Recombinant Human IL-12 Exerts Differential Effects on Circulating CD56bright and CD56dim NK Cells in Healthy Human Subjects
Authors: Thomas Simmy , Lawrence Chris E , Mar Vernon , Kha Hue , Basile Lena A 
Published: October 2019

Abstract
Interleukin-12 (IL-12) has potent immunoregulatory and hematopoietic properties, and exerts significant biological effects on natural killer (NK) cells, inducing IFNγ production and enhancing cytotoxicity. Two distinct NK cell populations correlate with their immunoregulatory functions. Mature CD56dimCD16bright NK cells represent 90% of the NK cells resident in the blood and can exert cytotoxic effects on transformed cells. Cytokine producing immature CD56brightCD16+/- NK cells exist in the blood (10% of total circulating NK cells) but are most prominently located in secondary lymphoid tissues.
In the continued clinical development of recombinant human IL-12 (HemaMax™, rHuIL-12), to be used in combination with radiotherapy or chemotherapy for the treatment of cancer patients, we have performed a clinical safety study in healthy human subjects. A single subcutaneous (sc) dose of rHuIL-12 (12μg) was administered to 17 healthy human subjects. Placebo was administered to 5 healthy subjects. Peripheral blood samples were collected before rHuIL-12 administration, and up to Day 14 post administration. Immunophenotyping of blood cell populations was conducted by FACS.
rHuIL-12 caused a transient decrease in peripheral blood CD56dimCD16bright NK cells, with a nadir (60% reduction from baseline) reached on Day 2 following rHuIL-12 administration. CD56dimCD16bright NK cell levels returned almost to baseline levels on Day 5. Placebo was without effect. Conversely rHuIL-12 caused an elevation in peripheral blood CD56brightCD16+/- NK cells, particularly between Days 2 and 3 after rHuIL-12 administration, which was sustained until a peak was reached on Day 5 (265% above baseline). Levels returned to baseline by Day 11, while placebo was without effect. rHuIL-12 did not impact the less functional CD56-CD16bright NK cell subset. CD56dimCD16bright NK cells expressing the IL-12 receptor β2 subunit (IL-12Rβ2+) showed a substantial, and transient, decrease in levels on Day 2. The plasma concentration of IFNγ was elevated to a peak over 35 fold above baseline level at 10hr. after rHuIL-12 administration.
Human NK cells were negatively selected from highly enriched leukapheresis-derived blood and stimulated in vitro with 10 pM rHuIL-12. After 16hr. incubation these predominantly CD56dimCD16brightNK cells showed enhanced release of IFNγ and the increased killing of K562 cells, a human erythroleukemic cell line, when compared with vehicle controls. qPCR analysis of the human NK cell lysates showed rHuIL-12-induced elevation of CD56 (302%) and IL-12Rβ2 (587%) mRNA, when compared with vehicle controls. rHuIL-12 did not influence CD16 mRNA expression, but did increase the level of CD62L (L selectin, 206%) mRNA.
The rapid 60% fall in circulating mature CD56dimCD16bright NK cells after rHuIL-12 administration to healthy human subjects suggests their immediate exit from peripheral blood into the tissue compartments. This could be mediated by the observed increase in NK cell CD62L mRNA expression seen in vitro. The sustained increase in immature CD56brightCD16+/- NK cell levels between Day 3 and 6 suggests their IL-12-induced development from CD34+ hematopoietic progenitor cells. In summary rHuIL-12 administration to healthy human subjects demonstrates differential effects on the two key NK cell populations in peripheral blood, increasing CD56brightCD16+/- NK cell numbers, potentially stimulating IFNγ release from and enhancing the cytotoxicity of the CD56dimCD16bright NK cells, and preparing this population for migration into tissues. rHuIL-12 thus shows excellent potential as an immunotherapeutic and hematopoietic agent for the treatment of cancer patients, by impacting the maturation, activation, immunoregulation, and cytolytic properties of NK cells.

Disclosures
Thomas: Neumedicines: Employment, Equity Ownership. Lawrence:Neumedicines: Employment, Equity Ownership. Mar:Neumedicines: Employment, Equity Ownership. Kha:Neumedicines: Employment, Equity Ownership. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.