Humanized immunoglobulins and methods of making the same

A method of producing a humanized immunoglobulin, the method comprising:

• providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having hypervariable regions from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 108 M−1 and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:
• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs, or
• (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences;
• expressing the DNA segments in the cell to produce the humanized immunoglobulin.

A method of producing a humanized immunoglobulin having hypervariable regions and framework regions, the method comprising:

• providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy or light chain frameworks, and each of these said donor amino acids:
• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs;
• and
• expressing the DNA segments in the cell to produce the immunoglobulin.

A method of producing a pharmaceutical composition, comprising;

• formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition, wherein the humanized immunoglobulin was produced by:
• (1) comparing the sequence of a donor immunoglobulin heavy chain variable region against a collection of sequences of human heavy chain variable regions;
• (2) selecting a human heavy chain variable region from the collection of human heavy chain variable regions to provide an acceptor heavy chain variable region, wherein the selected variable region framework is at least 65% identical to the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering;
• (3) synthesizing a DNA segment encoding a humanized heavy chain variable region, comprising complementarity determining regions (CDRs) from the donor immunoglobulin heavy chain variable region and a variable region framework from the selected acceptor heavy chain variable region;
• (4) introducing the DNA segment encoding the humanized immunoglobulin heavy chain variable region and a DNA segment encoding a humanized immunoglobulin light chain variable region into a cell;
• and
• (5) expressing the DNA segments in the cell to produce the humanized immunoglobulin,
• wherein the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acid residues identical to those in the acceptor immunoglobulin heavy chain variable region framework.

A method of producing a pharmaceutical composition, comprising:

• formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition, wherein the humanized antibody has complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 108 M−1 and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:
• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs, or
• (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences,
• with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

A method of producing a pharmaceutical composition, comprising:

• formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition,
• wherein the humanized antibody has complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:
• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs,
• with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy or light chain frameworks, and each of these said donor amino acids:

• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs, or
• (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences,
• wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab′)2.

A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor heavy chain immunoglobulin framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:

• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs, or
• (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences,
• with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

A method of producing a humanized immunoglobulin, the method comprising:

• providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:
• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs, or
• (III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences;
• and
• expressing the DNA segments in the cell to produce the humanized immunoglobulin, with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

A method of producing a humanized immunoglobulin, the method comprising:

• providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 108 M−1 and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:
• (I) is adjacent to a CDR in the donor immunoglobulin sequence, or
• (II) is capable of interacting with the CDRs;
• and expressing the DNA segments in the cell to produce the immunoglobulin, with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).