Method for selecting a single cell expressing a heterogeneous combination of antibodies

• US 7,919,257 B2
• Filed: 11/30/2005
• Issued: 04/05/2011
• Est. Priority Date: 05/30/2003
• Status: Active Grant

• Current Assignee: Merus N.V.
• Original Assignee: Merus Biopharmaceuticals B.V. I.O.
• Inventors: Hoogenboom, Hendricus Renerus Jacobus Mattheus, Logtenberg, Ton
• Primary Examiner(s): Peng; Bo
• Assistant Examiner(s): Blumel; Benjamin P
• Application Number:
  US11/292,414
• Publication Number:
  US 20060160184A1
• Time in Patent Office: 1,952 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/10 from RNA viruses
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/248 IL-6
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/64 : comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: G01N 33/56966 : Animal cells

Abstract

The present invention provides combinations of specific binding proteins, such as immunoglobulins, that are designed to be true combinations, essentially all components of the combination being functional and compatible with each other. The invention further provides a method for producing a composition comprising at least two different proteinaceous molecules comprising paired variable regions, the at least two proteinaceous molecules having different binding specificities, comprising paired variable regions, at least two proteinaceous molecules having different binding specificities, comprising contacting at least three different variable regions under conditions allowing for pairing of variable regions and harvesting essentially all proteinaceous molecules having binding specificities resulting from the pairing.

Claims

1. A method for selecting a single recombinant cell that expresses a heterogeneous combination of monospecific and bispecific antibodies, or antibody fragments thereof, wherein the monospecific and bispecific antibodies, or antibody fragments thereof, are human, humanized, or deimmunized, and wherein the heterogeneous combination has specific affinity for two target epitopes, said method comprising:

carrying out a process for producing the heterogeneous combination;

the process comprising

providing three different variable regions consisting of two heavy chain regions and one light chain variable region in recombinant cells, wherein antigen binding parts of the variable regions originate from an antibody from a single species and wherein one variable region is able to functionally pair with more than one other variable region, and under conditions allowing for pairing of variable regions and secretion of the paired regions from the recombinant cells resulting in the production of said heterogeneous combination, and

providing two target epitopes,

selecting a single recombinant cell from said recombinant cells that produces a heterogeneous combination that binds the two target epitopes, and

co-purifying the heterogeneous combination therefrom utilizing a shared feature of each of the monospecific and bispecific antibodies, or antibody fragments thereof.

2. The method according to claim 1, wherein said two target epitopes are associated with a disease and/or disorder.

3. The method according to claim 2, further comprising:

subjecting a heterogeneous combination to a biological assay indicative of an effect of the combination on the disease and/or disorder.

4. The method according to claim 1, wherein the one variable region able to functionally pair with more than one other variable region does not significantly contribute to the resulting binding specificity of the resulting paired regions.

5. The method according to claim 1, wherein each variable region can only pair with one other variable region.

6. The method according to claim 1, wherein two of the three variable regions are part of one single chain Fv.

7. The method according to claim 1, wherein the expression of two of the variable regions is under the direction of different control elements.

8. The method according to claim 7, wherein the different control elements lead to differential expression.

9. The method according to claim 8, wherein the differential expression is different in levels of expression and/or time of expression.

10. The method according to claim 1, wherein the combination comprises two monospecific antibodies produced in the single recombinant cell.

11. The method according to claim 1, further comprising

producing an expression system comprising nucleic acid sequences encoding variable regions;

said producing comprising;

synthesizing nucleic acid sequences encoding variable regions,

expressing said nucleic acid sequences and allowing the expression products to pair, and

selecting nucleic acid sequences encoding variable regions having desired pairing behavior, so as to produce nucleic acid sequences encoding variable regions.

12. A method for producing a heterogeneous combination of monospecific and bispecific antibodies, or antibody fragments thereof, the method comprising:

carrying out a selection method according to claim 1, and

allowing the selected cell to express the heterogeneous combination of monospecific and bispecific antibodies, or antibody fragments thereof, resulting in the production of said heterogeneous combination.

13. The method according to claim 1, wherein the shared feature is an Fc region.