Treatment with 177lu-HH1 Increases CD20 Expression in Non-Hodgkin Lymphoma Cells in Vitro and In Vivo

Immunotherapy (IT) with the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy has resulted in significantly improved response rate and survival in patients with various types of CD20 positive B-cell lymphoproliferative disorders. To be effective, rituximab depends on selective expression of a sufficient number of CD20 antigens per cell. Treatment with rituximab alone or in combination with chemotherapy can, however, result in disappearance of the CD20 expression, which may result in reduced clinical effect of subsequent CD20 targeted treatments. We have discovered that treatment of NHL in vitro and in vivo with the anti-CD37 antibody radionuclide conjugate (ARC) ¹⁷⁷Lu-DOTA-HH1 (¹⁷⁷Lu-HH1 or Betalutin™) results in an upregulation of the CD20 antigen expression, and therefore represents a rationale for a combination treatment with both agents.

The in vitro expression of CD20 in Burkitt’s Lymphoma, Daudi, cells 1-7 days after treatment with ¹⁷⁷Lu-HH1 increased up to 120 % when compared with cells treated with unlabeled mAb,  while Ramos (Burkitt’s Lymphoma) and Rec-1 (Mantle Cell Lymphoma) cells showed 10 to 30 % increase, indicating a variation of the antigen upregulation in vitro with different cell lines. An upregulation of CD20 at the same order of magnitude was observed when cells where treated with similar absorbed radiation doses of external beam radiation.

Treatment of  nude mice with Ramos xenografts with ¹⁷⁷Lu-HH1 resulted in a 3 times higher uptake of radiolabeled rituximab in tumor xenografts 5 days after start of treatment than in mice treated with unlabeled HH1 (p < 0.05) while uptake in normal organs was similar in both treatment groups (p > 0.05).

SCID mice with intravenously injected Rec-1 cells were treated with NaCl, 100 mg rituximab, 40 MBq/kg ¹⁷⁷Lu-HH1 or with the combination of 40 MBq/kg ¹⁷⁷Lu-HH1 followed with 100 mg rituximab 5 days later. The combination of  ¹⁷⁷Lu-HH1 and rituximab resulted in significantly improved survival as compared with NaCl or rituximab alone, and a strong therapeutic gain as compared with ¹⁷⁷Lu-HH1 alone (Table 1).

In conclusion, ¹⁷⁷Lu-HH1 treatment seems to improve uptake of rituximab and increase tumor suppression when used prior to anti-CD20 monoclonal antibody targeting in preclinical models. The reason for the upregulation of CD20 is probably related to the oxidative stress induced by the ARC-treatment, which will be evaluated in further studies. If the upregultation of CD20 is confirmed in clinical studies this effect could affect the way ARC and CD20 immunotherapy would be used in the future.

 Table 1. Therapy experiment groups and result

Group	Median ± SD	Surviving fraction at the end of the study	% Increase in symptom free survival compared to control
NaCl + NaCl	64 ± 2	0.1	----
NaCl + Rituximab	75 ± 10	0.3	15.4
177Lu-HH1 + NaCl	92 ± 14 *	0.3	43.8
177Lu-HH1 + Rituximab	> 132 *	0.7	> 106.3
*Significantly different from NaCl + NaCl group (p < 0.01)