Patent Application
20230272055
This application contains a Sequence Listing that has been submitted electronically as an XML file named 20443-0736001_SL.xml. The XML file, created on Dec. 7, 2022, is 388,039 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
Calreticulin (CALR) is a highly conserved chaperone protein that resides primarily in the endoplasmic reticulum and is involved in a variety of cellular processes including protein folding, calcium homeostasis, cell adhesion, and integrin signaling. CALR is also found in the nucleus, suggesting that it may have a role in transcription regulation. Mutations in the gene for CALR have been identified in patients with myeloproliferative neoplasms.
The present disclosure is based, at least in part, on the development of antibodies having high binding affinity and specificity to mutant CALR (“mutCALR”).
Accordingly, aspects of the present disclosure provide an antibody that binds to human mutant calreticulin (CALR), wherein the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein: the VH CDR1 comprises the amino acid sequence X1X2X3X4X5, wherein X1 is S, E, or D; wherein X2 is Y, L, or S; wherein X3 is A, S, or F; wherein X4 is I or M; and wherein X5 is S, Q, or H; the VH CDR2 comprises the amino acid sequence X6X7X8PX9X10X11X12X13X14YAX15X16X17X18G (SEQ ID NO:97), wherein X6 is L or G; wherein X7 is V, F, or I; wherein X8 is D or I; wherein X9 is E, D, or I; wherein X10 is D, G, F, A, S, or E; wherein X11 is G or A; wherein X12 is E or T; wherein X13 is T or A; wherein X14 is I, M, or N; wherein X15 is E or Q; wherein X16 is K or R; wherein X17 is F or L; and wherein X18 is R or Q; the VH CDR3 comprises the amino acid sequence X19X20X21X22X23X24X25X26X27X28X29X30X31X32X33X34X35X36X37X38 (SEQ ID NO:98), wherein X19 is P, E, or absent; wherein X20 is G, E, or absent; wherein X21 is G, W, S, or absent; wherein X22 is I, D, S, P, or absent; wherein X23 is S, L, I, T, G, or absent; wherein X24 is P, T, Q, I, D, R, or absent; wherein X25 is G, D, or absent; wherein X26 is E, Y, P, L, D, or S; wherein X27 is E, D, A, or G; wherein X28 is S, F, A, E, Y, or W; wherein X29 is Y or F; wherein X30 is G, D, or W; wherein X31 is P, Y, 1, or H; wherein X32 is Y or absent; wherein X33 is Y or absent; wherein X34 is Y or absent; wherein X35 is G or absent; wherein X36 is M or absent; wherein X37 is D or absent; wherein X38 is V or absent; wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence X39X40X41X42X43X44X45X46X47X48X49X50X51X52X53X54 (SEQ ID NO:99), wherein X39 is T, A, or absent; wherein X40 is G or absent; wherein X41 is Q, G, V, T, or S; wherein X42 is A, G, S, or N; wherein X43 is S, N, D, T, or Y; wherein X44 is Q, Y, N, D, S, or K; wherein X45 is D, I, F, V, S, or T; wherein X46 is N or absent; wherein X47 is I or absent; wherein X48 is I, G, or R; wherein X49 is S, G, A, D, I, R, or T; wherein X50 is Y or absent; wherein X51 is N, K, I, or E; wherein X52 is Y, S, N, D, H, F, R, or G; wherein X53 is L or V; and wherein X54 is N, H, S, D, or F; the VL CDR2 comprises the amino acid sequence X55X56X57X58X59X60X61, wherein X55 is T, D, E, Q, or R; wherein X56 is A, D, V, or N; wherein X57 is S, G, N, or R; wherein X58 is I, N, D, or K; wherein X59 is L, R, or W; wherein X60 is E or P; and wherein X61 is S, T, or L; the VL CDR3 comprises the amino acid sequence X62X63X64X65X66X67X68X69X70X71X72, wherein X62 is Q, S, C, or G; wherein X63 is Q, V, S, T, or A; wherein X64 is Q, L, W, or Y; wherein X65 is Q, N, D, I, T, A, or G; wherein X66 is S, P, G, N, or A; wherein X67 is N, Y, I, S, N, L, or D; wherein X68 is E, P, S, I, N, H, L, or T; wherein X69 is D, T, S, or absent; wherein X70 is P, H, L, R, F, A, Q, or absent; wherein X71 is W, L, V, Y, S, A, or E; and wherein X72 is T, V, or I.
In some embodiments, the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NOs:1-6; the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NOs:7-17 and 92-95; the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NOs:18-25; the VL CDR1 comprises the amino acid sequence of any one of SEQ ID NOs:26-52 or 118; the VL CDR2 comprises the amino acid sequence of any one of SEQ ID NOs:53-68; and the VL CDR3 comprises the amino acid sequence of any one of SEQ ID NOs:69-91.
In some embodiments, the VH CDR1, the VH CDR2, and the VH CDR3 each correspond to the VH CDRs set forth in Tables 1-2 for a single VH clone, and wherein the VL CDR1, the VL CDR2, and the VL CDR3 each correspond to the VL CDRs set forth in Tables 1-2 for a single VL clone.
In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:26; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:53; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:69;
In some embodiments, the VH is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs:165-208; and the VL is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs:264-318.
In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs:165-208; and the VL comprises the amino acid sequence of any one of SEQ ID NOs:264-318.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:165 and the VL comprises the amino acid sequence of SEQ ID NO:264;
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:165 and the VL comprises the amino acid sequence of SEQ ID NO:277.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:165 and the VL comprises the amino acid sequence of SEQ ID NO:279.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:165 and the VL comprises the amino acid sequence of SEQ ID NO:268.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:165 and the VL comprises the amino acid sequence of SEQ ID NO:315.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 190 and the VL comprises the amino acid sequence of SEQ ID NO:315.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:196 and the VL comprises the amino acid sequence of SEQ ID NO:315.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO 191 and the VL comprises the amino acid sequence of SEQ ID NO:277.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:165 and the VL comprises the amino acid sequence of SEQ ID NO:317.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:191 and the VL comprises the amino acid sequence of SEQ ID NO:318.
In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:195 and the VL comprises the amino acid sequence of SEQ ID NO:318.
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:119 and the light chain comprises the amino acid sequence of SEQ ID NO:222.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:224.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:142 and the light chain comprises the amino acid sequence of SEQ ID NO:213.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:142 and the light chain comprises the amino acid sequence of SEQ ID NO:260.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:145 and the light chain comprises the amino acid sequence of SEQ ID NO:260.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:151 and the light chain comprises the amino acid sequence of SEQ ID NO:260.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:146 and the light chain comprises the amino acid sequence of SEQ ID NO:222.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:142 and the light chain comprises the amino acid sequence of SEQ ID NO:262.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:146 and the light chain comprises the amino acid sequence of SEQ ID NO:263.
In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:150 and the light chain comprises the amino acid sequence of SEQ ID NO:263.
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO:1); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO:18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence GGX104X105X106GX107X108X109VX110 (SEQ ID NO:103; Group 1 clones), wherein X104 is N, D, or S; wherein X105 is Y, N, or D; wherein X106 is I or T; wherein X107 is S, D, I, R, or T; wherein X108 is K, E, or I; wherein X109 is S, I, R, G, N, or A; and wherein X110 is H, F, or N; the VL CDR2 comprises the amino acid sequence DDX111DRPX112 (SEQ ID NO:104; Group 1 clones), wherein X111 is G, S, or R; and wherein X112 is S or L; and the VL CDR3 comprises the amino acid sequence QVWDX113X114X115DX116X117X118 (SEQ ID NO:105; Group 1 clones), wherein X113 is S or A; wherein X114 is I or S; wherein X115 is S, I, or N; wherein X116 is H, L, or Q; wherein X117 is V or L; and wherein X118 is V or I.
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO:329); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO:18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence GGX104X105X106GX107X108X109VX110 (SEQ ID NO:103; Group 1 clones), wherein X104 is N, D, or S; wherein X105 is Y, N, or D; wherein X106 is I or T; wherein X107 is S, D, I, R, or T; wherein X108 is K, E, or I; wherein X109 is S, I, R, G, N, or A; and wherein X110 is H, F, or N; the VL CDR2 comprises the amino acid sequence DDX111DRPX112 (SEQ ID NO:104; Group 1 clones), wherein X111 is G, S, or R; and wherein X112 is S or L; and the VL CDR3 comprises the amino acid sequence QVWDX113X114X115DX116X117X118 (SEQ ID NO:105; Group 1 clones), wherein X113 is S or A; wherein X114 is I or S; wherein X115 is S, I, or N; wherein X116 is H, L, or Q; wherein X117 is V or L; and wherein X118 is V or I.
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO:1); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO:30); the VL CDR2 comprises the amino acid sequence X119VSX120RPS (SEQ ID NO:106; Group 1 clones); wherein X119 is E or D; and wherein X120 is N or K; and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO:329); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X110 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO:18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO:30); the VL CDR2 comprises the amino acid sequence X119VSX120RPS (SEQ ID NO:106; Group 1 clones); wherein X119 is E or D; and wherein X120 is N or K; and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence DYFIH (SEQ ID NO:2); the VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEX121FQG (SEQ ID NO:107; Group 2 clones), wherein X121 is K or R; the VH CDR3 comprises the amino acid sequence PGGILTDPDAFDI (SEQ ID NO:19); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence X122GTX123SDVGGYNX124VS (SEQ ID NO:108; Group 2 clones), wherein X122 is T or A; wherein X123 is S or G; and wherein X124 is Y or H; the VL CDR2 comprises the amino acid sequence X125VX126X127RPS (SEQ ID NO:109; Group 2 clones), wherein X125 is D or E; wherein X126 is N or S; and wherein X127 is K or N; and the VL CDR3 comprises the amino acid sequence SSYX128X129SSTX130X131V (SEQ ID NO:110; Group 2 clones), wherein X128 is I or T; wherein X129 is P or S; wherein X130 is R, P, F, or absent; and wherein X131 is W or Y.
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEKFX132G (SEQ ID NO:111; Group 3 clones), wherein X132 is R or Q; the VH CDR3 comprises the amino acid sequence EESYGP (SEQ ID NO:20); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence QASQDISNYLX133 (SEQ ID NO:112; Group 3 clones), X133 is N or D; the VL CDR2 comprises the amino acid sequence DASNLET (SEQ ID NO:61); and the VL CDR3 comprises the amino acid sequence QQLNSYPLT (SEQ ID NO:80).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence EX134SMH (SEQ ID NO:113; Group 4 clones), wherein X134 is S or L; the VH CDR2 comprises the amino acid sequence LVDPEDGETIYAQKFQG (SEQ ID NO:14); the VH CDR3 comprises the amino acid sequence EEWSGDGDDAFDI (SEQ ID NO:21); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence SGSSSNIGSYSVS (SEQ ID NO:46); the VL CDR2 comprises the amino acid sequence DX135NKRPS (SEQ ID NO:114; Group 4 clones), wherein X135 is N or D; and the VL CDR3 comprises the amino acid sequence GTWDSSLSAWV (SEQ ID NO:81).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 15); the VH CDR3 comprises the amino acid sequence SPLRGSGWYWHYYYGMDV (SEQ ID NO:22); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence GGNX136IX137X138KX139VH (SEQ ID NO:115; Group 6 clones), wherein X136 is N or K; wherein X137 is R or G; wherein X138 is A, S, R, or T; and wherein X139 is H or S; the VL CDR2 comprises the amino acid sequence X140DX141X142RPS (SEQ ID NO:116; Group 6 clones), wherein X140 is Q or R; wherein X141 is S or R; and wherein X142 is N or K; and the VL CDR3 comprises the amino acid sequence QX143WX144SX145TX146V (SEQ ID NO:117; Group 6 clones), wherein X143 is A or V; wherein X144 is D or G; wherein X145 is S or N; and wherein X146 is V, A, or E.
In some embodiments, the human mutant CALR is human Type 1 mutant CALR comprising the amino acid sequence of SEQ ID NO:320. In some embodiments, the human mutant CALR is human Type 2 mutant CALR comprising the amino acid sequence of SEQ ID NO:321.
In some embodiments, the anti-mutCALR antibody inhibits one or more signaling pathways downstream of thrombopoietin receptor (MPL) in a cell expressing human mutant CALR; inhibits oncogenic cell proliferation in a cell expressing human mutant CALR; and/or inhibits dimerization of MPL in a cell expressing human mutant CALR.
In some embodiments, the anti-mutCALR antibody inhibits one or more signaling pathways downstream of MPL in both a first cell expressing human Type 1 mutant CALR and in a second cell expressing human Type 2 mutant CALR; inhibits oncogenic cell proliferation in both a first cell expressing human Type 1 mutant CALR and in a second cell expressing human Type 2 mutant CALR; and/or inhibits dimerization of MPL in both a first cell expressing human Type 1 mutant CALR and in a second cell expressing human Type 2 mutant CALR.
In some embodiments, the one or more signaling pathways downstream of MPL are selected from the group consisting of Janus tyrosine kinase (JAK) and signal transducers and activators of transcription (STAT) signaling, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, serine/threonine kinase (AKT) signaling, and mammalian target of rapamycin (mTOR) signaling.
In some embodiments, the anti-mutCALR antibody has modulated Fc effector function. In some embodiments, the modulated Fc effector function is increased Fc effector function or reduced Fc effector function. In some embodiments, the anti-mutCALR antibody has reduced Fc effector function. In some embodiments, the Fc effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), or antibody dependent cellular phagocytosis (ADCP). In some embodiments, the anti-mutCALR antibody having reduced Fc effector function has increased binding affinity to human mutant CALR as compared to an antibody without reduced Fc effector function. In some embodiments, the Fc effector function is ADCC.
In some embodiments, the anti-mutCALR antibody is a human or humanized antibody. In some embodiments, the anti-mutCALR antibody is a full-length antibody. In some embodiments, the anti-mutCALR antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, the anti-mutCALR antibody is an IgG1 antibody. In some embodiments, the anti-mutCALR antibody is a bispecific antibody, a biparatopic antibody, a single chain antibody (scFv), an Fab fragment, an F(ab′)2 fragment, an Fab′ fragment, an Fsc fragment, an Fv fragment, an scFv, an sc(Fv)2, or a diabody.
In some embodiments, the anti-mutCALR antibody is a biparatopic antibody comprising two heavy chain-light chain pairs or one heavy chain-light chain pair. In some embodiments, the biparatopic antibody is a full-length antibody. In some embodiments, the biparatopic antibody comprises one heavy chain-light chain pair.
In some embodiments, the anti-mutCALR antibody is conjugated to a toxic substance. In some embodiments, the toxic substance is a radioisotope or a cytotoxic agent.
Aspects of the present disclosure provide a nucleic acid or a set of nucleic acids, which collectively encodes any one of the anti-mutCALR antibodies described herein.
Aspects of the present disclosure provide an expression vector or a set of expression vectors comprising the nucleic acid or the set of nucleic acids encoding any one of the anti-mutCALR antibodies described herein operably linked to a promoter.
Aspects of the present disclosure provide an isolated cell comprising the nucleic acid or the set of nucleic acids encoding any one of the anti-mutCALR antibodies or the expression vector or the set of expression vectors comprising the nucleic acid or the set of nucleic acids encoding any one of the anti-mutCALR antibodies described herein operably linked to a promoter.
Aspects of the present disclosure provide a method of making the anti-mutCALR antibody, comprising culturing the cell described herein and isolating the antibody.
Aspects of the present disclosure provide a pharmaceutical composition comprising the anti-mutCALR antibody, the nucleic acid or the set of nucleic acids, the expression vector or the set of expression vectors, or the isolated cell, and a pharmaceutically acceptable carrier.
Aspects of the present disclosure provide a method of treating a myeloproliferative neoplasm in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an anti-mutCALR antibody described herein or the pharmaceutical composition thereof. Other aspects of the present disclosure provide an anti-mutCALR antibody described herein for use in the treatment of a myeloproliferative neoplasm, or the use of an anti-mutCALR antibody described herein for the manufacture of a medicament for the treatment of a myeloproliferative neoplasm.
Aspects of the present disclosure provide a method of detecting a CALR exon 9 mutation in a biological sample, the method comprising obtaining a biological sample from a human subject who has or is suspected of having a myeloproliferative neoplasm and contacting the sample with an anti-mutCALR antibody described herein such that the anti-mutCALR antibody binds to a mutCALR protein if the mutCALR protein is present in the biological sample.
Another aspect of the present disclosure provides a method of diagnosing a human subject with a myeloproliferative neoplasm, the method comprising obtaining a biological sample from a human subject who has or is suspected of having a myeloproliferative neoplasm and contacting the sample with an anti-mutCALR antibody described herein such that the anti-mutCALR antibody binds to a mutCALR protein if the mutCALR protein is present in the biological sample.
In some embodiments, the myeloproliferative neoplasm is selected from the group consisting of chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, and chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.
In some embodiments, methods described herein further comprise administering to the human subject an additional therapy selected from the group consisting of a Janus tyrosine kinase (JAK) inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, a standard of care therapy, or a combination thereof. In some embodiments, the JAK inhibitor is ruxolitinib and itaticinib. In some embodiments, the PI3K inhibitor is parsaclisib. In some embodiments, the standard of care therapy is selected from the group consisting of IFN-alpha, hydroxyurea, thalidomide, lenalidomide, an androgen, an erythropoietin-stimulating agent, a chemotherapeutic agent, or a combination thereof.
In some embodiments, the administration of the antibody or the pharmaceutical composition thereof in combination with the JAK inhibitor produces a synergistic effect. In some embodiments, the JAK inhibitor is ruxolitinib.
Aspects of the present disclosure provide a kit comprising the anti-mutCALR antibody, the nucleic acid or the set of nucleic acids, the expression vector or the set of expression vectors, or the isolated cell, and instructions for use in treating a myeloproliferative neoplasm in a human subject in need thereof, optionally with instructions for use in combination with an additional therapy.
Provided herein are anti-mutCALR antibodies and related nucleic acids, expression vectors, cells, kits, and pharmaceutical compositions. The anti-mutCALR antibodies described herein are useful in the treatment or prevention or diagnosis of myeloproliferative neoplasms (e.g., chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.).
CALR is a highly conserved chaperone protein that resides primarily in the endoplasmic reticulum and is involved in a variety of cellular processes including protein folding, calcium homeostasis, cell adhesion, and integrin signaling. Mutations in the CALR gene have been identified in patients with myeloproliferative neoplasms. The two most frequent CALR mutations are a 52 base pair (bp) deletion and a 5 bp insertion, which are referred to as Type 1 and Type 2 mutations, respectively. Type 1 and Type 2 mutations cause a +1 frameshift within exon 9 that generates a novel, positively-charged C-terminal amino acid sequence that lacks the KDEL domain (SEQ ID NO:347) of the WT protein, thereby enabling the mutCALR to escape the ER and activate the thrombopoietin receptor (MPL) and induce constitutive activation of Janus kinase 2 (JAK2) signaling. The amino acid sequences of human WT CALR, Type 1 and Type 2 mutCALR, and the novel C-terminal sequence are shown below. The frameshift amino acid residues in Type 1 and Type 2 mutCALR are shown in bold and the novel C-terminal sequence is marked by underlining.
REACLQGWTEA
RTRRKMRRKMSPARPRTSCREACLQGWTEA
This disclosure provides anti-mutCALR antibodies that are useful in treating or diagnosing myeloproliferative neoplasms. Note that in the present disclosure, unless stated otherwise, amino acid positions assigned to CDRs and frameworks in a variable region of the anti-mutCALR antibodies are specified according to Kabat; see EA Kabat, Sequences of Proteins of Immunological Interest, U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, (OCoLC)1138727707.
In some embodiments, the anti-mutCALR antibody is an anti-mutCALR antibody that comprises one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein. In some embodiments, an anti-mutCALR antibody comprises (i) one, two, and/or three heavy chain CDRs of any one of the clones presented in Tables 1-2, and/or (ii) one, two, and/or three light chain CDRs from any one of the clones presented in Tables 1-2.
In some embodiments, an anti-mutCALR antibody comprises (i) three heavy chain CDRs from any one of the clones presented in Tables 4-5, and (ii) three light chain CDRs from any one of the clones presented in Tables 4-5.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, an anti-mutCALR antibody comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, an anti-mutCALR antibody comprises a mouse version, mouse variant, human version, human variant, humanized version, humanized variant, or affinity matured variant of an antibody described herein.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from any clone disclosed herein, a humanized version thereof, or variants thereof (including affinity matured variants). In some embodiments, an anti-mutCALR antibody comprises a heavy chain CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from any clone disclosed herein. In other embodiments, an anti-mutCALR antibody comprises a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from any clone disclosed herein. In certain embodiments, an anti-mutCALR antibody comprises a heavy chain CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is a mouse version of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is a mouse variant of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is a human version of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is a human variant of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is a humanized version of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is a variant of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody is an affinity matured variant of any clone disclosed herein.
In some embodiments, the anti-mutCALR antibody is a variant of an anti-mutCALR antibody described herein which comprises one to thirty conservative amino acid substitution(s), e.g., one to twenty-five, one to twenty, one to fifteen, one to ten, one to five, or one to three conservative amino acid substitution(s). In some embodiments, the conservative amino acid substitution(s) is in a CDR of the antibody. In some embodiments, the conservative amino acid substitution(s) is not in a CDR of the antibody. In some embodiments, the conservative amino acid substitution(s) is in a framework region of the antibody.
In some embodiments, a CDR comprises one amino acid substitution. In some embodiments, a CDR comprises two amino acid substitutions. In some embodiments, a CDR comprises three amino acid substitutions. In some embodiments, a CDR comprises four amino acid substitutions. In some embodiments, the one or more amino acid substitutions are conservative substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain variable region CDR2. In some embodiments, the CDR is a heavy chain variable region CDR3. In some embodiments, the CDR is a light chain variable region CDR1. In some embodiments, the CDR is a light chain variable region CDR2. In some embodiments, the CDR is a light chain variable region CDR3. In some embodiments, the one or more substitutions are made as part of a humanization process. In some embodiments, the one or more substitutions are made as part of a germline humanization process. In some embodiments, the one or more substitutions are made as part of an affinity maturation process. In some embodiments, the one or more substitutions are made as part of an optimization process.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3, each of which correspond to the heavy chain variable region CDRs set forth in Tables 1-2 for a single clone, and a light chain variable region CDR1, a light chain variable region VL CDR2, and a light chain variable region VL CDR3, each of which correspond to the VL CDRs set forth in Tables 1-2 for a single clone.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3, each of which correspond to the VH and VL CDRs set forth in Tables 1-2 for a single clone.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 comprises the amino acid sequence X1X2X3X4X5, wherein X1 is S, E, or D; wherein X2 is Y, L, or S; wherein X3 is A, S, or F; wherein X4 is I or M; and wherein X5 is S, Q, or H; the VH CDR2 comprises the amino acid sequence X6X7X8PX9X10X11X12X13X14YAX15X16X17X18G (SEQ ID NO:97), wherein X6 is L or G; wherein X7 is V, F, or I; wherein X8 is D or I; wherein X9 is E, D, or I; wherein X10 is D, G, F, A, S, or E; wherein X11 is G or A; wherein X12 is E or T; wherein X13 is T or A; wherein X14 is I, M, or N; wherein X15 is E or Q; wherein X16 is K or R; wherein X17 is F or L; and wherein X18 is R or Q; the VH CDR3 comprises the amino acid sequence X19X20X21X22X23X24X25X26X27X28X29X30X31X32X33X34X35X36X37X38 (SEQ ID NO:98), wherein X19 is P, E, or absent; wherein X20 is G, E, or absent; wherein X21 is G, W, S, or absent; wherein X22 is I, D, S, P, or absent; wherein X23 is S, L, I, T, G, or absent; wherein X24 is P, T, Q, I, D, R, or absent; wherein X25 is G, D, or absent; wherein X26 is E, Y, P, L, D, or S; wherein X27 is E, D, A, or G; wherein X28 is S, F, A, E, Y, or W; wherein X29 is Y or F; wherein X30 is G, D, or W; wherein X31 is P, Y, I, or H; wherein X32 is Y or absent; wherein X33 is Y or absent; wherein X34 is Y or absent; wherein X35 is G or absent; wherein X36 is M or absent; wherein X37 is D or absent; wherein X38 is V or absent; wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence X39X40X41X42X43X44X45X46X47X48X49X50X51X52X53X54 (SEQ ID NO:99), wherein X39 is T, A, or absent; wherein X40 is G or absent; wherein X41 is Q, G, V, T, or S; wherein X42 is A, G, S, or N; wherein X43 is S, N, D, T, or Y; wherein X44 is Q, Y, N, D, S, or K; wherein X45 is D, I, F, V, S, or T; wherein X46 is N or absent; wherein X47 is I or absent; wherein X48 is I, G, or R; wherein X49 is S, G, A, D, I, R, or T; wherein X50 is Y or absent; wherein X51 is N, K, I, or E; wherein X52 is Y, S, N, D, H, F, R, or G; wherein X53 is L or V; and wherein X54 is N, H, S, D, or F; the VL CDR2 comprises the amino acid sequence X55X56X57X58X59X60X61, wherein X55 is T, D, E, Q, or R; wherein X56 is A, D, V, or N; wherein X57 is S, G, N, or R; wherein X58 is I, N, D, or K; wherein X59 is L, R, or W; wherein X60 is E or P; and wherein X61 is S, T, or L; the VL CDR3 comprises the amino acid sequence X62X63X64X65X66X67X68X69X70X71X72, wherein X62 is Q, S, C, or G; wherein X63 is Q, V, S, T, or A; wherein X64 is Q, L, W, or Y; wherein X65 is Q, N, D, I, T, A, or G; wherein X66 is S, P, G, N, or A; wherein X67 is N, Y, I, S, N, L, or D; wherein X68 is E, P, S, I, N, H, L, or T; wherein X69 is D, T, S, or absent; wherein X70 is P, H, L, R, F, A, Q, or absent; wherein X71 is W, L, V, Y, S, A, or E; and wherein X72 is T, V, or I.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO:1); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO:18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence GGX104X105X106GX107X108X109VX110 (SEQ ID NO:103; Group 1 clones), wherein X104 is N, D, or S; wherein X105 is Y, N, or D; wherein X106 is I or T; wherein X107 is S, D, I, R, or T; wherein X108 is K, E, or I; wherein X109 is S, I, R, G, N, or A; and wherein X110 is H, F, or N; the VL CDR2 comprises the amino acid sequence DDX111DRPX112 (SEQ ID NO:104; Group 1 clones), wherein X111 is G, S, or R; and wherein X112 is S or L; and the VL CDR3 comprises the amino acid sequence QVWDX113X114X115DX116X117X118 (SEQ ID NO:105; Group 1 clones), wherein X113 is S or A; wherein X114 is I or S; wherein X115 is S, I, or N; wherein X116 is H, L, or Q; wherein X117 is V or L; and wherein X118 is V or I.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO:1); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO:18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO:30); the VL CDR2 comprises the amino acid sequence X119VSX120RPS (SEQ ID NO:106; Group 1 clones); wherein X119 is E or D; and wherein X120 is N or K; and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO:329); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO:102; Group 1 clones); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO:18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO:30); the VL CDR2 comprises the amino acid sequence X119VSX120RPS (SEQ ID NO:106; Group 1 clones); wherein X119 is E or D; and wherein X120 is N or K; and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence DYFIH (SEQ ID NO:2); the VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEX121FQG (SEQ ID NO:107; Group 2 clones), wherein X121 is K or R; the VH CDR3 comprises the amino acid sequence PGGILTDPDAFDI (SEQ ID NO: 19); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence X122GTX123SDVGGYNX124VS (SEQ ID NO:108; Group 2 clones), wherein X122 is T or A; wherein X123 is S or G; and wherein X124 is Y or H; the VL CDR2 comprises the amino acid sequence X125VX126X127RPS (SEQ ID NO:109; Group 2 clones), wherein X125 is D or E; wherein X126 is N or S; and wherein X127 is K or N; and the VL CDR3 comprises the amino acid sequence SSYX128X129SSTX130X131V (SEQ ID NO:110; Group 2 clones), wherein X128 is I or T; wherein X129 is P or S; wherein X130 is R, P, F, or absent; and wherein X131 is W or Y.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEKFX132G (SEQ ID NO: 111; Group 3 clones), wherein X132 is R or Q; the VH CDR3 comprises the amino acid sequence EESYGP (SEQ ID NO:20); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence QASQDISNYLX133 (SEQ ID NO:112; Group 3 clones), X133 is N or D; the VL CDR2 comprises the amino acid sequence DASNLET (SEQ ID NO:61); and the VL CDR3 comprises the amino acid sequence QQLNSYPLT (SEQ ID NO:80).
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence EX134SMH (SEQ ID NO:113; Group 4 clones), wherein X134 is S or L; the VH CDR2 comprises the amino acid sequence LVDPEDGETIYAQKFQG (SEQ ID NO:14); the VH CDR3 comprises the amino acid sequence EEWSGDGDDAFDI (SEQ ID NO:21); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence SGSSSNIGSYSVS (SEQ ID NO:46); the VL CDR2 comprises the amino acid sequence DX135NKRPS (SEQ ID NO:114; Group 4 clones), wherein X135 is N or D; and the VL CDR3 comprises the amino acid sequence GTWDSSLSAWV (SEQ ID NO:81).
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3); the VH CDR2 comprises the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO:15); the VH CDR3 comprises the amino acid sequence SPLRGSGWYWHYYYGMDV (SEQ ID NO:22); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence GGNX136IX137X138KX139VH (SEQ ID NO: 115; Group 6 clones), wherein X136 is N or K; wherein X137 is R or G; wherein X138 is A, S, R, or T; and wherein X139 is H or S; the VL CDR2 comprises the amino acid sequence X140DX141X142RPS (SEQ ID NO:116; Group 6 clones), wherein X140 is Q or R; wherein X141 is S or R; and wherein X142 is N or K; and the VL CDR3 comprises the amino acid sequence QX143WX144SX145TX146V (SEQ ID NO:117; Group 6 clones), wherein X143 is A or V; wherein X144 is D or G; wherein X145 is S or N; and wherein X146 is V, A, or E.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region with the C-terminal lysine removed. In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region comprising an amino acid sequence that has the three VH CDRs of any anti-mutCALR clone disclosed herein and which has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VH sequences set forth in Tables 4-5 and a light chain variable region comprising an amino acid sequence that has the three VL CDRs of any anti-mutCALR clone disclosed herein and which has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VL sequences set forth in Tables 4-5.
In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region comprising any one of the VH sequences set forth in Tables 4-5. In some embodiments, an anti-mutCALR antibody comprises a light chain variable region comprising any one of the VL sequences set forth in Tables 4-5. In some embodiments, an anti-mutCALR antibody comprises a heavy chain variable region comprising any one of the VH sequences set forth in Tables 4-5 and a light chain variable region comprising any one of the VL sequences set forth in Tables 4-5.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:264.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:265.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:166 and a VL comprising the amino acid sequence of SEQ ID NO:266.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:266.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:267.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:269.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:167 and a VL comprising the amino acid sequence of SEQ ID NO:270.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:271.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:272.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:168 and a VL comprising the amino acid sequence of SEQ ID NO:273.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:169 and a VL comprising the amino acid sequence of SEQ ID NO:274.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:275.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ ID NO:276.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:278.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:280.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:172 and a VL comprising the amino acid sequence of SEQ ID NO:281.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:282.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:173 and a VL comprising the amino acid sequence of SEQ ID NO:283.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:284.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:285.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:174 and a VL comprising the amino acid sequence of SEQ ID NO:286.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:287.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:288.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:289.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:290.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:291.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:292.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:175 and a VL comprising the amino acid sequence of SEQ ID NO:293.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:175 and a VL comprising the amino acid sequence of SEQ ID NO:294.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:175 and a VL comprising the amino acid sequence of SEQ ID NO:295.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:175 and a VL comprising the amino acid sequence of SEQ ID NO:296.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:176 and a VL comprising the amino acid sequence of SEQ ID NO:294.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:175 and a VL comprising the amino acid sequence of SEQ ID NO:297.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:175 and a VL comprising the amino acid sequence of SEQ ID NO:298.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:177 and a VL comprising the amino acid sequence of SEQ ID NO:299.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:178 and a VL comprising the amino acid sequence of SEQ ID NO:300.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:179 and a VL comprising the amino acid sequence of SEQ ID NO:301.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:180 and a VL comprising the amino acid sequence of SEQ ID NO:301.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:180 and a VL comprising the amino acid sequence of SEQ ID NO:302.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:181 and a VL comprising the amino acid sequence of SEQ ID NO:303.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:304.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:305.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:183 and a VL comprising the amino acid sequence of SEQ ID NO:306.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:307.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:308.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:309.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:184 and a VL comprising the amino acid sequence of SEQ ID NO:310.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:311.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:185 and a VL comprising the amino acid sequence of SEQ ID NO:312.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:186 and a VL comprising the amino acid sequence of SEQ ID NO:313.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:187 and a VL comprising the amino acid sequence of SEQ ID NO:314.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:268.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:315.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:316.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:277.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:317.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:165 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:188 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:189 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:190 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:191 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:192 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:193 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:194 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:195 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:197 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:198 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:199 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:200 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:201 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:279.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:170 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:203 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:204 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:205 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:206 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:207 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:208 and a VL comprising the amino acid sequence of SEQ ID NO:318.
In some embodiments, an anti-mutCALR antibody comprises a modification which modulates (e.g., reduces or increases) the Fc region-mediated effector function, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Depending on the therapeutic antibody or Fc fusion protein application, it may be desired to either reduce or increase the effector function.
In certain embodiments, an anti-mutCALR antibody has Fc effector function. In certain embodiments, an anti-mutCALR antibody has enhanced Fc effector function. In certain embodiments, an anti-mutCALR antibody exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). An anti-mutCALR antibody can be engineered to enhance the ADCC activity (for review, see Kubota T el al. Cancer Sci. 2009; 100(9):1566-72). For example, ADCC activity of an antibody can be improved when the antibody itself has a low ADCC activity, by slightly modifying the constant region of the antibody (Junttila T T. et al. Cancer Res. 2010; 70(11):4481-9). Changes are sometimes also made to improve storage or production or to remove C-terminal lysines (Kubota T el al. Cancer Sci. 2009; 100(9):1566-72). Another suitable method to improve ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway resulting in a reduced fucose (von Horsten H H. el al. Glycobiology. 2010; 20(12):1607-18). Alternatively, or additionally, other suitable methods can be used to achieve ADCC enhancement, for instance including glycoengineering (Kyowa Hakko/Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which seek to improve Fc binding to low-affinity activating FcγRIIIa, and/or to reduce binding to the low affinity inhibitory FcγRIIb. In certain embodiments, a binding moiety of the present disclosure exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, a binding moiety of the present disclosure is afucosylated.
In certain embodiments, an anti-mutCALR antibody has reduced Fc effector function. In certain embodiments, an anti-mutCALR antibody exhibits reduced or substantially no complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP). In certain embodiments, an anti-mutCALR antibody exhibits reduced or substantially no antibody-dependent cell-mediated cytotoxicity (ADCC). An anti-mutCALR antibody can be engineered to reduce effector function, for example ADCC activity, by any suitable method including removal of glycosylation sites in the Fc region. In certain embodiments, an anti-mutCALR antibody that has a reduced Fc effector function (e.g., a reduced ADCC effector function) comprises an N297A mutation on the heavy chain.
In some embodiments, an anti-mutCALR antibody is an IgG1 isotype (e.g., IgG1, IgG2, IgG3 or IgG4). In some embodiments, an anti-mutCALR antibody is an IgG1. In some embodiments, an IgG1, IgG2, IgG3 or IgG4 anti-mutCALR antibody has Fc-effector function. In some embodiments, an IgG1, IgG2, IgG3 or IgG4 anti-mutCALR antibody is Fc-effector function null. In some embodiments, an IgG1 anti-mutCALR antibody has Fc-effector function. In some embodiments, an IgG1 anti-mutCALR antibody is Fc-effector function null.
In some instances, the anti-mutCALR antibody is an antibody fragment. Fragments of the antibodies described herein (e.g., Fab, Fab′, F(ab′)2, Facb, and Fv) may be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin or the FabRICATOR® (IdeS) recombinant enzyme (Genovis AB) that digests IgG antibodies to produce a homogeneous pool of F(ab′)2 and Fc/2 fragments. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies yields F(ab′)2 or Fab′; and plasmin digestion of whole antibodies yields Facb fragments.
Alternatively, antibody fragments can be produced recombinantly. For example, nucleic acids encoding the antibody fragments of interest can be constructed, introduced into an expression vector, and expressed in suitable host cells. See, e.g., Co, M. S. et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, A. H., Methods in Enzymology, 178:476-496 (1989); Plueckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymology, (1989) 121:663-669 (1989); and Bird, R. E. et al., TIBTECH, 9:132-137 (1991)). Antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′)2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046.
In some instances, the anti-mutCALR antibody is a minibody. Minibodies of anti-mutCALR antibodies include diabodies, single chain (scFv), and single-chain (Fv)2 (sc(Fv)2).
A “diabody” is a bivalent minibody constructed by gene fusion (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. U.S.A, 90:6444-6448 (1993); EP 404,097; WO 93/11161). Diabodies are dimers composed of two polypeptide chains. The VL and VH domain of each polypeptide chain of the diabody are bound by linkers. The number of amino acid residues that constitute a linker can be between 2 to 12 residues (e.g., 3-10 residues or five or about five residues). The linkers of the polypeptides in a diabody are typically too short to allow the VL and VH to bind to each other. Thus, the VL and VH encoded in the same polypeptide chain cannot form a single-chain variable region fragment, but instead form a dimer with a different single-chain variable region fragment. As a result, a diabody has two antigen-binding sites.
An scFv is a single-chain polypeptide antibody obtained by linking the VH and VL with a linker (see, e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A, 85:5879-5883 (1988); and Plickthun, “The Pharmacology of Monoclonal Antibodies” Vol. 113, Ed Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)). The order of VHs and VLs to be linked is not particularly limited, and they may be arranged in any order. Examples of arrangements include: [VH] linker [VL]; or [VL] linker [VH]. The heavy chain variable domain and light chain variable domain in an scFv may be derived from any anti-mutCALR antibody described herein.
An sc(Fv)2 is a minibody in which two VHs and two VLs are linked by a linker to form a single chain (Hudson, et al., J. Immunol. Methods, (1999) 231: 177-189 (1999)). An sc(Fv)2 can be prepared, for example, by connecting scFvs with a linker. The sc(Fv)2 of the present invention include antibodies preferably in which two VHs and two VLs are arranged in the order of: VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]), beginning from the N terminus of a single-chain polypeptide; however the order of the two VHs and two VLs is not limited to the above arrangement, and they may be arranged in any order.
In some instances, the anti-mutCALR antibody is a bispecific antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the mutCALR protein. Other such antibodies may combine a mutCALR binding site with a binding site for another antigen. Bispecific antibodies can be prepared as full length antibodies or low molecular weight forms thereof (e.g., F(ab′)2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).
Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). In a different approach, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the proportions of the three polypeptide fragments. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields.
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods.
The “diabody” technology provides an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
In certain embodiments, the bispecific anti-mutCALR antibody is a biparatopic antibody A biparatopic antibody is antibody which recognizes two non-identical epitopes (overlapping or non-overlapping epitopes) on the same target antigen (e.g., the C-terminal of mutCALR domain). A biparatopic anti-mutCALR antibody can comprise two immunoglobulin heavy chain-light chain pairs or one immunoglobulin heavy chain-light chain pair. In some embodiments, a biparatopic anti-mutCALR antibody comprises one immunoglobulin heavy-chain-light chain pair. In some embodiments, a biparatopic anti-mutCALR antibody is a full-length antibody comprising one immunoglobulin heavy-chain-light chain pair.
In some instances, the anti-mutCALR antibody is a multivalent antibody. A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies describe herein can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. An exemplary dimerization domain comprises (or consists of) an Fc region or a hinge region. A multivalent antibody can comprise (or consist of) three to about eight (e.g., four) antigen binding sites. The multivalent antibody optionally comprises at least one polypeptide chain (e.g., at least two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, X1 and X2 represent an amino acid or peptide spacer, and n is 0 or 1.
In some instances, the anti-mutCALR antibody is a conjugated antibody. The antibodies disclosed herein may be conjugated antibodies, which are bound to various molecules including macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, radioactive materials (e.g., 90Y, 131I), fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, drugs, and toxins (e.g., calcheamicin, Pseudomonas exotoxin A, ricin (e.g., deglycosylated ricin A chain) and auristatins (e.g., auristatin E or auristatin F)).
In one embodiment, to improve the cytotoxic actions of anti-mutCALR antibodies and consequently their therapeutic effectiveness, the antibodies are conjugated with highly toxic substances, including radioisotopes and cytotoxic agents. These conjugates can deliver a toxic load selectively to the target site (i.e., cells expressing the antigen recognized by the antibody) while cells that are not recognized by the antibody are spared. In order to minimize toxicity, conjugates are generally engineered based on molecules with a short serum half-life (thus, the use of murine sequences, and IgG3 or IgG4 isotypes).
In certain embodiments, an anti-mutCALR antibody is modified with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, the anti-mutCALR antibody can be associated with (e.g., conjugated to) a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, the anti-mutCALR antibody can be conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides.
The above-described conjugated antibodies can be prepared by performing chemical modifications on the antibodies, respectively, or the lower molecular weight forms thereof described herein. Methods for modifying antibodies are well known in the art (see, e.g., U.S. Pat. Nos. 5,057,313 and 5,156,840).
Antibodies may be produced in bacterial or eukaryotic cells. Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS). In addition, antibodies (e.g., scFvs) can be expressed in a yeast cell such as Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hansenula, or Saccharomyces. To produce the antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in suitable host cells. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody.
If the antibody is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have characteristics that permit amplification of the vector in the bacterial cells. Additionally, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as a host, the vector must have a promoter, for example, a lacZ promoter (Ward et al., 341:544-546 (1989), araB promoter (Better et al., Science, 240:1041-1043 (1988)), or T7 promoter that can allow efficient expression in E. coli. Examples of such vectors include, for example, M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) may be used as the signal sequence for antibody secretion. For bacterial expression, calcium chloride methods or electroporation methods may be used to introduce the expression vector into the bacterial cell.
If the antibody is to be expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, for example, an SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or domain thereof, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
In one embodiment, antibodies are produced in mammalian cells. Exemplary mammalian host cells for expressing an antibody include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601 621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.
In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of an anti-mutCALR antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and the antibody is recovered from the culture medium.
Antibodies can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly. Animals are also provided comprising one or more of the nucleic acids described herein.
The antibodies of the present disclosure can be isolated from inside or outside (such as from the medium) of the host cell and purified as substantially pure and homogenous antibodies. Methods for isolation and purification commonly used for antibody purification may be used for the isolation and purification of antibodies, and are not limited to any particular method. Antibodies may be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A column and protein G column. Examples of columns using protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.
The disclosure also provides polynucleotides and vectors encoding an anti-mutCALR antibody or portion thereof (e.g., VH, VL, HC, or LC) described herein. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. In some instances, the polynucleotide is DNA. In some instances, the polynucleotide is complementary DNA (cDNA). In some instances, the polynucleotide is RNA. In some embodiments, a polynucleotide described herein is isolated. In some embodiments, a polynucleotide described herein is purified.
In some instances, the polynucleotide encodes a VH comprising the VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some instances, the polynucleotide encodes a VL comprising the VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some instances, the polynucleotide encodes a heavy chain comprising a VH comprising the VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some instances, the polynucleotide encodes a light chain comprising a VL comprising the VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some instances, the polynucleotide is operably linked to a promoter.
In some instances, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a VH comprising the VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (see, e.g., Tables 1-5); and (ii) a second nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a VL comprising the VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some instances, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a heavy chain comprising a VH comprising the VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein (see, e.g., Tables 1-5); and (ii) a second nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a light chain comprising a VL comprising the VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some instances, the first nucleic acid is operably linked to a first promoter and the second nucleic acid is operably linked to a second promoter.
In some instances, the polynucleotide encodes a VH described herein (see, e.g., Tables 4-5) or a variant thereof. In some instances, the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs:165-208. In some instances, the polynucleotide encodes a polypeptide comprising an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and/or deletions relative to the amino acid sequence set forth in any one of SEQ ID NOs:165-208. In some instances, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:165-208. In some instances, the polynucleotide is operably linked to a promoter.
In some instances, the polynucleotide encodes a VL described herein (see, e.g., Tables 4-5) or a variant thereof. In some instances, the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs:264-318. In some instances, the polynucleotide encodes a polypeptide comprising an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and/or deletions relative to the amino acid sequence set forth in any one of SEQ ID NOs:264-318. In some instances, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:264-318. In some instances, the polynucleotide is operably linked to a promoter.
In some instances, the polynucleotide comprises: (i) a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises a VH described herein (see, e.g., Tables 4-5) or a variant thereof; and (ii) a second nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises a VL described herein (see, e.g., Tables 4-5) or a variant thereof. In some instances, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs:165-208, and (ii) a second nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs:264-318. In some instances, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and/or deletions relative to the amino acid sequence set forth in any one of SEQ ID NOs:165-208; and (ii) a second nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and/or deletions relative to the amino acid sequence set forth in any one of SEQ ID NOs:264-318. In some instances, the first nucleic acid encodes the amino acid sequence set forth in any one of SEQ ID NOs:165-208 and the second nucleic acid encodes the amino acid sequence set forth in any one of SEQ ID NOs:264-318. In some instances, the first nucleic acid is operably linked to a first promoter and the second nucleic acid is operably linked to a second promoter.
Also provided herein are expression vectors encoding the anti-mutCALR antibodies or portions thereof (e.g., VH, VL, HC, and/or LC) described herein. Also provided herein are expression vectors comprising one or more polynucleotides described herein. Various types of expression vectors are known in the art and described herein.
Also provided herein are cells comprising the anti-mutCALR antibodies described herein. Also provided herein are cells comprising one or more polynucleotides described herein. Also provided herein are cells comprising one or more expression vectors described herein. Various types of cells are known in the art and described herein.
The anti-mutCALR antibodies of the present disclosure can inhibit the activity of mutCALR, inhibit the activity of one or more signaling pathways downstream of MPL, inhibit oncogenic cell proliferation, inhibit dimerization of MPL, compete with MPL for binding to mutCALR, or a combination thereof.
As used herein, an anti-mutCALR antibody that competes with MPL for binding to mutCALR means that the anti-mutCALR antibody binds to mutCALR with a greater affinity than MPL. In some embodiments, the anti-mutCALR antibody binds to mutCALR with about 10-fold, 50-fold, 100-fold, 500-fold or 1000-fold greater affinity than MPL. In some embodiments, the anti-mutCALR antibody binds to mutCALR with an IC50 of about 10-fold, 50-fold, 100-fold, 500-fold or 1000-fold less than MPL. In some embodiments, the anti-mutCALR antibody binds to mutCALR with an IC50 of between about 0.1 and 1 nM. In some embodiments, the anti-mutCALR antibody binds to mutCALR with an IC50 of about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM or 1 nM.
Accordingly, the antibodies or compositions described herein can be used in methods of inhibiting activity of mutCALR, inhibiting the activity of one or more signaling pathways downstream of MPL, inhibiting oncogenic cell proliferation, inhibiting dimerization of MPL, inhibit the binding of MPL to mutCALR, or a combination thereof in an individual/patient in need of the inhibition by administering an effective amount of an antibody described herein.
Non-limiting examples of signaling pathways downstream of MPL include Janus tyrosine kinase (JAK) and signal transducers and activators of transcription (STAT) signaling, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, serine/threonine kinase (AKT) signaling, and mammalian target of rapamycin (mTOR) signaling.
Another aspect of the present disclosure pertains to methods of treating a mutCALR-associated disease or disorder in an individual (e.g., patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of one or more antibodies of the present disclosure or a pharmaceutical composition thereof. A mutCALR-associated disease or disorder can include any disease, disorder or condition that is directly or indirectly linked to expression or activity of mutCALR.
Another aspect of the present disclosure pertains to methods of treating a myeloproliferative neoplasm in an individual (e.g., patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of one or more antibodies of the present disclosure or a pharmaceutical composition thereof.
Non-limiting examples of a myeloproliferative neoplasm include chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.
Anti-mutCALR antibodies disclosed herein can be used to treat, alone or in combination with other therapies, a myeloproliferative neoplasm, or can be used, alone or in combination with other therapies, for the manufacture of a medicament for the treatment of a myeloproliferative neoplasm. Non-limiting examples of other therapies include a JAK inhibitor (e.g., ruxolitinib, itaticinib), a PI3K inhibitor (e.g., parsaclisib), a standard of care therapy (e.g., IFN-alpha, hydroxyurea, thalidomide, lenalidomide, an androgen, an erythropoietin-stimulating agent, a chemotherapeutic agent), or a combination thereof.
Non-limiting examples of JAK inhibitors for use as described herein are provided in U.S. Pat. Nos. 7,335,667; 9,359,358; 8,691,807; 9,181,271; and 9,034,884, each of which is incorporated herein by reference in its entirety.
Non-limiting examples of PI3K inhibitors for use as described herein are provided in U.S. Pat. Nos. 9,108,984; 9,062,055; 8,759,359; and 9,434,746, each of which is incorporated herein by reference in its entirety.
The terms “individual” or “patient” or “subject”, used interchangeably, refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans (i.e., a human subject).
The phrase “therapeutically effective amount” refers to the amount of active antibody or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
As used herein, the term “treating” or “treatment” refers to one or more of (1) inhibiting the disease; e.g., inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; e.g., ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.
In some embodiments, the antibodies of the invention are useful in preventing or reducing the risk of developing any of the diseases referred to herein; e.g., preventing or reducing the risk of developing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease.
An anti-mutCALR antibody described herein can be formulated as a pharmaceutical composition for administration to a subject, e.g., to treat a disease or disorder described herein. Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19).
Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).
The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.
The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and liposomes. A suitable form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration or as a lyophilized preparation. Sterile injectable solutions can be prepared by incorporating an anti-mutCALR antibody described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that 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, the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts.
The anti-mutCALR antibodies can also be formulated as liposomes prepared by any suitable method known in the art.
Pharmaceutical compositions formulated for subcutaneous administration may be suitable in some circumstances because the subject can self-administer the pharmaceutical composition. Pharmaceutical formulations for subcutaneous administration can further comprise protein formulations comprising arginine-HCl, histidine, and/or polysorbate, which may confer increased potency, improved serum half-life, or enhanced solubility to the anti-mutCALR antibodies.
The anti-mutCALR antibody can be administered to a subject, e.g., a subject in need thereof, for example, a human subject, by a variety of methods. For many applications, the route of administration can be intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection.
The route and/or mode of administration of the antibody can also be tailored for the individual case, e.g., by monitoring the subject, e.g., using tomographic imaging, e.g., to visualize a tumor.
The anti-mutCALR antibody can be administered as a fixed dose, or in a mg/kg patient weight dose. The dose can also be chosen to reduce or avoid production of antibodies against the anti-mutCALR antibody. Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response or a combinatorial therapeutic effect. Generally, doses of the anti-mutCALR antibody (and optionally a second agent) can be used in order to provide a subject with the agent in bioavailable quantities.
Dosage unit form or “fixed dose” or “flat dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. Alternatively, or in addition, the antibody may be administered via continuous infusion.
The disclosure also provides a kit comprising one or more containers of an anti-mutCALR antibody or a pharmaceutical formulation thereof, optionally with one or more other prophylactic or therapeutic agents useful for the treatment of a disease or disorder, and optionally with instructions for using the anti-mutCALR antibody or a pharmaceutical formulation thereof.
The instructions relating to the use of an anti-mutCALR antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label package insert indicates that an anti-mutCALR antibody is used for treating, delaying the onset, and/or alleviating a myeloproliferative neoplasm.
Mutant CALR can be detected in a biological sample of a subject who has a myeloproliferative neoplasm. Thus an aspect of the present disclosure provides a method of detecting a CALR exon 9 mutation in a subject's biological sample, the method comprising obtaining a biological sample from a subject who has or is suspected of having a myeloproliferative neoplasm and contacting the sample with an anti-mutCALR antibody of described herein such that the anti-mutCALR antibody binds to the mutCALR protein if the mutCALR protein is present in the biological sample.
Another aspect of the present disclosure provides a method of diagnosing a subject with a myeloproliferative neoplasm, the method comprising obtaining a biological sample from a subject who has or is suspected of having a myeloproliferative neoplasm and contacting the sample with an anti-mutCALR antibody described herein such that the anti-mutCALR antibody binds to the mutCALR protein if the mutCALR protein is present in the biological sample.
The biological sample can be a blood sample, a bone marrow sample or a serum sample. In some embodiments, the biological sample is fresh or frozen. In some embodiments, the biological sample is fixed, for example in formaldehyde or paraformaldehyde.
Non-limiting examples of a myeloproliferative neoplasm that can be diagnosed with the present method include chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.
An anti-mutCALR antibody described herein for use in the present detection or diagnostic methods can also comprise (e.g., be conjugated to) a detectable label. Suitable detectable labels include a radioisotope, a nanoparticle, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as used in an ELISA), biotin, digoxigenin, or haptens. Suitable techniques for conjugating diagnostic agents to antibodies are known in the art (see, e.g., Jazayeri etl al., Sensing and Bio-Sensing Research (2016); 9:17-22 and Balasubramanya, Mater. Methods (2018); 8:2670).
An anti-mutCALR antibody described herein bound to mutCALR protein, whether labeled or unlabeled, can be detected by any suitable detection method, including immunologic techniques such as immunohistochemistry (IHC), immunocytochemistry, Western blot, or ELISA immunoassay; gel- or blot-based methods; mass spectrometry, flow cytometry; or fluorescent activated cell sorting (FACS).
The disclosure also provides a kit for the diagnosis of a myeloproliferative neoplasm comprising one or more containers of an anti-mutCALR antibody described herein or a diagnostic formulation thereof, and optionally with instructions for using the anti-mutCALR antibody or a diagnostic formulation thereof to detect a mutCALR exon 9 mutation or diagnose a myeloproliferative neoplasm.
The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
To generate anti-human mutCALR monoclonal antibodies, mice were immunized with one plasmid encoding human MPL and a second plasmid encoding human Type 1 mutCalR. The sequences for mutant CalR and MPL were cloned into the pVAC2 expression plasmid (Invivogen). The nucleotide sequences used in the vectors as well as the sequences of the encoded proteins are shown below.
Plasmablasts were isolated from the mouse spleen and lymph nodes by flow cytometry. Antibody sequences of the plasmablasts were determined using 10× Genomics VH/VL paired B cell sequencing. The murine VH/VL pairs were expressed as chimeras with huIgG1 Fc and tested for binding and functionality. An antibody designated clone 54 was produced by this process.
In addition, multiple selection rounds of single-donor and multi-donor phage display libraries were performed according to the method of Erasmus et al., Communications Biology 4, article no. 350 (2021). Phage libraries were enriched for either 2 or 3 rounds on biotinylated MBP-mutCalR Type 1 fusion protein (Cepter Biopartners) with deselection using MBP (Rockland Immunochemicals) and streptavidin beads (Thermo Fisher) each round (see, e.g., Example 2). scFv cassettes from the library pools showing the strongest specific enrichment for mutCalR were then recombined into a yeast display vector to create yeast display libraries. These were selected over four rounds using decreasing concentrations of MBP-mutCalR with and without blocking with either a scrambled peptide or oligo-lysine peptide (Alamanda Polymers). In the final round of selection, yeast were also selected for binding to a biotinylated mutCalR long peptide (Biot-LC-MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSSREASLQGWTEA; SEQ ID NO:332) or short peptide (Biot-LC-MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKM, SEQ ID NO:333). Unique sequences were obtained from the final sorting output by Sanger sequencing of yeast colonies. For screening, these scFv were reformatted by cloning into a human IgG1 expression vector, then expressed and purified from Expi293F cell (Thermo Fisher, cat. #A14635) supernatants. The MBP-mutCalR Type 1 fusion protein used in this experiment is:
The amino acid sequences of the six CDRs for each of the 54 unique clones are shown in Table 1. The heavy chain, VH, light chain, and VL sequences of each clone are shown in Table 4.
Mutations were introduced into three of the identified clones (clones 6, 15, and 17) to generate 161 unique mutant clones (clones 55-215). The amino acid sequences of the six CDRs for each of the mutant clones are shown in Table 2. Light chain and heavy chain sequences of the parental clones (clones 6, 15, and 17) and mutant clones (clones 55-215) are shown in Table 5. One such mutation to the heavy chain, N297A, resulted in Fc effector function “null” mutants, where the Fc effector function, particularly ADCC, was eliminated or substantially eliminated. As can be seen from the activity data in the Examples, those antibodies with N297A Fc effector null mutations have a generally increased binding affinity for mutCALR than those antibodies without an Fc effector null mutation.
The amino acid sequence of recombinant MBP-mutCALR Type 1 fusion protein used in Experiments 1 and 2 is:
Experiment 1: Recombinant MBP-mutCALR Type 1 fusion protein was custom made at Cepter (cepterbiopartners.com). Data for the interaction of antibodies with recombinant MBP-mutCALR Type 1 fusion protein were collected at 25° C. on a Biacore 8K instrument (Cytiva, Marlborough, Mass.), and all reagents were obtained from Cytiva unless otherwise specified. The experiments were conducted using Tris-buffered saline (pH 7.2), 0.005% Surfactant P20, 1 mM CaCl2) as running buffer. To prepare the capture surfaces, anti-human IgG (Fc) antibodies (Product #29234600) was amine-coupled onto a CM4 chip (Product #BR100534) under standard conditions using an Amine Coupling Kit (Product #BR100050). Anti-mutCALR antibodies were captured onto the chip surface by injecting over flow cell 2 only at a flow rate of 30 μL/min for 30 seconds. Typical capture levels were in the range of 10-30RU. Recombinant MBP-mutCALR Type 1 fusion protein was prepared at nominal concentrations of 0, 3.1, 9.3, 27.8, 83.3, and 250 nM in the running buffer and was injected over both flow cells 1 and 2 for 210 seconds at a flow rate of 50 μL/min, followed by a 510-second dissociation phase at the same flow rate. The kinetics parameters were obtained by applying 1:1 binding model to fit the data from multiple-cycle injection experiments using Biacore Insight Evaluation software. MBP protein at 250 nM concentration was injected as a control, which showed no binding to anti-mutCALR antibodies.
Experiment 2: Recombinant MBP-mutCALR Type 1 fusion protein was custom made at Cepter (cepterbiopartners.com). Data for the interaction of antibodies with recombinant mutCALR protein were collected at 25° C. on a Biacore 8K instrument (Cytiva, Marlborough, Mass.), and all reagents were obtained from Cytiva unless otherwise specified. The experiments were conducted using Tris-buffered saline (pH 7.2), 0.005% Surfactant P20, 1 mM CaCl2) as running buffer. To prepare the capture surfaces, anti-human IgG (Fc) antibodies (Product #29234600) was amine-coupled onto a CM4 chip (Product #BR100534) under standard conditions using an Amine Coupling Kit (Product #BR100050). Anti-mutCALR antibodies were captured onto the chip surface by injecting over flow cell 2 only at a flow rate of 10 μL/min for 30 seconds. Typical capture levels were in the range of 15-25RU. Recombinant MBP-mutCALR Type 1 fusion protein was prepared at nominal concentrations of 0, 0.75, 2.22, 6.67, 20, and 60 nM and injected over both flow cells 1 and 2 for 150 seconds at a flow rate of 69 L/min, followed by a 230-second dissociation phase at the same flow rate. The kinetics parameters were obtained by applying 1:1 binding model to fit the data from multiple-cycle injection experiments using Biacore Insight Evaluation software.
Experiment 3: The amino acid sequence of recombinant MBP-mutCALR Type 2 fusion protein used in this Experiment 3 is:
Recombinant MBP-mutCALR Type 2 fusion protein was custom made at Cepter (cepterbiopartners.com). Data for the interaction of antibodies with recombinant MBP-mutCALR Type 2 fusion protein were collected as described in Experiment 2. The chip used was a Biacore Series S Sensor Chip CM4 with anti-huFc immobilized onto the chip surface. Recombinant MBP-mutCALR Type 2 fusion protein was prepared at nominal concentrations of 1.1, 3.3, 10, 30, and 90 nM. Typical capture levels were in the range of 16-37RU. The results are shown in Table 8.
To assess binding of CALR antibodies to cell surface CALR, parental BaF3 (DSMZ) cells or BaF3 cells stably expressing only human MPL or both human MPL (Uniprot Number: P40238-1) and human mutCALR Type 1 and Type 2 were used. On the day of the assay, cells were washed and resuspended in assay buffer. Approximately 200,000 cells/well were added to 96-well plates and stained with the indicated concentration of antibodies for 30 minutes on ice. Cells were then washed and stained with goat anti-human secondary conjugated to R-phycoerythrin (R-PE) (Jackson Immuno Research Laboratories) for 30 minutes on ice. The cells were then washed and analyzed by flow cytometry. Geometric Mean Fluorescence Intensity (GMFI) of cell binding was graphed and EC50, hill slope, and area under the curve (AUC) were determined after four parameter curve fitting using GraphPad Prism Software (version 7.04).
To test the ability of anti-mutCALR antibodies to inhibit phosphorylation of STAT5, Ba/F3 cells (DSMZ) expressing MPL and Type 1 mutCALR variants were generated by nucleofection (Amaxa Cell Line Nucleofection Kit V, Lonza, Basel, Switzerland) and cultured in RPMI 1640+10% FBS+selection antibiotics. Prior (24 hours) to pSTAT5 assessment, cells were cultured in selection-free media and then plated at 200,000 cells per well (96 well plate) in RPMI 1640, 10% FBS. Antibodies were added to the cells and incubated for 2 hours followed by cell lysing and quantification of pSTAT-5 levels by MSD (Phospho-STAT5a,b Whole Cell Lysate Kit, MSD, Kenilworth, N.J.). Anti-mutCALR antibodies inhibit phosphorylation of STAT5 in a dose-dependent manner (
To test the ability of anti-mutCALR antibodies to inhibit cell proliferation, engineered Ba/F3 cells transfected with MPL and mutCALR Type 1 were plated at 5,000 cells per well in RPMI 1640+2% FBS, antibodies added, incubated for 72 hours, and followed by assessment of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.) and Top Count (Perkin Elmer, Boston, Mass.) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. Anti-mutCALR antibodies inhibit mutCALR-induced oncogenic cell proliferation in a dose-dependent manner in both the Ba/F3 engineered cells (
To test the ability of anti-mutCALR antibodies to inhibit oncogenic cell proliferation triggered by the Type 1 and Type 2 CALR mutations, Ba/F3 cells (DSMZ) were engineered to express MPL+mutCALR Type 1 (52 bp deletion, SEQ ID NO:320) or MPL+mutCALR Type 2 (5 bp insertion; SEQ ID NO:321). Cells were plated at 5,000 cells per well in RPMI 1640+2% FBS, antibodies added, incubated for 72 hours, and followed by assessment of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.) and Top Count (Perkin Elmer, Boston, Mass.) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. Anti-mutant CALR antibodies show potency to inhibit mutCALR-induced oncogenic cell proliferation associated with both Type 1 and Type 2 mutations (
Inhibition of MPL dimerization was tested as a potential mechanism of action of the anti-mutCALR antibodies. HAP1 cells knocked out for human JAK2 (Horizon Discovery, Ltd.) were transiently transfected with vectors encoding the MPL-LgBiT and MPL-smBiT fusion proteins (Promega Corp.). Also included in the transfection were vectors encoding the full-length human JAK2 and the full-length WT or mutant CALR Type 1 protein (full-length cds cloned into the pD2529 vector, ATUM Bio). The cells were transfected in 96 well plates using Trans-IT 2020 reagent (Mirus Bio LLC) with equivalent amounts of each plasmid. Cells were then incubated at 37° C., 5% CO2. 6 hours post-transfection, antibodies were diluted into growth medium (IMDM, 10% FBS) and added to the cells at the indicated concentrations. The plates were incubated overnight and the growth medium was replaced with 100 μl OPTI-MEM I, no phenol red, containing the same concentrations of antibodies. After 1 hour at 37° C., 5% CO2, 25 μl NanoGlo Live Cell Reagent (Promega) was added to each well. Plates were returned to the incubator for 30 minutes before luminescence was read on a PHERAstar FSX (BMG Labtech). Data was analyzed using GraphPad PRISM Software (version 7.04) and expressed as percent inhibition compared to isotype control. As shown in
The amino acid sequences for the MPL-LgBiT and MPL-smBiT fusion proteins used in this Example are:
To test the functional ability of anti-mutCALR antibodies in vivo, antibodies were evaluated in a mouse model of tumor growth. Engineered Ba/F3 tumor cells expressing MPL/mutCALR Type 1 were inoculated intravenously in NSG immunodeficient mice (NOD-scid IL2Rgammanull, The Jackson Laboratories, Bar Harbor, Me.). Tumors were allowed to grow for 10 days when mice were randomized into antibody or isotype control treatment groups. Different doses of antibodies were administered intraperitoneally and tumor growth was followed over time by assessing the presence and number of tumor cells in the blood (Sysmex, Kobe, Japan). Further, full hematology and tumor infiltration in the spleen and bone marrow were evaluated for assessment of antibody potencies. A representative in vivo study is shown in
The anti-mutCALR antibodies prolonged mouse survival (
To test the ability of anti-mutCALR antibodies to potentiate the therapeutic response of the JAK1/2 inhibitor ruxolitinib, Ba/F3 cells were engineered to express MPL+mutCALR Type 1 (52 bp deletion) or MPL+mutCALR Type 2 (5 bp insertion) as described above. Cells were plated at 5,000 cells per well in RPMI 1640+2% FBS, treated with 50 nM of ruxolitinib and/or anti-mutCALR antibodies. Cells were incubated for 72 hours followed by assessment of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.) and Top Count (Perkin Elmer, Boston, Mass.) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. Ruxolitinib used at a concentration of 50 nM inhibited the level of oncogenic cell proliferation by approximately 20% (dotted line). Anti-mutant CALR antibody clone 6 potentiated the ability of ruxolitinib to inhibit cell proliferation in cells carrying the CALR Type 1 (
The amino acid sequence of recombinant His tag_hMPL used in this example is:
The amino acid sequence of recombinant Flag tag_GFP_tev_hmutCALR Type 1 used in this example is:
Recombinant His tag_hMPL and recombinant Flag tag_GFP_tev_hmutCALR Type 1 proteins (80 nM MPL+5 nM mutCALR) were incubated in assay buffer (HEPES, pH 7.5 50 nM; Prionex 0.05%; NaCl 100 nM; Pluoronic F-127 0.01%; CaCl2 1 mM; MgCl2 1 mM; DTT) for 1 h at room temperature to allow for the formation of the mutCALR/MPL complex. In a 384-well plate, 5 μl of anti-mutCALR antibodies 6 and 32, non-functional CAL2 antibody (Dianova), or untagged mutCALR (competitor) was incubated with 5 μl of the mutCALR/MPL protein mix for 1 h at room temperature. HTRF detection solution (2 nM anti-FLAG-Europium, and 100 nM anti-6×His-SureLight® APC) was then added and the plate was read on BMG PHERAstar FSX using kinetic HTRF protocol, obtaining readings every 15 minutes for 120 minutes total. Data showed that binding equilibrium was reached by 90 min and the 90 min time point HTRF Ratio values were used for analysis. HTRF Ratio from each test well containing FLAG-GFP-tev-mutCALR+MPL-6×His solution was background-subtracted using control well (MPL_His tag not included). Percent-of-control values were obtained by dividing the background-subtracted HTRF Ratios for each test well by the buffer control well in the same plate row. The data shows that untagged mutCALR competes with FLAG tag_mutCALR for binding to MPL (positive control) while the isotype does not interfere with the mutCALR/MPL interaction (negative control). Whereas the non-functional CAL2 antibody showed some signal inhibition at high concentration (>100 nM), the anti-mutCALR antibodies 6 and 32 fully inhibited the interaction of MPL and mutCALR, indicating that the anti-mutCALR antibodies compete with MPL for binding to mutCALR (
For Examples 11 and 12, and
The Fab portion of anti-mutCALR antibody (clone 55; also referred to as antibody 55) was expressed and purified as follows:
The Fab portion of antibody 55 was expressed in Expi293F cells (Thermo Fisher, cat. #A14635) by transient transfection for 5 days. The Fab was purified from clarified supernatants by binding to CaptureSelect CHI-XL Affinity Matrix (ThermoFisher), washing with PBS buffer, and eluting with 50 mM sodium acetate, pH 4.0. After elution, Fab was buffer exchanged to PBS and concentrated using Ultra-15 centrifugal filter unit, 10 kDa MWCO (Amicon) and then stored at −80° C.
Crystallization was performed as follows:
Fab: The Fab portion of antibody 55 was concentrated to 10 mg/mL in TBS buffer. Sparse matrix crystallization screens were set up using an NT8 crystallization robot (Formulatrix, Bedford, Mass.). Drops containing 200 nl (Fab fragment)+200 nl (reservoir) were used for the setup and the plates were incubated at 4° C., 13° C. and 20° C. Crystals of the Fab portion of antibody 55 appeared in condition C10 of the JCSG Top96 Screen (0.1 M HEPES pH 7.5, 20% (w/v) PEG 8,000 (Rigaku, Bainbridge Island, Wash.) after five days incubation at 4° C.
Fab-mutCALR Peptide: The Fab portion of antibody 55 was mixed with mutCALR peptide (Acetyl-DEEQRTRRMMRTKMRMRRMRR-NH2; SEQ ID NO:339) at a ratio of 1:1.5 molar excess, and then concentrated to a final concentration of 35 mg/mL. Sparse matrix crystallization screens were set up using an NT8 crystallization robot (Formulatrix, Bedford, Mass.). Drops containing 200 nl (Fab plus mutCALR peptide)+200 nl (reservoir) were used for the setup and the plates were incubated at 4° C., 13° C. and 20° C. Initial crystal hits grew from condition C7 of the JCSG Top96 Screen (Rigaku, Bainbridge Island, Wash.) after 3 days incubation at 4° C. This initial hit condition was further refined, leading to the mutCALR peptide bound crystals. The final condition for Fab-mutCALR peptide was 0.1 M Bicine pH 7.94, 19% w/v PEG 6000. These crystals grew in approximately five days.
All crystals were flash cooled in liquid nitrogen for X-ray data collection.
Data Collection, Processing and Refinement was performed as follows:
Diffraction data was collected at 100K using synchrotron radiation at the Advanced Photon Source (IMCA-CAT beamline 17-ID). Diffraction data indexing, integration and scaling were performed with the AutoPROC package. Data collection statistics, phasing and refinement are given in Table 14.
For the Fab-Apo data, MoRda (Keegan and Winn. (2007) Acta Cryst. D63, 447-57; Keegan et al. (2018) Acta Cryst. D74, 167-82; and Winn et al. (2011) Acta. Cryst. D67, 235-42) was used to place two copies of a Fab fragment as a template (PDB ID: 5AZE). For the Fab-mutCALR peptide data Phaser was used to place fragments of the Fab-Apo model and subsequent rounds of manual model building were performed resulting in a model containing two Fabs and 3 peptides. For both datasets, subsequent cycles of density modifications, model building and refinement were carried out using Refmac (Murshudov et al. (1997) Acta Cryst. D53, 240-255), Coot (Emsley et al. (2010) Acta Crystallographica Section D—Biological Crystallography, 66, 486-501) and Phenix (Liebschner et al. (2019) Acta Cryst. (2019). D75, 861-77) until the structures converged at reasonable R-work and R-free (Table 14). The final models were analyzed for good stereochemistry, geometry and clash scores using MolProbity (Table 14).
Results:
The structure of Fab-Apo was determined to 2.9 Å and consists of residues Light chains 2-213, and Heavy chains 2-219 (2 Fabs) and has good electron density throughout, with the exception of residues Chain L: Asp28-Tyr34, Chain H: Ser99-Gly101, Lys134-Gly139, Chain M: Gly24-Tyr34, Chain I: Ser133-Gly138 which are not modelled due to weak electron density. The structure was refined to an R-work/R-free of 23% and 30%, respectively, and has good stereochemistry throughout with 4 Ramachandran outliers which is acceptable for this resolution (48th percentile for this residue range, Table 14).
The structure of Fab-mutCALR peptide was determined to 3.2 Å. The two Light chains consist of residues Chain L: 2-215, Chain M:2-216; and two Heavy chains H:2-219 and I:2-185. There are two Fabs and two peptides in the asymmetric unit. The model has good electron density throughout, with the exception of the following residues: Chain H, Ser132-Gly139; Chain M, Thr25-Gly31; and Chain I, Ala130-Ala142, which are not modelled due to weak electron density. The structure was refined to an R-work/R-free of 23% and 32%, respectively, and has good stereochemistry throughout with 7 Ramachandran outliers which is acceptable for this resolution (56th percentile for this residue range, Table 14).
As shown in
The Fab fragment having the sequences shown below was purified as described above for Example 11.
Crystallization was performed as follows: Recombinant Fab fragment was mixed with the 31-mer peptide at a ratio of 1:3 molar excess, and then concentrated to a final concentration of 21 mg/mL. Sparse matrix crystallization screens were set up using an NT8 crystallization robot (Formulatrix, Bedford, Mass.). Drops containing 200 nl (Fab fragment plus CalR peptide)+200 nl (reservoir) were used for the setup and the plates were incubated at 4° C., 13° C. and 20° C. Crystals grew from condition E7 of the JCSG Top96 Screen (Rigaku, Bainbridge Island, Wash.) after one day of incubation at 20° C. The final crystal condition for antibody X:31-mer peptide complex was TRIS 0.1M, pH8.5, Polyethylene glycol 400 40% v/v, and 0.2 M Lithium sulfate.
All crystals were flash cooled in liquid nitrogen for X-ray data collection.
Data Collection, Processing and Refinement was performed as follows: Diffraction data was collected at 100K at (λ=0.9201 Å) with an Eiger 9M detector using synchrotron radiation at beamline 17-ID-1 at the Center for BioMolecular Structure at the National Synchrotron Light Source II, Brookhaven, N.Y., USA. 1800 images were collected using a rotation of 0.1V per image. Diffraction data indexing, integration and scaling were performed using Fast_dp. Data collection statistics, phasing and refinement are given in Table 15.
For the Fab fragment-31-mer peptide data, Phaser was used to place fragments of the Fab fragment model and subsequent rounds of manual model building were performed resulting in a model containing 1 Fab and I peptide. Subsequent cycles of density modifications, model building and refinement were carried out using Refmac, Coot and Phenix until the structure converged at reasonable R-work and R-free (Table 15). The final model were analyzed for good stereochemistry, geometry and clash scores using MolProbity (Table 15).
Results:
The structure of Fab fragment-31-mer mutCalR peptide was determined at 2.0 Å with space group C 1 2 1. Each asymmetric unit contains one Fab molecule and one 31-mer peptide molecule. The Fab molecule consists of a light chain, Chain L: 2-215, and a heavy chain, Chain H:1-221. The model has well defined electron density throughout, with the exception of the following residues: Chain H, Gly42-Lys43, which are not modelled due to weak electron density. The structure was refined to an R-work/R-free of 20.1% and 22.6%, respectively, and has good stereochemistry throughout with 2 Ramachandran outliers.
To test the therapeutic potential of anti-mutCALR antibodies in vivo, an antibody was evaluated in a mouse model of essential thrombocythemia (ET). In this mouse model, a conditional allele that expresses a mutCALR protein with a C-terminal sequence (DEL52) identical to what found in MPN patients was knocked in to the mouse CALR sequence. The engineered mice (CALRdel/del) develop an ET-like disease with marked thrombocytosis, splenomegaly, and abnormal megakaryocytosis (Li et. al. Blood 2018; 131:649). The expression of mutCALR in the engineered mice was induced with intraperitoneal injections of polyinosinic:polycytidylic acid (poly I:C) (250 μg/dose; every other day for a total of 3 injections). Treatment initiated 19 weeks post-poly I:C induction and consisted of intravenous injections of anti-mutCALR antibody (clone 74) at 50 mg/kg QW for a total of 4 weeks. ET phenotype was confirmed by assessing the platelets counts in the blood (Sysmex, Kobe, Japan), spleen size and bone marrow histology. A representative study is shown in
The anti-mutCALR antibody restored normal platelet counts (
Taken together, these results demonstrate the efficacy of the anti-mutCALR antibodies in treating ET.
CD34+ cells isolated from MPN patients carrying the CALR mutation were used to characterize the ability of an anti-mutCALR antibody to inhibit the mutCALR-derived oncogenic function. Peripheral blood mononuclear cells (PBMCs) were isolated from non-identified blood samples from MPN patients by Ficoll gradient extraction (Fisher Scientific) and CD34+ cells were enriched using magnetic enrichment (Miltenyi Biotec). CD34+ cells were cultured for seven days in SFEM-II media (STEMCELL Technologies) containing hSCF, hFLT3L, TPO, LDL2698, SR1, and UM171.
CD34+ cells (50,000 cells/well) were then plated into 96-well plates and treated with a mutCALR or isotype control antibody for 2 hours. Following treatment, plates were centrifuged, the supernatant was aspirated and subsequently washed with PBS. After centrifugation the cell pellets were lysed using lysing buffer (Cell Signaling Technologies) supplemented with 1× Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were added to the Phospho (Tyr694)/Total STAT5a,b whole cell lysate kit (Meso Scale Diagnostics) and phospho-STAT5 levels were then quantitated using Meso Sector S 600 (Meso Scale Diagnostics). Clone 74 selectively inhibited pSTAT5 in CD34+ cells harboring mutCALR in a dose-dependent manner, while the isotype control (IgG) at 10 μg/mL had no impact on CD34+ cells. Moreover, pSTAT5 was not inhibited in CD34+ cells harboring the V617F JAK2 mutation (
To evaluate the ability of antibodies to inhibit mutCALR oncogenic function and imbalanced proliferation of megakaryocytes, CD34+ cells (50,000 cells/well) were added to a 12-well plate with SFEM-II supplemented with hSCF, hGCSF, hIL3, and hIL6 and treated with a mutCALR antibody or isotype control for 6 days. Cells were stained and analyzed by flow cytometry (LSRFortessam X-20 analyzer, BD Biosciences). Antibodies used were: APC anti-human CD38 antibody (BioLegend), FITC anti-human lineage cocktail (BioLegend), PE/Cyanine7 anti-human CD34 antibody (BioLegend), PE anti-human CD41 antibody (BioLegend), APC mouse anti-human CD42b antibody (BD Pharmingen). Megakaryocytes were identified as the CD41+CD42b+ cells. The anti-mutCALR antibodies (clones 74 and 65) selectively prevented the differentiation of mutCALR CD34+ cells into mature megakaryocytes in a dose-dependent manner, while isotype control (IgG) had no impact on this population. A representative experiment is shown in
In another experiment, the CD34+ cells described above were added at 50,000 cells/well to a 12-well plate containing 2.0 mL culture media with specified concentrations of clone 74 or isotype control. The treatment period was 12 hours. After 12 hours, cells were collected, washed and lysed using lysing buffer (Cell Signaling Technologies) supplemented with 1× Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Protein samples (6 μg) were separated in pre-casted 4-12% TrisGglycine gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes using iBlot 2 Dry Blotting System and iBlot™ 2 Transfer Stacks (Thermo Fisher Scientific). The nitrocellulose membranes were blocked with StartingBlock (Thermo Fisher Scientific) for one hour and probed with antibodies to detect pSTAT5 (Cell Signaling), STAT5 (Cell Signaling), pSTAT3 (Cell Signaling), STAT3 (Cell Signaling), and β-actin (Cell Signaling). Detection was performed using horseradish peroxidase (HRP)-conjugated secondary rabbit antibody (Cell Signaling) and chemiluminescence HRP substrate (Thermo Scientific). Clone 74 selectively inhibited pSTAT3 and pSTAT5 in CD34+ cells harboring mutCALR in a dose-dependent manner, while the isotype control (IgG) at 10 μg/mL had no impact on CD34+ cells (
In another experiment, Clone 74 and ruxolitinib were combined to determine the impact of co-treatment on megakaryopoiesis produced by CD34+ cells from human cord blood (WT cells) or an MPN patient with mutCALR (mutCALR cells). After six days in culture, 25 nM ruxolitinib alone did not impact the frequency of megakaryocytes produced by either WT or mutCALR-expressing CD34 cells. In contrast, 25 μg/mL of clone 74 alone selectively reduced the production of megakaryocytes from mutCALR+ CD34+ cells but not from WT CD34+ cells. Further, the combination of 25 nM ruxolitinib and 25 μg/mL of clone 74 led to further depletion of pathogenic megakaryocytes produced by mutCALR+ CD34+ cells (
Taken together, these results demonstrate the ability of the anti-mutCALR antibodies to inhibit mutCALR-derived oncogenic functions in MPN patient cells.
Engineered BaF3 cells (10,000 cells/well) were added to a 12-well plate containing 2.0 mL culture media with serially diluted antibodies. The treatment period was for 24 hours. After 22 hours of incubation, Ba/F3 cells were pulse-labeled with BrdU for 2 hours. After 24 hours, Ba/F3 cells were collected, washed with PBS and incubated with BD Cytofix/Cytoperm buffer for 20 minutes on ice. Cells were then washed with the BD Perm/Wash buffer and the cell pellets were resuspended in 100 μL of BD Cytoperm and Permeabilization buffer and incubated on ice for 10 minutes. Cells were then washed and resuspended in 100 μl of BD Cytofix/Cytoperm buffer and incubated at room temperature for 5 minutes. Cells were washed and treated with 100 μL of 300 μg/mL solution of DNase, and incubated for 1 hour at room temperature, and were then washed and resuspended in 50 μl of BD Perm/Wash buffer containing anti-BrdU-APC antibody for 20 minutes in the dark at room temperature. After additional washing, cells were resuspended in PBS containing 2% FBS and 7-AAD and analyzed for cell cycle profiles on the LSRFortessa™ X-20 analyzer (BD Biosciences). Clone 74 was screened to determine its effect on the cell cycle using WT BaF3 cells or Ba/F3-TPOR/mutCALR type 1. Clone 74 selectively induced apoptosis in BaF3 cells carrying mutCALR in a dose-dependent manner, whereas isotype control (IgG) had no effect on these cells. In contrast, clone 74 did not impact cell cycle profiles of WT BaF3 cells (
Cell binding of various anti-mutCALR antibodies as compared to clone 4 was assessed using engineered BaF3 cells stably expressing human MPL and human mutCALR Type 1. The antibodies used in this experiment were obtained commercially (AB1: CAL2 antibody, Dianova) or synthesized from sequences found in the literature (AB2: anti-mutCALR antibody including VH and VL regions of antibody B3 from WO2020175689 fused to human constant regions; AB3: anti-mutCALR antibody including the BJ095 VH region and the BJ097 VL region from WO2019178362 fused to human constant regions; and AB4: anti-mutCALR antibody including VH and VL regions of antibody 8B2-H6 from WO2016087514 fused to human constant regions).
Approximately 200,000 cells/well were added to 96-well plates and stained with the indicated concentration of antibodies for 30 minutes on ice. Cells were then washed and stained with goat anti-human secondary conjugated to R-phycoerythrin (R-PE) (Jackson Immuno Research Laboratories) for 30 minutes on ice. The cells were then washed again and analyzed by flow cytometry. Geometric Mean Fluorescence Intensity (GMFI) of cell binding was graphed using GraphPad Prism Software (version 7.04). As shown in
The ability of the five antibodies shown here to inhibit cell proliferation was tested. Inhibition of cell proliferation was assessed using engineered BaF3 cells expressing MPL/mutCALR Type 1. Cells were plated at 5,000 cells per well in RPMI 1640+2% FBS, antibodies were added and incubated for 72 hours, followed by assessment of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.) and Top Count (Perkin Elmer, Boston, Mass.) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. As shown in
The ability of other antibodies of the present disclosure to inhibit cell proliferation as compared to AB1, AB2, AB3 and AB4 was tested in the same manner as described above. The identified anti-mutCALR antibody clones listed below inhibited mutCALR-induced oncogenic cell proliferation in a dose-dependent manner, while AB1, AB2, AB3 and AB4 showed no functional activity. The IC50 values are shown in Table 16.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Patent Application No. 63/287,394, filed on Dec. 8, 2021, U.S. Provisional Patent Application No. 63/288,479, filed on Dec. 10, 2021, and U.S. Provisional Patent Application No. 63/421,052, filed on Oct. 31, 2022, each of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63287394 | Dec 2021 | US | |
| 63288479 | Dec 2021 | US | |
| 63421052 | Oct 2022 | US |
