Method for generating and screening an antibody library

Patent metadata

Field	Value
Publication number	US-9862759-B2
Application number	US-74546908-A
Country	US
Kind code	B2
Filing date	Dec 4, 2008
Priority date	Dec 4, 2007
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

Abstract

The invention relates to a method for generating a DNA sequence coding for the heavy chain or the light chain of at least one antibody from RNA from a cell capable of producing an antibody. More particularly, the invention relates to the generation of a monoclonal antibody library. The invention also relates to the use of an antibody library for screening monoclonal antibodies, preferably human antibodies for treating cancer.

First claim

The invention claimed is:

1. A method for generating a bank of DNAs coding for at least one heavy chain or a light chain of at least one antibody from an extract or mixture of RNA from a cell, characterized in that it applies a method for generating a DNA sequence coding for a heavy chain or a light chain of at least one antibody from an extract or mixture of RNA from a cell by converting RNA into said DNA sequence, characterized in that said method comprises at least the following steps:
a) generating two primers, primary primer (I) and secondary primer (II), wherein primary primer (I) is complementary to any region of any RNA that codes for a heavy or light chain of any region C of any antibody and wherein secondary primer (II) has reverse complementarity to any region of primer (I);
b) putting a primary primer (I) in contact with said RNA extract or mixture liable to contain at least one RNA, designated here as a sense RNA, coding for the heavy chain or the light chain of an antibody, this primary primer specifically hybridizing to a fragment of the sequence of said sense RNA, this fragment being comprised in the sequence coding for the constant region C of the heavy or light chain of said antibody;
c) synthesizing, from said primary primer (I), a single strand cDNA designated as anti-sense cDNA;
d) eliminating if necessary, the primary primer (I) which have not hybridized to the fragment of the sequence of said RNA in step a);
e) annealing said anti-sense cDNA by forming a covalent bond between its ends 5′ and 3′ to form an anti-sense circular cDNA;
f) putting a secondary primer (II), designated as sense secondary primer, in contact with said anti-sense circular cDNA obtained in step e), this sense secondary primer (II) hybridizing to said anti-sense circular cDNA;
g) amplifying said anti-sense cDNA from said secondary primer (II), wherein said amplification is applied with an enzyme that amplifies a sequence of single strand circular DNA, wherein said enzyme consists in the rolling circle polymerase of the bacteriophage φ29; and
h) recovering the thereby amplified sense complementary linear DNA strand, wherein the recovered amplified sense complementary linear DNA strand codes for a heavy chain or a light chain of at least one antibody from an extract or mixture of the RNA from a cell,
wherein said amplification step is carried out on a single strand sequence,
i) putting a tertiary primer (III), designated as anti-sense tertiary primer, into contact with said sense linear DNA obtained in step h), this anti-sense tertiary primer (III) specifically hybridizes to a fragment of the sequence of the linear DNA comprised between the end 3′ and the end 5′ of said linear DNA strand and corresponding to the sequence of this linear DNA coding for the constant region C,
j) generating from said tertiary primer (III), a concatemer by synthesis of a complementary anti-sense DNA strand, and
k) cloning said thereby obtained double strand DNA concatemer into a vector.

2. The method according to claim 1 , characterized in that said tertiary primer (III) is specific of a sequence located at or adjacent to the end 5′ of the sequence of the sense linear DNA corresponding to the constant region C.

3. The method for generating a bank of DNAs coding for heavy or light antibody chains according to claim 1 , characterized in that it comprises, prior to step k), a step consisting of segmenting the concatemer at the sequences corresponding to the primers used.

4. The method according to claim 3 , characterized in that the primary primer (I) comprises a restriction site.

5. The method according to claim 3 , characterized in that said step k) for segmenting the concatemer is applied by enzymatic digestion with a specific restriction endonuclease of the restriction site comprised in the primary primer (I).

6. The method according to anyone of claim 1 , characterized in that the vector used for step k) further comprises sequences coding for the constant domains of the heavy or light chain of an immunoglobulin.

7. The method according to claim 6 , characterized in that said sequence coding for the constant domain of the heavy chain preferentially consists in a sequence from an immunoglobulin with membrane anchoring, or comprising a transmembrane C-terminal region.

8. The method according to claim 1 , characterized in that it further comprises a step l) for transfecting with the vector obtained in step k) a host cell that expresses the heavy or light chain of the antibody coded by the double strand DNA fragment inserted within said vector.

9. The method according to claim 8 , characterized in that said host cell of step l) is a cell that expresses at its surface the antibody coded by the inserted double strand DNA fragment.

10. The method according to claim 9 , characterized in that said host cell is an eukaryotic cell.

11. The method according to claim 10 , characterized in that said eukaryotic cell is selected from CHO, COS, HEK and NIH-3T3 cells.

12. The method according to claim 1 , characterized in that the cells from which stem the extract or mixture of RNA are of human origin.

Assignees

• Lowe Peter
• Bes Cédric
• Boute Nicolas
• Pf Medicament

Inventors

• Lowe Peter
• Bes Cédric
• Boute Nicolas

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title