US5858725A
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Preparation of chimaeric antibodies using the recombinant PCR strategy
Assignee: Wellcome Foundation Ltd
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Filing: 1990-10-08
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Granted: 1999-01-12
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Expires: 2010-10-08
Abstract
Abstract PCT No. PCT/GB91/01744 Sec. 371 Date Jul. 29, 1993 Sec. 102(e) Date Jul. 29, 1993 PCT Filed Oct. 8, 1990 PCT Pub. No. WO92/07075 PCT Pub. Date Apr. 30, 1992The invention relates to a method of producing a chimaeric antibody in which the CDR of a first antibody is spliced between the framework regions of a second antibody. The method is performed using a template comprising two framework regions, AB and CD, and between them, the CDR which is to be replaced by a donor CDR. Primers A and B are used to amplify the framework region AB, and primers C and D used to amplify the framework region CD. However, the primers B and C also contain, at their 5' ends, additional sequence corresponding to all or at least part of the donor CDR sequence. Primers B and C overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a polymerase chain reaction to be performed and thereby incorporate all of the donor CDR sequence. The amplified regions AB and CD may undergo splice overlap extension to produce the chimaeric product in a single reaction.
Inventors
- 👤 James Scott Crowe
- 👤 Alan Peter Lewis
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 12:25
CPC Classifications
C; C07; C07K; C07K16/00; C07K16/46; C07K16/461; C07K16/464; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/686; C07K2317/00; C07K2317/20; C07K2317/24; C07K2319/00; C07K2319/01; C07K2319/02; Y; Y10; Y10S; Y10S530/00; Y10S530/867
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
418 words
A method for producing a double- or single-stranded DNA of formula 5'F1-M-F2 3' encoding an antibody chain or fragment thereof in which of the complementarity determining regions (CDRs) of the variable region of the antibody chain is derived from a first mammalian antibody, and the framework of the variable region is derived from a second, different mammalian antibody, wherein M comprises DNA encoding a CDR of the second antibody and F1 and F2 respectively encode 5' and 3' sequences flanking M, which method comprises; (i) preparing a single- or double-stranded DNA template of the formula 5'f1-H-f2 3' wherein H comprises DNA encoding a CDR of a different specificity from M and f1 and f2 are substantially homologous to F1 and F2 respectively; (ii) obtaining DNA oligonucleotide primers A, B, C and D wherein A comprises a sequence a 1 which has a 5' end corresponding to the 5' end of F1 and which is identical to a corresponding length of the sequence F1, is oriented in a 5' to 3' direction towards H; B consists of the sequence 5'b.sup.1 -b.sup.2 3' wherein b 1 comprises a sequence complementary to a corresponding length of M and has a 3' end which is complementary to the 5' end of M, and b 2 is complementary to a sequence of corresponding length in F 1 and has a 5' end which starts at the nucleotide complementary to the 3' end of F 1 ; C consists of the sequence 5'c.sup.1 -c.sup.2 3' wherein c 1 comprises a sequence identical to the corresponding length of M and has a 3' end which corresponds to the 3' end of M, and c 2 is identical to a sequence of corresponding length in F2 and has a 5' end which starts at the nucleotide corresponding to the 5' end of F2; D comprises a sequence d 1 which has a 5' end complementary to the 3' end of F2 and which is complementary to a corresponding length of F2, and is oriented in a 5' to 3' direction towards H; and wherein b 1 and c 1 overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a polymerase chain reaction (PCR) to be performed; (iii) performing, in any desired order, PCR reactions with primers pairs A,B and C,D on the template prepared in (i) above; and (iv) mixing the products obtained in (iii) above and performing a PCR reaction using primers A and D.
Claim 2
Independent Claim
418 words
A method for producing a double- or single-stranded DNA of formula 5'F1-M-F2 3' encoding an antibody chain or fragment thereof in which of the complementarity determining regions (CDRs) of the variable region of the antibody chain is derived from a first mammalian antibody, and the framework of the variable region is derived from a second, different mammalian antibody, wherein M comprises DNA encoding a CDR of the second antibody and F1 and F2 respectively encode 5' and 3' sequences flanking M, which method comprises; (i) preparing a single- or double-stranded DNA template of the formula 5'f1-H-f2 3' wherein H comprises DNA encoding a CDR of a different specificity from M and f1 and f2 are substantially homologous to F1 and F2 respectively; (ii) obtaining DNA oligonucleotide primers A, B, C and D wherein A comprises a sequence a 1 which has a 5' end corresponding to the 5' end of F1 and which is identical to a corresponding length of the sequence F1, is oriented in a 5' to 3' direction towards H; B consists of the sequence 5'b.sup.1 -b.sup.2 3' wherein b 1 comprises a sequence complementary to a corresponding length of M and has a 3' end which is complementary to the 5' end of M, and b 2 is complementary to a sequence of corresponding length in F 1 and has a 5' end which starts at the nucleotide complementary to the 3' end of F 1 ; C consists of the sequence 5'c.sup.1 -c.sup.2 3' wherein c 1 comprises a sequence identical to the corresponding length of M and has a 3' end which corresponds to the 3' end of M, and c 2 is identical to a sequence of corresponding length in F2 and has a 5' end which starts at the nucleotide corresponding to the 5' end of F2; D comprises a sequence d 1 which has a 5' end complementary to the 3' end of F2 and which is complementary to a corresponding length of F2, and is oriented in a 5' to 3' direction towards H; and wherein b 1 and c 1 overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a polymerase chain reaction (PCR) to be performed; (iii) performing, in any desired order, PCR reactions with primers pairs A,B and C,D on the template prepared in (i) above; and (iv) mixing the products obtained in (iii) above and performing a PCR reaction using primers A and D.
Claim 3
Dependent Claim
23 words
A method according to claim 1 wherein F1 and F2 each encode at least one human antibody framework region, and optionally further CDRs.
Claim 4
Dependent Claim
18 words
A method according to claim 1 or 2 wherein H encodes a CDR of the said first antibody.
Claim 5
Dependent Claim
13 words
A method according to claim 1 wherein M encodes a non-human CDR region.
Claim 6
Dependent Claim
14 words
A method according to claim 4 wherein M encodes a murine or rodent CDR.
Claim 7
Dependent Claim
28 words
A method according to any claim 1 wherein the primers A and D contain at least one restriction endonuclease recognition site within 10 nucleotides of their 5' ends.
Claim 8
Dependent Claim
26 words
A method according to claim 1 wherein, in the primers B and C, b 1 and c 1 are the same number of nucleotides in length.
Claim 9
Dependent Claim
22 words
A method according to claim 1 wherein primers A, B, C and D are each from 15 to 200 nucleotides in length.
Claim 10
Dependent Claim
35 words
A method according to claim 8 wherein a 1 , b 2 , c 2 and d 1 of primers A, B, C and D respectively are each from 15 to 30 nucleotides in length.
References Cited (Backward Citations)
WO1990007861A1
Cited
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