CD47-CD138 Bispecific Antibody Exhibits Selective Targeting of Multiple Myeloma

CD47 is a “do not eat me signal” that is overexpressed on malignant tumors including multiple myeloma (MM). CD47 on tumor cells interacts with SIRP-α on macrophages and inhibits its phagocytosis enabling tumor progression. Clinical studies with antibodies blocking CD47-SIRP-α interaction to promote tumor phagocytosis have shown varying success. CD47 is widely expressed on normal tissues thus, monoclonal antibodies binding to these can lead to loss of treatment efficacy due to “sink effect” and to off-tumor on-target toxicity. In addition, when used as an adjuvant, CD47 antibody had a deleterious effect on CAR T cell survival and efficacy (Yamada-Hunter et al., Nature 2024).

To overcome these limitations and improve the on-tumor effect of CD47 blockade in MM, we generated a bispecific antibody (BsAb) inhibitor wherein an anti-CD47 single chain variable fragment (scFv) is combined with an anti-CD138 scFv. Since CD138 expression is mostly limited to MM we hypothesized that CD47-CD138 BsAb will preferentially bind to MM cells, thereby improving anti-tumor efficacy and at the same time minimizing off-tumor toxicity.

We generated CD47-CD138 BsAb using different linkers in Expi293 mammalian expression system. The expression of BsAb was best when human serum albumin (HSA, AA 403-427) was used as a linker compared to others like (G₄S)₃ or CBH-1. As these antibodies have been reformatted into a BsAb, we determined whether the BsAb was able to independently bind to its cognate antigens. As expected, BsAb showed strong binding to either immobilized recombinant CD47 or CD138, similar to the corresponding monospecific scFv in solid phase time resolved fluorescence assay.

We determined whether the BsAb can bind simultaneously to both CD47 and CD138 using a bridging ELISA assay. Recombinant CD138 was immobilized on a plate and incubated with either CD47 scFv or CD47-CD138 BsAb. After washing, the plate was incubated with biotinylated CD47, and binding was determined using streptavidin-HRP. Compared to PBS control (OD 0.048) or CD47 scFv (OD 0.078), CD47-CD138 BsAb (OD 2.95) was able to bind both antigens simultaneously.

We determined whether CD47-CD138 BsAb had increased binding to MM cells compared to CD47 scFv. OPM2 myeloma cell line was incubated with CD47 scFv or CD47-CD138 BsAb and binding was determined by flow cytometry. CD47-CD138 BsAb had a significantly increased binding to OPM2 cells compared to CD47 scFv (median fluorescence intensity (MFI) 7.82E+05 vs 7.07E+04, p <0.0001, n=3). We then determined whether this increased binding to MM cells will lead to increase phagocytosis. Monocytes isolated from peripheral blood were cultured in media with human serum for 7 days and then co-cultured with carboxyfluorescein succinimidyl ester (CFSE) labelled OPM2 cells for 4 hours in the presence of equimolar concentrations of CD47 scFv or CD47-CD138 BsAb and phagocytosis was determined by flow cytometry. There was increased phagocytosis of OPM2 cells in the presence of CD47-CD138 BsAb compared to CD47 scFv (12.2% vs 9.5%, p<0.05, n=2).

Next, we compared the binding of CD47 scFv or CD47-CD138 BsAb to T cells and, as expected, the MFI values were similar (3.82E+04 vs 3.54E+04, n=3) with no significant difference. We then determined whether CD47-CD138 BsAb would preferentially bind to MM cells in the presence of T cells. We mixed equal number of T cells and OPM2 cells and incubated them with CD47 scFv or CD47-CD138 BsAb and determined antibody binding by flow cytometry. There was significantly increased binding of CD47-CD138 to OPM2 cells compared to T cells (MFI 6.25E+05 vs 4.82E+04, p<0.0001, n=3). We then determined whether CD47-CD138 BsAb would preferentially induce phagocytosis of MM cells over T cells. T cells labelled with Cell Trace Far Red (CTFR) dye and OPM2 cells labelled with CFSE were co-cultured with macrophages in the presence of CD47-CD138 BsAb. Macrophages demonstrated preferential phagocytosis of OPM2 cells compared to T cells in the presence of CD47-CD138 BsAb (6.1% vs 1.9%, p<0.03, n=2).

We have developed a CD47-CD138 bispecific antibody that has enhanced and preferential binding to MM cells. We have demonstrated that this preferential binding translates into augmented phagocytosis of MM cells while relatively sparing CD47 expressing T cells. Sparing normal cells expressing CD47 will possibly reduce off-tumor toxicities associated with CD47 targeted therapies, therefore, enhancing their efficacy.