Bispecific fab fusion proteins comprising a cd3-binding fab fragment with n-terminal fusion to a binding domain and methods of use

Abstract

The present disclosure relates generally to multi-specific Fab fusion proteins (MSFP) which comprise an antibody Fab fragment with both N-termini fused to a fusion moiety (fusion moiety A or B). MSFP containing the Fab fragment exhibit significantly reduced binding ability of the Fab fragment to the Fab target. Binding of the Fab to its target is restored when the MSFP is clustered on a cell surface by binding of the fusion moieties to their target. The reduced binding of the Fab to its target, especially when presented on a cell surface in its native state, absent fusion moiety binding provides advantages such as: reduced side effects and allows desirable pharmacological effects of selectivity and specificity in a controlled manner.

1 . A bispecific Fab fusion protein comprising:
 (i) a Fab fragment that binds to the N-terminus of CD3 epsilon (“anti-CD3 Fab fragment”), wherein the anti-CD3 Fab fragment comprises a first chain comprising an immunoglobulin light chain variable (VL) domain and a second chain comprising an immunoglobulin heavy chain variable (VH) domain; and   (ii) a fusion moiety A linked to the N-terminus of the VL domain of the anti-CD3 Fab fragment via an optional first linker disposed between the C-terminus of the fusion moiety A and the N-terminus of the VL domain, or a fusion moiety B linked to the N-terminus of the VH domain of the anti-CD3 Fab fragment via an optional second linker disposed between the C-terminus of the fusion moiety B and the N-terminus of the VH domain;   wherein the fusion moiety A comprises a cell surface antigen binding domain comprising an antigen binding fragment, and the fusion moiety B comprises a cell surface antigen binding domain comprising an antigen binding fragment; and   wherein the VL domain of the anti-CD3 Fab fragment comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and the VH domain of the anti-CD3 Fab fragment comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25.   
     
     
         2 . The bispecific Fab fusion protein of  claim 1 , wherein the anti-CD3 Fab fragment binds to an epitope within amino acids 1-27 of CD3 epsilon. 
     
     
         3 - 9 . (canceled) 
     
     
         10 . The bispecific Fab fusion protein of  claim 1 , wherein each of the antigen binding fragment of the fusion moiety A and the fusion moiety B is an scFv. 
     
     
         11 . (canceled) 
     
     
         12 . The bispecific Fab fusion protein of  claim 1 , wherein the fusion moiety A and the fusion moiety B bind to a cell surface antigen selected from the group consisting of: FcγRIIb, CD28, CTLA-4, FAS, FGFR1, FGFR2, FGFR3, FGFR4, GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor 2, CEA, PSMA, BCMA, CAIX, cMet, EGFR1, Her2/neu, ErbB3, EpCAM, Folate receptor, Ephrin receptor, CD19, CD20, CD30, CD33, CD40, CD37, CD38, and CD138. 
     
     
         13 . (canceled) 
     
     
         14 . The bispecific Fab fusion protein of  claim 1 , wherein the fusion moiety A and the fusion moiety B are generated from phage display, yeast display, or a human antibody gene transgenic mouse. 
     
     
         15 . (canceled) 
     
     
         16 . The bispecific Fab fusion protein of  claim 1 , wherein the constant region of the light chain (CL region) of the anti-CD3 Fab fragment comprises a knob or hole mutation, and the heavy chain constant region 1 (CH1 region) of the anti-CD3 Fab fragment comprises a corresponding knob or hole mutation such that the CL region and the CH1 region stably interact. 
     
     
         17 . (canceled) 
     
     
         18 . Isolated polynucleotide or polynucleotides encoding the bispecific Fab fusion protein of  claim 1 . 
     
     
         19 . An isolated expression vector comprising the isolated polynucleotide or polynucleotides of  claim 18 . 
     
     
         20 . An isolated host cell comprising the vector of  claim 19 . 
     
     
         21 . A method of expressing a bispecific Fab fusion protein by culturing the host cell of  claim 20  under conditions in which the vector expresses the encoded bispecific Fab fusion protein. 
     
     
         22 . A pharmaceutical composition comprising the bispecific Fab fusion protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         23 . A method for treating a cancer in a subject, comprising administering an effective amount of the pharmaceutical composition of  claim 22  to the subject having the cancer, wherein the cancer expresses a cell surface antigen to which the bispecific Fab fusion protein can bind. 
     
     
         24 . The bispecific Fab fusion protein of  claim 1 , wherein the VH domain of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, 50, and 54; and/or wherein the VL domain of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 58, 62, 66, and 70. 
     
     
         25 . The bispecific Fab fusion protein of  claim 1 , wherein the moiety A and the fusion moiety B bind to EpCAM. 
     
     
         26 . The bispecific Fab fusion protein of  claim 1 , wherein the moiety A and the fusion moiety B bind to CD19. 
     
     
         27 . The bispecific Fab fusion protein of  claim 25 , wherein each antigen binding fragment of the fusion moiety A and the fusion moiety B:
 1) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 139, a CDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 141; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 142, a CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 144;   2) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 147; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 148, a CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 150;   3) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 78 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 78;   4) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 88 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 88;   5) comprises a VH domain comprising amino acid residues 1-116 of SEQ ID NO: 94 and a VL domain comprising amino acid residues 132-241 of SEQ ID NO: 94; or   6) comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 88, and 94.   
     
     
         28 . A bispecific Fab fusion protein comprising:
 (i) a Fab fragment that binds to the N-terminus of CD3 epsilon (“anti-CD3 Fab fragment”), wherein the anti-CD3 Fab fragment comprises a first chain comprising an immunoglobulin VL domain and a second chain comprising an immunoglobulin VH domain; and   (ii) a fusion moiety A linked to the VL domain of the anti-CD3 Fab fragment via an optional first linker disposed between the C-terminus of the fusion moiety A and the N-terminus of the VL domain, or a fusion moiety B linked to the VH domain of the anti-CD3 Fab fragment via an optional second linker disposed between the C-terminus of the fusion moiety B and the N-terminus of the VH domain;   wherein the fusion moiety A comprises an EpCAM-binding fragment, wherein the fusion moiety B comprises an EpCAM-binding fragment, and wherein each EpCAM-binding fragment of the fusion moiety A and the fusion moiety B:
 1) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 139, a CDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 141; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 142, a CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 144; 
 2) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 147; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 148, a CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 150; 
 3) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 78 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 78; 
 4) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 88 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 88; 
 5) comprises a VH domain comprising amino acid residues 1-116 of SEQ ID NO: 94 and a VL domain comprising amino acid residues 132-241 of SEQ ID NO: 94; or 
 6) comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 88, and 94. 
   
     
     
         29 . The bispecific Fab fusion protein of  claim 28 , wherein each EpCAM-binding fragment of the fusion moiety A and the fusion moiety B is an scFv. 
     
     
         30 . The bispecific Fab fusion protein of  claim 28 , wherein the anti-CD3 Fab fragment is humanized. 
     
     
         31 . A pharmaceutical composition comprising the bispecific Fab fusion protein of  claim 28  and a pharmaceutically acceptable carrier.