Lambodies with high affinity and selectivity for glycans and uses therefor

Patent metadata

Field	Value
Publication number	US-10036747-B2
Application number	US-201214359653-A
Country	US
Kind code	B2
Filing date	Nov 21, 2012
Priority date	Nov 22, 2011
Publication date	Jul 31, 2018
Grant date	Jul 31, 2018

Abstract

The invention relates to dimeric proteins comprised of subunits having (i) recombinant lamprey variable lymphocyte receptor (VLR) diversity regions linked to (ii) multimerization domains. The dimeric proteins exhibit binding specificity for glycosylated antigens, and they may be used in methods of detecting or isolating glycans from a sample, and in methods of disease diagnosis, prognosis, progression monitoring, treatment, and imaging.

First claim

What is claimed is:

1. A lambody, wherein the lambody comprises a first recombinant lambody subunit and a second recombinant lambody subunit, wherein each recombinant lambody subunit is a fusion protein consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, wherein the lambody subunits are dimerized via their multimerization domains, and wherein the lambody exhibits binding specificity for a glycan, glycolipid or glycoprotein.

2. The lambody of claim 1 , wherein the VLR diversity regions of the first and second recombinant lambody subunits have identical amino acid sequences.

3. The lambody of claim 1 , wherein the VLR diversity regions of the first and second recombinant lambody subunits have different amino acid sequences.

4. The lambody of claim 1 , wherein the lambody exhibits a binding affinity (K D ) of at least about 1×10 −7 M for a target against which it exhibits binding specificity.

5. The lambody of claim 1 , wherein the multimerization domain is selected from the group consisting of a yeast leucine zipper dimerization domain, a partial antibody hinge region and leucine zipper dimerization domain, a coil-coiled dimerization peptide, and an antibody Fc fragment.

6. A lambody multimer, wherein the lambody multimer comprises three or more multimerized recombinant lambody subunits, wherein the recombinant lambody subunits are each fusion proteins consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, wherein the lambody subunits are multimerized via their multimerization domains, and wherein the lambody multimer exhibits binding specificity for a glycan, glycolipid or glycoprotein.

7. The lambody multimer of claim 6 , wherein the multimerization domain is selected from the group consisting of a yeast leucine zipper dimerization domain, a partial antibody hinge region and leucine zipper dimerization domain, a coil-coiled dimerization peptide, and an antibody Fc fragment.

8. A recombinant lambody subunit, wherein the recombinant lambody subunit is a fusion protein consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, wherein the lambody subunit exhibits binding specificity for a glycan, glycolipid or glycoprotein.

9. The recombinant lambody subunit of claim 8 , wherein the multimerization domain is selected from the group consisting of a yeast leucine zipper dimerization domain, a partial antibody hinge region and leucine zipper dimerization domain, a coil-coiled dimerization peptide, and an antibody Fc fragment.

10. A method for isolating a glycan-bearing element from a sample, said method comprising (i) adhering a recombinant lambody subunit, lambody or lambody multimer to a support, (ii) contacting the support with a sample under conditions permitting binding of a glycan of the glycan-bearing element in the sample by a VLR diversity region of a recombinant lambody subunit, lambody or lambody multimer adhered to the support, (iii) washing unbound sample from the support, (iv) eluting the glycan-bearing element from the support, and (v) collecting the glycan-bearing element,
wherein the recombinant lambody subunit is a fusion protein consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, and wherein the lambody subunit exhibits binding specificity for a glycan, glycolipid or glycoprotein;
wherein the lambody comprises a first recombinant lambody subunit and a second recombinant lambody subunit, wherein each recombinant lambody subunit is a fusion protein consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, wherein the lambody subunits are dimerized via their multimerization domains, and wherein the lambody exhibits binding specificity for a glycan, glycolipid or glycoprotein; and
wherein the lambody multimer comprises three or more multimerized recombinant lambody subunits, wherein the recombinant lambody subunits are each fusion proteins consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, wherein the lambody subunits are multimerized via their multimerization domains, and wherein the lambody multimer exhibits binding specificity for a glycan, glycolipid or glycoprotein.

11. The method of claim 10 , wherein the support is a bead in a column.

12. A method for detecting a glycan, glycolipid or glycoprotein in a biological sample from a subject, said method comprising (i) contacting a biological sample from a subject with a recombinant lambody subunit, lambody or lambody multimer under conditions permitting binding of the glycan, glycolipid or glycoprotein by a VLR diversity region of the recombinant lambody subunit, lambody or lambody multimer, and (ii) detecting binding by the VLR diversity region of the recombinant lambody subunit, lambody or lambody multimer to the glycan, glycolipid or glycoprotein in the sample,
wherein the recombinant lambody subunit is a fusion protein consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, and wherein the lambody subunit exhibits binding specificity for a glycan, glycolipid or glycoprotein;
wherein the lambody comprises a first recombinant lambody subunit and a second recombinant lambody subunit, wherein each recombinant lambody subunit is a fusion protein consisting of a lamprey variable lymphocyte receptor (VLR) diversity region linked via an amide bond to a multimerization domain, wherein the VLR diversity region is SEQ ID NO:2, SEQ ID NO:4, or the variant of SEQ ID NO:2 with the five amino acid mutations S19N, S86G, H105R, K112M and T208S, said variant differing from SEQ ID NO:2 only by the five amino acid mutations, wherein the lambody subunits are dimerized via their mu

Assignees

• Univ Maryland

Inventors

• Pancer Zeev
• Mao Li
• Hong Xia
• Ma Mark Z

Classifications

• G01N33/575

  for cancer · CPC title

• G01N2400/02

  involving antibodies to sugar part of glycoproteins · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C07K14/461

  from fish · CPC title