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US-12624122-B2 - Affinity maturated TAG72 specific single chain antibodies

US12624122B2US 12624122 B2US12624122 B2US 12624122B2US-12624122-B2

Abstract

Disclosed are compositions and methods for targeted treatment of TAG-72-expressing cancers. In particular, affinity maturated single chain variable fragment (scFv) antibodies that bind TAG-72 on cancer cells are disclosed.

Inventors

  • Hatem Soliman

Assignees

  • H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC.

Dates

Publication Date
20260512
Application Date
20220328

Claims (14)

  1. 1 . A single chain variable fragment (scFv) TAG-72 antibody, comprising a variable heavy (V H ) domain having CDR1, CDR2 and CDR3 sequences and a variable light (V L ) domain having CDR1, CDR2 and CDR3 sequences, wherein the CDR1 sequence of the V H domain comprises the amino acid sequence DHAIH (SEQ ID NO:1), wherein the CDR2 sequence of the V H domain has the amino acid sequence WIGYFSPGNDDFRYNERFKG (SEQ ID NO:3), wherein the CDR3 sequence of the V H domain comprises the amino acid sequence LNMAY (SEQ ID NO:2), wherein the CDR1 sequence of the V L domain has the amino acid sequence KSSQSLLYSGNHKNYLA (SEQ ID NO:12), wherein the CDR2 sequence of the V L domain comprises the amino acid sequence WASARES (SEQ ID NO:10), and wherein the CDR3 sequence of the V_domain comprises the amino acid sequence QQYYSYPLT (SEQ ID NO:11).
  2. 2 . The antibody of claim 1 , wherein the V H domain comprises the amino acid sequence of SEQ ID NO:6.
  3. 3 . The antibody of claim 1 , wherein the V L domain comprises the amino acid sequence of SEQ ID NO: 13.
  4. 4 . The antibody of claim 1 , comprising a peptide linker between the V H and V L domains comprising the amino acid sequence of SEQ ID NO: 15.
  5. 5 . The antibody of claim 1 , comprising the amino acid sequence of SEQ ID NO: 16.
  6. 6 . A bispecific antibody comprising a single polypeptide chain comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region is capable of recruiting the activity of a human immune effector cell by specifically binding to an immune cell antigen located on the human immune effector cell, and wherein the second antigen-binding region comprises the antibody of claim 1 .
  7. 7 . The bispecific antibody of claim 6 , wherein the human effector cell is a member of the human lymphoid lineage.
  8. 8 . The bispecific antibody of claim 6 , wherein the effector cell is capable of exerting a cytotoxic or an apoptotic effect on a target cell.
  9. 9 . The bispecific antibody of claim 8 , wherein the immune cell antigen is selected from the group consisting of human CD3 antigen, the human CD16 antigen, the human NKG2D antigen, the human CD2 antigen, the human CD28 antigen and the human CD25 antigen.
  10. 10 . The bispecific antibody of claim 6 , wherein the human effector cell is a member of the human myeloid lineage.
  11. 11 . The bispecific antibody of claim 10 , wherein the human effector cell is capable of exerting a cytotoxic or an apoptotic effect on a target cell.
  12. 12 . The bispecific antibody of claim 11 , wherein the immune cell antigen is chosen from one or more of the human CD64 antigen or the human CD89 antigen.
  13. 13 . A pharmaceutical composition comprising the antibody of claim 1 in a pharmaceutically acceptable carrier.
  14. 14 . An isolated nucleic acid encoding the antibody of claim 1 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of copending application Ser. No. 16/318,589, filed Jan. 17, 2019, which is a National Stage of International Application No. PCT/US2017/045533, filed Aug. 4, 2017, which claims benefit of U.S. Provisional Application No. 62/371,018, filed Aug. 4, 2016, and Application No. 62/449,892, filed Jan. 24, 2017, which are hereby incorporated herein by reference in their entirety. SEQUENCE LISTING This application contains a sequence listing filed in electronic form as an ASCll.txt file entitled “320314-1011 Sequence Listing_ST25” created on Marcy 28, 2022, having 58,515 bytes. The content of the sequence listing is incorporated herein in its entirety. BACKGROUND Surgery, radiation therapy, and chemotherapy have been the standard accepted approaches for treatment of cancers including leukemia, solid tumors, and metastases. Immunotherapy (sometimes called biological therapy, biotherapy, or biological response modifier therapy), which uses the body's immune system, either directly or indirectly, to shrink or eradicate cancer has been studied for many years as an adjunct to conventional cancer therapy. It is believed that the human immune system is an untapped resource for cancer therapy and that effective treatment can be developed once the components of the immune system are properly harnessed. SUMMARY Disclosed herein are affinity maturated single chain variable fragment (scFv) antibodies that bind TAG-72 on cancer cells with higher affinity than prior TAG-72 scFv antibodies. In some cases, the bispecific antibody has a dissociation constant (KD) for TAG-72 that is less than 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, or 10 nM. The affinity/specificity of the binding construct is driven in large part by specific sequences within complementarity determining regions (CDRs) in the heavy (VH) and light (VL) chain. Each VH and VL sequence will have three CDRs (CDR1, CDR2, CDR3). In some embodiments, the TAG-72 VH domain comprises the following CDR domains: CDR1: DHAIH (SEQ ID NO:1), and CDR3: LNMAY (SEQ ID NO:2). In some cases, the CDR2 domain is selected from the group consisting of WIGYFSPGNDDFRYNERFKG (SEQ ID NO:3), WIGYFSPGNDDFKYNERYKG (SEQ ID NO:4), and WIGYFSPGNNDFKYNERFKG (SEQ ID NO:5). In some embodiments, the VH domain of the disclosed TAG-72 scFv antibodies comprises the amino acid sequence: QVQLX1X2SX3X4X5X6X7X8PX9X10X11X12X13X14X15CX16X17SGYTFTX18DHAIHWVX19Q X20PX21X22X23LWEX24GYX25SPX26NX27DX28X29YX30X31X32X33X34G35X36TX37X38X39D X40X41X42X43X44X45X46X47X48LX49SX50X51X52X53DX54AVYX55CX56RSX57X58X59X60X61WGQGXE62X63X64TVSS (SEQ ID NO:47), wherein at least one of X27=D or N, X29=K or R, X33=F or Y, or any combination thereof; and wherein X1=V or Q; X2=Q or E; X3=D or G; X4=A or P; X5=E or G; X6=L or V; X7=V or K; X8=K or R; X9=G or S; X10=A or Q; X11=S or T; X12=V or L; X13=K or S; X14=I, L or V; X15=S or T; X16=K or T; X17=A or V; X18=T or nothing; X19=K or R; X20=N, K, P, or A; X21=E or G; X22=Q or R; X23=G or R; X24=I or M; X25=F or I; X26=G or Q; X28=F or I; X30=N or S; X31=E or Q; X32=R or K; X34=K or Q; X35=K or R; X36=A or V; X37=L, M, or I; X38=T or L; X39=A, V, or R; X40=K or T; X41=S or P; X42=S, A, or K; X43=S or N; X44=T or Q; X45=A, V, or F; X46=Y or S; X47=V, M, or L; X48=Q, E, or R; X49=N or S; X50=L or V; X51=T, P, or R; X52=S or A; X53=E, N, or A; X54=S or T; X55=F or Y; X56=T, R, or A; X57=L, F, or Y; X58=N, Y, or S; X59=M or G; X60=A, N, D or H; X61=Y, S, or nothing; X62=T or S; X63=S, T, or L; and X64=V or L. Therefore, in some cases, the CDR2 domain comprises the amino acid sequence WX24G YX25SPX26NX27DX28X29YX30X31X32X33X34G (SEQ ID NO:23), wherein at least one of X27=D or N, X29=K or R, X33=F or Y, or any combination thereof; and wherein X24=I or M; X25; =F or I; X26=G or Q; X28=F or I; X30=N or S; X31=E or Q; X32=R or K; X34=K or Q. In some cases, the TAG-72 VH domain is selected from the group consisting of: (SEQ ID NO: 24)QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYF SPGNX1DFX2YNERX3KGKATLTADKSSSTAYVQLNSLTSEDSAVYFCTRSLNMAYW GQGTSVTVSS; (SEQ ID NO: 25)QVQLQQSDAELVKPGASVKISCKASGYTFTTDHAIHWVKQNPEQGLEWIGY FSPQNX1DFX2YNERX3KGKATLTADKSSSTAYVQLNSLTSNDSAVYFCTRSLNMAY WGQGTSVTVSS; (SEQ ID NO: 26)QVQLQQSDAELVKPGASVKISCKASGYTFTTDHAIHWVKQNPEQGLEWIGY ISPQNX1DIX2YNEKX3KGKATLTADKSSSTAYMQLNSLTSNDSAVYFCRRSFYGN- WGQGTTLTVSS; (SEQ ID NO: 27)QVQLQQSDAELVKPGASVKISCKASGYTFTTDHAIHWVKQKPEQGLEWIGY ISPQNX1DIX2YNEKX3KGKATLTADKPSNTVYMQLNSLTSNDSAVYFCTRSLSGDSW GQGTTLTVSS; (SEQ ID NO: 28)QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYF SPGNX1DFX2YNERX3KGKATLTADTSASTAYVELSSLPSEDTAVYFCTRSLNMAYW GQGTLVTVSS; (SEQ ID NO: 29)QVQLQESGPGLVRPSQTLSLTCTVSGYTFTDHAIHWVRQPPGRGLEWIGYI SPGNX1DIX2YNEKX3KGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARSYYGH- WGQGSLVTVSS; (SEQ ID NO: 30)QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYF SPGNX1DFX2YSQKX3QGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYW GQGTLVTVSS; (SEQ ID NO: 31)QVQLVQSGAEVVKPGASVKVSCKASGYTFTDHAIHWVRQNPGQRLEW