Compositions and methods for NK-92 cells expressing native CD16
A recombinant NK-92 cell has a constitutive active promotor that effects expression of native CD16, and most preferably homogenous CD16 158V. Further contemplated recombinant NK-92 cells also include a recombinant nucleic acid that encodes an intracellularly retained interleukin (e.g., IL-2 or er-IL-2), wherein the recombinant NK-92 cell will secrete no more than 5,000 pg/mL IL-2 into a culture medium. The recombinant NK-92 cells presented herein have a significantly improved signal-to-noise ratio and exhibit reduced non-ADCC cytotoxicity.
1 . An isolated recombinant NK-92 cell that is natively heterozygous for CD16, comprising:
a native CD16 gene that is natively heterozygous for CD16, wherein the native CD16 gene has a CD16 158V allele and a CD16 158F allele;
a recombinant constitutive promotor sequence upstream of the native CD16 gene that is functionally in place of a native promotor sequence of the native CD16 gene;
wherein the recombinant promotor sequence is operably coupled to the native CD16 gene to thereby enable expression of the native CD16; and
wherein the recombinant promoter sequence leads to detectable expression of the CD16 158V allele, but not the 158F allele.
2 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant promotor sequence is a non-human promotor sequence.
3 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant promotor sequence is a spleen focus forming virus (SFFV) promotor sequence.
4 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant promotor sequence replaces an inactive native promotor sequence.
5 . The isolated recombinant NK-92 cell of claim 1 , wherein the NK-92 cell remain viable for at least 6 months and/or wherein the NK-92 cell express CD16 consistently for at least 6 months.
6 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant NK-92 cell further comprises a recombinant nucleic acid that encodes an intracellularly retained cytokine.
7 . The isolated recombinant NK-92 cell of claim 6 , wherein the intracellularly retained cytokine is an IL-2 or er-IL-2.
8 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant nucleic acid further encodes an antibiotic resistance gene and/or a reporter gene.
9 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant NK-92 cell has antibody-dependent cellular cytotoxicity (ADCC) against a target cell expressing an antigen, and wherein the antigen is bound by an antibody that is bound to the CD16 expressed on the surface of the recombinant NK-92 cell.
10 . The isolated recombinant NK-92 cell of claim 1 , wherein the recombinant NK-92 cell, compared to a non-recombinant NK-92 cell, has a reduced signal-to-noise ratio for ADCC and/or reduced non-antibody-dependent cellular cytotoxicity (ADCC) cytotoxicity as compared to a non-recombinant NK-92 cell.
11 . A method of testing an antibody, comprising:
providing a plurality of target cells expressing an antigen,
providing a plurality of recombinant NK-92 cells of claim 1 ;
combining the target cells and the recombinant NK-92 cells with an antibody that binds to the antigen; and
measuring ADCC against the target cells.
12 . The method of claim 11 , wherein the antibody is an IgG for therapy in human.
13 . The method of claim 11 , wherein a ratio of recombinant NK-92 cells to target cells is between 0.1:1 and 50:1.
14 . The method of claim 11 , wherein the method generates results that are more predictive than the same method using NK-92 cells expressing a transgene homozygous high-affinity CD16.
15 . A method of making a recombinant NK-92 cell, comprising:
providing a NK-92 cell;
introducing to the NK-92 cell a recombinant constitutive promotor sequence upstream of a gene encoding a native CD16 allele to thereby make the recombinant NK-92 cell;
wherein the step of introducing the recombinant promotor sequence comprises functional replacement of a native promotor sequence of the native CD16 allele by homologous recombination with the recombinant promotor sequence;
wherein the recombinant promotor sequence is operably coupled to the native CD16 allele to thereby enable expression of the native CD16 allele; and
wherein the recombinant promoter sequence leads to detectable expression of the CD16 158V allele, but not the 158F allele.
16 . The method of claim 15 , wherein the recombinant promotor sequence is a non-human promotor sequence.
17 . The method of claim 15 , wherein the recombinant promoter sequence is introduced to the NK-92 cell via electroporation.