Re-evaluation of CD37 as target for of Non-Hodgkin Jostein Dahle1, Ada Repetto1, Camilla Mollatt2, Katrine B. Melhus2, Øyvind S. Bruland3,4, Arne Kolstad4 and Roy H. Larsen5

1Nordic Nanovector AS, Kjelsåsveien 168 B, 0884 Oslo, Norway, 2Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway. 3Insititute for Clinical Medicine, University of Oslo. 4Department of Oncology, The Norwegian Radium Hospital, Oslo, Norway. 5Sciencons Ltd., Oslo, Norway.

NaCl NaCl Internalization of in Daudi cells 96 h incubation after wash Introduction 20 8 tetulomab tetulomab tetulomab The monoclonal anti-CD20 rituximab alone or as part of combination therapy, is rituximab rituximab 1,0 50 Mbq/kg 177Lu-tetulomab 177 100 Mbq/kg 177Lu-tetulomab 50 MBq/kg Lu-tetraxetan-tetulomab 15 6 considered standard therapy for non-Hodgkin . However, significantly better 100 MBq/kg 177Lu-tetraxetan-tetulomab A 0,8 clinical results have been obtained for beta-emitting anti –CD20 radioimmunoconjugates 7 10 4 /L) 9 (RICs) than for rituximab. RICs targeting CD20 may be problematic because of antigenic 6 0,6 5 2 drift and antigen blocking caused by previous treatments with rituximab. Therefore, novel 5 fgtetulomab/cell

therapeutic approaches targeting other antigens might be more effective after Survival 4 0,4

Internalized (fg/cell) antibody 0 0 20 40 60 80 100 120 140 160 180 200 0 3 111In-tetulomab 125I-tetulomab rituximab treatment failure than a new anti-CD20 treatment. In the present study, we have Time after start of incubation (min) 177 2 compared the therapeutic effect of the novel anti-CD37 RIC Lu-tetraxetan-tetulomab 0,2 Four days after washing there was almost three 1 177 (10 Number WBC of times more 111In-tetulomab than 125I-tetulomab with the anti-CD20 RIC Lu-tetraxetan-rituximab as well as the cold antibodies. D The tetulomab antibody was internalized 0,0 0 associated to Daudi cells. The result indicate that faster and to a larger extent than the 0 50 100 150 200 -2 0 2 4 6 8 10 12 14 metallic nuclides like 111In are retained inside the rituximab antibody. Weeks after injection cells to a larger extent than halogen nuclides. Time after injection (days) NaCl tetulomab The mean number of antigens (B ) on Daudi cells, the equilibrium rituximab max 1,0 50 MBq/kg 177Lu-tetraxetan-tetulomab dissociation constant (Kd) and the association rate constant (ka) for the B 177 antibodies HH1 and rituximab. The affinity of HH1 to CD37 was similar to the 100 MBq/kg Lu-HH1 affinity of rituximab to CD20. 0,8

-1 -1 2000 2 h incubation 18 h incubation Antibody Bmax(Ag/cell) Kd (nM) ka (nM h ) /L)

0,6 NaCl 9 Cell bound activity Cell bound activity 20000 tetulomab 340 000 ± 5 000 2.7 ± 0.3 0.72 ± 0.07 tetulomab 1500 35000 rituximab tetulomab tetulomab 50 MBq/kg 177Lu-tetulomab 30000 tetulomab, blocked tetulomab, blocked rituximab 307 000 ± 12 000 5.8 ± 1.5 0.14 ± 0.01 0,4 Survival (%) 100 MBq/kg 177Lu-tetulomab rituximab 15000 rituximab 1000 25000 rituximab, blocked rituximab, blocked

20000 0,2 Platelets (10 10000 Lymphoma biopsies tested for CD37 expression. 500 Lu atoms per cell per atoms Lu Lu atoms per cell per atoms Lu

15000 177 177 Diagnosis Number of samples % CD37 + E 10000 5000 0,0 0

5000 0 50 100 150 200 -2 0 2 4 6 8 10 Number of

Number of B-lymphoblastic lymphoma 1 100,0 0 Days after start of treatment Time after injection (weeks) 0 1 2.5 10 20 Antibody concentration (g/ml) Burkitts lymphoma 4 100,0 NaCl Antibody concentration (g/ml) tetulomab Diffuse large B cell lymphoma 25 100,0 Biodistribution of 177Lu-tetulomab in rituximab 10 µg/ml 1 µg/ml No treatment 50 MBq/kg 177Lu-tetraxetan-tetulomab No treatment 177 Lu-tetulomab SCID mice with injected Daudi cells 177 antibody 177 antibody 100 MBq/kg Lu-tetraxetan-tetulomab Lu-tetulomab 177 concentration 177 concentration Lu-Rituximab DLBCL transformed from low grade 19 100,0 30 Lu-Rituximab 177 16 177 Lu-tetulomab, blocked Lu-tetulomab, blocked 177 20 h 177 Lu-Rituximab, blocked 25 14 1200000 Lu-Rituximab, blocked 92 100,0 120 h 600000 192 h 12 1000000

20 /L) Low-grade unspecified 2 100,0 12 10 800000 C 400000 15 Maginal zone lymphoma 9 100,0 8 600000 ID/g % 10 6

400000 200000 RBC (10 28 100,0 4

200000 cells tumor of Number

Number of tumor cells tumor of Number 5 2 0 0 Small lymphocytic lymphoma 37 97,3 F 0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400 0 0 Time after seeding (h) Time after seeding (h) Total 217 99,5 -2 0 2 4 6 8 10 Heart Liver Brain Skull Blood Bowel Femur Spleen Kidney Muscle Time after injection (weeks) SCID mice were intravenously injected with 10 million Daudi cells one week before treatment with 20 µg/ml No treatment 2.5 µg/ml No treatment 177 177 177 antibody Lu-tetulomab antibody Lu-tetulomab 50 and 100 MBq/kg Lu-tetulomab, 50 µg HH1, 50 µg rituximab or NaCl. The Daudi cells will 177 177 concentration Lu-Rituximab concentration Lu-Rituximab Conclusions 177Lu-tetulomab, blocked 177Lu-tetulomab, blocked cause hind leg paralysis. A) Survival without hind leg paralysis. B) Survival without radiation toxicity, 177 177 177 9 177 Lu-Rituximab, blocked Lu-Rituximab, blocked • A significantly increased survival of mice treated with the Lu-tetulomab as compared i.e. WBC > 1.5·10 cells/L and no hind leg paralysis. C) Biodistribution of Lu-tetulomab. D) 600000 600000 with rituximab or tetulomab treatment was observed. Number of white blood cells. E) Number of platelets. F) Number of red blood cells. • The maximum tolerated dosage of the 177Lu-tetulomab treatment was between 50 and Spine CD20+ Spine HE Kidney CD20+ Kidney HE Ovary CD20+ Ovary HE 400000 400000 100 Mbq/kg for SCID mice.

200000 200000 • The CD37-tetulomab complex was internalized to a significantly larger extent than Number of tumor cells tumor of Number Number of tumor cells tumor of Number CD20-rituximab. 0 0 177 0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400 • The biodistribution of Lu-tetulomab in SCID mice showed higher retention times than Immunohistological preparations of mice tissue stained with HE and anti-CD20 antibody to Time after seeding (h) Time after seeding (h) usual and was different from nude mice. visualize infiltration of Daudi cells, which show a strong brown staining of the . • At the same antibody concentration 177Lu-tetulomab was significantly more effective in Dadui cells were incubated for 2 h and 18 h with different concentrations of 177Lu-tetulomab and 177Lu-rituximab. inhibiting cell growth in vitro than 177Lu-rituximab although the cell bound activity of At the same antibody concentration 177Lu-tetulomab was significantly more effective in inhibiting cell growth in vitro than 177Lu-rituximab although the cell bound activity of 177Lu-rituximab was higher. 177Lu-rituximab was higher. • This work warrants further preclinical and clinical studies with 177Lu-tetulomab