HEMOGLOBIN G-MAKASSAR BINDING POLYPEPTIDES AND ANTIBODIES AND METHODS OF USING THE SAME

1 . A binding polypeptide, anti-HbG-Makassar antibody, or an antigen binding portion thereof that specifically binds to an hemoglobin G (HbG) Makassar variant polypeptide, or a peptide thereof, but fails to detectably bind or binds at reduced levels to a wild-type beta (β)-globin polypeptide and/or a sickle cell globin (HbS) polypeptide, or a peptide thereof.

2 . The binding polypeptide or the antibody of claim 1 , comprising one or more complementarity determining regions (CDRs) which comprise or consist of heavy chain variable region (VH) CDRs and/or light chain variable region (VL) CDRs selected from the following:

A)

VH CDR1:

GIDFSRYW;

VH CDR2:

INIDSSTI;

VH CDR3:

ARAYDGYSLDY;

VL CDR1:

SSVSY;

VL CDR2:

DTS;

VL CDR3:

RQWSSYPLT;

B)

VH CDR1:

GYTFTNYF;

VH CDR2:

INPKNGGI;

VH CDR3:

ARGSANWGAY;

VL CDR1:

QRTNC;

VL CDR2:

HDL;

VL CDR3:

QQWSSYPLT;

or

C)

VH CDR1:

GYTFTSDW;

VH CDR2:

IYPRSGST;

VH CDR3:

ARGTYYGSRSYYFDY;

VL CDR1:

SSVSY;

VL CDR2:

DTS:

VL CDR3:

RQWSSYPLT,

3 . The binding polypeptide or the antibody of claim 1 , which comprises or consists of:

VL CDR1

SSVSY,

VL CDR2:

DTS,

and

VL CDR3:

RQWSSYPLT

and

VH CDR1:

GIDFSRYW;

VH CDR2:

INIDSSTI;

VH CDR3:

ARAYDGYSLDY

or

VH CDR1:

GYTFTSDW;

VH CDR2:

IYPRSGST;

VH CDR3:

ARGTYYGSRSYYFDY;

VL CDR1

QRTNC;

VL CDR2:

HDL;

VL CDR3:

QQWSSYPLT

and

VH CDR1:

GYTFTNYF;

VH CDR2:

INPKNGGI;

VH CDR3:

ARGSANWGAY.

a variable heavy chain (VH) domain comprising a CDR1 comprising amino acid sequence GIDFSRYW, a CDR2 comprising amino acid sequence INIDSSTI, and a CDR3 comprising amino acid sequence ARAYDGYSLDY; or

a variable heavy chain (VH) domain comprising a CDR1 comprising amino acid sequence GYTFTSDW, a CDR2 comprising amino acid sequence IYPRSGST, and a CDR3 comprising amino acid sequence ARGTYYGSRSYYFDY; and/or

a variable light chain (VL) domain comprising a CDR1 comprising amino acid sequence SSVSY, a CDR2 comprising amino acid sequence DTS, and a CDR3 comprising amino acid sequence RQWSSYPLT.

4 . The binding polypeptide or the antibody of claim 1 , comprising a heavy chain variable domain (VH) sequence having at least 85%, 90%, or 95% amino acid sequence identity to the amino acid sequence:

EVQLQESGGGLVQPGGSLKLSCAASGIDFSRYWMSWVRRAPGKGL

EWIGEINIDSSTINYAPSLKDKFIISRDNAKNTLYLQMSKVRSED

TALYYCARAYDGYSLDYWGQGTSVTVSS,

and/or comprising a light chain variable domain (VL) sequence having at least 85%, 90%, or 95% amino acid sequence identity to the amino acid sequence:

QIVLTQSPAIMSASPGEKVTMTCSTSSSVSYMFWYQQKPGSSPRL

LIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCRQW

SSYPLTFGAGTKLELK.

5 . The binding polypeptide or the antibody of claim 1 , comprising a heavy chain variable domain (VH) sequence having at least 85%, 90%, or 95% amino acid sequence identity to the amino acid sequence:

EVLLQQSGPELVKPGASVKISCKASGYTFTNYFMNWVKQSHGKSL

EWIGDINPKNGGISYNQKFKGKATLIVDKSSSTAYMELRSLTSED

SAVYYCARGSANWGAYWGQGTLVTVSA,

and/or comprising a light chain variable domain (VL) sequence having at least 85%, 90%, or 95% amino acid sequence identity to the amino acid sequence:

WWEDGYSWCSISHFQLPANQCLSHTVQRTNCSRPVSSNHVCISRG

EGHHDLQCQLKFPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQW

SSYPLTFGAGTKLELK.

6 . The binding polypeptide or the antibody of claim 1 , comprising a heavy chain variable domain (VH) sequence having at least 85%, 90%, or 95% amino acid sequence identity to the amino acid sequence:

QVQLQQPGAELVKPGASVKMSCKASGYTFTSDWITWVKQRPGQGL

EWIGDIYPRSGSTNYNEKFKSKATLTVDISSNTAYMQLSSLTSED

SAVFYCARGTYYGSRSYYFDYWGQGTTLTVSS,

and/or comprising a light chain variable domain (VL) sequence having at least 85%, 90%, or 95% amino acid sequence identity to the amino acid sequence:

QIVLTQSPAIMSASPGEKVTMTCSTSSSVSYMFWYQQKPGSSPRL

LIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCROW

SSYPLTFGAGTKLELK.

7 . The binding polypeptide or the antibody of claim 1 , wherein the binding polypeptide or the antibody, or a binding portion thereof, comprises an affinity tag or a detectable amino acid sequence.

8 . A method of identifying an HbG-Makassar variant polypeptide or peptide, the method comprising contacting a sample with the binding polypeptide or the antibody of claim 1 for a time sufficient for the polypeptide or the antibody to bind to the HbG-Makassar variant polypeptide or peptide in the sample.

9 . An isolated nucleic acid molecule that encodes the binding polypeptide or the antibody of claim 1 .

10 . The isolated nucleic acid molecule of claim 9 , comprising a nucleic acid sequence having at least 85%, 90, 95, or 100% sequence identity to the heavy chain variable domain

(VH) nucleic acid sequence

gaggtgcagctgcaggagtctggaggtggcctggtgcagcctgga

ggatccctgaaactctcctgtgcagcctcaggaatcgattttagt

agatactggatgagttgggttcggcgggctccagggaaaggacta

gaatggattggagaaattaatatagatagcagtacaataaactat

gcaccatctctaaaggataaattcatcatctccagagacaacgcc

aaaaatacgctgtacctgcaaatgagcaaagtgagatctgaggac

acagccctttattactgtgcaagggcctatgatggttattcgttg

gactactggggtcaaggaacctcagtcaccgtctcctcag

and/or comprising a nucleic acid sequence having at least 85% sequence identity to the light chain variable domain (VL) nucleic acid sequence

caaattgttctcacccagtctccagcaatcatgtctgcatctcca

ggggagaaggtcaccatgacctgcagtaccagctcaagtgtaagt

tacatgttctggtaccagcagaagccaggatcctcccccagactc

ctgatttatgacacatccaacctggcttctggagtccctgttcgc

ttcagtggcagtgggtctgggacctcttactctctcacaatcagc

cgaatggaggctgaagatgctgccacttattactgccggcagtgg

agtagttaccccctcacgttcggtgctgggaccaagctggagctg

aaac;

gaggtcctgctgcaacaatctggacctgagctggtgaagcctggg

gcttcagtgaagatttcctgtaaggcttctggatacacgttcact

aactacttcatgaactgggtgaagcagagccatggaaagagcctt

gagtggattggagatattaatcctaagaatggtggtattagttac

aaccagaaatttaagggcaaggccacattgattgtagacaagtcc

tccagcacagcctacatggagctccgcagcctgacttctgaggac

tctgcagtctattattgtgcaagagggtcagctaactggggggct

tactggggccaagggactctggtcactgtctctgcag

and/or comprising a nucleic acid sequence having at least 85% sequence identity to the light chain variable domain (VL) nucleic acid sequence

tggtgggaagatggatacagttggtgcagcatcagccattttcag

cttcctgctaatcagtgcctcagtcatactgtccagaggacaaat

tgttctcgcccagtctccagcaatcatgtctgcatctccagggga

gaaggtcaccatgacctgcagtgccagctcaagttccctgttcgc

ttcagtggcagtgggtctgggacctcttactctctcacaatcagc

cgaatggaggctgaagatgctgccacttattactgccagcagtgg

agtagttaccccctcacgttcggtgctgggaccaagctggaactg

aaac;

or

caggtccagctgcagcagcctggggctgagcttgtgaagcctggg

gcttcagtgaagatgtcctgcaaggcttctggctacaccttcacc

agcgactggataacctgggtgaagcagaggcctggacaaggcctt

gagtggattggagatatttatcctcgtagtggtagtactaactac

aatgagaagttcaagagcaaggccacactgactgtagatatatcc

tccaacacagcctacatgcagctcagcagcctgacatctgaggac

tctgcggtcttttactgtgcaagagggacttactacggtagtagg

tcctactactttgactactggggccaaggcaccactctcacagtc

tcctcag

and/or comprising a nucleic acid sequence having at least 85% sequence identity to the light chain variable domain (VL) nucleic acid sequence

caaattgttctcacccagtctccagcaatcatgtctgcatctcca

ggggagaaggtcaccatgacctgcagtaccagctcaagtgtaagt

tacatgttctggtaccagcagaagccaggatcctcccccagactc

ctgatttatgacacatccaacctggcttctggagtccctgttcgc

ttcagtggcagtgggtctgggacctcttactctctcacaatcagc

cgaatggaggctgaagatgctgccacttattactgccggcagtgg

agtagttaccccctcacgttcggtgctgggaccaagctggagctg

aaac.

11 . A method of identifying and/or selecting a subject expressing an HbG-Makassar polypeptide, the method comprising:

(a) contacting a sample obtained from the subject with the binding polypeptide, the antibody, or an antigen binding portion thereof, of claim 1 ;

(b) detecting specific binding between the binding polypeptide, the antibody, or the antigen binding portion thereof, and an HbG-Makassar polypeptide in the sample; and

(c) identifying and/or selecting the subject as expressing an HbG-Makassar polypeptide based on the detecting step (b).

12 . A method of monitoring a subject for the production of HbG-Makassar polypeptide, the method comprising:

(a) contacting a sample obtained from the subject with the binding polypeptide, the antibody, or an antigen binding portion thereof, of claim 1 at a first time point and detecting specific binding between the binding polypeptide, the antibody, or the antigen binding portion thereof, and an HbG-Makassar polypeptide in the sample;

(b) contacting a sample obtained from the subject with the binding polypeptide, the antibody, or the antigen binding portion thereof, of claim 1 at one or more additional time points and detecting specific binding between the binding polypeptide or the antibody and an HbG-Makassar polypeptide in the sample; and

(c) monitoring that the subject is expressing the HbG-Makassar polypeptide by detecting the same level or a greater level of the HbG-Makassar polypeptide in the subject's sample in step (b) versus step (a).

13 . A method of assessing a relative or absolute level of HbG-Makassar hemoglobin in a subject expressing an HbG-Makassar polypeptide, the method comprising:

(a) contacting a sample obtained from the subject with the binding polypeptide, the antibody, or an antigen binding portion thereof, of claim 1 ;

(b) detecting specific binding between the binding polypeptide, the antibody, or the antigen binding portion thereof, and an HbG-Makassar polypeptide in the sample; and

(c) assessing a relative or absolute level of at least 30% of HbG-Makassar hemoglobin in the sample based on the detecting step (b); wherein said level of HbG-Makassar in the subject is sufficient to prevent sickling by HbG-S hemoglobin in the subject.

14 . The method of claim 13 , wherein the anti-HbG-Makassar antibody comprises 1C10.E3.G7, 1C10.C1.C7, or 5D6.F6.D2, or an antigen binding portion thereof.

15 . A composition comprising the binding polypeptide or the antibody of claim 1 , or an antigen binding fragment thereof, or the nucleic acid encoding the binding polypeptide or the antibody.

16 . A vector comprising a nucleic acid molecule that encodes the binding polypeptide or the antibody of claim 1 .

17 . A cell comprising the vector of claim 15 .

18 . A kit comprising the binding polypeptide, the antibody, or an antigen binding portion thereof, of claim 1 or the nucleic acid molecule encoding the binding polypeptide or the antibody.

19 . The binding polypeptide, the anti-HbG-Makassar antibody, or an antigen binding portion thereof, of claim 1 , wherein the polypeptide, antibody, or the antigen binding portion thereof specifically binds to a hemoglobin G (HbG) Makassar peptide comprising the amino acid sequence VHLTPAEKSAVTA.

20 . The binding polypeptide, the anti-HbG-Makassar antibody, or an antigen binding portion thereof, of claim 18 , wherein the polypeptide, antibody, or the antigen binding portion thereof specifically binds to a hemoglobin G (HbG) Makassar peptide comprising the amino acid sequence VHLTPAEKSAVTA, but fails to detectably bind or binds at reduced levels to a sickle cell HbS peptide comprising the amino acid sequence VHLTPVEKSAVTA and/or to a wildtype beta-globin peptide comprising the amino acid sequence VHLTPEEKSAVTA.