METHOD FOR SELECTING A SINGLE CELL EXPRESSING A HETEROGENEOUS COMBINATION OF ANTIBODIES

• US 20160238600A1
• Filed: 04/27/2016
• Published: 08/18/2016
• Est. Priority Date: 05/30/2003
• Status: Active Grant

• Current Assignee: Merus N.V.
• Original Assignee: Merus BV (Merus N.V.)
• Inventors: Hoogenboom, Hendricus Renerus, Logtenberg, Ton, Throsby, Mark, Kramer, Robert A., Pinto, Rui Daniel, de Kruif, Cornelis A., Houtzager, Erwin
• Granted Patent: US 10,605,808 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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

Described are 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. Further provided are methods 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, the 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 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,

providing two target epitopes, and

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

wherein the one light chain variable region comprises a rearranged human immunoglobulin light chain variable region consisting essentially of a germline V and a germline J and encoded by a human immunoglobulin light chain V gene segment joined to a human immunoglobulin light chain J gene segment.

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 polynucleotides encoding variable regions, said producing comprising;

synthesizing polynucleotides encoding variable regions,

expressing said polynucleotides and allowing the expression products to pair, and

selecting polynucleotides encoding variable regions having desired pairing behavior, so as to produce polynucleotides encoding variable regions.

12. The method according to claim 1, further comprising:

expressing the heterogeneous combination of monospecific and bispecific antibodies, or antibody fragments thereof, resulting in the production of said heterogeneous combination in the single recombinant cell.

13. The method according to claim 1, wherein the human immunoglobulin light chain V gene segment joined to the human immunoglobulin light chain J gene segment is obtained from a transgenic mouse.

14. The method according to claim 13, wherein the three different variable regions consisting of two heavy chain regions and one light chain variable region are encoded by a transgenic mouse.

15. The method according to claim 14, wherein the genome of the transgenic mouse comprises a transgene comprising the human immunoglobulin light chain V gene segment joined to the human immunoglobulin light chain J gene segment,

wherein the joined human V/J gene segments encode the rearranged human immunoglobulin light chain variable region, and

wherein the transgenic mouse, in response to the two target epitopes, produces antibodies with immunoglobulin light chains comprising the rearranged human light chain variable region and a murine light chain constant region, paired with a diversity of immunoglobulin heavy chains which bind the two target epitopes.

16. The method according to claim 1, wherein a first heavy chain of the two heavy chain regions is obtained by immunizing a first mouse with a first target epitope of the two target epitopes and wherein a second heavy chain of the two heavy chain regions is obtained by immunizing a second mouse with a second target epitope of the two target epitopes.

17. The method according to claim 1, wherein the three different variable regions consisting of two heavy chain regions and one light chain variable region are encoded by a transgenic mouse.

18. The method according to claim 17,

wherein the genome of the transgenic mouse comprises a transgene comprising the human immunoglobulin light chain V gene segment joined to the human immunoglobulin light chain J gene segment,

wherein the joined human V/J gene segments encode the rearranged human immunoglobulin light chain variable region, and

wherein the transgenic mouse, in response to the two target epitopes, produces antibodies with immunoglobulin light chains comprising the rearranged human light chain variable region and a murine light chain constant region, paired with a diversity of immunoglobulin heavy chains which bind the two target epitopes.

19. 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, the method comprising:

producing a set of antibody combinations from a set of recombinant cells by a process comprising;

pairing, in the set of recombinant cells, three different variable regions consisting of two heavy chain variable regions, and

one light chain variable region comprising a rearranged human immunoglobulin light chain variable region encoded by a human immunoglobulin light chain V gene segment joined to a human immunoglobulin light chain J gene segment,

wherein antigen binding parts of the variable regions originate from a single species and

wherein one variable region is able to functionally pair with more than one other variable region, and

secreting the paired regions from the recombinant cells to produce an antibody combination,

testing the set antibody combinations to identity a single heterogeneous combination that has specific affinity for the two target epitopes, and

isolating from the set of recombinant cells a single recombinant cell that produces the single heterogeneous combination.