Targeting C3d in Combination with Daratumumab:a Strategy to Enhance Therapeutic Potential in Multiple Myeloma

The monoclonal antibody (mAb) daratumumab (DARA) targeting CD38 can induce minimal residual disease negative remissions in patients with multiple myeloma (MM). However, majority of patients subsequently relapse or become refractory to treatment (RRMM). Rapid reduction of surface CD38 levels in MM cells following DARA infusion due to trogocytosis by monocytes and granulocytes leads to reduced complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), effector functions that rely on antigen density. High expression of complement regulatory proteins, CD55 and CD59 on MM cell surface can also impede CDC activity. Therefore, modalities to overcome these limitations and improve clinical outcome for patients with RRMM are needed.

Daratumumab is a strong activator of complement. While a subset of cells may be lysed by the membrane attack complex, all cells reacted with daratumumab are opsonized by C3d, the terminal component of C3. C3d remains covalently attached at the cell surface. Thus, the newly acquired membrane bound C3d constitutes a target for additional intervention, particularly when the cell surface CD38 is lost or low following treatment with DARA. Using phage display technology, we generated and characterized recombinant rabbit/human chimeric mAbs that selectively bind C3 fragments including C3d but not full length C3. We selected two high affinity anti-C3d mAbs (clones 5S8 and d301 with kD values 3.35 nM and 0.99 nM, respectively) that showed reactivity against both human and mouse C3d. To deposit C3d on myeloma cell lines (MM1-S, MM1-R, RPMI8226, NCI-H929, KMS-12-PE) without activating CDC, the cells were reacted with DARA in C5 depleted normal human serum. Both 5S8 and d301 bound to the C3d opsonized cells. Based on SPR analysis these two mAbs do not compete for C3d binding suggesting that they recognize distinct epitopes. In normal human serum, the anti-C3d mAbs enhanced killing by CDC of DARA-opsonized target cells compared to DARA alone (P<0.0001). The non-targeting IgG1 isotype control mAb trastuzumab (TRA) did not bind C3d or enhance DARA-mediated killing.

To test the combination of DARA with anti-C3d mAbs in vivo, we used Balb/c-SCID mice bearing subcutaneous (s. c.) MM1-R tumors. First, using clones 5S8 and d301, we demonstrated in vivo binding of mouse C3d on MM1-R tumor cells harvested 24 h after a single intraperitoneal (i. p.) injection of DARA. Next, we challenged the mice s. c. with MM1-R cells and randomly divided them into the following treatment groups: 20 mg/kg DARA single agent (n=7), or combination treatments of 10 mg/kg of each of two mAbs: DARA + TRA (n=10), DARA + 5S8 (n=13), DARA + d301 (n=13). The mAbs were given via i. p. injections weekly starting on day 7 post tumor challenge and continued for 6 weeks. Palpable tumors were noticed in mice between days 7 and 14, and tumor growth was monitored by caliper measurements twice weekly. Mice were euthanized when tumors reached pre-defined limits. All the mice that received DARA + TRA showed progressive tumor growth with median survival of 38 days and all mice had to be euthanized by day 50. Doubling the dose of DARA to 20mg/kg slightly increased median survival to 43 days (P = 0.12 vs DARA+TRA). Two of 7 mice that received DARA alone (at 20 mg/kg) survived to day 175. Median survival for mice treated with DARA (10mg/kg) plus an anti-C3d mAb was 162 days (P = 0.006 for comparison to DARA 20mg/kg), and 13 of 26 (50%) mice in this cohort survived tumor-free beyond 200 days. There was no statistically significant difference between groups treated with 5S8 or d301 in combination with DARA (P = 0.23). All the surviving mice showed no evidence of tumor, weight loss or other morbidity indicating that they are essentially tumor-free. In summary, these results demonstrate that combination treatment of DARA with anti-C3d mAbs improves efficacy over single agent DARA leading to long-term survival in the MM.1R xenograft model. The combination of C3d targeting mAbs with DARA could improve clinical efficacy and overcome resistance to single agent therapy.

Rader:T-CURX: Current equity holder in publicly-traded company; Pyxis Oncology: Membership on an entity's Board of Directors or advisory committees; RiverVest Ventures: Membership on an entity's Board of Directors or advisory committees; Alnylam Pharmaceuticals: Research Funding. Wiestner:Acerta Pharma: Research Funding; Pharmacyclics: Research Funding; Abbvie company: Research Funding; Merck: Research Funding; Nurix: Research Funding; GenMab: Research Funding; Verastem: Research Funding.