Therapeutic efficacy of FcγRI/CD64-directed bispecific antibodies in B-cell lymphoma by Jamie Honeychurch, Alison L. Tutt, Thomas Valerius, Ingmar A. F. M. Heijnen, Jan G. J. Van de Winkel, and Martin J. Glennie Blood Volume 96(10):3544-3552 November 15, 2000 ©2000 by American Society of Hematology

Levels of huCD64 expression on PMN Levels of huCD64 expression on PMN.HuCD64+ mice were treated with murine G-CSF, 2 μg/d, subcutaneously, for 4 days. Levels of huCD64 expression on PMN.HuCD64+ mice were treated with murine G-CSF, 2 μg/d, subcutaneously, for 4 days. Levels of huCD64 on PMN were determined by FACS analysis of whole blood incubated with FITC-labeled M22 (anti-huCD64). The comparative expression levels on nontransgenic (A), unprimed (B), and primed (C) transgenic mice are shown. The level of staining given by a control irrelevant FITC-labeled IgG on PMN from Tg and nonTg was the same as that shown by curve A. The inset shows the scatter profiles, with the percentages of PMN (gated) in the total leukocyte population before and after the course of G-CSF. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

RCC of A31 and BCL1 using isolated murine PMN RCC of A31 and BCL1 using isolated murine PMN.Chromium-labeled target cells were mixed with BsAb of varying specificity (as indicated) at 1 μg/mL, and then murine PMN were added at an E:T ratio of 50:1. RCC of A31 and BCL1 using isolated murine PMN.Chromium-labeled target cells were mixed with BsAb of varying specificity (as indicated) at 1 μg/mL, and then murine PMN were added at an E:T ratio of 50:1. PMN isolated from huCD64 Tg animals either without (solid bars) or with G-CSF priming (hatched bars) were compared to PMN from huCD64-non Tg littermates, again either unprimed (open bars) or G-CSF treated (lined bars). BsAb were of the specificity indicated. Control BsAb contained the alternative, nonbinding anti-Id Fab' arm for each tumor (ie, anti-BCL1 for A31 and anti-A31 for BCL1) and were ineffective. Similar results were obtained in 2 separate experiments. All determinations were performed in triplicate. Error bars represent the SD of the triplicates. ■, nonTg −G-CSF; ▨, nonTg +G-CSF; ▪, Tg −G-CSF; ⊠, Tg +G-CSF. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

Immunotherapy of A31- or BCL1-bearing mice with BsAb directed against huCD64 on the effector cells and Id on the tumors.Groups of 5 huCD64+ age-matched mice received 2 μg/d murine G-CSF subcutaneously, 4 days before tumor inoculation, and throughout the cou... Immunotherapy of A31- or BCL1-bearing mice with BsAb directed against huCD64 on the effector cells and Id on the tumors.Groups of 5 huCD64+ age-matched mice received 2 μg/d murine G-CSF subcutaneously, 4 days before tumor inoculation, and throughout the course of therapy (10 days in total). Mice were inoculated with either 105 (BCL1) or 2 × 105 (A31) tumor cells intraperitoneally on day 0 and were treated with twice-daily doses of 5 μg BsAb intraperitoneally on days 1 to 5 (50 μg/mouse in total). (This treatment schedule is shown in the inset.) Treatment was as indicated: PBS (●), anti-huCD64 F(ab')2 (⋄), anti-Id F(ab')2 (■), a mixture of anti-Id and anti-huCD64 F(ab')2 (♦), [huCD64 × Id] (○). Symbol labeling on plots shows antibody specificity. Anti-Id Ab preparations were selected for reaction with A31 or BCL1 Id Ig, as appropriate. Survival was recorded daily. Only cohorts treated with [huCD64 × Id] BsAb showed a significant increase in survival over all control groups (P < .02). Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

Immunotherapy of lymphoma-bearing mice with BsAb directed to Id, MHC II, or CD19 on the tumors.Groups of 5 huCD64+ age-matched mice received a treatment schedule as shown in Figure 3. Immunotherapy of lymphoma-bearing mice with BsAb directed to Id, MHC II, or CD19 on the tumors.Groups of 5 huCD64+ age-matched mice received a treatment schedule as shown in Figure 3. Treatment was as indicated: PBS (●); [huCD64 × CD19] (▵); [huCD64 × MHC II] (■); [huCD64 × Id] (○). Survival was recorded daily. In the A31 model all treatment modalities resulted in a statistically significant increase in survival over PBS-treated controls (P < .02), though only [huCD64 × Id] BsAb–treated cohorts showed long-term survival. Similar results were obtained in at least 3 similar experiments. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

Only huCD64 Tg mice treated with G-CSF are protected by [huCD64 × Id] BsAb.Groups of 5 age-matched mice received a treatment schedule as shown in Figure 3 using BCL1 lymphoma cells. Only huCD64 Tg mice treated with G-CSF are protected by [huCD64 × Id] BsAb.Groups of 5 age-matched mice received a treatment schedule as shown in Figure 3 using BCL1 lymphoma cells. However, mice were either huCD64 Tg (c, d) or nonTg littermates (a, b) that were (b, d) or were not (a, c) given 10 days of recombinant G-CSF. Antibody-treatment was as indicated: PBS (●), anti-Id IgG (▵), [huCD64 × Id] (○). Survival was recorded daily. Similar results were obtained with the A31 model, but in this case the IgG anti-Id gave approximately 50 days protection in all groups (not shown). Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

Comparison of the immunotherapy achieved against BCL1 with anti-Id IgG and BsAb.Groups of 5 huCD64+ age-matched mice received the treatment schedule shown in Figure 3. Comparison of the immunotherapy achieved against BCL1 with anti-Id IgG and BsAb.Groups of 5 huCD64+ age-matched mice received the treatment schedule shown in Figure 3. However, mice were inoculated with 105 BCL1 tumor cells intraperitoneally on day 0 and were treated with twice-daily doses of (a) 0.1, (b) 1, (c) 10, or (d) 100 μg mAb or BsAb intraperitoneally on days 1 to 5 (either 1, 10, 100, or 1000 μg in total/mouse). Treatment was as indicated: PBS (●), anti-Id IgG (▵), or [huCD64 × Id] BsAb (○). Symbols on plots show antibody specificity. Survival was recorded daily, and similar results were obtained in at least 3 similar experiments. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

In vivo tracking of BCL1 tumor in peritoneal cavity In vivo tracking of BCL1 tumor in peritoneal cavity.HuCD64+ mice received 2 μg murine G-CSF/d subcutaneously 4 days before tumor and throughout the course of therapy. In vivo tracking of BCL1 tumor in peritoneal cavity.HuCD64+ mice received 2 μg murine G-CSF/d subcutaneously 4 days before tumor and throughout the course of therapy. Mice were then inoculated with 107BCL1 tumor cells intraperitoneally on day 0 and were treated with twice daily injections of 50 μg BsAb intraperitoneally on days 1 to 5 (500 μg/mouse total). One animal per group was killed each day of therapy, and the percentage tumor cells in the peritoneum was analyzed using 2-color flow cytometry with PE-labeled anti-Id and FITC-labeled anti-CD22. In mice treated with [huCD64 × Id] BsAb, tumor cells were detected using FITC-labeled anti-CD22 and PE-labeled anti-CD19 (see “Materials and methods”). The graph shows the percentage of tumor cells in the peritoneal cavity present on each day of therapy after treatment with PBS (●), [huCD64 × Id] BsAb (○), [huCD64 × MHC II] BsAb (■), or [huCD64 × CD19] BsAb (▵). Symbols on plots show BsAb specificity. The inset shows scatter profiles of tumor (gated) on day 5, from mice receiving control PBS (top) or anti-MHC II BsAb (bottom). Similar results were obtained in at least 2 similar experiments. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

In vivo tracking of BCL1 tumor in the spleen In vivo tracking of BCL1 tumor in the spleen.HuCD64+ mice received 2 μg murine G-CSF/d subcutaneously 4 days before tumor and continuing throughout the course of therapy (10 days in total). In vivo tracking of BCL1 tumor in the spleen.HuCD64+ mice received 2 μg murine G-CSF/d subcutaneously 4 days before tumor and continuing throughout the course of therapy (10 days in total). Mice were then inoculated with 107 BCL1 tumor cells intraperitoneally on day 0 and were treated with twice daily injections of 50 μg BsAb intraperitoneally on days 1 to 5 (500 μg/mouse in total). One animal per group was killed on days 5, 10, and 15, and the percentages tumor cells in spleen were determined using flow cytometry with PE-labeled anti-Id versus FITC-labeled anti-CD22. Shown are dot plots from a representative mouse at days 10 and 15, treated with BsAb of the specificities indicated. Control BsAb was the nonbinding [huCD64 × A31 Id]. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

Measurement of the levels of intracellular free calcium in lymphoma cells treated with BsAb.πBCL1 were loaded with Indo-1-am fluorescent dye, warmed to 37°C for 5 minutes, and then incubated with either BsAb alone (2 μg/mL) or BsAb (2 μg/mL) plus appropriat... Measurement of the levels of intracellular free calcium in lymphoma cells treated with BsAb.πBCL1 were loaded with Indo-1-am fluorescent dye, warmed to 37°C for 5 minutes, and then incubated with either BsAb alone (2 μg/mL) or BsAb (2 μg/mL) plus appropriate polyclonal anti-IgG antibody (20 μg/mL), and analyzed immediately using a FACS Vantage. Shown are plots of FL5/ FL4 versus time. An increase in the ratio of FL5/FL4 indicates an increase in the level of intracellular free calcium. BsAb specificity is as indicated. Control BsAb was the non-binding [huCD64 × A31 Id]. Similar results were obtained in at least 3 separate experiments. Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology

Therapeutic efficacy of [huCD64× Id] BsAb against BCL1 is dependent on CD4 and CD8 T cells. Therapeutic efficacy of [huCD64× Id] BsAb against BCL1 is dependent on CD4 and CD8 T cells. This figure represents a composite of 2 identical independent experiments. For each experiment, groups of 5 huCD64+, age-matched mice received 2 μg/d murine G-CSF subcutaneously 4 days before tumor inoculation and throughout the course of therapy (10 days in total). Mice were inoculated with 105 BCL1 tumor cells intraperitoneally on day 0 and were treated with twice-daily doses of 5 μg BsAb intraperitoneally on days 1 to 5 (50 μg/mouse in total). To deplete T cells, mice received either anti-CD8 (YTS169; 0.5 mg), anti-CD4 (GK1.5; 1 mg) or a mixture of both YTS169 (0.5 mg) and GK1.5 (1 mg) intraperitoneally 1 day before tumor and again on days 2 and 5 after the tumor inoculation. (The full treatment schedule is shown in the inset.) Treatments were as indicated: PBS (●), anti-CD4 + [huCD64 × Id] (▵), anti-CD8 + [huCD64 × Id] (▴), a mixture of anti-CD4 and anti-CD8 + [huCD64 × Id] (♦), control rat IgG + [huCD64 × Id] (○). Survival was recorded daily. Only cohorts treated without T-cell depletion and [huCD64 × Id] BsAb showed a significant increase in survival over all control groups (P ≤ .01). Jamie Honeychurch et al. Blood 2000;96:3544-3552 ©2000 by American Society of Hematology