Fig 1.
In vivo hCD38-hAtt treatment results in robust anti-tumor activity in xenograft tumor models of multiple myeloma.
Immunocompromised NCG mice were inoculated with A, ANBL-6, B, LP-1, C, MM1.S, or D, JJN-3 tumor cells, at cell numbers outlined in the Materials and Methods section. When tumors reached an average volume of ~ 100 mm3 (day 0), mice were randomized into treatment groups and treated via intraperitoneal injection with vehicle or 10 mg/kg hCD38-hAtt twice weekly for up to six doses. Tumor volumes were measured twice weekly until humane endpoint was reached. E, Tabulated data summary of CD38 and IFNAR2 expression (mean fluorescence intensity) of each tumor cell line, as measured by flow cytometry.
Fig 2.
NK cells, CD4 T cells, and CD8 T cells contribute to the anti-tumor activity of hCD38-mAtt and in extending overall survival of treated mice in the 38C13-hCD38 tumor model.
C3H (C3H/HeNCrl) mice bearing 38C13-hCD38 tumors were randomized into different treatment groups when tumors reached an average volume of ~ 100 mm3. One day prior to treatment (day -1), and every 7 days thereafter, mice were administered depleting αCD4, αCD8, or αAsialo GM-1 antibodies at doses shown to effectively and specifically deplete these cell populations [15–17]. On day 0, mice were administered 7 mg/kg hCD38-mAtt or PBS vehicle (equivalent final volume of 200 µ L) twice weekly by intraperitoneal injection for up to six doses. P-values were calculated using Log-rank (Mantel-Cox) test comparing each depletion treatment arm to vehicle or hCD38-mAtt, as indicated.
Fig 3.
Increased intra-tumoral infiltration and activation of immune cells driven by hCD38-mAtt.
C3H (C3H/HeNCrl) mice were inoculated with 38C13-hCD38 tumor cells and, when tumors reached an average volume of ~ 300 mm3, treated with vehicle (PBS, 200 µ L final volume) or treatment agent hCD38-mAtt on days 1 and 4. Tumors were collected at 2, 4, and 6 days post treatment initiation (day 0) from pre-specified groups of mice. The intra-tumoral numbers of A, NK cells, B, conventional CD4 T cells, and C, CD8 T cells as a percentage of live cells, was determined by flow cytometry. Gene expression of D, CD8α, E, granzyme B, and F, TGFβ1 was captured at day 6 post treatment in the tumor. Samples from each animal were run in triplicate. GzB, granzyme B.
Fig 4.
In vivo administration of mCD38-mAtt demonstrates differential anti-tumor activity across a range of syngeneic mouse tumor models.
Different strains of immunocompetent mice (see Materials and Methods) were inoculated with A, A20, B, B16F10, C, CT26, or D, MC38 tumor cells, and when tumors reached an average volume of ~ 100 mm3, mice were randomized into treatment groups and treated with a single intraperitoneal dose of 8 mg/kg mIgG1 isotype control, 2 mg/kg mAtt alone (i.e., no antibody), or 10 mg/kg mCD38-mAtt. These dose levels were equilibrated based on dosing the molar equivalence of antibody (mIgG1 isotype to mCD38-mAtt) or mIFNα6 (mAtt alone to mCD38-mAtt). Tumor volume was measured until humane endpoint was reached in the vehicle-treated groups. E, Tabulated summary documenting percent GRI, and statistical significance thereof, following treatment with mCD38-mAtt versus vehicle control for each tumor model. CI, confidence interval; mIFNα6, murine interferon alpha 6; mIgG1, murine immunoglobulin G1.
Fig 5.
Depletion of T cells or NK cells negatively impacts the anti-tumor efficacy of mCD38-mAtt.
Immunocompetent BALB/c mice were inoculated with A–C, A20, or D–F, CT26 cells and when tumors reached an average volume of ~ 100 mm3, mice were randomized into treatment groups and administered αAsialo GM-1- (A, D), αCD8- (B, E), or αCD4- (C, F) depleting antibodies to deplete specific immune cell subtypes, starting 1 day prior to drug treatment (day -1), and continuing weekly thereafter through the duration of the experiment. On day 0, mice were treated with a single dose of vehicle (PBS, 200 µ L final volume) or 10 mg/kg mCD38-mAtt and tumor volume was measured twice weekly until humane endpoint for vehicle control groups was reached.
Fig 6.
Treatment with mCD38-mAtt induces the proliferation and activation of CD8 T cells.
Immunocompetent BALB/c mice were inoculated with CT26 tumor cells, and once tumors reached an average volume of ~ 300 mm3, mice were administered vehicle or mCD38-mAtt, at 10 or 30 mg/kg. Six days post treatment initiation, mice were euthanized, and tumors were collected. Proliferation of CD8 T cells was measured by Ki67 staining A, expressed as a percentage of total tumor CD8 T cells. B, The ratio of CD8:Tregs within tumors was also assessed. C–E, Immunocompetent BALB/c mice were inoculated with CT26 tumor cells, and once tumors reached an average volume of ~ 300 mm3, mice were administered vehicle, 2 mg/kg mIFNα, or 10 mg/kg mCD38-mAtt, and tumors were harvested 7 days post treatment. C, Tumor antigen AH1-specific CD8 T cells were measured by dextramer staining and are expressed as a percentage of total tumor CD8+ T cells. D, Dissociated tumor cells were stimulated ex vivo with or without AH1 peptide. The frequency of AH1-specific cells producing granzyme B was assessed by subtracting the background (percentage of granzyme B+ cells without AH1 restimulation) from AH1-stimulated cells. Correlation between the number of granzyme B+ E, AH1-specific, or F, non-specific CD8 T cells with tumor mass at the time of take down. Linear regression was used to assess goodness of fit. GzB, granzyme B; mIFNα, murine interferon alpha.