Binding molecules that modulate a biological activity expressed by a cell

Abstract

The invention provides means and methods for inhibiting a biological activity of cells. In one embodiment the invention is concerned with a method of inhibiting a biological activity in a first or second cell mediated by the binding of two membrane proteins that are binding partners for each other. The mentioned biological activity is inhibited by providing the cells with an antibody or antibody like molecule that can bind to each of the mentioned binding partners and the binding blocks the binding of the two binding partners thereby inhibiting the mentioned biological activity.

1 - 33 . (canceled) 
     
     
         34 . A method of inhibiting a biological activity in a first or second cell mediated by the binding of a member of the CD28 family (first membrane protein) on a first cell to a member of the B7 family (second membrane protein) on a second cell, wherein said first and second membrane protein are binding partners (i.e. a ligand and receptor pair), the method comprising:
 providing a system comprising said first and second cell with an antibody or a variant of said antibody that maintains the binding specificity of the antibody comprising a variable domain that can bind to an extracellular part of said first membrane protein and a variable domain that can bind to an extracellular part of said second membrane protein; and   incubating said system under conditions that are permissive for the first or second cell to express said biological activity mediated by the binding of said first membrane protein to said second membrane protein in the absence of said antibody or variant thereof;   wherein the binding of the variable domain that can bind to an extracellular part of said first membrane protein blocks the binding of said first membrane protein to said second membrane protein and/or the binding of the variable domain that can bind to an extracellular part of said second membrane protein blocks the binding of said first membrane protein to said second membrane protein.   
     
     
         35 . The method of  claim 34 , wherein the binding of the antibody or variant thereof reduces an inhibitory activity of the binding of the binding partners in said first cell. 
     
     
         36 . The method of  claim 34 , wherein said first membrane protein is Programmed Cell Death 1 protein (PD-1); cytotoxic T-lymphocyte-associated protein 4 (CTLA-4); B- and T-lymphocyte attenuator (BTLA); or Transmembrane And Immunoglobulin Domain Containing 2 (TMIGD2). 
     
     
         37 . The method of  claim 34 , wherein said second membrane protein is Programmed Cell Death 1 Ligand 1 protein (PD-L1); Programmed Cell Death 1 Ligand 2 protein (PD-L2); CD80; CD86; B7-H4; TNFRSF14; or B7-H7. 
     
     
         38 - 52 . (canceled) 
     
     
         53 . The method of  claim 37 , wherein the first membrane protein is PD-1 and the second membrane protein is PD-L1. 
     
     
         54 . The method of  claim 53 , wherein the binding of the variable domain that binds PD-1 blocks the binding of PD-1 to PD-L1 and/or the binding of the variable domain that binds PD-L1 blocks the binding of PD-L1 to PD-1. 
     
     
         55 . The method of  claim 53 , wherein the binding of the variable domain that binds PD-1 blocks the binding of PD-1 to PD-L2. 
     
     
         56 . The method of  claim 53 , wherein the binding of the variable domain that binds PD-L1 blocks the binding of PD-L1 to CD80. 
     
     
         57 . The method of  claim 53 , wherein the antibody or variant thereof has a stronger CD4+ T cell activation potential in a  Staphylococcus  enterotoxin B (SEB) assay as compared to an equimolar mix of:
 bivalent monospecific antibodies that comprise two of said variable domains that bind PD-1, and   bivalent monospecific antibodies that comprise two of said variable domains that bind PD-L1; and/or   is able to activate T cells in an antigen-specific CD4+ T cell assay more strongly than benchmark antibody 5C4 or benchmark antibody YW243.55.S70 or a combination of benchmark antibodies 5C4 and YW243.55.S70; and/or   has a stronger CD4+ T cell activation potential in a mixed lymphocyte reaction (MLR) assay as compared to benchmark antibody 5C4 or benchmark antibody YW243.55.S70.   
     
     
         58 . The method of  claim 53 , wherein the antibody or variant thereof comprises a variable domain that has a binding affinity for an extracellular part of PD-L1 with an equilibrium dissociation constant (K D ) of lower than or equal to 4.27 nM, preferably of lower than or equal to 1.31 nM, preferably of lower than or equal to 1.27 nM, as measured by SPR. 
     
     
         59 . The method of  claim 53 , wherein the antibody or variant thereof is capable of enhancing the proliferation of CD4+ and/or CD8+ tumor-infiltrating T cells; or
 is capable of inducing a stronger T cell mediated anti-tumor response in vivo as compared to a combination of benchmark antibodies MK-3475 and YW243.55.S70.   
     
     
         60 . The method of  claim 53 , wherein the variable domain that binds an extracellular part of PD-1 is defined as a variable domain that when in a bivalent monospecific antibody format that comprises two of said variable domains that bind PD-1, inhibits PD-1/PD-L1 mediated inhibition of T cell receptor mediated activation of a Jurkat cell in a range of 20-150% when compared to the inhibition obtained with the antibody Nivolumab on a Jurkat cell; and/or
 wherein the variable domain that binds an extracellular part of PD-L1 is defined as a variable domain that when in a bispecific antibody that has a second variable domain that binds an irrelevant antigen such as Tetanus Toxoid, provides the bispecific antibody with a Kd of 0.1-14 nM for PD-L1 binding (as measured by Biacore).   
     
     
         61 . The method of  claim 53 , wherein said variable domain that can bind to an extracellular part of PD-1 comprises a heavy chain variable region with a CDR3 region that comprises the amino acid sequence of the CDR3 of a heavy chain variable region of one of the VH depicted for MF6076 (SEQ ID NO: 44); MF6236 (SEQ ID NO: 45); MF6256 (SEQ ID NO: 46); MF6226 (SEQ ID NO: 48); MF6930 (SEQ ID NO: 47); MF6932 (SEQ ID NO: 43); MF6935 (SEQ ID NO: 42); MF6936 (SEQ ID NO: 41); MF6972 (SEQ ID NO: 40); MF6974 (SEQ ID NO: 39); MF6982 (SEQ ID NO: 38); MF6929 (SEQ ID NO: 56); MF7699 (SEQ ID NO: 50); MF7698 (SEQ ID NO: 51); MF7687 (SEQ ID NO: 52); MF7686 (SEQ ID NO: 53); MF7685 (SEQ ID NO: 54); or MF7684 (SEQ ID NO: 55);
 in particular comprises
 a heavy chain variable region with a CDR3 region that comprises the amino acid sequence of the CDR3 of a heavy chain variable region of one of the VH depicted for MF6974 (SEQ ID NO: 39), MF6076 (SEQ ID NO: 44) or MF7686 (SEQ ID NO: 53). 
   
     
     
         62 . The method of  claim 53 , wherein said variable domain that binds PD-1 comprises a heavy chain variable region that comprises the amino acid sequence of the CDR1, CDR2 and CDR3 of a heavy chain variable region of one of the VH depicted for MF6076 (SEQ ID NO: 44); MF6236 (SEQ ID NO: 45); MF6256 (SEQ ID NO: 46); MF6226 (SEQ ID NO: 48); MF6930 (SEQ ID NO: 47); MF6932 (SEQ ID NO: 43); MF6935 (SEQ ID NO: 42); MF6936 (SEQ ID NO: 41); MF6972 (SEQ ID NO: 40); MF6974 (SEQ ID NO: 39); MF6982 (SEQ ID NO: 38); MF6929 (SEQ ID NO: 56); MF7699 (SEQ ID NO: 50); MF7698 (SEQ ID NO: 51); MF7687 (SEQ ID NO: 52); MF7686 (SEQ ID NO: 53); MF7685 (SEQ ID NO: 54); or MF7684 (SEQ ID NO: 55);
 in particular comprises
 a heavy chain variable region that comprises the amino acid sequence of the CDR1, CDR2 and CDR3 of a heavy chain variable region of one of the VH depicted for MF6974 (SEQ ID NO: 39), MF6076 (SEQ ID NO: 44) or MF7686 (SEQ ID NO: 53). 
   
     
     
         63 . The method of  claim 62 , that comprises a variable domain that can bind to an extracellular part of PD-1 and comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region as depicted for MF6076 (SEQ ID NO: 44); MF6236 (SEQ ID NO: 45); MF6256 (SEQ ID NO: 46); MF6226 (SEQ ID NO: 48); MF6930 (SEQ ID NO: 47); MF6932 (SEQ ID NO: 43); MF6935 (SEQ ID NO: 42); MF6936 (SEQ ID NO: 41); MF6972 (SEQ ID NO: 40); MF6974 (SEQ ID NO: 39); MF6982 (SEQ ID NO: 38); MF6929 (SEQ ID NO: 56); MF7699 (SEQ ID NO: 50); MF7698 (SEQ ID NO: 51); MF7687 (SEQ ID NO: 52); MF7686 (SEQ ID NO: 53); MF7685 (SEQ ID NO: 54); or MF7684 (SEQ ID NO: 55), having at most 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably having 0, 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the amino acid sequence of the VH as depicted for the indicated MF;
 in particular comprises:
 a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region as depicted for MF6974 (SEQ ID NO: 39) or MF6076 (SEQ ID NO: 44) or MF7686 (SEQ ID NO: 53), or 
 a heavy chain variable region having a sequence that is at least 80% identical to the amino acid sequence of the heavy chain variable region as depicted for MF6974 (SEQ ID NO: 39) or MF6076 (SEQ ID NO: 44) or MF7686 (SEQ ID NO: 53). 
   
     
     
         64 . The method of  claim 53 , that comprises a variable domain that can bind to an extracellular part of PD-L1 and that comprises a heavy chain variable region with a CDR3 region that comprises the amino acid sequence of the CDR3 region of the heavy chain variable region as depicted for MF5359 (SEQ ID NO: 36); MF5361 (SEQ ID NO: 37); MF5377 (SEQ ID NO: 35); MF5382 (SEQ ID NO: 34); MF5424 (SEQ ID NO: 33); MF5426 (SEQ ID NO: 32); MF5439 (SEQ ID NO: 31); MF5442 (SEQ ID NO: 30); MF5553 (SEQ ID NO: 29); MF5557 (SEQ ID NO: 28); MF5561 (SEQ ID NO: 27); MF5576 (SEQ ID NO: 26); MF5594 (SEQ ID NO: 25); MF5708 (SEQ ID NO: 24); MF7691 (SEQ ID NO: 57); MF7690 (SEQ ID NO: 58); MF7689 (SEQ ID NO: 59); MF7688 (SEQ ID NO: 60); MF7700 (SEQ ID NO: 61); MF7701 (SEQ ID NO: 62); MF7703 (SEQ ID NO: 63); MF7694 (SEQ ID NO: 64); MF7693 (SEQ ID NO: 65); MF7692 (SEQ ID NO: 66); MF7697 (SEQ ID NO: 67); MF7696 (SEQ ID NO: 68); or MF7695 (SEQ ID NO: 69);
 in particular comprises:
 a heavy chain variable region with a CDR3 region of the heavy chain variable region as depicted for MF7689 (SEQ ID NO: 59) or MF7703 (SEQ ID NO: 63). 
 
 
     
     
         65 . The method of  claim 53 , wherein said variable domain that binds to an extracellular part of PD-L1 comprises a heavy chain variable region that comprises the amino acid sequence of the CDR1, CDR2 and CDR3 of a heavy chain variable region of one of the VH depicted for MF5359 (SEQ ID NO: 36); MF5361 (SEQ ID NO: 37); MF5377 (SEQ ID NO: 35); MF5382 (SEQ ID NO: 34); MF5424 (SEQ ID NO: 33); MF5426 (SEQ ID NO: 32); MF5439 (SEQ ID NO: 31); MF5442 (SEQ ID NO: 30); MF5553 (SEQ ID NO: 29); MF5557 (SEQ ID NO: 28); MF5561 (SEQ ID NO: 27); MF5576 (SEQ ID NO: 26); MF5594 (SEQ ID NO: 25); MF5708 (SEQ ID NO: 24); MF7691 (SEQ ID NO: 57); MF7690 (SEQ ID NO: 58); MF7689 (SEQ ID NO: 59); MF7688 (SEQ ID NO: 60); MF7700 (SEQ ID NO: 61); MF7701 (SEQ ID NO: 62); MF7703 (SEQ ID NO: 63); MF7694 (SEQ ID NO: 64); MF7693 (SEQ ID NO: 65); MF7692 (SEQ ID NO: 66); MF7697 (SEQ ID NO: 67); MF7696 (SEQ ID NO: 68); or MF7695 (SEQ ID NO: 69);
 in particular comprises:
 a heavy chain variable region that comprises the amino acid sequence of the CDR1 and CDR2 and CDR3 of a heavy chain variable region of one of the VH depicted for MF7689 (SEQ ID NO: 59) or MF7703 (SEQ ID NO: 63). 
   
     
     
         66 . The method of  claim 65 , that comprises a variable domain that can bind to an extracellular part of PD-L1 and comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region as depicted for MF5359 (SEQ ID NO: 36); MF5361 (SEQ ID NO: 37); MF5377 (SEQ ID NO: 35); MF5382 (SEQ ID NO: 34); MF5424 (SEQ ID NO: 33); MF5426 (SEQ ID NO: 32); MF5439 (SEQ ID NO: 31); MF5442 (SEQ ID NO: 30); MF5553 (SEQ ID NO: 29); MF5557 (SEQ ID NO: 28); MF5561 (SEQ ID NO: 27); MF5576 (SEQ ID NO: 26); MF5594 (SEQ ID NO: 25); MF5708 (SEQ ID NO: 24); MF7691 (SEQ ID NO: 57); MF7690 (SEQ ID NO: 58); MF7689 (SEQ ID NO: 59); MF7688 (SEQ ID NO: 60); MF7700 (SEQ ID NO: 61); MF7701 (SEQ ID NO: 62); MF7703 (SEQ ID NO: 63); MF7694 (SEQ ID NO: 64); MF7693 (SEQ ID NO: 65); MF7692 (SEQ ID NO: 66); MF7697 (SEQ ID NO: 67); MF7696 (SEQ ID NO: 68); or MF7695 (SEQ ID NO: 69), having at most 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably having 0, 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof with respect to the amino acid sequence of the indicated MF;
 in particular comprises:
 a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region as depicted for MF7689 (SEQ ID NO: 59) or MF7703 (SEQ ID NO: 63), or 
 a heavy chain variable region having a sequence that is at least 80% identical to the amino acid sequence of the heavy chain variable region as depicted for MF7689 (SEQ ID NO: 59) or MF7703 (SEQ ID NO: 63). 
   
     
     
         67 . The method of  claim 53 , which comprises a light chain variable region having the CDR1 (SEQ ID NO: 70), CDR2 and CDR3 (SEQ ID NO: 71) sequences of the light chain variable region according to SEQ ID NO: 6. 
     
     
         68 . The method of  claim 67 , which comprises a light chain variable region having a sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 6. 
     
     
         69 . The method of  claim 53 , wherein the antigen binding sites of said antibody consist of one immunoglobulin variable domain that can bind an extracellular part of PD-1 and one immunoglobulin variable domain that can bind an extracellular part of PD-L1 or PD-L2. 
     
     
         70 . The method of  claim 53 , wherein said antibody is a full length bispecific antibody. 
     
     
         71 . The method of  claim 53 , wherein said antibody is an IgG.