The CD37-targeted antibody-drug conjugate IMGN529 is highly active against human CLL and in a novel CD37 transgenic murine leukemia model

Abstract

Therapeutic regimens for chronic lymphocytic leukemia (CLL) have increasingly utilized monoclonal antibodies since the chimeric anti-CD20 antibody rituximab was introduced. Despite improved clinical outcomes, current CLL therapies are not curative. Therefore, antibodies with greater efficacy and novel targets are desirable. One promising target is CD37, a tetraspanin protein highly expressed on malignant B-cells in CLL and non-Hodgkin lymphoma. Although several novel CD37-directed therapeutics are emerging, detailed preclinical evaluation of these agents is limited by lack of appropriate animal models with spontaneous leukemia expressing the human CD37 (hCD37) target. To address this, we generated a murine CLL model that develops transplantable hCD37+ leukemia. Subsequently, we engrafted healthy mice with this leukemia to evaluate IMGN529, a novel hCD37-targeting antibody-drug conjugate. IMGN529 rapidly eliminated peripheral blood leukemia and improved overall survival. In contrast, the antibody component of IMGN529 could not alter disease course despite exhibiting substantial in vitro cytotoxicity. Furthermore, IMGN529 is directly cytotoxic to human CLL in vitro, depletes B-cells in patient whole blood and promotes killing by macrophages and natural killer cells. Our results demonstrate the utility of a novel mouse model for evaluating anti-human CD37 therapeutics and highlight the potential of IMGN529 for treatment of CLL and other CD37-positive B-cell malignancies.

Figure 1. The anti-CD37 antibody-drug conjugate IMGN529 demonstrates in vitro activity against neoplastic B-cells from human CLL patients

(A) Viability of freshly isolated CLL patient B-cells (n=16) following 24 hour treatment with 10 μg/ml IMGN529 or its antibody component K7153A +/− 50 μg/ml crosslinking antibody (αFc). Anti-HER2/neu antibody trastuzumab included as an additional negative control for n=11 patients. Data are normalized to untreated controls. Significance is indicated by asterisks (*p<0.05, ***p<0.0001, or NS if p>0.05).(B) NK cell mediated antibody-dependent cytotoxicity as measured by ⁵¹Cr release assay. CLL target cells (n=7) were incubated with 10 μg/ml antibody for 30 minutes, followed by incubation with healthy donor NK cells (n=8) for 4 hours. Mean specific lysis displayed for n=18 NK/CLL combinations with error bars indicating standard error of the mean (SEM). (C) Induced phagocytosis of CLL cells by monocyte-derived macrophages (MDMs) following 1 hour incubation with 10 μg/ml antibody. Relative phagocytosis displayed for a total of n=10 MDM/CLL combinations (7 MDMs, 5 CLL) with error bars indicating SEM. (D) Complement-dependent cytotoxicity assay for B-CLL incubated with 10 μg/ml antibody and autologous plasma (+/− heat inactivation) for 1 hour. Mean values displayed for n=9 CLL patients with error bars indicating SEM. (E,F) CLL patient whole blood was treated with 10 μg/ml antibody for 1 hour at 37°C. B-cells and T-cells were stained with anti-CD19 and CD3 antibodies respectively. Counts were obtained using CountBright absolute counting beads and normalized to untreated control. Mean percent depletion of B-cells (E) and T-cells (F) are displayed for n=23 CLL patients with error bars indicating SEM. Greater than 97% of the CD19+ cells in these patients were confirmed to be malignant CD5+ B-cells.

Figure 2. IMGN529 exhibits superior cytotoxicity compared to K7153A and induces mitotic arrest in a proliferating B-cell line in vitro

(A) Raji cells were treated with 1 μg/ml K7153A, IMGN529, or relevant controls for 72 hours and viability assayed by Annexin V and propidium iodide staining. Data are normalized to untreated control and reported as mean with error bars indicating SEM for n=5 replicate cultures from two independent experiments. (B) Cell cycle analysis of Raji cells that were synchronized with the DNA polymerase inhibitor aphidicolin for 24 hours, released from this inhibition, and then treated with 1 μg/ml antibody for 24 hours.

Figure 3. Generation and characterization of the human CD37 transgenic mouse

(A) Schematic representation of the construct used to generate the hCD37-Tg mouse. (B) PCR genotyping of founder lines with human CD37 specific primers. (C) Surface expression of human CD37 on transgenic (TG; top) and non-transgenic (NTG; bottom) splenocytes. Cells stained for B220 and CD3 to analyze B and T lymphocytes, respectively. (D) Surface expression of human CD37 on transgenic (TG; top) and non-transgenic (NTG; bottom) peripheral blood leukocytes. Whole blood stained for CD19, CD4, CD8, and human CD37. After staining, RBCs were lysed and samples analyzed by flow cytometry. (E) Example of flow cytometry performed on purified splenic B-cells stained with Annexin V and propidium iodide to assess viability after 24 hour treatment with 10 μg/ml anti-CD37 antibody (K7153A) in the presence of 50 μg/ml goat anti-human Fc antibody (αFc) for crosslinking. (F) Average viability of purified B-cells from transgenic or non-transgenic spleens (n=7/group) after 24 hour treatment with 10 μg/ml anti-CD37 antibody (K7153A) in the presence of 50 μg/ml goat anti-human Fc antibody (αFc) for crosslinking. Mean percentage of viable cells is displayed with error bars indicating SEM.

Figure 4. CD37xTCL1 double transgenic mice retain transgene expression and develop hCD37+CD19+CD5+ leukemia

(A) Flow cytometry performed on peripheral blood from a CD37xTCL1 double transgenic mouse and a TCL1 littermate. Cells were stained with antibodies against mouse B220 to label B-cells and human CD37. (B) Western blot using protein lysates from purified B-cells obtained from spleens of CD37xTCL1 mice or TCL1 littermates. Western blot is probed with anti-human TCL1 antibody. Lysates from C57BL/6 and leukemic TCL1 mice are included as controls in the first two lanes. (C) Flow cytometry performed on samples obtained from peripheral blood, spleen, and lymph nodes of a leukemic CD37xTCL1 mouse. Cells were stained for CD5, CD19, and human CD37. The right side of the panel demonstrates staining with K7153A-PE antibody and is gated on either CD5+CD19+ B-cells (dark gray histogram) or hCD37-negative T-cells (light gray histogram).

Figure 5. IMGN529 demonstrates in vivo efficacy against hCD37+ mouse leukemia

(A) Schematic outline of the hCD37×TCL1 engraftment experiment. Healthy hCD37-Tg recipients were injected i.v. with 1×10⁷ splenocytes from a leukemic hCD37×TCL1 donor. Mice were randomly assigned (n=6-7/group) for treatment with 10 mg/kg IMGN529 ADC, its K7153A antibody component, IgG-DM1 ADC control, or trastuzumab control. Treatment began following leukemia development, which was defined as when >20% of CD45+ events were CD5+CD19+ B-cells. Animals received a 10 mg/kg i.p. treatment on the day of leukemia diagnosis and repeat doses twice weekly for three weeks (70 mg/kg total). (B) Kaplan-Meier plot comparing the overall survival of engrafted mice. IMGN529 significantly improved survival relative to its IgG-DM1 control (p=0.042), while parent antibody K7153A had no survival benefit compared with trastuzumab control (p=0.94). Log-rank tests were used for statistical analysis. Arrow indicates day 20, when mice received their last injection. (C) Representative examples of flow cytometry performed on mice 24h after receiving their third dose. Whole blood was stained for CD45/CD19/CD5 and plots are gated on singlet CD45+ events. (D) Absolute concentration of the CD5+CD19+ B-cell population to which the leukemia belongs. Data expressed as mean +/− SEM. Arrow indicates day 20, when mice received their last injection. (E) Concentration of T-cells in the IMGN529 treated group. Mean T-cell concentration for n=6 mice is displayed with error bars indicating SEM.

Figure 6. IMGN529 targets proliferating mouse leukemia cells within lymphoid tissues

(A-E) Healthy hCD37-Tg mice engrafted with hCD37×TCL1 leukemia. Upon leukemia development (day 0), mice were randomized (n=5 mice/group) to receive 10 mg/kg i.p. doses of IMGN529 or IgG-DM1 control on days 3 and 5. To detect in vivo proliferation 100 μg EdU was administered on day 6 and mice were euthanized on day 7. (Mean is displayed for n=5 mice/group with error bars indicating SEM. ***p<0.0001, **p<0.01, *p<0.05) (A) Percentage CD5+CD19+ leukemia cells (of total CD45+ events) in peripheral blood on day 0 and day 7. (***p<0.0001 for change in cell percentage). (B) Percentage CD5+CD19+ leukemia cells (of total CD45+ events) in the blood, spleen, and bone marrow on day 7. (C) Examples of flow cytometry performed on peripheral blood, spleen, and bone marrow from mice treated with IMGN529 or IgG-DM1. Plots are gated on singlet, live, CD45+ cells. (D) Examples of spleens removed from mice receiving IMGN529 or IgG-DM1 (left panel) and average spleen length of all mice in the study (n=5/group; right panel). (E) Percentage of EdU+ cells among CD5+CD19+ events in the spleen and bone marrow.