Immunoglobulins devoid of light chains
AssigneeVrije Universiteit Brussel
InventorsCecile Casterman, Raymond Hamers
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Abstract
The invention relates to an immunoglobulin having determined antigen specificity, and having a variable fragment which is derived from a so-called heavy-chain immunoglobulin having two heavy polypeptide chains capable of recognizing and binding one or several antigens and which is further naturally devoid of light chains, wherein the immunoglobulin having determined antigen specificity is devoid of CH1 constant region between the variable region and the hinge region and has within its constant region at least part of a constant region of a human antibody.
Claims14
1-17. (canceled)
A cDNA library comprising nucleotide sequences coding for a fragment of an immunoglobulin, said immunoglobulin comprising two heavy polypeptide chains capable of recognizing and binding one or several antigens, said immunoglobulin containing a variable (VHH) region and a constant region, said constant region being devoid of a first domain (CH1), said immunoglobulin being devoid of light polypeptide chains, which fragment forms a determined antigen binding site, wherein said cDNA library is obtained by performing the following steps:
- (a) treating a sample containing lymphoid cells from a healthy animal selected among the Camelids in order to separate the lymphoid cells;
- (b) separating polyadenylated RNA from other nucleic acids and components of the B-lymphocytes;
- (c) reacting the obtained RAN with a reverse transcriptase in order to obtain the corresponding cDNA;
- (d) contacting the obtained cDNA with 5′ primers located in the promoter, leader or framework sequences of the VHH sequence of said heavy chain immunoglobulin, and with 3′ primers located in the hinge, CH2, CH3, 3′ untranslated region or polyA tail;
- (e) amplifying the DNA,
- (f) cloning the amplified DNA in a vector;
- and
- (g) recovering the obtained clones.
A cDNA library comprising nucleotide sequences coding for a fragment of an immunoglobulin comprising two heavy polypeptide chains capable of recognizing and binding one or several antigens, said immunoglobulin containing a variable (VHH) region and a constant region, said constant region being devoid of a first domain (CH1), said immunoglobulin being devoid of light polypeptide chains, which fragment forms a determined antigen binding site, wherein said cDNA library is obtained by performing the following steps:
- (a) treating a sample containing lymphoid cells from a healthy animal selected among the Camelids in order to separate the lymphoid cells;
- (b) separating polyadenylated RNA from other nucleic acids and components of the B-lymphocytes;
- (c) reacting the obtained RNA with a reverse transcriptase in order to obtain the corresponding cDNA;
- (d) contacting the obtained cDNA with 5′ primers corresponding to mouse VH domain of four-chain immunoglobulins, which primer contains a determined restriction site, and with 3′ primers corresponding to the N-terminal part of a CH2 domain containing a KpnI site;
- (e) amplifying the DNA;
- (f) cloning the amplified DNA in a vector;
- and
- (g) recovering the obtained clones.
A cDNA library comprising nucleotide sequences coding for a fragment of an immunoglobulin, said immunoglobulin comprising two heavy polypeptide chains capable of recognizing and binding one or several antigens, said immunoglobulin containing a variable (VHH) region and a constant region, said constant region being devoid of a first domain (CH1), said immunoglobulin being devoid of light polypeptide chains, which fragment forms a determined antigen binding site, wherein said cDNA library is obtained by performing the following steps:
- (a) treating a sample containing lymphoid cells from a healthy animal selected among the Camelids in order to separate the lymphoid cells;
- (b) separating polyadenylated RNA from other nucleic acids and components of the B-lymphocytes;
- (c) reacting the obtained RAN with a reverse transcriptase in order to obtain the corresponding cDNA;
- (d) contacting the obtained cDNA with 5′ primers located in the promoter, leader or framework sequences of the VHH sequence of said heavy chain immunoglobulin, and with 3‘primers located in the ’ hinge, CH2, CH3, 3′ untranslated region or polyA tail;
- (e) cloning the amplified DNA in a vector;
- and
- (f) recovering the obtained clones.
A cDNA library comprising nucleotide sequences coding for a fragment of an immunoglobulin comprising two heavy polypeptide chains capable of recognizing and binding one or several antigens, said immunoglobulin containing a variable (VHH) region and a constant region, said constant region being devoid of a first domain (CH1), said immunoglobulin being devoid of light polypeptide chains, which fragment forms a determined antigen binding site, wherein said cDNA library is obtained by performing the following steps:
- (a) treating a sample containing lymphoid cells from a healthy animal selected among the Camelids in order to separate the lymphoid cells;
- (b) separating polyadenylated RNA from other nucleic acids and components of the B-lymphocytes;
- (c) reacting the obtained RNA with a reverse transcriptase in order to obtain the corresponding cDNA;
- (d) contacting the obtained cDNA with 5′ primers corresponding to mouse VH domain of four-chain immunoglobulins, which primer contains a determined restriction site, and with 3′ primers corresponding to the N-terminal part of a CH2 domain containing a KpnI site;
- (e) cloning the amplified DNA in a vector;
- and
- (f) recovering the obtained clones.
Dependencies are inferred from the claim text.