Sanofi Ref.: PAT24161 CEREBLON LIGASE MODULATOR AND BCMA NK CELL ENGAGER COMBINATION THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS 5 This application claims the benefit of European Priority Application No. 24306234.6, filed July 23, 2024, which is incorporated by reference in its entirety for all purposes. BACKGROUND An exemplary trifunctional anti-BCMA NK Cell Engager (BCMA NKCE), is 10 currently in a phase 1/2 trial in patients with relapsed/refractory (R/R) multiple myeloma (MM) and R/R light chain amyloidosis (LCA) (NCT05839626). The BCMA NKCE acts by targeting BCMA antigen expressed on MM cells and by co-engaging NKp46 and CD16a activating receptors on NK cells, facilitating the formation of an immunological synapse between NK and BCMA-positive tumor cells. This leads to NK cell activation, degranulation 15 and tumor cell killing. The anti-tumor activity of BCMA NKCE in preclinical models has been previously described (Tang et al. American Association for Cancer Research (2023) 83 (7_Supplement): 2960). Cereblon modulating agents, such as lenalidomide, pomalidomide, iberdomide and mezigdomide, have been demonstrated for their therapeutic role in MM. 20 None-the-less, there exists a need in the art for potent combination therapies leveraging the activities of BCMA NKCEs with cereblon modulating agent. SUMMARY In one aspect, the disclosure provides a method of treating cancer in a subject, 25 comprising administering to the subject: i) a binding protein comprising a first antigen binding domain (ABD) with binding specificity to BCMA and a second ABD with binding specificity to NKp46; and ii) a cereblon modulating agent, thereby treating the cancer in the subject. In certain embodiments, (a) the first ABD comprises: (a1) a first 30 immunoglobulin heavy chain variable domain (VH1) comprising an HCDR1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), an HCDR2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and an 1
Sanofi Ref.: PAT24161 HCDR3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and (a2) a first immunoglobulin light chain variable domain (VL1) comprising an LCDR1 sequence comprising the amino acid sequence of CX1SSTGX2VTPX3X4YAN (SEQ ID NO: 4), wherein X1 is R or A, X2 is T or A, X3 is S or G, and X4 is N or Y, an 5 LCDR2 sequence comprising the amino acid sequence of DNNX5X6PP (SEQ ID NO: 5), wherein X5 is S, I, or N and X6 is R or K, and an LCDR3 sequence comprising the amino acid sequence of ALX7X8GX9QWV (SEQ ID NO: 6), wherein X7 is W or Y, X8 is F or Y, and X9 is N or G; and (b) the second ABD comprises binding specificity to NKp46. In certain embodiments, (b) the second ABD comprises: (b1) a second 10 immunoglobulin heavy chain variable domain (VH2) comprising: - an HCDR1 sequence comprising DYVIN, an HCDR2 sequence comprising EIYPGSGTNYYNEKFKA, and an HCDR3 sequence comprising RGRYGLYAMDY; - an HCDR1 sequence comprising GYTFSDYVIN (SEQ ID NO: 19), an HCDR2 sequence comprising EIYPGSGTN (SEQ ID NO: 20), and an HCDR3 sequence comprising RGRYGLYAMDY (SEQ ID NO: 21); - an 15 HCDR1 sequence comprising SDYAWN (SEQ ID NO: 22), an HCDR2 sequence comprising YITYSGSTSYNPSLES (SEQ ID NO: 23), and an HCDR3 sequence comprising GGYYGSSWGVFAY (SEQ ID NO: 24); - an HCDR1 sequence comprising EYTMH (SEQ ID NO: 25), an HCDR2 sequence comprising GISPNIGGTSYNQKFKG (SEQ ID NO: 26), and an HCDR3 sequence comprising RGGSFDY (SEQ ID NO: 27); - an HCDR1 sequence 20 comprising SFTMH (SEQ ID NO: 28), an HCDR2 sequence comprising YINPSSGYTEYNQKFKD (SEQ ID NO: 29), and an HCDR3 sequence comprising GSSRGFDY (SEQ ID NO: 30); or - an HCDR1 sequence comprising SDYAWN (SEQ ID NO: 31), an HCDR2 sequence comprising YITYSGSTNYNPSLKS (SEQ ID NO: 32), and an HCDR3 sequence comprising CWDYALYAMDC (SEQ ID NO: 33); and (b2) a second 25 immunoglobulin light chain variable domain (VL2) comprising: - an LCDR1 sequence comprising RASQDISNYLN (SEQ ID NO: 34), an LCDR2 sequence comprising YTSRLHS (SEQ ID NO: 35), and an LCDR3 sequence comprising QQGNTRPWT (SEQ ID NO: 36); - an LCDR1 sequence comprising RVSENIYSYLA (SEQ ID NO: 37), an LCDR2 sequence comprising NAKTLAE (SEQ ID NO: 38), and an LCDR3 sequence 30 comprising QHHYGTPWT (SEQ ID NO: 39); - an LCDR1 sequence comprising RASQSISDYLH (SEQ ID NO: 40), an LCDR2 sequence comprising YASQSIS (SEQ ID NO: 41), and an LCDR3 sequence comprising QNGHSFPLT (SEQ ID NO: 42); - an LCDR1 2
Sanofi Ref.: PAT24161 sequence comprising RASENIYSNLA (SEQ ID NO: 43), an LCDR2 sequence comprising AATNLAD (SEQ ID NO: 44), and an LCDR3 sequence comprising QHFWGTPRT (SEQ ID NO: 45); or - an LCDR1 sequence comprising RTSENIYSYLA (SEQ ID NO: 46), an LCDR2 sequence comprising NAKTLAE (SEQ ID NO: 47), and an LCDR3 sequence 5 comprising QHHYDTPLT (SEQ ID NO: 48). In certain embodiments, the VL1 comprises: - an LCDR1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 8), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 9); - an 10 LCDR1 sequence comprising the amino acid sequence of CRSSTGTVTPSNYAN (SEQ ID NO: 10), an LCDR2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 11), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 12); - an LCDR1 sequence comprising the amino acid sequence of CASSTGAVTPSNYAN (SEQ ID NO: 13), an LCDR2 sequence comprising the amino acid 15 sequence of DNNIKPP (SEQ ID NO: 14), and an LCDR3 sequence comprising the amino acid sequence of ALWYGGQWV (SEQ ID NO: 15); or - an LCDR1 sequence comprising the amino acid sequence of CASSTGAVTPGYYAN (SEQ ID NO: 16), an LCDR2 sequence comprising the amino acid sequence of DNNNKPP (SEQ ID NO: 17), and an LCDR3 sequence comprising the amino acid sequence of ALYYGGQWV (SEQ ID NO: 18). 20 In certain embodiments, (a) the first ABD comprises: (a1) a first immunoglobulin heavy chain variable domain (VH1) comprising an HCDR1 sequence comprising the amino acid sequence of GFTFSNFG (SEQ ID NO: 112), an HCDR2 sequence comprising the amino acid sequence of IWSDETNR (SEQ ID NO: 113), and an HCDR3 sequence comprising the amino acid sequence of ARDQQYCSSDSCFTWFDP 25 (SEQ ID NO: 114); and (a2) a first immunoglobulin light chain variable domain (VL1) comprising an LCDR1 sequence comprising the amino acid sequence of TGTVTPSNY (SEQ ID NO: 118), an LCDR2 sequence comprising the amino acid sequence of DNN (SEQ ID NO: 119), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 120). 30 In certain embodiments, (a) the first ABD comprises: (a1) a first immunoglobulin heavy chain variable domain (VH1) comprising an HCDR1 sequence comprising the amino acid sequence of NFGMH (SEQ ID NO: 115), an HCDR2 sequence 3
Sanofi Ref.: PAT24161 comprising the amino acid sequence of VIWSDETNRYYADSVKG (SEQ ID NO: 116), and an HCDR3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 117); and (a2) a first immunoglobulin light chain variable domain (VL1) comprising an LCDR1 sequence comprising the amino acid sequence of 5 ASSTGTVTPSNYAN (SEQ ID NO: 121), an LCDR2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 122), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 123). In certain embodiments, - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the 10 VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; - the VH1 comprises an amino acid sequence that is at least 15 about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 51; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid 20 sequence of SEQ ID NO: 52; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 53; or - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 25 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 54. In certain embodiments, - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 55; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 30 comprises an amino acid sequence of SEQ ID NO: 50; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 51; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and 4
Sanofi Ref.: PAT24161 wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 52; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 53; or - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 54. 5 In certain embodiments, - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 64; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 57, and wherein the VL2 10 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 65; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 58, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 66; - the VH2 comprises an amino acid sequence that is at least 15 about 90% identical to the amino acid sequence of SEQ ID NO: 59, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 67; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 60, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid 20 sequence of SEQ ID NO: 68; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 61, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 69; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 62, and wherein the VL2 25 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 70; or - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 63, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 71. 30 In certain embodiments, - the VH2 comprises an amino acid sequence of SEQ ID NO: 56, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 64; - the VH2 comprises an amino acid sequence of SEQ ID NO: 57, and wherein the VL2 5
Sanofi Ref.: PAT24161 comprises an amino acid sequence of SEQ ID NO: 65; - the VH2 comprises an amino acid sequence of SEQ ID NO: 58, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 66; - the VH2 comprises an amino acid sequence of SEQ ID NO: 59, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 67; - the VH2 comprises 5 an amino acid sequence of SEQ ID NO: 60, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 68; - the VH2 comprises an amino acid sequence of SEQ ID NO: 61, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 69; - the VH2 comprises an amino acid sequence of SEQ ID NO: 62, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 70; or - the VH2 comprises an amino acid sequence of 10 SEQ ID NO: 63, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 71. In certain embodiments, the binding protein further comprises all or part of an immunoglobulin Fc domain or variant thereof. In certain embodiments, the Fc domain is an IgG1 Fc domain. In certain embodiments, the IgG1 Fc domain is a human IgG1 Fc domain. 15 In certain embodiments, all or part of the immunoglobulin Fc domain or variant thereof binds to a human Fc-γ receptor. In certain embodiments, all or part of the immunoglobulin Fc domain or variant thereof binds to a human CD16A (FcγRIII) polypeptide. In certain embodiments, the Fc domain comprises a native glycan at amino acid position 297, according to EU numbering. 20 In certain embodiments, the binding protein is N-glycosylated. In certain embodiments, the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain. In certain embodiments, at least one Fc heavy chain comprises an engineered intrachain disulfide bond mediated by a pair of cysteines (C) that substitute for: (i) a leucine 25 (L) at amino acid position 242 and a lysine (K) at amino acid position 334; or (ii) an arginine (R) at amino acid position 292 and a valine (V) at amino acid position 302; wherein the amino acid positions are according to EU numbering. In certain embodiments, the first Fc heavy chain or the second Fc heavy chain comprises the pair of cysteines. In certain embodiments, the first and the second Fc heavy 30 chain each comprise the pair of cysteines. In certain embodiments, the first and the second Fc heavy chain each comprise the L242C / K334C substitutions. In certain embodiments, the first and the second Fc heavy chain each comprise the R292C / V302C substitutions. 6
Sanofi Ref.: PAT24161 In certain embodiments, at least one Fc heavy chain comprises a substitution at amino acid position 332, according to EU numbering. In certain embodiments, the substitution at amino acid position 332 is a glutamic acid (E). In certain embodiments, at least one Fc heavy chain further comprises one or 5 more substitutions at amino acid positions 236, 239, or 330, according to EU numbering. In certain embodiments, the substitution at amino acid position 236 is an alanine (A). In certain embodiments, the substitution at amino acid position 239 is an aspartic acid (D). In certain embodiments, the substitution at amino acid position 330 is a leucine (L). In certain embodiments, at least one Fc heavy chain further comprises an aspartic 10 acid (D) at amino acid position 239, and a glutamic acid (E) at amino acid position 332, according to EU numbering. In certain embodiments, at least one Fc heavy chain further comprises an alanine (A) at amino acid position 236, an aspartic acid (D) at amino acid position 239, and a glutamic acid (E) at amino acid position 332, according to EU numbering. In certain embodiments, at least one Fc heavy chain further comprises an alanine (A) at 15 amino acid position 236, an aspartic acid (D) at amino acid position 239, a leucine (L) at amino acid position 330, and a glutamic acid (E) at amino acid position 332, according to EU numbering. In certain embodiments, the binding protein comprises at least two polypeptide chains that form at least two antigen-binding sites, wherein at least one polypeptide chain 20 comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I]; and at least one polypeptide chain comprises a structure represented by the formula: VH2-L3-VH1-L4-CH1 [II]; 25 wherein: CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and 30 wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 7
Sanofi Ref.: PAT24161 In certain embodiments, the binding protein comprises three polypeptide chains that form two antigen-binding sites, wherein one polypeptide chain comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I]; 5 one polypeptide chain comprises a structure represented by the formula: VH2-L3-VH1-L4-CH1-hinge-CH2-CH3 [III]; and one polypeptide chain comprises a structure represented by the formula: hinge-CH2-CH3 [IV] wherein: 10 CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; 15 and L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 20 In certain embodiments: (a) L1, L2, L3, and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS, GGGGSGGGGSGGGGS (SEQ ID NO: 82), S, RT, TKGPS (SEQ ID NO: 83), GQPKAAP (SEQ ID NO: 84), and GGSGSSGSGG (SEQ ID NO: 85); or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting 25 of GGGGSGGGGS, GGGGSGGGGSGGGGS, S, RT, TKGPS, GQPKAAP, and GGSGSSGSGG. In certain embodiments, L1 and L2 each comprise the amino acid sequence GGGGSGGGGS. In certain embodiments, L3 and L4 are each absent. In certain embodiments, (a) the first ABD of the binding protein comprises a 30 first immunoglobulin heavy chain variable domain (VH1) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and a first immunoglobulin light chain variable domain (VL1) comprising an amino acid 8
Sanofi Ref.: PAT24161 sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; and (b) the second ABD of the binding protein comprises a second immunoglobulin heavy chain variable domain (VH2) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56, and a second immunoglobulin light 5 chain variable domain (VL2) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 64. In certain embodiments, the binding protein comprises: (i) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 72; (ii) a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 73; and (iii) a third polypeptide chain 10 comprising an amino acid sequence of SEQ ID NO: 74. In certain embodiments, the cereblon modulating agent is a cereblon E3 ligase modulator (CELMoD) or an immunomodulatory drug (IMiD). In certain embodiments, the CELMoD is iberdomide or mezigdomide. In certain embodiments, the IMiD is pomalidomide, lenalidomide, or 15 thalidomide. In certain embodiments, the binding protein is administered to the subject prior to the cereblon modulating agent. In certain embodiments, the cereblon modulating agent is administered to the subject prior to the binding protein. 20 In certain embodiments, the binding protein and the cereblon modulating agent are administered concurrently to the subject. In certain embodiments, the cancer is multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS 25 The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. FIG. 1 depicts a distinct NKp46-BCMA NKCE format that engages BCMA at the surface of the tumor cell while also recruiting NK cells through the dual engagement of 30 both NKp46 and a Fcγ receptor, CD16a, inducing ADCC activity with an enhanced Fc competent format. The enhanced Fc competent format (hereinafter, termed “CODV-OL1- ADE-DSB”) includes: (1) ADE mutations (G236A/S239D/I332E) in CH2 to enhance 9
Sanofi Ref.: PAT24161 ADCC-activity; (2) DSB (R292C/V302C) in CH2 for thermal stabilization and productivity (e.g., expressability in cells and overall yield) ; (3) knob-into-hole mutations (KIH) in CH3 to favor heterodimer formation in Fc - (3a) knob (in heavy chain containing the VH/VL domains): S354C / T366W and (3b) hole (in heavy chain lacking the VH/VL domains): 5 Y349C / T366S / L368A / Y407V; and (4) RF mutations (H435R/Y436F) in one CH3 to favor purification of heterodimers in Fc. All Fc domain amino acid numbering is according to EU. This NKp46-BCMA NKCE_Fc CODV-OL1-ADE-DSB additionally contains two linkers GGGGSGGGGS in the light chain: one between VL anti-BCMA and VL anti-NKp46 and between VL NKp46 and CL. NKp46 binding site: 3D9; BCMA binding site: CA10v7; 10 CD16 binding through ADCC-competent Fc domain. FIG. 2 depicts a scatterplot of area under the curve (AUC) for each recited treatment group. FIG. 3 depicts a scatterplot of percent inhibition of AUC for each recited treatment group. 15 FIG. 4A – FIG. 4C depict RFP-expressing H929 cell cytotoxicity over time, normalized to time 0 with the exemplary BCMA NKCE in combination with iberdomide (FIG.4A, denoted “iber”), pomalidomide (FIG.4B, denoted “pom”) and mezigdomide (FIG. 4C, denoted “mez”). 20 DETAILED DESCRIPTION The disclosure provides methods of treating cancer (e.g., multiple myeloma) with the combination of i) binding proteins that bind one surface biomarker on immune NK cells, i.e., NKp46 and one antigen of interest on the cell membrane of normal and malignant plasma cells i.e., BCMA, and is capable of redirecting NK cells to lyse a target cell that 25 expresses the BCMA surface biomarker; and ii) cereblon modulating agents. It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. 30 Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker and are explained fully 10
Sanofi Ref.: PAT24161 in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring 5 Harbor (2013, 2nd edition). Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include 10 pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid 15 chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed 20 according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical 25 preparation, formulation, and delivery, and treatment of patients. That the disclosure may be more readily understood, select terms are defined below. The term “polypeptide” refers to any polymeric chain of amino acids and encompasses native or artificial proteins, polypeptide analogs or variants of a protein 30 sequence, or fragments thereof, unless otherwise contradicted by context. A polypeptide may be monomeric or polymeric. A polypeptide fragment comprises at least about 5 contiguous 11
Sanofi Ref.: PAT24161 amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids, for example. The term “isolated protein” or “isolated polypeptide” refers to a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally 5 associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein or polypeptide may also be 10 rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. As used herein, the term “binding protein” or “binding polypeptide” shall refer to a protein or polypeptide (e.g., an antibody or immunoadhesin) that contains at least one binding site which is responsible for selectively binding to a target antigen of interest (e.g., 15 a human target antigen). Exemplary binding sites include an antibody variable domain, a ligand binding site of a receptor, or a receptor binding site of a ligand. In certain aspects, the binding proteins or binding polypeptides comprise multiple (e.g., two, three, four, or more) binding sites. In certain aspects, the binding protein or binding polypeptide is a therapeutic enzyme. 20 The term “ligand” refers to any substance capable of binding, or of being bound, to another substance. Similarly, the term “antigen” refers to any substance to which an antibody may be generated. Although “antigen” is commonly used in reference to an antibody binding substrate, and “ligand” is often used when referring to receptor binding substrates, these terms are not distinguishing, one from the other, and encompass a wide 25 range of overlapping chemical entities. For the avoidance of doubt, antigen and ligand are used interchangeably throughout herein. Antigens/ligands may be a peptide, a polypeptide, a protein, an aptamer, a polysaccharide, a sugar molecule, a carbohydrate, a lipid, an oligonucleotide, a polynucleotide, a synthetic molecule, an inorganic molecule, an organic molecule, and any combination thereof. 30 The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor 12
Sanofi Ref.: PAT24161 matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD” as used herein refers to the dissociation 5 constant of the interaction between a particular binding protein and a target antigen. The term “specifically binds” as used herein, refers to the ability of an antibody or an immunoadhesin to bind to a target (e.g., an antigen) with a dissociation constant (KD) of at most about 1 x 10-6 M, about 1 x 10-7 M, about 1 x 10-8 M, about 1 x 10-9 M, about 1 x 10-10 M, about 1 x 10-11 M, about 1 x 10-12 M or less, and/or to bind to an antigen with an 10 affinity that is at least about two-fold greater than its affinity for a nonspecific antigen. Specific binding of an antibody can be to a target antigen through the CDR sequences. An antibody can also specifically bind to FcRs, such as FcRn or FcγRIIIa through the Fc region. As used herein, the term “antibody” refers to such assemblies (e.g., intact antibody molecules, immunoadhesins, or variants thereof) which have significant known 15 specific immunoreactive activity to an antigen of interest (e.g. a tumor associated antigen). Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood. As will be discussed in more detail below, the generic term “antibody” comprises 20 five distinct classes of antibody that can be distinguished biochemically. While all five classes of antibodies are clearly within the scope of the current disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000- 25 70,000. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. Light chains of immunoglobulin are classified as either kappa (κ) or lambda (λ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, 30 the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells, or 13
Sanofi Ref.: PAT24161 genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), with some subclasses among them (e.g., γl-γ4). It 5 is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA, or IgE, respectively. The immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well-characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the current 10 disclosure. Both the light and heavy chains are divided into regions of structural and functional homology. The term “region” refers to a part or portion of an immunoglobulin or antibody chain and includes constant region or variable regions, as well as more discrete parts or portions of said regions. For example, light chain variable regions include 15 “complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs,” as defined herein. The regions of an immunoglobulin heavy or light chain may be defined as “constant” (C) region or “variable” (V) regions, based on a relative lack of sequence variation within the regions of various class members in the case of a “constant region,” or 20 based on a significant variation within the regions of various class members in the case of a “variable regions.” The terms “constant region” and “variable region” may also be used functionally. In this regard, it will be appreciated that the variable regions of an immunoglobulin or antibody determine antigen recognition and specificity. Conversely, the constant regions of an immunoglobulin or antibody confer important effector functions such 25 as secretion, trans-placental mobility, Fc receptor binding, complement binding, and the like. The subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well-known. The constant and variable regions of immunoglobulin heavy and light chains are folded into domains. The term “domain” refers to a globular region of a heavy or light chain 30 comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by β-pleated sheet and/or an intra-chain disulfide bond. Constant region domains on the light chain of an immunoglobulin are referred to interchangeably as “light chain constant region 14
Sanofi Ref.: PAT24161 domains,” “CL regions,” “CL domains” or “CK domains.” Constant domains on the heavy chain (e.g., hinge, CH1, CH2 or CH3 domains) are referred to interchangeably as “heavy chain constant region domains,” “CH” region domains or “CH domains.” Variable domains on the light chain are referred to interchangeably as “light chain variable region domains,” 5 “VL region domains” or “VL domains.” Variable domains on the heavy chain are referred to interchangeably as “heavy chain variable region domains,” “VH region domains” or “VH domains.” By convention, the numbering of the amino acids of the variable constant region domains increases as they become more distal from the antigen-binding site or amino- 10 terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is a variable region and the C-terminus is a constant region. The CH3 and CL domains comprise the carboxy-terminus of the heavy and light chain, respectively. Accordingly, the domains of a light chain immunoglobulin are arranged in a VL-CL orientation, while the domains of the heavy chain are arranged in the VH-CH1-hinge-CH2- 15 CH3 orientation. The assignment of amino acids to each variable region domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between 20 different heavy chain variable regions or between different light chain variable regions are assigned the same number. CDRs 1, 2 and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2 and CDR-L3. CDRs 1, 2 and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR- H2 and CDR-H3. If so noted, the assignment of CDRs can be in accordance with IMGT® (Lefranc et al., Developmental & 25 Comparative Immunology 27:55-77; 2003) in lieu of Kabat. Numbering of the heavy chain constant region is via the EU index as set forth in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. 30 (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. 15
Sanofi Ref.: PAT24161 Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol.262, 732-745. (“Contact” numbering scheme), Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” 5 numbering scheme), and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme). The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while 10 the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on 15 analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. As used herein, the CDRs of an antibody can be determined according to the numbering system called, “IMGT” described in Lefranc (1999), The Immunologist, vol. 7:132-136 and Lefranc et al. (1999), Nucleic Acids Res., vol. 27:209-212. 20 As used herein, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops. Chothia and Lesk (1987), J. Mol. Biol., vol.196:901-917; Al-Lazikani et al. (1997), J. Mol. Biol., vol. 273:927-948; Chothia et al. (1992), J. Mol. Biol., vol. 227:799-817; Tramontano A et al. (1990), J. Mol. Biol. vol. 215(1):175-82. 25 As used herein, the CDRs of an antibody can be determined according to the Honegger-Pluckthun numbering scheme described in Honnegger and Pluckthun (2001), J. Mol. Biol.,vol. 309(3):657-670. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term “VL domain” includes the amino 30 terminal variable domain of an immunoglobulin light chain. As used herein, the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain that extends, e.g., from about 16
Sanofi Ref.: PAT24161 positions 114-223 in the Kabat numbering system (EU positions 118-215). The CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule and does not form a part of the Fc region of an immunoglobulin heavy chain. 5 As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998, 161 :4083). 10 As used herein, the term “CH2 domain” includes the portion of a heavy chain immunoglobulin molecule that extends, e.g., from about positions 244-360 in the Kabat numbering system (EU positions 231-340). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. In one 15 embodiment, a binding polypeptide of the current disclosure comprises a CH2 domain derived from an IgG1 molecule (e.g. a human IgG1 molecule). As used herein, the term “CH3 domain” includes the portion of a heavy chain immunoglobulin molecule that extends approximately 110 residues from N-terminus of the CH2 domain, e.g., from about positions 361-476 of the Kabat numbering system (EU 20 positions 341-445). The CH3 domain typically forms the C-terminal portion of the antibody. In some immunoglobulins, however, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the e chain of IgE). In one embodiment, a binding polypeptide of the current disclosure comprises a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule). 25 As used herein, the term “CL domain” includes the constant region domain of an immunoglobulin light chain that extends, e.g., from about Kabat position 107A to about Kabat position 216. The CL domain is adjacent to the VL domain. In one embodiment, a binding polypeptide of the current disclosure comprises a CL domain derived from a kappa light chain (e.g., a human kappa light chain). 30 The variable regions of an antibody allow it to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region (Fv) that defines a three dimensional antigen binding 17
Sanofi Ref.: PAT24161 site. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the heavy and light chain variable regions. As used herein, the term “antigen binding site” includes a site that specifically binds an antigen (e.g., a cell surface or soluble antigen). The antigen binding site includes an immunoglobulin 5 heavy chain and light chain variable region and the binding site formed by these variable regions determines the specificity of the antibody. An antigen binding site is formed by variable regions that vary from one antibody to another. The altered antibodies of the current disclosure comprise at least one antigen binding site. In certain embodiments, binding polypeptides of the current disclosure comprise 10 at least two antigen binding domains that provide for the association of the binding polypeptide with the selected antigen. The antigen binding domains need not be derived from the same immunoglobulin molecule. In this regard, the variable region may or be derived from any type of animal that can be induced to mount a humoral response and generate immunoglobulins against the desired antigen. As such, the variable region of a binding 15 polypeptide may be, for example, of mammalian origin e.g., may be human, murine, rat, goat, sheep, non-human primate (such as cynomolgus monkeys, macaques, etc.), lupine, or camelid (e.g., from camels, llamas and related species). In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned 20 to form the antigen binding site as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the framework regions. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, these 25 framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. 30 Exemplary binding polypeptides include antibody variants. As used herein, the term “antibody variant” includes synthetic and engineered forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two 18
Sanofi Ref.: PAT24161 heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multi-specific forms of antibodies (e.g., bi-specific, tri-specific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules and the like. In addition, the term “antibody 5 variant” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen). As used herein the term “valency” refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide comprises more than one target 10 binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes on the same antigen). The subject binding polypeptides typically has at least one binding site specific for a human antigen molecule. For example, a typical IgG1 monoclonal antibody is specific for one target antigen. A bivalent antibody is one comprising antigen binding domains that 15 targets two different antigens, or two antigen binding domains that target one antigen. Similarly, a trivalent antibody may be a monospecific antibody with three targeting domains to a single antigen. A trivalent antibody may be bispecific if it binds a first antigen with two binding domains and a second antigen with another binding domain. A trivalent antibody maybe trispecific and bind to three different targets. 20 The term “specificity” refers to the ability to specifically bind (e.g., immunoreact with) a given target antigen (e.g., a human target antigen). A binding polypeptide may be monospecific and contain one or more binding sites which specifically bind a target or a polypeptide may be multi-specific and contain two or more binding sites which specifically bind the same or different targets. In certain embodiments, a binding polypeptide is specific 25 for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, a binding polypeptide is specific for more than one target. Exemplary binding polypeptides (e.g., antibodies) which comprise antigen binding sites that bind to antigens expressed on tumor cells are known in the art and one or more CDRs from such antibodies can be included in an antibody as described herein. 30 The term “antigen” or “target antigen,” as used herein, refers to a molecule or a portion of a molecule that is capable of being bound by the binding site of a binding polypeptide. A target antigen may have one or more epitopes. 19
Sanofi Ref.: PAT24161 The term “about” or “approximately” means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range. As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an isolated 5 binding polypeptide provided herein) into a patient, such as by, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular, subcutaneous delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being managed or treated, administration of the substance typically occurs after the onset of the disease or symptoms 10 thereof. When a disease, or symptom thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof and may be continued chronically to defer or reduce the appearance or magnitude of disease-associated symptoms. As used herein, the term “composition” is intended to encompass a product 15 containing the specified ingredients (e.g., an isolated binding polypeptide provided herein) in, optionally, the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in, optionally, the specified amounts. “Effective amount” means the amount of active pharmaceutical agent (e.g., an 20 isolated binding polypeptide of the present disclosure) sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors. 25 As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In certain embodiments, the term “subject,” as used herein, refers to a vertebrate, such as a mammal. Mammals include, without limitation, humans, non-human primates, wild animals, feral animals, farm animals, 30 sport animals, and pets. As used herein, the term “therapy” refers to any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of a disease 20
Sanofi Ref.: PAT24161 or a symptom related thereto. In some embodiments, the term “therapy” refers to any protocol, method and/or agent that can be used in the modulation of an immune response to an infection in a subject or a symptom related thereto. In some embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other 5 therapies useful in the prevention, management, treatment and/or amelioration of a disease or a symptom related thereto, known to one of skill in the art such as medical personnel. In other embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the modulation of an immune response to an infection in a subject or a symptom related thereto known to one of skill in the art such 10 as medical personnel. As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or a symptom related thereto, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic 15 agents, such as an isolated binding polypeptide provided herein). The term “treating,” as used herein, can also refer to altering the disease course of the subject being treated. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptom(s), diminishment of direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, 20 amelioration or palliation of the disease state, and remission or improved prognosis. BCMA As used herein, the term "BCMA" refers to B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis receptor 25 (TNFR) family and is predominantly expressed on terminally differentiated B cells, e.g., memory B cells, and plasma cells. Its ligands are called B-cell activator of the TNF family (BAFF) and a proliferation inducing ligand (APRIL). BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The gene for BCMA is encoded on chromosome 16 producing a primary 35 mRNA transcript of 994 nucleotides 30 in length (NCBI accession NM_001192.2) that encodes a protein of 184 amino acids (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described, which may play a role in regulating BCMA expression. (Laabi Y. et al., Nucleic 21
Sanofi Ref.: PAT24161 Acids Res., 1994, 22:1147-1154.) Additional transcript variants have been described with unknown significance (Smirnova A S et al. Mol Immunol., 2008, 45(4):1179-1183 A second isoform, also known as TV4, has been identified (Uniprot identifier Q02223-2). “BCMA” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, 5 deletions, and splice variants of full length wild-type BCMA. Natural Killer Cells As used herein, “natural killer cells” or “NK cells” refers to a sub-population of lymphocytes that is involved in innate immunity. NK cells can be identified by virtue of 10 certain characteristics and biological properties, such as the expression of specific surface antigens including CD16, CD56 and/or CD57, NKp46 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express “self” MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK 15 activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein “active” NK cells designate biologically active NK cells, including NK cells having the capacity of lysing 20 target cells or enhancing the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press. pp. 219-238 (2000). 25 NKp46 As used herein, the “NKp46” marker, or “natural cytotoxicity triggering receptor 1,” also known as “CD335” or “NKP46” or “NK-p46” or “LY94” refers to a protein or polypeptide encoded by the Ncr1 gene. A reference sequence of full-length human NKp46 30 protein is available from the NCBI database under the accession number NP_004820. The human NKp46 mRNA sequence is described in NCBI accession number NM_004829. 22
Sanofi Ref.: PAT24161 NK Cell Engager As used herein, the isolated effector-competent polypeptide comprises a multispecific antibody in an NK cell engager format. An “NK cell engager” refers to binding proteins comprising monoclonal antibody domains targeting activating NK cell receptors, 5 antigen-specific targeting regions, and optionally an Fc region (Gauthier, et al. (2019) Cell, 177: 1701-13). NK cells express CD16a, also known as FcγRIIIa, which binds with low affinity to the Fc parts of antibodies (Cerwenka and Lanier (2018) Science 359:6383). Engagement of CD16a is less demanding compared to CD3 engagement due to lower steric hindrances and is additionally facilitated by the lack of accessory molecules. Upon 10 recognizing a target cell decorated with antibodies, NK cells mediate antibody-dependent cellular cytotoxicity (ADCC) resulting in killing of target cells (Lo Nigro (2019) Ann Transl Med 7:105). This naturally occurring mechanism can be utilized to engage CD16a when in conjunction with NKp46 (another activating NK cell receptor) to generate a trifunctional natural killer cell engager (i.e., NKCE) yielding impressive therapeutic outcomes. For a 15 review of NK cell engagers, see Demaria et al. (2021) Eur. J. Immunology 51(8): 1934, incorporated in its entirety by reference herein. Trifunctional NKCEs are more potent in vitro than clinical therapeutic antibodies targeting the same antigen and they also have similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects. See International Application No. 20 PCT/IB21/62494 incorporated in its entirety by reference herein. Accordingly, the disclosure provides trifunctional anti-BCMA NK Cell Engagers (BCMA NKCEs or NKp46-BCMA NKCEs), used in combination with cereblon modulating agents described herein. 25 Fc Domains In certain aspects of the disclosure, Fc domains, e.g., Fc domain variants, are provided. As used herein, the term “Fc region” or “Fc domain” refers to the portion of a heavy chain constant region beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of heavy chain constant region 30 to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge domain, a CH2 domain, and a CH3 domain. 23
Sanofi Ref.: PAT24161 The Fc region of an antibody is involved in non-antigen binding and can mediate effector function by binding to a Fc receptor. There are several different types of Fc receptors, which are classified based on the type of antibody that they recognize. For example, Fc-gamma receptors (FcγR) bind to IgG class antibodies, Fc-alpha receptors 5 (FcαR) bind to IgA class antibodies, and Fc-epsilon receptors (FcεR) bind to IgE class antibodies. The neonatal Fc receptor (FcRn) interacts with the Fc region of an antibody to promote antibody recycling through rescue of normal lysosomal degradation. The FcγRs belong to a family that includes several members, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. 10 The term “native Fc” or “wild-type Fc,” as used herein, refers to a molecule corresponding to the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is typically of human origin and can be any of the immunoglobulins, such as IgG1 and IgG2. 15 Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a disulfide-bonded 20 dimer resulting from papain digestion of an IgG. The term “native Fc,” as used herein, is generic to the monomeric, dimeric, and multimeric forms. The term “Fc domain variant,” “Fc variant” or “modified Fc,” as used herein, refers to a molecule or sequence that is modified from a native/wild-type Fc but still comprises a binding site for a FcR. Thus, the term “Fc variant” can comprise a molecule or 25 sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activities that are not required for the antibody-like binding polypeptides described herein. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has been modified, that affect or are 30 involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) 24
Sanofi Ref.: PAT24161 interaction with complement, (6) binding to a Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). As used herein, an “effector-competent Fc variant” or “effector-competent polypeptide” refers to a Fc domain that has one or more Fc effector functions as described 5 further herein. In certain exemplary embodiments, a Fc variant featured herein has one or more of increased serum half-life, enhanced FcRn binding affinity, enhanced FcRn binding affinity at acidic pH, enhanced FcγRIIIa binding affinity, and/or similar thermal stability, as compared to a wild-type Fc. 10 FcγRIIIa V176, or FcγRIIIa V158, or human CD16a-V receptor, or CD16aV, refers to a polypeptide construct comprising a fragment of the CD16 human receptor binding to a Fc region of a natural antibody, mediating antibody-dependent cellular cytotoxicity and bearing a Valine (V) on position 176 or position 158, which is also reported in the literature as allotype CD16a V176 or allotype CD16a V158. 15 FcγRIIIa F176, or FcγRIIIa F158, or human CD16a-F receptor, or CD16aF, refers to a polypeptide construct comprising a fragment of the CD16 human receptor binding to a Fc region of a natural antibody, mediating antibody-dependent cellular cytotoxicity and bearing a Phenylalanine (F) on position 176 or position 158, which is also reported in the literature as allotype CD16a F176 or allotype CD16a F158. 20 The term “Fc domain” as used herein encompasses native/wild-type Fc and Fc variants and sequences as defined herein. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. In certain exemplary embodiments, a Fc domain as described herein is thermally 25 stabilized. In certain exemplary embodiments, a Fc domain as described herein is glycosylated (e.g., via N--linked glycosylation). In certain exemplary embodiments, a Fc domain comprises N-linked glycosylation, e.g., at an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS (X being any amino acid residue except 30 proline). In certain exemplary embodiments, a Fc domain is glycosylated at amino acid position 297, according to EU numbering. 25
Sanofi Ref.: PAT24161 In certain exemplary embodiments, a Fc domain as described herein is effector- competent. In certain exemplary embodiments, a Fc domain as described herein is any combination of thermally stabilized, glycosylated, and effector-competent. 5 Thermally-Stabilized Fc Domain Variants The structure of constant antibody domains is similar to that of the variable domains consisting of β-strands connected with loops and short helices. The CH2 domain of the heavy constant regions exhibits weak carbohydrate-mediated interchain protein-protein 10 interactions in contrast to the extensive interchain interactions exhibited in other domains. Isolated murine CH2 domains are relatively unstable at physiological temperature (Feige et al., 2004, J. Mol. Biol. 344:107-118), but previous efforts demonstrate that the thermostability of CH2 domains may be enhanced with the addition of intrachain disulfide bonds, and that these could be used as scaffolds for binders (Gong et al., 2009, J. Biol. Chem. 15 284:14203-210). Effector-enhancing Fc domain variants that exhibit increased thermal instability (i.e., decreased thermal stability) relative to a wild-type Fc domain are known. For example, S239D/I332E and S239D/I332E/A330L variants lead to decreased stability of the CH2 domain as indicated by the lowering of melting temperature (Tm) in differential scanning 20 calorimetry (DSC) analysis. G236A/S239D/A330L/I332E has a reduced protein thermal shift measurement when compared to wild-type, as well as a considerably reduced half-life in hFcγR transgenic mice. (See Liu et al. (2014) J. Biol. Chem. 289(6): 3571, and Liu et al. (2020) Antibodies 9(4): 64 for review.) Effector-enhancing Fc domain variants having improved FcγR binding wherein 25 stability is not significantly reduced as compared to wild-type are known. (See, e.g., Igawa et al., EP 2940135) It has been further discovered that thermostabilized Fc domain variants may be produced by introducing one or more disulfide bonds in the Fc domain. Accordingly, in one aspect, the present disclosure provides a Fc domain variant comprising one or more 30 engineered (e.g., non-native) disulfide bonds, e.g., intrachain disulfide bonds mediated, e.g., by one or more pairs of cysteines. 26
Sanofi Ref.: PAT24161 In certain exemplary embodiments, a disulfide bond is an intrachain disulfide bond between the two CH2 regions of a Fc domain. In certain exemplary embodiments, a disulfide bond is an intrachain disulfide bond between the two CH3 regions of a Fc domain. In certain exemplary embodiments, two or more intrachain disulfide bonds are present in 5 between the two CH2 regions of a Fc domain and/or between the two CH3 regions of a Fc domain. Thermal stability, or the propensity of a Fc domain (e.g., a Fc domain with or without a binding polypeptide) to unfold, may be determined using a variety of methods known in the art. For example, the unfolding or denaturation temperature can be measured 10 by nano-format differential scanning calorimetry (nanoDSC) or nano-format differential scanning fluorimetry (nanoDSF) (Wen et al., 2020 Anal. Biochem. 593:113581). The detectable temperature at which a protein begins to unfold is the Tonset. In certain exemplary embodiments, the Tonset of a thermally-stabilized Fc domain variant (e.g., having one or more engineered disulfide bonds) is increased relative to 15 a Fc domain variant that is not thermally stabilized. In certain exemplary embodiments, the Tonset of a thermally-stabilized Fc domain variant is increased by about 1.0 oC, about 1.5 oC, about 2.0 oC, about 2.5 oC, about 3.0 oC, about 3.5 oC, about 4.0 oC, about 4.5 oC, about 5.0 oC, about 5.5 oC, about 6.0 oC, about 6.5 oC, about 7.0 oC, about 7.5 oC, about 8.0 oC, about 8.5 oC, about 9.0 oC, about 9.5 oC, about 10.0 oC, about 10.5 oC, about 11.0 oC, about 20 11.5 oC, about 12.0 oC, about 12.5 oC, about 13.0 oC, about 13.5 oC, about 14.0 oC, about 14.5 oC, about 15.0 oC, about 15.5 oC, about 16.0 oC, about 16.5 oC, about 17.0 oC, about 17.5 oC, about 18.0 oC, about 18.5 oC, about 19.0 oC, about 19.5 oC, about 20.0 oC, about 20.5 oC, about 21.0 oC, about 21.5 oC, about 22.0 oC, about 22.5 oC, about 23.0 oC, about 23.5 oC, about 24.0 oC, about 24.5 oC or about 25.0 oC relative to a Fc domain variant that 25 is not thermally stabilized. In certain exemplary embodiments, a thermally-stabilized Fc domain variant has one or more amino acid substitution pairs selected from the group consisting of cysteine substitutions at: amino acid positions 242 and 334; amino acid positions 240 and 334; amino acid positions 287 and 306; amino acid positions 292 and 302; amino acid positions 323 and 30 332; amino acid positions 259 and 306; amino acid positions 350 and 441; amino acid positions 343 and 431; amino acid positions 375 and 404; amino acid positions 375 and 396; and amino acid positions 348 and 439, according to EU numbering. (See Wozniak-Knopp 27
Sanofi Ref.: PAT24161 et al., 2012, PLoS One 7: e30083; Jacobsen et al., 2017 J. Biol. Chem. 202:1865-75; WO2014153063 for reviews.) In certain exemplary embodiments, a thermally-stabilized Fc domain variant comprises an engineered (e.g., a non-native) intrachain disulfide bond mediated by a pair of 5 cysteines that substitute for (i) a leucine (L) at amino acid position 242 and a lysine (K) at amino acid position 334; (ii) an alanine (A) at amino acid position 287 and a leucine (L) at amino acid position 306; or (iii) an arginine (R) at amino acid position 292 and a valine (V) at amino acid position 302, according to EU numbering. In certain exemplary embodiments, a thermostabilized Fc domain variant 10 comprises an engineered (e.g., a non-native) intrachain disulfide bond mediated by a pair of cysteines that substitute for (i) a leucine (L) at amino acid position 242 and a lysine (K) at amino acid position 334; (ii) an arginine (R) at amino acid position 292 and a valine (V) at amino acid position 302, according to EU numbering. In certain exemplary embodiments, a thermally-stabilized Fc domain variant 15 comprises an engineered (e.g., a non-native) intrachain disulfide bond mediated by a pair of cysteines that substitute for a leucine (L) at amino acid position 242 and a lysine (K) at amino acid position 334. In certain exemplary embodiments, a thermally-stabilized Fc domain variant comprises an engineered (e.g., a non-native) intrachain disulfide bond mediated by a pair of cysteines that substitute an alanine (A) at amino acid position 287 and a leucine (L) 20 at amino acid position 306. In certain exemplary embodiments, a thermally-stabilized Fc domain variant comprises an engineered (e.g., a non-native) intrachain disulfide bond mediated by a pair of cysteines that substitute for an arginine (R) at amino acid position 292 and a valine (V) at amino acid position 302. In certain exemplary embodiments, a thermally- stabilized Fc domain variant may comprise at least one engineered intrachain disulfide bond. 25 In certain exemplary embodiments, a thermally-stabilized Fc domain variant may comprise more than one engineered intrachain disulfide bond. Effector-Enhancing Fc Domain Variants In one aspect, the present disclosure provides a Fc domain variant comprising 30 effector-enhancing amino acid substitutions. In one embodiment, a Fc domain variant with altered FcγRIIIa binding comprising one or more amino acid substitutions as disclosed herein. In one embodiment, a 28
Sanofi Ref.: PAT24161 Fc domain variant with enhanced FcγRIIIa binding affinity having one or more amino acid substitutions as disclosed herein. In one embodiment, a Fc domain variant with enhanced FcγRIIIa binding affinity comprises two or more amino acid substitutions as disclosed herein. In one embodiment, a Fc domain variant with enhanced FcγRIIIa binding affinity 5 comprises three or more amino acid substitutions as disclosed herein. In one embodiment, a Fc domain variant with enhanced FcγRIIIa binding affinity comprises four or more amino acid substitutions as disclosed herein. In one embodiment, a Fc domain variant with altered FcRn binding comprises a Fc domain having one or more amino acid substitutions as disclosed herein. In one 10 embodiment, a Fc domain variant with enhanced FcRn binding affinity comprises a Fc domain having one or more amino acid substitutions as disclosed herein. In one embodiment, a Fc domain variant with enhanced FcRn binding affinity comprises a Fc domain having two or more amino acid substitutions as disclosed herein. In one embodiment, a Fc domain variant with enhanced FcRn binding affinity comprises a Fc domain having three or more 15 amino acid substitutions as disclosed herein. In some embodiments, a Fc domain variant may exhibit a species-specific FcRn binding affinity. In one embodiment, a Fc domain variant may exhibit hum a FcRn binding affinity. In one embodiment, a Fc domain variant may exhibit cyno FcRn binding affinity. In some embodiments, a Fc domain variant may exhibit cross-species FcRn binding affinity. 20 Such a Fc domain variants are said to be cross-reactive across one or more different species. In one embodiment, a Fc domain variant may exhibit both human and cyno FcRn binding affinity. The neonatal Fc receptor (FcRn) interacts with the Fc region of antibodies to promote recycling through rescue of normal lysosomal degradation. This process is a pH- 25 dependent process that occurs in the endosomes at acidic pH (e.g., a pH less than 6.5) but not under the physiological pH conditions of the bloodstream (e.g., a non-acidic pH). In some embodiments, a Fc domain variant has enhanced FcRn binding affinity at an acidic pH compared to a wild-type Fc domain. In some embodiments, a Fc domain variant has enhanced FcRn binding affinity at pH less than 7.0, e.g., at about pH 6.5, at about pH 6.0, at 30 about pH 5.5, at about pH 5.0, compared to a wild-type Fc domain. In some embodiments, a Fc domain variant has enhanced FcRn binding affinity at pH less than 7.0, e.g., at about pH 6.5, at about pH 6.0, at about pH 5.5, at about pH 5.0, compared to the FcRn binding 29
Sanofi Ref.: PAT24161 affinity of a wild-type Fc domain at an elevated non-acidic pH. An elevated non-acidic pH can be, e.g., pH greater than 7.0, about pH 7.0, about pH 7.4, about pH 7.6, about pH 7.8, about pH 8.0, about pH 8.5, about pH 9.0. In certain embodiments, it may be desired for a Fc domain variant to exhibit 5 approximately the same FcRn binding affinity at non-acidic pH as a wild-type Fc domain. In some embodiments, it may be desired for a Fc domain variant to exhibit less FcRn binding affinity at non-acidic pH than a binding polypeptide comprising a modified Fc domain having the double amino acid substitution M428L/N434S, according to EU numbering (See USPN 8,088,376). Accordingly, it may be desired a Fc domain variant to exhibit minimal 10 perturbation to pH-dependent FcRn binding. In some embodiments, a Fc domain variant having enhanced FcRn binding affinity at an acidic pH, has a reduced (i.e., slower) FcRn off-rate as compared to a wild- type Fc domain. In some embodiments, a Fc domain variant having enhanced FcRn binding affinity at an acidic pH compared to the FcRn binding affinity of the binding polypeptide at 15 an elevated non-acidic pH, has a slower FcRn off-rate at the acidic pH compared to the FcRn off-rate of a wild-type Fc domain at the elevated non-acidic pH. Certain embodiments include Fc domain variants in which at least one amino acid in one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced or enhanced effector 20 functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity. In certain other embodiments, a Fc domain variant comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more of a human 25 IgG1, IgG2, IgG3, or IgG4). In other embodiments, a Fc domain variant comprises a chimeric hinge (i.e., a hinge comprising hinge portions derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule and an IgG1 middle hinge domain). In certain embodiments, the Fc domain may be mutated to increase or decrease effector function using techniques known in the art. 30 In some embodiments, a Fc domain variant has altered binding affinity to a Fc receptor. There are several different types of Fc receptors, which are classified based on the type of antibody that they recognize. For example, Fc-gamma receptors (FcγR) bind to IgG 30
Sanofi Ref.: PAT24161 class antibodies, Fc-alpha receptors (FcαR) bind to IgA class antibodies, and Fc-epsilon receptors (FcεR) bind to IgE class antibodies. The FcγRs belong to a family that includes several members, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, a Fc domain variant has altered FcγRIIIa binding affinity, compared to a wild- 5 type Fc domain. In some embodiments, a Fc domain variant has reduced FcγRIIIa binding affinity, compared to a wild-type Fc domain. In some embodiments, a Fc domain variant has enhanced FcγRIIIa binding affinity, compared to a wild-type Fc domain. In some embodiments, a Fc domain variant modified Fc domain has approximately the same FcγRIIIa binding affinity, compared to a wild-type Fc domain. 10 In certain embodiments, a Fc domain variant comprises an antibody constant region (e.g., an IgG constant region e.g., a human IgG constant region, e.g., a human IgG1 constant region) which mediates one or more effector functions. For example, binding of the C1-complex to an antibody constant region may activate the complement system. Activation of the complement system is important in the opsonization and lysis of cell pathogens. The 15 activation of the complement system also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to receptors on various cells via the Fc domain (Fc receptor binding sites on the antibody Fc region bind to Fc receptors (FcRs) on a cell). There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA 20 (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of 25 immunoglobulin production. In some embodiments, a Fc domain variant, e.g., a binding polypeptide (e.g., an antibody, immunoadhesin or an antibody variant) binds to a Fc-gamma receptor. In alternative embodiments, a Fc domain variant comprised a constant region which is devoid of one or more effector functions (e.g., ADCC activity) and/or is unable to bind Fcγ receptor. 30 In certain exemplary embodiments, an effector-enhancing Fc domain variant has one or more amino acid substitutions selected from the group consisting of: an aspartic acid (D) at amino acid position 221; a cysteine (C) at amino acid position 222; a tyrosine (Y) at 31
Sanofi Ref.: PAT24161 amino acid position 234; an alanine (A) at amino acid position 236; a tryptophan (W) at amino acid position 236; an aspartic acid (D) at amino acid position 239; a leucine (L) at amino acid position 243; a glutamic acid (E) at amino acid position 267; a phenylalanine (F) at amino acid position 268; a proline (P) at amino acid position 292; an alanine (A) at amino 5 acid position 298; a leucine (L) at amino acid position 300; an isoleucine (I) at amino acid position 305; a threonine (T) at amino acid position 324; a tryptophan (W) at amino acid position 326; an alanine (A) at amino acid position 326; a leucine (L) at amino acid position 330; a glutamic acid (E) at amino acid position 332; an alanine (A) at amino acid position 333; a serine (S) at amino acid position 333; an alanine (A) at amino acid position 334; an 10 alanine (A) at amino acid position 336; an arginine (R) at amino acid position 345; and a leucine (L) at amino acid position 396, according to EU numbering. (See Saunders, 2009, Front. Immunol. doi: 10.3389/fimmu.2019.01296, for a review.) In some embodiments, a Fc domain variant may comprise an amino acid substitution at positions selected from amino acid positions 236, 239, 330, and 332, 15 according to EU numbering. In some embodiments, the substitutions may comprise an alanine (A) at amino acid position 236, an aspartic acid (D) at amino acid 239, a leucine (L) at amino acid position 330, and a glutamic acid (E) at amino acid position 332, according to EU numbering. In some embodiments, a Fc domain variant may comprise a double amino acid substitution at any two amino acid positions selected from an alanine (A) at amino acid 20 position 236, aspartic acid (D) at amino acid 239, a leucine (L) at amino acid position 330, and a glutamic acid (E) at amino acid position 332. In some embodiments, a Fc domain variant may comprise a triple amino acid substitution at any three amino acid positions selected from an alanine (A) at amino acid position 236, an aspartic acid (D) at amino acid 239, a leucine (L) at amino acid position 330, and a glutamic acid (E) at amino acid position 25 332. In some embodiments, a Fc domain variant may comprise a quadruple amino acid substitution at any four amino acid positions selected from an alanine (A) at amino acid position 236, an aspartic acid (D) at amino acid 239, a leucine (L) at amino acid position 330, and a glutamic acid (E) at amino acid position 332. In some embodiments, a Fc domain variant may comprise the combination of amino acid substitutions comprising an aspartic 30 acid (D) at amino acid 239 and a glutamic acid (E) at amino acid position 332. In some embodiments, a Fc domain variant may comprise the combination of amino acid 32
Sanofi Ref.: PAT24161 substitutions comprising an alanine (A) at amino acid position 236, an aspartic acid (D) at amino acid position and a glutamic acid at position 332. In some embodiments, a Fc domain variant may further comprise an amino acid substitution at amino acid positions 256 and/or 307, according to EU numbering. In some 5 embodiments, a Fc domain variant may comprise the combination of amino acid substitutions comprising an aspartic acid (D) at amino acid positions 256 and a glutamine (Q) at amino acid position 307 (See Mackness et al., 2019 MAbs 11:1276-88; WO2019147973A1, incorporated in its entirety by reference herein). 10 Glycosylated Fc Domain Variants In certain exemplary embodiments, a binding protein is glycosylated. Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on antibody function, particularly effector functioning such as those described above, see for example, Boyd et al (Mol. Immunol, 32: 1311-1318, 1996). 15 Glycosylation of a binding protein of the present disclosure wherein one or more carbohydrate moiety is added, substituted, deleted or modified are contemplated. In some embodiments, the glycosylation of the Fc domain of the binding protein is an N-linked glycosylation. Introduction of an asparagine-X-serine or asparagine-X-threonine motif creates a potential site for enzymatic attachment of carbohydrate moieties and may therefore 20 be used to manipulate the glycosylation of a Fc domain variant. In Raju et al. (Biochemistry 40: 8868-8876, 2001) the terminal sialylation of a TNFR-IgG immunoadhesin was increased through a process of re-galactosylation and/or re-sialylation using β-1,4- galactosyltransferace and/or alpha, 2,3 sialyltransferase. Increasing the terminal sialylation is believed to increase the half-life of the immunoglobulin. 25 Antibodies, in common with most glycoproteins, are typically produced as a mixture of glycoforms. This mixture is particularly apparent when antibodies are produced in eukaryotic, particularly mammalian cells. A variety of methods have been developed to manufacture defined glycoforms (see Zhang et al.2004, Science 303: 371; Sears et al, 2001, Science 291: 2344; Wacker et al., 2002, Science 298: 1790; Davis et al. 2002, Chem. Rev. 30 102: 579; Hang et al., 2001, Acc. Chem. Res. 34: 727). In some embodiments, the glycosylated Fc domain comprises a native glycan at amino acid position 297, according to EU numbering. Glycosylation of the asparagine at amino acid position 297 in the CH2 33
Sanofi Ref.: PAT24161 domain of IgG1 is known to facilitate interaction between the Fc domain and FcγR. Elimination of this glycosylation site eliminates effector function (Leabman, et al., 2013, MAbs 5:896-903). In particularly exemplary embodiments, a Fc domain comprises wild-type levels, or near wild-type levels, of glycosylation at amino acid position 297, according to 5 EU numbering. In some embodiments, the glycosylated Fc domain variant comprises an engineered or non-native glycan. In some embodiments, the engineered or non-native glycan is a modified glycan that can be conjugated to a therapeutic molecule (e.g., antibody-drug conjugate). 10 Fc-Containing Binding Polypeptides In one aspect, the present disclosure provides an isolated Fc domain variant comprising or complexed with (e.g., fused to) at least one binding domain (e.g., at least one binding polypeptide). In certain embodiments, the binding domain comprises one or more 15 antigen binding domains. The antigen binding domains need not be derived from the same molecule as the parental Fc domain. In certain embodiments, the Fc domain variant is present in an antibody. In one embodiment, a Fc domain variant is present in an antibody or is complexed with an antibody. Any antibody from any source or species can be employed 20 with a Fc domain variant disclosed herein. Suitable antibodies include without limitation, chimeric antibodies, humanized antibodies, or human antibodies. Suitable antibodies include without limitation, full-length antibodies, monoclonal antibodies, polyclonal antibodies, or single-domain antibodies, such as VHH antibodies. In certain exemplary embodiments, a Fc domain variant may be bound to or 25 complexed with an antigen-binding fragment of an antibody. The term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody which binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). Antigen-binding fragments can be produced by recombinant or biochemical methods that are well known in the art. Exemplary 30 antigen-binding fragments include Fv, Fab, Fab', and (Fab')2. In certain exemplary embodiments, a binding polypeptide of the current disclosure comprises at least one antigen- binding fragment and a Fc domain variant. 34
Sanofi Ref.: PAT24161 In some embodiments, the binding polypeptide comprises a single chain variable region sequence (ScFv). Single chain variable region sequences comprise a single polypeptide having one or more antigen binding sites, e.g., a VL domain linked by a flexible linker to a VH domain. ScFv molecules can be constructed in a VH-linker-VL orientation or 5 VL-linker-VH orientation. The flexible hinge that links the VL and VH domains that make up the antigen binding site includes from about 10 to about 50 amino acid residues. Connecting peptides are known in the art. Binding polypeptides may comprise at least one scFv and/or at least one constant region. In one embodiment, a binding polypeptide of the current disclosure may comprise at least one scFv linked or fused to a Fc domain variant. 10 In some embodiments, a binding polypeptide of the current disclosure is a multivalent (e.g., tetravalent) antibody which is produced by fusing a DNA sequence encoding an antibody with a ScFv molecule (e.g., an altered ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (e.g., an altered ScFv molecule) is linked at its N-terminus or C-terminus to a Fc domain variant via 15 a flexible linker (e.g., a gly/ser linker). In another embodiment a tetravalent antibody of the current disclosure can be made by fusing a ScFv molecule to a connecting peptide, which is fused to a Fc domain variant to construct a ScFv-Fab tetravalent molecule. In another embodiment, a binding polypeptide of the current disclosure is an altered minibody. An altered minibody of the current disclosure is a dimeric molecule made 20 up of two polypeptide chains each comprising a ScFv molecule which is fused to a Fc domain variant via a connecting peptide. Minibodies can be made by constructing a ScFv component and connecting peptide components using methods described in the art (see, e.g., US patent 5,837,821 or WO 94/09817Al). In another embodiment, a tetravalent minibody can be constructed. Tetravalent minibodies can be constructed in the same manner as minibodies, 25 except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then joined to a Fc domain variant. In another embodiment, a binding polypeptide of the current disclosure comprises a diabody. Diabodies are dimeric, tetravalent molecules each having a polypeptide similar to scFv molecules, but usually having a short (less than 10, e.g., about 1 to about 5) 30 amino acid residue linker connecting both variable domains, such that the VL and VH domains on the same polypeptide chain cannot interact. Instead, the VL and VH domain of one polypeptide chain interact with the VH and VL domain (respectively) on a second 35
Sanofi Ref.: PAT24161 polypeptide chain (see, for example, WO 02/02781). Diabodies of the current disclosure comprise a scFv-like molecule fused to a Fc domain variant. In another embodiment, a binding polypeptide of the current disclosure comprises a single-domain antibody (sdAb), also referred to as a VHH or a nanobody. 5 Nanobody® is registered trademark of Ablynx. VHHs comprise variable heavy chain domains devoid of light chains. Similar to conventional VH domains, VHHs contain four FRs and three CDRs. VHHs have advantages over conventional antibodies. As they are about ten times smaller than IgG molecules, properly folded functional VHHs can be produced by in vitro expression while achieving high yield. Furthermore, VHHs are very 10 stable, and resistant to the action of proteases. The properties and production of VHHs have been reviewed by Harmsen and De Haard H J (Appl. Microbiol. Biotechnol. 2007 November; 77(1):13-22). In certain exemplary embodiments, a Fc domain is fused with one or more VHHs. 15 In other embodiments, binding polypeptides comprise multi-specific or multivalent antibodies comprising one or more variable domain in series on the same polypeptide chain, e.g., tandem variable domain (TVD) polypeptides. Exemplary TVD polypeptides include the “double head” or “dual-Fv” configuration described in U.S. Patent No.5,989,830. In the dual-Fv configuration, the variable domains of two different antibodies 20 are expressed in a tandem orientation on two separate chains (one heavy chain and one light chain), wherein one polypeptide chain has two VH domains in series optionally separated by a peptide linker (VH1-linker-VH2) and the other polypeptide chain consists of complementary VL domains optionally connected in series by a peptide linker (VL1-linker- VL2). In the cross-over double head configuration, the variable domains of two different 25 antibodies are expressed in a tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), wherein one polypeptide chain has two VH domains in series optionally separated by a peptide linker (VH1-linker-VH2) and the other polypeptide chain consists of complementary VL domains optionally connected in series by a peptide linker in the opposite orientation (VL2-linker-VL1). Additional antibody variants based on 30 the “dual-Fv” format include the dual-variable-domain IgG (DVD-IgG) bispecific antibody (see U.S. Patent No. 7,612,181 and the TBTI format (see US 2010/0226923 A1). In some embodiments, binding polypeptides comprise multi-specific or multivalent antibodies 36
Sanofi Ref.: PAT24161 comprising one or more variable domain in series on the same polypeptide chain fused to a Fc domain variant. In another embodiment, a binding polypeptide comprises a cross-over dual variable domain IgG (CODV-IgG) bispecific antibody based on a “double head” 5 configuration (see US20120251541 A1, which is incorporated by reference herein in its entirety). In other embodiments, a binding polypeptide comprises a CrossMab or a CrossMab-Fab multispecific format (see WO2009080253 and Schaefer, et al., PNAS (2011), 108: 11187-1191). Antibody variants based on the CrossMab format have a 10 crossover of antibody domains within one arm of a bispecific IgG antibody enabling correct chain association. In other embodiments, the glycosylated effector-competent polypeptide comprises a multispecific antibody in a T cell engager format. A “T cell engager” refers to binding proteins directed to a host’s immune system, more specifically the T cells’ cytotoxic 15 activity as well as directed to a tumor target protein. In some embodiments, the isolated effector-competent polypeptide comprises a multispecific antibody in a NK cell engager format. A “NK cell engager” refers to binding proteins comprising monoclonal antibody fragments targeting activating NK cell receptors, antigen-specific targeting regions, and a Fc region (Gauthier, et al. Cell (2019), 177: 1701-13). 20 A binding polypeptide of the present disclosure, comprising a Fc domain variant described herein, can include the CDR sequences or the variable domain sequences of a known “parent” antibody. In some embodiments, the parent antibody and the antibody of the disclosure can share similar or identical sequences except for modifications to the Fc domain as disclosed herein. 25 Cross-Over Dual Variable In a particular embodiment, “cross-over dual variable” or “CODV” refers to an antigen-binding domain that specifically binds to at least one target antigen or at least one target epitope, and comprises at least two polypeptide chains that form at least two antigen- 30 binding sites, wherein at least one polypeptide chain comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I] 37
Sanofi Ref.: PAT24161 and at least one polypeptide chain comprises a structure represented by the formula: VH2-L3-VH1-L4-CH1 [II] wherein: 5 VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; 10 CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 15 In certain exemplary embodiments, the binding protein of the disclosure comprises a “CODV-OL1” format, comprising three polypeptide chains that form two antigen-binding sites, wherein one polypeptide chain comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I]; 20 one polypeptide chain comprises a structure represented by the formula: VH2-L3-VH1-L4-CH1-hinge-CH2-CH3 [III]; and one polypeptide chain comprises a structure represented by the formula: hinge-CH2-CH3 [IV] wherein: 25 CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; 30 and L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and 38
Sanofi Ref.: PAT24161 wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. In a particular embodiment, a CODV antigen-binding domain specifically binds to at least one target antigen or at least one target epitope, and comprises four polypeptide 5 chains that form four antigen-binding sites, wherein two polypeptide chains each comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I] and two polypeptide chains each comprises a structure represented by the formula: 10 VH2-L3-VH1-L4-CH1-Fc [II] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; 15 VH2 is a second immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; Fc is an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and 20 L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair, wherein the VH1/VL1 pair comprises a first antigen binding specificity and the 25 VH2/VL2 pair comprises a second antigen binding specificity. In a particular embodiment, an antigen-binding protein described herein is a trispecific and/or a trivalent antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more different antigen targets, wherein the first polypeptide chain comprises a structure represented by the formula: 30 VL2-L1-VL1-L2-CL [I] the second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II] 39
Sanofi Ref.: PAT24161 the third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III] and the fourth polypeptide chain comprises a structure represented by the formula: 5 VL3-CL [IV], wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VL3 is a third immunoglobulin light chain variable domain; 10 VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; 15 CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, 20 L2, L3, and L4 are optionally absent, and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. In certain embodiments, the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen-binding sites. In some 25 embodiments, the VH1 and VL1 form a binding pair and form the first antigen-binding site. In some embodiments, the VH2 and VL2 form a binding pair and form the second antigen- binding site. In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen-binding site. In some embodiments, the VH3 and VL3 form a binding pair and form the third antigen-binding site. 30 Such antigen-binding protein comprises at least three antigen-binding sites. It is at least a trivalent antigen-binding molecule. In a particular embodiment, it specifically binds to one antigen target, i.e., it is a monospecific antigen-binding molecule. In another 40
Sanofi Ref.: PAT24161 embodiment, it specifically binds to two different antigen targets, i.e., it is a bispecific antigen-binding molecule. In another embodiment, it specifically binds to three different antigen targets, i.e., it is a trispecific antigen-binding molecule. The examples listed above are not intended to limit the scope of the disclosure 5 in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the antibody-like binding proteins described herein. In certain embodiments of the binding protein, (a) L1, L2, L3, and L4 each 10 independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS, GGGGSGGGGSGGGGS, S, RT, TKGPS, GQPKAAP, and GGSGSSGSGG; or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS, GGGGSGGGGSGGGGS, S, RT, TKGPS, GQPKAAP, and GGSGSSGSGG. 15 In certain embodiments, L1 and L2 each comprise the amino acid sequence GGGGSGGGGS. In certain embodiments, wherein L3 and L4 are each absent. The CODV antibody format, the various permutations of the CODV antibody format, and additional details regarding linkers is further described in WO 2012/135345A1, 20 and WO 2017/180913A2, which are incorporated herein by reference in their entireties. Cereblon Modulating Agents As used herein, the term “cereblon modulating agent” refers to a class of compounds that bind to and alter the activity of the protein cereblon, encoded by the CRBN 25 gene. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1) (see, Angers et al. Nature. 443 (7111): 590–3. 2006). By binding to cereblon, cereblon modulating agents change the substrate specificity of the CRBN E3 ubiquitin ligase complex, which, among other things, results in the breakdown of internal Ikaros and Aiolos proteins. Cereblon modulating agents 30 have been shown to be effective in the treatment of multiple myeloma. Cereblon modulating agents may be divided into two subclasses, cereblon E3 ligase modulators (CELMoDs) and immunomodulatory drugs (IMiDs). Both subclasses of 41
Sanofi Ref.: PAT24161 agents bind to cereblon, modulating the activity of cereblon, and are derived from the compound thalidomide. Compounds of both classes share a glutarimide core structure of the formula (CH2)3(CO)2NH, also known as 2,6-piperidinedione. Accordingly, the disclosure provides for cereblon modulating agents in 5 combination with a BCMA NKCE described herein for the treatment of cancer (e.g., multiple myeloma). In certain embodiments, the cereblon modulating agent is a cereblon E3 ligase modulator (CELMoD) or an immunomodulatory drug (IMiD). In certain embodiments, the CELMoD comprises mezigdomide or iberdomide. In certain embodiments, the IMiD comprises pomalidomide, lenalidomide, or thalidomide. Each of the 10 specific cereblon modulating agents are described in greater detail below in the following paragraphs. Iberdomide As used herein, the term “iberdomide” refers to a specific cereblon modulating agent 15 (CAS Registry Number 1323403-33-3) with the following structure:
. In certain embodiments, iberdomide as described herein is referred to as (S)-3-(4- ((4-(Morpholinomethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, (3s)-3-[4- ({4-[(Morpholin-4-Y1)methyl]phenyl (methoxy)-1-Oxo-1,3-Dihydro-2h-Isoindol-2-20 Y1]piperidine-2,6-Dione,(3S)-3-[7-[[4-(morpholin-4-ylmethyl)phenyl]methoxy]-3-oxo- 1H-isoindol-2-y1]piperidine-2,6-dione, (S)-3-[4-(4-Morpholin-4-y1-methyl-benzyloxy)-1- oxo-1,3-dihydro-isoindol-2-y1]-piperidine-2,6-dione, or (3S)-3-[7-[[4-(morpholin-4- ylmethyl)phenyl]methoxy]-3-oxo-1H-isoindol-2-yl]piperidine-2,6-dione. Methods of preparing iberdomide are described, e.g., in US 20110196150, the entirety of which is 25 incorporated herein by reference. 42
Sanofi Ref.: PAT24161 In certain embodiments, iberdomide as described herein can be in a form of hydrochloride salt. In certain embodiments, iberdomide hydrochloride is referred to as (35)- 3 -(4- { [4-(morpholin-4-ylmethyl)benzyl] oxy} - 1 -oxo- 1,3 -dihydro-2H-i soindol-2- yl)piperidine-2, 6-dione hydrochloride (1 :1) or 2,6-piperidinedione, 3-[1,3-dihydro-4-[[4- 5 (4- morpholinylmethyl)phenyl]methoxy]-1-oxo-2H-isoindol-2-yl]-, (3S)-, hydrochloride (1 : 1). Mezigdomide As used herein, the term “mezigdomide” or “CC-92480” refers to a specific cereblon 10 modulating agent (CAS Registry Number 2259648-80-9) with the following structure:
In certain embodiments, mezigdomide as described herein is referred to as (S)-4-(4- (4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzyl)piperazin-1-yl)- 3- fluorobenzonitrile, 4-[4-[[4-[[2-[(3S)-2,6-dioxopiperidin-3-yl]-1-oxo-3H-isoindol-4- 15 yl]oxymethyl]phenyl]methyl]piperazin-1-yl]-3-fluorobenzonitrile, or (83S)-12-fluoro- 71,82,86-trioxo-71,73-dihydro-6-oxa-7(4,2)-isoindola-2(1,4)-piperazina-8(3)-piperidina- 1(1),4(1,4)-dibenzenaoctaphane-14-carbonitrile. 20 43
Sanofi Ref.: PAT24161 Pomalidomide As used herein, the term “pomalidomide” refers to a specific cereblon modulating agent (CAS Registry Number 19171-19-8) with the following structure:
5 In certain embodiments, pomalidomide as described herein is referred to as (RS)-4- Amino-2-(2,6-dioxopiperidin-3-yl)-isoindoline-1,3-dione. Lenalidomide As used herein, the term “lenalidomide” refers to a specific cereblon modulating 10 agent (CAS Registry Number 191732-72-6) with the following structure:
. In certain embodiments, lenalidomide as described herein is referred to as 3-(4- amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl) piperidine-2,6-dione. 15 Thalidomide As used herein, the term “thalidomide” refers to a specific cereblon modulating agent (CAS Registry Number 50-35-1) with the following structure: 44
Sanofi Ref.: PAT24161
In certain embodiments, thalidomide as described herein is referred to as 2-(2,6- dioxopiperidin-3-yl)isoindoline-1,3-dione. 5 Combination Therapy for the Treatment of Cancer The disclosure provides methods of treating cancer with the combination of binding proteins that bind NKp46 on NK cells and BCMA on cancer cells (BCMA NKCEs) and cereblon modulating agents. The combination of the BCMA NKCEs of the disclosure 10 and the cereblon modulating agents of the disclosure enhance cancer cell cytotoxicity. Thus, in one aspect, the disclosure provides a method of treating cancer in a subject, comprising administering to the subject a combination comprising: i) a binding protein comprising a first antigen binding domain (ABD) with binding specificity to BCMA and a second ABD with binding specificity to NKp46 (including any binding protein 15 described herein); and ii) at least one cereblon modulating agent (including any cereblon modulating agent described herein), thereby treating the cancer in the subject. The term “combination”, “combination therapy,” “co-administration,” or “co- administered” (or minor variations of these terms) refers to the administration of two or more therapeutic agents (e.g., the binding proteins and cereblon modulating agents of the 20 disclosure) to treat a therapeutic condition or disorder described in the present disclosure (e.g., cancer, such as multiple myeloma). Such administration encompasses co- administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate containers (e.g., capsules and/or intravenous formulations) for each active ingredient. In addition, such 25 administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. For example, the first agent can be dosed daily, every week, every 21 days, every 28 days, or every two months 45
Sanofi Ref.: PAT24161 while the second agent can, for example, be dosed daily. In any case, the treatment regimen of the drug combination will provide beneficial effects in treating the conditions or disorders described herein. In certain embodiments, a first agent (e.g., the binding protein of the disclosure) 5 is administered simultaneously with a second agent (e.g., the cereblon modulating agent of the disclosure). Simultaneous administration typically means that both compounds enter the subject at precisely the same time. However, simultaneous administration also includes the possibility that the first and second agents enter the subject at different times, but the difference in time is sufficiently miniscule that the first administered compound is not 10 provided the time to take effect on the subject before entry of the second administered compound. Such delayed times typically correspond to less than 1 minute, and more typically, less than 30 seconds. In one example, wherein the compounds are in solution, simultaneous administration can be achieved by administering a solution containing the combination of compounds. In another example, simultaneous administration of separate 15 solutions, one of which contains the first agent and the other of which contains the second agent, can be employed. In one example wherein the compounds are in solvated form, simultaneous administration can be achieved by administering a composition containing the combination of compounds. In another embodiment, the first agent and the second agent are administered 20 sequentially. According to this embodiment, the first agent is administered first, followed immediately thereafter by administration of the second agent. In another embodiment, the second agent is administered, a time interval occurs and then the first agent is administered. The time interval can be one or more hour(s), one or more day(s) or one or more week(s). In an embodiment of the invention, the first agent is administered first, followed immediately 25 thereafter by administration of the second. In another embodiment, the first agent is administered, a time interval occurs and then the second agent is administered. In certain embodiments, the binding protein is administered to the subject at a time interval prior to the cereblon modulating agent. n certain embodiments, the cereblon modulating agent is administered to the subject at a time interval prior to the binding protein. 30 46
Sanofi Ref.: PAT24161 In certain embodiments, two or more different cereblon modulating agents are administered, such as mezigdomide and iberdomide, iberdomide and pomalidomide, iberdomide and lenalidomide, mezigdomide and pomalidomide, or mezigdomide and lenalidomide. 5 In certain embodiments, the cancer is a hematological cancer. In certain embodiments, the hematological cancer is a plasma cells malignancy. In certain embodiments, the plasma cell malignancy is multiple myeloma. In certain embodiments, the multiple myeloma is relapsed/refractory multiple myeloma, newly diagnosed multiple myeloma, or smoldering multiple myeloma. In certain other embodiments, the disease is 10 light chain amyloidosis (LCA). In certain embodiments, the LCA is relapsed/refractory LCA, newly diagnosed LCA, or smoldering LCA. Method of Treatment/Use for Multiple Myeloma In one aspect, the disclosure is directed to the treatment and prevention of multiple 15 myeloma with the combination of a BCMA NKCE described herein and a cereblon modulating agent described herein. The term “Multiple Myeloma (MM)” (also known as plasma cell myeloma, myelomatosis, or Kahler's disease) is a progressive hematologic cancer of the plasma cell, a type of white blood cell normally responsible for producing antibodies. The condition is 20 characterized by excessive numbers of plasma cells in the bone marrow and overproduction of intact monoclonal immunoglobulin or free monoclonal light chains. Clinically the disease is diagnosed, staged, and treated based on a variety of parameters which include the myeloma tumor cell mass on the basis of the amount of monoclonal (or myeloma) protein (M protein) in the serum and/or urine, along with hemoglobin and serum calcium concentrations, the 25 number of lytic bone lesions based on a skeletal survey, and the presence or absence of renal failure. Additional approaches to characterizing the condition include the detection of greater than ten percent (10%) of plasma cells on a bone marrow examination, the presence of soft tissue plasmacytomas and the detection of free kappa and lambda serum immunoglobulin light chain. Bone marrow examination is done using standard histology and 30 immunohistochemistry techniques. Additional cytogenetics of bone marrow samples may be conducted to determine prognosis. Follow up surveillance consists of chemistry and bone marrow evaluations if clinically indicated due to its invasive nature. 47
Sanofi Ref.: PAT24161 In certain embodiments, the methods of the invention include treatment of patients having relapsed and/or refractory MM or patients having MM who have received one or more prior therapies for MM. In certain embodiments, the multiple myeloma is relapsed/refractory multiple myeloma (RR/MM). In some embodiments, the patient has 5 received at least one or two previous therapies for multiple myeloma (e.g., a thalidomide analog such as lenalidomide, a proteasome inhibitor, or autologous stem cell transplant (ASCT), and had demonstrated disease progression on last therapy or after completion of the last therapy. “Relapsed MM” refers to multiple myeloma which has been previously treated and 10 which progresses and requires the initiation of additional treatment but does not meet the criteria for either primary' refractory' or relapsed and refractory MM. Clinical criteria for determining relapse are well known to those of skill in the art. For example, clinical criteria developed by the International Myeloma Working Group (IMWG) include serum M- component increases of >1 gm/dL, development of new soft tissue plasmacytomas or bone 15 lesions, and increases in the size of existing plasmacytomas or bone lesions. “Refractory MM” refers to multiple myeloma which is non-responsive (e.g., fails to achieve a minimal response or develops progressive disease while on therapy). In certain embodiments, the multiple myeloma is non-responsive while on primary or salvage therapy, or progresses within 60 days of the last therapy. 20 In certain embodiments, the MM is “relapsed and refractory MM”. “Relapsed and refractory MM” is non-responsive while on salvage therapy (e.g., therapy that is administered after treatment with first line of therapy has failed) or disease that progresses within 60 days of last therapy in patients who have achieved minimal response or better at some point prior to progressing in their current disease course. 25 In certain embodiment, the MM is Primary Refractory MM. Primary refractory MM is MM disease that is non-responsive in patients who have never achieved minimal response or better with any therapy. In certain embodiments, the MM is precancerous or “smoldering” MM. Smoldering multiple myeloma is a precancerous condition that alters certain proteins in blood and/or 30 increases plasma cells in bone marrow, but it does not cause symptoms of disease. About half of those diagnosed with the condition, however, will develop multiple myeloma within 5 years. Patients are closely monitored for evidence of progression to active multiple 48
Sanofi Ref.: PAT24161 myeloma. Patients are diagnosed with smoldering multiple myeloma if they meet certain criteria: a blood test showing an M protein of >3g/dL of blood or a 24-hour urine test showing 500 mg or more of protein or a bone marrow biopsy that shows that plasma cells make up between 10% and 59% of blood cells in the bone marrow; and no sign of abnormal 5 bone lesions or kidney damage that active myeloma may cause. There are currently no approved treatments for smoldering multiple myeloma. Method of Treatment/Use for Light Chain Amyloidosis In one aspect, the disclosure is directed to the treatment and prevention of light chain 10 amyloidosis (LCA) with the combination of a BCMA NKCE described herein and a cereblon modulating agent described herein. In certain embodiments, the present disclosure provides kits and methods for the treatment of diseases and disorders, e.g., an amyloidosis in a mammalian subject in need of such treatment. Current standards of care for LCA are limited due to tolerability in this 15 population with frequent organ dysfunction. As such, there is still an unmet need for additional therapeutics for LCA that are effective and safe. In another aspect, the disclosure provides a method of treating or preventing light chain amyloidosis in a subject in need thereof, the method comprising administering to the subject a binding protein as disclosed herein, comprising a first antigen binding domain with 20 binding specificity to BCMA and a second antigen binding domain with binding specificity to a Natural Killer (NK) cell marker. In certain embodiments, the disease is an amyloidosis or an amyloid disease. The term “amyloidosis” or “amyloid disease”, as used herein, refers to diseases or disorders that fall under the umbrella of plasma cell dyscrasias. Amyloidosis or amyloid disease occurs 25 when amyloid builds up in organs and interferes with normal physiological function. The term “amyloid”, as used herein, refers to the abnormal fibrous, extracellular, proteinaceous deposits found in organs and tissues. Amyloid is not normally found in the body, but it can be formed from several different types of protein. Amyloid is typically insoluble and is structurally dominated a by β-sheet structure. Organs that may be affected include the heart, 30 kidneys, liver, spleen, nervous system, skin, and digestive tract. The term “amyloidosis” is used to refer to a cluster of diseases which share a common feature, i.e., the extracellular deposition of pathologic insoluble fibrillar proteins in organs and tissues. 49
Sanofi Ref.: PAT24161 A hallmark feature of amyloid disease is the production of amyloids characterized by a fibrillar morphology of about 5-15 nm in diameter which bind the dye congo red and display fluorescence birefringence when bound to the dye thioflavin T. The fibers can form secondary structures called protofilaments made of pleated β-sheets which make them 5 resistant to degradation. Toyama and Weissman (2011), Annu Rev Biochem, 80:557-585; Picken (2020), Acta Haematol, vol. 143: 322-334. In certain embodiments, the amyloidosis or the amyloid disease is light chain amyloidosis or amyloid light-chain amyloidosis. The current classification of amyloid is based on the type of amyloid protein. For instance, amyloid is termed “A” (for amyloid) 10 followed by an abbreviation of the protein type: AL (amyloid derived from immunoglobulin light chain). The term “amyloid light-chain amyloidosis” or “light chain amyloidosis” or “LCA”; also called AL amyloidosis, AL, ALA, or AL primary amyloidosis, is the most common form of systemic amyloidosis in the United States and in developed countries. Picken (2020), Acta Haematol, vol. 143: 322-334. The patient may present with a first 15 instance of LCA (newly diagnosed LCA), or the LCA may be relapsed and/or refractory (relapsed/refractory LCA). LCA is the most common form of systemic amyloidosis and is associated with an underlying plasma cell dyscrasia. Aberrant plasma cells are derived from a single plasma cell source and secrete toxic fibril-forming free light chains. These plasma cells have been shown to be BCMA-positive (Godara et al. 2019. Blood 134 (Supplement 20 _1): 4409). In certain embodiments, the patient with LCA is treatment-naïve. In some embodiments, the patient with LCA has received or is receiving one or more therapeutics for light chain amyloidosis (e.g., chemotherapy, autologous stem cell transplant, immunomodulatory drugs, immunotherapies, proteasome inhibitors and any combinations thereof). 25 LCA is a hematological disorder caused primarily by clonal plasma cells that produce misfolded immunoglobulin light chains. These aberrant light chains form a toxic aggregate in plasma cells and deposit fibrils (amyloids) in organs and tissue which result in significant and sometimes permanent organ dysfunction. LCA can impact any organ other than the brain. Li et al. (2019), J Int Med Res., 47(4): 1778-1786. The mechanisms by which 30 amyloidogenic immunoglobulin light chains result in organ dysfunction are not well characterized but likely due to both amyloid deposits and prefibrillar aggregates cytotoxic 50
Sanofi Ref.: PAT24161 effects on the underlying organ(s). Primary LCA is that which is neither associated with nor considered a complication of multiple myeloma. Pharmaceutical Compositions and Administration Thereof 5 Methods of preparing and administering the binding protein of the current disclosure and the cereblon modulating agent of the disclosure to a subject are well known to or are readily determined by those skilled in the art. The route of administration of the binding polypeptides of the current disclosure and the cereblon modulating agent of the disclosure may be oral, parenteral, by inhalation or topical. The term parenteral as used 10 herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the current disclosure, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip or for subcutaneous administration. Usually, a suitable pharmaceutical composition for 15 injection may comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e.g., human albumin), etc. In some embodiments, Fc domain variants can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent. Preparations for parenteral administration include sterile aqueous or non- 20 aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the compositions and methods of the current disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, e.g., 0.05 M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer’s 25 dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer’s dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile 30 aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It 51
Sanofi Ref.: PAT24161 should be stable under the conditions of manufacture and storage and will typically be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and 5 suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, isotonic agents will be included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged 10 absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a binding protein of the disclosure) in the required amount in an 15 appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include 20 vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit. Such 25 articles of manufacture will typically have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from or predisposed to autoimmune or neoplastic disorders. Effective doses of the compositions of the present disclosure, for the treatment of the above-described conditions vary depending upon many different factors, including 30 means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals including 52
Sanofi Ref.: PAT24161 transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. A pharmaceutical composition in accordance with the present disclosure can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological 5 saline, nontoxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of the binding protein shall be held to mean an amount sufficient to achieve effective binding to an antigen and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. In the case of tumor cells, the polypeptide can interact with selected antigens on neoplastic or 10 immunoreactive cells and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the modified binding polypeptide. In keeping with the scope of the present disclosure, the binding proteins of the 15 disclosure and the cereblon modulating agent of the disclosure may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The binding proteins of the disclosure and the cereblon modulating agent of the disclosure can be administered to such human or other animal in a conventional dosage form prepared by combining the 20 antibody of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail 25 comprising one or more species of binding polypeptides described in the current disclosure may prove to be particularly effective. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby 30 incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and 53
Sanofi Ref.: PAT24161 information from any such articles, patents, patent applications, or other physical and electronic documents. While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes 5 may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, 10 composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. 15 EMBODIMENTS OF THE DISCLOSURE 1. A method of treating cancer in a subject, comprising administering to the subject: i) a binding protein comprising a first antigen binding domain (ABD) with 20 binding specificity to BCMA and a second ABD with binding specificity to NKp46; and ii) a cereblon modulating agent, thereby treating the cancer in the subject. 2. The method of embodiment 1, wherein: 25 (a) the first ABD comprises: (a1) a first immunoglobulin heavy chain variable domain (VH1) comprising an HCDR1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), an HCDR2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and an HCDR3 sequence comprising the amino acid sequence of 30 DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and (a2) a first immunoglobulin light chain variable domain (VL1) comprising an LCDR1 sequence comprising the amino acid sequence of CX1SSTGX2VTPX3X4YAN (SEQ 54
Sanofi Ref.: PAT24161 ID NO: 4), wherein X1 is R or A, X2 is T or A, X3 is S or G, and X4 is N or Y, an LCDR2 sequence comprising the amino acid sequence of DNNX5X6PP (SEQ ID NO: 5), wherein X5 is S, I, or N and X6 is R or K, and an LCDR3 sequence comprising the amino acid sequence of ALX7X8GX9QWV (SEQ ID NO: 6), wherein X7 is W or Y, X8 is F or Y, and X9 is N or 5 G; and (b) the second ABD comprises binding specificity to NKp46. 3. The method of embodiment 1 or 2, wherein (b) the second ABD comprises: 10 (b1) a second immunoglobulin heavy chain variable domain (VH2) comprising: - an HCDR1 sequence comprising DYVIN, an HCDR2 sequence comprising EIYPGSGTNYYNEKFKA, and an HCDR3 sequence comprising RGRYGLYAMDY; - an HCDR1 sequence comprising GYTFSDYVIN (SEQ ID NO: 19), an HCDR2 sequence comprising EIYPGSGTN (SEQ ID NO: 20), and an HCDR3 sequence 15 comprising RGRYGLYAMDY (SEQ ID NO: 21); - an HCDR1 sequence comprising SDYAWN (SEQ ID NO: 22), an HCDR2 sequence comprising YITYSGSTSYNPSLES (SEQ ID NO: 23), and an HCDR3 sequence comprising GGYYGSSWGVFAY (SEQ ID NO: 24); - an HCDR1 sequence comprising EYTMH (SEQ ID NO: 25), an HCDR2 20 sequence comprising GISPNIGGTSYNQKFKG (SEQ ID NO: 26), and an HCDR3 sequence comprising RGGSFDY (SEQ ID NO: 27); - an HCDR1 sequence comprising SFTMH (SEQ ID NO: 28), an HCDR2 sequence comprising YINPSSGYTEYNQKFKD (SEQ ID NO: 29), and an HCDR3 sequence comprising GSSRGFDY (SEQ ID NO: 30); or 25 - an HCDR1 sequence comprising SDYAWN (SEQ ID NO: 31), an HCDR2 sequence comprising YITYSGSTNYNPSLKS (SEQ ID NO: 32), and an HCDR3 sequence comprising CWDYALYAMDC (SEQ ID NO: 33); and (b2) a second immunoglobulin light chain variable domain (VL2) comprising: 30 - an LCDR1 sequence comprising RASQDISNYLN (SEQ ID NO: 34), an LCDR2 sequence comprising YTSRLHS (SEQ ID NO: 35), and an LCDR3 sequence comprising QQGNTRPWT (SEQ ID NO: 36); 55
Sanofi Ref.: PAT24161 - an LCDR1 sequence comprising RVSENIYSYLA (SEQ ID NO: 37), an LCDR2 sequence comprising NAKTLAE (SEQ ID NO: 38), and an LCDR3 sequence comprising QHHYGTPWT (SEQ ID NO: 39); - an LCDR1 sequence comprising RASQSISDYLH (SEQ ID NO: 40), an 5 LCDR2 sequence comprising YASQSIS (SEQ ID NO: 41), and an LCDR3 sequence comprising QNGHSFPLT (SEQ ID NO: 42); - an LCDR1 sequence comprising RASENIYSNLA (SEQ ID NO: 43), an LCDR2 sequence comprising AATNLAD (SEQ ID NO: 44), and an LCDR3 sequence comprising QHFWGTPRT (SEQ ID NO: 45); or 10 - an LCDR1 sequence comprising RTSENIYSYLA (SEQ ID NO: 46), an LCDR2 sequence comprising NAKTLAE (SEQ ID NO: 47), and an LCDR3 sequence comprising QHHYDTPLT (SEQ ID NO: 48). 4. The method of embodiment 2 or 3, wherein the VL1 comprises: 15 - an LCDR1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 8), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 9); - an LCDR1 sequence comprising the amino acid sequence of 20 CRSSTGTVTPSNYAN (SEQ ID NO: 10), an LCDR2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 11), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 12); - an LCDR1 sequence comprising the amino acid sequence of CASSTGAVTPSNYAN (SEQ ID NO: 13), an LCDR2 sequence comprising the amino acid 25 sequence of DNNIKPP (SEQ ID NO: 14), and an LCDR3 sequence comprising the amino acid sequence of ALWYGGQWV (SEQ ID NO: 15); or - an LCDR1 sequence comprising the amino acid sequence of CASSTGAVTPGYYAN (SEQ ID NO: 16), an LCDR2 sequence comprising the amino acid sequence of DNNNKPP (SEQ ID NO: 17), and an LCDR3 sequence comprising the amino 30 acid sequence of ALYYGGQWV (SEQ ID NO: 18). 5. The method of embodiment 1, wherein: 56
Sanofi Ref.: PAT24161 (a) the first ABD comprises: (a1) a first immunoglobulin heavy chain variable domain (VH1) comprising an HCDR1 sequence comprising the amino acid sequence of GFTFSNFG (SEQ ID NO: 112), an HCDR2 sequence comprising the amino acid sequence of IWSDETNR (SEQ ID NO: 5 113), and an HCDR3 sequence comprising the amino acid sequence of ARDQQYCSSDSCFTWFDP (SEQ ID NO: 114); and (a2) a first immunoglobulin light chain variable domain (VL1) comprising an LCDR1 sequence comprising the amino acid sequence of TGTVTPSNY (SEQ ID NO: 118), an LCDR2 sequence comprising the amino acid sequence of DNN (SEQ ID NO: 119), and 10 an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 120). 6. The method of embodiment 1, wherein: (a) the first ABD comprises: 15 (a1) a first immunoglobulin heavy chain variable domain (VH1) comprising an HCDR1 sequence comprising the amino acid sequence of NFGMH (SEQ ID NO: 115), an HCDR2 sequence comprising the amino acid sequence of VIWSDETNRYYADSVKG (SEQ ID NO: 116), and an HCDR3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 117); and 20 (a2) a first immunoglobulin light chain variable domain (VL1) comprising an LCDR1 sequence comprising the amino acid sequence of ASSTGTVTPSNYAN (SEQ ID NO: 121), an LCDR2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 122), and an LCDR3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 123). 25 7. The method of any one of embodiments 2-6, wherein: - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 30 55; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino 57
Sanofi Ref.: PAT24161 acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 50; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino 5 acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 51; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 52; - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 53; or 15 - the VH1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 54. 20 8. The method of any one of embodiments 2-7, wherein: - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 55; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 50; 25 - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 51; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 52; - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein 30 the VL1 comprises an amino acid sequence of SEQ ID NO: 53; or - the VH1 comprises an amino acid sequence of SEQ ID NO: 49, and wherein the VL1 comprises an amino acid sequence of SEQ ID NO: 54. 58
Sanofi Ref.: PAT24161 9. The method of any one of embodiments 3-8, wherein: - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56, and wherein the VL2 comprises an amino 5 acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 64; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 57, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 65; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 58, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 66; 15 - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 59, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 67; - the VH2 comprises an amino acid sequence that is at least about 90% identical 20 to the amino acid sequence of SEQ ID NO: 60, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 68; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 61, and wherein the VL2 comprises an amino 25 acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 69; - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 62, and wherein the VL2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 30 70; or - the VH2 comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 63, and wherein the VL2 comprises an amino 59
Sanofi Ref.: PAT24161 acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 71. 10. The method of any one of embodiments 3-9, wherein: 5 - the VH2 comprises an amino acid sequence of SEQ ID NO: 56, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 64; - the VH2 comprises an amino acid sequence of SEQ ID NO: 57, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 65; - the VH2 comprises an amino acid sequence of SEQ ID NO: 58, and wherein 10 the VL2 comprises an amino acid sequence of SEQ ID NO: 66; - the VH2 comprises an amino acid sequence of SEQ ID NO: 59, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 67; - the VH2 comprises an amino acid sequence of SEQ ID NO: 60, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 68; 15 - the VH2 comprises an amino acid sequence of SEQ ID NO: 61, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 69; - the VH2 comprises an amino acid sequence of SEQ ID NO: 62, and wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 70; or - the VH2 comprises an amino acid sequence of SEQ ID NO: 63, and wherein 20 the VL2 comprises an amino acid sequence of SEQ ID NO: 71. 11. The method of any one of embodiments 1-10, wherein the binding protein further comprises all or part of an immunoglobulin Fc domain or variant thereof. 25 12. The method of embodiment 11, wherein all or part of the immunoglobulin Fc domain or variant thereof binds to a human Fc-γ receptor. 13. The method of embodiment 11 or 12, wherein all or part of the immunoglobulin Fc domain or variant thereof binds to a human CD16A (FcγRIII) 30 polypeptide. 60
Sanofi Ref.: PAT24161 14. The method of any one of embodiments 11-13, wherein the Fc domain comprises a native glycan at amino acid position 297, according to EU numbering. 15. The method of any one of embodiments 11-14, wherein the binding protein 5 is N-glycosylated. 16. The method of any one of embodiments 11-15, wherein the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain. 10 17. The method of embodiment 16, wherein at least one Fc heavy chain comprises an engineered intrachain disulfide bond mediated by a pair of cysteines (C) that substitute for: (i) a leucine (L) at amino acid position 242 and a lysine (K) at amino acid position 334; 15 or (iii) an arginine (R) at amino acid position 292 and a valine (V) at amino acid position 302; wherein the amino acid positions are according to EU numbering. 20 18. The method of embodiment 17, wherein the first and the second Fc heavy chain each comprise the L242C / K334C substitutions. 19. The method of embodiment 17, wherein the first and the second Fc heavy chain each comprise the R292C / V302C substitutions. 25 20. The method of any one of embodiments 16-19, wherein at least one Fc heavy chain comprises a substitution at amino acid position 332, according to EU numbering. 21. The method of embodiment 20, wherein the substitution at amino acid 30 position 332 is a glutamic acid (E). 61
Sanofi Ref.: PAT24161 22. The method of embodiment 20 or 21, wherein the at least one Fc heavy chain further comprises one or more substitutions at amino acid positions 236, 239, or 330, according to EU numbering. 5 23. The method of embodiment 22, wherein the substitution at amino acid position 236 is an alanine (A), the substitution at amino acid position 239 is an aspartic acid (D), and the substitution at amino acid position 330 is a leucine (L). 24. The method of any one of embodiments 16-23, wherein at least one Fc 10 heavy chain comprises an aspartic acid (D) at amino acid position 239, and a glutamic acid (E) at amino acid position 332, according to EU numbering. 25. The method of any one of embodiments 16-24, wherein at least one Fc heavy chain comprises an alanine (A) at amino acid position 236, an aspartic acid (D) at amino 15 acid position 239, and a glutamic acid (E) at amino acid position 332, according to EU numbering. 26. The method of any one of embodiments 16-25, wherein at least one Fc heavy chain further comprises an alanine (A) at amino acid position 236, an aspartic acid (D) at 20 amino acid position 239, a leucine (L) at amino acid position 330, and a glutamic acid (E) at amino acid position 332, according to EU numbering. 27. The method of any one of embodiments 3-26, wherein the binding protein comprising at least two polypeptide chains that form at least two antigen-binding sites, 25 wherein at least one polypeptide chain comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I]; and at least one polypeptide chain comprises a structure represented by the formula: 30 VH2-L3-VH1-L4-CH1 [II]; wherein: 62
Sanofi Ref.: PAT24161 CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; and L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and 5 wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 28. The method of any one of embodiments 3-27, wherein the binding protein comprises three polypeptide chains that form two antigen-binding sites, wherein one 10 polypeptide chain comprises a structure represented by the formula: VL1-L1-VL2-L2-CL [I]; one polypeptide chain comprises a structure represented by the formula: VH2-L3-VH1-L4-CH1-hinge-CH2-CH3 [III]; and 15 one polypeptide chain comprises a structure represented by the formula: hinge-CH2-CH3 [IV] wherein: CL is an immunoglobulin light chain constant domain; 20 CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and 25 L1, L2, L3, and L4 are amino acid linkers, wherein any one or more of L1, L2, L3, and L4 are optionally absent, and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. 30 29. The method of embodiment 27 or 28, wherein: 63
Sanofi Ref.: PAT24161 (a) L1, L2, L3, and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS, GGGGSGGGGSGGGGS, S, RT, TKGPS, GQPKAAP, and GGSGSSGSGG; or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from 5 the group consisting of GGGGSGGGGS, GGGGSGGGGSGGGGS, S, RT, TKGPS, GQPKAAP, and GGSGSSGSGG. 30. The method of any one of embodiments 1-29, wherein: (a) the first ABD of the binding protein comprises a first immunoglobulin heavy 10 chain variable domain (VH1) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 49, and a first immunoglobulin light chain variable domain (VL1) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 55; and (b) the second ABD of the binding protein comprises a second immunoglobulin 15 heavy chain variable domain (VH2) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 56, and a second immunoglobulin light chain variable domain (VL2) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 64. 20 31. The method of any one of embodiments 1-30, wherein the binding protein comprises: (i) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 72; (ii) a second polypeptide chain comprising an amino acid sequence of SEQ ID 25 NO: 73; and (iii) a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74. 32. The method of any one of embodiments 1-31, wherein the cereblon 30 modulating agent is a cereblon E3 ligase modulator (CELMoD) or an immunomodulatory drug (IMiD). 64
Sanofi Ref.: PAT24161 33. The method of embodiment 32, wherein the CELMoD is iberdomide or mezigdomide. 34. The method of embodiment 32, wherein the IMiD is pomalidomide, 5 lenalidomide, or thalidomide. 35. The method of any one of embodiments 1-34, wherein the binding protein is administered to the subject prior to the cereblon modulating agent. 10 36. The method of any one of embodiments 1-34, wherein the cereblon modulating agent is administered to the subject prior to the binding protein. 37. The method of any one of embodiments 1-34, wherein the binding protein and the cereblon modulating agent are administered concurrently to the subject. 15 38. The method of any one of embodiments 1-37, wherein the cancer is multiple myeloma. EXAMPLES 20 The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. Example 1: Combination of NKp46-BCMA NKCE with cereblon modulating agents Introduction 25 The aim of this study was to evaluate the cytotoxic effect of an exemplary BCMA NKCE, NKP46-BCMA_FC-ADE-DSB NKCE, in combination with one of the three cereblon modulating agents, pomalidomide, iberdomide or mezigdomide, in the presence of NK cells against a multiple myeloma (MM) cell line (H929 RFP). The NKP46-BCMA_FC-ADE-DSB NKCE used in the Examples is schematically 30 depicted in FIG. 1 and comprises: a first antigen binding domain (ABD) with binding specificity to BCMA and a second ABD with binding specificity to NKp46, wherein: 65
Sanofi Ref.: PAT24161 (a) the first ABD of the binding protein comprises a first immunoglobulin heavy chain variable domain (VH1) comprising an amino acid sequence of SEQ ID NO: 49, and a first immunoglobulin light chain variable domain (VL1) comprising an amino acid sequence of SEQ ID NO: 55; and 5 (b) the second ABD of the binding protein comprises a second immunoglobulin heavy chain variable domain (VH2) comprising an amino acid sequence of SEQ ID NO: 56, and a second immunoglobulin light chain variable domain (VL2) comprising an amino acid sequence of SEQ ID NO: 64. The NKP46-BCMA_FC-ADE-DSB NKCE has three polypeptide chains, (i) the 10 first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 72; (ii) the second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 73; and (iii) the third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74. Cell count was studied over a period of 96 hours with a measurement every 4 15 hours. The following groups were evaluated: - H929 (blank wells with tumor cell line and without NK cells) - H929 + NK cells - isotype Control at 1 nM 20 - NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM alone - iberdomide at 50 nM alone - iberdomide at 50 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM - mezigdomide at 0.5 nM alone - mezigdomide at 0.5 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM 25 - pomalidomide at 200 nM alone - pomalidomide at 200 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM The cell count (fluorescent cells) was measured for four healthy NK cell donors (NK922, NK184, NK860 and NK617) except for iberdomide alone and in combination for 30 which the measures were not done on NK860 donor and for pomalidomide alone and in combination for which the measures were not done on NK922 donor. 66
Sanofi Ref.: PAT24161 For each donor, all groups were measured in duplicates excepted for H929 alone for which it was measured in four replicates. Due to technical issues, there were only two replicates for NK922 donor and three replicates for NK184 donor for H929 group and one replicate for donor NK860 for mezigdomide at 0.5 nM + NKP46-BCMA_FC-ADE-DSB 5 NKCE at 1 nM and pomalidomide at 200 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM groups. The measured parameter was the count of H929 cells normalized by baseline value (hour 0) expressed in percentage. 10 Statistical Methods The objectives of the statistical analysis were the following: - Objective 1: comparison of each treated group to the isotype control group - Objective 2: comparison of each combination versus the single compounds 15 The Area Under the Curve (AUC) was computed for each replicate from 0h to 96h according to the following formula :
Where: “h” is hour increasing in steps of 4; 20 “h−1” is the previous measured timepoint; and “Count” is the number of normalized H929 cells measured For each donor, all groups were measured at least in duplicates, the mean of AUC was calculated for each group and donor. 25 A global mixed model including all groups except H929 and H929 + NK cells groups (not needed for these objectives) with group as fixed factor and donor as random factor was applied on log-transformed AUC. For objective 1, it was followed by a Dunnett’s test to compare all treated groups to the isotype control group. 67
Sanofi Ref.: PAT24161 For objective 2, it was followed by contrast analyses to compare combinations versus single compounds with a Bonferroni-Holm correction for multiplicity. An heterogeneity of variance was taken into account for the group pomalidomide at 200 nM alone versus the other. 5 Results Iberdomide, pomalidomide, mezigdomide, NKP46-BCMA_FC-ADE-DSB NKCE and isotype Control were respectively abbreviated as iber, pom, mezig, NKCE1 and IC1 to lighten the graphs and the tables. 10 Table 1 – Descriptive statistics of FIG. 2 – AUC
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Table 2 – Descriptive statistics of FIG. 3 – percent inhibition of AUC
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Sanofi Ref.: PAT24161 Table 3 – Objectives 1 and 2: comparison of AUC versus isotype control and comparison of the combinations versus single compounds
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As shown in FIG. 2 and FIG. 3 and Tables 1-3, the combination of NKP46- BCMA_FC-ADE-DSB NKCE and any one of the compounds iberdomide, pomalidomide, or mezigdomide, consistently lead to greater H929 MM cell death compared to either agent 5 alone. There was a significant difference versus the isotype control for all treated groups, except for pomalidomide at 200 nM alone for which a high variability was observed. For the combinations iberdomide at 50 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM and mezigdomide at 0.5 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 10 nM, there was a significant difference versus the single compounds. For the combination pomalidomide at 200 nM + NKP46-BCMA_FC-ADE-DSB NKCE at 1 nM, there was a significant difference versus NKP46-BCMA_FC-ADE-DSB NKCE alone but not versus the pomalidomide at 200 nM alone. 15 Additional cytotoxic assays: Further experiments were performed to assess the in vitro cytotoxicity of the combination of NKP46-BCMA_FC-ADE-DSB NKCE with one of three cereblon modulating agents. The in vitro cytotoxicity assays were performed with fluorescent H929-RFP MM 20 cells, having a BCMA cell surface density of about 15,000 sites/cell. The assays were performed with an effector : target (E : T) ratio of 1:2 during 6 days by live-cell imaging analysis (Incucyte system). NKP46-BCMA_FC-ADE-DSB NKCE was used at a 71
Sanofi Ref.: PAT24161 concentration of 0.1 nM, pomalidomide at 100 nM, iberdomide at 10 nM, and mezigdomide at 0.1 nM. As shown in FIG. 4A – FIG. 4C, the combination of NKP46-BCMA_FC-ADE- DSB NKCE with any one of the cereblon modulating agents greatly enhanced in vitro 5 cytotoxicity of the multiple myeloma cell line, compared to either NKP46-BCMA_FC- ADE-DSB NKCE or the cereblon modulating agentalone. NKP46-BCMA_FC-ADE-DSB NKCE and the different IMiD/CELMoDs demonstrated potent MM cell killing in a concentration-dependent manner as single agents against H929 cells as compared to an isotype control that does not bind to BCMA. When 10 NKP46-BCMA_FC-ADE-DSB NKCE was combined with either pomalidomide, iberdomide or mezigdomide, a higher cytotoxic activity was observed as compared to single agent pomalidomide (p=0.1197) and significantly higher as compared to single agent iberdomide (p=0.0035) or mezigdomide (p=0.0026). NKP46-BCMA_FC-ADE-DSB NKCE shows the highest potency in combination with 15 mezigdomide. Sequence listing
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