Binding polypeptides and methods of making the same

Patent metadata

Field	Value
Publication number	US-2018201926-A1
Application number	US-201715852718-A
Country	US
Kind code	A1
Filing date	Dec 22, 2017
Priority date	Dec 23, 2016
Publication date	Jul 19, 2018
Grant date	—

Abstract

Polypeptides, such as antibody molecules and TCR molecules, and methods of making the same, are disclosed. The polypeptides can be used to treat, prevent, and/or diagnose disorders.

First claim

What is claimed is:

1 . A method of making a nucleic acid sequence comprising a sequence that encodes a heavy chain element (HC element) of an antibody heavy chain variable region (HCVR) and a light chain element (LC element) of an antibody light chain variable region (LCVR), and wherein the HCVR and LCVR are matched, the method comprising:
a) acquiring an isolated production reaction site, comprising:
i) a heavy chain (HC) strand, wherein the HC strand is a strand of a heavy chain double-stranded cDNA (HC ds cDNA) comprising a segment that encodes the HC element of the HCVR from a cell; and
ii) a light chain (LC) strand, wherein the LC strand is a strand of a light chain double-stranded cDNA (LC ds cDNA) comprising a segment that encodes the LC element of the LCVR from the cell, and
b) covalent linking of the HC strand to the LC strand, wherein the isolated production reaction site does not comprise a nucleic acid encoding an HCVR or an LCVR from a cell other than the cell, thereby making the nucleic acid sequence.

2 . The method of claim 1 , wherein the HC element comprises, or consists of, a heavy chain variable region sequence (HCVRS), or an antigen binding fragment thereof.

3 . The method of claim 1 , wherein the LC element comprises, or consists of, a light chain variable region sequence (LCVRS), or an antigen binding fragment thereof.

4 . The method of claim 1 , wherein the nucleic acid sequence is configured such that, when expressed, the HC element and the LC element form a functional antigen binding molecule in vitro, ex vivo, or in vivo.

5 . The method of claim 1 , wherein acquiring the isolated production reaction site comprises:
a) acquiring a capture substrate bound to:
(i) a first double-stranded cDNA (ds cDNA) comprising a strand that is complementary to a first mRNA that encodes the HCVR from the cell; and
(ii) a second ds cDNA comprising a strand complementary to a second mRNA encoding the LCVR from the cell, to produce a loaded capture substrate, and
b) maintaining the isolated production reaction site under conditions that allow amplification of the first and second ds cDNAs, to produce: a plurality of HC ds cDNAs comprising the segment that encodes the HC element of the HCVR from the cell; and a plurality of LC ds cDNAs comprising the segment that encodes an LC element of the LCVR from the cell.

6 . The method of claim 1 , wherein the capture substrate comprises a bead and a moiety which binds to cDNA.

7 . The method of claim 1 , wherein the isolated production reaction site comprises a reagent mixture suitable for producing, from the first and second mRNAs, a first ds cDNA comprising the segment that encodes the HC element of the HCVR of the cell, and a second ds cDNA comprising a segment that encodes the LC element of the LCVR of the cell.

8 . The method of claim 5 , wherein the first and second ds cDNAs are amplified in the presence of primers, wherein at least one of the primers comprises a first member, a second member, and a nucleotide modification between the first and second members, wherein the nucleotide modification reduces DNA synthesis.

9 . The method of claim 8 , wherein the nucleotide modification comprises an insertion of a spacer between two adjacent nucleotides or a modification to a ribose.

10 . The method of claim 8 , wherein the first member is capable of annealing with the second member in the same primer or a different primer, forming a double-stranded structure comprising a duplex region of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, more basepairs.

11 . The method of claim 8 , wherein at least one of the primers is phosphorylated and comprises a sequence encoding at least a portion of a linker sequence, or a complementary sequence thereof.

12 . The method of claim 1 , wherein the HC ds cDNA comprises a 5′ overhang and a blunt end and the LC ds cDNA comprises a 5′ overhang and a blunt end.

13 . The method of claim 1 , wherein the HC strand and the LC strand are covalently linked to produce a single stranded nucleic acid sequence, wherein the HC and LC strands are both sense strands or both antisense strands.

14 . The method of claim 1 , wherein the covalent linking occurs in an isolated linkage reaction site comprising a ligase.

15 . The method of claim 1 , wherein the HC strand and the LC strand are covalently linked in the presence of a splint oligonucleotide, wherein the splint oligonucleotide is hybridized to a sequence comprising the junction of the HC strand and the LC strand to form a duplexed region at the site of linkage.

16 . The method of claim 14 , wherein the splint oligonucleotide comprises a modification that inhibits DNA synthesis.

17 . The method of claim 1 , further comprising, prior to acquiring the isolated production reaction site, acquiring an mRNA loaded capture substrate comprising:
a) acquiring an isolated cell reaction site, comprising:
i) a cell; and
ii) a capture substrate capable of binding a first mRNA encoding an HCVR from the cell and a second mRNA encoding an LCVR from the cell; and
b) maintaining the isolated cell reaction site under conditions that allow lysis of the cell and binding of the capture substrate with the first mRNA and the second mRNA to form the mRNA loaded capture substrate, wherein the isolated cell reaction site does not include a nucleic acid encoding an HCVR or an LCVR from a cell other than the cell.

18 . The method of claim 17 , further comprising releasing the mRNA loaded capture substrate from the isolated cell reaction site in the presence of a poly(dA) or poly(dT) oligonucleotide.

19 . The method of claim 1 , further comprising amplifying the nucleic acid sequence.

20 . The method of claim 1 , further comprising sequencing all or a portion of the nucleic acid sequence.

21 . The method of claim 1 , further comprising inserting all or a portion of nucleic acid sequence into a vector.

22 . The method of claim 1 , comprising expressing the nucleic acid sequence to produce a polypeptide comprising the segment that encodes the HC element of the HCVR, and the segment that encodes the LC element of the LCVR.

23 . The method of claim 22 , further comprising contacting the polypeptide with an antigen and determining if the polypeptide binds the antigen.

24 . A method of making a library comprising a plurality of unique members, the method comprising:
making the plurality of members by the method of claim 1 , wherein each of the members comprises a sequence that encodes a heavy chain element (HC element) of a heavy chain variable region (HCVR) and a light chain element (LC element) of a light chain variable region (LCVR), wherein the HCVR and the LCVR are matched, and wherein each unique nucleic acid sequence of the plurality comprises an HC element and an LC element from a different unique cell, thereby making the library.

25 . The method of claim 24 , wherein the library comprises one, two, three, or all of the following properties:
a) the plurality of unique members comprises at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 unique members; b) the plurality of unique members comprises 10 4 to 10 9 , 10 4 to 10 8 , 10 4 to 10 7 , 10 4 to 10 6 , 10 4 to 10 5 , 10 8 to 10 9 , 10 7 to 10 9 , 10 6 to 10 9 , 10 5 to 10 9 , 10 5 to 10 8 , 10 6 to 10 7 , 10 4 to 10 5 , 10 5 to 10 6 , 10 6 to 10 7 , 10 7 to 10 8 , or 10

Assignees

• Visterra Inc

Inventors

• Shriver Zachary
• Babcock Gregory
• Robinson Luke

Classifications

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K2317/10

  characterized by their source of isolation or production · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title