A Novel Class of Bifunctional Immunotherapeutic That Exploit a Universal Antibody Binding Terminus (uABT) to Recruit Endogenous Antibodies to Cells Expressing CD38 Demonstrates Anti-Multiple Myeloma Activity in Vitro and Ex Vivo against Patient Tumor Cells

Background:

Antibody recruiting molecules (ARM) are novel, immunotherapeutic bifunctional molecules composed of two active termini connected by a linker. One of the termini binds to a target molecule on a cancer cell. The other terminus, called universal antibody binding terminus (uABT), recruit all endogenous IgG antibodies independent of their antigen binding specificity. As a result, the target cell is "opsonized" by antibodies which then bring the immune effector cells to eliminate the target through various antibody-dependent destruction mechanisms.

Kleo Pharmaceuticals has developed a series of CD38-ARM mlecules which target human CD38 highly expressed by multiple myeloma cells. CD38-ARM compounds are able to mediate ADCC without depleting CD38 expressing immune effector cells like existing therapeutic antibodies such as Daratumumab.

Methods:

Cyclized peptides containing natural and non-natural amino-acid that selectively bind to human CD38 were identified using Peptidream Flexizyme-based, cell free Peptide Discovery Translation System. These peptides were linked to uABT antibody binder via a linker to generate the final CD38-ARM molecules

Binding of CD38-ARM was tested by ternary complex formation between CD38 expressing cells, CD38-ARM and labelled human IgG1. To confirm the activity of CD38-ARM, surrogate CD16a binding and signaling assays were performed using the NFAT Promega system. Antibody dependent cellular cytotoxicity (ADCC) assays using purified NK cells from multiple donors with polymorphism variants (V/V, F/F, and V/F) of CD16a were performed to confirm activity. Live cell imaging was utilized to assess the dynamics of NK-RAJI cell interactions mediated by CD38-ARM +/- IgG. We evaluated the ability of compounds to mediate complement dependent cytotoxicity (CDC). We tested the effect of CD38-ARM on human immune cell populations within PBMC and whole bone marrow (WBM) by flow cytometry.

Lastly, ex vivo samples from WBM of MM patients at diagnosis or relapse were used to evaluate CD38-ARM anti-tumor activity as well as off-target effects, without the addition of external source of IgG, through multiparametric flow-cytometry (CD45, CD19, CD38, CD138, CD56, CD27, CD8, CD117).

Results:

The CD38-ARM were shown to have the ability to bind to CD38 with a 7nM affinity and to human IgG1 and IgG2 with affinity of 15nM and 11nM by SPR.

Activity of KP compounds was observed in all assays except for CDC. In ternary assay, KP-6 had an apparent EC50 of 16nM while KP-7's EC50 was 6nM. Both KP-6 and 7 had comparable EC50s in the single digit nanamolar range in the NFAT activation assay induced by CD16a binding was confirmed using human IgG to induce, while Daratumumab had an apparent EC50 of 0.04nM. In the ADCC assay, both KP-6 & KP-7 had EC50s of 7 & 6nM respectively, while Daratumumab had an EC50 of 0.1nM. In addition, no NK cell depletion was observed when PBMC were treated with KP compounds, whereas a profound reduction in both percentages and absolute numbers in this cell subset was observed with Daratumumab treatment.

Increasing dose of CD38-ARM (range 0.1uM- 25uM) were tested in ex vivo WBM samples from MM patients together with a negative control and Daratumumab. At concentrations of 10uM and 25uM, CD38-ARM induced a significant reduction of MM cells achieving results comparable to those of Daratumumab activity (p >0.05 in both cases), while sparing all other CD38+ normal cells such as NK, T lymphocytes, monocytes and granulocytes, which are always reduced in the presence of Daratumumab.

Conclusions:

CD38-ARMs are able to kill MM cells by ADCC without depleting CD38 expressing immune cells contrary to existing antibodies such as Daratumumab. CD38-ARMs do not activate complement, which might be involved in the infusion reaction seen with Daratumumab. Most importantly, CD38-ARMs kill multiple myeloma cells ex vivo in patient bone marrow samples as well as plasma cell leukemia in patient blood. Combined with the in vivo efficacy data presented elsewhere, this data establishes the therapeutic potential of CD38-ARM. They also represent the first demonstration of the ARM platform ability to generate therapeutic agents tailored to a specific indication, by varying target binding moiety of the molecule.