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US 2026/0201046 A1Published application
A heterohybridoma-based method of generating recombinant rabbit monoclonal antibodies and antibodies produced by method
AssigneeBio-Rad Laboratories, Inc.
InventorsYu PENG, Chune ZHANG
Life of this patent
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Abstract
Provided is a heterohybridoma-based method of generating recombinant rabbit monoclonal antibodies, recombinant anti-IL-6 receptor-alpha (IL-6Rα) antibodies generated using such methods, and immune assay methods kits employing such antibodies.
Claims14
A method of identifying a rabbit monoclonal antibody, the method comprising:
- (a) generating heterohybridomas comprising splenocytes obtained from a rabbit immunized with an antigen of interest fused to mouse myeloma cell;
- (b) screening heterohybridomas generated in (a) to identify antibodies that bind to the antigen of interest;
- (c) selecting heterohybridomas that bind to the antigen of interest to provide a subpopulation for further screening
- (d) obtaining VH and VL CDNAs from RNA isolated from each heterohybridoma identified in (b)
- (e) sequencing the VH and VL CDNA, and aligning VH sequences to prepare a phylogenetic tree for VH regions and aligning VL regions to prepare a phylogenetic tree for VL regions for each heteorhybridoma;
- (f) selecting a VH region that comprises a CDR3 that is 8-20 residues in length;
- and
- (i) does not contain greater than 3 consecutive residues of the same amino acid;
- (ii) comprises a at least one aromatic residue;
- (iii) does not comprise three of one of the following amino acids, or three of the following amino acids in combination:
- P, C, K or Q;
- (g) selecting a VL region that comprises a CDR3 that is 8-20 residues in length;
- and
- (i) does not contain more than 3 consecutive residues of the same amino acid;
- (ii) comprises an aromatic amino acid;
- (iii) does not comprise three of one of the following amino acids, or three of the following amino acids in combination:
- P, C, K or Q;
- (h) independently expressing a VH region selected in (f) with each VL region selected in (g) to provide VH and VL region pairs;
- and independently expressing each VL region selected in (g) with each VH region selected (f) to provide VH and VL pairs;
- (i) analyzing the VH and VL region pairs to identify a pair that binds to the antigen of interest with the highest affinity relative to the other pairs.
17 more elements
An anti-IL-6 receptor-alpha (IL-6Rα) antibody that binds to a soluble IL-6Rα (sIL-6Rα) polypeptide, said anti-IL-6Rα antibody comprising:
- (a)
- HCDR1:
- (SEQ ID NO:
- 10)
- GFSFSGDYD
- HCDR2:
- (SEQ ID NO:
- 11)
- TDSGFSGTT
- HCDR3:
- (SEQ ID NO:
- 12)
- ARDFDSSGSYYWDL
- LCDR1:
- (SEQ ID NO:
- 13)
- ESVSSNNR
- LCDR2:
- AAS
- LCDR3:
- (SEQ ID NO:
- 14)
- AGYKGLYSDGRA;
- (b)
- HCDR1:
- (SEQ ID NO:
- 15)
- GFSFISYHYY
- HCDR2:
- (SEQ ID NO:
- 16)
- IDAISSGST
- HCDR3:
- (SEQ ID NO:
- 17)
- ARAPYYTYDGVYYALTL
- LCDR1:
- (SEQ ID NO:
- 18)
- ENIYNF
- LCDR2:
- RAS
- LCDR3:
- (SEQ ID NO:
- 19)
- QRNAYSSWA;
- (c)
- HCDR1:
- (SEQ ID NO:
- 20)
- GFSFSSGYY
- HCDR2:
- (SEQ ID NO:
- 21)
- IYTGSDTT
- HCDR3:
- (SEQ ID NO:
- 22)
- ARDLGSRGNL
- LCDR1:
- (SEQ ID NO:
- 23)
- ENIYSF
- LCDR2:
- RAS
- LCDR3:
- (SEQ ID NO:
- 19)
- QRNAYSSWA;
- or
- (d)
- HCDR1:
- (SEQ ID NO:
- 24)
- GFSFSSNYW
- HCDR2:
- (SEQ ID NO:
- 25)
- IYLFSVGNT
- HCDR3:
- (SEQ ID NO:
- 26)
- ARAPYYLDGARAYYAFNL
- LCDR1:
- (SEQ ID NO:
- 27)
- QSIGSD
- LCDR2:
- FAS
- LCDR3:
- (SEQ ID NO:
- 28)
- AGGYNSADIFA.
90 more elements
Dependencies are inferred from the claim text.