# US 12,180,280 B2 · Antibodies binding human and monkey CD3 and uses thereof

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- Number: US 12,180,280 B2
- Document ID: US12180280B2
- Status: Granted
- Kind: B2
- Application number: 17/460,591
- Filed: 2021-08-30
- Published: 2022-06-23
- Granted: 2024-12-31
- Earliest claimed: 2020-12-23
- Assignees: Beijing Mabworks Biotech Co., Ltd.
- Applicants: Beijing Mabworks Biotech Co. Ltd.
- Inventors: Jiangmei Li; Fangjie Liu; Chunyang Jin; Yu Liu; Sijia Huang; Feng Li
- Web page: https://patentgrep.com/patent/US12180280B2

## CPC

- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- A61P 35/00: Antineoplastic agents
- C07K 16/2887: {against CD20}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/35: Valency
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/74: Inducing cell proliferation
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Disclosed is a monoclonal antibody that binds human and monkey CD3, or an antigen-binding portion thereof, as well as the use of the antibody or antigen-binding portion thereof in treatment of inflammatory diseases and in preparation of bispecific antibodies. A bispecific antibody against e.g., CD3 and CD20 comprising the antibody or antigen-binding portion thereof of the disclosure, and the use of the bispecific antibody in treatment of diseases such as cancers are also provided.

## Claims

### Claim 1

An isolated monoclonal antibody, or an antigen-binding portion thereof, capable of binding to CD3, comprising a heavy chain variable region comprising a VH CDR1 region, a VH CDR2 region and a VH CDR3 region, and a light chain variable region comprising a VL CDR1 region, a VL CDR2 region and a VL CDR3 region, wherein the VH CDR1 region comprises the amino acid sequence X1YAMN (SEQ ID NO: 1), wherein X1 is Thr (T),
the VH CDR2 region comprises the amino acid sequence RIRSKYNNYATYYAX1SV (SEQ ID NO:2), wherein X1 is Be (I),
the VH CDR3 region comprises the amino acid sequence HGNFGNSYX1SX2WAY (SEQ ID NO: 3), wherein X1 and X2 are Leu (L) and Tyr (Y), respectively,
the VL CDR1 region comprises the amino acid sequence X1SSTGAVTTX2NYAN (SEQ ID NO: 4), wherein X1 and X2 are Gln (Q) and Asn (N), respectively,
the VL CDR2 region comprises the amino acid sequence GTX1X2X3AP (SEQ ID NO: 5), wherein X1, X2 and X3 are Lys (K), Gln (Q) and Arg (R), respectively, and
the VL CDR3 region comprises the amino acid sequence X1LWYSNLWV (SEQ ID NO: 6), wherein X1 is Val (V).

### Claim 2 (depends on 1)

The isolated monoclonal antibody, or the antigen-binding portion thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% identity to EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYAISVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYLSYWAYW GQGTLVTVSA (SEQ ID NO: 8), EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYAISVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYLSYWAY WGQGTLVTVSS (SEQ ID NO: 9), or EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYAISVKDRFTISRDDSKNTLYLQMNSLRAEDTAMYYCVRHGNFGNSYLSYWAY WGQGTLVTVSS (SEQ ID NO: 10).

### Claim 3 (depends on 1)

The isolated monoclonal antibody, or the antigen-binding portion thereof of claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 95% identity to QAVVTQESALTTSPGETVTLTCQSSTGAVTTNNYANWVQEKPDHLFTGLIGGTKQRAPG VPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 16), QTVVTQEPSLTVSPGGTVTLTCQSSTGAVTTNNYANWVQQKPGQAPRALIGGTKQRAP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 17) or QAVVTQEPSLTVSPGGTVTLTCQSSTGAVTTNNYANWVQQKPGHAFRGLIGGTKQRAP GVPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNLWVFGGGTKLTVL(SEQ ID NO: 18).

### Claim 4 (depends on 2)

The isolated monoclonal antibody, or the antigen-binding portion thereof of claim 2, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 95% identity to
(1) EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYL SYWAYWGQGTLVTVSA (SEQ ID NO: 8) and QAVVTQESALTTSPGETVTLTCQSSTGAV TTNNYANWVQEKPDHLFTGLIGGTKQRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYF CVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 16), respectively;
(2) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 9) and QTVVTQEPSLTVSPGGTVTLTCQSSTGAV TTNNYANWVQQKPGQAPRALIGGTKQRAPGTPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 17), respectively;
(3) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 9) and QAVVTQEPSLTVSPGGTVTLTCQSSTGAV TTNNYANWVQQKPGHAFRGLIGGTKQRAPGVPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNLWVFGGGTKLTVL(SEQ ID NO: 18), respectively;
(4) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDDSKNTLYLQMNSLRAEDTAMYYCVRHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 10) and QTVVTQEPSLTVSPGGTVTLTCQSSTGA VTTNNYANWVQQKPGQAPRALIGGTKQRAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 17), respectively; or
(5) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDDSKNTLYLQMNSLRAEDTAMYYCVRHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 10) and QAVVTQEPSLTVSPGGTVTLTCQSSTGA VTTNNYANWVQQKPGHAFRGLIGGTKQRAPGVPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 18), respectively.

### Claim 5 (depends on 1)

The isolated monoclonal antibody, or the antigen-binding portion thereof of claim 1, comprising
i) a heavy chain constant region having the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEX1X2G GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YX3STYRVVSVLTVLHQDWLNGKEYKCKVSNKALX4APIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) wherein X1, X2, X3 and X4 are A, A, N and P, respectively; A, A, N and G, respectively; A, A, A and P, respectively; or A, A, A and G, respectively, linked to the heavy chain variable region, and
ii) a light chain constant region having the amino acid sequence GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO: 23) or GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT (SEQ ID NO: 32), linked to the light chain variable region.

### Claim 6 (depends on 1)

A bispecific antibody, comprising
i) an antigen binding domain against CD3, which comprises the isolated monoclonal antibody or the antigen binding portion thereof of claim 1, and
ii) an antigen binding domain against a disease associated antigen, wherein the disease associated antigen is a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen,
wherein the antigen binding domain against CD3 is linked to the antigen binding domain against a disease associated antigen.

### Claim 7 (depends on 6)

The bispecific antibody of claim 6, wherein the disease associated antigen is CD20 protein, and the antigen binding domain against CD20 protein comprises an antibody or an antigen-binding portion thereof that binds CD20 protein, wherein the antibody or antigen-binding portion thereof that binds CD20 protein comprises a heavy chain variable region and a light chain variable region.

### Claim 8 (depends on 7)

The bispecific antibody of claim 7, comprising one antigen binding domain against CD3 and two antigen binding domains against CD20 protein.

### Claim 9 (depends on 8)

The bispecific antibody of claim 8, comprising
i) a first polypeptide, comprising an anti-CD20 heavy chain variable region and a heavy chain constant region,
ii) a second polypeptide, comprising an anti-CD20 light chain variable region,
iii) a third polypeptide, comprising an anti-CD20 heavy chain variable region, an anti-CD20 light chain variable region, an anti-CD3 heavy chain variable region, and a heavy chain constant region, and
iv) a fourth polypeptide, comprising an anti-CD3 light chain variable region,
wherein the anti-CD20 heavy chain variable region in the first polypeptide and the anti-CD20 light chain variable region in the second polypeptide associate to form the antigen binding fragment against CD20 protein,
wherein the anti-CD20 heavy chain variable region and the anti-CD20 light chain variable region in the third polypeptide associate to form the antigen binding fragment against CD20 protein,
wherein the anti-CD3 heavy chain variable region in the third polypeptide and the anti-CD3 light chain variable region in the fourth polypeptide associate to form an antigen binding fragment against CD3,
wherein the heavy chain constant region in the first polypeptide and the heavy chain constant region in the third polypeptide are associated together.

### Claim 10 (depends on 9)

The bispecific antibody of claim 9, wherein the anti-CD20 heavy chain variable region contained in the first and third polypeptides comprises the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGD TDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS (SEQ ID NO: 26), and the anti-CD20 light chain variable region contained in the second and third polypeptides comprises the amino acid sequence DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK (SEQ ID NO: 27).

### Claim 11 (depends on 9)

The bispecific antibody of claim 9, wherein the heavy chain constant region in the first polypeptide comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33), and the heavy chain constant region in the third polypeptide comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34).

### Claim 12 (depends on 9)

The bispecific antibody of claim 9, wherein the first polypeptide comprises, from N terminus to C terminus, the anti-CD20 heavy chain variable region and the heavy chain constant region, and the third polypeptide comprises, from N terminus to C terminus, the anti-CD20 heavy chain variable region, the anti-CD20 light chain variable region, the anti-CD3 heavy chain variable region, and the heavy chain constant region; or
the first polypeptide comprises, from N terminus to C terminus, the anti-CD20 heavy chain variable region and the heavy chain constant region, and the third polypeptide comprises, from N terminus to C terminus, the anti-CD20 light chain variable region, the anti-CD20 heavy chain variable region, the anti-CD3 heavy chain variable region, and the heavy chain constant region.

### Claim 13 (depends on 12)

The bispecific antibody of claim 12, wherein the anti-CD20 heavy chain variable region is linked, via a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 28), to the anti-CD20 light chain variable region, and the anti-CD20 light chain variable region is linked, via a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 28), to the anti-CD3 heavy chain variable region.

### Claim 14 (depends on 13)

The bispecific antibody of claim 13, wherein the third polypeptide comprises the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGD TDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYW YLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLEL PYTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFNT YAMNWVRQAPGKGLEWVARIRSKYNNYATYYAISVKDRFTISRDNSKNTLYLQMNSL RAEDTAVYYCVKHGNFGNSYLSYWAYWGQGTLVTVSS (SEQ ID NO: 29) or QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGD TDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYW YLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLEL PYTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFNT YAMNWVRQAPGKGLEWVARIRSKYNNYATYYAISVKDRFTISRDDSKNTLYLQMNSL RAEDTAMYYCVRHGNFGNSYLSYWAYWGQGTLVTVSS (SEQ ID NO: 30).

### Claim 15 (depends on 9)

The bispecific antibody of claim 9, wherein the second polypeptide further comprises at the C terminus a light chain constant region comprising the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 31), and the fourth polypeptide further comprises at the C terminus a light chain constant region comprising the amino acid sequence GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT (SEQ ID NO: 32).

### Claim 16 (depends on 1)

A pharmaceutical composition comprising a pharmaceutically effective amount of the isolated monoclonal antibody or the antigen-binding portion thereof of claim 1, and a pharmaceutically acceptable carrier.

### Claim 17

An isolated monoclonal antibody, or an antigen-binding portion thereof, capable of binding to CD3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences
(1) EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYL SYWAYWGQGTLVTVSA (SEQ ID NO: 8) and QAVVTQESALTTSPGETVTLTCQSSTGAV TTNNYANWVQEKPDHLFTGLIGGTKQRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYF CVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 16), respectively;
(2) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 9) and QTVVTQEPSLTVSPGGTVTLTCQSSTGAV TTNNYANWVQQKPGQAPRALIGGTKQRAPGTPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 17), respectively;
(3) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 9) and QAVVTQEPSLTVSPGGTVTLTCQSSTGAV TTNNYANWVQQKPGHAFRGLIGGTKQRAPGVPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNLWVFGGGTKLTVL(SEQ ID NO: 18), respectively;
(4) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDDSKNTLYLQMNSLRAEDTAMYYCVRHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 10) and QTVVTQEPSLTVSPGGTVTLTCQSSTGA VTTNNYANWVQQKPGQAPRALIGGTKQRAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNLWVFGGGTKLTVL (SEQ ID NO: 17), respectively; or
(5) EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYAISVKDRFTISRDDSKNTLYLQMNSLRAEDTAMYYCVRHGNFGNSYL SYWAYWGQGTLVTVSS (SEQ ID NO: 10) and QAVVTQEPSLTVSPGGTVTLTCQSSTGA VTTNNYANWVQQKPGHAFRGLIGGTKQRAPGVPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNLWVFGGGTKLTVL(SEQ ID NO: 18), respectively.