COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc-ANTIGEN BINDING DOMAIN CONSTRUCTS
20220267460 · 2022-08-25
Inventors
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
C07K2317/72
CHEMISTRY; METALLURGY
C07K2317/60
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/64
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain.
Claims
1. A polypeptide comprising an antigen binding domain; a linker; a first IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein at least one Fc domain monomer comprises mutations forming an engineered protuberance, and wherein at least one other Fc domain monomer comprises at least one, two or three reverse charge mutations.
2.-54. (canceled)
55. A polypeptide complex comprising a polypeptide of claim 1 joined to a second polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein the polypeptide and the second polypeptide are joined by disulfide bonds between cysteine residues within the hinge domain of the first, second or third IgG1 Fc domain monomer of the polypeptide and the hinge domain of the second polypeptide.
56.-78. (canceled)
79. The polypeptide complex of claim 55, wherein the polypeptide complex is further joined to a third polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein the polypeptide and the third polypeptide are joined by disulfide bonds between cysteine residues within the hinge domain of the first, second or third IgG1 Fc domain monomer of the polypeptide and the hinge domain of the third polypeptide, wherein the second and third polypeptides join to different IgG1 Fc domain monomers of the polypeptide.
80.-82. (canceled)
83. The polypeptide complex of claim 55, wherein the second polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions.
84. The polypeptide complex of claim 79, wherein the third polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions.
85.-93. (canceled)
94. An Fc-antigen binding domain construct comprising: a) a first polypeptide comprising i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a linker joining the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) an antigen binding domain joined to the first polypeptide and to the second polypeptide; wherein the first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain and the second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain.
95.-99. (canceled)
100. The Fc-antigen binding domain construct of claim 94, wherein each of the Fc domain monomers independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43, 45, and 47 having up to 10 single amino acid substitutions.
101.-116. (canceled)
117. An Fc-antigen binding domain construct comprising: a) a first polypeptide comprising i) a first Fc domain monomer, ii) a second Fc domain monomer, iii) a third Fc domain monomer, iii) a linker joining the first Fc domain monomer and the second Fc domain monomer; and iv) a linker joining the second Fc domain monomer to the third Fc domain monomer; b) a second polypeptide comprising a fourth Fc domain monomer; c) a third polypeptide comprising a fifth Fc domain monomer; and d) an antigen binding domain joined to the first polypeptide and to the second polypeptide; wherein the first Fc domain monomer and the fourth Fc domain monomer combine to form a first Fc domain; wherein the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain; and wherein the third Fc domain monomer and the fifth Fc domain monomer combine to form a third Fc domain.
118.-121. (canceled)
122. The Fc-antigen binding domain construct of claim 117, wherein each of the Fc domain monomers independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43, 45, and 47 having up to 10 single amino acid substitutions.
123.-142. (canceled)
143. An Fc-antigen binding domain construct comprising: a) a first polypeptide comprising: i) a first Fc domain monomer, ii) a second Fc domain monomer iii) a first heavy chain binding domain, and iv) a linker joining the first and second Fc domain monomers; b) a second polypeptide comprising: i) a third Fc domain monomer, iii) a second heavy chain binding domain and iv) a linker joining the third and fourth Fc domain monomers; c) a third polypeptide comprising a first light chain binding domain; d) a fourth polypeptide comprising a second light chain binding domain; e) a fifth polypeptide comprising a fourth Fc domain monomer; and wherein the first and fourth Fc domain monomers together form a first Fc domain, the second and third Fc domain monomers together form a second Fc domain, the first heavy chain binding domain and first light chain binding domain together form a first Fab; and the second heavy chain binding domain and second light chain binding domain together form a second Fab.
144.-145. (canceled)
146. The Fc-antigen binding domain construct of claim 143, wherein each of the Fc domain monomers independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions.
147.-150. (canceled)
151. An Fc-antigen binding domain construct comprising: a) a first polypeptide comprising: i) a first Fc domain monomer, ii) a second Fc domain monomer, iii) a third Fc domain monomer, iv) a first heavy chain binding domain, and iv) a linker joining the first and second Fc domain monomers; v) a linker joining the second and third Fc domain monomers; b) a second polypeptide comprising: i) a sixth Fc domain monomer, iii) a second heavy chain binding domain; c) a third polypeptide comprising a fourth Fc domain monomer; d) a fourth polypeptide comprising a fifth Fc domain monomer; e) a fifth polypeptide comprising a first light chain binding domain; and f) a sixth polypeptide comprising a second light chain binding domain wherein the first and fourth Fc domain monomers together form a first Fc domain, the second and fifth Fc domain monomers together form a second Fc domain, the third and sixth Fc domain monomers together form a third Fc domain, the first heavy chain binding domain and first light chain binding domain together form a first Fab; and the second heavy chain binding domain and second light chain binding domain together form a second Fab.
152.-153. (canceled)
154. The Fc-antigen binding domain construct of claim 151, wherein each of the Fc domain monomers independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions.
155.-158. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0110] Many therapeutic antibodies function by recruiting elements of the innate immune system through the effector function of the Fc domains, such as antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In some instances, the present disclosure contemplates combining an antigen binding domain of a known single Fc-domain containing therapeutic, e.g., a known therapeutic antibody, with at least two Fc domains to generate a novel therapeutic with unique biological activity. In some instances, a novel therapeutic disclosed herein has a biological activity greater than that of the known Fc-domain containing therapeutic, e.g., a known therapeutic antibody. The presence of at least two Fc domains can enhance effector functions and to activate multiple effector functions, such as ADCC in combination with ADCP and/or CDC, thereby increasing the efficacy of the therapeutic molecules. The methods and compositions described herein allow for the construction of antigen-binding proteins with multiple Fc domains by introducing multiple orthogonal heterodimerization technologies (e.g., two different sets of mutations selected from Tables 3 and 4) into the polypeptides that join together to form the same protein. The design principles described herein, which introduce multiple heterodimerizing mutations into the polypeptides that assemble into the same protein, allow for the creation of a great diversity of protein configurations, including, e.g., antibody-like proteins with tandem Fc domains, symmetrically branched proteins, and asymmetrically branched proteins.
[0111] The orthogonal Fc-antigen binding domain constructs described herein contain at least one antigen-binding domain and at least two Fc domains that are joined together by a linker, wherein at least two of the Fc domains differ from each other, e.g., at least one Fc domain of the construct is joined to an antigen-binding domain and at least one Fc domain of the construct is not joined to an antigen-binding domain, or two Fc domains of the construct are joined to different antigen-binding domains. The orthogonal Fc-antigen binding domain constructs are manufactured by expressing one long peptide chain containing two or more Fc monomers separated by linkers and expressing two or more different short peptide chains that each contain a single Fc monomer that is designed to bind preferentially to one or more particular Fc monomers on the long peptide chain. Any number of Fc domains can be connected in tandem in this fashion, allowing the creation of constructs with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains.
[0112] The orthogonal Fc-antigen binding domain constructs are created using the Fc engineering methods for assembling molecules with two or more Fc domains described in PCT/US2018/012689 and in International Publication Nos. WO/2015/168643, W02017/151971, WO 2017/205436, and WO 2017/205434, which are herein incorporated by reference in their entirety. The engineering methods make use of two sets of heterodimerizing selectivity modules to accurately assemble orthogonal Fc-antigen binding domain constructs (constructs 43 and 44;
[0113] In some embodiments, assembly of an orthogonal Fc-antigen binding domain construct described herein can be accomplished using different electrostatic steering mutations between the two sets of heterodimerizing mutations as described herein. One example of orthogonal electrostatic steering mutations is E357K in a first knob of an Fc monomer and K370D in a first hole of an Fc monomer, wherein these Fc monomers associate to form a first Fc domain, and D399K in a second knob of an Fc monomer and K409D in a second hole of an Fc monomer, wherein these Fc monomers associate to form a second Fc domain.
[0114] In some embodiments, the Fc-antigen binding domain construct has at least two antigen-binding domains (e.g., two, three, four, five, or six antigen-binding domains) with different binding characteristics, such as different binding affinities (for the same or different targets) or specificities for different target molecules. Bispecific constructs may be generated from the above Fc scaffolds in which two or more of the polypeptides of the Fc-antigen binding domain construct include different antigen-binding domains, e.g., a long chain includes one antigen-binding domain of a first specificity and a short chain includes a different antigen-binding domain of a second specificity. The different antigen binding domains may use different light chains, or a common light chain, or may consist of scFv domains.
[0115] Bi-specific and tri-specific constructs may be generated by the use of two different sets of heterodimerizing mutations, i.e., orthogonal heterodimerizing mutations. Such heterodimerizing sequences need to be designed in such a way that they disfavor association with the other heterodimerizing sequences. Such designs can be accomplished using different electrostatic steering mutations between the two sets of heterodimerizing mutations, and/or different protuberance-into-cavity mutations between the two sets of heterodimerizing mutations, as described herein. One example of orthogonal electrostatic steering mutations is E357K in the first knob Fc, K370D in first hole Fc, D399K in the second knob Fc, and K409D in the second hole Fc.
I. Fc Domain Monomers
[0116] An Fc domain monomer includes at least a portion of a hinge domain, a C.sub.H2 antibody constant domain, and a C.sub.H3 antibody constant domain (e.g., a human IgG1 hinge, a CH2 antibody constant domain, and a C.sub.H3 antibody constant domain with optional amino acid substituions). The Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer may also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomers may also be hybrids, e.g., with the hinge and C.sub.H2 from IgG1 and the CH3 from IgA, or with the hinge and C.sub.H2 from IgG1 but the C.sub.H3 from IgG3. A dimer of Fc domain monomers is an Fc domain (further defined herein) that can bind to an Fc receptor, e.g., FcγRIIIa, which is a receptor located on the surface of leukocytes. In the present disclosure, the CH3 antibody constant domain of an Fc domain monomer may contain amino acid substitutions at the interface of the C.sub.H3-C.sub.H3 antibody constant domains to promote their association with each other. In other embodiments, an Fc domain monomer includes an additional moiety, e.g., an albumin-binding peptide or a purification peptide, attached to the N- or C-terminus. In the present disclosure, an Fc domain monomer does not contain any type of antibody variable region, e.g., VH, VL, a complementarity determining region (CDR), or a hypervariable region (HVR).
[0117] In some embodiments, an Fc domain monomer in an Fc-antigen binding domain construct described herein (e.g., an Fc-antigen binding domain construct having three Fc domains) may have a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequence of SEQ ID NO:42. In some embodiments, an Fc domain monomer in an Fc-antigen binding domain construct described herein (e.g., an Fc-antigen binding domain construct having three Fc domains) may have a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 43, 44, 46, 47, 48, and 50-53. In certain embodiments, an Fc domain monomer in the Fc-antigen binding domain construct may have a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 42-53.
TABLE-US-00002 SEQ ID NO: 42 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 44 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 46 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 48 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVD GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 50 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 51 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 52 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 53 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK
II. Fc Domains
[0118] As defined herein, an Fc domain includes two Fc domain monomers that are dimerized by the interaction between the C.sub.H3 antibody constant domains. An Fc domain forms the minimum structure that binds to an Fc receptor, e.g., Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcϵ receptors (FcϵR)), and/or the neonatal Fc receptor (FcRn). In some embodiments, an Fc domain of the present disclosure binds to an Fcγ receptor (e.g., FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and/or FcγRIV and/or the neonatal Fc receptor (FcRn).
III. Antigen Binding Domains
[0119] An antigen binding domain may be any protein or polypeptide that binds to a specific target molecule or set of target molecules. Antigen binding domains include one or more peptides or polypeptides that specifically bind a target molecule. Antigen binding domains may include the antigen binding domain of an antibody. In some embodiments, the antigen binding domain may be a fragment of an antibody or an antibody-construct, e.g., the minimal portion of the antibody that binds to the target antigen. An antigen binding domain may also be a synthetically engineered peptide that binds a target specifically such as a fibronectin-based binding protein (e.g., a FN3 monobody). In some embodiments, an antigen binding domain cab be a ligand or receptor. A fragment antigen-binding (Fab) fragment is a region on an antibody that binds to a target antigen. It is composed of one constant and one variable domain of each of the heavy and the light chain. A Fab fragment includes a V.sub.H, V.sub.L, C.sub.H1 and CL domains.
[0120] The variable domains VH and VL each contain a set of 3 complementarity-determining regions (CDRs) at the amino terminal end of the monomer. The Fab fragment can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fab fragment monomer may also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). In some embodiments, a Fab fragment may be covalently attached to a second identical Fab fragment following protease treatment (e.g., pepsin) of an immunoglobulin, forming an F(ab′).sub.2 fragment. In some embodiments, the Fab may be expressed as a single polypeptide, which includes both the variable and constant domains fused, e.g. with a linker between the domains.
[0121] In some embodiments, only a portion of a Fab fragment may be used as an antigen binding domain. In some embodiments, only the light chain component (V.sub.L+C.sub.L) of a Fab may be used, or only the heavy chain component (V.sub.H+C.sub.H) of a Fab may be used. In some embodiments, a single-chain variable fragment (scFv), which is a fusion protein of the the V.sub.H and V.sub.L chains of the Fab variable region, may be used. In other embodiments, a linear antibody, which includes a pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1), which, together with complementary light chain polypeptides form a pair of antigen binding regions, may be used.
[0122] Antigen binding domains may be placed in various numbers and at various locations within the Fc-containing polypeptides described herein. In some embodiments, one or more antigen binding domains may be placed at the N-terminus, C-terminus, and/or in between the Fc domains of an Fc-containing polypeptide. In some embodiments, a polypeptide or peptide linker can be placed between an antigen binding domain, e.g., a Fab domain, and an Fc domain of an Fc-containing polypeptide. In some embodiments, multiple antigen binding domains (e.g., 2, 3, 4, or 5 or more antigen binding domains) joined in a series can be placed at any position along a polypeptide chain (Wu et al., Nat. Biotechnology, 25:1290-1297, 2007).
[0123] In some embodiments, two or more antigen binding domains can be placed at various distances relative to each other on an Fc-domain containing polypeptide or on a protein complex made of numerous Fc-domain containing polypeptides. In some embodiments, two or more antigen binding domains are placed near each other, e.g., on the same Fc domain, as in a monoclonal antibody). In some embodiments, two or more antigen binding domains are placed farther apart relative to each other, e.g., the antigen binding domains are separated from each other by 1, 2, 3, 4, or 5, or more Fc domains on the protein structure.
[0124] In some embodiments, an antigen binding domain of the present disclosure includes for a target or antigen listed in Table 1A and 1B, one, two, three, four, five, or all six of the CDR sequences listed in
[0125] Table 1A and 1B for the listed target or antigen, as provided in further detail below Table 1A and 1B.
TABLE-US-00003 TABLE 1A CDR1-IMGT CDR2-IMGT CDR3-IMGT CDR1-IMGT CDR2-IMGT CDR3-IMGT Target Antibody Name (heavy) (heavy) (heavy) (light) (light) (light) B7-H3 Enoblitzumab GFTFSSFG ISSDSSAI GRGRENIYY QNVDTN SAS QQYNNYPF (SEQ ID NO: (SEQ ID NO: GSRLDY (SEQ ID NO: T 76) 106) (SEQ ID NO: 171) (SEQ ID NO: 137) 201) beta-amyloid Gantenerumab GFTFSSYA INASGTRT ARGKGNTH QSVSSSY GAS LQIYNMPIT (SEQ ID NO: (SEQ ID NO: KPYGYVRYF (SEQ ID NO: (SEQ ID NO: 77) 107) DV 172) 202) (SEQ ID NO: 138) CCR4 Mogamulizumab GFIFSNYG ISSASTYS GRHSDGNF RNIVHINGD KVS FQGSLLPW (SEQ ID NO: (SEQ ID NO: AFGY TY T 78) 108) (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 139) 173) 203) CD19 Inebilizumab GFTFSSSW IYPGDGDT ARSGFITTV ESVDTFGIS EAS QQSKEVPFT (SEQ ID NO: (SEQ ID NO: RDFDY F (SEQ ID NO: 79) 109) (SEQ ID NO: (SEQ ID NO: 204) 140) 174) CD20 Obinutuzumab GYAFSYSW IFPGDGDT ARNVFDGY KSLLHSNGI QMS AQNLELPYT (SEQ ID NO: (SEQ ID NO: WLVY TY (SEQ ID NO: 80) 110) (SEQ ID NO: (SEQ ID NO: 205) 141) 175) CD20 Ocaratuzumab GRTFTSYN AIYPLTGDT ARSTYVGG SSVPY ATS QQWLSNPP MH (SEQ ID NO: DWQFDV (SEQ ID NO: T (SEQ ID NO: 111) (SEQ ID NO: 176) (SEQ ID NO: 81) 142) 206) CD20 Rituximab GYTFTSYN IYPGNGDT CARSTYYG SSVSY ATS QQWTSNPP (SEQ ID NO: (SEQ ID NO: GDWYFNV (SEQ ID NO: T 82) 112) (SEQ ID NO: 177) (SEQ ID NO: 143) 207) CD20 Ublituximab GYTFTSYN IYPGNGDT ARYDYNYA SSVSY ATS QQWTFNPP (SEQ ID NO: (SEQ ID NO: MDY (SEQ ID NO: T 82) 112) (SEQ ID NO: 177) (SEQ ID NO: 144) 208) CD20 Veltuzumab GYTFTSYN IYPGNGDT ARSTYYGG SSVSY ATS QQWTSNPP (SEQ ID NO: (SEQ ID NO: DWYFDV (SEQ ID NO: T 82) 112) (SEQ ID NO: 177) (SEQ ID NO: 145) 207) CD22 Epratuzumab GYTFTSYW INPRNDYT ARRDITTFY QSVLYSANH WAS HQYLSS (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: KNY (SEQ ID NO: 83) 113) 146) (SEQ ID NO: 209) 178) CD37 Otlertuzumab GYSFTGYN IDPYYGGT ARSVGPFD ENVYSY FAK QHHSDNPW (SEQ ID NO: (SEQ ID NO: S (SEQ ID NO: T 84) 114) (SEQ ID NO: 179) (SEQ ID NO: 147) 210) CD38 Daratumumab GFTFNSFA ISGSGGGT AKDKILWFG QSVSSY DAS QQRSNWPP (SEQ ID NO: (SEQ ID NO: EPVFDY (SEQ ID NO: T 85) 115) (SEQ ID NO: 180) (SEQ ID NO: 148) 211) CD38 Isatuximab GYTFTDYW IYPGDGDT ARGDYYGS QDVSTV SAS QQHYSPPY (SEQ ID NO: (SEQ ID NO: NSLDY (SEQ ID NO: T 86) 109) (SEQ ID NO: 181) (SEQ ID NO: 149) 212) CD3epsilon Foralumab GFKFSGYG IWYDGSKK ARQMGYWH QSVSSY DAS QQRSNWPP (SEQ ID NO: (SEQ ID NO: FDLW (SEQ ID NO: LT 87) 116) (SEQ ID NO: 180) (SEQ ID NO: 150) 213) CD52 Alemtuzumab GFTFTDFY IRDKAKGYT AREGHTAA QNIDKY NTN LQHISRPRT (SEQ ID NO: T PFDY (SEQ ID NO: (SEQ ID NO: 88) (SEQ ID NO: (SEQ ID NO: 182) 214) 117) 151) CD105 Carotuximab GFTFSDAW IRSKASNHA TRWRRFFD SSVSY ATS QQWSSNPL (SEQ ID NO: T S (SEQ ID NO: T 89) (SEQ ID NO: (SEQ ID NO: 177) (SEQ ID NO: 118) 152) 215) CD147 cHAb18 GFTFSDAW IRSANNHAP TRDSTATH QSVIND TAS QQDTSPP (SEQ ID NO: T (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 89) (SEQ ID NO: 153) 183) 216) 119) c-Met ABT-700 GYIFTAYT IKPNNGLA ARSEITTEF ESVDSYANS RAS QQSKEDPLT (SEQ ID NO: (SEQ ID NO: DY F (SEQ ID NO: 90) 120) (SEQ ID NO: (SEQ ID NO: 217) 154) 184) CTLA-4 Ipilimumab GFTFSSYT ISYDGNNK ARTGWLGP QSVGSSY GAF QQYGSSPW (SEQ ID NO: (SEQ ID NO: FDY (SEQ ID NO: T 91) 121) (SEQ ID NO: 185) (SEQ ID NO: 155) 218) EGFR2 Margetuximab GFNIKDTY IYPTNGYT SRWGGDGF QDVNTA SAS QQHYTTPPT (SEQ ID NO: (SEQ ID NO: YAMDY (SEQ ID NO: (SEQ ID NO: 92) 122) (SEQ ID NO: 186) 219) 156) EGFR3 Lumretuzumab GYTFRSSY IYAGTGSP ARHRDYYS QSVLNSGN WAS QSDYSYPYT (SEQ ID NO: (SEQ ID NO: NSLTY QKNY (SEQ ID NO: 93) 123) (SEQ ID NO: (SEQ ID NO: 220) 157) 187) EphA3 Ifabotuzumab GYTFTGYW IYPGSGNT ARGGYYED QGIISY AAS GQYANYPY (SEQ ID NO: (SEQ ID NO: FDS (SEQ ID NO: T 94) 124) (SEQ ID NO: 188) (SEQ ID NO: 158) 221) GD3 Ecromeximab GFAFSHYA ISSGGSGT TRVKLGTYY QDISNY YSS HQYSKLP (SEQ ID NO: (SEQ ID NO: FDS (SEQ ID NO: (SEQ ID NO: 95) 125) (SEQ ID NO: 189) 222) 159) GPC3 Codrituzumab GYTFTDYE LDPKTGDT TRFYSYTY QSLVHSNR KVS SQNTHVPPT (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: NTY (SEQ ID NO: 96) 126) 160) (SEQ ID NO: 223) 190) KIR2DL1/2/3 Lirilumab GGTFSFYA FIPIFGAA ARIPSGSYY QSVSSY DAS QQRSNWMY (SEQ ID NO: (SEQ ID NO: YDYDMDV (SEQ ID NO: T 97) 127) (SEQ ID NO: 180) (SEQ ID NO: 161) 224) MUC5AC Ensituximab GFSLSKFG IWGDGST VKPGGDY SSISY DTS HQRDSYPW (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: T 98) 128) 162) 191) (SEQ ID NO: 225) phosphatidyl- Bavituximab GYSFTGYN IDPYYGDT VKGGYYGH QDIGSS ATS LQYVSSPPT serine (SEQ ID NO: (SEQ ID NO: WYFDV (SEQ ID NO: (SEQ ID NO: 84) 129) (SEQ ID NO: 192) 226) 163) RHD Roledumab GFTFKNYA ISYDGRNI ARPVRSRW QDIRNY AAS QQYYNSPP (SEQ ID NO: (SEQ ID NO: LQLGLEDAF (SEQ ID NO: T 99) 130) HI 193) (SEQ ID NO: (SEQ ID NO: 227) 164) SLAMF7 Elotuzumab GFDFSRYW INPDSSTI ARPDGNYW QDVGIA WAS QQYSSYPY (SEQ ID NO: (SEQ ID NO: YFDV (SEQ ID NO: T 100) 131) (SEQ ID NO: 194) (SEQ ID NO: 165) 228) HER2 Trastuzumab GFNIKDTY IYPTNGYT SRWGGDGF QDVNTA SAS QQHYTTPPT (SEQ ID NO: (SEQ ID NO: YAMDY (SEQ ID NO: (SEQ ID NO: 92) 122) (SEQ ID NO: 186) 219) 156) OX40 Oxelumab GFTFNSYA ISGSGGFT AKDRLVAPG QGISSW AAS QQYNSYPY (SEQ ID NO: (SEQ ID NO: TFDY (SEQ ID NO: T 101) 132) (SEQ ID NO: 195) (SEQ ID NO: 166) 229) PD-L1 Avelumab GFTFSSYI IYPSGGIT ARIKLGTVT SSDVGGYN DVS SSYTSSSTR (SEQ ID NO: (SEQ ID NO: TVDY Y V 102) 133) (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 167) 196) 230) CD135 4G8-SDIEM SYWMH EIDPSDSYK AITTTPFDF RASQSISNN YSQSIS QQSNTWPY (SEQ ID NO: DYNQKFKD (SEQ ID NO: LH (SEQ ID T 103) (SEQ ID NO: 168) (SEQ ID NO: NO: 200) (SEQ ID NO: 134) 197) 231) HIV1 VRCO1LS GYTFLNCPI GWMKPRG ARYFFGSSP SQYGSLAW GGS QQYEFFGQ (SEQ ID NO: GAVN NWYFD (SEQ ID NO: GT 104) (SEQ ID NO: (SEQ ID NO: 198) (SEQ ID NO: 135) 169) 232) HER3 KTN3379 GFTFSYYYM IGSSGGVTN ARVGLGDA SLSNIGLN SRN AAWDDSPP Q (SEQ ID NO: FDIWQQ (SEQ ID NO: G (SEQ ID NO: 136) (SEQ ID NO: 199) (SEQ ID NO: 105) 170) 233) CD38 MOR 202 GFTFSSYYM GISGDPSNT DLPLVYTGF SGDNLRHY GDSKRPS QTYTGGAS N (SEQ ID YYADSVKG AY (SEQ ID YVY (SEQ ID (SEQ ID (SEQ ID NO: NO: 245) (SEQ ID NO: NO: 247) NO: 248) NO: 249) 250) 246)
TABLE-US-00004 TABLE 1B Variable Domain Sequences Antibody VH/CH1 VL Atezolizumab EVQLVESGGGLVQPGGSLRLSCAAS DIQMTQSPSSLSASVGDRVTITCRASQDV PD-L1 GFTFSDSWIHWVRQAPGKGLEWVA STAVAWYQQKPGKAPKLLIYSASFLYSGV WISPYGGSTYYADSVKGRFTISADTS PSRFSGSGSGTDFTLTISSLQPEDFATYYC KNTAYLQMNSLRAEDTAVYCARRHW QQYLYHPATFGQGTKVEIKRTVAAPSVFIF PGGFDYWGQGTLVTVSSASTKGPSV PPSDEQLKSGTASVVCLLNNFYPREAKVQ FPLAPSSKSTSGGTAALGCLVKDYFP WKVDNALQSGNSQESVTEQDSKDSTYSL EPVTVSWNSGALTSGVHTFPAVLQSS SSTLTLSKADYEKHKVYACEVTHQGLSSP GLYSLSSVVTVPSSSLGTQTYICNVN VTKSFNRGEC (SEQ ID NO: 256) HKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYASTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 251) Durvalumab EVQLVESGGGLVQPGGSLRLSCAAS EIVLTQSPGTLSLSPGERATLSCRASQRVS PD-L1 GFTFSRYWMSWVRQAPGKGLEWVA SSYLAWYQQKPGQAPRLLIYDASSRATGI NIKQDGSEKYYVDSVKGRFTISRDNA PDRFSGSGSGTDFTLTISRLEPEDFAVYYC KNSLYLQMNSLRAEDTAVYYCAREG QQYGSLPWTFGQGTKVEIKRTVAAPSVFI GWFGELAFDYWGQGTLVTVSSASTK FPPSDEQLKSGTASVVCLLNNFYPREAKV GPSVFPLAPSSKSTSGGTAALGCLVK QWKVDNALQSGNSQESVTEQDSKDSTYS DYFPEPVTVSWNSGALTSGVHTFPAV LSSTLTLSKADYEKHKVYACEVTHQGLSS LQSSGLYSLSSVVTVPSSSLGTQTYIC PVTKSFNRGEC (SEQ ID NO: 257) NVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEFEGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKAL PASIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 252) Tremelimumab QVQLVESGGG VVQPGRSLRL DIQMTQSPSSLSASVGDRVTITCRASQSIN CTLA-4 SCAASGFTFS SYGMHWVRQA SYLDWYQQKPGKAPKLLIYAASSLQSGVP PGKGLEWVAV IWYDGSNKYY SRFSGSGSGTDFTLTISSLQPEDFATYYC ADSVKGRFTI SRDNSKNTLY QQYYSTPFTFGPGTKVEIKRTVAAPSVFIF LQMNSLRAED TAVYYCARDP PPSDEQLKSGTASVVCLLNNFYPREAKVQ RGATLYYYYY GMDVWGQGTT WKVDNALQSGNSQESVTEQDSKDSTYSL VTVSSASTKG PSVFPLAPCS SSTLTLSKADYEKHKVYACEVTHQGLSSP RSTSESTAAL GCLVKDYFPE VTKSFNRGEC (SEQ ID NO: 258) PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K (SEQ ID NO: 253) Isatuximab QVQLVQSGAEVAKPGTSVKLSCKAS DIVMTQSHLSMSTSLGDPVSITCKASQDV CD38 GYTFTDYWMQWVKQRPGQGLEWIG STVVAWYQQKPGQSPRRLIYSASYRYIGV TIYPGDGDTGYAQKFQGKATLTADKS PDRFTGSGAGTDFTFTISSVQAEDLAVYY SKTVYMHLSSLASEDSAVYYCARGDY CQQHYSPPYTFGGGTKLEIKRTVAAPSVFI YGSNSLDYWGQGTSVTVSSASTKGP FPPSDEQLKSGTASVVCLLNNFYPREAKV SVFPLAPSSKSTSGGTAALGCLVKDY QWKVDNALQSGNSQESVTEQDSKDSTYS FPEPVTVSWNSGALTSGVHTFPAVLQ LSSTLTLSKADYEKHKVYACEVTHQGLSS SSGLYSLSSVVTVPSSSLGTQTYICNV PVTKSFNRGEC (SEQ ID NO: 259) NHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 254) MOR 202 QVQLVESGGGLVQPGGSLRLSCAAS DIELTQPPSVSVAPGQTARISCSGDNLRHY CD38 GFTFSSYYMNWVRQAPGKGLEWVS YVYWYQQKPGQAPVLVIYGDSKRPSGIP GISGDPSNTYYADSVKGRFTISRDNS ERFSGSNSGNTATLTISGTQAEDEADYYC KNTLYLQMNSLRAEDTAVYYCARDLP QTYTGGASLVFGGGTKLTVLGQ (SEQ ID LVYTGFAYWGQGTLVTV (SEQ ID NO: NO: 260) 255) (VH Only)
[0126] The antigen binding domains of Fc-antigen binding domain construct 43s (4304/4310 and 4312/4314 in
[0127] The antigen binding domains of Fc-antigen binding domain construct 44 (4404/4412 and 4414/4416 in
[0128] In some embodiments, the antigen binding domain (e.g., a Fab or a scFv) includes the VH and VL chains of an antibody listed in Table 2 or Table 1 B. In some embodiments, the Fab includes the CDRs contained in the V.sub.H and V.sub.L chains of an antibody listed in Table 2 or Table 1 B. In some embodiments, the Fab includes the CDRs contained in the V.sub.H and V.sub.L chains of an antibody listed in Table 2 and the remainder of the V.sub.H and V.sub.L sequences are at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V.sub.H and V.sub.L sequences of an antibody in Table 2. In some embodiments, the Fab includes the CDRs contained in the VH and VL chains of an antibody listed in Table 1B and the remainder of the VH and VL sequences are at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the VH and VL sequences of an antibody in Table 1B.
TABLE-US-00005 TABLE 2 Target Antibody Name AbGn-7 antigen AbGn-7 AMHR2 GM-102 B7-H3 DS-5573a CA19-9 MVT-5873 CAIX Anti-CAIX CD19 XmAb5871 CD33 BI-836858 CD37 BI-836826 CD38 MOR-202 CD47 Anti-CD47 CD70 ARGX-110 CD70 ARGX-110 CD98 IGN-523 CD147 Metuzumab CD157 MEN-1112 c-Met ARGX-111 EGFR2 GT-Mab 7.3-GEX EphA2 DS-8895a FGFR2 FPA-144 GM2 BIW-8962 HPA-1a NAITgam ICAM-1 BI-505 IL-3Ralpha Talacotuzumab JL-1 Leukotuximab kappa myeloma MDX-1097 antigen KIR32DL2 IPH-4102 LAG-3 GSK-2381781 P. aeruginosa AR-104 serotype O1 pGlu-abeta PBD-C06 TA-MUC1 GT-MAB 2.5-GEX
[0129] The antigen binding domains of Fc-antigen binding domain construct 43 (4304/4310 and 4312/4314 in
[0130] The antigen binding domains of Fc-antigen binding domain construct 44 (4404/4412 and 4414/4416 in
[0131] The antigen binding domains of Fc-antigen binding domain construct 43 (4304/4310 and 4312/4314 in
[0132] The antigen binding domains of Fc-antigen binding domain construct 44 (4404/4412 and 4414/4416 in
[0133] The antigen binding domains of Fc-antigen binding domain construct 43 (4304/4310 and 4312/4314 in
[0134] The antigen binding domains of Fc-antigen binding domain construct 44 (4404/4412 and 4414/4416 in
IV. Dimerization Selectivity Modules
[0135] In the present disclosure, a dimerization selectivity module includes components or select amino acids within the Fc domain monomer that facilitate the preferred pairing of two Fc domain monomers to form an Fc domain. Specifically, a dimerization selectivity module is that part of the C.sub.H3 antibody constant domain of an Fc domain monomer which includes amino acid substitutions positioned at the interface between interacting C.sub.H3 antibody constant domains of two Fc domain monomers. In a dimerization selectivity module, the amino acid substitutions make favorable the dimerization of the two C.sub.H3 antibody constant domains as a result of the compatibility of amino acids chosen for those substitutions. The ultimate formation of the favored Fc domain is selective over other Fc domains which form from Fc domain monomers lacking dimerization selectivity modules or with incompatible amino acid substitutions in the dimerization selectivity modules. This type of amino acid substitution can be made using conventional molecular cloning techniques well-known in the art, such as QuikChange® mutagenesis.
[0136] In some embodiments, a dimerization selectivity module includes an engineered cavity (described further herein) in the CH3 antibody constant domain. In other embodiments, a dimerization selectivity module includes an engineered protuberance (described further herein) in the C.sub.H3 antibody constant domain. To selectively form an Fc domain, two Fc domain monomers with compatible dimerization selectivity modules, e.g., one C.sub.H3 antibody constant domain containing an engineered cavity and the other C.sub.H3 antibody constant domain containing an engineered protuberance, combine to form a protuberance-into-cavity pair of Fc domain monomers. Engineered protuberances and engineered cavities are examples of heterodimerizing selectivity modules, which can be made in the C.sub.H3 antibody constant domains of Fc domain monomers in order to promote favorable heterodimerization of two Fc domain monomers that have compatible heterodimerizing selectivity modules.
[0137] In other embodiments, an Fc domain monomer with a dimerization selectivity module containing positively-charged amino acid substitutions and an Fc domain monomer with a dimerization selectivity module containing negatively-charged amino acid substitutions may selectively combine to form an Fc domain through the favorable electrostatic steering (described further herein) of the charged amino acids. In some embodiments, an Fc domain monomer may include one or more of the following positively-charged and negatively-charged amino acid substitutions: K392D, K392E, D399K, K409D, K409E, K439D, and K439E. In one example, an Fc domain monomer containing a positively-charged amino acid substitution, e.g., D356K or E357K, and an Fc domain monomer containing a negatively-charged amino acid substitution, e.g., K370D or K370E, may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In another example, an Fc domain monomer containing E357K and an Fc domain monomer containing K370D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In another example, an Fc domain monomer containing E356K and D399K and an Fc domain monomer containing K392D and K409D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In some embodiments, reverse charge amino acid substitutions may be used as heterodimerizing selectivity modules, wherein two Fc domain monomers containing different, but compatible, reverse charge amino acid substitutions combine to form a heterodimeric Fc domain. Specific dimerization selectivity modules are further listed, without limitation, in Tables 3 and 4 described further below.
[0138] In other embodiments, two Fc domain monomers include homodimerizing selectivity modules containing identical reverse charge mutations in at least two positions within the ring of charged residues at the interface between C.sub.H3 domains. Homodimerizing selectivity modules are reverse charge amino acid substitutions that promote the homodimerization of Fc domain monomers to form a homodimeric Fc domain. By reversing the charge of both members of two or more complementary pairs of residues in the two Fc domain monomers, mutated Fc domain monomers remain complementary to Fc domain monomers of the same mutated sequence, but have a lower complementarity to Fc domain monomers without those mutations. In one embodiment, an Fc domain includes Fc domain monomers including the double mutants K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E. In another embodiment, an Fc domain includes Fc domain monomers including quadruple mutants combining any pair of the double mutants, e.g., K409D/D399K/E357K/K370E. Examples of homodimerizing selectivity modules are further shown in Tables 5 and 6. Homodimerizing Fc domains can be used to create symmetrical branch points on an Fc-antigen binding domain construct. In one embodiment, an Fc-antigen binding domain construct described herein has one homodimerizing Fc domain. In one embodiment, an Fc-antigen binding domain construct has two or more homodimerizing Fc domains, e.g., two, three, four, or five or more homodimerizing domains. In one embodiment, an Fc-antigen binding domain construct has three homodimerizing Fc domains. In some embodiments, an Fc-antigen binding domain construct has one homodimerizing selectivity module. In some embodiments, an Fc-antigen binding domain construct has two or more homodimerizing selectivity modules, e.g., two, three, four, or five or more homodimerizing selectivity modules.
[0139] In further embodiments, an Fc domain monomer containing (i) at least one reverse charge mutation and (ii) at least one engineered cavity or at least one engineered protuberance may selectively combine with another Fc domain monomer containing (i) at least one reverse charge mutation and (ii) at least one engineered protuberance or at least one engineered cavity to form an Fc domain. For example, an Fc domain monomer containing reversed charge mutation K370D and engineered cavities Y349C, T366S, L368A, and Y407V and another Fc domain monomer containing reversed charge mutation E357K and engineered protuberances S354C and T366W may selectively combine to form an Fc domain.
[0140] The formation of such Fc domains is promoted by the compatible amino acid substitutions in the C.sub.H3 antibody constant domains. Two dimerization selectivity modules containing incompatible amino acid substitutions, e.g., both containing engineered cavities, both containing engineered protuberances, or both containing the same charged amino acids at the C.sub.H3-C.sub.H3 interface, will not promote the formation of a heterodimeric Fc domain.
[0141] Multiple pairs of heterodimerizing Fc domains can be used to create Fc-antigen binding domain constructs with multiple asymmetrical branch points, multiple non-branching points, or both asymmetrical branch points and non-branching points. Multiple, distinct heterodimerization technologies (see, e.g., Tables 3 and 4) are incorporated into different Fc domains to assemble these Fc domain-containing constructs. The heterodimerization technologies have minimal association (orthogonality) for undesired pairing of Fc monomers. Two different Fc heterodimerization methods, such as knobs-into-holes (Table 3) and electrostatic steering (Table 4), can be used in different Fc domains to control the assembly of the polypeptide chains into the desired construct. Alternatively, two different variants of knobs-into-holes (e.g., two distinct sets of mutations selected from Table 3), or two different variants of electrostatic steering (e.g., two distinct sets of mutations selected from Table 4), can be used in different Fc domains to control the assembly of the polypeptide chains into the desired construct. Asymmetrical branches can be created by placing the Fc domain monomers of a heterodimerizing Fc domain on different polypeptide chains, polypeptide chain having multiple Fc domains. Non-branching points can be created by placing one Fc domain monomer of the heterodimerizing Fc domain on a polypeptide chain with multiple Fc domains and the other Fc domain monomer of the heterodimerizing Fc domain on a polypeptide chain with a single Fc domain.
[0142] In some embodiments, the Fc-antigen binding domain constructs described herein are linear. In some embodiments, the Fc-antigen binding domain constructs described herein do not have branch points. For example, an Fc-antigen binding domain construct can be assembled from one large peptide with two or more Fc domain monomers, wherein at least two Fc domain monomers are different (i.e., have different heterodimerizing mutations), and two or more smaller peptides, each having a different single Fc domain monomer (i.e., two or more small peptides with Fc domain monomers having different heterodimerizing mutations). The Fc-antigen binding domain constructs described herein can have two or more dimerization selectivity modules that are incompatible with each other, e.g., at least two incompatible dimerization selectivity modules selected from Tables 3 and/or 4, that promote or facilitate the proper formation of the Fc-antigen binding domain constructs, so that the Fc domain monomer of each smaller peptide associates with its compatible Fc domain monomer(s) on the large peptide. In some embodiments, a first Fc domain monomer or first subset of Fc domain monomers on a long peptide contains amino acids substitutions forming part of a first dimerization selectivity module that is compatible to a part of the first dimerization selectivity module formed by amino acid substitutions in the Fc domain monomer of a first short peptide. A second Fc domain monomer or second subset of Fc domain monomers on the long peptide contains amino acids substitutions forming part of a second dimerization selectivity module that is compatible to part of the second dimerization selectivity module formed by amino acid substitutions in the Fc domain monomer of a second short peptide. The first dimerization selectivity module favors binding of a first Fc domain monomer (or first subset of Fc domain monomers) on the long peptide to the Fc domain monomer of a first short peptide, while disfavoring binding between a first Fc domain monomer and the Fc domain monomer of the second short peptide. Similarly, the second dimerization selectivity module favors binding of a second Fc domain monomer (or second subset of Fc domain monomers) on the long peptide to the Fc domain monomer of the second short peptide, while disfavoring binding between a second Fc domain monomer and the Fc domain monomer of the first short peptide.
[0143] In certain embodiments, an Fc-antigen binding domain construct can have a first Fc domain with a first dimerization selectivity module, and a second Fc domain with a second dimerization selectivity module. In some embodiments, the first Fc domain is assembled from one Fc monomer with at least one protuberance-forming mutations selected from Table 3 and/or at least one reverse charge mutation selected from Table 4 (e.g., the Fc monomer can have S354C and T366W protuberance-forming mutations and an E357K reverse charge mutation), and one Fc monomer with at least one cavity-forming mutation from selected from Table 3 and/or at least one reverse charge mutation selected from Table 4 (e.g., the Fc monomer can have Y349C, T366S, L368A, and Y407V cavity-forming mutations and a K370D reverse charge mutation. In some embodiments, the second Fc domain is assembled from one Fc monomer with at least one protuberance-forming mutations selected from Table 3 and/or at least one reverse charge mutation selected from Table 4 (e.g., the Fc monomer can have D356K and D399K reverse charge mutations), and one Fc monomer with at least one cavity-forming mutation from selected from Table 3 and/or at least one reverse charge mutation selected from Table 4 (e.g., the Fc monomer can have K392D and K409D reverse charge mutations).
[0144] Furthermore, other methods used to promote the formation of Fc domains with defined Fc domain monomers include, without limitation, the LUZ-Y approach (U.S. Patent Application Publication No. WO2011034605) which includes C-terminal fusion of a monomer α-helices of a leucine zipper to each of the Fc domain monomers to allow heterodimer formation, as well as strand-exchange engineered domain
[0145] (SEED) body approach (Davis et al., Protein Eng Des Sel. 23:195-202, 2010) that generates Fc domain with heterodimeric Fc domain monomers each including alternating segments of IgA and IgG CH3 sequences.
V. Engineered Cavities and Engineered Protuberances
[0146] The use of engineered cavities and engineered protuberances (or the “knob-into-hole” strategy) is described by Carter and co-workers (Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J Mol Biol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-681, 1998). The knob and hole interaction favors heterodimer formation, whereas the knob-knob and the hole-hole interaction hinder homodimer formation due to steric clash and deletion of favorable interactions. The “knob-into-hole” technique is also disclosed in U.S. Pat. No. 5,731,168.
[0147] In the present disclosure, engineered cavities and engineered protuberances are used in the preparation of the Fc-antigen binding domain constructs described herein. An engineered cavity is a void that is created when an original amino acid in a protein is replaced with a different amino acid having a smaller side-chain volume. An engineered protuberance is a bump that is created when an original amino acid in a protein is replaced with a different amino acid having a larger side-chain volume. Specifically, the amino acid being replaced is in the C.sub.H3 antibody constant domain of an Fc domain monomer and is involved in the dimerization of two Fc domain monomers. In some embodiments, an engineered cavity in one C.sub.H3 antibody constant domain is created to accommodate an engineered protuberance in another C.sub.H3 antibody constant domain, such that both C.sub.H3 antibody constant domains act as dimerization selectivity modules (e.g., heterodimerizing selectivity modules) (described above) that promote or favor the dimerization of the two Fc domain monomers. In other embodiments, an engineered cavity in one C.sub.H3 antibody constant domain is created to better accommodate an original amino acid in another C.sub.H3 antibody constant domain. In yet other embodiments, an engineered protuberance in one C.sub.H3 antibody constant domain is created to form additional interactions with original amino acids in another CH3 antibody constant domain. An engineered cavity can be constructed by replacing amino acids containing larger side chains such as tyrosine or tryptophan with amino acids containing smaller side chains such as alanine, valine, or threonine. Specifically, some dimerization selectivity modules (e.g., heterodimerizing selectivity modules) (described further above) contain engineered cavities such as Y407V mutation in the C.sub.H3 antibody constant domain. Similarly, an engineered protuberance can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains. Specifically, some dimerization selectivity modules (e.g., heterodimerizing selectivity modules) (described further above) contain engineered protuberances such as T366W mutation in the C.sub.H3 antibody constant domain. In the present disclosure, engineered cavities and engineered protuberances are also combined with inter-CH3 domain disulfide bond engineering to enhance heterodimer formation. In one example, an Fc domain monomer containing engineered cavities Y349C, T366S, L368A, and Y407V may selectively combine with another Fc domain monomer containing engineered protuberances S354C and T366W to form an Fc domain. In another example, an Fc domain monomer containing an engineered cavity with the addition of Y349C and an Fc domain monomer containing an engineered protuberance with the addition of S354C may selectively combine to form an Fc domain. Other engineered cavities and engineered protuberances, in combination with either disulfide bond engineering or structural calculations (mixed HA-TF) are included, without limitation, in Table 3.
TABLE-US-00006 TABLE 3 Fc heterodimerization methods (Knobs-into-holes) Mutations (Chain A) Mutations (Chain B) (CH3 antibody (CH.sub.3 antibody constant domain constant domain of Fc domain of Fc domain Method monomer 1) monomer 2) Reference Knobs-into- Y407T T336Y U.S. Pat. No. Holes (Y-T) 8,216,805 Knobs-into- Y407A T336W U.S. Pat. No. Holes 8,216,805 Knobs-into- F405A T394W U.S. Pat. No. Holes 8,216,805 Knobs-into- Y407T T366Y U.S. Pat. No. Holes 8,216,805 Knobs-into- T394S F405W U.S. Pat. No. Holes 8,216,805 Knobs-into- T394W, Y407T T366Y, F406A U.S. Pat. No. Holes 8,216,805 Knobs-into- T394S, Y407A T366W, F405W U.S. Pat. No. Holes 8,216,805 Knobs-into- T366W, T394S F405W, T407A U.S. Pat. No. Holes 8,216,805 Knobs-into- F405T T394Y Holes Knobs-into- S354C, T366W Y349C, T366S, Holes L368A, Y407V Knobs-into- Y349C, T366S, S354C, T366W Merchant et al., Holes (CW- L368A, Y407V Nat. Biotechnol. CSAV) 16(7): 677-81, 1998 HA-TF S364H, F405A Y349T, T394F WO2011028952 Note: All residues numbered per the EU numbering scheme (Edelman et al, Proc Natl Acad Sci USA, 63: 78-85, 1969)
[0148] Replacing an original amino acid residue in the C.sub.H3 antibody constant domain with a different amino acid residue can be achieved by altering the nucleic acid encoding the original amino acid residue. The upper limit for the number of original amino acid residues that can be replaced is the total number of residues in the interface of the C.sub.H3 antibody constant domains, given that sufficient interaction at the interface is still maintained.
[0149] Combining Engineered Cavities and Engineered Protuberances with Electrostatic Steering
[0150] Electrostatic steering can be combined with knob-in-hole technology to favor heterominerization, for example, between Fc domain monomers in two different polypeptides. Electrostatic steering, described in greater detail below, is the utilization of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher ordered protein molecules. Electrostatic steering can be used to promote either homodimerization or heterodimerization, the latter of which can be usefully combined with knob-in-hole technology. In the case of heterodimerization, different, but compatible, mutations are introduced in each of the Fc domain monomers which are to heterodimerize. Thus, an Fc domain monomer can be modified to include one of the following positively-charged and negatively-charged amino acid substitutions: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E. For example, one Fc domain monomer, for example, an Fc domain monomer having a cavity (Y349C, T366S, L368A and Y407V), can also include K370D mutation and the other Fc domain monomer, for example, an Fc domain monomer having a protuberance (S354C and T366W) can include E357K.
[0151] More generally, any of the cavity mutations (or mutation combinations): Y407T, Y407A, F405A, Y407T, T394S, T394W:Y407A, T366W:T394S, T366S:L368A:Y407V:Y349C, and S3364H:F405 can be combined with a mutation in Table 4 and any of the protuberance mutations (or mutation combinations):
[0152] T366Y, T366W, T394W, F405W, T366Y:F405A, T366W:Y407A, T366W:S354C, and Y349T:T394F can be combined with a mutation in Table 4 that is paired with the Table 4 mutation used in combination with the cavity mutation (or mutation combination).
VI. Electrostatic Steering
[0153] Electrostatic steering is the utilization of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher ordered protein molecules. A method of using electrostatic steering effects to alter the interaction of antibody domains to reduce for formation of homodimer in favor of heterodimer formation in the generation of bi-specific antibodies is disclosed in U.S. Patent Application Publication No. 2014-0024111.
[0154] In the present disclosure, electrostatic steering is used to control the dimerization of Fc domain monomers and the formation of Fc-antigen binding domain constructs. In particular, to control the dimerization of Fc domain monomers using electrostatic steering, one or more amino acid residues that make up the C.sub.H3-C.sub.H3 interface are replaced with positively- or negatively-charged amino acid residues such that the interaction becomes electrostatically favorable or unfavorable depending on the specific charged amino acids introduced. In some embodiments, a positively-charged amino acid in the interface, such as lysine, arginine, or histidine, is replaced with a negatively-charged amino acid such as aspartic acid or glutamic acid. In other embodiments, a negatively-charged amino acid in the interface is replaced with a positively-charged amino acid. The charged amino acids may be introduced to one of the interacting C.sub.H3 antibody constant domains, or both. By introducing charged amino acids to the interacting C.sub.H3 antibody constant domains, dimerization selectivity modules (described further above) are created that can selectively form dimers of Fc domain monomers as controlled by the electrostatic steering effects resulting from the interaction between charged amino acids.
[0155] In some embodiments, to create a dimerization selectivity module including reversed charges that can selectively form dimers of Fc domain monomers as controlled by the electrostatic steering effects, the two Fc domain monomers may be selectively formed through heterodimerization or homodimerization.
[0156] Heterodimerization of Fc Domain Monomers
[0157] Heterodimerization of Fc domain monomers can be promoted by introducing different, but compatible, mutations in the two Fc domain monomers, such as the charge residue pairs included, without limitation, in Table 4. In some embodiments, an Fc domain monomer may include one or more of the following positively-charged and negatively-charged amino acid substitutions: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E, e.g., 1, 2, 3, 4, or 5 or more of D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E. In one example, an Fc domain monomer containing a positively-charged amino acid substitution, e.g., D356K or E357K, and an Fc domain monomer containing a negatively-charged amino acid substitution, e.g., K370D or K370E, may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In another example, an Fc domain monomer containing E357K and an Fc domain monomer containing K370D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In another example, an Fc domain monomer containing E356K and D399K and an Fc domain monomer containing K392D and K409D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids.
[0158] A “heterodimeric Fc domain” refers to an Fc domain that is formed by the heterodimerization of two Fc domain monomers, wherein the two Fc domain monomers contain different reverse charge mutations (heterodimerizing selectivity modules) (see, e.g., mutations in Table 4) that promote the favorable formation of these two Fc domain monomers. In one example, in an Fc-antigen binding domain construct having three Fc domains, two of the three Fc domains may be formed by the heterodimerization of two Fc domain monomers, as promoted by the electrostatic steering effects.
TABLE-US-00007 TABLE 4 Fc heterodimerization methods (electrostatic steering) Mutations (Chain B) Mutations (Chain A) (CH.sub.3 of Fc domain Method (CH.sub.3 of Fc domain monomer 1) monomer 2) Reference Electrostatic K409D D399K US 2014/0024111 Steering Electrostatic K409D D399R US 2014/0024111 Steering Electrostatic K409E D399K US 2014/0024111 Steering Electrostatic K409E D399R US 2014/0024111 Steering Electrostatic K392D D399K US 2014/0024111 Steering Electrostatic K392D D399R US 2014/0024111 Steering Electrostatic K392E D399K US 2014/0024111 Steering Electrostatic K392E D399R US 2014/0024111 Steering Electrostatic K392D, K409D E356K, D399K Gunasekaran et Steering (DD- al., J Biol Chem. KK) 285: 19637-46, 2010 Electrostatic K370E, K409D, K439E E356K, E357K, D399K WO 2006/106905 Steering Knobs-into- S354C, E357K, T366W Y349C, T366S, L368A, WO 2015/168643 Holes plus K370D, Y407V Electrostatic Steering Electrostatic K370D E357K US 2014/0024111 Steering Electrostatic K370D E357R US 2014/0024111 Steering Electrostatic K370E E357K US 2014/0024111 Steering Electrostatic K370E E357R US 2014/0024111 Steering Electrostatic K370D D356K US 2014/0024111 Steering Electrostatic K370D D356R US 2014/0024111 Steering Electrostatic K370E D356K US 2014/0024111 Steering Electrostatic K370E D356R US 2014/0024111 Steering Electrostatic K370E, K409D, K439E E356K, E357K, D399K Steering Note: All residues numbered per the EU numbering scheme (Edelman et al, Proc Natl Acad Sci USA, 63: 78-85, 1969)
[0159] Homodimerization of Fc Domain Monomers
[0160] Homodimerization of Fc domain monomers can be promoted by introducing the same electrostatic steering mutations (homodimerizing selectivity modules) in both Fc domain monomers in a symmetric fashion. In some embodiments, two Fc domain monomers include homodimerizing selectivity modules containing identical reverse charge mutations in at least two positions within the ring of charged residues at the interface between C.sub.H3 domains. By reversing the charge of both members of two or more complementary pairs of residues in the two Fc domain monomers, mutated Fc domain monomers remain complementary to Fc domain monomers of the same mutated sequence, but have a lower complementarity to Fc domain monomers without those mutations. Electrostatic steering mutations that may be introduced into an Fc domain monomer to promote its homodimerization are shown, without limitation, in Tables 5 and 6. In one embodiment, an Fc domain includes two Fc domain monomers each including the double reverse charge mutants (Table 5), e.g., K409D/D399K. In another embodiment, an Fc domain includes two Fc domain monomers each including quadruple reverse mutants (Table 6), e.g., K409D/D399K/K370D/E357K.
[0161] For example, in an Fc-antigen binding domain construct having three Fc domains, one of the three Fc domains may be formed by the homodimerization of two Fc domain monomers, as promoted by the electrostatic steering effects. A “homodimeric Fc domain” refers to an Fc domain that is formed by the homodimerization of two Fc domain monomers, wherein the two Fc domain monomers contain the same reverse charge mutations (see, e.g., mutations in Tables 5 and 6). In an Fc-antigen binding domain construct having three Fc domains—one carboxyl terminal “stem” Fc domain and two amino terminal “branch” Fc domains—the carboxy terminal “stem” Fc domain may be a homodimeric Fc domain (also called a “stem homodimeric Fc domain”). A stem homodimeric Fc domain may be formed by two Fc domain monomers each containing the double mutants K409D/D399K.
TABLE-US-00008 TABLE 5 Fc homodimerization (electrostatic steering with 2 mutations) Mutations (Chains A and B) (CH.sub.3 of Fc domain monomers 1 Method and 2) Reference Wild Type None Electrostatic Steering (KD) D399K, K409D Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering D399K, K409E Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering E357K, K370D Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering E357K, K370E Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering D356K, K439D Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering D356K, K439E Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering K392D, D399K Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering K392E, D399K Gunasekaran et al., J Biol Chem. 285: 19637-46, 2010, WO 2015/168643 Electrostatic Steering D399R, K409D Electrostatic Steering D399R, K409E Electrostatic Steering D399R, K392D Electrostatic Steering D399R, K392E Electrostatic Steering E357K, K370D Electrostatic Steering E357R, K370D Electrostatic Steering E357K, K370E Electrostatic Steering E357R, K370E Electrostatic Steering D356K, K370D Electrostatic Steering D356R, K370D Electrostatic Steering D356K, K370E Electrostatic Steering D356R, K370E Note: All residues numbered per the EU numbering scheme (Edelman et al, Proc Natl Acad Sci USA, 63: 78-85, 1969)
TABLE-US-00009 TABLE 6 Fc homodimerization (electrostatic steering 4 mutations) Mutations (Chains A and B) Mutations (Chains A and B) (CH.sub.3 of Fc domain (CH.sub.3 of Fc domain monomers 1 and 2) monomers 1 and 2) K409D/D399K/K370D/E357K K392D/D399K/K370D/E357K K409D/D399K/K370D/E357R K392D/D399K/K370D/E357R K409D/D399K/K370E/E357K K392D/D399K/K370E/E357K K409D/D399K/K370E/E357R K392D/D399K/K370E/E357R K409D/D399K/K370D/D356K K392D/D399K/K370D/D356K K409D/D399K/K370D/D356R K392D/D399K/K370D/D356R K409D/D399K/K370E/D356K K392D/D399K/K370E/D356K K409D/D399K/K370E/D356R K392D/D399K/K370E/D356R K409D/D399R/K370D/E357K K392D/D399R/K370D/E357K K409D/D399R/K370D/E357R K392D/D399R/K370D/E357R K409D/D399R/K370E/E357K K392D/D399R/K370E/E357K K409D/D399R/K370E/E357R K392D/D399R/K370E/E357R K409D/D399R/K370D/D356K K392D/D399R/K370D/D356K K409D/D399R/K370D/D356R K392D/D399R/K370D/D356R K409D/D399R/K370E/D356K K392D/D399R/K370E/D356K K409D/D399R/K370E/D356R K392D/D399R/K370E/D356R K409E/D399K/K370D/E357K K392E/D399K/K370D/E357K K409E/D399K/K370D/E357R K392E/D399K/K370D/E357R K409E/D399K/K370E/E357K K392E/D399K/K370E/E357K K409E/D399K/K370E/E357R K392E/D399K/K370E/E357R K409E/D399K/K370D/D356K K392E/D399K/K370D/D356K K409E/D399K/K370D/D356R K392E/D399K/K370D/D356R K409E/D399K/K370E/D356K K392E/D399K/K370E/D356K K409E/D399K/K370E/D356R K392E/D399K/K370E/D356R K409E/D399R/K370D/E357K K392E/D399R/K370D/E357K K409E/D399R/K370D/E357R K392E/D399R/K370D/E357R K409E/D399R/K370E/E357K K392E/D399R/K370E/E357K K409E/D399R/K370E/E357R K392E/D399R/K370E/E357R K409E/D399R/K370D/D356K K392E/D399R/K370D/D356K K409E/D399R/K370D/D356R K392E/D399R/K370D/D356R K409E/D399R/K370E/D356K K392E/D399R/K370E/D356K K409E/D399R/K370E/D356R K392E/D399R/K370E/D356R Note: All residues numbered per the EU numbering scheme (Edelman et al, Proc Natl Acad Sci USA, 63: 78-85, 1969)
[0162] Other Heterodimerization Methods
[0163] Numerous other heterodimerization technologies have been described. Any one or more of these technologies (Table 7) can be combined with any knobs-into-holes and/or electrostatic steering heterodimerization and/or homodimerization technology described herein to make an Fc-antigen binding domain construct.
TABLE-US-00010 TABLE 7 Other Fc heterodimerization methods Method Mutations (Chain A) Mutations (Chain B) Reference ZW1 (VYAV- T350V, L351Y, F405A, Y407V T350V, T366L, K392L, Von Kreudenstein VLLW) T394W et al, MAbs, 5: 646- 54, 2013 IgG1 hinge/CH3 D221E, P228E, L368E D221R, P228R, K409R Strop et al, J Mol charge pairs Biol, 420: 204-19, (EEE-RRR) 2012 Method Mutations (Chain A) Mutations (Chain B) Reference EW-RVT K360E, K409W Q347R, D399V, F405T Choi et al, Mol Cancer Ther, 12:2748-59, 2013 EW-RVT.sub.S-S K360E, K409W, Y349C Q347R, D399V, F405T, Choi et al, Mol S354C Immunol, 65: 377- 83, 2015 Charge L351D T366K De Nardis, J Biol Introduction (DK Chem, 292: 14706- Biclonic) 17, 2017 Charge L351D, L368E L351K, T366K De Nardis, J Biol Introduction Chem, 292: 14706- (DEKK Biclonic) 17, 2017 DuoBody (L-R) F405L K409R Labrijn et al, Proc Natl Acad Sci USA, 110: 5145- 50, 2013 SEEDbody IgG/A chimera IgG/A chimera Davis et al, Protein Eng Des Sel, 23: 195-202, 2010 BEAT (A/B) S364K, T366V, K370T, K392Y, Q347E, Y349A, L351F, Skegro et al, J Biol F405S, Y407V, K409W, T411N S364T, T366V, K370T, Chem, 292: 9745- T394D, V397L, D399E, 59, 2017 F405A, Y407S, K409R, T411R BEAT (A/B min) S364K, T366V, K370T, K392Y, F405A, Y407S Skegro et al, J Biol K409W, T411N Chem, 292: 9745- 59, 2017 BEAT (A/B + Q) Q347A, S364K, T366V, K370T, Q347E, Y349A, L351F, Skegro et al, J Biol K392Y, F405S, Y407V, S364T, T366V, K370T, Chem, 292: 9745- K409W, T411N T394D, V397L, D399E, 59, 2017 F405A, Y407S, K409R, T411R BEAT (A/B − T) S364K, T366V, K370T, K392Y, Q347E, Y349A, L351F, Skegro et al, J Biol F405S, Y407V, K409W, T411N S364T, T366V, K370T, Chem, 292: 9745- T394D, V397L, D399E, 59, 2017 F405A, Y407S, K409R 7.8.60 (DMA- K360D, D399M, Y407A E345R, Q347R, T366V, Leaver-Fay et al, RRVV) K409V Structure, 24: 641- 51,2016 20.8.34 (SYMV- Y349S, K370Y, T366M, K409V E356G, E357D, S364Q, Leaver-Fay et al, GDQA) Y407A Structure, 24: 641- 51,2016 Note: All residues numbered per the EU numbering scheme (Edelman et al, Proc Natl Acad Sci USA, 63:78-85, 1969)
VII. Linkers
[0164] In the present disclosure, a linker is used to describe a linkage or connection between polypeptides or protein domains and/or associated non-protein moieties. In some embodiments, a linker is a linkage or connection between at least two Fc domain monomers, for which the linker connects the C-terminus of the CH3 antibody constant domain of a first Fc domain monomer to the N-terminus of the hinge domain of a second Fc domain monomer, such that the two Fc domain monomers are joined to each other in tandem series. In other embodiments, a linker is a linkage between an Fc domain monomer and any other protein domains that are attached to it. For example, a linker can attach the C-terminus of the C.sub.H3 antibody constant domain of an Fc domain monomer to the N-terminus of an albumin-binding peptide.
[0165] A linker can be a simple covalent bond, e.g., a peptide bond, a synthetic polymer, e.g., a polyethylene glycol (PEG) polymer, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. In the case that a linker is a peptide bond, the carboxylic acid group at the C-terminus of one protein domain can react with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. Specifically, the peptide bond can be formed from synthetic means through a conventional organic chemistry reaction well-known in the art, or by natural production from a host cell, wherein a polynucleotide sequence encoding the DNA sequences of both proteins, e.g., two Fc domain monomer, in tandem series can be directly transcribed and translated into a contiguous polypeptide encoding both proteins by the necessary molecular machineries, e.g., DNA polymerase and ribosome, in the host cell.
[0166] In the case that a linker is a synthetic polymer, e.g., a PEG polymer, the polymer can be functionalized with reactive chemical functional groups at each end to react with the terminal amino acids at the connecting ends of two proteins.
[0167] In the case that a linker (except peptide bond mentioned above) is made from a chemical reaction, chemical functional groups, e.g., amine, carboxylic acid, ester, azide, or other functional groups commonly used in the art, can be attached synthetically to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups can then react to through synthetic chemistry means to form a chemical bond, thus connecting the two proteins together. Such chemical conjugation procedures are routine for those skilled in the art.
[0168] Spacer
[0169] In the present disclosure, a linker between two Fc domain monomers can be an amino acid spacer including 3-200 amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-120, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200 amino acids). In some embodiments, a linker between two Fc domain monomers is an amino acid spacer containing at least 12 amino acids, such as 12-200 amino acids (e.g., 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-60, 12-50, 12-40, 12-30, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, or 12-13 amino acids) (e.g., 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids). In some embodiments, a linker between two Fc domain monomers is an amino acid spacer containing 12-30 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids). Suitable peptide spacers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or SGGG (SEQ ID NO: 3). In certain embodiments, a spacer can contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO: 4), GSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7), or GSGSGSGSGSGS (SEQ ID NO: 8). In certain other embodiments, a spacer can contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), and GGSGGSGGSGGS (SEQ ID NO: 11). In yet other embodiments, a spacer can contain 4 to 20 amino acids including motifs of GGSG (SEQ ID NO: 2), e.g., GGSGGGSG (SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG (SEQ ID NO: 14), or GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 15). In other embodiments, a spacer can contain motifs of GGGGS (SEQ ID NO: 1), e.g., GGGGSGGGGS (SEQ ID NO: 16) or GGGGSGGGGSGGGGS (SEQ ID NO: 17). In certain embodiments, a spacer is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18).
[0170] In some embodiments, a spacer between two Fc domain monomers contains only glycine residues, e.g., at least 4 glycine residues (e.g., 4-200 (SEQ ID NO: 270), 4-180 (SEQ ID NO: 271), 4-160 (SEQ ID NO: 272), 4-140 (SEQ ID NO: 273), 4-40 (SEQ ID NO: 274), 4-100 (SEQ ID NO: 275), 4-90 (SEQ ID NO: 276), 4-80 (SEQ ID NO: 277), 4-70 (SEQ ID NO: 278), 4-60 (SEQ ID NO: 279), 4-50 (SEQ ID NO: 280), 4-40 (SEQ ID NO: 274), 4-30 (SEQ ID NO: 264), 4-20 (SEQ ID NO: 265), 4-19 (SEQ ID NO: 281), 4-18 (SEQ ID NO: 282), 4-17 (SEQ ID NO: 283), 4-16 (SEQ ID NO: 284), 4-15 (SEQ ID NO: 285), 4-14 (SEQ ID NO: 286), 4-13 (SEQ ID NO: 287), 4-12 (SEQ ID NO: 288), 4-11 (SEQ ID NO: 289), 4-10 (SEQ ID NO: 290), 4-9 (SEQ ID NO: 291), 4-8 (SEQ ID NO: 292), 4-7 (SEQ ID NO: 293), 4-6 (SEQ ID NO: 294) or 4-5 (SEQ ID NO: 295) glycine residues) (e.g., 4-200 (SEQ ID NO: 270), 6-200 (SEQ ID NO: 296), 8-200 (SEQ ID NO: 297), 10-200 (SEQ ID NO: 298), 12-200 (SEQ ID NO: 299), 14-200 (SEQ ID NO: 300), 16-200 (SEQ ID NO: 301), 18-200 (SEQ ID NO: 302), 20-200 (SEQ ID NO: 303), 30-200 (SEQ ID NO: 304), 40-200 (SEQ ID NO: 305), 50-200 (SEQ ID NO: 306), 60-200 (SEQ ID NO: 307), 70-200 (SEQ ID NO: 308), 80-200 (SEQ ID NO: 309), 90-200 (SEQ ID NO: 310), 100-200 (SEQ ID NO: 311), 120-200 (SEQ ID NO: 312), 140-200 (SEQ ID NO: 313), 160-200 (SEQ ID NO: 314), 180-200 (SEQ ID NO: 315), or 190-200 (SEQ ID NO: 316) glycine residues). In certain embodiments, a spacer has 4-30 glycine residues (SEQ ID NO: 264) (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 glycine residues (SEQ ID NO: 264)). In some embodiments, a spacer containing only glycine residues may not be glycosylated (e.g., 0-linked glycosylation, also referred to as 0-glycosylation) or may have a decreased level of glycosylation (e.g., a decreased level of O-glycosylation) (e.g., a decreased level of 0-glycosylation with glycans such as xylose, mannose, sialic acids, fucose (Fuc), and/or galactose (Gal) (e.g., xylose)) as compared to, e.g., a spacer containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)).
[0171] In some embodiments, a spacer containing only glycine residues may not be 0-glycosylated (e.g., O-xylosylation) or may have a decreased level of O-glycosylation (e.g., a decreased level of O-xylosylation) as compared to, e.g., a spacer containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)).
[0172] In some embodiments, a spacer containing only glycine residues may not undergo proteolysis or may have a decreased rate of proteolysis as compared to, e.g., a spacer containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)).
[0173] In certain embodiments, a spacer can contain motifs of GGGG (SEQ ID NO: 19), e.g., GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21), GGGGGGGGGGGGGGGG (SEQ ID NO: 22), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). In certain embodiments, a spacer can contain motifs of GGGGG (SEQ ID NO: 24), e.g., GGGGGGGGGG (SEQ ID NO: 25), or GGGGGGGGGGGGGGG (SEQ ID NO: 26). In certain embodiments, a spacer is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).
[0174] In other embodiments, a spacer can also contain amino acids other than glycine and serine, e.g., GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30),
[0175] RPACKIPNDLKQKVMNH (SEQ ID NO: 31), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 32), AAANSSIDLISVPVDSR (SEQ ID NO: 33), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34).
[0176] In certain embodiments in the present disclosure, a 12- or 20-amino acid peptide spacer is used to connect two Fc domain monomers in tandem series, the 12- and 20-amino acid peptide spacers consisting of sequences GGGSGGGSGGGS (SEQ ID NO: 35) and SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18), respectively. In other embodiments, an 18-amino acid peptide spacer consisting of sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36) may be used. In some embodiments, a spacer between two Fc domain monomers may have a sequence that is at least 75% identical (e.g., at least 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 1-36 described above. In certain embodiments, a spacer between two Fc domain monomers may have a sequence that is at least 80% identical (e.g., at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 17, 18, 26, and 27. In certain embodiments, a spacer between two Fc domain monomers may have a sequence that is at least 80% identical (e.g., at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 99.5%) to the sequence of SEQ ID NO: 18 or 27.
[0177] In certain embodiments, the linker between the amino terminus of the hinge of an Fc domain monomer and the carboxy terminus of a Fc monomer that is in the same polypeptide (i.e., the linker connects the C-terminus of the CH3 antibody constant domain of a first Fc domain monomer to the N-terminus of the hinge domain of a second Fc domain monomer, such that the two Fc domain monomers are joined to each other in tandem series) is a spacer having 3 or more amino acids rather than a covalent bond (e.g., 3-200 amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-120, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200 amino acids) or an amino acid spacer containing at least 12 amino acids, such as 12-200 amino acids (e.g., 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-60, 12-50, 12-40, 12-30, 12-20, 12-19, 12-18, 12-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids)).
[0178] A spacer can also be present between the N-terminus of the hinge domain of a Fc domain monomer and the carboxy terminus of a CD38 binding domain (e.g., a CH1 domain of a CD38 heavy chain binding domain or the CL domain of a CD38 light chain binding domain) such that the domains are joined by a spacer of 3 or more amino acids (e.g., 3-200 amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-120, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200 amino acids) or an amino acid spacer containing at least 12 amino acids, such as 12-200 amino acids (e.g., 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-60, 12-50, 12-40, 12-30, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, or 12-13 amino acids) (e.g., 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids)).
VII. Serum Protein-Binding Peptides
[0179] Binding to serum protein peptides can improve the pharmacokinetics of protein pharmaceuticals, and in particular the Fc-antigen binding domain constructs described here may be fused with serum protein-binding peptides
[0180] As one example, albumin-binding peptides that can be used in the methods and compositions described here are generally known in the art. In one embodiment, the albumin binding peptide includes the sequence DICLPRWGCLW (SEQ ID NO: 37). In some embodiments, the albumin binding peptide has a sequence that is at least 80% identical (e.g., 80%, 90%, or 100% identical) to the sequence of SEQ ID NO: 37.
[0181] In the present disclosure, albumin-binding peptides may be attached to the N- or C-terminus of certain polypeptides in the Fc-antigen binding domain construct. In one embodiment, an albumin-binding peptide may be attached to the C-terminus of one or more polypeptides in Fc constructs containing an antigen binding domain. In another embodiment, an albumin-binding peptide can be fused to the C-terminus of the polypeptide encoding two Fc domain monomers linked in tandem series in Fc constructs containing an antigen binding domain. In yet another embodiment, an albumin-binding peptide can be attached to the C-terminus of Fc domain monomer (e.g., Fc domain monomers 114 and 116 in
VIII. Fc-Antigen Binding Domain Constructs
[0182] In general, the disclosure features Fc-antigen binding domain constructs having 2-10 Fc domains and one or more antigen binding domains attached. These may have greater binding affinity and/or avidity than a single wild-type Fc domain for an Fc receptor, e.g., FcγRIIIa. The disclosure discloses methods of engineering amino acids at the interface of two interacting C.sub.H3 antibody constant domains such that the two Fc domain monomers of an Fc domain selectively form a dimer with each other, thus preventing the formation of unwanted multimers or aggregates. An Fc-antigen binding domain construct includes an even number of Fc domain monomers, with each pair of Fc domain monomers forming an Fc domain. An Fc-antigen binding domain construct includes, at a minimum, two functional Fc domains formed from dimer of four Fc domain monomers and one antigen binding domain. The antigen binding domain may be joined to an Fc domain e.g., with a linker, a spacer, a peptide bond, a chemical bond or chemical moiety. In some embodiments, the disclosure relates to methods of engineering one set of amino acid substitutions selected from Tables 3 and 4 at the interface of a first pair of two interacting C.sub.H3 antibody constant domains, and engineering a second set of amino acid substitutions selected from Tables 3 and 4, different from the first set of amino acid substitutions, at the interface of a second pair of two interacting CH3 antibody constant domains, such that the first pair of two Fc domain monomers of an Fc domain selectively form a dimer with each other and the second pair of two Fc domain monomers of an Fc domain selectively form a dimer with each other, thus preventing the formation of unwanted multimers or aggregates.
[0183] The Fc-antigen binding domain constructs can be assembled in many ways. The Fc-antigen binding domain constructs can be assembled from asymmetrical tandem Fc domains. The Fc-antigen binding domain constructs can be assembled from singly branched Fc domains, where the branch point is at the N-terminal Fc domain. The Fc-antigen binding domain constructs can be assembled from singly branched Fc domains, where the branch point is at the C-terminal Fc domain. The Fc-antigen binding domain constructs can be assembled from singly branched Fc domains, where the branch point is neither at the N- or C-terminal Fc domain. The Fc-antigen binding domain constructs can be assembled to form bispecific constructs using long and short chains with different antigen binding domain sequences. The Fc-antigen binding domain constructs can be assembled to form bispecific and trispecific constructs using chains with different sets of heterodimerization mutations and different antigen binding domains. A bispecific Fc-antigen binding domain construct includes two different antigen biding domains. A trispecific Fc-antigen binding domain construct includes three different antigen binding domains.
[0184] The antigen binding domain can be joined to the Fc-antigen binding domain construct in many ways. The antigen binding domain can be expressed as a fusion protein of an Fc chain. The heavy chain component of the antigen can be expressed as a fusion protein of an Fc chain and the light chain component can be expressed as a separate polypeptide (
[0185] In some embodiments, one or more Fc polypeptides in an Fc-antigen binding domain construct lack a C-terminal lysine residue. In some embodiments, all of the Fc polypeptides in an Fc-antigen binding domain construct lack a C-terminal lysine residue. In some embodiments, the absence of a C-terminal lysine in one or more Fc polypeptides in an Fc-antigen binding domain construct may improve the homogeneity of a population of an Fc-antigen binding domain construct (e.g., an Fc-antigen binding domain construct having three Fc domains), e.g., a population of an Fc-antigen binding domain construct having three Fc domains that is at least 85%, 90%, 95%, 98%, or 99% homogeneous.
[0186] In some embodiments, the N-terminal Asp in one or more of the Fc-antigen binding domain polypeptides described herein may be mutated to Gln.
[0187] For the exemplary Fc-antigen binding domain constructs described in the Examples herein, Fc-antigen binding domain constructs may contain the E357K and K370D charge pairs in the Knobs and Holes subunits, respectively. Fc-antigen binding domain constructs 29-42 can use orthogonal electrostatic steering mutations that may contain E357K and K370D pairings, and also could include additional steering mutations. For Fc-antigen binding constructs 29-42 with orthogonal knobs and holes electrostatic steering mutations are required all but one of the orthogonal pairs, and may be included in all of the orthogonal pairs.
[0188] In some embodiments, if two orthogonal knobs and holes are required, the electrostatic steering modification for Knob1 may be E357K and the electrostatic steering modification for Hole1 may be K370D, and the electrostatic steering modification for Knob2 may be K370D and the electrostatic steering modification for Hole2 may be E357K. If a third orthogonal knob and hole is needed (e.g. for a tri-specific antibody) electrostatic steering modifications E357K and D399K may be added for Knob3 and electrostatic steering modifications K370D and K409D may be added for Hole3 or electrostatic steering modifications K370D and K409D may be added for Knob3 and electrostatic steering modifications E357K and D399K may be added for Hole3.
[0189] Any one of the exemplary Fc-antigen binding domain constructs described herein (e.g. Fc-antigen binding domain constructs 1-42) can have enhanced effector function in an antibody-dependent cytotoxicity (ADCC) assay, an antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) assay relative to a construct having a single Fc domain and the antigen binding domain, or can include a biological activity that is not exhibited by a construct having a single Fc domain and the antigen binding domain.
IX. Host Cells and Protein Production
[0190] In the present disclosure, a host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the polypeptides and constructs described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells can be of mammalian, bacterial, fungal or insect origin. Mammalian host cells include, but are not limited to, CHO (or CHO-derived cell strains, e.g., CHO-K1, CHO-DXB11 CHO-DG44), murine host cells (e.g., NSO, Sp2/0), VERY, HEK (e.g., HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells. Host cells can also be chosen that modulate the expression of the protein constructs, or modify and process the protein product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of protein products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the protein expressed.
[0191] For expression and secretion of protein products from their corresponding DNA plasmid constructs, host cells may be transfected or transformed with DNA controlled by appropriate expression control elements known in the art, including promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods for expression of therapeutic proteins are known in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 edition (Jul. 20, 2004); Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 edition (Jun. 28, 2012).
[0192] In some embodiments, at least 50% of the Fc-antigen binding domain constructs that are produced by a host cell transfected with DNA plasmid constructs encoding the polypeptides that assemble into the Fc construct, e.g., in the cell culture supernatant, are structurally identical (on a molar basis), e.g., 50%, 60%, 70%, 80%, 90%, 95%, 100% of the Fc constructs are structurally identical.
X. Afucosylation
[0193] Each Fc monomer includes an N-glycosylation site at Asn 297. The glycan can be present in a number of different forms on a given Fc monomer. In a composition containing antibodies or the antigen-binding Fc constructs described herein, the glycans can be quite heterogeneous and the nature of the glycan present can depend on, among other things, the type of cells used to produce the antibodies or antigen-binding Fc constructs, the growth conditions for the cells (including the growth media) and post-production purification. In various instances, compositions containing a construct described herein are afucosylated to at least some extent. For example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 95% of the glycans (e.g., the Fc glycans) present in the composition lack a fucose residue. Thus, 5%-60%, 5%-50%, 5%-40%, 10%-50%, 10%-50%, 10%-40%, 20%-50%, or 20%-40% of the glycans lack a fucose residue. Compositions that are afucosylated to at least some extent can be produced by culturing cells producing the antibody in the presence of 1,3,4-Tri-O-acetyl-2-deoxy-2-fluoro-L-fucose inhibitor. Relatively afucosylated forms of the constructs and polypeptides described herein can be produced using a variety of other methods, including: expressing in cells with reduced or no expression of FUT8 and expressing in cells that overexpress beta-1,4-mannosylglycoprotein 4-beta-N-acetylglucosaminyltransferase (GnT-III).
XI. Purification
[0194] An Fc-antigen binding domain construct can be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity (e.g., Protein A affinity), and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, an Fc-antigen binding domain construct can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column with chromatography columns, filtration, ultra filtration, salting-out and dialysis procedures (see, e.g., Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; and Subramanian (ed.) Antibodies-Volume I-Production and Purification, Kluwer Academic/Plenum Publishers, New York (2004)).
[0195] In some instances, an Fc-antigen binding domain construct can be conjugated to one or more purification peptides to facilitate purification and isolation of the Fc-antigen binding domain construct from, e.g., a whole cell lysate mixture. In some embodiments, the purification peptide binds to another moiety that has a specific affinity for the purification peptide. In some embodiments, such moieties which specifically bind to the purification peptide are attached to a solid support, such as a matrix, a resin, or agarose beads. Examples of purification peptides that may be joined to an Fc-antigen binding domain construct include, but are not limited to, a hexa-histidine peptide (SEQ ID NO: 38), a FLAG peptide, a myc peptide, and a hemagglutinin (HA) peptide. A hexa-histidine peptide (SEQ ID NO: 38) (HHHHHH (SEQ ID NO: 38)) binds to nickel-functionalized agarose affinity column with micromolar affinity. In some embodiments, a FLAG peptide includes the sequence DYKDDDDK (SEQ ID NO: 39). In some embodiments, a FLAG peptide includes integer multiples of the sequence DYKDDDDK (SEQ ID NO: 39) in tandem series, e.g., 3xDYKDDDDK (SEQ ID NO: 261). In some embodiments, a myc peptide includes the sequence EQKLISEEDL (SEQ ID NO: 40). In some embodiments, a myc peptide includes integer multiples of the sequence EQKLISEEDL (SEQ ID NO: 40) in tandem series, e.g., 3xEQKLISEEDL (SEQ ID NO: 262). In some embodiments, an HA peptide includes the sequence YPYDVPDYA (SEQ ID NO: 41). In some embodiments, an HA peptide includes integer multiples of the sequence YPYDVPDYA (SEQ ID NO: 41) in tandem series, e.g., 3xYPYDVPDYA (SEQ ID NO: 263). Antibodies that specifically recognize and bind to the FLAG, myc, or HA purification peptide are well-known in the art and often commercially available. A solid support (e.g., a matrix, a resin, or agarose beads) functionalized with these antibodies may be used to purify an Fc-antigen binding domain construct that includes a FLAG, myc, or HA peptide.
[0196] For the Fc-antigen binding domain constructs, Protein A column chromatography may be employed as a purification process. Protein A ligands interact with Fc-antigen binding domain constructs through the Fc region, making Protein A chromatography a highly selective capture process that is able to remove most of the host cell proteins. In the present disclosure, Fc-antigen binding domain constructs may be purified using Protein A column chromatography as described in Examples 2-3.
[0197] In some embodiments, use of the heterodimerizing and/or homodimerizing domains described herein allow for the preparation of an Fc-antigen binding domain construct with 60% or more purity, i.e., wherein 60% or more of the protein construct material produced in cells is of the desired Fc construct structure, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the protein construct material in a preparation is of the desired Fc construct structure. In some embodiments, less than 30% of the protein construct material in a preparation of an Fc-antigen binding domain construct is of an undesired Fc construct structure (e.g., a higher order species of the construct, as described in Example 1), e.g., 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of the protein construct material in a preparation is of an undesired Fc construct structure. In some embodiments, the final purity of an Fc-antigen binding domain construct, after further purification using one or more known methods of purification (e.g., Protein A affinity purification), can be 80% or more, i.e., wherein 80% or more of the purified protein construct material is of the desired Fc construct structure, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the protein construct material in a preparation is of the desired Fc construct structure. In some embodiments, less than 15% of protein construct material in a preparation of an Fc-antigen binding domain construct that is further purified using one or more known methods of purification (e.g., Protein A affinity purification) is of an undesired Fc construct structure (e.g., a higher order species of the construct, as described in Example 1), e.g.,15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of the protein construct material in the preparation is of an undesired Fc construct structure.
XII. Pharmaceutical Compositions/Preparations
[0198] The disclosure features pharmaceutical compositions that include one or more Fc-antigen binding domain constructs described herein. In one embodiment, a pharmaceutical composition includes a substantially homogenous population of Fc-antigen binding domain constructs that are identical or substantially identical in structure. In various examples, the pharmaceutical composition includes a substantially homogenous population of any one of Fc-antigen binding domain constructs 1-42.
[0199] A therapeutic protein construct, e.g., an Fc-antigen binding domain construct described herein (e.g., an Fc-antigen binding domain construct having three Fc domains), of the present disclosure can be incorporated into a pharmaceutical composition. Pharmaceutical compositions including therapeutic proteins can be formulated by methods know to those skilled in the art. The pharmaceutical composition can be administered parenterally in the form of an injectable formulation including a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated by suitably combining the Fc-antigen binding domain construct with pharmaceutically acceptable vehicles or media, such as sterile water for injection (WFI), physiological saline, emulsifier, suspension agent, surfactant, stabilizer, diluent, binder, excipient, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided.
[0200] The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™ HCO-50, and the like commonly known in the art. Formulation methods for therapeutic protein products are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2d ed.) Taylor & Francis Group, CRC Press (2006).
XIII. Methods of Treatment and Dosage
[0201] The constructs described herein can be used to treat disorders that are treated by the antibody from which the antigen binding domain is derived. For example, when the construct has an antigen binding domain that recognizes CD38, the construct can be used to treat a variety of cancers (e.g., hematologic malignancies and solid tumors) and autoimmune diseases. The cancer can be one that is resistant to a therapeutic anti-CD38 monoclonal antibody treatment. The cancer can be selected from: gastric cancer, breast cancer, colon cancer, lung cancer, mantle cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, NK cell leukemia, NK/T-cell lymphoma, chronic lymphocytic leukemia, plasma cell leukemia, and multiple myeloma. The constructs can also be used to treat: Amyloid light chain Amyloidosis, Castleman's disease, Monoclonal gammopathy of undetermined significance (MGUS), Biclonal gammopathy of undetermined significance, Heavy chain diseases, Solitary plasmacytome, Extramedullary plasmacytoma. In some cases, the constructs can be used to augment immunoregulatory functions against cancer cells by immune complex mediated induction of preventative and/or therapeutic vaccinal effects. CD38 targeted constructs can also be used to treat: plasma cell dyscrasias or monoclonal gammopathies such as: Light chain deposition disease, Membranoproliferative Glomerulonephritis (MGRS), Autoimmune hemolytic anemia, Tempi Syndrome (Telangiectasia-Erythrocytosis-Monoclonal Gammopathy Perinephric-Fluid Collections-Intrapulmonary Shunting), Rheumatoid Arthritis, Lupus Erythematosus POEMS Syndrome (Polyneuropathy-Organomegaly-Endocrinopathy-Monoclonal plasmaproliferative disorder-Skin) and Waldenström Macroglobulinemia
[0202] The constructs can be used to treat autoantibody-mediated diseases such as: Myasthenia Gravis (MG), MuSK-MG, Myocarditis, Lambert Eaton, Myasthenic Syndrome, Neuromyotonia, Neuromyelitis optica, Narcolepsy, Acute motor axonal neuropathy, Guillain-Barré syndrome, Fisher Syndrome, Acute Sensory Ataxic Neuropathy, Paraneoplastic Stiff Person Syndrome, Chronic Neuropathy, Peripheral Neuropathy, Acute disseminated encephalomyelitis, Multiple sclerosis, Goodpasture Syndrome, Membranous Nephropathy, Glomerulonephritis, Pulmonary Alveolar Proteinosis, CIPD, Autoimmune hemolytic anemia, Autoimmune Thrombocytopenic purpura, Pemphigus vulgaris, Pemphigus foliaceus, Bullous pemphigoid, pemphigoid gestationis, Epidermolysis bullosa aquisita, Neonatal lupus erythematosus, Dermatitis herpetiformis, Graves Disease, Addison's Disease, Ovarian insufficiency, Autoimune Orchitis, Sjogren's Disease, Autoimmune gastritis, Rheumatoid Arthritis, SLE, Dry eye disease, Vasulitis (Acute), Carditis, and Antibody-mediated rejection.
[0203] The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, oral dosage forms such as ingestible solutions, drug release capsules, and the like. The appropriate dosage for the individual subject depends on the therapeutic objectives, the route of administration, and the condition of the patient. Generally, recombinant proteins are dosed at 1-200 mg/kg, e.g., 1-100 mg/kg, e.g., 20-100 mg/kg. Accordingly, it will be necessary for a healthcare provider to tailor and titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
XIV. Complement-Dependent Cytotoxicity (CDC)
[0204] Fc-antigen binding domain constructs described in this disclosure are able to activate various Fc receptor mediated effector functions. One component of the immune system is the complement-dependent cytotoxicity (CDC) system, a part of the innate immune system that enhances the ability of antibodies and phagocytic cells to clear foreign pathogens. Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway, all of which entail a set of complex activation and signaling cascades. In the classical complement pathway, IgG or IgM trigger complement activation. The C1q protein binds to these antibodies after they have bound an antigen, forming the C1 complex. This complex generates C1s esterase, which cleaves and activates the C4 and C2 proteins into C4a and C4b, and C2a and C2b. The C2a and C4b fragments then form a protein complex called C3 convertase, which cleaves C3 into C3a and C3b, leading to a signal amplification and formation of the membrane attack complex.
[0205] The Fc-antigen binding domain constructs of this disclosure are able to enhance CDC activity by the immune system.
[0206] CDC may be evaluated by using a colorimetric assay in which Raji cells (ATCC) are coated with a serially diluted antibody, Fc-antigen binding domain construct, or IVIg. Human serum complement (Quidel) can be added to all wells at 25% v/v and incubated for 2 h at 37° C. Cells can be incubated for 12 h at 37° C. after addition of WST-1 cell proliferation reagent (Roche Applied Science). Plates can then be placed on a shaker for 2 min and absorbance at 450 nm can be measured.
XV. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
[0207] The Fc-antigen binding domain constructs of this disclosure are also able to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) activity by the immune system. ADCC is a part of the adaptive immune system where antibodies bind surface antigens of foreign pathogens and target them for death. ADCC involves activation of natural killer (NK) cells by antibodies. NK cells express Fc receptors, which bind to Fc portions of antibodies such as IgG and IgM. When the antibodies are bound to the surface of a pathogen-infected target cell, they then subsequently bind the NK cells and activate them. The NK cells release cytokines such as IFN-γ, and proteins such as perforin and granzymes. Perforin is a pore forming cytolysin that oligomerizes in the presence of calcium. Granzymes are serine proteases that induce programmed cell death in target cells. In addition to NK cells, macrophages, neutrophils and eosinophils can also mediate ADCC.
[0208] ADCC may be evaluated using a luminescence assay. Human primary NK effector cells (Hemacare) are thawed and rested overnight at 37° C. in lymphocyte growth medium-3 (Lonza) at 5×10.sup.5/mL. The next day, the human lymphoblastoid cell line Raji target cells (ATCC CCL-86) are harvested, resuspended in assay media (phenol red free RPMI, 10% FBSΔ, GlutaMAX™), and plated in the presence of various concentrations of each probe of interest for 30 minutes at 37° C. The rested NK cells are then harvested, resuspended in assay media, and added to the plates containing the anti-CD20 coated Raji cells. The plates are incubated at 37° C. for 6 hours with the final ratio of effector-to-target cells at 5:1 (5×10.sup.4 NK cells: 1×10.sup.4 Raji).
[0209] The CytoTox-Glo™ Cytotoxicity Assay kit (Promega) is used to determined ADCC activity. The CytoTox-Glo™ assay uses a luminogenic peptide substrate to measure dead cell protease activity which is released by cells that have lost membrane integrity e.g. lysed Raji cells. After the 6 hour incubation period, the prepared reagent (substrate) is added to each well of the plate and placed on an orbital plate shaker for 15 minutes at room temperature. Luminescence is measured using the PHERAstar F5 plate reader (BMG Labtech). The data is analyzed after the readings from the control conditions (NK cells+Raji only) are subtracted from the test conditions to eliminate background.
XVI. Antibody-Dependent Cellular Phagocytosis (ADCP)
[0210] The Fc-antigen binding domain constructs of this disclosure are also able to enhance antibody-dependent cellular phagocytosis (ADCP) activity by the immune system. ADCP, also known as antibody opsonization, is the process by which a pathogen is marked for ingestion and elimination by a phagocyte. Phagocytes are cells that protect the body by ingesting harmful foreign pathogens and dead or dying cells. The process is activated by pathogen-associated molecular patterns (PAMPS), which leads to NF-κB activation. Opsonins such as C3b and antibodies can then attach to target pathogens. When a target is coated in opsonin, the Fc domains attract phagocytes via their Fc receptors. The phagocytes then engulf the cells, and the phagosome of ingested material is fused with the lysosome. The subsequent phagolysosome then proteolytically digests the cellular material.
[0211] ADCP may be evaluated using a bioluminescence assay. Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. ADCP can be mediated by monocytes, macrophages, neutrophils and dendritic cells via FcγRIIa (CD32a), FcγRI (CD64), and FcγRIIIa (CD16a). All three receptors can participate in antibody recognition, immune receptor clustering, and signaling events that result in ADCP; however, blocking studies suggest that
[0212] FcγRIIa is the predominant Fcγ receptor involved in this process.
[0213] The FcγRIIa-H ADCP Reporter Bioassay is a bioluminescent cell-based assay that can be used to measure the potency and stability of antibodies and other biologics with Fc domains that specifically bind and activate FcγRIIa. The assay consists of a genetically engineered Jurkat T cell line that expresses the high-affinity human FcγRIIa-H variant that contains a Histidine (H) at amino acid 131 and a luciferase reporter driven by an NFAT-response element (NFAT-RE).
[0214] When co-cultured with a target cell and relevant antibody, the FcγRIIa-H effector cells bind the Fc domain of the antibody, resulting in FcγRIIa signaling and NFAT-RE-mediated luciferase activity. The bioluminescent signal is detected and quantified with a Luciferase assay and a standard luminometer.
EXAMPLES
[0215] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.
Example 1. Use of Orthogonal Heterodimerizing Domains to Control the Assembly of Linear Fc-Antigen Domain Containing Polypeptides
[0216] A variety of approaches to appending Fc domains to the C-termini of antibodies have been described, including in the production of tandem Fc constructs with and without peptide linkers between Fc domains (see, e.g., Nagashima et al., Mol Immunol, 45:2752-63, 2008, and Wang et al. MAbs, 9:393-403, 2017). However, methods described in the scientific literature for making antibody constructs with multiple Fc domains are limited in their effectiveness because these methods result in the production of numerous undesired species of Fc domain containing proteins. These species have different molecular weights that result from uncontrolled off-register association of polypeptide chains during product production, resulting in a ladder of molecular weights (see, e.g., Nagashima et al., Mol Immunol, 45:2752-63, 2008, and Wang et al. MAbs, 9:393-403, 2017).
[0217] The use of orthogonal heterodimerization domains allowed for the production of antibody-like structures with tandem Fc extensions without also generating large amounts of higher order species (HOS).
[0218] Examples 2 and 3 describe the production of orthogonal linear Fc-antigen domain binding constructs that correspond to the structures depicted in the schematics of
Example 2. Design and Purification of Fc-Antigen Binding Domain Construct 43 with an Anti-CD20 Antigen Binding Domain or an Anti-PD-L1 Antigen Binding Domain
[0219] Fc-antigen binding domain constructs are designed to increase folding efficiencies, to minimize uncontrolled association of subunits, which may create unwanted high molecular weight oligomers and multimers, and to generate compositions for pharmaceutical use that are substantially homogenous (e.g., at least 85%, 90%, 95%, 98%, or 99% homogeneous). With these goals in mind, an unbranched construct formed from tandem Fc domains (
[0220] and two copies of either an anti-CD20 light chain polypeptide (SEQ ID NO: 61) or an anti-PD-L1 light chain polypeptide (SEQ ID NO: 49), respectively. The long Fc chain contains two Fc domain monomers in a tandem series, each with a different protuberance-forming mutations selected from Table 3 (heterodimerization mutations), and/or different reverse charge mutation selected from Table 4, in a tandem series with an antigen binding domain at the N-terminus. The first short Fc chain contains an Fc domain monomer with a first set of cavity-forming mutations selected from Table 3 and/or one or more reverse charge mutation selected from Table 4 (wherein the mutations are different from mutations in the second short Fc chain). The second short Fc chain contains an Fc domain monomer with a second set of cavity-forming mutations selected from Table 3 and/or one or more reverse charge mutation selected from Table 4 (wherein the mutations are different from the first set off mutations in the first short Fc chain), and an antigen binding domain at the N-terminus.
[0221] In this case, the long Fc chain contains an Fc domain monomer with D356K and D399K charge mutations in a tandem series with an Fc domain monomer with S354C and T366W protuberance-forming mutations and a E357K charge mutation, and either anti-CD20 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 43 (CD20) or anti-PD-L1 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 43 (PD-L1)). The first short Fc chain contains an Fc domain monomer with K392D and K409D charge mutations. The second short Fc chain contains an Fc domain monomer with Y349C, T366S, L368A and Y407V cavity-forming mutations and a K370D charge mutation, and either anti-CD20 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 43 (CD20)) or anti-PD-L1 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 43 (PD-L1)).
TABLE-US-00011 TABLE 8 Construct 43 (CD20) and Construct 43 (PD-L1) sequences Long Fc chain Second Short Fc chain (with anti-CD20 or (with anti-CD20 or anti-PD-L1 VH and anti-PD-L1 VH and Construct Light chain CH1) First Short Fc chain CH1) Construct SEQ ID NO: 61 SEQ ID NO: 234 SEQ ID NO: 236 SEQ ID NO: 67 43 (CD20) DIVMTQTPLSLPVTPGE QVQLVQSGAEVKKPGS DKTHTCPPCPAPELLGG QVQLVQSGAEVKKPGS PASISCRSSKSLLHSNGI SVKVSCKASGYAFSYSW PSVFLFPPKPKDTLMISR SVKVSCKASGYAFSYSW TYLYWYLQKPGQSPQL INWVRQAPGQGLEW TPEVTCVVVDVSHEDP INWVRQAPGQGLEW LIYQMSNLVSGVPDRFS MGRIFPGDGDTDYNGK EVKFNWYVDGVEVHN MGRIFPGDGDTDYNGK GSGSGTDFTLKISRVEA FKGRVTITADKSTSTAY AKTKPREEQYNSTYRVV FKGRVTITADKSTSTAY EDVGVYYCAQNLELPYT MELSSLRSEDTAVYYCA SVLTVLHQDWLNGKEY MELSSLRSEDTAVYYCA FGGGTKVEIKRTVAAPS RNVFDGYWLVYWGQG KCKVSNKALPAPIEKTIS RNVFDGYWLVYWGQG VFIFPPSDEQLKSGTASV TLVTVSSASTKGPSVFPL KAKGQPREPQVYTLPPS TLVTVSSASTKGPSVFPL VCLLNNFYPREAKVQW APSSKSTSGGTAALGCL RDELTKNQVSLTCLVKG APSSKSTSGGTAALGCL KVDNALQSGNSQESVT VKDYFPEPVTVSWNSG FYPSDIAVEWESNGQP VKDYFPEPVTVSWNSG EQDSKDSTYSLSSTLTLS ALTSGVHTFPAVLQSSG ENNYDTTPPVLDSDGSF ALTSGVHTFPAVLQSSG KADYEKHKVYACEVTH LYSLSSVVTVPSSSLGTQ FLYSDLTVDKSRWQQG LYSLSSVVTVPSSSLGTQ QGLSSPVTKSFNRGEC TYICNVNHKPSNTKVDK NVFSCSVMHEALHNHY TYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCP TQKSLSLSPG KVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKP APELLGGPSVFLFPPKPK KDTLMISRTPEVTCVVV DTLMISRTPEVTCVVVD DVSHEDPEVKFNWYVD VSHEDPEVKFNWYVDG GVEVHNAKTKPREEQY VEVHNAKTKPREEQYN NSTYRVVSVLTVLHQD STYRVVSVLTVLHQDW WLNGKEYKCKVSNKAL LNGKEYKCKVSNKALPA PAPIEKTISKAKGQPREP PIEKTISKAKGQPREPQ QVYTLPPCRDKLTKNQ VCTLPPSRDELTKNQVS VSLWCLVKGFYPSDIAV LSCAVDGFYPSDIAVEW EWESNGQPENNYKTTP ESNGQPENNYKTTPPV PVLDSDGSFFLYSKLTV LDSDGSFFLVSKLTVDKS DKSRWQQGNVFSCSV RWQQGNVFSCSVMHE MHEALHNHYTQKSLSL ALHNHYTQKSLSLSPG SPGKGGGGGGGGGGG GGGGGGGGGDKTHTC PPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFN WYVDGVEVHNAKTKP REEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVS NKALPAPIEKTISKAKG QPREPQVYTLPPSRKEL TKNQVSLTCLVKGFYPS DIAVEWESNGQPENNY KTTPPVLKSDGSFFLYSK LTVDKSRWQQGNVFSC SVMHEALHNHYTQKSL SLSPGQ Construct SEQ ID NO: 49 SEQ ID NO: 235 SEQ ID NO: 236 SEQ ID NO: 68 43 (PD-L1) QSALTQPASVSGSPGQ EVQLLESGGGLVQPGG DKTHTCPPCPAPELLGG EVQLLESGGGLVQPGG SITISCTGTSSDVGGYNY SLRLSCAASGFTFSSYIM PSVFLFPPKPKDTLMISR SLRLSCAASGFTFSSYIM VSWYQQHPGKAPKLM MWVRQAPGKGLEWV TPEVTCVVVDVSHEDP MWVRQAPGKGLEWV IYDVSNRPSGVSNRFSG SSIYPSGGITFYADTVKG EVKFNWYVDGVEVHN SSIYPSGGITFYADTVKG SKSGNTASLTISGLQAE RFTISRDNSKNTLYLQM AKTKPREEQYNSTYRVV RFTISRDNSKNTLYLQM DEADYYCSSYTSSSTRVF NSLRAEDTAVYYCARIK SVLTVLHQDWLNGKEY NSLRAEDTAVYYCARIK GTGTKVTVLGQPKANP LGTVITVDYWGQGTLV KCKVSNKALPAPIEKTIS LGTVTIVDYWGQGTLV TVTLFPPSSEELQANKA TVSSASTKGPSVFPLAPS KAKGQPREPQVYTLPPS TVSSASTKGPSVFPLAPS TLVCLISDFYPGAVTVA SKSTSGGTAALGCLVKD RDELTKNQVSLTCLVKG SKSTSGGTAALGCLVKD WKADGSPVKAGVETTK YFPEPVTVSWNSGALTS FYPSDIAVEWESNGQP YFPEPVTVSWNSGALTS PSKQSNNKYAASSYLSL GVHTFPAVLQSSGLYSL ENNYDTTPPVLDSDGSF GVHTFPAVLQSSGLYSL TPEQWKSHRSYSCQVT SSVVTVPSSSLGTQTYIC FLYSDLTVDKSRWQQG SSVVTVPSSSLGTQTYIC HEGSTVEKTVAPTECS NVNHKPSNTKVDKKVE NVFSCSVMHEALHNHY NVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPE TQKSLSLSPG PKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTL LLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVS MISRTPEVTCVVVDVSH HEDPEVKFNWYVDGV EDPEVKFNWYVDGVEV EVHNAKTKPREEQYNS HNAKTKPREEQYNSTY TYRVVSVLTVLHQDWL RVVSVLTVLHQDWLNG NGKEYKCKVSNKALPAP KEYKCKVSNKALPAPIEK IEKTISKAKGQPREPQV TISKAKGQPREPQVCTL YTLPPCRDKLTKNQVSL PPSRDELTKNQVSLSCA WCLVKGFYPSDIAVEW VDGFYPSDIAVEWESN ESNGQPENNYKTTPPV GQPENNYKTTPPVLDS LDSDGSFFLYSKLTVDKS DGSFFLVSKLTVDKSRW RWQQGNVFSCSVMHE QQGNVFSCSVMHEAL ALHNHYTQKSLSLSPGK HNHYTQKSLSLSPG GGGGGGGGGGGGGG GGGGGGDKTHTCPPCP APELLGGPSVFLFPPKP KDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVD GVEVHNAKTKPREEQY NSTYRVVSVLTVLHQD WLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREP QVYTLPPSRKELTKNQV SLTCLVKGFYPSDIAVE WESNGQPENNYKTTPP VLKSDGSFFLYSKLTVDK SRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG
[0222] Cell Culture
[0223] DNA sequences were optimized for expression in mammalian cells and cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains were encoded by multiple plasmids.
[0224] Protein Purification
[0225] The expressed proteins were purified from the cell culture supernatant by Protein A-based affinity column chromatography, using a Poros MabCapture A (LifeTechnologies) column. Captured Fc-antigen binding domain constructs were washed with phosphate buffered saline (PBS, pH 7.0) after loading and further washed with intermediate wash buffer 50 mM citrate buffer (pH 5.5) to remove additional process related impurities. The bound Fc construct material was eluted with 100 mM glycine, pH 3 and the eluate was quickly neutralized by the addition of 1 M TRIS pH 7.4 then centrifuged and sterile filtered through a 0.2 μm filter.
[0226] The proteins were further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample was diluted (1:3) in the equilibration buffer for loading. The sample was eluted using a 12-15CV's linear gradient from 50 mM MES (100% A) to 400 mM sodium chloride, pH 6 (100% B) as the elution buffer. All fractions collected during elution were analyzed by analytical size exclusion chromatography (SEC) and target fractions were pooled to produce the purified Fc construct material. After ion exchange, the target fraction was buffer exchanged into 1X-PBS buffer using a 30 kDa cut-off polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. The samples were concentrated to approximately 10-15 mg/mL and sterile filtered through a 0.2 μm filter.
[0227] Non-Reducing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Samples were denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95° C. for 10 min. Samples were run on a Criterion TGX stain-free gel (4-15% polyacrylamide, Bio-Rad). Protein bands were visualized by UV illumination or Coommassie blue staining. Gels were imaged by ChemiDoc MP Imaging System (Bio-Rad). Quantification of bands was performed using Imagelab 4.0.1 software (Bio-Rad).
Example 3. Design and Purification of Fc-Antigen Binding Domain Construct 44 with an Anti-CD20 Antigen Binding Domain or an Anti-PD-L1 Antigen Binding Domain
[0228] An unbranched construct formed from tandem Fc domains (
[0229] In this case, the long Fc chain contains two Fc domain monomers, each with D356K and D399K charge mutations in a tandem series with an Fc domain monomer with S354C and T366W protuberance-forming mutations and a E357K charge mutation, and either anti-CD20 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 44 (CD20)) or anti-PD-L1 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 44 (PD-L1)). The first short Fc chain contains an Fc domain monomer with K392D and K409D charge mutations. The second short Fc chain contains an Fc domain monomer with Y349C, T366S, L368A and Y407V cavity-forming mutations and a K370D charge mutation, and either anti-CD20 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 44 (CD20)) or anti-PD-L1 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct 44 (PD-L1)).
TABLE-US-00012 TABLE 9 Construct 44 (CD20) and Construct 44 (PD-L1) sequences Long Fc chain Second Short Fc chain (with anti-CD20 or (with anti-CD20 or anti-PD-L1 VH and anti-PD-L1 VH and Construct Light chain CH1) First Short Fc chain CH1) Construct SEQ ID NO: 61 SEQ ID NO: 237 SEQ ID NO: 63 SEQ ID NO: 67 44 (CD20) DIVMTQTPLSLPVTPGE QVQLVQSGAEVKKPGS DKTHTCPPCPAPELLGG QVQLVQSGAEVKKPGS PASISCRSSKSLLHSNGI SVKVSCKASGYAFSYSW PSVFLFPPKPKDTLMISR SVKVSCKASGYAFSYSW TYLYWYLQKPGQSPQL INWVRQAPGQGLEW TPEVTCVVVDVSHEDP INWVRQAPGQGLEW LIYQMSNLVSGVPDRFS MGRIFPGDGDTDYNGK EVKFNWYVDGVEVHN MGRIFPGDGDTDYNGK GSGSGTDFTLKISRVEA FKGRVTITADKSTSTAY AKTKPREEQYNSTYRVV FKGRVTITADKSTSTAY EDVGVYYCAQNLELPYT MELSSLRSEDTAVYYCA SVLTVLHQDWLNGKEY MELSSLRSEDTAVYYCA FGGGTKVEIKRTVAAPS RNVFDGYWLVYWGQG KCKVSNKALPAPIEKTIS RNVFDGYWLVYWGQG VFIFPPSDEQLKSGTASV TLVTVSSASTKGPSVFPL KAKGQPREPQVCTLPP TLVTVSSASTKGPSVFPL VCLLNNFYPREAKVQW APSSKSTSGGTAALGCL SRDELTKNQVSLSCAVD APSSKSTSGGTAALGCL KVDNALQSGNSQESVT VKDYFPEPVTVSWNSG GFYPSDIAVEWESNGQ VKDYFPEPVTVSWNSG EQDSKDSTYSLSSTLTLS ALTSGVHTFPAVLQSSG PENNYKTTPPVLDSDGS ALTSGVHTFPAVLQSSG KADYEKHKVYACEVTH LYSLSSVVTVPSSSLGTQ FFLVSKLTVDKSRWQQ LYSLSSVVTVPSSSLGTQ QGLSSPVTKSFNRGEC TYICNVNHKPSNTKVDK GNVFSCSVMHEALHN TYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCP HYTQKSLSLSPG KVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKP APELLGGPSVFLFPPKPK KDTLMISRTPEVTCVVV DTLMISRTPEVTCVVVD DVSHEDPEVKFNWYVD VSHEDPEVKFNWYVDG GVEVHNAKTKPREEQY VEVHNAKTKPREEQYN NSTYRVVSVLTVLHQD STYRVVSVLTVLHQDW WLNGKEYKCKVSNKAL LNGKEYKCKVSNKALPA PAPIEKTISKAKGQPREP PIEKTISKAKGQPREPQ QVYTLPPCRDKLTKNQ VCTLPPSRDELTKNQVS VSLWCLVKGFYPSDIAV LSCAVDGFYPSDIAVEW EWESNGQPENNYKTTP ESNGQPENNYKTTPPV PVLDSDGSFFLYSKLTV LDSDGSFFLVSKLTVDKS DKSRWQQGNVFSCSV RWQQGNVFSCSVMHE MHEALHNHYTQKSLSL ALHNHYTQKSLSLSPG SPGKGGGGGGGGGGG GGGGGGGGGDKTHTC PPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFN WYVDGVEVHNAKTKP REEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVS NKALPAPIEKTISKAKG QPREPQVYTLPPSRKEL TKNQVSLTCLVKGFYPS DIAVEWESNGQPENNY KTTPPVLKSDGSFFLYSK LTVDKSRWQQGNVFSC SVMHEALHNHYTQKSL SLSPGQKGGGGGGGG GGGGGGGGGGGGDK THTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEV KFNWYVDGVEVHNAK TKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRK ELTKNQVSLTCLVKGFY PSDIAVEWESNGQPEN NYKTTPPVLKSDGSFFL YSKLTVDKSRWQQGN VFSCSVMHEALHNHYT QKSLSLSPGQ Construct SEQ ID NO: 49 SEQ ID NO: 238 SEQ ID NO: 63 SEQ ID NO: 68 44 (PD-L1) QSALTQPASVSGSPGQ EVQLLESGGGLVQPGG DKTHTCPPCPAPELLGG EVQLLESGGGLVQPGG SITISCTGTSSDVGGYNY SLRLSCAASGFTFSSYIM PSVFLFPPKPKDTLMISR SLRLSCAASGFTFSSYIM VSWYQQHPGKAPKLM MWVRQAPGKGLEWV TPEVTCVVVDVSHEDP MWVRQAPGKGLEWV IYDVSNRPSGVSNRFSG SSIYPSGGITFYADTVKG EVKFNWYVDGVEVHN SSIYPSGGITFYADTVKG SKSGNTASLTISGLQAE RFTISRDNSKNTLYLQM AKTKPREEQYNSTYRVV RFTISRDNSKNTLYLQM DEADYYCSSYTSSSTRVF NSLRAEDTAVYYCARIK SVLTVLHQDWLNGKEY NSLRAEDTAVYYCARIK GTGTKVTVLGQPKANP LGTVITVDYWGQGTLV KCKVSNKALPAPIEKTIS LGTVTIVDYWGQGTLV TVTLFPPSSEELQANKA TVSSASTKGPSVFPLAPS KAKGQPREPQVCTLPP TVSSASTKGPSVFPLAPS TLVCLISDFYPGAVTVA SKSTSGGTAALGCLVKD SRDELTKNQVSLSCAVD SKSTSGGTAALGCLVKD WKADGSPVKAGVETTK YFPEPVTVSWNSGALTS GFYPSDIAVEWESNGQ YFPEPVTVSWNSGALTS PSKQSNNKYAASSYLSL GVHTFPAVLQSSGLYSL PENNYKTTPPVLDSDGS GVHTFPAVLQSSGLYSL TPEQWKSHRSYSCQVT SSVVTVPSSSLGTQTYIC FFLVSKLTVDKSRWQQ SSVVTVPSSSLGTQTYIC HEGSTVEKTVAPTECS NVNHKPSNTKVDKKVE GNVFSCSVMHEALHN NVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPE HYTQKSLSLSPG PKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTL LLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVS MISRTPEVTCVVVDVSH HEDPEVKFNWYVDGV EDPEVKFNWYVDGVEV EVHNAKTKPREEQYNS HNAKTKPREEQYNSTY TYRVVSVLTVLHQDWL RVVSVLTVLHQDWLNG NGKEYKCKVSNKALPAP KEYKCKVSNKALPAPIEK IEKTISKAKGQPREPQV TISKAKGQPREPQVCTL YTLPPCRDKLTKNQVSL PPSRDELTKNQVSLSCA WCLVKGFYPSDIAVEW VDGFYPSDIAVEWESN ESNGQPENNYKTTPPV GQPENNYKTTPPVLDS LDSDGSFFLYSKLTVDKS DGSFFLVSKLTVDKSRW RWQQGNVFSCSVMHE QQGNVFSCSVMHEAL ALHNHYTQKSLSLSPGK HNHYTQKSLSLSPG GGGGGGGGGGGGGG GGGGGGDKTHTCPPCP APELLGGPSVFLFPPKP KDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVD GVEVHNAKTKPREEQY NSTYRVVSVLTVLHQD WLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREP QVYTLPPSRKELTKNQV SLTCLVKGFYPSDIAVE WESNGQPENNYKTTPP VLKSDGSFFLYSKLTVDK SRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG KGGGGGGGGGGGGG GGGGGGGDKTHTCPP CPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWY VDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPRE PQVYTLPPSRKELTKNQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPP VLKSDGSFFLYSKLTVDK SRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG
[0230] Cell Culture
[0231] DNA sequences were optimized for expression in mammalian cells and cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains were encoded by multiple plasmids.
[0232] Protein Purification
[0233] The expressed proteins were purified from the cell culture supernatant by Protein A-based affinity column chromatography, using a Poros MabCapture A (LifeTechnologies) column. Captured Fc-antigen binding domain constructs were washed with phosphate buffered saline (PBS, pH 7.0) after loading and further washed with intermediate wash buffer 50 mM citrate buffer (pH 5.5) to remove additional process related impurities. The bound Fc construct material was eluted with 100 mM glycine, pH 3 and the eluate was quickly neutralized by the addition of 1 M TRIS pH 7.4 then centrifuged and sterile filtered through a 0.2 μm filter.
[0234] The proteins were further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample was diluted (1:3) in the equilibration buffer for loading. The sample was eluted using a 12-15CV′s linear gradient from 50 mM MES (100% A) to 400 mM sodium chloride, pH 6 (100%B) as the elution buffer. All fractions collected during elution were analyzed by analytical size exclusion chromatography (SEC) and target fractions were pooled to produce the purified Fc construct material.
[0235] After ion exchange, the target fraction was buffer exchanged into 1X-PBS buffer using a 30 kDa cut-off polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. The samples were concentrated to approximately 10-15 mg/mL and sterile filtered through a 0.2 pm filter.
[0236] Non-reducing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Samples were denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95 ° C. for 10 min. Samples were run on a Criterion TGX stain-free gel (4-15% polyacrylamide, Bio-Rad). Protein bands were visualized by UV illumination or Coommassie blue staining. Gels were imaged by ChemiDoc MP Imaging System (Bio-Rad). Quantification of bands was performed using Imagelab 4.0.1 software (Bio-Rad).
Example 4. Experimental Assays Used to Characterize Fc-Antigen Binding Domain Constructs
[0237] Peptide and Glycopeptide Liquid Chromatography-MS/MS
[0238] The proteins (Fc constructs) were diluted to 1 μg/pL in 6M guanidine (Sigma). Dithiothreitol (DTT) was added to a concentration of 10 mM, to reduce the disulfide bonds under denaturing conditions at 65° C. for 30 min. After cooling on ice, the samples were incubated with 30 mM iodoacetamide (IAM) for 1 h in the dark to alkylate (carbamidomethylate) the free thiols. The protein was then dialyzed across a 10-kDa membrane into 25 mM ammonium bicarbonate buffer (pH 7.8) to remove IAM, DTT and guanidine. The protein was digested with trypsin in a Barocycler (NEP 2320; Pressure Biosciences, Inc.). The pressure was cycled between 20,000 psi and ambient pressure at 37° C. for a total of 30 cycles in 1 h. LC-MS/MS analysis of the peptides was performed on an Ultimate 3000 (Dionex) Chromatography System and an Q-Exactive (Thermo Fisher Scientific) Mass Spectrometer. Peptides were separated on a BEH PepMap (Waters) Column using 0.1% FA in water and 0.1% FA in acetonitrile as the mobile phases.
[0239] Intact Mass Spectrometry
[0240] 50 μg of the protein (Fc construct) was buffer exchanged into 50 mM ammonium bicarbonate (pH 7.8) using 10 kDa spin filters (EMD Millipore) to a concentration of 1 μg/μL. 30 units PNGase F (Promega) was added to the sample and incubated at 37° C. for 5 hours. Separation was performed on a Waters Acquity C4 BEH column (1×100 mm, 1.7 um particle size, 300A pore size) using 0.1% FA in water and 0.1% FA in acetonitrile as the mobile phases. LC-MS was performed on an Ultimate 3000 (Dionex) Chromatography System and an Q-Exactive (Thermo Fisher Scientific) Mass Spectrometer. The spectra were deconvoluted using the default ReSpect method of Biopharma Finder (Thermo Fisher Scientific).
[0241] Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) Assay
[0242] Samples were diluted to 1 mg/mL and mixed with the HT Protein Express denaturing buffer (PerkinElmer). The mixture was incubated at 40° C. for 20 min. Samples were diluted with 70 μL of water and transferred to a 96-well plate. Samples were analyzed by a Caliper GXII instrument (PerkinElmer) equipped with the HT Protein Express LabChip (PerkinElmer). Fluorescence intensity was used to calculate the relative abundance of each size variant.
[0243] Non-Reducing SDS-PAGE
[0244] Samples are denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95° C. for 10 min. Samples are run on a Criterion TGX stain-free gel (4-15% polyacrylamide, Bio-Rad). Protein bands are visualized by UV illumination or Coommassie blue staining. Gels are imaged by ChemiDoc MP Imaging System (Bio-Rad). Quantification of bands is performed using Imagelab 4.0.1 software (Bio-Rad).
[0245] Complement Dependent Cytotoxicity (CDC)
[0246] CDC was evaluated by a colorimetric assay in which Raji cells (ATCC) were coated with serially diluted Rituximab, an Fc construct, or IVIg. Human serum complement (Quidel) was added to all wells at 25% v/v and incubated for 2 h at 37° C. Cells were incubated for 12 h at 37° C. after addition of WST-1 cell proliferation reagent (Roche Applied Science). Plates were placed on a shaker for 2 min and absorbance at 450 nm was measured.
Example 5. Complement-Dependent Cytotoxicity (CDC) Activation by Anti-CD20 Fc Constructs
[0247] A CDC assay was developed to test the degree to which anti-CD20 Fc constructs enhance CDC activity relative to an anti-CD20 monoclonal antibody, obinutuzumab. Anti-CD20 Fc constructs 43 and 44 having the Fab sequence (VL+CL, VH+CH1) of obinutuzumab were produced as described in Examples 2 and 3. Each anti-CD20 Fc construct, and the obinutuzumab monoclonal antibody, was tested in a CDC assay performed as follows:
[0248] Daudi cells grown in RPMI-1640 supplemented with 10% heat-inactivated FBS were pelleted, washed 1× with ice-cold PBS and resuspended in RPMI-1640 containing 0.1% BSA at a concentration of 1.0×10.sup.6 viable cells per mL. Fifty microliters of this cell suspension was added to all wells (except plate edges) of 96-well plates. Plates were kept on ice until all additions had been made. Test articles were serially diluted four-fold from a starting concentration of 450 nM in RPMI-1640+BSA. A total of ten concentrations was tested for each test article. Fifty microliters each was added to plated Daudi cells. Normal or C1q-depleted human complement serum (Quidel, San Diego, Calif.) was diluted 1:5 in RPMI-1640 +BSA. Fifty microliters each was added to plated Daudi cells. Six normal serum control wells received cells, media only (no treatment) and ⅕ normal serum (Normal Background). Three of these wells also received 16.5 μL Triton X-100 (Promega, Madison, Wis.) (Normal Lysis Control). C1q-depleted Background and Lysis Controls were similarly prepared. PBS was added to all plate edge wells. Plates were incubated for 2 h at 37° C. After 2 h, 50 μL pre-warmed Alamar blue (Thermo, Waltham, Mass.) was added to all wells (expect plate edges). Plates were returned to the incubator overnight (18 h at 37° C.). After 18 h fluorescence was measured in a FlexStation 3. Plates were top-read using 544/590 Ex/Em filters and Auto Cut-Off. Means were calculated for Normal Background, Normal Lysis Control, C1q-depleted Background and C1q-depleted Lysis Control wells. Percent cell lysis was calculated as:
% Cell Lysis=(RFU Test−RFU Background)/(RFU Lysis Control−RFU Background)*100. The EC50 (nM) was determined for each construct.
[0249] As depicted in Table 10, anti-CD20 Fc constructs induced CDC in Daudi cells and demonstrated greater potency in enhancing cytotoxicity relative to the obinutuzumab monoclonal antibody, as evidenced by lower EC50 values.
TABLE-US-00013 TABLE 10 Potency of anti-CD20 Fc constructs to induce CDC in Daudi cells EC50 (nM) Construct.sup.1 n Range Mean SD IgG1 Antibody, 5 38-65 47 11 Fucosylated Obinutuzumab S2L-AT-OBI 2 1.6-2.5 2.1 0.59 Construct 43 (anti-CD20) S3L-A22-OBI 2 9.8-12 11 1.5 Construct 44 (anti-CD20) .sup.1All constructs included G20 (SEQ ID NO: 23) linkers unless otherwise noted.
Example 6. Antibody-Dependent Cellular Phagocytosis (ADCP) activation by anti-CD20 Fc constructs
[0250] ADCP Reporter Assay
[0251] An ADCP reporter assay was developed to test the degree to which anti-CD20 Fc constructs activate FcγRIIa signaling, thereby enhancing ADCP activity, relative to an anti-CD20 monoclonal obinutuzumab antibody. Anti-CD20 Fc constructs 43 and 44 having the CDRs of obinutuzumab were produced as described in Examples 2 and 3. Each anti-CD20 Fc construct, and fucosylated and afucosylated obinutuzumab monoclonal antibodies, were tested in an ADCC reporter assay performed as follows:
[0252] Raji target cells (1.5×10.sup.4 cells/well) and Jurkat/FcγRIIa-H effector cells (Promega) (3.5×10.sup.4 cells/well) were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-CD20 Fc constructs. After incubation for 6 h at 37° C. in 5% CO2, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer's protocol using a PHERAstar FS luminometer (BMG LABTECH).
[0253] As depicted in Table 11, the anti-CD20 Fc constructs induced FcγRIIa signaling in an ADCP reporter assay and demonstrated greater potency in enhancing ADCP activity relative to the fucosylated obinutuzumab monoclonal antibody, as evidenced by lower EC50 values. Construct 44 also exhibited greater potency in the ADCP assay relative to afucosylated obinutuzumab monoclonal antibody.
TABLE-US-00014 TABLE 11 Potency of anti-CD20 Fc constructs to induce FcγRIIa signaling in an ADCP reporter assay EC50 (nM) Construct.sup.1 n Range Mean SD IgG1 Antibody, 6 4.5-10.8 7.1 2.2 Fucosylated IgG1 Antibody, 3 5.5-6.1 5.8 0.3 Afucosylated S2L-AT-OBI 1 7.8 7.8 N/A Construct 43 (anti-CD20) S3L-A22-OBI 1 0.17 0.17 N/A Construct 44 (anti-CD20) .sup.1All constructs included G20 (SEQ ID NO: 23) linkers unless otherwise noted.
Example 7. Antibody-Dependent Cellular Phagocytosis (ADCP) Activation by Anti-PD-L1 Fc Constructs
[0254] ADCP Reporter Assay
[0255] An ADCP reporter assay was developed to test the degree to which anti-PD-L1 Fc constructs activate FcγRIIa signaling, thereby enhancing ADCP activity, relative to an anti-PD-L1 monoclonal antibody, avelumab (Bavencio). Anti-PD-L1 Fc constructs 43 and 44 having the Fab sequence (VL+CL, VH+CH1) of avelumab were produced as described in Examples 2 and 3. Each anti-PD-L1 Fc construct, and fucosylated and afucosylated avelumab monoclonal antibodies, were tested in an ADCC reporter assay performed as follows:
[0256] Target HEK-PD-L1 cells (1.5×10.sup.4 cells/well) and effector Jurkat/FcγRIIa-H cells (Promega) (3.5×10.sup.4 cells/well) were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-PD-L1 Fc constructs. After incubation for 6 hours at 37° C. in 5% CO2, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer's protocol using a PHERAstar FS luminometer (BMG LABTECH).
[0257] As depicted in Table 12, anti-PD-L1 Fc constructs induced FcγRIIa signaling in an ADCP reporter assay.
TABLE-US-00015 TABLE 12 Potency of anti-PD-L1 Fc constructs to induce FcγRIIa signaling in an ADCP reporter assay Construct EC50 (nM) Number.sup.1 n Range Mean SD IgG1 Antibody, 6 No effect.sup.2 No N/A Fucosylated effect.sup.2 IgG1 Antibody, 1 No effect.sup.2 No N/A Afucosylated effect.sup.2 S2L-AA-AVE 1 0.037 0.037 N/A Construct 43 (anti-PD-L1) S3L-AA-AVE 1 0.033 0.033 N/A Construct 44 (anti-PD-L1) .sup.1All constructs included G20 (SEQ ID NO: 23) linkers unless otherwise noted. .sup.2Construct did not induce measurable FcyRIIa signaling under the assay conditions.
Example 8. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activation by Anti-CD20 Fc Constructs
[0258] ADCC Reporter Assay
[0259] An ADCC reporter assay was developed to test the degree to which anti-CD20 Fc constructs induce FcγRIIIa signaling and enhance ADCC activity relative to an anti-CD20 monoclonal antibody obinutuzumab. Anti-CD20 Fc constructs 43 and 44 having the Fab sequence (VL+CL, VH+CH1) of obinutuzumab were produced as described in Examples 2 and 3. Each anti-CD20 Fc construct, and fucosylated and afucosylated obinutuzumab monoclonal antibodies, were tested in an ADCC reporter assay performed as follows:
[0260] Raji target cells (1.25×10.sup.4 cells/well) and Jurkat/FcγRIIIa effector cells (Promega) (7.45×104 cells/well) were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-CD20 Fc constructs. After incubation for 6 hours at 37° C. in 5% CO2, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer's protocol using a PHERAstar FS luminometer (BMG LABTECH).
[0261] As depicted in Table 13, anti-CD20 Fc constructs induced FcγRIIIa signaling in an ADCC reporter assay.
TABLE-US-00016 TABLE 13 Potency of anti-CD20 Fc constructs to induce FcγRIIIa signaling in an ADCC reporter assay EC50 (nM) Construct.sup.1 n Range Mean SD IgG1 Antibody, 6 0.039-0.150 0.08 0.04 Fucosylated S2L-AT-OBI 1 0.86 0.86 N/A Construct 43 (anti-CD20) S3L-AA-OBI 1 0.055 0.055 N/A Construct 44 (anti-CD20) .sup.1All constructs included G20 (SEQ ID NO: 23) linkers unless otherwise noted
Example 9. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activation by Anti-PD-L1 Fc Constructs
[0262] ADCC Reporter Assay
[0263] An ADCC reporter assay was developed to test the degree to which anti-PD-L1 Fc constructs induce FcγRIIIa signaling and enhance ADCC activity relative to an anti-PD-L1 monoclonal antibody, avelumab (Bavencio). Anti-PD-L1 Fc constructs 43 and 44 having the Fab sequence (VL+CL, VH+CH1) of avelumab were produced as described in Examples 2 and 3. Each anti-PD-L1 Fc construct, and fucosylated and afucosylated avelumab monoclonal antibodies, were tested in an ADCC reporter assay performed as follows:
[0264] Target HEK-PD-L1 cells (1.25×10.sup.4 cells/well) and effector Jurkat/FcγRIIIa cells (Promega) (7.45 ×10.sup.4 cells/well) were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-PD-L1 constructs. After incubation for 6 hours at 37° C. in 5% CO2, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer's protocol using a PHERAstar FS luminometer (BMG LABTECH).
[0265] As depicted in Table 14, Fc construct 43 induced FcγRIIIa signaling in an ADCC reporter assay. Induction of FcγRIIIa signaling could not be determined for Fc construct 44 and the afucosylated monoclonal antibody using this assay.
TABLE-US-00017 TABLE 14 Potency of anti-PD-L1 Fc constructs to induce FcγRIIIa signaling in an ADCC reporter assay Construct EC50 (nM) Number.sup.1 n Range Mean SD IgG1 Antibody, 5 0.037-0.056 0.049 0.008 Fucosylated IgG1 Antibody, 1 Not Not N/A Afucosylated determined.sup.2 determined.sup.2 S2L-AA-AVE 1 0.028 0.028 N/A Construct 43 (anti-PD-L1) S3L-AA-AVE 1 Not Not N/A Construct 44 determined.sup.2 determined.sup.2 (anti-PD-L1) .sup.1All constructs included G20 (SEQ ID NO: 23) linkers unless otherwise noted. .sup.2Data could not be reliably fit to a four parameter logistic (4PL) curve.
Example 10: Activity of Anti-PD-L1 and Anti-CD20 Fc Constructs
[0266]
[0267]
[0268]
[0269]
[0270] The following methods were used in the studies described in Example 10.
[0271] SDS PAGE: Media supernatants and purified Fc constructs were denatured for 10 min at 95 ° C. in the presence of Laemmli buffer (Bio-Rad, Hercules, Calif.). Samples were separated on 4%-1 5% TGX stainfree acrylamide pre-cast gels (Bio-Rad) using the Bio-Rad Criterion gel electrophoresis vertical cell following the manufacturers instructions. Proteins were visualized by either rapid fluorescent detection or by staining with Coomassie R-250 brilliant blue stain (Bio-Rad). Images were acquired with the ChemiDoc MP imaging system (Bio-Rad).
[0272] Analytical size exclusion chromatography (SEC): Samples were analyzed at 1 mg/mL concentration on an Agilent 1200 system (Agilent Technologies, Santa Clara, Calif.) using a Zenix-C 4.6 c 300 mm 3 m/h particle size column (Sepax Technologies, Newark, Del.) at an isocratic flow of 0.35 mL/min with 150 mM sodium phosphate (pH 7.0) as the running buffer and column thermostated to 30° C. The total run time was around 12-1 5 min with UV detection at 280 nm. The totally excluded volume was at approximately 4 min.
[0273] CDC assay: The target cells used in the anti-CD20 CDC assay are the Daudi lymphoblastoid human B cell line. Daudi cells were removed from suspension culture by centrifugation and resuspended in X-VIVO 15 media at 6×105 cells/ml. Daudi cells were transferred to a 96 well flat-bottom assay plate in a volume of 100 m l per well (6×104 cells/well).Each of the anti-CD20 monoclonal antibodies (mAbs) and SIF Bodies were diluted to 3.33 mM in XVIVO15 media. Serial 1:3 dilutions were then performed with each of the anti-CD20 mAbs and SIFBodies in 1.5 ml polypropylene tubes resulting in an 11 point dilution series. Each dilution of the anti-CD20 mAbs and SIF Bodies was transferred at 50 m l/well to the appropriate wells in the assay plate. Immediately following the transfer of the anti-CD20 mAbs and SIF Bodies, 50 m l of normal human serum complement were transferred to each well of the assay plate. The assay plate was incubated at 37° C. and 5% CO2 for 2 h. Following the 2 h incubation, 20 m l of WST-1 proliferation reagent was added to each well of the assay plate. The plate was returned to the 37° C., 5% CO2 incubator for 14 h. Following the 14 h incubation, the plate was shaken for 1 min on a plate shaker and the absorbance of the wells was immediately determined at 450 nm with 600 nm correction using a spectrophotometer.
[0274] Antibody-Dependent Cellular Cytotoxicity Reporter (ADCP): Jurkat/FcγRIIIa-H effector cells (Promega) (3.5×104 cells/well) and Raji (for CD20) or HEK-PD-L1 (Crown-Bio transfected for PD-L1) cells were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-CD20 or PD-L1 constructs. After incubation for 6 h at 37° C. in 5% CO2, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer's protocol using a PHERAstar FS luminometer (BMG LABTECH).
[0275] Antibody-Dependent Cellular Cytotoxicity Reporter (ADCC): Jurkat/FcγRIIIa effector cells (Promega) (7.45×104 cells/well) and Raji (for CD20) or HEK-PD-L1 (Crown-Bio transfected for PD-L1) cells were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-CD20 or anti-PD-L1 constructs. After incubation for 6 h at 37° C. in 5% CO2, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer's protocol using a PHERAstar FS luminometer (BMG LABTECH).
[0276] Antibody-Dependent Cellular Cytotoxicity (KILR ADCC): A549 cells (ATCC) were obtained and cultured in F-12K media (Gibco), 10% FBS (Hyclone), and 2 mM glutamax (Gibco). Twenty-four hours before the experiment, 150,000 cells/mL of A549 cells were cultured in growth media, with 50 ng/mL of
[0277] IFN-y added to stimulate PD-L1 expression. Hemacare NK cells were used as the effector cells in this assay and were rested overnight in a non-tissue culture treated flask (Falcon). The A549 cells were then harvested with 3ml of Accutase (Corning) for 5 min. The cells were resuspended at 0.2×10{circumflex over ( )}6 cells/mL. Fifty μL of A549 cells were added to each well of a 96 well Tissue culture treated white flat bottom plate (Costar). Without any incubation time, 10 μL of constructs were added to each well. Immediately after, 50 μL of NK cells at 1×10{circumflex over ( )}6 cells/mL were added to each well of the plate. The plate was incubated at 37° C. for 5 hours. Then 50 μL of Cytotox glo reagent (Promega) was added followed by incubation at 37° C. for 15 minutes. The luminescence was read using a PHERAstar FS (BMG Labtech).
OTHER EMBODIMENTS
[0278] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
[0279] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the disclosure that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
[0280] Other embodiments are within the claims.