG2D receptor dimer incorporated in miniGNC format and potency of tri, tetra and penta molecules in killing MDA- MB-231 tumor cells

Table 8. The characterization of bispecific miniGNC molecules with different IgG subtypes in the Fc domain.

Table 9. Annotation of binding domains for their binding specificity, domain structure, origin, and sequence identification number (SEQ ID).

SEQUENCE LISTING

Claims

CLAIMS What is claimed is:

1. A multi-specific antibody-like protein having a N-terminus and a C-terminus, comprising, a first monomer, comprising, from the N-terminus to the C-terminus, a first binding monomer, a CHI domain, a first hinge, a first CH2 domain, and a first CH3 domain, wherein the first monomer comprises optionally a first binding domain (Dl) linked to the N-terminus, a fourth binding domain (D4) linked to the C-terminus, or both, a second monomer, comprising, from the N-terminus to the C-terminus, a second binding monomer, a CL domain, a second hinge, a second CH2 domain, and a second CH3 domain, wherein the second monomer comprises optionally a second binding domain (D2) linked to the N-terminus, a fifth binding domain (D5) linked to the C-terminus, or both, wherein the first binding monomer and a second binding monomer are configured to form a dimer, wherein the first monomer and the second monomer are covalently paired through at least one disulfide bond between the CHI domain and the CL domain and at least one disulfide bond between the first hinge and the second hinge, and wherein the multi-specific antibody-like protein is at least bi-specific.

2. The multi-specific antibody-like protein of Claim 1, wherein the first binding monomer comprises a VH domain, a second binding monomer comprises a VL domain, and wherein the VH, CHI, VL, CL domains form a Fab region as a third binding domain (D3).

3. The multi-specific antibody-like protein of Claim 1, wherein the first binding monomer and the second binding monomer form a NKG2D receptor as a third binding domain (D3).

4. The multi-specific antibody-like protein of Claim 1, wherein the first CH3 domain is configured to form a hole structure, wherein the second CH3 domain is configured to form a knob structure, and wherein the first CH2 and CH3 domains and the second CH2 and CH3 domains are configured to heterodimerize to form a complementary Fc domain.

5. The multi-specific antibody-like protein of Claim 1, wherein the Dl, D2, D4, and D5 is independently a scFv domain, a VHH domain, a receptor, or a ligand.

6. The multi-specific antibody-like protein of Claim 1, wherein the Dl, D2, D3, D4, and D5 each independently has a binding specificity against an antigen selected, EGFR, HER2, HER3, EGFRvlll, ROR1, CD3, CD28, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D, NKG2D ligand, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, PD-L1, PD1, 0X40, 4-1BB, GITR, TIG IT, TIM-3, LAG-3, CTLA4, CD40, CD40L, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73, CD39, CLDN18.2, DLL3, HLA-G, FcRH5, GPRC5D, LIV-1, MUC1, CD138, CD70, CD16, uPAR, Siglec-15, CD47, CD38, , NKp46, PD-L2, CD160, LOX-1, SIRPa, CD27 and wherein the Fc domain comprises a human IgG Fc domain.

7. The multi-specific antibody-like protein of Claim 1, wherein D1 has a binding specificity to CD3, CD20, CEA, HER2, EGFR, or NKG2D ligand.

8. The multi-specific antibody-like protein of Claim 1, wherein D2 has a binding specificity to HER3, EGFR, CD3, or CD19.

9. The multi-specific antibody-like protein of Claim 1, wherein D3 has a binding specificity to HER3, EGFR, CD3, or NKG2D ligands.

10. The multi-specific antibody-like protein of Claim 1, wherein D4 has a binding specificity to 4-1BB, or EGFR.

11. The multi-specific antibody-like protein of Claim 1, wherein D5 has a binding specificity to PD-L1 or HER3.

12. The multi-specific antibody-like protein of Claim 1, wherein the antibody is a tri-specific antibody having the binding specificity to EGFR, HER3, and CD3.

13. The multi-specific antibody-like protein of Claim 1, wherein the antibody is a tetra-specific antibody having the binding specificity to EGFR, HER3, CD3, and 4-1BB.

14. The multi-specific antibody-like protein of Claim 1, wherein the antibody is a tetra-specific antibody having the binding specificity to EGFR, HER3, CD3, and PD-L1.

15. The multi-specific antibody-like protein of Claim 1, wherein the antibody is a penta- specific antibody having the binding specificity to EGFR, HER3, CD3, 4-1BB and PD-L1.

16. A complimentary determining region (CDR) having an affinity to CEA, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 301, 302, 303, 304, 305, or 306.

17. A protein having an affinity to CEA, comprising the CDR of Claim 16.

18. A multi-specific antibody-like protein having a binding affinity to CEA, wherein the antibody-like protein comprises an amino acid sequence having at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO. 279, 280, 281 or 282.

19. A complimentary determining region (CDR) having a binding affinity to CD3, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 307, 308, 309,

310, 311 or 312.

20. A protein having an affinity to CD3, comprising CDR of Claim 19.

21. A multi-specific antibody-like protein having a binding affinity to CD3, wherein the antibody-like protein comprises an amino acid sequence having at least 98% sequence identity to an amino acid sequence selected from SEQ ID NO. 227-230, 231-234, 235-238, 239-242 and 291-294.

22. An isolated nucleic acid sequence encoding the multi-specific antibody-like protein of Claim 1.

23. An expression vector comprising the isolated nucleic acid sequences of Claim 22.

24. A host cell comprising the isolated nucleic acid sequence of Claim 22.

25. An immunoconjugate comprising the multi-specific antibody-like protein of Claim 1 and a cytotoxic agent.

26. A pharmaceutical composition, comprising the multi-specific antibody-like protein of Claim 1 and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition, comprising the immunoconjugate of Claim 25 and a pharmaceutically acceptable carrier.

28. A method for treating or preventing a cancer, an autoimmune disease, or an infectious disease in a subject, said method comprising administering to the subject a pharmaceutical composition comprising a purified multi-specific antibody-like protein of Claim 1.

29. A method for producing the multi-specific antibody-like protein of Claim 1, comprising culturing a host cell such that the DNA sequence encoding the multi-specific antibody-like protein of Claim 1 is expressed, and purifying said multi-specific antibody-like protein.

30. A solution comprising an effective concentration of the multi-specific antibody-like protein of Claim 1, wherein the solution is blood plasma in a subject.