Bifunctional chimeric antibodies

Claim 2

A recombinant DNA compound comprising a first DNA sequence which encodes for the human carcino­embryonic antigen-specific light chain variable region of a chimeric monoclonal antibody and a second DNA sequence which encodes for the human carcinoembryonic antigen-specific heavy chain variable region of a chimeric monoclonal antibody, wherein the antibody light chain variable region amino acid sequence comprises: Asp - Ile - Val - Met - Thr - Gln - Ser - Gln - Lys Phe - Met - Ser - Thr - Ser - Val - Gly - Asp - Arg Val - Ser - Ile - Thr - Cys - Lys - Ala - Ser - Gln Asn - Val - Arg - Thr - Ala - Val - Ala - Trp - Tyr Gln - Gln - Lys - Pro - Gly - Gln - Ser - Pro - Lys Ala - Leu - Ile - Tyr - Leu - Ala - Ser - Asn - Arg Tyr - Thr - Gly - Val - Pro - Asp - Arg - Phe - Thr Gly - Ser - Gly - Ser - Gly - Thr - Asp - Phe - Thr Leu - Thr - Ile - Thr - Asn - Val - Gln - Ser - Glu Asp - Leu - Ala - Asp - Tyr - Phe - Cys - Leu - Gln His - Trp - Asn - Tyr - Pro - Leu - Thr - Phe - Gly Ala - Gly - Thr - Lys - Leu - Glu - Leu - Lys - Arg and wherein the antibody heavy chain variable region amino acid sequence comprises: Asp - Val - Gln - Leu - Val - Glu - Ser - Gly - Gly Gly - Leu - Val - Gln - Pro - Gly - Gly - Ser - Arg Lys - Leu - Ser - Cys - Ala - Ala - Ser - Gly - Phe Thr - Phe - Ser - Asn - Phe - Gly - Met - His - Trp Ile - Arg - Gln - Ala - Pro - Glu - Lys - Gly - Leu Glu - Trp - Val - Ala - Tyr - Ile - Ser - Gly - Gly Ser - Ser - Thr - Ile - Tyr - Tyr - Ala - Asp - Thr Val - Lys - Gly - Arg - Phe - Thr - Ile - Ser - Arg Asp - Asn - Pro - Arg - Asn - Thr - Leu - Phe - Leu Gln - Met - Thr - Ser - Leu - Arg - Ser - Glu - Asp Thr - Ala - Met - Phe - Tyr - Cys - Ala - Arg - Asp Tyr - Tyr - Ala - Asn - Asn - Tyr - Trp - Tyr - Phe Asp - Val - Trp - Gly - Ala - Gly - Thr - Thr - Val Thr - Val - Ser - Ser - Ala.

Claim 3

The recombinant DNA compound according to Claim 1 wherein the first DNA strand coding sequence comprises: GAC - ATT - GTG - ATG - ACC - CAG - TCT - CAA - AAA TTC - ATG - TCC - ACA - TCA - GTA - GGA - GAC - AGG GTC - AGC - ATC - ACC - TGC - AAG - GCC - AGT - CAG AAT - GTT - CGT - ACT - GCT - GTT - GCC - TGG - TAT CAA - CAG - AAA - CCA - GGG - CAG - TCT - CCT - AAA GCA - CTG - ATT - TAC - TTG - GCA - TCC - AAC - CGG TAC - ACT - GGA - GTC - CCT - GAT - CGC - TTC - ACA GGC - AGT - GGA - TCT - GGG - ACA - GAT - TTC - ACT CTC - ACC - ATT - ACC - AAT - GTG - CAA - TCT - GAA GAC - CTG - GCA - GAT - TAT - TTC - TGT - CTG - CAA CAT - TGG - AAT - TAT - CCG - CTC - ACG - TTC - GGT GCT - GGG - ACC - AAG - CTG - GAG - CTG - AAA - CGG and wherein the second DNA strand coding sequence comprises: GAT - GTG - CAG - CTG - GTG - GAG - TCT - GGG - GGA GGC - TTA - GTG - CAG - CCT - GGA - GGG - TCC - CGG AAA - CTC - TCC - TGT - GCA - GCC - TCT - GGA - TTC ACT - TTC - AGT - AAC - TTT - GGA - ATG - CAC - TGG ATT - CGT - CAG - GCT - CCA - GAG - AAG - GGA - CTG GAG - TGG - GTC - GCA - TAC - ATT - AGT - GGT - GGC AGT - AGT - ACC - ATC - TAC - TAT - GCA - GAC - ACA GTG - AAG - GGC - CGA - TTC - ACC - ATC - TCC - AGA GAC - AAT - CCC - AGG - AAC - ACC - CTC - TTC - CTG CAA - ATC - ACC - AGT - CTA - AGG - TCT - GAG - GAC ACG - GCC - ATG - TTT - TAC - TGT - GCA - AGA - GAT TAC - TAC - GCT - AAC - AAC - TAC - TGG - TAC - TTC GAT - GTC - TGG - GGC - GCA - GGG - ACC - ACG - GTC ACC - GTC - TCC - TCA - GCC.

Claim 4

The recombinant DNA compound according to Claim 1 or 2 wherein the DNA construct further comprises a third DNA strand sequence which codes for the light chain constant region of the chimeric monoclonal anti­body and a fourth DNA strand sequence which codes for the heavy chain constant region of the chimeric mono­clonal antibody.

Claim 5

The recombinant DNA compound according to any of Claims 1, 2 or 3 wherein the first and second DNA strand coding sequences are derived from a murine hybridoma.

Claim 6

The recombinant DNA compound according to Claim 4 wherein the murine hybridoma is CEM 231.6.7.

Claim 7

The recombinant DNA compound according to Claim 3 wherein the third and fourth DNA strand coding sequences are derived from the human lymphocyte.

Claim 8

The recombinant DNA compound of Claim 6 that is plasmid pNCEMKG1 as shown in Figure 5.

Claim 9

The recombinant DNA compound of Claim 6 that is plasmid pNCEMKG1(E-) as described in Example 6F.

Claim 10

A recombinant DNA compound comprising a first DNA sequence which encodes for the chelate-­specific light chain variable region of a chimeric monoclonal antibody and a second DNA sequence which encodes for the chelate-specific heavy chain variable region of a chimeric monoclonal antibody, wherein the antibody light chain variable region amino acid sequences comprises: Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Arg Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly and wherein the antibody heavy chain variable region amino acid sequence comprises: Glu Val Thr Leu Val Glu Ser Gly Gly Asp Ser Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Glu Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Thr Leu Ser Gly Gly Gly Phe Thr Phe Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Asn Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys Ala Ser His Arg Phe Val His Trp Gly His Gly Thr Leu Val Thr Val Ser Ala Ala.

Claim 11

A recombinant DNA compound comprising a first DNA sequence which encodes for the chelate-­specific light chain variable region of a chimeric monoclonal antibody and a second DNA sequence which encodes for the chelate-specific heavy chain variable region of a chimeric monoclonal antibody, wherein the antibody light chain variable region amino acid sequences comprises: Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Arg Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly and wherein the antibody heavy chain variable region amino acid sequence comprises: Glu Val Thr Leu Val Glu Ser Gly Gly Asp Ser Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Glu Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Thr Leu Ser Gly Gly Gly Phe Thr Phe Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Asn Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys Ala Ser His Arg Phe Val His Trp Gly His Gly Thr Leu Val Thr Val Ser Ala Ala.

Claim 12

The recombinant DNA compound according to Claim 8 wherein the first DNA strand coding sequence comprises: CAG GCT GTT GTG ACT CAG GAA TCT GCA CTC ACC ACA TCA CCT GGT GAA ACA GTC ACA CTC ACT TGT CGC TCA AGT ACT GGG GCT GTT ACA ACT AGT AAC TAT GCC AAC TGG GTC CAA GAA AAA CCA GAT CAT TTA TTC ACT GGT CTA ATA GGT GGT ACC AAT AAC CGG GCT CCA GGT GTT CCT GCC AGA TTC TCA GGC TCC CTG ATT GGA GAC AAG GCT GCC CTC ACC ATC ACA GGG GCA CAG ACT GAA GAT GAG GCA AGA TAT TTC TGT GCT CTA TGG TAC ACC AGC CTC TGG GTA TTC GGT GGA GGA ACC AAA CTG ACT GTC CTA GGG and wherein the second DNA strand coding sequence comprises: GAA GTG ACG CTG GTG GAG TCT GGG GGA GAC TCA GTG AAG CCT GGA GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA TTC ACT TTA AGT GGT GAA ACC ATG TCT TGG GTT CGC CAG ACT CCG GAG AAG AGG CTG GAG TGG GTC GCA ACC ACT CTT AGT GGT GGT GGT TTC ACC TTC TAT TCA GCC AGT GTG AAG GGT CGT TTC ACC ATC TCC AGA GAC AAT GCC CAG AAC AAC CTC TAT CTA CAA CTG AAT AGT CTG AGG TCT GAG GAC ACG GCC TTG TAT TTC TGT GCA AGT CAT CGG TTT GTT CAC TGG GGC CAC GGG ACT CTG GTC ACT GTC TCT GCA GCC.

Claim 13

The recombinant DNA compound according to Claim 1 or 2 wherein the DNA construct further com­prise a third DNA strand sequence which codes for the light chain constant region of the chimeric monoclonal antibody and a fourth DNA strand sequence which codes for the heavy chain constant region of the chimeric monoclonal antibody.

Claim 14

The recombinant DNA compound according to any of Claims 9, 10 or 11 wherein the first and second DNA strand coding sequences are derived from a murine hybridoma.

Claim 15

The recombinant DNA compound according to Claim 12 wherein the murine hybridoma is CHA 255.5.

Claim 16

The recombinant DNA compound according to Claim 11 wherein the third and fourth DNA strand coding sequences are derived from a human lymphocyte.

Claim 17

The recombinant DNA compound of Claim 14 that is plasmid pGCHAKG1 as shown in Figure 10.

Claim 18

The recombinant DNA compound of Claim 14 that is plasmid pGCHAKG1(E-) as described in Example 6G.

Claim 19

The recombinant DNA compound of Claim 14 that is plasmid pGCHAKG1-2 as described in Example 7B.

Claim 20

The recombinant DNA compound of Claim 14 that is plasmid pGCHAKG1-3 as described in Example 7B.

Claim 21

A transformed host cell which secretes bifunctional chimeric CEM/CHA.

Claim 22

A transformed host cell which secretes bifunctional chimeric CEM/CHA.

Claim 23

The host cell of Claim 19 that is SP2/0/pNCEMKG1/pGCHAKG1.

Claim 24

The host cell of Claim 19 that is SP2/0/pNCEMKG1/pGCHAKG1(E-).

Claim 25

The host cell of Claim 19 that is SP2/0/pNCEMKG1/pGCHAKG1-2.

Claim 26

The host cell of Claim 19 that is SP2/0/pNCEMKG1/pGCHAKG1-3.

Claim 27

The host cell of Claim 19 that is SP2/0/pNCEMKG1(E-)/pGCHAKG1.

Claim 28

The host cell of Claim 19 that is SP2/0/pNCEMKG1(E-)/pGCHAKG1(E-).

Claim 29

The host cell of Claim 19 that is SP2/0/pNCEMKG1(E-)/pGCHAKG1-2.

Claim 30

The host cell of Claim 19 that is SP2/0/pNCEMKG1(E-)/pGCHAKG1-3.

Claim 31

A bifunctional chimeric monoclonal anti­body wherein one light chain variable region and one corresponding heavy chain variable region comprise light and heavy chain variable regions which specifically recognize a first antigen and the second light chain variable region and second corresponding heavy chain variable region comprise light and heavy chain variable regions which specifically recognize a second and different antigen and wherein the constant regions of all light and heavy chains comprise human antibody constant regions.

Claim 32

A bifunctional chimeric monoclonal anti­body wherein one light chain variable region and one corresponding heavy chain variable region comprise light and heavy chain variable regions which specifically recognize a first antigen and the second light chain variable region and second corresponding heavy chain variable region comprise light and heavy chain variable regions which specifically recognize a second and different antigen and wherein the constant regions of all light and heavy chains comprise human antibody constant regions.

Claim 33

The bifunctional chimeric monoclonal anti­body of Claim 28 wherein one light chain variable region and one corresponding heavy chain variable region comprise light and heavy chain variable regions which specifically recognize human carcinoembryonic antigen and the second light chain variable region and second corresponding heavy chain variable region comprise light and heavy chain variable regions which specifically recognize metal chelates and wherein the constant regions of all light and heavy chains comprise human antibody constant regions.

Claim 34

The bifunctional chimeric monoclonal antibody of Claim 29 wherein the light chain variable region which recognize human carcinoembryonic antigen comprises the amino acid sequence: Asp - Ile - Val - Met - Thr - Gln - Ser - Gln - Lys Phe - Met - Ser - Thr - Ser - Val - Gly - Asp - Arg Val - Ser - Ile - Thr - Cys - Lys - Ala - Ser - Gln Asn - Val - Arg - Thr - Ala - Val - Ala - Trp - Tyr Gln - Gln - Lys - Pro - Gly - Gln - Ser - Pro - Lys Ala - Leu - Ile - Tyr - Leu - Ala - Ser - Asn - Arg Tyr - Thr - Gly - Val - Pro - Asp - Arg - Phe - Thr Gly - Ser - Gly - Ser - Gly - Thr - Asp - Phe - Thr Leu - Thr - Ile - Thr - Asn - Val - Gln - Ser - Glu Asp - Leu - Ala - Asp - Tyr - Phe - Cys - Leu - Gln His - Trp - Asn - Tyr - Pro - Leu - Thr - Phe - Gly Ala - Gly - Thr - Lys - Leu - Glu - Leu - Lys - Arg and heavy chain variable region which recognizes human carcinoembryonic antigen comprises the amino acid sequence: Asp - Val - Gln - Leu - Val - Glu - Ser - Gly - Gly Gly - Leu - Val - Gln - Pro - Gly - Gly - Ser - Arg Lys - Leu - Ser - Cys - Ala - Ala - Ser - Gly - Phe Thr - Phe - Ser - Asn - Phe - Gly - Met - His - Trp Ile - Arg - Gln - Ala - Pro - Glu - Lys - Gly - Leu Glu - Trp - Val - Ala - Tyr - Ile - Ser - Gly - Gly Ser - Ser - Thr - Ile - Tyr - Tyr - Ala - Asp - Thr Val - Lys - Gly - Arg - Phe - Thr - Ile - Ser - Arg Asp - Asn - Pro - Arg - Asn - Thr - Leu - Phe - Leu Gln - Met - Thr - Ser - Leu - Arg - Ser - Glu - Asp Thr - Ala - Met - Phe - Tyr - Cys - Ala - Arg - Asp Tyr - Tyr - Ala - Asn - Asn - Tyr - Trp - Tyr - Phe Asp - Val - Trp - Gly - Ala - Gly - Thr - Thr - Val Thr - Val - Ser - Ser - Ala the light chain variable region which recognizes metal chelates comprises the amino acid sequence: Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Arg Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly and the heavy chain variable region which recognizes metal chelates comprises the amino acid sequence: Glu Val Thr Leu Val Glu Ser Gly Gly Asp Ser Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Glu Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Thr Leu Ser Gly Gly Gly Phe Thr Phe Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Asn Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys Ala Ser His Arg Phe Val His Trp Gly His Gly Thr Leu Val Thr Val Ser Ala Ala.

Claim 35

The bifunctional chimeric monoclonal antibody of Claim 30 that is bifunctional chimeric CEM/CHA.